A claudin-based molecular signature identifies high-risk, chemoresistant colorectal cancer patients

Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse

Abstract 4143: Mastl, a novel therapeutic target in colon cancer

Abstract Combining chemoradiation with agents that modulate tumor-specific pathways such as cell cycle checkpoints has shown immense promise in preclinical and clinical studies. To ensure that only healthy cells proliferate, checkpoints have evolved that induce cell-cycle arrest in response to the detection of defects that may have arisen during DNA replication or other steps leading to mitosis. The G2/M checkpoint Mastl in mammals (and Greatwall in Xenopus and Drosophila) prevents cells from premature entry into mitosis, and thus minimizes chromosome mis-segregation. Our preliminary data demonstrate that Mastl not only plays a key role in regulating cell cycle and mitosis but also regulates key oncogenic signaling pathways and assists therapy resistance in cancer cells. Interestingly, genetic elimination of Mastl expression in vivo compromised survival in young mice, however, only mild alterations were found when deleted in adult mice. Here we show using two clinical and genomic databases using a total of 265 patient samples that Mastl expression increases in colon cancer across all cancer stages compared with the normal colon tissue (P &amp;lt; 0.001). Moreover, high Mastl expression associates with high-risk colorectal cancer patients and poor prognosis. Silencing of Mastl expression using gene-specific shRNA, in two different colon cancer cells with high endogenous expression of Mastl, induced cell death/apoptosis supporting important role of Mastl in colon cancer cell survival. To further determine molecular mechanism/s by which Mastl regulates cell survival, we screened a panel of oncogenes. It was interesting that the knock-down of Mastl expression sharply inhibited β-catenin/c-myc signaling, key regulators of colon carcinogenesis, in both cell types. To further expand the breadth of this investigation and to identify additional key players in Mastl dependent regulation of colon cancer cell survival, we performed an oncogenic array analysis using above cell lines with or without stable inhibition of Mastl expression. Here, we observed robust effects of inhibiting Mastl upon anti-apoptotic proteins Bcl-xl and survivin. Notably, c-myc regulates survivin and Bcl-xl expressions. Additional studies, where colon cancer cells were subjected to 5-FU treatment sharply induced Mastl expression and combinational therapy using 5FU and (targeting) Mastl resulted in significantly increased death among these cells (P&amp;gt;0.0025; versus control). Taken together, we here report a novel and previously undescribed role of Mastl in colon cancer progression, malignancy and therapy resistance potentially by regulating cell survival in manners dependent on β-catenin/c-Myc/Survivin/Bcl-xl signaling. Citation Format: JayaPrakash Uppada, SaiPrasad Gowrikumar, Rizwan Ahmad, Steven Chen, J Joshua Smith, Amar B. Singh, Punita Dhawan. Mastl, a novel therapeutic target in colon cancer [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 4143. doi:10.1158/1538-7445.AM2017-4143</jats:p

MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/β-catenin signaling

Abstract Background Chemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood. Methods We utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer. Results Here, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the β-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression. Conclusion Overall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment

PIK3C3 Inhibition Promotes Sensitivity to Colon Cancer Therapy by Inhibiting Cancer Stem Cells

Background: Despite recent advances in therapies, resistance to chemotherapy remains a critical problem in the clinical management of colorectal cancer (CRC). Cancer stem cells (CSCs) play a central role in therapy resistance. Thus, elimination of CSCs is crucial for effective CRC therapy; however, such strategies are limited. Autophagy promotes resistance to cancer therapy; however, whether autophagy protects CSCs to promote resistance to CRC-therapy is not well understood. Moreover, specific and potent autophagy inhibitors are warranted as clinical trials with hydroxychloroquine have not been successful. Methods: Colon cancer cells and tumoroids were used. Fluorescent reporter-based analysis of autophagy flux, spheroid and side population (SP) culture, and qPCR were done. We synthesized 36-077, a potent inhibitor of PIK3C3/VPS34 kinase, to inhibit autophagy. Combination treatments were done using 5-fluorouracil (5-FU) and 36-077. Results: The 5-FU treatment induced autophagy only in a subset of the treated colon cancer. These autophagy-enriched cells also showed increased expression of CSC markers. Co-treatment with 36-077 significantly improved efficacy of the 5-FU treatment. Mechanistic studies revealed that combination therapy inhibited GSK-3β/Wnt/β-catenin signaling to inhibit CSC population. Conclusion: Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3β/Wnt/β-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy