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Background: Ovarian clear cell carcinomas (OCCC) constitute a rare subtype of epithelial ovarian cancer with distinct clinical features. Resistance to first-line platinum-based treatment is common. We recently reported deregulated Rho GTPase signaling in OCCC. Rho GTPases are small signaling G proteins, and a subfamily of the RAS superfamily. Rho GTPases are reported to regulate actin organization and cytoskeletal organization. We hypothesized that inhibiting Rho GTPases would have a potent cytotoxic effect on OCCC cell lines. Materials and Methods: Sulphorhodamine B (SRB) assays were used to assess the antiproliferative effects of 72h of treatment with the cholesterol-lowering agent simvastatin, the pan GTPase inhibitor CID1067700 (CID) and carboplatin (as control) in the two OCCC cell lines JHOC5 and OVMANA. Single effects (IC50) and combination effects were calculated using the Chou-Talalay method. We assessed cytoskeletal integrity using phalloidin staining of actin and DAPI for cell death. Western blotting was used to assess the expression of cleaved PARP (cPARP) after 4h and 24h of treatment. Results: All three drugs had similar IC50 values in both OCCC cell lines; however, they were significantly different from the high-grade serous ovarian cancer control cell line CAOV3. JHOC5 and OVMANA were more resistant to carboplatin (IC50: 65.3 µM and 76.2 µM, respectively) compared to CAOV3 (IC50: 16.4 µM). JHOC5 and OVAMANA were more sensitive to simvastatin and CID (JHOC5: 6.7 µM/69.2 µM, OVMANA: 10.3 µM/84.9 µM, simvastatin/CID1067700) compared to CAOV3 (27.7 µM/122.5 µM). The combination index (CI) for either CID or simvastatin in combination with carboplatin was moderately antagonistic for all cell lines (CI: 1.6-2.4). Combinations of CID and simvastatin were mildly to moderately synergistic (CI: 0.6-0.8) for all cell lines. Simvastatin and CID were found to interfere with actin organization, compared to carboplatin, with simvastatin also interfering with cell membrane integrity. A small increase in cPARP was observed after 24h of treatment with all drugs in single regimens. Conclusion: Cytotoxic effects of simvastatin and CID both in single regimens or in combination were achieved with lower doses in OCCC cell lines than in the high-grade serous cells; however, combinations of simvastatin and CID were found to be mildly synergistic in all cell lines. Simvastatin was found to be more potent than CID. Interestingly, all combinations with carboplatin were found to be moderately antagonistic. Actin organization was more extensively disrupted with simvastatin than CID. An initial assessment of cell death mechanisms suggested a mechanism working through cleaved PARP. However, these findings provide a basis for further studies into the molecular mechanisms of simvastatin and CID in OCCC. These studies are currently under way, including an in-depth assessment of the underlying signaling and cell death mechanisms. Citation Format: Nicolai S. Arildsen, Ingrid Hedenfalk. Targeting Rho GTPases in ovarian clear cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2880.

High grade serous ovarian cancer, the most lethal subtype of ovarian cancer, can originate in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). PAX8 is a lineage specific transcription factor that is ubiquitously expressed in HGSOC. We have shown that knockdown of PAX8 using shRNA in multiple ovarian tumor cells lines leads to apoptosis, suggesting that PAX8 plays an essential role in cancer survival. In this study, we used CRISPR technology to delete PAX8 from the OVCAR8 cell line. PAX8 deletion led to a decrease in migration and invasion in vitro and an increase in survival and a reduction in tumor volume in vivo. Previous work using RNA-sequencing and ChIP-sequencing identified cell adhesion as a top differentially expressed gene between malignant ovarian cancer and benign fallopian tube cell lines. We performed quantitative proteomic analysis of the OVCAR8-PAX8-/- cell line to define PAX8 altered proteins. We also performed quantitative proteomics and transcriptomic analyses on a previously generated murine OSE cell line with forced PAX8 expression (MOSE-PAX8). These analyses identified several genes that contribute to an increase in migration and EMT. Specifically, our data indicates PAX8 upregulates key drivers involved in altering cell morphology including the GTPases: RhoA, Cdc42, and Ras. Inhibition of RhoA led to a greater decrease in migration for both OVCAR8-PAX8-/- and MOSE-PAX8 when compared to control. Inhibition of Ras had a greater effect in OVCAR8-PAX8-/- while inhibition of Cdc42 had a greater effect in MOSE-PAX8. These data provide a mechanistic explanation for the role of PAX8 on increasing migration and metastasis in ovarian cancer. Citation Format: Laura Hardy, Melissa Pergande, Angel Hernandez, Stephanie Cologna, Joanna Burdette. PAX8 increases migration and metastasis of high grade serous ovarian cancer through upregulation of Rho GTPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4039.

Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)–induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action. [Cancer Res 2009;69(2):518–25]

The epithelial cell transforming sequence 2 (ECT2), a member of the Dbl family of guanine nucleotide exchange factor for Rho GTPases, is required for cytokinesis. The tumor suppressor p53 plays a crucial role in coordinating cellular processes, such as cell cycle arrest and apoptosis, in response to stress signals. Here, we showed that ECT2 is negatively regulated by wild-type p53 but not tumor-derived mutant p53 or other p53 family members. In addition, ECT2 is down-regulated in multiple cell lines by DNA damage agents and Nutlin-3, an MDM2 antagonist, in a p53-dependent manner. We also showed that the activity of the ECT2 promoter is repressed by wild-type p53, and to a lesser extent, by p21. In addition, the second activation domain in p53 is necessary for the efficient repression of ECT2. Importantly, we found that the ECT2 gene is bound by p53 in vivo in response to DNA damage and Nutlin-3 treatment. Furthermore, we provided evidence that inhibition of protein methyltransferases, especially arginine methyltransferases, relieve the repression of ECT2 induced by DNA damage or Nutlin-3 in a p53-dependent manner. Finally, we generated multiple cell lines in which ECT2 is inducibly knocked down and found that ECT2 knockdown triggers cell cycle arrest in G1. Taken together, we uncovered a novel function for ECT2 and provided a novel mechanism by which p53 represses gene expression via protein methyltransferases. (Cancer Res 2006; 66(12): 6271-9)

Development of cutting edge molecular techniques greatly enhanced the knowledge of the biology of the prostate cancer (CaP). These results helped us for early detection of the disease and also developing targeted therapies of CaP. It is a fact that other than Androgen Receptor (AR), there is no specific gene candidate that could be either linked for initiation, progression or treatment of CaP. Emerging evidences suggest that a small set of microRNAs (miRNA) linked to pathogenesis of CaP especially leading to castration resistant prostate cancer (CRPC). The current conventional therapies have not proven to be successful for CRPC that compelled us to identify new targets or potent small molecules to efficiently suppress the growth of advance stages of CaP. In genome wide miRNA profiling, we have found that hsa-miR-301a-3p is expressed at stage specific manner in prostate tumor as compared to controls. These results encouraged us to dissect the role of miRNA-301a in preclinical models of CaP. miR-301-3P was highly expressed (10-14 folds) in CaP cell lines (LNCaP, C4-2B, DU-145 and PC-3) when compared to normal prostate epithelial cell lines (PZHPV-7 and PrEC). Silencing specifically miR-301a-3p resulted in inhibition of proliferation, colony forming abilities, invasion, migration and cell adhesion of CaP cells. At molecular levels we found, inhibition of miR-301a-3p upregulated its direct target of tumor suppressor TXNIP/VDUP1 expression. In addition, silencing miR-301a-3p negatively activated Rho GTPases family proteins; specifically Rho1 and Rac-1 ubiquitous signaling, resulted in transcriptional down-regulation of Epithelial-Mesenchymal-Transition (EMT) markers like slug and β-catenin. Our immunofluorescence studies suggest E-cadherin is highly expressed in the membranes and at the cellular junctions when miR-301a-3p were inhibited in CRPC cells. On the contrary, over expression of miR-301-3P, increased proliferation of CaP cells and activated Rho GTPase signaling (Rho A/B/C, Rac1 and CDC42) and mesenchymal markers like slug, b-catenin resulted in invasion and migration of CaP cells. Currently, we are interrupting Rho signaling pathways, especially upstream events of RhoA effectors Rho-associated kinases (ROCK) in miR301 over expressed CaP cells and dissecting the downstream events and correlate with phenotypic changes of CaP cells. Our in vivo assays may reveal whether silencing miR-301-3p could be a potential target for the treatment of CRPC cells. Citation Format: Vittal Kurisetty, Trinath P. Das, Rama S. Reddy, Jessica Stiles, Brad Bryan, Chendil Damodaran. The role of miR-301-3P in the regulation of Rho GTPases mediated EMT signaling in castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5329. doi:10.1158/1538-7445.AM2013-5329 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Tumor metastasis causes the majority of cancer deaths. Dysregulation of small GTPases such as K-Ras and Rho family GTPases (RhoA, RhoC, Rac1 and Cdc42) is critical to drive the invasion and metastasis of a variety of cancers including breast carcinomas. In order to fulfill their biological functions, these GTPases must properly localize subcellularly and associate with cellular membrane. The association of these proteins to membranes is regulated by a series of post-translational modifications including either farnesylation or geranylgeranylation of the C-terminal CaaX motif, thus providing the rationale to target the farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) as the potential anti-cancer drugs. However, FTase inhibitors (FTIs) failed in clinic due to alternative prenylation, which causes K-Ras to be geranylgeranylated, and their inability to target geranylgeranylated proteins such as Rho family GTPases. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are synthetic farnesol or geranylgeraniol analogs that act as alternative substrates for FTase or GGTase but do not support normal prenyl function. Here, we test the ability of our lead PFIs anilinogeraniol (AGOH; farnesol analog) and anilinofarnesol (AFOH; geranylgeraniol analog) to impact the migratory and invasive capacities of breast carcinoma cells. We found that AGOH treatment effectively decreased the invasion of MDA-MBA-231 in a two-dimensional invasion assay at 100 mM while it blocked invasive growth in three-dimensional (3D) culture model in as little as 20 μM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase, which ribosylates and inactivates Rho proteins. The results demonstrate that Rho proteins are essential for the invasive growth of MDA-MB-231 cells. To determine how AGOH impacts Rho signaling, we performed RhoA and RhoC activity assays in MDA-MB-231 and MDA-MB-468 cells. We determined that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA/C activation and invasive growth where it inhibited RhoA and RhoC activation completely at 20 μM and blocked the invasive growth of MDA-MB-231 cells at ≤ 5 μM. Interestingly, both AGOH and AFOH had no effect on the growth of MCF10A cells in 3D culture. Collectively, this study demonstrates that our lead PFI compounds dramatically inhibited breast cancer invasion, at least in part, through affecting RhoA and RhoC function; thus, suggesting that targeting the function of Rho prenylation by PFIs will offer a promising mechanism for the treatment of breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-11.

Tumor dormancy has been a very real problem in breast cancer survivors where recurrences are common and usually fatal. Molecular mechanisms involved in this process are often poorly understood and are a hot spotin breast cancer research today. We have found that Rho GTPases, specifically RhoC, is hyperactivated in low metastatic dormant breast tumor cells while RhoA is downregulated. We also found RhoC causes phosphorylation of c-jun NH2 terminal kinase (JNK) in these cells which in turn activates the process of autophagy, a possible mode of cell survival in dormant tumor cells. Modulating RhoC affected phosphorylation of JNK. Also, abrogating JNK by a pharmacological inhibitor SP600125 inhibited expression of the autophagy marker LC3B-II. Autophagy genes like Atg 5 and Atg 12 were expressed along with the autophagy marker LC3B-II. On inhibiting autophagy by a specific inhibitor 3-Methyladenine (3-MA), cells did not readily undergo apoptosis but on stressing them doubly with LY294002, an inhibitor of Class I PI3K, along with 3-MA (inhibitor of Class III PI3K), significant number of dormant cells underwent apoptosis as observed by TUNEL staining. We also found that p190RhoGAP was activated in these dormant cells which may lead to the observed downregulation of RhoA activation levels. Involvement of beta-1 integrin along with c-src in activation of RhoC was observed in these dormant cells. We are in the process of further elucidating these signal transduction processes that might be involved in cellular dormancy of breast cancer. A better understanding of some of the cellular mechanisms of induction and sustenance of dormancy may aid in formulating therapies for annihilating these dormant tumor cells thus preventing the often fatal relapses in breast cancer survivors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-16.

