Search

Background: Immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD). While the increase in cancer incidence with age and age-associated changes in immune function have been well characterized, age-related differences in the intratumoral immune populations and how this affects immunotherapy response remain unknown. Methods: We collected clinical data from 3,544 samples treated with immune checkpoint blockade (ICB) at the pan-cancer level. Single-cell RNA sequencing data from 19 LUAD samples were integrated and analyzed. Cell types were identified based on canonical marker gene expression. Results: Aged individuals responded more efficiently to ICB treatment at the pan-cancer level. By focusing on LUAD, we showed that patients aged above 46 responded better and had longer progression-free survival than younger counterparts. Further, we presented a 58,031-cell catalog of age-associated single-cell transcriptomes from LUAD patients. Our analysis revealed the accumulation of immune response and chronic inflammation with increasing age. Aged tissues (> 60 years) displayed dominant exhausted natural killer (NK)/T cells and T regulatory cells and were enriched with two subtypes of tumour-associated macrophages marked by SPP1 and CX3CR1 expression, respectively. Furthermore, the ageing dynamics of stroma cells created a pro-tumor microenvironment reflected by increased angiogenesis and collagen formation. Conclusions: These results revealed an increasing immune-suppressive tumour microenvironment with age, supporting an observation of improved ICB response in older LUAD patients. Our extensive single-cell analysis enhances the understanding of age-dependent molecular and cellular dynamics in LUAD. Our results highlight the importance of considering age as a factor for ICB response and provide a rich resource for developing therapeutic targets in LUAD-microenvironment interactions. Citation Format: Xiaoqiang Zhu, Hai Wang, Xuefeng Pan, Jie Hong, Jason Wing Hon Wong, Haoyan Chen, Youwei Zhang. Single-cell RNA sequencing of lung adenocarcinoma patients reveals age-dependent tumour microenvironment alterations that facilitate response to immunotherapy [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 6102.

Background: Targeting programmed cell death-1 (PD-1) or programmed cell death ligand-1 (PD-L1) has been shown improved clinical efficacy in a wide range of tumor types. We evaluated serum interleukin 14α (IL14α) as a biomarker to predict the response of anti- PD-1 therapy. Patients and methods: Thirty advanced cancer patients treated with PD-1 inhibitor were enrolled in this study. Serum levels of IL-14α were tested at baseline and after 2 cycles of treatment. Result: Among these 30 patients, the mean expression level of IL14α before treatment was 2.1±1.21, whereas the mean level of IL14α after 2 cycles was 1.99±0.82. There were no association between the expression levels of IL14α and clinical outcome. Early change of IL14α after 2-cycles of anti-PD-1 therapy was calculated as delta IL14α % change = (IL14α level after 2 cycles - IL14α level before treatment)/IL14α level before treatment*100%. Receiver operating characteristic (ROC) was analyzed to get a cutoff point of delta IL14α % change as 2.46% (Sensitivity 85.71%, Specificity 62.5%; AUC=0.7277, P=0.034). Using this cutoff to subgroup the patients, we found higher delta IL14α % change was significantly associated with superior overall response to anit-PD-1 therapy (p=0.0072) and had a better progression free survival (p=0.0039). Conclusion: Early changes of serum IL14α level may be a useful predicting factor in advanced cancer patients with anti-PD-1 therapy. Keywords: IL14α, serum biomarker, anti-PD-1 therapy response, cancer Citation Format: Bu Hai Wang, Cai Yue Chen, Xian Zhang, Yu Xiang Huang, Yi Chun Zeng, Lei Li, Mao Qi Wang, Jing Liang Guo, Qiu Xian Li, Long Shen, Juan J. Gu, Yi Chen Liang. The early change of serum interleukin 14α levels predicts the response to anti-PD-1 therapy in 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 5091.

Although metabolic reprogramming is recognized as a hallmark of tumorigenesis and progression, little is known about metabolic enzymes and oncometabolites that regulate breast cancer metastasis, and very few metabolic molecules have been identified as potential therapeutic targets. In this study, the transketolase (TKT) expression correlated with tumor size in the 4T1/BALB/c syngeneic model. In addition, TKT expression was higher in lymph node metastases compared with primary tumor or normal tissues of patients, and high TKT levels were associated with poor survival. Depletion of TKT or addition of alpha-ketoglutarate (αKG) enhanced the levels of tumor suppressors succinate dehydrogenase and fumarate hydratase (FH), decreasing oncometabolites succinate and fumarate, and further stabilizing HIF prolyl hydroxylase 2 (PHD2) and decreasing HIF1α, ultimately suppressing breast cancer metastasis. Reduced TKT or addition of αKG mediated a dynamic switch of glucose metabolism from glycolysis to oxidative phosphorylation. Various combinations of the TKT inhibitor oxythiamine, docetaxel, and doxorubicin enhanced cell death in triple-negative breast cancer (TNBC) cells. Furthermore, oxythiamine treatment led to increased levels of αKG in TNBC cells. Together, our study has identified a novel TKT-mediated αKG signaling pathway that regulates breast cancer oncogenesis and can be exploited as a modality for improving therapy. Significance: These findings uncover the clinical significance of TKT in breast cancer progression and metastasis and demonstrate effective therapy by inhibiting TKT or by adding αKG. Cancer Res; 78(11); 2799–812. ©2018 AACR.

