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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.

Malignant gliomas are one of the most aggressive and deadly forms of cancer and can affect any age group. In glioblastoma multiforme (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Patients with malignant gliomas respond poorly to the standard therapeutic regimen of radiotherapy and chemotherapy that follow tumor resection and have only a 16-month median survival. It is therefore imperative to identify new approaches to specifically attack GBM cell survival, proliferation and invasion. The cellular mechanisms driving GBM-mediated migration and invasion are not fully understood. RSK2 (p90 ribosomal S6 kinase 2) is a kinase that regulates proliferation and adhesion and can promote metastasis. We find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. We demonstrate that active RSK2 regulates GBM adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. We further show that the effects of RSK2 on GBM invasion are mediated in part through activation of Rho GTPases. Rho family of GTPases are key regulators of cell polarity, migration and invasion. Here, we identify Rho A, B and C as downstream effectors of (RSK2) in GBM cell migration and invasion. Here, we identified a unique signaling pathway in which RSK2 confers invasiveness through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 and Cdc42. Our data support a fundamental importance of residue of Thr577 to the invasive signaling of RSK2. The invasive signaling of RSK2 appears to depend on RhoA and B, but not RhoC. RSK2 activates RhoA through activation of LARG by phosphorylation at Ser1288. Intriguingly, LARG activity is only required for RSK2-T577E, but not RSK2-Y707A-mediated RhoA activation and cell invasion. This further supports a pivotal role of Thr577 in invasive RSK2 signaling. These results establish a unique RSK2-dependent LARG-Rho signaling cascade as an uncompensated factor key to GBM cell migration and invasion. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy. Citation Format: Geng-Xian Shi, Ling Jin, Michelle L. Matter, Santosh Kesari, Joe W. Ramos. RSK2 provokes invasive signaling in glioblastoma through LARG-dependent activation of Rho GTPases [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 1363. doi:10.1158/1538-7445.AM2017-1363

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

High-grade brain tumors such as grade III astrocytoma and glioblastoma represent among the most difficult to manage malignancies facing oncological practice. Diffuse migration and invasion into adjacent healthy brain tissue yields complete surgical resection of these tumors unfeasible. As a result, despite receiving maximal surgical resection and an aggressive course of chemoradiation, the majority of high-grade brain tumor patients suffer from recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in high-grade brain tumors have been correlated with poorer outcomes. HFE is known to influence cellular iron metabolism by inhibiting transferrin-mediated iron uptake yet little is known regarding how HFE or iron impact the migratory capabilities of high-grade brain tumor cells. In order to better understand how HFE expression and cellular iron metabolism influence cell migration in high grade brain tumors, we utilized brain tumor cell lines that had been genetically manipulated to express different levels of HFE. We observed that knocking down HFE in KR158 or LN229 glioma cell lines resulted in significantly decreased migratory capacity. Since HFE is known to inhibit transferrin mediated iron uptake, we studied how directly modulating the iron status of glioma cells impacted their ability to migrate. Treatment of a panel of glioma cell lines: LN229, T98G, U87, KR158, with iron in the form of hemin or ferric ammonium citrate resulted in significantly reduced migration. Furthermore, the iron-induced reduction in migration could be rescued by the addition of deferoxamine, an iron chelator. Cell viability in response to the iron treatments was assayed and found to not be significantly altered - suggesting that cellular iron status was influencing migratory capacity independent of cell viability. To gain mechanistic insights into HFE and iron-induced effects on cell migration, we analyzed the Chinese Glioma Genome Atlas (CGGA) for correlations between HFE and the Rho GTPases RHOA, RAC1, and CDC42 – genes which are known to play a crucial role in determining the migratory capacity of cancer cells. Interestingly, we found statistically significant correlations between the Rho GTPases RHOA, RAC1, CDC42 and HFE in both grade III astrocytoma and glioblastoma patient cohorts. Immunoblotting of iron treated glioma cell lines demonstrated that expression of RhoA and Cdc42 was reduced suggesting that alterations in Rho GTPase expression and signaling may play a role in iron-induced effects on cell migration. Our results demonstrate that targeting cancer cell iron metabolism as an addition to existing treatment regimens may be a promising avenue for further investigation. Citation Format: Ganesh Shenoy, Katie Troike, Kondaiah Palsa, Madison Kuhn, Quinn Wade, Becky Slagle-Webb, Amanda Snyder, Chachrit Khunsriraksakul, Justin D. Lathia, Dhimant Desai, Hong-Gang Wang, Elizabeth Proctor, James R. Connor. The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2430.

Although initially responsive to chemotherapy, patients with advanced breast cancer often relapse, generally with incurable metastatic disease. This may be due to a subpopulation of tumor cells, called cancer-initiating cells, or cancer stem cells (CSCs), which are relatively resistant to chemotherapy and have self-renewing and tumor-initiating capacities. Prior studies in our laboratory found that CSCs may express ROR1, an onco-embryonic, tyrosine-kinase-like orphan receptor, which we found could bind Wnt5a to activate non-canonical Wnt-signaling (Proc Nat Acad Sci USA 111:17266, 2014). Interrogation of the transcriptomes of breast-cancer cells obtained from patients before and after paclitaxel therapy revealed that chemotherapy treatment enhanced cancer-cell expression of ROR1, along with genes induced by activation of Rho-GTPases (e.g. RhoA, cdc42, and Rac1). We found that primary breast-cancer patient-derived xenografts with high-level expression of ROR1 were enriched for cells that had activated Rho-GTPases and stem-cell-like gene-expression signatures. Furthermore, we found that treatment of breast cancer cell lines with Wnt5a induced ROR1-dependent activation of Rho-GTPases andAKT and induced high-level protein expression of BMI1, also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51); Wnt5a also enhanced the capacity of breast cancer cell lines to form spheroids. All these effects could be inhibited by cirmtuzumab, a humanized high-affinity anti-ROR1 mAb, which can block Wnt5a signaling. We find that ROR1-positive breast cancer cells have a greater capacity to form spheroids or engraft immune-deficient mice than did ROR1-negative cancer cells isolated from the same PDX tumor. Treatment of immune-deficient mice bearing breast-cancer PDX with paclitaxel reduced tumor volumes but enhanced expression of ROR1 and other CSC markers, such as aldehyde dehydrogenase 1 (ALDH1). Moreover, the breast cancer cells surviving such paclitaxel treatment had increased activation of Rho-GTPases and AKT, and increased expression of BMI1, relative to that of breast cancer cells obtained from the same primary tumor prior to therapy. On the other hand, treatment of such mice with cirmtuzumab also reduced breast cancer PDX tumor volumes, but the remaining cells had reduced expression of ROR1 and CSC markers and had impaired capacity to re-engraft immune-deficient mice. Finally, therapy with cirmtuzumab and paclitaxel was more effective in eradicating breast-cancer PDX than treatment with either agent alone. Collectively, these findings support use of cirmtuzumab in combination with conventional anti-cancer drugs to improve the outcome of patients with advanced breast cancer. Citation Format: Zhang S, Zhang H, Ghia EM, Liu G, Tripple V, Xu S, Cui B, Widhopf G, Yu J, Schwab R, Messer K, Parker BA, Kipps TJ. Breast cancer initiating cells express functional ROR1, which can be targeted by cirmtuzumab to potentially mitigate the risk of relapse after therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-07.

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.