Activation of the small GTPase RHOA has strong oncogenic effects in many tumor types, although its role in colorectal cancer remains unclear. We show that RHOA inactivation contributes to colorectal cancer progression/metastasis, largely through the activation of Wnt/β-catenin signaling. RhoA inactivation in the murine intestine accelerates the tumorigenic process and in human colon cancer cells leads to the redistribution of β-catenin from the membrane to the nucleus and enhanced Wnt/β-catenin signaling, resulting in increased proliferation, invasion and de-differentiation. In mice, RHOA inactivation contributes to colon cancer metastasis and reduced RHOA levels were observed at metastatic sites compared to primary human colon tumors. Therefore, we have identified a new mechanism of activation of Wnt/β-catenin signaling and characterized the role of RHOA as a novel gene with tumor suppressor activity in colorectal cancer. These results constitute a shift from the current paradigm and demonstrate that RHO GTPases can suppress tumor progression and metastasis. Citation Format: Paulo Rodrigues, Irati Macaya, Sarah Bazzocco, Elena Andretta, Rocco Mazzolini, Higinio Dopeso, Silvia Mateo-Lozano, Josipa Bilic, Fernando Cartón-García, Rocio Nieto, Lucia Suárez-López, Elsa Afonso, Stefania Landolfi, Javier Hernandez-Losa, Kazuto Kobayashi, Santiago Ramón y Cajal, Josep Tabernero, Niall C. Tebbutt, John M. Mariadason, Simo Schwartz Jr, Diego Arango. RHOA inactivation enhances Wnt signaling and promotes colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2015-2058

Rho GTPases activated in cancer cells drive proliferation, migration, and metastasis. Thus, RhoGAP proteins, which negatively regulate Rho GTPases, are generally thought to function as tumor suppressors. Here this expectation was challenged by characterization of ARHGAP18, a RhoGAP family member that is selectively overexpressed in highly migratory triple-negative breast cancer (TNBC) cells. In human breast tumors, higher ARHGAP18 levels associated with worse overall survival, recurrence-free survival, and metastasis-free survival. In TNBC cells, ARHGAP18 deletion increased RhoA activation but reduced growth, migration, and metastatic capacity. Mechanistic investigations revealed that ARHGAP18 levels were controlled by miR-200b, the enforced expression of which was sufficient to activate RhoA, enhanced formation of focal adhesions and actin stress fibers, and reduced migration and metastasis. Enforced elevation of ARHGAP18 where miR-200b was stably expressed reduced RhoA activity but increased cell migration. Pharmacologic inhibition of the Rho effector kinase ROCK blocked RhoA signaling and reversed the inhibitory effect of miR-200b on cell migration. Finally, ARHGAP18 overexpression or ROCK inhibition was sufficient to overcome metastatic blockade by miR-200b. Taken together, these results define opposing roles for oncogenic ARHGAP18 and tumor suppressive miR-200b in determining TNBC cell migration and metastatic prowess. Cancer Res; 77(15); 4051–64. ©2017 AACR.

Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers. Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression. Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells. Citation Format: Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5153.

Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a metastasis suppressor in bladder and possibly other cancers. This protein is a member of a family of proteins that maintain Rho GTPases in the cytoplasm and inhibit their activation and function. To understand the mechanism of metastasis suppression, we compared effects of RhoGDI1 and RhoGDI2. Despite showing much stronger inhibition of metastasis, RhoGDI2 is a weak inhibitor of Rho GTPase membrane targeting and function. However, point mutants that increase or decrease the affinity of RhoGDI2 for GTPases abolished its ability to inhibit metastasis. Surprisingly, metastasis suppression correlates with increased rather than decreased Rac1 activity. These data show that RhoGDI2 metastasis inhibition works through Rho GTPases but via a mechanism distinct from inhibition of membrane association. [Cancer Res 2009;69(7):2838–44]

Metastatic disease is the major cause of breast cancer–related death and despite many advances, current therapies are rarely curative. Tumor cell migration and invasion require actin cytoskeletal reorganization to endow cells with capacity to disseminate and initiate the formation of secondary tumors. However, it is still unclear how these migratory cells colonize distant tissues to form macrometastases. The E6-associated protein, E6AP, acts both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors. We report that E6AP suppresses breast cancer invasiveness, colonization, and metastasis in mice, and in breast cancer patients, loss of E6AP associates with poor prognosis, particularly for basal breast cancer. E6AP regulates actin cytoskeletal remodeling via regulation of Rho GTPases, acting as a negative regulator of ECT2, a GEF required for activation of Rho GTPases. E6AP promotes ubiquitination and proteasomal degradation of ECT2 for which high expression predicts poor prognosis in breast cancer patients. We conclude that E6AP suppresses breast cancer metastasis by regulating actin cytoskeleton remodeling through the control of ECT2 and Rho GTPase activity. These findings establish E6AP as a novel suppressor of metastasis and provide a compelling rationale for inhibition of ECT2 as a therapeutic approach for patients with metastatic breast cancer. Cancer Res; 76(14); 4236–48. ©2016 AACR.

Tumor dormancy has been a very real problem in breast cancer survivors where recurrences are common and usually fatal. Molecular mechanisms involved in this process are often poorly understood and are a “hot-spot” in breast cancer research today. Ongoing efforts in our laboratory has shown that Rho GTPases, specifically RhoA and RhoC, play a significant role in dormancy of breast cancer. We have currently established two in vitro models of breast tumor dormancy — one involving growth of the breast cancer cell line MCF7 on fibronectin and subsequent treatment with basic fibroblast growth factor and the other involving treatment of MCF7 cells with the farnesyl transferase inhibitor FTI-L-744,832. In both these models, we find high levels of active RhoC and almost undetectable levels of active RhoA GTPases in quiescent breast tumor cells. Investigating further downstream we found c-jun N-terminal kinase (JNK) is phosphorylated in these dormant cells but not p38mitogen activated protein kinase(MAPK) or extracellular signalregulated kinase(ERK1/2). On inhibiting JNK with a pharmacological inhibitor specific for JNK, SP600125, we found dormancy could be interrupted and cell proliferation was induced. JNK has been implicated in inducing autophagy in cells by activating autophagy gene Atg5 and Beclin1 (BCN1) [Byun et al., Carcinogenesis, 2009]. Since autophagy can be a mode of survival for the dormant tumor cell [Matthew et al., Cancer Cell, 2006] we explored the possibility of autophagy being induced in these dormant tumor cells via JNK by employing the autophagy marker LC3-II. We found upregulation of the autophagy marker, thus revealing autophagy as a strategy for survival for these tumor cells in unfavorable tumor microenvironment. We are currently investigating the molecular mechanisms underlying the induction of dormancy in breast tumor cells by Rho GTPases through JNK and the dormant tumor survival strategy through autophagy. We believe that targeting these implicated molecules like RhoA and RhoC GTPases, JNK or some of the autophagy genes might yield therapeutic benefits for breast cancer patients in remission and wipe out fears of deadly metastatic recurrences thus prolonging survival. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-10.

Background and Objective: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. Although the primary cause of tumor-related death in ES is relapse at distant sites, the mechanisms underlying metastatic relapse remain to be fully elucidated. In the current study we are investigating the role of the CXCR4/CXCL12 chemokine axis as a mediator of ES metastasis. Methods: Expression of CXCR4 transcript and surface protein were quantified using qRT-PCR and flow cytometry. Label-retaining, putative cancer stem cells were isolated 10-12 days following PKH-dye incorporation into cell membranes. Chemotactic migration and invasion to SDF-1α were evaluated in unmodified cells and in cells following CXCR4 knockdown or exposure to the CXCR4 antagonist AMD3100. Small molecule inhibitors of Rho-GTPases were used to assess the contribution of these proteins to CXCR4-dependent migration. Results: Baseline expression of CXCR4 transcript and surface protein varied widely among ES cell lines. Immunohistochemical staining of primary tumors also revealed marked variability of CXCR4, ranging from completely undetectable to high-level expression. Interestingly, CXCR4 transcript was increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells implicating CXCR4 as a potential marker of ES cancer stem cells. To determine if CXCR4 expression is static or responsive to changes in the microenvironment we measured transcript levels and the frequency of CXCR4+ cells under different experimental conditions. Significantly, serum deprivation, hypoxia and growth constraint (cell confluence) all resulted in upregulation of CXCR4 mRNA and CXCR4+ cell frequency in each of 4 different ES cell lines. This upregulation of CXCR4 was rapidly reversed upon removal of each of the stressors indicating the plastic and dynamic nature of CXCR4 regulation. In addition, within 3 weeks of FACS sorting, TC-32 ES cells that had been sorted into CXCR4+ and CXCR4- populations reverted to their baseline state, demonstrating that CXCR4+ cells can change into CXCR4- cells and vice versa. Functionally, CXCR4+ ES cells migrated and invaded towards SDF-1α, the ligand for CXCR4 and this chemotactic migration and invasion were impeded pharmacologically by AMD3100, and genetically by CXCR4 knockdown. Interestingly, we observed complete abrogation of invasion through Matrigel in CXCR4 knockdown and AMD3100 treated cells suggesting a critical role for CXCR4 in mediating ES cell invasion through basement membranes. To begin to define the mechanism of CXCR4-mediated invasion we exposed ES cells to small molecule inhibitors of the Rho-GTPases CDC42 and RAC1. Importantly, sub-cytotoxic doses of both compounds suppressed ES cell migration towards SDF-1α. Conclusions: We have demonstrated the highly dynamic nature of CXCR4 expression in ES cells and have shown that this molecular plasticity allows ES to switch back and forth between CXCR4- and CXCR4+ states in response to changes in the microenvironment. Significantly, ES cells that express high levels of CXCR4 are highly migratory and invasive in response to chemotactic signaling from SDF-1α and preliminary data suggest that Rho-GTPases CDC42 and/or RAC1 mediate this aggressive phenotype. We propose a model in which stressors in the microenvironment induce ES cells to upregulate CXCR4 thus contributing to cellular migration and invasion. Ongoing work is testing whether CXCR4+ ES cells are more metastatic than CXCR4- cells in vivo. The CXCR4/CXCL12 axis may thus be a novel therapeutic target for the prevention of ES metastasis. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Christopher A. Scannell, Rashmi Chugh, Dafydd G. Thomas, Elizabeth R. Lawlor. Expression of CXCR4 is highly dynamic in Ewing sarcoma and its upregulation in response to microenvironmental stress promotes cell migration and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A56.

Nasopharyngeal carcinoma (NPC) has distinctive ethnic and geographic distributions, with the highest incidence in Southern China. Infection with the human herpesvirus, Epstein-Barr virus, genetic predisposition, dietary, and environmental factors are high-risk factors for the development of NPC. Evidence indicates that loss of heterozygosity on chromosome 3 is one of the early events contributing to the cancerization of normal nasopharyngeal epithelial cells. By using a chromosome 3 NotI genomic microarray, Fibulin-2 (FBLN2) mapping to chromosome 3p25.1, was previously identified as a candidate tumor suppressor. FBLN2, which is an extracellular matrix (ECM) protein, contains four domains, regions consisting of cysteine-rich and cysteine-free, anaphylatoxin-like domain, serial EGF-like repeats, and a fibulin module. FBLN2 is silenced by promoter hypermethylation in NPC. Re-expression of FBLN2 in NPC cell lines significantly reduces colony formation, cell proliferation, migration, invasion, and in vivo tumor formation and angiogenesis. In this study, the aim is to investigate the molecular mechanisms and signaling pathways of FBLN2-mediated tumor suppressive and anti-angiogenesis effects. Full-length FBLN2 was subcloned into inducible tetracycline-regulated and lentivirus systems. Phosphorylated forms of the key signaling molecules, Erk1/2 and NFκB, were found to be significantly reduced in FBLN2 stably-expressing NPC cell lines after phosphorylation target screening using Western blot analysis. FBLN2 also induces changes in the cytoskeleton arrangement, possibly through the regulation of Rho GTPases. Thus, the activities of Rho GTPases were determined by the Rho pull-down assays and immunofluorescence staining with confocal microscopy. FBLN2 is able to reduce the tumor formation ability via inhibiting Erk1/2 and NFκB pathways and to inhibit the cell migration and invasion via regulating Rho GTPase activities. FBLN2 is a secretory protein and is generally silenced in NPC, suggesting the possibility that it may be useful as a diagnostic marker and/or new target for therapeutics of NPC. Therefore, these studies provide strategic new insights into the understanding of the mechanistic role of FBLN2 in NPC and enhance our understanding of the molecular genetic basis of NPC tumorigenesis. Citation Format: Wai Ho Shuen, Maria Li Lung. Fibulin-2 suppresses tumor growth and angiogenesis through the inhibition of Erk1/2 and p65 pathways in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2013-4309

Breast cancer development and progression is enabled by alterations in the surrounding stroma originating from stromal interactions with cancerous epithelium. Rho GTPases and their regulators have been implicated as important mediators of stromal-epithelial interactions in mammary epithelial cells (MECs), and alterations in the microenvironment profoundly affect MEC morphogenesis in a Rho GTPase-dependent manner. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP), a major inhibitor of Rho GTPases, has pro-tumorigenic functions during MMTV-Neu induced mammary tumorigenesis. We hypothesized that p190B overexpression in the epithelium promotes mammary tumor development by altering stromal-epithelial interactions to drive MEC proliferation and disrupt tissue architecture. To investigate this hypothesis, we examined the effects of p190B overexpression in the developing mammary gland (MG) using Tet-regulatable p190B overexpressing mice. Interestingly, overexpression of p190B in the mammary epithelium during postnatal MG development causes hyperbranching and disorganization of the ductal tree. Using a combination of in vivo morphometric and immunohistochemical analyses and in vitro stromal-epithelial co-cultures, we have demonstrated that p190B overexpression in MECs activates fibroblasts in the adjacent microenvironment leading to increased extracellular matrix (ECM) deposition, elevated expression of the collagen crosslinking enzyme lysyl oxidase, and increased expression of potent regulators of branching morphogenesis, including FGF and TGFβ growth factors. These stromal alterations correlated with increased proliferation of p190B overexpressing MECs in vivo. Taken together, these data suggest that p190B overexpression may contribute to mammary tumorigenesis by creating a pro-tumorigenic stromal environment leading to increased MEC proliferation and altered tissue architecture. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 32. doi:1538-7445.AM2012-32

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. The 5-year survival rate of HNSCC has remained stagnant despite advances in the clinical management of this disease. Understanding the molecular pathways that lead to aggressive HNSCC is crucial to identify new “druggable” targets for anti-cancer drug development. Protein kinase Cepsilon (PKCε) is elevated in HNSCC and regulates cell invasion and motility thorough activation of Rho GTPases. Our laboratory and others provide evidence that PKCε is a transforming oncogene that phosphorylates and modulates a cascade of signal transduction pathways, including Akt, EGFR, Rho GTPases, and Stat3. Thus, targeting PKCε may be an attractive therapeutic approach to dampen multiple hyper-active signaling pathways in concert. At this time, the molecular mechanism of PKCε dysregulation in HNSCC remains to be elucidated. In silico analysis with TargetScan, MiRBase, and miRanda identified numerous putative microRNA (miR) binding sites in the 3’-UTR region of PKCε. We screened a panel of miRs using qPCR and miR-107 was identified to have an inverse association with PKCε in HNSCC cell lines with high endogenous PKCε. Ectopic expression of miR-107 in several HNSCC cell lines significantly reduced PKCε mRNA expression and protein levels. The expression of a luciferase reporter construct containing the 3’-UTR of PKCε was down-regulated by miR-107 and deletion/mutation of the three cognate miR-107 binding sites completely abolished the regulation by miR-107. These observations confirm PKCε as a validated target of miR-107. Ectopic expression of miR-107 in HNSCC cell lines significantly inhibited cell proliferation, DNA replication, clonogenic survival, invasion and migration. Tumor take and tumor growth of HNSCC cells was suppressed by miR-107 in a pre-clinical nude mouse model of HNSCC. Lastly, miR-107 was significantly reduced and showed an inverse expression relationship with PKCε in human primary HNSCC tumors. These results indicate that miR-107 regulates PKCε levels to control the tumorigenicity of HNSCC cells. Taken together, our data substantiate the potential application of miR-107 as a novel anti-cancer therapeutic for HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 144. doi:10.1158/1538-7445.AM2011-144

Breast cancer metastasis, when cancer cells move and establish tumors in distant organs, confounds treatment options. Therefore, there is an unmet need for targeted therapeutics to address metastasis, especially in the intractable HER2 positive (+) breast cancer type. HER2 (+) breast cancer is treated with the monoclonal HER2 antibody trastuzumab and the HER2/EGFR targeting small molecule lapatinib. However, ˜20% of early stage breast cancer patients and ˜70% of patients with metastatic disease are resistant to trastuzumab. Trastuzumab-resistant breast cancers circumvent HER2 inhibition via bypass signaling, which includes the PI3-K/Akt/mTOR pathway. HER2/EGFR and PI-3K/Akt signaling activate the Rho GTPases Rac and Cdc42, and overexpression of Rac or its downstream effector p21-activated kinase (PAK) significantly diminishes response to anti HER2 therapy and patient survival. Therefore, targeting Rac and Cdc42, pivotal regulators of cancer cell migration/invasion, and thus, metastasis, is a viable option to overcome HER2/EGFR therapy resistance. First, we designed and developed the Rac inhibitor Ehop-016 (US patents 8,884,006 B2, 9,278,956B1), which inhibits Rac activation with an IC50 of 1 μM and reduces breast cancer growth and metastasis in mouse models of trastuzumab resistant HER2 (+) breast cancer. A more potent dual Rac and Cdc42 inhibitor, MBQ-167 (US Patent 9,981,980 B2), inhibits Rac and Cdc42 activation with IC50s of 103 nM and 78 nM, respectively. In vivo, MBQ-167 inhibits trastuzumab resistant HER2 (+) mammary tumor growth and metastasis by ˜90-100% (Humphries-Bickley et al. 2017, Mol Cancer Therap). Therefore, Rac/Cdc42 inhibition blocks cell proliferation, cell cycle progression, migration, and induces apoptosis to ultimately impede tumor growth and metastasis in trastuzumab resistant HER2 (+) breast cancer models. To further investigate the role of Rac/Cdc42 inhibitors in overcoming HER2 therapy resistance, we developed a lapatinib resistant variant of the SKBR3 HER2 (+) breast cancer cell line and found that Rac was overexpressed and over activated in the therapy resistant variant compared to the parental cells. Accordingly, the Rac/Cdc42 inhibitors overcame lapatinib resistance by decreasing cell viability and inducing apoptosis in parental and SKBR3 therapy resistant variants. Pharmacokinetic analysis in mouse plasma demonstrated that the bioavailability of EHop-016 and MBQ-167 is ˜30% with a half-life of 3-4 h. Therefore, to increase the bioavailability of Rac/Cdc42 inhibitors and to facilitate sensitization of trastuzumab therapy, we designed and developed novel nanoliposomal formulations containing Rac/Cdc42 inhibitors with trastuzumab on the outer surface to target HER2 (+) breast cancer cells. This data demonstrates the utility of developing Rac/Cdc42 inhibitors as mono or combined therapy with current targeted therapeutics for HER2 (+) breast cancer. Citation Format: Rivera-Robles MJ, Medina-Vlazquez JI, Borrero-Garcia LD, Velazquez-Vega LE, Maldonado MdM, Ruiz-Calderon J, Castillo-Pichardo L, Vivas-Mejia P, Dharmawardhane S. Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-14.

Aggressive metastatic neuroblastoma confers a poor clinical prognosis. Stathmin is a microtubule destabilizing protein highly expressed in advanced stage (metastatic) neuroblastoma (1) that promotes metastasis of non-neuronal cancers (2). However, stathmin's role in neuroblastoma metastasis is unclear. The aims of this study were to determine the functional and mechanistic role of stathmin in neuroblastoma metastasis. During metastasis, restructuring of the cell cytoskeleton, in particular the actin network is necessary to drive cell migration/invasion. The Rho-GTPase/LIM kinase/cofilin pathway regulates actin remodeling. Activation of LIM kinases (LIMK1/2) by Rho-GTPases induces actin polymerization via phosphorylation (inactivation) of cofilin. Interestingly, stathmin is also regulated downstream of the Rho-GTPases. We therefore investigated this pathway as a potential mechanism underlying stathmin's role in neuroblastoma cell migration/invasion. Methods: Studies were performed using siRNA to knockdown stathmin in 2 independent neuroblastoma cell lines, BE(2)-C and SY5Y. Stathmin knockdown was confirmed by qPCR and Western blot. SiRNA-treated cell lines were subjected to in vitro migration and invasion assays using fetal calf serum as a chemo-attractant. The expression of actin-regulatory proteins LIMK1/2 and the actin-severing protein cofilin were analyzed by Western blot. Actin filaments within neuroblastoma cells were visualized by phalloidin staining. Results: Stathmin knockdown significantly reduced the migration of BE(2)C (47% reduction p Conclusion: Stathmin plays a pro-migratory and invasive role via regulation of the actin network in neuroblastoma. These identify stathmin as a promising new therapeutic target to treat metastatic neuroblastoma. 1. N. Hailat et al. J Clin Invest 1991; 88: 341 2. B. Belletti et al. Mol Biol Cell. 2008: 19: 2003 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5141.