Pancreatic cancer is the third most common cause of death from cancer in the US. The disease has a poor prognosis with a five-year survival rate under 10%. Mutations in KRAS are driving tumor growth in about 95% of patients with pancreatic cancer. Currently there are no approved drugs that target KRAS. Recently there has been progress targeting a specific mutation, KRAS G12C with small molecules in lung cancer, but this mutation accounts for only 1% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). RNA interference has the potential to directly target KRAS, but delivery to pancreas has been challenging. We have developed a lipid nanoparticle formulation that is able to distribute to pancreatic tumor tissue. The proprietary lipid nanoparticle (LNP) is comprised of an ionizable, non-immunogenic, biodegradable lipid and the composition has been tuned to favor enrichment in pancreatic tumor by screening in orthotopic pancreatic tumor mouse models. A potent siRNA with IC50 in the range of 10pM targeting KRAS directly has been identified and the activity on downstream pathways and kinetics have been demonstrated. Transfection of ND-05-1192 led to more impeded cell growth in pancreatic cancer cells (PANC-1, AsPC-1, Capan-1, SW1990 and BxPC-3) than normal cells (human hepatocyte, pancreatic cells and HUVEC cells). Western blot results demonstrated effective downregulations of key proteins involved in MAPK and PI3K signaling pathways and upregulated activities of proteins involved in apoptotic signaling pathways, such as BIM, caspase 3/7 and cleaved-PARP following inhibition of KRAS protein expression. In vivo biodistribution and tumor cell uptake study results demonstrated that LNP transported ND-05-1192 siRNA molecules to the pancreas site and pancreatic orthotopic tumor tissues with the majority of tumor cells showing LNP product uptake. The application of ND-05-1192/LNP product significantly suppressed tumor growth in PANC-1 and AsPC-1 bioluminescent orthotopic pancreatic xenograft models. Inhibition efficiency revealed by tumor weight and bioluminescent response (BLR) as the treatment end points reached up to 50% in the PANC-1 orthotopic xenografts following once weekly intravenous administration. In the orthotopic AsPC-1 model, the average BLR was significantly reduced in a dose-dependent manner. Additionally, in AsPC-1 orthotopic xenografts, the survival rate of the ND-05-1192/LNP treatment group was significantly prolonged at a 4 mg/kg dose compared to the vehicle control group. In summary, we conclude that ND-05-1192/LNP is delivered to the orthotopic pancreatic tumor and its considerable uptake by tumor cells results in KRAS target downregulation, tumor growth inhibition and overall survival benefit as demonstrated in our orthotopic xenograft models. Citation Format: Zhihong (Julie) OBrien, Li Wang, Cima Cina, Bharat Majeti, James Chu, Xiaoen Wang, Robiel Baclig, James Lin, Jose Ibarra, Dhrubamitra Chatterjee, Jung-Kang Jin, Jean-Pierre Clamme, Jennifer Daniels, Hai Wang, Jens Harborth, Wenbin Ying. Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer [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 4060.

TGFβ signaling inhibits cell proliferation to block cancer initiation, yet it also enhances metastasis to promote malignancy during breast cancer development. The mechanisms underlying these differential effects are still unclear. Here, we report that HER2/EGFR signaling switches TGFβ function in breast cancer cells from antiproliferation to cancer promotion. Inhibition of HER2/EGFR activity attenuated TGFβ–induced epithelial–mesenchymal transition and migration but enhanced the antiproliferative activity of TGFβ. Activation of HER2/EGFR induced phosphorylation of Smad3 at Ser208 of the linker region through AKT, which promoted the nuclear accumulation of Smad3 and subsequent expression of the genes related to EMT and cell migration. In contrast, HER2/EGFR signaling had no effects on the nuclear localization of Smad2. Knockdown of Smad3, but not Smad2, blocked TGFβ–induced breast cancer cell migration. We observed a positive correlation between the nuclear localization of Smad3 and HER2 activation in advanced human breast cancers. Our results demonstrate a key role for HER2/EGFR in differential regulation of Smad3 activity to shift TGFβ function from antitumorigenic to protumorigenic during breast cancer development. Significance: TGFβ signaling can shift from inhibiting to promoting breast cancer development via HER2/EGFR AKT-mediated phosphorylation of Smad3 at S208, enhancing its nuclear accumulation and upregulation of EMT-related genes. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6073/F1.large.jpg. Cancer Res; 78(21); 6073–85. ©2018 AACR.

14-3-3ζ is overexpressed in more than 40% of breast cancers, but its pathophysiologic relevance to tumorigenesis has not been established. Here, we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter-driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors, whereas control mice did not. Whey acidic protein promoter-driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared with MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling, leading to increased miR-221 transcription, which inhibited p27 CDKI translation and, consequently, promoted cell proliferation. Importantly, this 14-3-3ζ–miR-221–p27 proliferation axis is also functioning in breast tumors in patients and is associated with high-grade cancers. Taken together, our findings show that overexpression of 14-3-3ζ has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation. Cancer Res; 74(1); 363–73. ©2013 AACR.

As a validated therapeutic target in several human cancers, the EGF receptor (EGFR) provides a focus to gain deeper insights into cancer pathophysiology. In this study, we report the identification of a naturally occurring and widely expressed EGFR isoform termed EGFRvA, which substitutes a Ser/Thr-rich peptide for part of the carboxyl-terminal regulatory domain of the receptor. Intriguingly, EGFRvA expression relates more closely to histopathologic grade and poor prognosis in patients with glioma. Ectopic expression of EGFRvA in cancer cells conferred a higher invasive capacity than EGFR in vitro and in vivo. Mechanistically, EGFRvA stimulated expression of STAT3, which upregulated heparin-binding EGF (HB-EGF). Reciprocally, HB-EGF stimulated phosphorylation of EGFRvA at Y845 along with STAT3, generating a positive feedback loop that may reinforce invasive function. The significance of EGFRvA expression was reinforced by findings that it is attenuated by miR-542-5p, a microRNA that is a known tumor suppressor. Taken together, our findings define this newfound EGFR isoform as a key theranostic molecule. Cancer Res; 73(23); 7056–67. ©2013 AACR.

Despite better control of early-stage disease and improved overall survival of patients with breast cancer, the incidence of life-threatening brain metastases continues to increase in some of these patients. Unfortunately, other than palliative treatments there is no effective therapy for this condition. In this study, we reveal a critical role for Src activation in promoting brain metastasis in a preclinical model of breast cancer and we show how Src-targeting combinatorial regimens can treat HER2+ brain metastases in this model. We found that Src was hyperactivated in brain-seeking breast cancer cells derived from human cell lines or from patients' brain metastases. Mechanistically, Src activation promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood–brain barrier. When combined with the EGFR/HER2 dual-targeting drug lapatinib, an Src-targeting combinatorial regimen prevented outgrowth of disseminated breast cancer cells through the induction of cell-cycle arrest. More importantly, this combinatorial regimen inhibited the outgrowth of established experimental brain metastases, prolonging the survival of metastases-bearing mice. Our results provide a rationale for clinical evaluation of Src-targeting regimens to treat patients with breast cancer suffering from brain metastasis. Cancer Res; 73(18); 5764–74. ©2013 AACR.

Trastuzumab is an iconic rationally designed targeted therapy for HER2-positive breast cancers. However, the low response rate and development of resistance call for novel approaches for the treatment of patients. Here, we report that concurrent targeting of tumor cells and activation of T cells in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes resistance in two distinct PTEN loss–mediated trastuzumab-resistant mammary tumor mouse models. In vivo combination treatment with HER2/Neu antibody and Akt inhibitor triciribine effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and mitogen-activated protein kinase signaling accompanied by increased T-cell infiltration in the tumor microenvironment. We showed that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of this combination treatment in an IFN-γ–dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and triciribine combination treatment were further improved when coinhibitory receptor cytotoxic T-lymphocyte–associated antigen 4 was blocked to enhance the T-cell response. Our data indicate that multitargeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment could cooperate to exert maximal therapeutic activity, suggesting a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies. Cancer Res; 72(17); 4417–28. ©2012 AACR.