Metastasis continues to be the primary cause of cancer-related death in women with breast cancer, needing targeted therapies to inhibit this process. Therefore, we developed inhibitors targeting the homologous Rho GTPases Rac1 and Cdc42, which direct the actin cytoskeletal changes required for cell migration/invasion; and thus, metastasis. We characterized the potent inhibitor, MBQ-167 that blocks both Rac1 and Cdc42 at IC50s 103 nM and 78 nM, respectively, in human HER2 (+) and triple-negative breast cancer (TNBC) cells. Consequently, MBQ-167 inhibits tumor growth and metastasis by ~90% in pre-clinical mouse models of breast cancer (Humphries-Bickley, et al., 2017). To improve the activity and solubility of MBQ-167, we synthesized a panel of derivatives, maintaining the 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole as the core for each derivative. Of the derivatives tested, MBQ-168 demonstrated improved solubility and comparable efficacy. Similar to MBQ-167, MBQ-168 induced actin cytoskeletal disintegration, cell rounding, and detachment to ultimately undergo anoikis. In 120hrs, MBQ-168 significantly inhibited the viability of MDA-MB-231 at a GI50 of 228nM and HER2+ MDA-MB-435 cells with a GI50 of 137 nM, without affecting the viability of Human Mammary Epithelial Cells (HMEC). In apoptosis assays, MBQ-168 treatment, at 500nM for 48 hrs, demonstrated a similar response to MBQ-167 by >4-fold increase in Caspase 3/7 activity in HER2 (+), and MDA-MB-231 and MDA-MB-468 TNBC cells. In wound healing assays, MBQ-168 also responded similar to MBQ-167 by a ~80% inhibition of breast cancer cell migration at 250nM and 500nM for 24hrs. Moreover, as quantified from Rac1.GTP pulldown assays, following 250nM treatment for 24hrs, MBQ-168 inhibited Rac1 activation in the attached cell population by ~70%, and in the detached cell population by ~100%, demonstrating a potent inhibition of Rac1 activation. In a preliminary study for relative efficacy in mice, MBQ-168 significantly reduced HER2+ MDA-MB-435 mammary tumor growth (~90%) at 5mg/kg BW via IP administration. In conclusion, MBQ-168 is an effective Rac inhibitor that reduces breast cancer cell viability, induces apoptosis, and inhibits breast cancer cell migration, actin cytoskeletal extensions, and Rac1 activation to result in a drastic reduction in mammary tumor growth in mice. We predict that the increased solubility of MBQ-168 will make it more bioavailable than MBQ-167. Thus, we have demonstrated that small structural modifications of MBQ-167 can affect the cytotoxic activity of carbazole derivatives with potential as anti-cancer drugs, and as tools to block Rac/Cdc42 activities in biological systems. Citation Format: Julia Medina, Tatiana Matos, Luis Velazquez, Michael Rivera, Ailed Cruz-Collazo, Eliud Hernandez, Cornelis Vlaar, Suranganie Dharmawardhane. Characterization of the Rac/Cdc42 inhibitor MBQ-168 as an anti-cancer compound [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-09.

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.

To colonize distant organ sites, cancer cells originating in solid tissues must traverse the circulatory system. How circulating tumor cells (CTCs) survive various biological and mechanical insults during this journey remains unclear. Mechanical challenges to CTCs include exposure to fluid shear stress (FSS) which ranges over several orders of magnitude throughout the circulatory system. To assess the effects of FSS on cancer cells, we developed a simple model to apply brief pulses of fluid shear stress to cancer cells in suspension. Surprisingly, we found that cancer cell lines, originating from many different tissues are more resistant to destruction by FSS than normal epithelial counterparts. FSS resistance is conferred on normal epithelial cells by transforming oncogenes and is evident in cancer cells acutely isolated from primary tumor tissues from mice and humans. Thus, FSS resistance is a conserved phenotype of viable transformed cells. FSS resistance is an active biological mechanism in viable cancer cells, dead and dying cancer cells are readily destroyed by FSS. In cancer cells, initial exposure to FSS results in rapid cell stiffening unlike non-transformed cells. This change in contractility of cancer cells exposed to FSS is associated with activation of RhoA/C GTPases. Correspondingly, in cancer cells exposed to FSS, there is a rapid increase in cortical F-actin that depends on formin activity, and myosin II activity is elevated. Both F-actin remodeling and myosin II activity are required for FSS resistance in vitro. Myosin II activity is also required for required for FSS resistance of experimental CTCs in vivo. Treatment of PC-3 cells with blebbistatin, a myosin II inhibitor, reduced the number of intact CTCs lodged in the lung and increased the number of CTCs destroyed immediately following tail vein injection. Moreover, treatment of mice bearing orthotopic prostate tumors with a non-toxic dose of blebbistatin reduced the levels of CTCs over 20-fold within 3h of treatment. As FSS exposure activates Rho GTPases, which are known to influence invasive and proliferative capacity of metastatic cells, more recently we have been investigating how FSS exposure may prime CTCs for metastasis. Exposure of both PC-3 and MDA-MB-231 cancer cell lines to FSS enhances metastatic colonization, invasive behavior in vitro and anchorage-independent proliferation. Ongoing efforts are to define the mechanisms that underly these emergent behaviors in response to FSS. In summary, we have found that viable CTCs actively resist destruction by FSS via a RhoA-dependent mechano-adaptive mechanism and that surviving cancer cells exposed to FSS exhibit enhanced metastatic potential. Citation Format: Michael Henry. Effects of fluid shear stress on circulating tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA014.