A tumor suppressor commonly targeted in human cancers is the retinoblastoma (pRb) protein, a regulator of the G1/S phase transition of the cell cycle. pRb itself is inactivated with a frequency of over 90% in a subset of human tumors such as retinoblastomas, osteosarcomas, and small cell lung carcinomas. Ours studies suggest a novel role of pRb as a regulator of the activity of the small Rho/GTPase Rac1. Our data show that pRb can regulate Rac1 activity by controlling the levels of the p21-activated protein kinase 1 (PAK1), a main Rac-1 effector. qRT-PCR, immunoblot and immunofluorescence analyses showed that pRb loss in murine osteoblasts results in a dramatic increase of PAK1 mRNA and protein. In order to determine if this increase is due to increased mRNA half-we performed mRNA stability assays using culturing cells with actinomycin D to block new transcription and to assess the half-life of pre-existing PAK1 mRNA molecules. We did not find a significant change in PAK1 mRNA half-life when comparing pRb-expressing vs pRb-deficient osteoblasts, suggesting that pRb regulation of PAK1 is not post-We next tested whether PAK1 regulation by pRb occurs at the trancriptional level, a scenario that would be consistent with pRb's well characterized role as a transcriptional regulator. We used a genomic PCR strategy to amplify several fragments of the PAK1 promoter spanning an area from − 601 to + 159 downstream into the coding region. Fragments were cloned into a luciferase reporter plasmid and the resulting constructs were transfected into pRb-expressing and pRb-osteoblasts, followed by luciferase activity assays. We identified a pRb-responsive element in the promoter fragment spanning from −201 to +159 that seems to be responsible for PAK1 transcriptional repression in pRb-expressing osteoblasts, suggesting the presence of pRb-sensitive elements within this Pak1 promoter fragment. We next performed bioinformatics analyses aimed at identifying binding sites for transcription factor known for their capacity to interact with pRb. We found that this fragment of the PAK1 promoter contains binding sites for several well known transcriptional factors, notable among this being E2F transcription factors. This observation makes it tempting to speculate that pRb represses PAK1 transcription by blocking the E2F mediated transcriptional of the PAK1 gene. Taken together our results implicate pRb in the regulation of the activity of the small Rho/GTPases Rac1. This in turn brings forth the interesting possibility that pRb, acting via small Rho/GTPases, could have additional novel roles as a regulator of processes such as changes in cell shape, migration, and cytoskeletal reorganization, processes in which small Rho GTPases are known to play a role. This project is supported by PSM Institutional Funds, by the U56 Partnership between PSM and the H. Lee Moffitt Cancer Center (Pilot Grant No. 10-14352-02-03), and RISE Program 1R25GMO82406 from NIH-NIGMS. Citation Information: Cancer Res 2009;69(23 Suppl):A68.

Tumors generally possess high levels of hyaluronan (HA), but have a reduced size compared with normal tissue. Experimentally increased HA induces tumor growth and metastasis in xenograft models (review by Toole, BP, 2004). Our study sought to elucidate the possible molecular mechanism of HA-mediated cell motility in a human epithelial cell line, HeLaS3, using a Boyden chamber system. We showed that among the different molecular masses of HA (3, 23, 230 and 940 kDa) (Seikagaku Kogyo Co.) 23 kDa HA was the most potent in chemokinesis, while 230 and 940 kDa HAs had no detectable effect on chemokinesis. With a drop of 23-kDa HA, sparsely adhered HeLaS3 cells spread, exhibiting multiple surface extensions in various directions, and altered lamellipodia. HeLaS3 cells exhibited fine cortical F-actin at the lamellipodia and formed many small focal complexes instead of large focal adhesions at the base of membrane protrusions at 1–20 minutes after 23 kDa HA stimulation. These morphological alterations are reminiscent of Rac1 activated fibroblasts (Rottner, 1999. To properly address the modulation of Rho GTPases in HA-induced chemokinesis, we evaluated cells carrying dominant negative mutants of either RhoA cDNA (dnRhoA) or Rac1 cDNA (dnRac1). The transduction of either dnRhoA or dnRac1 into HeLaS3 cells caused a dramatic decrease in chemokinesis with or without HA stimulation, compared with mock-transduced control. To biochemically confirm Rho GTPase activation, we measured GTP-RhoA and GTP-Rac1 levels after stimulation with 23 kDa HA using ELISA based Rho GTPases activation assay kits (Cytoskeleton, Inc.). GTP-Rho A increased after 23kD HA ligation, had a small peak at 1 minute, decreased, and then returned to baseline by 60 min. After 23kD HA stimulation, Rac1 activation was observed in dual peaks of 1.8–2 fold increases at 3 and 15 minutes; thereafter, it became near to baseline by 60 minutes. It has been shown that HA can modulate cell migration involving hyaladherin-mediated signaling through ERK1/2 (Vigetti, 2008, Tolg, 2006). Initially, we examined the effect of the ERK inhibitor PD98059 in our system. A treatment of 6.6 µg/ ml PD98059 suppressed 23 kDa HA-induced chemokinesis compared with the control group. Western blotting of the cell lysates showed sustained ERK1/2 phosphorylation over 120 minutes. This study indicates that sustained ERK1/2 phosphorylation is required for 23 kDa HA-induced chemokinesis. Here we show that fragmented 23 kDa HA elicits enhanced chemokinesis and changes the shape of epithelial cells by weak Rho A activation prior to the recurrent Rac1 activation and the sustained phosphorylation of ERK1/2. Our unpublished study also indicates that hyaluronidase-2-mediated catabolism could play a significant role in HA oligosaccharide generation with the association of CD44 in epithelial cells. This indicates the possibility that fragmented HA acts as a mediator in the autocrine/paracrine mechanism. Our study showed that signals initiated by fragmented HA-hyaladherin interactions induce chemokinesis of epithelial cells, expanding our understanding of the functions of HA in cancer cell biology. Citation Information: Cancer Res 2009;69(23 Suppl):C74.

Invasive motility limits treatment efficacy and is a significant contributor to poor outcomes in glioblastoma (GBM). GBM tumor microtubes are actin- and microtubule-enriched membrane tubes that facilitate invasive motility and underlie many components of GBM pathophysiology. Rho-GTPases mediate GBM invasion through localized activation of cytoskeletal effector proteins, such as mammalian Diaphanous-related formins (mDia) and Rho-associated protein kinase (ROCK). Active mDia nucleates and polymerizes F-actin and independently stabilizes microtubules, while active ROCK phosphorylates myosin phosphatase and myosin light chain to induce the actomyosin crosslinking required for contractility. GBM invasion depends on a delicate balance between mDia-mediated extension of leading-edge structures (such as tumor microtubes) and ROCK-mediated contraction of trailing cell bodies. In this study, we assessed the roles of ROCK and mDia in tumor microtube-associated GBM invasion using a 3D patient-derived neurosphere model of GBM invasion. Neurospheres were embedded in 3D matrices and treated with the small molecule inhibitor of ROCK (Y-27632) and the small molecule agonist of mDia (IMM-02). Treatment with Y-27632 alone reduced the total distance of cell body migration and increased tumor microtube length without effecting the total area of neurosphere invasion. Tumor microtubes extending from Y-27632 treated neurospheres displayed an atypical undulant morphology, further suggesting that ROCK inhibition modifies primary invasion programs. Treatment with IMM-02 alone profoundly reduced total area of neurosphere invasion, distance of cell body migration, and length of tumor microtubes. Combination treatment effects (Y-27632 + IMM-02) were time dependent. At 24 hours, combination treatment did not significantly reduce total area of invasion over either individual treatment. However, tumor microtubes were shorter with combination treatment than Y-27632 alone and longer than IMM-02 alone. At 96 hours, combination treatment reduced total area invaded and length of tumor microtubes in comparison to Y-27632 alone, but no significant difference was observed in comparison to IMM-02 alone. Western blot analysis of both free-floating patient-derived 3D neurospheres and semi-adherent “2.5D” monolayer cultures demonstrated that agonism of mDia formins with IMM-02 (96-hrs) results in the progressive loss of mDia1 and mDia2 protein expression. Therefore, endogenous mDia regulatory mechanisms triggered in response to continuous agonist-mediated mDia activation may inhibit mDia function more effectively than direct antagonism strategies in GBM. Conclusively, both IMM-02 and Y-27632 treatment disrupt the tumor microtube mechanism of GBM neurosphere invasion. Further studies are warranted to evaluate these small molecule compounds for potential anti-tumor microtube therapeutic effect in vivo. Citation Format: Kathryn N. Becker, Krista M. Pettee, Amanda Sugrue, Kevin A. Reinard, Jason L. Schroeder, Kathryn M. Eisenmann. ROCK and mDia have dynamic roles in glioblastoma tumor microtube invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2887.

The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826–37. ©2016 AACR.

While the Ras GTPases are frequently mutated, especially at codons 12, 13, or 61, the structurally related Rho GTPases, although they are implicated in tumor invasion and metastasis, are rarely mutated in solid tumors, except for diffuse gastric cancer. The vast majority of cancer-associated RHO mutations are not located in codons 14, 15, or 63, which correspond to the 3 canonical mutant Ras codons. Here we have evaluated the biological and biochemical phenotypes of several cancer-associated RhoA mutants, which indicated that some RhoA mutants have unusual phenotypes and therefore prompted us to generalize our findings to other small GTPases by constructing and analyzing the analogous mutants in K-Ras. We constructed 8 cancer-associated RhoA mutants from the TCGA database, and found that each was gain-of-function biologically. Using the Rhotekin binding assay to infer RhoGTP levels, 5 mutants (Y42C, L57V, D59G, A61D, D76G), had higher binding than WT RhoA. However, 3 mutants (G62E. L69P and L69R) did not bind Rhotekin, but did bind strongly to Citron, a Rho-dependent kinase implicated in cytokinesis and cell cycle regulation. Because the three mutants were highly transforming, we directly determined GTP/GDP binding levels, the “gold-standard” assay, for all mutants, which indicated G62E had high GTP-binding and low GDP-binding, in contrast to its negative binding to Rhotekin, while L69P and L69R had high GDP-binding and low GTP-binding similar to WT. Purified GDP-bound L69P and L69R formed a complex with Citron more efficiently than did WT GTP-bound or GDP-bound RhoA. Endogenous Citron and CDK1 contribute to anchorage-independent growth, as siRNA knockdown of either gene in lines transformed by G62E, L69R or L69P inhibited this phenotype. The cancer-associated K-Ras mutants G60E and M67L (which correspond to RhoA G62E and L69R) and also K-Ras M67R displayed gain-of-function biologically, and the GTP/GDP-binding phenotype of each was analogous to the respective RhoA mutant: high GTP-binding for G60E, and low Raf-RBD binding and high GDP-binding for M67L and M67R. The low GTP K-Ras mutants bound more strongly to the RBD domain of Ras effectors other than Raf, resulting in activation of RAP and AKT in transfected cells. Our microarray data revealed that these GDP-bound mutant K-Ras or RhoA can up-regulate cell-cycle progression genes and down-regulate tumor suppressor genes compared to WT, which may account for their transforming phenotype. We conclude that some cancer-associated RhoA mutants are transforming when GDP-bound, a phenotype that has not been described for any small GTPase. The equivalent K-Ras mutants display a similar phenotype, which implies the findings should be extrapolatable to many small GTPases. Such mutants can be strongly transforming, although they only bind a subset of preferred downstream effectors and activate their respective pathways. In addition, the widely used Rhotekin binding assay to infer RhoGTP levels may give misleading results for some mutants. Citation Format: Xiaolan Qian, Alex Papageorge, Dunrui Wang, Brajendra Tripathi, Yucheng Gao, Marian Durkin, Beatriz Sanchez-Solana, Dominic Esposito, Timothy Waybright, Andrew Stephen, Douglas R. Lowy. Identification of cancer-associated RhoA mutants and analogous K-Ras mutants that induce gain-of function despite low GTP binding [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2586.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer that accounts for about 10% of breast cancer mortality annually in US. Poor prognosis is largely due to the high propensity of IBC tumors to develop distant metastasis. Owing to the complex metastatic process, the molecular basis of IBC aggressiveness is poorly understood and no specific therapeutic target has been identified. Among the few signaling pathways that differ between IBC and non-IBC is the Rho GTPase that is hyperactivated in many IBC tumors and is associated with cell motility in IBC cells. However, Rho GTPases are not druggable factors and the pathways that activate these proteins in IBC are not fully defined. Another difference is that up to 83% of IBC tumors lack estrogen receptor α (ERα) expression compared with other forms of breast cancer that are mostly ERα-positive. Despite the lack of ERα in the majority of IBC tumors, estrogen may still play a role in these cancers through pathways that involve ERβ. Our tissue staining reveals expression of ERβ in a significant number of IBCs that is reproduced in IBC cell lines. Furthermore, analysis of IBC datasets indicates correlation of receptor expression with good prognosis. We studied this association in preclinical models of IBC by knocking out ERβ in IBC cells. This promotes migration and invasion through cytoskeleton remodeling whereas re-expression of the receptor in knockout cells restores the cytoskeletal structure and migration to the levels of control cells. Consistent with increased migration, deletion of ERβ activates large gene networks of cell de-differentiation including cell surface receptor signaling that enhances Rho GTPase activity to promote the motile phenotype of IBC cells. In contrast, ligands that activate ERβ inhibit the same signaling that contributes to metastasis in IBC. Analysis of orthotopic xenograft models shows that IBC tumors lacking ERβ have higher propensity for metastasis compared with the ERβ-proficient tumors. Conversely, ERβ agonists decrease the metastatic potential of IBC tumors supporting the anti-metastatic activity of the receptor. Our findings point towards a role of ERβ in preventing distant metastases by inhibiting dissemination of IBC cells. This function combined with distinct expression indicates the potential of ERβ to represent a unique prognostic marker and therapeutic target that can be utilized to repress IBC metastasis and eliminate its associated mortality. Citation Format: Christoforos Thomas, Ilias Karagounis, Ratnesh Srivastava, Sushil Kumar, Noelle Francois, Fotis Nikolos, Igor Bado, Hann-Hsiang Chao, Constantinos Koumenis, Savitri Krishnamurthy, Naoto T Ueno, Rumela Chakrabarti, Amit Maity. Estrogen receptor β suppresses metastasis of inflammatory breast cancer by regulating signaling that promotes cytoskeleton remodeling [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-02.