Therapeutics targeting known oncoproteins have been applied for pancreatic cancer treatment but clinical outcomes are not promising. Hence, there is an urgent need to identify novel targets and develop effective drugs to improve pancreatic cancer therapeutic index. Antibody-drug conjugates (ADC) represent a promising class of drugs for targeted cancer therapy. Here we developed a novel ADC targeting RON receptor tyrosine kinase for potential pancreatic cancer treatment. To this end, we have synthesized a bis-alkylating linker (BL), attached to a lysosomal protease-cleavable dipeptide with payload Monomethyl auristatin E (MMAE). The BL-MMAE was then conjugated to Zt/g4 (anti-RON mAb) through cysteine bridging technology to produce Zt/g4-BL-MMAE with a homogeneous conjugation profile and an antibody to drug ratio of 1:4. Zt/g4-BL-MMAE showed significant improvement in drug conjugation homogeneity and serum stability over conventional ADCs prepared through maleimide based linkers. In pancreatic cancer cell lines overexpressing RON, Zt/g4-BL-MMAE specifically targeted RON-expressing tumor cells and was effective in rapid induction of cell surface RON endocytosis. Functional analysis revealed that Zt/g4-BL-MMAE caused cell cycle arrest at G2/M phase, reduction of cell viability and subsequently resulted in massive cell death. The calculated IC50 is in the range of 1 to 2 µg/ml. We conclude that Zt/g4-BL-MMAE is a novel anti-RON ADC with excellent conjugation profile, serum stability, and selective cytotoxicity for pancreatic cancer cells. This work provides a pharmaceutical opportunity for evaluating potentials of RON-targeted ADCs in pancreatic cancer treatment in the future. Citation Format: Sreedhar Reddy Suthe, Hang-Ping Yao, Paul C. Trippier, Ming-Hai Wang. Development of a novel ron receptor targeted antibody-drug conjugates using cysteine bridging technology for potential treatment of pancreatic 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 745.

Metastasis, the foremost cause of mortality in cancer patients, is increasingly recognized as a coordinated biological process. The multistep process of metastasis posts difficulty in studying its mechanism and molecular basis. Recent works have shown that the basic helix-loop-helix transcriptional factor Twist and the serine/threonine kinase AKT play pivotal roles in tumor development and progression. Our recent study has shown that AKT2 is a transcriptional regulatory target of Twist and acts downstream of Twist to promote cancer cell survival, migration, and invasion. Functional convergence of Twist and AKT2 underscores the importance of this signaling pathway in tumor development and progression and as a potential therapeutic target. [Cancer Res 2008;68(4):957–60]

Metastasis, the cardinal feature of malignant tumors, is an important clinical variable in patient prognosis. To understand the basis for metastasis, we systematically selected for highly invasive cells from breast cancer cell lines, MCF7 and MDA-MB-453, with moderate to low invasive ability using Boyden chamber invasion assay. The four-cycle selected invasive lines, named MCF7-I4 and MDA-MB-453-I4, respectively, displayed epithelial-mesenchymal transition (EMT) and dramatically enhanced invasive ability. EMT changes were corroborated with decreased level of E-cadherin and increased vimentin, fibronectin, and β1 integrin. Twist, a basic helix-loop-helix transcription factor, and AKT2, a known proto-oncogene, were found to be elevated in the invasive cells compared with the parental. Ectopic expression and knockdown of Twist by short interference RNA resulted in significant increase and reduction, respectively, of AKT2 protein and mRNA expression. Twist bound to E-box elements on AKT2 promoter and enhanced its transcriptional activity. Moreover, silencing AKT2 decreased Twist-promoted migration, invasion, and paclitaxel resistance. Reintroducing AKT2 largely rescued the phenotype resulted from knockdown of Twist in I4 cells, suggesting that AKT2 is a downstream target and functional mediator of Twist. Finally, we observed a 68.8% correlation of elevated Twist and AKT2 expression in late-stage breast cancers as oppose to 13% in early-stage breast cancers. Our study identifies Twist as a positive transcriptional regulator of AKT2 expression, and Twist-AKT2 signaling is involved in promoting invasive ability and survival of breast cancer cells. [Cancer Res 2007;67(5):1979–87]

MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols from poorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR-491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein—coupled receptor kinase-interacting protein 1 (GIT1)—as a direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-491-5p–mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal–regulated kinase (ERK1/2) activation, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer. Cancer Res; 74(3); 751–64. ©2013 AACR.

Tamoxifen and aromatase inhibitors (AIs) have shown efficacy to prevent estrogen receptor positive (ER+) breast cancer; however, there is no effective prevention strategy for ER- breast cancer. HER2 overexpression (HER2+) can be detected in up to 40% of ER- breast cancers and it has been reported to promote the progression of early stage breast cancer. Here, we tested the feasibility of lapatinib to prevent ER-/HER2+ early stage breast disease. Our data showed that lapatinib reduced the proliferation and increased the apoptosis in an in vitro ER-/HER2+ early breast disease model. Furthermore, lapatinib treatment in the HER2-driven MMTV-neu mouse model from early stage of disease development (atypia, at 10 weeks of age) significantly delayed tumor initiation and progression as detected by mammary gland biopsy and fiber optic micro-endoscope (FOME) imaging. Mechanistically, we found that lapatinib inhibited GLUT4 upregulation and increased glucose up-taking in HER2-overexpressing cells. These results suggested that lapatinib is a promising agent for the prevention/intervention of ER- and HER2+ breast cancers. Our study brings better understanding of the mechanism underlying ER-/HER2+ early stage breast disease progression to cancer and our strategy of cancer prevention with low-dose kinase inhibitor could be rapidly translated into the clinic for other cancer types in the near future. Citation Format: Sunil Acharya, Jia Xu, Xiao Wang, Shalini Jain, Hai Wang, Qingling Zhang, Joseph Bower, Rebecca Richards-Kortum, Dihua Yu. Preventing the progression of estrogen receptor-negative/HER2-overexpressing early stage breast disease by lapatinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 832.

Metastasis is the number one cause of cancer-related mortality. Major neoplastic diseases such as melanoma, lung, breast, and colon cancers have high incidences of brain metastases. One-year survival after diagnosis of brain metastasis is less than 20%. Cancer cells dynamically interacts with specific organ microenvironments to establish metastasis as depicted by the “seed and soil” hypothesis. Yet it is unclear when and how disseminated tumor cells acquire the essential traits from the brain microenvironment that primes their subsequent metastatic outgrowth. Here we found that primary tumor cells with normal PTEN expression lose PTEN after dissemination to the brain, but not to other organs. Metastatic brain tumor cells that have experienced PTEN loss have PTEN levels restored once they leave the brain. This brain microenvironment-dependent, reversible PTEN mRNA and protein down-regulation is epigenetically regulated by microRNAs (miRNAs) from astrocyte-derived exosomes. Furthermore, this adaptive PTEN loss in brain metastatic tumor cells leads to an increased secretion of cytokine chemokine (C-C motif) ligand 2 (CCL2), which recruits Iba1+ myeloid cells that reciprocally enhance outgrowth of brain metastatic tumor cells via enhanced proliferation and reduced apoptosis. Our findings signify the dynamic and reciprocal cross-talk between tumor cells and other brain stromal cells. Disseminated tumor cells acquire the essential traits from the microenvironment of brain that prime their outgrowth. Importantly, our finding provides new opportunities for effective anti-metastasis therapies: inhibiting CCL2 might be an effective therapeutic intervention of life-threatening brain metastases. Citation Format: Lin Zhang, Siyaun Zhang, Jun Yao, Frank J. Lowery Lowery, Qingling Zhang, Wen-Chien Huang, Ping Li, Min Li, Xiao Wang, Chenyu Zhang, Hai Wang, Kenneth Ellis, Mujeeburahiman Cheerathodi, Joseph McCarty, Diane Palmieri, Patricia Steeg, Jodi S Saunus, Sunil Lakhani, Suyun Huang, Aysegul Sahin, Kenneth Aldape, Dihua Yu. Brain microenvironment induced PTEN loss by microRNAs promotes brain metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 907.

Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby facilitating cellular adaptation to the adverse environment and sustaining rapid proliferation. Recently, new evidence has emerged that abnormal metabolic reprogramming may occur at the early-stages of neoplasia and critically contribute to early transformation and cancer initiation. Previously, we reported that 14-3-3ζ overexpression is an early event in human breast cancer, taking place as early as in the atypical ductal hyperplasia (ADH) stage. We therefore hypothesized that 14-3-3ζ overexpression in breast epithelial cells may trigger metabolic reprogramming by elevating glycolysis that contributes to cancer initiation. In the present study, our bioinfomatics analysis of microarray profiling data from the early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of genes of the canonical glycolytic pathway, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human non-transformed mammary epithelial cells (hMECs), MCF10A and MCF12A, transcriptionally up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation of hMECs. Conversely, knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis by activating the MEK-ERK-CREB signaling axis and transcriptionally up-regulating LDHA, thereby critically contributes to human breast cancer initiation. Citation Format: Chia-Chi Chang, Chenyu Zhang, Ozgur Sahin, Qingling Zhang, Hai Wang, Jian Zhang, Sumaiyah Rehman, Ping Li, Dihua Yu. Upregulation of lactate dehydrogenase A by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-313.

Prostate cancer remains the second leading cause of cancer death in men in the Western world. Yet current therapies do not significantly improve the long-term survival of patients with distant metastasis. In this study, we investigated the role of the guanine nucleotide exchange factor Vav3 in prostate cancer progression and metastasis and found that Vav3 expression correlated positively with prostate cancer cell migration and invasion. Stimulation of the receptor tyrosine kinase EphA2 by ephrinA1 resulted in recruitment and tyrosine phosphorylation of Vav3, leading to Rac1 activation as well as increased migration and invasion in vitro. Reduction of Vav3 resulted in fewer para-aortic lymph nodes and bone metastasis in vivo. Clinically, expression of Vav3 and EphA2 was elevated in late-stage and metastatic prostate cancers. Among patients with stage IIB or earlier prostate cancer, higher Vav3 expression correlated with lower cumulative biochemical failure-free survival, suggesting that Vav3 may represent a prognostic marker for posttreatment recurrence of prostate cancer. Together, our findings provide evidence that the Vav3-mediated signaling pathway may serve as a therapeutic target for prostate cancer metastasis. Cancer Res; 72(12); 3000–9. ©2012 AACR.

The 14-3-3zeta protein, a member of the 14-3-3 family regulates multiple cellular processes during development and genomic amplification of 14-3-3zeta is observed in multiple cancers. To dissect the in vivo function of 14-3-3zeta in development and in cancer, we generated a 14-3-3zeta knockout (14-3-3zeta-/-) mouse model. Targeted deletion of 14-3-3zeta in the C57Bl/6 murine genetic background led to neonatal lethality due to respiratory distress and could be rescued by out-breeding to the CD-1 or backcrossing to the FVB/NJ congenic background. Hematoxylin and eosin (H&E) staining performed on lung sections of 18.5 days post conception embryos showed that 14-3-3zeta-/- lungs lacked saccular structures and exhibited increased mesenchymal compartments with thickened saccular septae indicating that the lung development of 14-3-3zeta-/- mice is arrested at the pseudoglandular stage. Interestingly, we found that the blood vessels in the lungs of the 14-3-3zeta-/- mice were not integrated and were surrounded by leukocyte aggregates compared with their wild-type counterparts. Immuno-histochemistry studies revealed no significant difference in pneumocyte proliferation or pneumocyte differentiation. The expression of miR-126, a miRNA known to regulate lung vascular integrity was down-regulated in the lungs of the 14-3-3zeta-/- embryos in the C57Bl/6 background as compared to their wild-type counterparts. Mir-126 was also down-regulated in endothelial cells stably expressing 14-3-3zeta targeting shRNA. Loss of 14-3-3zeta in endothelial cells inhibited the angiogenic capability of the endothelial cells as determined by both trans-well migration assays and tube formation assays and these defects could be rescued by re-expressing miR-126. Mechanistically, loss of 14-3-3zeta was found to suppress Erk1/2 phosphorylation resulting in attenuated binding of the transcription factor Ets2 on the miR-126 promoter ultimately resulting in reduced expression of miR-126. Collectively, our data demonstrates that miR-126 is an important angiogenesis regulator that functions downstream of 14-3-3zeta and downregulation of miR-126 plays a critical role in 14-3-3zeta-loss induced defects in lung vasculature observed in a the C57Bl/6 background. Targeting of 14-3-3zeta in various cancers can exhibit anti-tumor activity and our results suggest that identifying the genes that contributed to 14-3-3zeta mediated lethality in the C57Bl/6 mouse strain may provide insights on predicting patients’ response to anti-14-3-3zeta therapy and optimizing personalized therapy in the future. Citation Format: Sonali Joshi, Jun Yang, Qingfei Wang, Ping Li, Hai Wang, Brian Pickering, Yan Xiong, Jan Parker-Thornburg, Richard Behringer, Dihua Yu. Loss of 14-3-3zeta attenuates miR-126 expression and results in neonatal lethality. [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 2310. doi:10.1158/1538-7445.AM2015-2310