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

The proposed study focuses on Inflammatory Breast Cancer (IBC), which is one of the most aggressive forms of locally advanced breast cancer. IBC is an understudied disease in terms of identifying various molecular entities that could potentially be responsible for the aggressiveness of this disease. Recent advancements reveal that IBC has a unique molecular profile when compared with non-IBC. Caveolin-1 is found to have opposite expression pattern in IBC versus non-IBC. Our lab has shown that Caveolin-1 is found to be over-expressed in IBC whereas in non-IBC its expression is much reduced compared to normal mammary epithelial cells. Caveolin-1 is known to be associated with specialized lipid rafts in the plasma membrane called ‘caveolae’. ‘Caveolin scaffolding domain (CSD)’ from amino acids 82–101 concentrates variety of signaling molecules such as growth factor receptors, protein kinases, heterotrimeric G proteins and Rho GTPases. Thus it is known as a potential regulator of many signaling pathways. Rho GTPases are members of the Ras superfamily of small GTP binding proteins involved in cytoskeletal rearrangements during cell motility and invasion. Our lab has previously shown that RhoC GTPase is over expressed in IBC and that exogenous expression of RhoC GTPase produces motile and invasive phenotype in human mammary epithelial cells (HMECs) similar to SUM149 IBC cells. Thus the overall objective of this study is to determine if caveolin-1 over expression mediates the RhoC GTPase driven invasive phenotype of IBC via its scaffolding action. The first objective of this study is to elucidate the effect of caveolin-1 over expression on SUM149 IBC cell invasion. This was done by down regulating caveolin-1 to a level that is comparable to human mammary epithelial cells (HMECs) by using si caveolin-1. With the same si caveolin-1 or exogenous CSD synthetic peptide, we performed Matrigel invasion assays followed by cell viability and proliferation assay. Si caveolin-1 did not produce significant changes in cell viability over the period of four days as well as in Ki-67 staining which was used as a proliferation marker. Caveolin-1 down regulation reduced cell invasion by 90% compared to untreated cells. Similarly, exogenous CSD synthetic peptide treatment reduced SUM149 cell invasion significantly. In order to understand caveolin-1 localization in SUM149 cells, we performed immuno gold labeling of caveolin-1 by transmission electron microscopy as well as sucrose gradient centrifugation. Both these methods showed distinct pool of cytoplasmic caveolin-1 in addition to plasma membrane associated caveolin-1. Co-localization of caveolin-1 and RhoC GTPase was studied by co-transfecting cells with GFP-caveolin-1 and RFP-RhoC GTPase. Thus, this data suggests potential role of caveolin-1 over expression in regulating IBC cell invasion. Due to its ability to concentrate variety of signaling molecules, caveolin-1 could act as a principle regulator of several downstream events that make cells motile and invasive through RhoC GTPase activation. Thus studying caveolin-1 localization and effect of its regulation on the signaling pathways could help understand the important molecular mechanisms in IBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-03.

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.

Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor a (ERa) negativity. We previously explored the second ER subtype, (ERb) and reported expression in IBC tumors and its correlation with reduced metastasis in patients. We have also seen decreased metastasis of IBC tumors when ERb is expressed or activated by chemical agonists. In contrast, ablation of ERb in IBC cells increased migration and stimulated signaling pathways that activate Rho GTPases promoting actin reorganization. We have now mapped all ERb binding sites in IBC cells and by interrogating this information together with gene expression data we identified genomic targets that mediate its tumor repressive effects. Through a combined analysis of cells that express endogenous ERb and knockout cells engineered to re-express the receptor we have defined how a biologically relevant ERb interacts with the cancer cell genome to control metastatic signals in IBC. We reveal key binding motifs and cis-regulatory chromatin sites and associate target genes with clinical outcomes to strengthen the relevance of ERb signaling for the progression of IBC. Our findings thus offer an opportunity to better understand the mechanism of ERb action that is critical step in validating its tumor repressive role in breast cancer. Citation Format: Ilias V. Karagounis, Spyros Tastsoglou, Anna Karavangeli, Amit Maity, Artemis Chatzigeorgiou, Christoforos Thomas. Genome-wide analysis of estrogen receptor b binding sites: Implications for endogenous anti-metastatic activity in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 974.