Despite a high number of neoantigens and T cell infiltration, most locally advanced and metastatic bladder cancers (BC) (>70%) do not respond to anti-PD1 treatment. Thus, targeting additional checkpoints may play an important role in treatment of BC. We sought to identify negative regulators of T cell activity and evaluate their function using a previously validated pre-clinical carcinogen-induced mouse model of BC. To identify regulators of T cell activity, we first investigated the timing and infiltration of T cells during tumor development in the BBN mouse BC model. We found that numbers of CD4+ and CD8+ T cell increased dramatically within the first 1-2 weeks, but then decreased over the next six weeks decreasing to the lowest levels at 8 weeks before increasing again coordinating with increasing tumor volume. During the decrease in T cell numbers, macrophages increased and by transcriptome profiling we identified increased levels of the T cell inhibitory protein B7-H4. B7-H4+CD11b+ macrophages increased over 4 weeks with opposite expression of CD4+ and CD8+ T cells by FACS. By IHC we localized B7-H4 expression in macrophages adjacent to tumor. A bioinformatic analysis of the human TCGA identified highest B7-H4 expression in luminal infiltrated subtypes of MIBCs, but did not correlate with CD4, CD8 or overall tumor mutation burden. MIBCs with elevated B7-H4 expression had significantly worse survival. In vitro, addition of anti-B7-H4 significantly increased proliferation and IFN-γ production by human T cells co-cultured with B7-H4 expressing APCs. To determine the role of B7-H4 in BC, we treated mice with overt BC (beginning at 4 months of carcinogen administration) with vehicle, anti-PD1 or anti-B7-H4 antibody. Compared to vehicle treated animals, anti-B7-H4 treated mice had a reduction in the number of pT3 tumors (50% vs. 73%), but less than anti-PD1 treated tumors (27%). However, anti-B7-H4 treated mice had significantly increased tumor infiltrating CD8+ T cells per histological field (77.1+34.3 vs. 11.5+7.8; p=0.0121), as well as a significant increase splenic CD8+IFN-γ+ T cells and levels of secreted IFN-γ upon anti-CD3 stimulation. Transcriptome profiling of tumors that responded to anti-B7-H4 antibody by RNA-Seq identified a decrease in mitotic cell cycle, cell cycle checkpoints, RHO GTPases and mitotic spindle checkpoint pathways in responsive tumors. In summary, we confirm the expression of B7-H4 in macrophages found in a murine model of BC and correlate expression to poor survival in the TCGA of MIBC. Inhibition of B7-H4 results in lymphocyte proliferation in vitro and inhibition of B7-H4 in mice resulted in decreased tumor stage and increased CD8+ TILs. This data suggests that B7-H4 may be a candidate alternative checkpoint that can be targeted in humans with BC unresponsive to PD1 therapy. Citation Format: Joshua Meeks, Alexander P. Glaser, Damiano Fantini, Yanni Yu, Valerie Eaton, Joseph R. Podojil, Stephen D. Miller. B7H4 as a T cell inhibitory regulator in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2405.

The high metastatic potential of inflammatory and other aggressive breast cancer is the major determinant of mortality. Recently, it has become clear that signals from the tumor microenvironment (TME) harbor the capacity to enhance cancer cell dissemination. Of the various cells that comprise the TME, macrophages are the most abundant in solid tumors. Therefore, we aimed to better understand the relationship between macrophage-breast cancer cell crosstalk. Intriguingly, in our previous work we showed that inflammatory breast cancer (IBC) cells are hyper-migratory in response to macrophage conditioned media. Additionally, we found this process is regulated by the Rho-GTPase, RhoC. Recent advances in tumor associated macrophage (TAM) biology have led to the understanding that TAM populations in the TME are diverse, primarily due to directional differentiation promoted by unique secreted components from the various cells in the TME. Therefore, this study aimed to understand which subpopulation of M2-like TAMs had the greatest impact on breast cancer cell aggressiveness. Our data show that stimulation of the triple negative breast cancer (TNBC) cell model, MDA-MB-231, and the IBC TNBC cell line, SUM-149, with media extracts from IL-4/IL-13 stimulated M2 macrophages (M2a) elicit the strongest migratory and invasive responses. Subsequently, we stimulated RhoA or RhoC knockout (CRISPR) MDA-231 and SUM-149 cells with M2a conditioned media to address their role in regulating migratory/invasive responses. In brief, we find that RhoA and RhoC, in part, regulate M2a-TAM induced responses. Secretome analysis of M2a-TAM conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Functional studies suggest that VEGF and CCL-18 synergistically enhance cellular invasiveness. Moreover, pretreatment with the ROCK inhibitor Y-27632 or GSK429286A drastically inhibited VEGF, CCL-18, or M2a induced migratory responses. These data suggest that the Rho-GTPases regulate M2a-mediated breast cancer cell invasion and potentially offer a novel approach for the treatment of metastatic breast cancer through potential prevention of progression. Citation Format: Andrew C. Little, Pragathi Pathanjeli, Zhifen Wu, Liwei Bao, Laura Goo, Joel A. Yates, Sofia D. Merajver. IL-4/IL-13 stimulated tumor-associated macrophages enhance breast cancer cell invasion through Rho-GTPase signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4518.

Synovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a chimeric transcriptional modifier. Several receptor tyrosine kinases have been found activated in synovial sarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of the results of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of the SRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK and PTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence of SRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcoma xenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC was detected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for the activation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated with increased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway. Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation, associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure of cells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasion were dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA. Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. In summary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovial sarcoma. Cancer Res; 73(8); 2518–28. ©2013 AACR.

Introduction. Renal cell carcinoma (RCC) is the one of the most common cancers diagnosed in adult men with more than 40000 new cases annually. Recurrence or metastasis of the disease occurs in more than 30% of patients and death from disease occurs in most of these patients due to limited therapeutic options. Clearly, a better understanding of factors contributing to RCC metastasis might improve therapeutic development and patient outcomes. One of the steps required for tumor cell progression from localized disease to metastatic disease is the acquisition of invasive abilities through epithelial to mesenchymal transition (EMT). EMT promotes loss of epithelial markers by downregulation of E-cadherin, gain of mesenchymal markers like Vimentin, and N-cadherin, and cytoskeletal rearrangement that enables cells to leave the primary site and intravasate into the blood or lymphatic systems and thereby colonize distant sites. Metastasis and colonization are highly directed mechanisms in which cancer cells migrate toward specific preferred organs that express specific protein receptors. One of these receptors is CXCR4, which is upregulated in advanced cancer including RCC and is correlated with poor survival. In this study we explored the hypothesis that CXCR4/CXCL12 signaling promotes migratory ability by inducing EMT in vitro in the RCC cell lines Caki-2 and 786-0. Methods. To pursue these studies we utilized in vitro mammalian cell culture, cell proliferation (WST) assays, cell migration (scratch) assays, and protein analysis methods including immunoblot and immunofluorescence. Results. These studies showed that RCC cell lines express CXCR4 that is activated by CXCL12 to induce cellular proliferation and promote a wound healing migratory response dependent on EMT cytoskeletal rearrangement by Rho GTPases. Moreover, CXCR4/CXCL12 induces down-regulation of E-cadherin, up-regulation of vimentin, N-cadherin and the upregulation of EMT transcription factors SLUG/SNAIL, consistent with EMT signaling activation. Lastly, CXCR4/CXCL12 activation promotes phosphorylation of AKT and ERK, suggesting that these signaling pathways are coupled to CXCR4/CXCL12-mediated RCC progression. Conclusions. Our findings suggest that CXCR4/CXCL12 activation in RCC has a pivotal role in EMT induction by the acquisition of a migratory and invasive phenotype. Understanding the underlying signaling pathways employed by the CXCL12/CXCR4 axis may shed new light on the migration/ invasion mechanisms that allow RCC cells to spread to distant organs and assist with the design and testing of new therapeutic strategies to treat advanced metastatic RCC. Citation Format: Alisa Zhilin-Roth, Jill A. Macoska. The CXCL12/CXCR4 axis drives epithelial-mesenchymal transition in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2021.

Estrogen mediates the growth and differentiation of breast epithelial cells and plays an important role in breast cancer progression. The upregulation of estrogen receptors has been linked to p190B RhoGAP in vivo. p190B regulates the activity of several Rho GTPases in vitro. To better understand the molecular mechanism of p190B's role in tumor progression, we sought to develop a cell culture model that would recapitulate the effects of p190B on various growth factor signaling pathways. Protein expression of p190B was knocked down using siRNA in MCF-7 breast cancer cells cultured in the absence of steroid hormones. Upon stimulation with estrogen, cell proliferation did not significantly differ between MCF-7 cells expressing reduced p190B and those expressing normal levels of p190B, suggesting that the effects of p190B on estrogen receptor expression are not cell autonomous and that additional components of the tumor microenvironment are required to recapitulate the in vivo findings. Citation Format: Eduardo Castro, Aaron Preston, Mykal Quinlan, Peter McHenry. p190B RhoGAP modifies growth factor signaling in breast cancer in a non-cell autonomous manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4493.

While Hippo pathway signaling undoubtedly plays an important role in cancer, the role of its effector YAP1 as an oncogene or tumor suppressor is context dependent. The precise conditions that support the tumor-promoting or tumor-suppressing roles of YAP1 remain largely undefined. While YAP1 can drive oncogenic programs through its nuclear function as a transcriptional coactivator of TEAD and other transcription factors, previous findings from our lab and others suggest that loss of YAP1 function can also have tumor promoting consequences. One well-characterized role of YAP1 is that of a nuclear relay of signals triggered by mechanical forces that lead to activation of Rho GTPases. Our new studies using a combination of genetic, biochemical, and microscopy techniques provide evidence that YAP1 is not only activated by Rho, but can also regulate Rho activation and the actin cytoskeleton. Knockdown of YAP1 in MCF10A immortalized mammary epithelial cells resulted in increased lamellipodia formation in culture as seen by time-lapse microscopy, and increased protrusive, invasive activity in 3D Matrigel culture. YAP1 knockdown also induced a scattering phenotype, characterized by cell dissociation from the leading edge of a wounded monolayer. While E-cadherin protein levels were unaffected by YAP1 knockdown, adherens junction maturation at cell-cell junctions was compromised. These morphological alterations were associated with decreased levels of active RhoA, as well as a decrease in downstream myosin light chain phosphorylation. Our results indicate loss of YAP1 decreases active RhoA, leading to reduced cell-cell adhesion and increased protrusive cellular behavior. Our findings raise the possibility that YAP1 is not only a Rho-sensor, but also mediates a feedforward regulatory loop to maintain Rho-regulated cytoskeletal structures, and that loss of YAP1 through genetic deletion in tumor cells could promote invasive behavior. Citation Format: Natalie M. Hendrick, Marcin P. Iwanicki, Carman Man-Chung Li, Duyen Amy Bui, Laura M. Selfors, Joan S. Brugge. Loss of YAP1 dysregulates RhoA signaling and promotes cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3155.