Multinucleation is associated with malignant neoplasm; however, the molecular mechanism underlying the nuclear abnormality remains unclear. Loss or mutation of PTEN is frequently implicated in the development of malignancies. We now demonstrate that increased expression of the oncogene MCT-1 antagonizes PTEN gene expression, protein stability and activation, thereby further promoting PI3K/AKT signaling and survival rate of the PTEN-deficient cells. MCT-1 interacts with p190B and Src in the triple negative breast cancer cells, supporting they are in proximity of the signaling complexes in vivo. MCT-1 overexpression and PTEN loss synergistically augmented the Src/p190B signaling function leading to inhibition of RhoA activity. Under such condition, the incidence of mitotic catastrophes including spindle multipolarity and cytokinesis failure were enhanced, driving a Src/p190B-dependent neoplastic multinucleation. Reduced MCT-1 expression by shRNA dramatically repressed the Src/p190B function and improved the nuclear structure, as well as suppressed tumorigenicity of the PTEN-null triple negative breast cancer cells. Consistent with the oncogenic effects in vitro, clinical evidence has confirmed that MCT-1 and p190B genes are highly expressed in human breast cancers. Accordingly, MCT-1 gene induction is recognized as a potentially indicator of breast tumor development. Abrogating MCT-1 function may be a promising strategy for treatment of the triple negative breast cancer involving aberrant activation of Src and/or dysfunction of PTEN. Citation Format: Meng-Hsun Wu, Yen-An Chen, Hsiao-Huei Chen, Ko-Wei Chang, I-Shou Chang, Lu-Hai Wang, Hsin-Ling Hsu. MCT-1 expression promotes neoplastic multinucleation through the Src/p190B signaling activation in triple-negative breast cancer cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A69. doi:10.1158/1538-7445.CHTME14-A69

INTRODUCTION: Helicobacter pylori (HP) is a type 1 carcinogen for gastric cancer (GC), the second most common cause of cancer-related death worldwide. The limited benefit of HP eradication or nutritional intervention in risk reduction and the dismal prognosis of GC underlie the urgent need for new preventive strategies. We have recently shown that Hp causes aberrant DNA methylation of tumor suppressor genes to promote gastric carcinogenesis (Cheng et al. 2013). However, the functional and mechanistic relationships between aberrant DNA methylation and GC remain elusive. AIMS&METHODS: We investigated the effect of 5-aza-2′-deoxycytidine (5′Aza-dC), a FDA-approved demethylating agent in a murine GC model induced by N-Nitroso-N-methylurea (MNU). Using MethylCap-microarray, quantitative RT-PCR and Western blot, we identified novel DNA methylation-controlled genes in paired GC tumors/adjacent tissues (4), 5′Aza-dC-treated (3) and -untreated (3) normal mucosa samples, followed by pyrosequencing and gene expression validation in human GC samples and cell lines. Gene functions were investigated by cell cycle and colony formation assays. RESULTS: At 52 weeks-post MNU exposure, GC was developed in 8 out of 19 mice. Administration of 5′Aza-dC for 24 weeks significantly reduced GC incidence from 42.1 to 11.1% (2/18 mice; p1.5-fold increase compared to tumor-adjacent tissues) was observed in 58.3% (14 out of 24) cases, which was accompanied with significant GDF1 down-regulation in both mRNA (p CONCLUSIONS: Epigenetic silencing of GDF1 may abrogate the growth-inhibitory effects of TGF-β signaling and render selective growth advantage to gastric epithelial cells during carcinogenesis. Our findings demonstrate a causal relationship between DNA methylation and GC development and lend support to demethylating drugs for GC chemoprevention trial. This study was supported by RFCID (08070172) and CUHK Direct Grant. REFERENCE: Cheng AS, et al. Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis. Gastroenterology 2013;144:122-33.e9. Citation Format: Wei Qin Yang, May S. M. Li, Wei Kang, Li Han Zeng, Tian Hai Wang, Anthony W. Chan, Enders K. W. Ng, Ka F. To, Francis K. L. Chan, Jun Yu, Michael W.Y. Chan, Joseph J. Y. Sung, Alfred S. L. Cheng. Reactivation of growth/differentiation factor 1 contributes to the chemopreventive effect of 5-aza-2′deoxycytidine in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2014-1245

The receptor tyrosine kinase RON, a member of the MET family, is critical in epithelial tumorigenesis. RON also is a validated drug target for cancer therapy due to its high levels of expression in cancer cells. At present, monoclonal antibodies (mAb) and tyrosine kinase inhibitors have been developed, preclinically tested, and evaluated under clinical trials. However, accumulated evidences have indicated that the efficacy of RON-targeted therapy alone is weak. Thus, there is an urgent need to develop a strategy to improve the effectiveness of RON-targeted cancer therapy. Here we report the development and therapeutic efficacy of anti-RON mAb Zt/g4-maytansinoid conjugates (Zt/g4-DM1) with enhanced cytotoxicity against colorectal cancer cells (CRC) in mouse xenograft tumor models. Zt/g4 (IgG1a/κ) was selected among several anti-RON mAbs as the drug carrier for its ability to induce RON endocytosis in CRC cells. Zt/g4 was conjugated to DM1 via the thioether link to form anti-RON antibody-drug conjugate Zt/g4-DM1 with an antibody-drug ratio of 1:4 molecules. Using HCT116, HT29, and SW620 cells as the model, we found that Zt/g4-DM1 retains the intrinsic activity that causes rapid RON internalization. Targeted payload delivery significantly induced cell cycle arrest at G2/M phase, reduced cell viability with IC50 values at ∼1.2 μg/ml, which resulted in massive cell death within a period of 72h. The cytotoxic effect of Zt/g4-DM1 was negligible in cancer cells with minimal RON expression. Studies from the mouse CRC xenograft model revealed that Zt/g4-DM1 is highly effective in delaying HT29 cell-mediated tumor growth in athymic nude mice. In a single cycle of q 4 days x 5 with Zt/g4-DM1 at 1, 3, 7, 10, and 15 mg/kg body weight, we observed significant tumor inhibition (>70% tumor growth delay) when Zt/g4-DM1 was used at 7 mg/kg. Significantly, more than 95% of tumor growth was inhibited at 10 mg/kg or 15 mg/kg of Zt/g4-DM1. Continued observation after last treatment showed that the inhibitory effect of Zt/g4-DM1 is long lasting up to 20 days without signs of tumor regrowth. The prepared Zt/g4-DM1 is stable in vitro and has a minimal toxic effect in vivo when Zt/g4-DM1 was used below 40 mg/kg. Zt/g4 has been sequenced and a 3D model of antigen-binding domains in variable regions in both heavy and light chains has been created. Zt/g4 is currently under humanization for potential clinical trials. In conclusion, Zt/g4-DM1 is a highly effective biotherapeutics for inhibition of CRC growth in mouse xenograft models. The therapeutic potential of Zt/g4-DM1 provides a novel strategy for targeted CRC therapy in the future. Moreover, the development of humanized Zt/g4-DM1 opens an avenue for testing its therapeutic effect on various types of cancers harboring aberrant RON expression. (This project is support in part by NIH grant CA91980 to MHW). Citation Format: Ming-Hai Wang, Liang Feng, Hang-Ping YAO, Wei Wang, Sharad Sharma, Ruiwen Zhang. Anti-RON antibody-drug maytansinoid conjugates (Anti-RON ADC) as potential biotherapeutics for targeted colorectal cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-269. doi:10.1158/1538-7445.AM2014-LB-269