Metastasis continues to be the most difficult phase for cancer treatment due to few targeted therapy options. Therefore, we focus on targeting the Rho GTPases Rac1 and Cdc42, key drivers of the primary steps of metastasis: cell migration and invasion. We developed MBQ-167 as a small molecule dual inhibitor for Rac1 and Cdc42 activation with IC50s of 103 and 78 nM, respectively. In metastatic human breast cancer cells, MBQ-167 inhibits the Rac/Cdc42 downstream effector p21-activated kinase (PAK)/LIM kinase/Cofilin signaling, lamellipodia extension, and thus, cell polarity, and migration. In addition, in metastatic breast cancer cells, MBQ-167 inhibits cell proliferation and cell cycle progression to ultimately induce apoptosis via anoikis. MBQ-167 inhibits tumor growth and metastasis in experimental and spontaneous HER-positive and Triple negative breast cancer (TNBC) mouse models. MBQ-167 has 35% bioavailability and is not toxic in rodent or canine models up to 1000 mg/kg. Therefore, we are further developing MBQ-167 as a lead anti metastatic cancer compound. The objective in this study is to elucidate potential activity of MBQ-167 metabolites. Prior work has identified MBQ-167 metabolites via LC MS/MS from a mammalian liver microsome (rats and human) assay. Four of the MBQ-167 metabolites with the highest MS peak area, were analyzed in metastatic breast cancer cell lines, for cell viability via MTT assays and apoptosis via caspase 3/7 assays. We report that the metabolites M6, M7, M8, and M9 did not inhibit cell viability or induce apoptosis at 250 or 500 nM in HER2-type MDA-MB-435, and TNBC MDA-MB-231 and MDA-MB-468 breast cancer cell lines, compared to MBQ-167. We next analyzed whether these metabolites could inhibit the activation of Rac and Cdc42 by analyzing the phosphorylation status of its downstream effector p-21 activated kinase isoforms 1/2/3 (PAK1/2/3). When MBQ-167 and metabolites at 250nM were incubated for 24h in HER2-type MDA-MB-435 metastatic cancer cells, and the lysates western blotted with antibodies to total PAK1/2/3 or phospho-Pak1/2/3, we found a decrease in the phosphorylation levels of PAK in response to MBQ-167 and metabolite M6. However, since the peak area of M6 in the microsome assay is only 6% compared to MBQ-167, we conclude that MBQ-167 is the major active compound with anti-metastatic cancer properties. Citation Format: Julia I. Medina, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane. Comparative activity of MBQ-167 metabolites in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 347.

Background: KRASG12C inhibitors sotorasib and adagrasib have shown impressive results in clinical studies. These inhibitors efficiently block KRAS downstream effectors such as RAF, MEK, and ERK/p-ERK. However, factors such as feedback reactivation or bypass of KRAS dependence are emerging, resulting in the need for rational combination approaches. KRASG12C inhibitors have limited impact on the parallel oncogenic signaling driven by PI3K/AKT, its regulator molecules such as the Rho GTPases, and their effectors. This signaling pathway can collectively promote p-ERK re-activation thereby blunting the efficacy of KRASG12C targeted drugs. The p21-activated kinase (PAK) family are crucial effectors of the Rho family of GTPases and act as regulatory switches that control important cellular processes including motility, proliferation, and survival. PAK4 is the main effector of Rho GTPase, cell division control protein 42 homolog (Cdc42), and has an established role in mutant RAS-dependent tumor metastasis. In this study, we rationally targeted two parallel signaling pathways by combining KRASG12C inhibitors with the PAK4 inhibitor KPT-9274 in KRASG12C mutant PDAC and NSCLC in vitro and in vivo models. Methods: We evaluated the cytotoxicity and molecular profile of KRASG12C inhibitors in combination with PAK4 inhibitor KPT-9274 as well as positive control PF-3758309 in KRASG12C mutant 2D and 3D cellular/spheroid models. The anti-tumor activity of the combinations was evaluated in KRASG12C mutant cell line-derived xenograft. Tumor volumes were assessed throughout the treatment period using an ultrasound scanner. Results: PAK4 inhibitor KPT-9274 synergized with both sotorasib and adagrasib yielding suppressed growth of KRASG12C mutant PDAC and NSCLC cells in 2D and 3D cultures, but not in KRASwt, KRASG12V, and KRASG12D cell lines (NCI RASless MEFs). In addition, the combination reduced the clonogenic potential of KRASG12C mutant cancer cells. PAK4-KRASG12C inhibitor combinations resulted in enhanced and prolonged inhibition of KRAS downstream effector pathways. In xenograft studies, oral administration of KPT-9274 at sub-optimal dose (100 mg/kg QD x 5 x 3 weeks) and sotorasib at one-fourth of MTD (25 mg/kg QD x 5 x 3 weeks) demonstrated remarkable inhibition of KRASG12C mutant MiaPaCa-2 tumor burden (p Conclusion: This is the first study showing that KRASG12C inhibitors can synergize with PAK4 inhibitors. This combination can simultaneously target direct KRAS downstream effectors and the parallel PI3K/AKT oncogenic signaling warranting further clinical investigations. Citation Format: Husain Yar Khan, Sahar Bannoura, Hirva Mamdani, Amro Aboukameel, Yousef Mzannar, Yiwei Li, Mohammad Najeeb Al-Hallak, Ibrahim Azar, Steve Kim, Rafic Beydoun, Ramzi Mohammad, Yosef Landesman, Yue Zhang, Erkan Baloglu, William Senapedis, Ammar Sukari, Misako Nagasaka, Anthony Shields, Asfar S. Azmi. Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5315.