Studies demonstrated loss of connexin 43 (Cx43) expression in breast cancer cells in-vitro and in-vivo. Our previous studies have shown that silencing Cx43 in HMT-3522 S1 cells, non-neoplastic human mammary epithelial cells (S1 cells), altered apical polarity and mitotic spindle orientation under 3-dimensional (3-D) culture conditions. The purpose of this study is to investigate the effects of Cx43 loss on proliferation and invasion of non-neoplastic breast epithelium. Cx43 shRNA-transfected and control S1 cells were cultured under 2-D and 3-D conditions. Cx43-knockdown S1 cells exhibited enhanced proliferation both in 2-D and 3-D throughout the culture period, as shown by the increased cell counts and acinar size, respectively. In addition, cell cycle entry was enhanced, as indicated by the increased percentages of cells in S and G2/M phases and the reduced percentage of cells in G0/G1 phase. Cx43-silenced S1 cells upregulated the expression of proliferation genes, c-Myc and cyclin D1, in 2-D and 3-D, but maintained β-catenin levels. Furthermore, immunoprecipitation studies showed that Cx43 associated with β-catenin and ZO-2 in 2-D and 3-D cultures of S1 cells, and co-localized with β-catenin in 3-D, suggesting assembly of gap junction complexes in S1 cells. The loss of Cx43 caused mis-localization of β-catenin in 3-D cultures of S1 cells, suggesting involvement of the Wnt/β-catenin pathway downstream of Cx43 signaling. β-Catenin primarily exhibited apico-lateral distribution in S1 cells and re-localized to basolateral membrane domains in Cx43 knockdown cells. Silencing Cx43 enhanced migration of S1 cells by 40%, as shown by time-lapse imaging. In addition, Cx43-silenced S1 cells exhibited loss of the characteristic spheroid morphology and acquired a migratory phenotype in acini. Matrigel-invasion and MMP-9 activity were also enhanced. Cx43-silenced S1 cells were capable of invading the matrigel by two-fold compared to control cells. This was concomitant with enhanced expression and activity levels of Rho GTPases (RhoA, Rac1 and Cdc42), suggesting that Cx43 regulates non-canonical Wnt signaling, as a mechanism to induce migration and invasion. We propose a role for Cx43 loss in triggering proliferation and invasion events in non-neoplastic breast epithelium downstream of disrupted Wnt signaling. Citation Format: Sabreen F. Fostok, Dana Bazzoun, Sophie A. Lelievre, Mirvat El-Sibai, Rabih S. Talhouk. Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4505.

Much of the recent focus of melanoma targeted therapy has been on the ERK pathway, which is aberrantly activated in approximately 90% of melanoma tumors. Over half of these express BRAFV600E. Current targeted therapies such as Vemurafenib (BRAFV600E inhibitor), or a combination therapy of BRAF + MEK inhibitors show profound initial effects in a majority of BRAFV600E expressing tumors. However, these responses are often short-lived and resistance typically develops within months. The goal of this work is to identify pharmacologically targetable resistance mechanisms so that effective combination therapies can be developed. Preliminary bioinformatics analysis suggests that the RhoA subfamily of Rho GTPases is activated in BRAFi-resistant cancer cell lines and BRAFi/MEKi-resistant human melanoma tumors. Thus, we hypothesize that RhoA promotes BRAFi resistance, so simultaneously targeting RhoA and BRAF may reverse drug resistance. Using cell line models of acquired BRAFi-resistance we demonstrated that the RhoA pathway is activated in a subset of resistant cell lines. The cell lines with increased RhoA activation have increased sensitivity to multiple ROCK inhibitors. In addition to regulating the cytoskeleton, RhoA can also regulate gene transcription through activation of multiple transcriptional co-activators, including MRTF and YAP. MRTF and YAP regulated gene transcription through their role as transcriptional co-activators and their interaction with chromatin remodeling complexes. MRTF and YAP are both activated in BRAFi-resistant cell lines. Indirectly targeting MRTF with CCG-222740 or YAP with the YES1 inhibitor Dasatinib effectively kills BRAFi-resistant melanoma cells. Sox10 was previously identified as a gene which promotes BRAFi resistance. Expression of Sox10 is downregulated, and Sox9 is upregulated, 100-1000-fold exclusively in the resistant cell lines which have RhoA activation. Further, a Sox10 gene signature is inversely correlated with a RhoA/C signature in the TCGA dataset and melanoma scRNA-seq data. Sox10 loss is a major drug resistance melanoma in melanoma. These data suggest that targeting RhoA-regulated gene transcription may be an effective mechanism to target Sox9High/Sox10Low cells, and ultimately prevent or reverse drug resistance. Taken together these data suggest that MRTF and YAP promote Vemurafenib resistance in Sox9High/Sox10Low melanoma cells. Citation Format: Sean A. Misek, Kathleen A. Gallo, Richard R. Neubig. Targeting RhoA-regulated gene transcription in drug-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5895.

The fragile histidine triad (FHIT) gene has been shown to function as a tumor suppressor gene in vitro and in vivo. However, the mechanism of its action is still largely unknown. To elucidate the molecular mechanism and biological pathway in FHIT-mediated tumor suppression, we used a complementary gene and protein expression profiling with DNA microarray and ProteinChip technologies to quantitatively monitor cellular changes in gene and protein expression and discover the molecular targets of FHIT in non–small cell lung carcinoma (NSCLC) cells. The Ras/Rho signaling pathway was identified as one of the unique biological pathways associated with FHIT activity. A significantly down-regulated expression of genes and proteins of multiple key components in the Ras/Rho GTPases molecular switch, including Ran, Rab, Rac, Rap, and Ral, was observed on gene and protein expression profiles and further validated by Western blot analysis. Ectopic activation of FHIT in FHIT-deficient H1299 cells also significantly reduced the invasive potential of tumor cells by down-regulating expression of RhoC, a potential marker of tumor cell invasion and metastases. A simultaneous knockdown of the expression of several key Ras/Rho signaling molecules using gene-specific small interfering RNAs (RHO-siRNA) targeting selected Rab11, Rac1, and Rap1 genes significantly inhibited tumor cell growth and induced apoptosis in NSCLC cells in vitro, and a local injection of RHO-siRNAs complexed with N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate:cholesterol nanoparticles inhibited tumor growth in A549 tumor xenografts in mice, mimicking the AdFHIT-mediated tumor-suppressing effect. These results suggest a new role of FHIT in down-regulating the Ras/Rho GTPase-associated oncogenic signaling pathway. [Cancer Res 2007;67(21):10379–88]

Hepatocellular carcinoma (HCC) is one of the major malignancies in the world. The prognosis of HCC is poor, due to frequent intrahepatic metastasis and tumor recurrence. P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, Rac1 and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Here, we show that Pak1 gene was overexpressed in human HCCs. Overexpression of Pak1 in human HCCs was associated with more aggressive tumor behavior in terms of more metastatic phenotype and more advanced tumor stages. In addition, HCC cell line stably expressing Pak1 displayed increased cell motility rates and, conversely, knockdown of endogenous Pak1 expression by small interfering RNA reduced the migration rates of HCC cells. In an established metastatic HCC cell line, we found that Pak1 was overexpressed compared with its primary HCC cell line and this overexpression was associated with higher cell motility. Importantly, we found that c-Jun NH2-terminal kinase (JNK) was activated in HCC cell lines overexpressing Pak1. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of HCC cells via attenuation of paxillin phosphorylation at Ser178. In conclusion, our results document that Pak1 is overexpressed in HCCs and plays an important role in the metastasis of HCC. The mechanism by which Pak1 induces cancer metastasis may involve activation of JNK and phosphorylation of paxillin. [Cancer Res 2007;67(8):3601–8]

Deleted in liver cancer 1 (DLC1) is a recently identified tumor suppressor gene frequently underexpressed in hepatocellular carcinoma (HCC). DLC1 encodes a Rho GTPase-activating protein domain that exhibits growth-suppressive activity in HCC cell lines. Our recent finding has revealed that inhibition of Rho-mediated actin stress fiber formation by DLC1 is associated with its growth inhibitory activity. In the present study, we identified tensin2 as the novel binding partner of DLC1. Tensin2 belongs to a new family of focal adhesion proteins that play key roles in cytoskeleton organization and signal transduction. Dysregulation of tensin proteins has previously been implicated in human cancers. Tensin2 is highly expressed in human liver. Introduction of tensin2 into HCC cell lines with low expression of tensin2 caused significant growth inhibition and induction of apoptosis. Tensin2 directly interacted with DLC1 in vitro and in vivo. Both proteins localized to punctate structures in the cytoplasm. Sequence analysis of DLC1 and tensin2 identified caveolin-1 binding motif in both proteins. In vivo immunoprecipitation study confirmed that both proteins indeed interacted with endogenous caveolin-1, which is the major structural component of caveolae. Our findings presented here suggest a new model for the action of DLC1 in hepatocytes, whereby DLC1-tensin2 complex interacts with Rho GTPases in caveolae to effect cytoskeletal reorganization. (Cancer Res 2006; 66(17): 8367-72)

Melanoma cells express the chemokine receptor CXCR4, which confers invasive signals on binding to its ligand CXCL12. We show here that knocking down membrane-type matrix metalloproteinase (MT1-MMP) expression translates into a blockade of invasion across reconstituted basement membranes and type I collagen gels in response to CXCL12, which is the result of lack of MMP-2 activation. Interference with MMP-2 expression further confirms its important role during this invasion. Vav proteins are guanine-nucleotide exchange factors for Rho GTPases that regulate actin dynamics and gene expression. We show that melanoma cells express Vav1 and Vav2, which are activated by CXCL12 involving Jak activity. Blocking Vav expression by RNA interference results in impaired activation of Rac and Rho by CXCL12 and in a remarkable inhibition of CXCL12-promoted invasion. Importantly, up-regulation of MT1-MMP expression by CXCL12, a mechanism contributing to melanoma cell invasion, is blocked by knocking down Vav expression or by inhibiting Jak. Together, these data indicate that activation of Jak/Vav/Rho GTPase pathway by CXCL12 is a key signaling event for MT1-MMP/MMP-2–dependent melanoma cell invasion. (Cancer Res 2006; 66(1): 248-58)

Progression of tumors to invasive and metastatic forms requires that tumor cells undergo dramatic morphologic changes, a process regulated by Rho GTPases. Elevated expression of RhoA and RhoC, as well as the Rho effector proteins ROCK I and ROCK II, are commonly observed in human cancers and are often associated with more invasive and metastatic phenotypes. To examine how ROCK contributes to the progression of solid tumors, we established a conditionally activated form of ROCK II by fusing the kinase domain to the estrogen receptor hormone-binding domain (ROCK:ER). ROCK:ER-expressing colon carcinoma cells grown as tumors in immunocompromised nude mice organized into discrete clusters surrounding blood vessels. However, ROCK:ER activation resulted in the aggressive dissemination of tumor cells into the surrounding stroma, indicating that increased ROCK signaling is sufficient to promote invasion from solid tumors. In addition, tumors in which ROCK:ER was activated were more highly vascularized, indicating that ROCK contributes to tumor angiogenesis. ROCK:ER activation resulted in changes to epithelial morphology and organization that facilitated motility in vitro, likely by inducing the redistribution of proteins such as ezrin, as well as adherens junction and extracellular matrix-binding proteins. These results suggest that ROCK inhibitors would be useful antimetastatic and antiangiogenic chemotherapeutic agents in tumors associated with elevated RhoA, RhoC, ROCK I, or ROCK II expression.