The Warburg effect is a distinct cancer hallmark that shifts the metabolic pathway in cancer cells through elevated aerobic glycolysis for energy supply and biomass. However, the mechanism of early stage metabolic dysregulation still remains unclear. 14-3-3ζ was found to be over-expressed in the early stages of breast disease and cancer (Atypical ductal hyperplasia and Ductal carcinoma in situ) and in >40% of breast cancer patients, correlating with poor patient prognosis. We found that 14-3-3ζ over-expression in non-transformed human mammary epithelial cells (HMECs), MCF10A and MCF12A, positively correlated with changes in the cellular glycolytic activity (increased glucose uptake, increased lactate production and reduced oxygen consumption). Additionally, 14-3-3ζ over-expression in HMECs induced soft agar colony formation and disorganized acini in three dimensional (3D) culture. Interestingly, we identified that lactate dehydrogenase A (LDHA) was up-regulated in these 14-3-3ζ overexpressing HMECs. LDHA knockdown in 14-3-3ζ overexpressing cells reduced aerobic glycolysis, decreased soft agar colonies and partially rescued the abnormal acinar structures, indicating LDHA upregulation contributed to 14-3-3ζ-mediated aerobic glycolysis and early transformation. Taken together, our data provide new insight into metabolic deregulation during the early stages of breast cancer transformation. Citation Format: Chia-Chi Chang, Jia Xu, Hai Wang, Chenyu Zhang, Jian Zhang, Sumaiyah K. Rehman, Ping Li, Dihua Yu. Increased glycolysis by 14-3-3ζ-mediated LDHA up-regulation contributes to transformation of early mammary epithelial cells. [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 5427. doi:10.1158/1538-7445.AM2013-5427

The 14-3-3 family proteins are highly conserved in eukaryotes and can interact with signaling proteins by binding phosphorylated Ser/Thr motifs. Studies in our laboratory have demonstrated that 14-3-3zeta is overexpressed in greater than 40% of breast cancer patients and is a strong indicator of poor patient prognosis, however the role of 14-3-3zeta in an in vivo context, especially its role in cancer, remains to be examined. We generated a 14-3-3zeta knockout mouse model to characterize the role of 14-3-3zeta in breast cancer progression. Loss of 14-3-3zeta resulted in neonatal lethality in the C57Bl/6 genetic background due to respiratory failure. Outbreeding to CD-1 or backcrossing to FVB/N background rescued the neonatal lethality. Crossing 14-3-3zeta knockout mice with MMTV-PyMT and MMTV-neu transgenic mice revealed that loss of 14-3-3zeta prolonged tumor latency and reduced lung metastasis as compared to MMTV-PyMT and MMTV-neu mice. Mechanistically, loss of 14-3-3zeta suppressed tumor proliferation and angiogenesis and promoted apoptosis. Reverse phase protein array analysis suggested that loss of 14-3-3zeta suppressed the Akt and Erk pathway and upregulated the expression of the tumor suppressor p53. Our results demonstrate an important role for 14-3-3zeta in normal development and cancer. Additionally our 14-3-3zeta knockout mouse model will also enable future studies on the role of 14-3-3zeta in other physiological and pathological processes. Citation Format: Sonali Joshi, Jun Yang, Qingfei Wang, Ping Li, Hai Wang, Brian Pickering, Yan Xiong, Jan Parker-Thornburg, Richard Behringer, Dihua Yu. Loss of 14-3-3zeta impairs oncogene-induced mammary tumorigenesis and metastasis. [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 2328. doi:10.1158/1538-7445.AM2013-2328

The RON receptor tyrosine kinase is a therapeutic target for cancer treatment. Here we report therapeutic effect and phenotypic change of breast cancer cells in response to BMS-777607, a RON tyrosine kinase inhibitor. Treatment of breast cancer cells with BMS-777607 at therapeutic doses inhibited cancerous clonogenic growth but had only minimal effect on cell apoptosis. Significantly, BMS-777607 induced extensive polyploidy with multiple sets of chromosomes in cancer cells. This effect is independent of RON expression. Knockdown of RON in T-47D and ZR-75-1 cells by specific siRNA did not prevent polyploid formation. Immunofluorescent analysis of α-tubulin and γ-tubulin expression in polyploid cells revealed that BMS-777607 disrupts bi-polar spindle formation and causes multi-polar-like microtubule assembly. Also, both metaphase equatorial alignment and chromosomal segregation were absent in polyploid cells. These results suggest that cellular mitosis arrests at prophase/pro-metaphase and fails to undergo cytokinesis. By analyzing kinase-inhibitory profiles, aurora kinase B was identified as the target molecule inhibited by BMS-777607. In BMS-777607 treated cells, aurora kinase B was inhibited followed by protein degradation. Moreover, BMS-777607 inhibited Ser10 phosphorylation of histone H3, a substrate of aurora kinase B. Chemosensitivity analysis indicated the resistance of polyploid cells towards chemotherapeutics. Treatment with doxorubicin, bleomycin,cisplatin, methotrexate, and paclitaxel significantly increased cellular IC50 values. These findings highlight the theory that BMS-777607 acts as a multi-kinase inhibitor at therapeutic doses and is capable of inducing polyploidy by inhibiting aurora kinase B. Increased resistance of polyploid cells to cytotoxic chemotherapeutics could have a negative impact on targeted cancer therapy using BMS-777607. Citation Format: Sharad Sharma, Jun-Yin Zeng, Chunmei Zhunag, Ruiwen Zhang, Ming-hai Wang. Small-molecule inhibitor BMS-777607 induces breast cancer cell polyploidy with increased resistance to cytotoxic chemotherapy agents. [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 921. doi:10.1158/1538-7445.AM2013-921