NGS studies implicate dysregulation of the splicing machinery in the development of diverse cancers. The X-chromosome RBM10 gene encodes an RNA-binding protein that modulates transcriptome-wide cassette exon splicing. Truncation and missense RBM10 mutations are enriched (11%) in non-anaplastic thyroid cancers of patients who died of metastatic disease. MSK-MET, an integrated pan-cancer cohort of tumor genomic and clinical outcome data showed RBM10 alterations associate with metastatic burden in thyroid cancer. Within the MSK-IMPACT thyroid cancer cohort, 44% of RBM10 alterations co-occur with RAS mutations. We developed a murine Rbm10 floxed allele, which results in a non-functional transcript. Thyroid-specific Rbm10 inactivation through Tpo-Cre did not induce a phenotype. When crossed with FR-HrasG12V mice, which upon recombination generate endogenous expression of HrasG12V, Hras/Rbm10 mice developed thyroid cancers, ~ 20% of which were metastatic to lung. Cell lines derived from these tumors showed high penetrance of lung metastases after tail vein or orthotopic implantation into the thyroid. We identified splicing targets of RBM10 by high depth RNAseq of 5 isogenic human thyroid cancer cell lines (2 RBM10-null with dox-induced RBM10; 3 RBM10 WT with RBM10 shRNA). The common abnormalities associated with RBM10 loss were exon inclusion events. Ingenuity Pathway Analysis of global transcriptomes in RBM10 isogenic human thyroid cancer cell lines showed enrichment in pro-migratory, aberrant integrin and RHO/RAC signaling expression signatures. Enriched GO analysis confined to genes subject to aberrant splicing by RBM10 loss extended these findings, with the top terms being cell adhesion, cytoskeleton, cadherin and integrin binding. RBM10 loss was associated with increased cell migration velocity in vitro as measured by time lapse imaging. Key cytoskeletal and extracellular matrix genes subject to exon inclusion events included vinculin (VCL), tenascin C (TNC), CD44, fibronectin (FN1) and tropomyosin 1 (TPM1). Isoform-specific knockdown of the VCL splice inclusion cassette exon that leads to illegitimate expression of metavinculin (mVCL) reduced cell migration, whereas isoform-specific knockdown of TNC and CD44 exon inclusion events reduced cell invasiveness in vitro. Rho GTPases transduce signals from the ECM to integrin receptors to regulate cell adhesion, migration, and invasiveness. Consistent with this, RBM10 overexpression in RBM10 null cells reduced RAC1-GTP levels. Finally, RBM10 re-expression in RBM10 null cells reversed metastatic competency in vivo. In conclusion, RBM10 loss alters the ratio of cassette exon inclusion events in a subset of transcripts that regulate interactions between the ECM and the cytoskeleton, leading to RHO/RAC activation and governing a process favoring increased cell movement and metastatic competence. Citation Format: Gnana P. Krishnamoorthy, Anthony Glover, Brian Untch, Mahesh Saqcena, Dina Vukel, Katherine Berman, Omar Abdel-Wahab, Robert K. Bradley, Jeffrey A. Knauf, James A. Fagin. RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 986.