Nasopharyngeal carcinoma (NPC) is categorized into three different differentiated subtypes by World Health Organization (WHO). Based on an earlier comparative proteomic database of the three histological subtypes, the study was to deepen our understanding of molecular mechanisms associated with NPC differentiation through bio-information mining. Among the three subtypes were 194 differentially expressed proteins (DEPs) of 725 identified proteins. Two DEPs, heat shock protein family B (small) member 1 (HSPB1) and keratin 5 (KRT5), were validated in a series of NPC tissue samples by using immunohistochemistry. Quantified protein families including keratins, S100 proteins (S100s) and heat shock proteins exhibited characteristic expression alterations. Comparisons of predicted bio-function activation states among different subtypes, including formation of cellular protrusion, metastasis, cell death, and viral infections, were conducted. Canonical pathway analysis inferred that Rho GTPases related signaling pathways regulated the motility and invasion of dedifferentiated NPC. In conclusion, the study explored the proteomic characteristics of NPC differentiation, which could deepen our knowledge of NPC tumorigenesis and allow the development of novel targets of therapeutic and prognostic value in NPC. Citation Format: Zhefeng Xiao, Guiying Zhang, Yongheng Chen. Proteomic characterization reveals a molecular portrait of nasopharyngeal carcinoma differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-073. doi:10.1158/1538-7445.AM2017-LB-073

Inflammatory breast cancer is a highly metastatic form of locally advanced breast cancer. Its uniqueness lies in the fact that it is found to be highly metastatic from the inception. Given the clinical differences between IBC and non-IBC, it is hypothesized that IBC has a characteristic molecular profile. Microarray data reveals interesting differences in the genetic makeup of these two cancer types. Caveolin-1, 2 proteins typically have much reduced expression in other breast cancers; however, this is not the case in inflammatory breast cancer. Caveolins, primarily caveolin-1 and 2 are over-expressed in IBC cells SUM 149. Caveolin-1 is membrane-associated protein located in lipid rafts called ‘caveolae'. Its unique scaffolding domain is known to concentrate several signaling molecules including PDGFR, Rho GTPases etc. leading to unique signaling cascades. Due to this characteristic feature, caveolin-1 has become one of the potential regulators of many cellular signaling and trafficking pathways that control cell growth and migration. Previously, our laboratory demonstrated that RhoC GTPase is responsible for the invasive phenotype of IBC. However, the mechanism of RhoC activation has not been elucidated yet. Preliminary data from Translational cancer research group Antwerp, Belgium shows over-expression of PDGFR alfa at mRNA level in IBC patient samples. Therefore, primary objectives of our study are to determine whether the highly invasive phenotype of IBC is due to increased PDGFR signaling through Akt1 leading to increased RhoC activation and what role does caveolin-1 play in regulating the interaction of PDGFRA and RhoC GTPase in Inflammatory breast cancer. We first determined the down-regulation of caveolin-1 at the mRNA level using varying doses of caveolin-1-specific siRNA in the inflammatory breast cancer cell line, SUM 149. QPCR showed a maximum down regulation of 50% at the mRNA level 72 h post siRNA treatment; bringing caveolin-1 mRNA levels close to the levels in normal human mammary epithelial cells. Next we quantitatively analyzed the effect of caveolin-1 down-regulation on SUM 149 cell invasiveness through a Matrigel coated filter. After 72 h caveolin-1 siRNA treatment, the percent invasion of SUM149 IBC cells was significantly reduced to 10% as compared to the control SUM 149 cells. We also determined that siRNA treatment does not affect cell viability or proliferation. This data supports our hypothesis that over-expression of caveolin-1 is involved in the invasive phenotype of IBC and is possibly due to interactions with RhoC GTPase. We are particularly interested in PDGFR that is found to be associated with caveolin-1 at plasma membrane. So far, we have found mRNA and protein expression of PDGFR in SUM 149 cells. However, instead of doublet at 150 and 180kDa in control NIH3T3 cells, SUM 149 cells produce only single band at 140-150kDa. Data from our lab shows that SUM 149 cells express high levels of active Akt1 and selective downregulation of Akt1 significantly reduces SUM 149 cells invasion. These results support our hypothesis that high levels of PDGFR in SUM 149 cells act through Akt1 causing RhoC activation at caveolin-1 and thus regulate invasive and metastatic behavior of IBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-19.

Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID–DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells. Cancer Res; 73(22); 6793–803. ©2013 AACR.

The Rho GTPases, which are frequently activated in advanced cancer, stimulate their downstream effectors to regulate a variety of fundamental cellular process, e.g., cell cycle, cell migration, cell-cell adhesion, and cytoskeleton organization. The down-regulation of RhoGAPs is one mechanism of increased Rho activity, and members of the tumor suppressor DLC Rho-GAP family (DLC1-3) are frequently down-regulated by gene deletion or DNA methylation in human cancers. Here we report that, in addition, point mutations in the DLC1-3 coding regions occur frequently in certain cancers, most of the mutants we have analyzed have reduced tumor suppressor activity, at least two distinct mechanisms can account for this reduction, and one mechanism appears to be Rho-independent. In the TCGA database, DLC1 was mutated in 11% of gastric cancer (GAC), 10% colorectal cancer (CRC), 7% of melanomas (MEL), and 6% of lung adenocarcinoma (LAD). Most alterations were point mutations encoding a single amino acid change. Mutation of DLC2 and DLC3 also occurred in these tumor types, but were less frequent. The mutation frequency of p190-A, which is a Rho-GAP from a different gene family, was even less frequent, ranging from 4% in LAD to zero in GAC. Taken together, mutation of at least one DLC gene or p190-A was found in about 20% of LAD, CRC, and MEL, and 15% of GAC. In LAD, the DLC1 expression levels from the patients with DLC1 mutations were lower than the patients with DLC1 wild type, suggesting selection for decreased DLC1 expression in tumors harboring mutant DLC1. We constructed expression vectors for many of the DLC1 mutants and analyzed their phenotypes in human tumor cell lines. Most were found to be loss of tumor suppressor function mutants, as they were deficient for reducing growth in soft agar, cell migration, and focal adhesion turnover. One class of deficient mutants had lesions in the RhoGAP domain, which directly inactivated its RhoGAP activity and increased the activity of Rho downstream effectors. A second class had lesions in the DLC1 START (StAR-related lipid-transfer) domain, which is known to bind caveolin. although the RhoGAP activity was normal, complex formation with caveolin was much lower than with wild type DLC1. We conclude that, in addition to the frequent down-regulation of DLC1 expression via gene deletion or DNA methylation in tumors, point mutation of its coding sequence commonly inactivates the tumor suppressor activity of DLC1 at the protein level by at least two alternate mechanisms: attenuation of it binding to caveolin or of its RhoGAP activity, which up-regulates RhoA and its downstream effectors. (# Contributed equally) Citation Format: Xiaolan Qian, Dunrui Wang, Beatriz Sanchez-Solana, Brajendra K. Tripathi, Marian E. Durkin, Alex Papageorge, Douglas R. Lowy. Inactivation of the DLC1 RhoGAP tumor suppressor by point mutation occurs commonly in human cancer and can result from Rho-dependent or Rho-independent mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 888.

Introduction. The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Materials and methods. To evaluate the mesenchymal ameboid transition we performed RhoA and Rac1 activation assay, immunofluorescence analysis of protein involved in cytoskeleton organization, and collagen degradation assay. For a clear visualization of collagen fiber breakdown in the process of proteolytic migration we used reconstruction by time-lapse video microscopy. Cell invasion was studied in Boyden chambers using filters coated with Matrigel. uPAR gene expression was inhibited using a 18-mer phosphorothioate aODN, and as a negative control we used a degenerated oligodeoxynucleotide, which is a mixture of all possible combinations of bases that compose the aODN. Results and discussion. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the “path generating” mesenchymal to the “path finding” amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. Conclusion. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly. Citation Format: Anastasia Chillà, Francesca Margheri, Anna Laurenzana, Cristina Luciani, Alessio Biagioni, Gabriella Fibbi, Mario Del Rosso. The receptor for urokinase-plasminogen activator controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5063.

Triple-negative breast cancers (TNBC: negative for oestrogen-receptor, progesterone-receptor and human-epidermal-growth-factor receptor 2 [HER2]) account for 15-20% of breast cancers and are characterized by an extremely aggressive phenotype. Currently, there is no efficient targeted therapy that can prevent the metastatic dissemination of TNBC. The serine threonine kinase PKCθ has recently been identified as a potential new marker of TNBC and as a strong inducer of cancer invasion. However, how PKCθ controls tumour invasion in TNBC is still unclear. Here our results suggest that PKCθ can promote invasion by activating the focal adhesion kinase (FAK) signalling that regulates the cycle of focal contact formation and disassembly. As a consequence of this activation, PKCθ enhances the dynamic of adhesion turnover, Rho GTPases activity and protrusions formation. We also have unravelled the molecular mechanisms by which PKCθ controls FAK: PKCθ interacts with and phosphorylates FAK thus leading to its opening and activation. We believe that the identified phosphorylation sites are implicated in the PKCθ controlled invasion. In the long term, this study could help develop new targeted therapy for TNBC such as PKCθ specific inhibitors that would limit metastasis formation. Citation Format: Lucie Chadelle, Valérie Cadamuro, Jiaying Liu, Xiaobo Wang, Karine Belguise. Control of invasion by PKC theta in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B59.