Aberrant RON expression contributes significantly to breast cancer tumorigenic progression. This observation has provided the basis for targeting RON in potential breast cancer treatment. In the study of breast cancer tumorigenesis, we discovered that inhibition of RON by small molecule inhibitor BMS-777607 causes polyploidy followed by apoptosis in various breast cancer cells including T-47D and ZR-75-1. BMS-777607 is a tyrosine kinase inhibitor specific to RON and MET receptor tyrosine kinases and blocks cancer cell proliferation in vitro and tumor growth in xenograft model in vivo. Using breast cancer cells as the model, we observed that BMS-777607 at experimental doses induces extensive polyploidy and apoptosis in breast cancer cells. The effect of BMS-777607 has the following features. First, BMS-777607 treatment dramatically altered cell morphology, increased cell size, and created multinucleate structures. Chromosome spreading at metaphase and flow cytometric analysis confirmed polyploid cells with 6N to 16N chromosome content, indicating that abortive mitosis and endoreduplication are responsible for the observed polyploidy. Second, the effect of BMS-777607 was highly specific and dependent on RON or MET expression. MCF-7 cells that do not express RON/MET showed only low levels of polyploidy, suggesting that RON and MET expression are required for the observed polyploid activity. Third, the effect of BMS-777607 is time and dose-dependent. A substantial polyploid population of cells accumulated within 24 h of drug treatment. Continuous cycle of abortive mitosis further increased cell ploidy with increased 16N cell fractions. Moreover, an experimental dose of 500 nM is sufficient to induce polyploidy. The maximal effect occurred when BMS-777607 was used at 5.0 μM for 72 h. Fourth, polyploidy induction promoted apoptosis and senescence of breast cancer cells, which led to significant changes in cellular sensitivity towards cytotoxic activity of chemoagents. Finally, we demonstrated that Aurora B kinase is the major signaling molecule responsible for BMS-777607-induced cancer cell polyploidy. BMS-777607 inhibited Aurora B kinase activity, which affected mitosis leading to polyploidy. In summary, BMS-777607 is a potent inducer of polyploidy in breast cancer cells. By blocking RON/MET-mediated signaling events, BMS-777607 impaired Aurora B kinase activity leading to polyploidy and apoptosis. Such observations provide mechanistic and therapeutic insight in BMS-777607 targeted cancer therapy (*supported in part by NIH grant R01 CA91980 and funds from Amarillo Area Foundation). 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 2809. doi:1538-7445.AM2012-2809

Breast cancer is one of the most commonly diagnosed cancers and the leading causes of cancer death in women worldwide. Natural product-oriented synthetic derivatives have played an important role in cancer drug discovery. JKA97, a benzylidene analog of harmine, is a novel anti-cancer candidate. The initial studies have shown its inhibitory effects on tumor growth in experimental models. However, the underlying molecular mechanisms are not fully elucidated. In this study, we determined the anti-cancer effects of JKA97 on human breast cancer in vitro and in vivo. Our data showed that, in a dose-dependent manner, JKA97 efficiently inhibited the growth and proliferation of human breast cancer cells with different p53 status, including MCF7 (p53 wild-type), MCF7 (p53 knockdown) and MDA-MB-468 (p53 mutant) cells. We also found that JKA97 induced apoptosis in human breast cancer cells independent of p53 and that JKA97 treatment resulted in G1 phase arrest in all the three breast cancer cell lines. In vivo, JKA97 significantly suppressed the growth of both breast cancer MCF7 and MDA-MB-468 xenograft tumors. Mechanistically, JKA97 modulated the levels of various cell-cycle regulators involved in G1 to S phase progression, such as p21, p27, cyclinE, and cyclinD1. Our results indicated that p21 and p27were up-regulated while cyclinE and cyclinD1 were down-regulated by JKA97 treatment. Using quantitative PCR and luciferase reporter assays, we demonstrated that the major mechanism for G1 phase arrest by JKA97 was induction of p21 at the transcriptional level, independent of p53. We further confirmed the role of p21 in JKA97-induced G1 phase arrest by using HCT116 cells with or without p21 expression. Taken together, these findings suggest that JKA97 inhibits human breast cancer by inducing cell cycle arrest through up-regulation of p21, regardless of p53 status. Considering that more than 50% human cancers have no or mutant p53 expression, our results provide a basis for further development of this compound as potential drug for breast cancer therapy. (This work was supported in part by NIH/NCI grants R01 CA112029 and R01 CA121211, R15 CA100102 and a grant (BCTR070731) from Susan G Komen for the Cure.) 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 3834. doi:1538-7445.AM2012-3834

ISG15 (interferon-stimulated gene 15), an ubiquitin-like protein (UBL), is a new tumor marker whose expression has been correlated with disease progression in bladder cancer and suggested to be prognostic for breast cancer. However, the molecular basis for, and the function of, ISG15 overexpression in tumors remain unclear. Here, we show that ISG15 overexpression in both breast cancer cells and oncogenic Ras-transformed mammary epithelial cells (MCF-10A) is due to elevated secretion of interferon-β. Moreover, interferon-β-ISG15 autocrine signaling in oncogenic Ras-transformed MCF-10A cells results in increased colony formation efficiency. Interestingly, gain of epithelial-mesenchymal transition (EMT) characteristics, including appearance of spindle-shaped cell morphology and EMT markers, as well as increased cell migration, in Ras-transformed MCF-10A cells is also dependent on interferon-β-ISG15 autocrine signaling. Our results demonstrate for the first time that tumor cell-secreted interferon-β may modulate tumor cell growth and migration through ISG15, suggesting potential cancer therapeutic approaches by antagonizing interferon autocrine signaling. 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 3438. doi:10.1158/1538-7445.AM2011-3438

Pancreatic adenocarcinoma (PAC) is a malignant disease characterized by metastasis and chemo-resistance. Accumulated evidence has suggested that PAC malignancies arise due to functional presence of cancer stem cells that have high potential for self renewal, differentiation, and resistance to cytotoxic agents. Currently, therapeutics targeting PAC stem cells are not available. The study in this project describes a novel strategy and potential therapeutics targeting PAC stem cells for enhanced cytotoxicity. PAC stem like cells (PSLC) were obtained from human L3.6pl pancreatic cancer line under stem cell culture conditions. These cells show a typical sphere-like morphology in suspension and re-differentiate into epithelial appearance when subjected to serum inclusive adherent conditions. PSLC express transcription factor Bmi-1, a unique self-renewal marker, Aldh-1a, a metabolic enzyme expressed in stem-like cells, and cell-surface markers including CD24, CD44, and ESA as evidenced by Western blot and flow cytometry analyses. PSLC also displayed a phenotype characterized as epithelial to mesenchymal transition. Analysis of a panel of receptor tyrosine kinases has revealed that expression of EGFR, VEGFR, and MET was significantly reduced in PSLC. In contrast, RON receptor tyrosine kinase expression was sustained, which provides the molecular basis for antibody-directed approach for targeting PSLC. To achieve this goal, Zt/c9 monoclonal antibody was post-inserted onto doxorubicin liposomes to form anti-RON immunoliposomes (Zt/c9-Dox-IL). In vitro analysis indicated that; a) Zt/c9 was highly effective in inducing receptor internalization, which facilitates intracellular delivery of chemoagents; b) Zt/c9-Dox-IL was effective in killing Panc 0327 and L3.6pl PAC cells. An average of 7 fold decrease in IC50 value was obtained compared to control Dox-IL inserted with mouse IgG (mIgG-Dox-IL); and c) Zt/c9-Dox-IL displayed increased cytotoxicity against PSLC with the IC50 at 8 µM compared to 69 µM for mIgG-Dox-IL. In conclusion, PSLC enriched from L3.6pl cells display a phenotype similar to cancer stem cells with properties of increased self renewal and EMT. RON expression was maintained in PSLC, which was used as the drug delivery moiety for enhanced cytotoxicity. Increased PSLC cytotoxicity by Zt/c9-Dox-IL suggests that antibody-directed RON targeted delivery of cytotoxic agents is a rational approach for targeting PSLC to achieve enhanced cytotoxicity. 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 2466. doi:10.1158/1538-7445.AM2011-2466

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss of function of tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Hence, inhibition of the key targets in the pathway, e.g. PI3K, AKT, mTOR, offers great potential for the treatment of cancer. In an effort to discover compounds that inhibit PI3Kα, a high throughput screen was carried out, and 4-methyl-pyrido-pyrimidine (MPP) derivatives were identified as potent and selective inhibitors of PI3Kα. For example, PF-00271897, 8-cyclopentyl-6-[3-(hydroxymethyl)phenyl]-4-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one demonstrated PI3Kα Ki of 2.2 nM. Multiple crystal structures of inhibitors bound to PI3K gamma were determined to inform design and optimization of the ADMET properties of this lead series. Crystallographic studies with PI3K gamma protein indicated that the aminopyrimidine moiety forms two hydrogen bonds to the kinase backbone, and the aromatic moiety at the 6 position binds in a hydrophobic pocket. The X-ray structure suggested that the 4-methyl group on the MPP core structure conferred the excellent overall kinase selectivity to the series. The structure and SAR suggested optimization could come from keeping N-R group at 2 position very small and maintaining aromatic moiety at 6 position for hydrophobic interaction. Introduction of polar groups to the 8N side chains that are located in the ribose binding pocket increased both metabolic stability and solubility. Based on the overall properties, PF-04691502, 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one, was selected as a clinical candidate. PF-04691502 demonstrated Ki's of 1.2-2.2 nM against PI3K α, β, γ and δ isoforms, and Ki of 9.1 nM against recombinant mTOR. PF-04691502 inhibited AKT phosphorylation at S473 in BT20 breast cancer line with IC50 of 12 nM. PF-04691502 is highly selective for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the class III PI3K hVps34. In the in vivo rat PK studies, PF-04691502 demonstrated the following properties: Clearance = 5.2 ml/min/kg, Vdss = 1.4 L/kg, T1/2 = 3.1 h, F% = 63%. The design, synthesis, in vitro potency SAR, selectivity, ADMET of the MPP derivatives will be discussed. The crystal structure of PF-04691502 in PI3K gamma will also be presented. 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 5779.

Hypoxia is a pathogenic factor observed in solid tumors, which plays a critical role in tumor malignancy and drug resistance. The MET and RON receptor tyrosine kinases belong to a unique proto-oncogene family and contribute to tumorigenic progression and malignant phenotypes in various epithelial cancers. To address their pathogenic significance under chronic hypoxic condition (1% O2, 5% CO2 for 5 days), functional analysis of MET and RON were conducted using colon cancer HCT116 and HT29 cells as models. Contrary to previous reports, we consistently observed that levels of MET and RON were significantly decreased over a period of 5 days under chronic hypoxic condition as documented by Western blot and cell-surface fluorescent analysis. The reduction was also accompanied with decreased in both spontaneous and ligand (HGF/MSP)-induced tyrosine phosphorylation manners, which affected the downstream signaling cascades. Moreover, we observed that hypoxia-induced HIF-1α expression (known as a positive regulator for both receptors) is unable to sustain MET or RON expression, suggesting that the receptor down-regulation is mediated through a HIF-1α-independent mechanism. RT-PCR analysis revealed that diminished MET and RON expression occurs at the mRNA transcription level. However, increase in protein degradation also contributed to the observed reduction. Functional studies further demonstrated that chronic hypoxia facilitates colon cancer cells to develop a unique pathogenic phenotype. This phenotype was featured by reduced cell migration, decreased cell proliferation, and lack of S phase in cell cycle profile. Tumor cells were also insensitive in response towards HGF or MSP-induced cell movement and replication. These cancer cells were resistant to cytotoxic effects induced by MET-RON duel small molecule inhibitors such as PHA665752 and compound I. As shown by cytotoxicity assay, the IC50 values of these inhibitors in cell survival were significantly increased in cancer cells under chronic hypoxic treatment compared to normoxic cells. In conclusion, our results demonstrate that under chronic hypoxia colon cancer cells developed a pathogenic phenotype characterized by diminished expression of MET and RON. Such a change contributes to the insensitivity of tumor cells towards cytotoxic activities of specifically targeted drugs. Thus, diminished expression of MET and RON under chronic hypoxia is a potential mechanism that affects the effectiveness of targeted therapy in the treatment colon cancer. 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 442.