The role of soy foods in cancer prevention is known but their effects on cancer metastasis remain to be understood. The purpose of this study is to investigate the effects and mechanism of action of dietary soy isoflavones in established carcinomas. This objective was achieved by testing the effect of the individual and combined soy isoflavones on progression of mammary fat pad tumors in nude mice created from green fluorescent protein (GFP) tagged-MDA-MB-435 metastatic cancer cells. Following tumor establishment, mice were orally gavaged with vehicle or soy isoflavones: genistein (10 mg/kg body weight (BW)), daidzein (10 mg/kg BW), or genistein (50%), daidzein (40%), and glycitein (10%) three times per week. Tumor progression was quantified by whole body fluorescence image analysis twice a week followed by microscopic image analysis of excised organs for metastases. Results show that daidzein significantly increased while genistein decreased mammary tumor growth by 38% and 33% respectively. Daidzein increased lung, heart, and kidney metastases while genistein decreased bone and liver metastases. Combined soy isoflavones did not affect primary mammary tumor growth but increased metastasis to all organs tested; i.e. lung, liver, heart, kidney, and bones. Phosphoinositide-3-kinase (PI3-K) pathway real time PCR array analysis on excised tumors demonstrates that genistein significantly downregulated 26% of the genes tested, including Rho GTPases RHOA, RAC1, and CDC42, and their downstream effector PAK1 that are known regulators of cancer cell invasion, and thus, metastasis. Daidzein upregulated 14% of the genes including CCND1, GRB2, MAPK1, JUN, CTNNB1, IRS1, EIF4G1, and GSK3B that regulate proliferation and protein synthesis. Combined soy treatment significantly increased EIF4E and decreased TIRAP expression. Western blotting of mammary tumor extracts following genistein, daidzein, or combined soy isoflavones show that genistein downregulates RhoA, Rac1, and Cdc42 while daidzein upregulates Rac1, GSK3, and eIF4G1 protein expression. Combined soy isoflavones did not show significant differences in expression of Rho GTPases or PAK. In conclusion, downregulation of Rho GTPases by genistein may reduce mammary tumor growth and metastasis while upregulation of eIF4G and Rac1 by daidzein may increase mammary tumor growth and metastasis. Consumption of soy foods that contain both genistein and daidzein may neutralize their differential effects on primary tumor growth. The increased metastasis but not primary tumor growth in response to combined soy isoflavones indicates that even though dietary soy has been implicated in cancer prevention, caution must be exercised in dietary decisions of soy foods for cancer patients and survivors. This research was supported by AICR IIG 03-31-06 and NIH/NIGMS SC3GM084824 to SD and NCCR/NIH 2G12RR003035 and S06GM050695 to UCC. 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 5725.

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.

Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor α (ERα) negativity. Here we explored the role of the second ER subtype, ERβ, and report expression in IBC tumors and its correlation with reduced metastasis. Ablation of ERβ in IBC cells promoted cell migration and activated gene networks that control actin reorganization, including G-protein–coupled receptors and downstream effectors that activate Rho GTPases. Analysis of preclinical mouse models of IBC revealed decreased metastasis of IBC tumors when ERβ was expressed or activated by chemical agonists. Our findings support a tumor-suppressive role of ERβ by demonstrating the ability of the receptor to inhibit dissemination of IBC cells and prevent metastasis. On the basis of these findings, we propose ERβ as a potentially novel biomarker and therapeutic target that can inhibit IBC metastasis and reduce its associated mortality. Significance: These findings demonstrate the capacity of ERβ to elicit antimetastatic effects in highly aggressive inflammatory breast cancer and propose ERβ and the identified associated genes as potential therapeutic targets in this disease.

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.