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the development of TSC and lymphangioleiomyomatosis (LAM). The tumor suppressor effect of tuberin lies in its GTPase-activating protein activity toward Ras homologue enriched in brain (Rheb), a Ras GTPase superfamily member. The statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have pleiotropic effects which may involve interference with the isoprenylation of Ras and Rho GTPases. We show that atorvastatin selectively inhibits the proliferation of Tsc2−/− mouse embryo fibroblasts and ELT-3 smooth muscle cells in response to serum and estrogen, and under serum-free conditions. The isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse atorvastatin-induced inhibition of Tsc2−/− cell growth, suggesting that atorvastatin dually targets a farnesylated protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which have elevated activity in Tsc2−/− cells. Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also inhibited the constitutive phosphorylation of mammalian target of rapamycin, S6 kinase, and S6 found in Tsc2−/− cells in an FPP-reversible manner and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin, but not rapamycin, attenuated the increased levels of activated RhoA in Tsc2−/− cells, and this was reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-insensitive mechanisms of tuberin-null cell growth, likely via Rheb and Rho inhibition, respectively. Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM. [Cancer Res 2007;67(20):9878–86]

The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal cells. Our previous studies indicated that amidated gastrin (G-17) induced gastric cancer cell migration through two separate signaling axes. These revealed that inhibition of Glycogen Synthase Kinase-3β (GSK3β) was necessary to activate G17-induced migratory pathways in gastric cancer cells. Incubation of AGSE gastric cancer cells overexpressing CCK2 receptor (CCK2R) with G17 resulted in a dose and time dependent increase of GSK3βSer9 phosphorylation, indicative of an inhibition of the kinase. Treatment with G-17 was also associated with increased Snail expression, a downstream target of GSK3β and p21-activated protein kinases (PAKs). In a second pathway, G-17 also induced JNK phosphorylation through activation of Mixed Lineage Kinase 3 (MLK3). This axis also promotes gastric cancer cell migration via CCK2R. In the present study our goal was to determine whether PAKs, which are effectors for the Rho GTPases Cdc42 and Rac and are central players in cell proliferation, survival and motility, are involved in the G-17 effects on gastric cancer cells. Our studies showed that in AGSE cells when pretreated with a commercially available PAK4 inhibitor PF 3758309, G-17 was unable to phosphorylate GSK3β and G-17-induced Snail expression was markedly inhibited. G17-induced p-JNK expression was also significantly attenuated with PAK4 inhibitor pretreatment. The inhibitory effect was stronger when PAK4 inhibitor was used in combination with a pan-MLK inhibitor CEP11004. The involvement of PAK4 in G17-induced events were also confirmed by using the PAK4 siRNA. Knockdown of PAK4 inhibited G17-induced p-JNK expression which was more pronounced when both PAK4 and MLK3 were knocked down in AGSE gastric cancer cells. However, knocking down PAK4 alone does-not seem to inhibit G-17-induced p-GSK3β expression completely, which suggest the potential involvement of other PAK family members. Studies are currently underway to determine which PAK(s) is involved in mediating G17-pGSK3β-Snail axis and also to elucidate any crosstalk of PAK4 and MLK3 in this pathway. Elucidation of the signaling pathways that mediate G17-induced cellular events will be very important in designing therapeutic approaches in the future to restrict gastric cancer cell migration and metastasis. Citation Format: Aninda Basu, Goutam Sondarva, Sreevidya Santha, Ajay Rana, Basabi Rana. Elucidation of signaling pathways that mediate gastrin-induced JNK activation and pGSK3β/Snail induction in gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1967. doi:10.1158/1538-7445.AM2015-1967

Glioblastoma (GBM) is the most aggressive and deadliest primary human brain tumor in adults due to the extensive tumor cell migration throughout surrounding brain parenchyma. The capacity to form new tumors, distant from the original location, resides in a specific cell subpopulation called brain tumor initiating cells (BTICs). Combined local and systemic therapies have been ineffective at targeting these invasive cells; furthermore, the mechanisms that confer GBM cells their invasive behavior have not been fully elucidated. The electroneutral Na+-K+-Cl- cotransporter 1 (NKCC1) is an important cell volume regulator that is implicated in cell migration and overexpressed in GBM compared to non-cancer brain tissue. We have shown that both downregulation and pharmacological inhibition (using Bumetanide, BMT) of NKCC1 in BTICs lead to decreased cell migration, in vitro and in vivo. In addition, we reported that NKCC1 knockdown cells (NKCC1 KD) show significantly larger focal adhesions, suggesting a potential role of NKCC1 in cell adhesion. We now report the role of NKCC1 on cytoskeletal dynamics. We found that glioma cells display a significant decrease in cell spreading capacity upon NKCC1 KD or inhibition by BMT. Further, F-actin organization (observed with phalloidin staining) showed dramatic changes upon NKCC1 KD in BTICs. These changes include concentration of actin filaments on the membrane periphery in a ring shaped form and decreased bundled actin content. To analyze actin changes in a dynamic model, we transduced BTICs with the Lifeact-RFP construct for live imaging of F-actin. We observed that upon EGF stimulation, control cells responded by uniform spreading, as reflected by an increased cell circularity within seconds after stimulation, while NKCC1 KD cells did not respond. This suggests that the dynamic response of the actin cytoskeleton to external stimuli is decreased when NKCC1 is down-regulated. To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition we studied the small Rho-GTPases, RhoA and Rac1. We observed that the levels of active (GTP-bound) forms of RhoA and Rac1 were decreased in NKCC1 KD cells. Moreover, when BTICs were treated with the ROCK1 inhibitor, Y-27632, they showed a dose-dependent decrease in cell migration and activity of MLC (ROCK substrate). These effects were significantly weaker when the ROCK inhibitor was applied on NKCC1 KD or BMT-treated cells, suggesting that decreased RhoA and ROCK1 activity mediates the effects of NKCC1 inhibition. In summary, NKCC1 regulates BTIC migration beyond cell volume changes, by modulating the cytoskeleton through multiple targets that include F-actin regulation and RhoA and Rac1 activity. Due to its essential role in cell migration and high expression in GBM, NKCC1 may serve as a specific therapeutic target to decrease BTIC cell invasion and increase survival in patients with primary brain cancer. Citation Format: Paula V. Schiapparelli, Roxana Magaña-Maldonado, Susan Hamilla, Eric Goulin Lippi Fernandes, Sara Ganaha, Chuan-Hsiang Huang, Hugo Guerrero-Cazares, Helim Aranda-Espinoza, Peter N. Devreotes, Alfredo Quiñones-Hinojosa. NKCC1 regulates migration of glioblastoma tumor initiating cells by interacting with the actin cytoskeleton. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2015-4152

The hydrolysis of glutamine to glutamate plus ammonia, catalyzed in the mitochondria by the enzyme glutaminase (Gls), plays a crucial role in the metabolism of glutamine by cancer cells. Recently, we discovered a novel class of allosteric inhibitors of a splice variant of Gls, known as glutaminase C (GAC), which inhibits the growth of Rho-GTPase transformed cells without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we took advantage of mouse embryonic fibroblasts (MEFs) transformed by the induced expression of an oncogenic guanine nucleotide exchange factor (GEF) named Dbl, which hyper-activates the Rho GTPases Rac, Rho, and CDC42, together with 13C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis. We then determined how 968 inhibits the catalytic activity of GAC using biophysical approaches on purified GAC enzyme. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrated how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays, together with novel GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC, and that the dose-dependent binding directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. We then investigated the 968 structure-activity relationship, and to this end, characterized a robust real-time direct binding and inhibition assay for testing a panel of 968 derivatives. Additionally, we have developed novel 968-derivatives with unique functional moieties, such as fluorescent and photo-cross linking groups, to further elucidate the mechanism of GAC inhibition using recombinant GAC in vitro as well as in cancer cells. Together, these findings represent recent advances in our understanding of the regulation of GAC activity both in vitro and in cells, and highlight the potential future of targeting glutamine metabolism with novel small molecule therapeutics. Citation Format: Clint Stalnecker, Scott Ulrich, Jon Erickson, Sekar Ramachandran, Ralph DeBerardinis, Rick Cerione. Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1155. doi:10.1158/1538-7445.AM2015-1155

Myelodysplastic syndromes (MDS) are characterized by complex cytogenetic abnormalities, including loss of all or part of the long arm of chromosome 5 (5q). Known 5q tumor suppressors include APC1 and EGR1. APC is associated with familial colon cancer where it functions to suppress canonical Wnt signaling. Egr1 is a transcription factor that governs expression of tumor suppressor genes ranging from the p53 family to PTEN1. Adjacent to EGR1 is the DIAPH1 gene for the Rho GTPase effector formin DIAPH1. Knockout of Diaph1, Egr, or Apc in mice creates a range of MDS phenotypes. Likewise, mice with the multiple intestinal neoplasia (min/Apcmin) allele develop anemia and MDS most similar to the Diaph1 knockout phenotype. DIAPH1 builds linear actin filaments and stabilizes microtubule dynamics to establish and maintain cell polarity during adhesion, migration, and asymmetric cell division when activated by Rho GTPases, such as RhoB. Combinatorial deficiencies in RhoB (also a known tumor susceptibility gene) with Diaph1 exacerbates the Diaph1 MDS phenotype but not the rate of pathogenesis. RhoB-activated DIAPHs also signal and activate new gene expression by the myocardin-related (MRTF) and Mcm10/Agamous deficiens/ [serum response factor (SRF)] (MADS)-box transcription factors. MRTF-MADS registers changes in actin dynamics downstream of DIAPH1 to activate EGR1 expression through multiple serum response elements (SREs; recognized by SRF) within the EGR1 promoter. DIAPH1 also collaborates with APC in cytoskeletal remodeling as both proteins physically associate with and stabilize microtubules. APC can also nucleate new actin filaments that become processively elongated by DIAPH1. We examined interactions between APC and DIAPH1 signaling to SRF and in suppressing MDS pathogenesis. First, we found that both DIAPH1 and APC activate SRF- regulated gene expression. Consistent with biochemical data showing that DIAPH1 elongates APC-nucleated filaments, activation of SRF by APC relies upon DIAPH1. We bred Apcmin mice to our Diaph1 knockout mice and found that the MDS phenotype is significantly accelerated in Apcmin/Diaph1 knockout mice when compared to either the Apcmin or Diaph1 mutant phenotype. Pathological defects (dysplasia and anemia in peripheral blood and myeloid organs (spleen/bone marrow) requires at least 200 days to develop in either single mutant; combined mutations create aberrant peripheral blood cell pathology by 100 days of age. Consistent with early disease onset, mortality is accelerated in Apcmin /Diaph1-deficient mice. These novel studies show that genetic defects in Diaph1 and Apc synergize to effectively mimic 5q- MDS. Further, the data supports the idea that RhoB - DIAPH1/APC and MRTF-MADS comprise a novel tumor suppression pathway that is essential for hematopoiesis. Apcmin/Diaph1-deficient mice may prove to be a useful pre-clinical model for the development of more effective theranostic tools to attack MDS. Citation Format: Julie D. Turner, Susan M. Kitchen-Goosen, Andrew M. Howard, Heather L. Schumacher, Arthur S. Alberts. APC and DIAPH1 collaborate to suppress myelodysplastic syndrome (MDS) pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2015-4194

Prostate Specific Antigen (PSA) screening contributes as a biomarker for diagnosis of prostate cancer; however, PSA screening has been considered an over diagnosis in some cases. This has lead to the identification of novel invasive markers which could eventually differentiate patients with lethal prostate cancer from those with indolent disease. Emerging evidences suggest that a small set of microRNAs (miRNA) are linked to prostate cancer pathogenesis leading to castration resistant prostate cancer (CRPC). In our studies of genome wide miRNA profiling, we have found that miR-301a is highly expressed in prostate cancer (CaP) cell lines as compared to normal prostate epithelial cells. While analyzing CaP samples, we found higher expression of miR-301a at specific stages. Interestingly, the miR-301a induction varies from 10 to 250 fold in aggressive tumors which clearly correlate with metastases. To confirm these findings, we utilized metastatic lesions from transgenic mice model of Trans Adenocarcinoma of Mouse Prostate (TRAMP) tissues, where higher levels of miR301a expression were observed in prostate tumors in comparison with normal prostate. Further, micro dissecting the tumors (metastasis) from lung showed ∼15 fold induction of miR301a expression compared to the normal lung components. Silencing specifically miR-301a resulted in the inhibition of proliferation, colony forming abilities, invasion, and migration and cell adhesion properties in CaP cell lines. Our molecular studies also suggest that miR-301 regulates Rho GTPases family proteins and transcriptionally activates Epithelial-Mesenchymal-Transition (EMT) markers like slug and β-catenin. Our ongoing studies may not only reveal whether miR-301 regulates Rho-associated kinases (ROCK) but also the potential role of EMT in CaP. Furthermore, we intend to perform a case control study by screening an additional 100 CaP cases besides indolent controls. As mentioned earlier, besides revealing the functional role in CaP pathogenesis, results from these findings will also suggest whether miR-301 expression distinguishes lethal CaP from indolent disease. Citation Format: Trinath P. Das, Suman Suman, Arokya Ms PapuJohn, Akhila Ankhem, Sandhya R. Rao, Jamie C. Messer, Houda Alatassi, Murali M. Ankhem, Chendil Damodaran. Expression of miR-301a regulates metastasis and act as a lethal marker for prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2015-3067