The Rho GTPases Rac and Cdc42 are potential targets against metastatic disease. Our group characterized the small molecule MBQ-167 as an effective dual Rac/Cdc42 inhibitor that reduces HER2 positive and triple negative tumor growth and metastasis in mice by ~90%. To continue validating this drug for FDA approval, we recently elucidated the plasma pharmacokinetics and the tissue distribution of MBQ-167 in mice (Maldonado et al., 2019). However, further studies are needed to characterize the tumor pharmacokinetics of MBQ-167 to determine drug exposure, time-to-peak, and the length of time that MBQ-167 remains in tumors after administration. The purpose of this study was to determine the tumor pharmacokinetics of MBQ-167 in a single dose input scheme (10 mg/kg BW) following intraperitoneal (IP) administration. Thirty-five female BALB/c mice (5 mice/group) were injected with 2.5 × 105 4T1 murine metastatic breast cancer cells at the mammary fat pad. After tumor establishment, a single dose of MBQ-167 (10 mg/kg BW) was administered via IP. Tumors were collected at 0.5, 1, 3, 6, 9,12, and 24 hours following drug administration. We used a validated bioanalytical method using supercritical fluid chromatography coupled with tandem mass spectrometry (SFC-MS/MS) to quantify MBQ-167 in tumors. Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNolin® software. Pharmacokinetic analysis revealed that MBQ-167 has an elimination half-life (t1/2) of 8.64 hours in tumors, which is much longer than its half-life in plasma (2.17 hours), indicating preferential absorption and retention of this lipophilic small molecule in tumor tissues. The area under the curve (AUC0-t) was 4.39 %ID⋅hr/g with a maximum concentration (Cmax) of 1268.9 ng/g in tumors. Moreover, the time-to-peak concentration in tumors was 0.5 hours. Hence, this study supports the continued development of MBQ-167 as a potential anti-cancer therapeutic. Citation Format: Maria Del Mar Maldonado, Ailed M. Cruz-Collazo, Luis D. Borrero-García, Gabriela T. Rosado-González, Jean F. Ruiz-Calderon, Jorge Duconge, Suranganie Dharmawardhane. Tumor pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice [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 3020.

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.

Metastasis is a major cause of death in epithelial cancers, largely because of a lack of effective treatments. The metastatic inhibitor MBQ-167 was developed to target the Rho GTPases Rac and Cdc42, which promote cell cycle progression, proliferation, survival, migration/invasion, and thus, metastasis. Our published data show that MBQ-167 inhibits the activation of Rac and Cdc42 with ~100 nM IC50, and subsequently inhibits their effector p21-activated kinase (PAK) activation by autophosphorylation. In metastatic cancer cells, MBQ-167 induces a loss of cell polarity, followed by detachment from the substrate, and subsequent apoptosis (anoikis). The objective herein was to determine the potential off-target effects of MBQ-167. Therefore, the effect of MBQ-167 on the triple negative breast cancer kinome was investigated using a Human Phospho-Kinase Array for 45 phospho proteins. MDA-MB-231 cells treated with MBQ-167 for 24 h at 200 nM demonstrated no difference in phosphorylation of off-target kinases. However, phospho (p)-CREB and p-c-Jun transcription factor levels increased by ~3-fold in the cells that remained attached following MBQ-167 treatment. This was confirmed by Western blot for p-CREB and p-c-Jun as well as their transcriptional targets (Cyclin D1, survivin, ZEB1). However, a time-course analysis via Western blot revealed that this increase in CREB and c-Jun activity is transient. Western blotting of the attached and detached cell populations of MDA-MB-231 cells following MBQ-167 treatment for 24, 48, or 96 h demonstrated decreased c-Jun/p-c-jun, and CREB and Jun targets in the detached cell population. Moreover, the detached cells demonstrated additional decreases in phospho forms of ERK1/2, Akt, mTOR, beta catenin and STAT3, which have all been implicated in Rac/PAK signaling; as well as reduced expression of the anti-apoptotic proteins Bcl2, BCL-XL, and MCl-1. Therefore, we posit that the initial upregulation of transcription factors in response to MBQ-167 is a transient reaction to its strong Rac/Cdc42/PAK inhibition. Prolonged treatment with MBQ-167 causes a massive deregulation of cancer drivers and pro-survival proteins to ultimately result in anoikis. We attempted to create a MBQ-167 resistant cell population by treating MDA-MB-231 cells with ascending concentrations of MBQ-167 over time, but were unable to recover any live cells. Therefore, we conclude that MBQ-167 is a direct and specific inhibitor of Rac and Cdc42 and that potential off-target effects of MBQ-167 are only temporary, where 100% of the metastatic cancer cells treated with MBQ-167 ultimately undergo apoptosis. Collectively, our data on the mechanism of action of MBQ-167 and its efficacy in vitro and in vivo in mouse models of metastatic cancer demonstrate that MBQ-167 is a viable anti-metastatic cancer inhibitor for further development as an experimental therapeutic. Citation Format: Hector M. Picon, Maria Del Mar Maldonado, Surangani F. Dharmawardhane Flanagan. Investigating potential off-target effects of the dual Rac and Cdc42 inhibitor MBQ-167 [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 623.

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.