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DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)–induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin/+). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment.Implications:This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.

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Supplementary Figures and Legends 1-3

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor–positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo, limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. Significance: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.

In spite of the many advances in cancer diagnosis and treatment, breast cancer remains a devastating disease among women worldwide. Although the critical role of estrogen (E2) in breast cancer has been unequivocally established and antiestrogens have been successfully used to treat this disease, there are about 2.6 million women in the United States who are continue to suffer from this disease. Further, approximately 80% of breast cancers are initially E2-dependent and respond well to antiestrogens, but many of them develop resistance and become unresponsive to antiestrogen therapy. Despite through investigation, the molecular mechanisms underlying this loss of responsiveness to antiestrogen therapy and the precise signaling molecules that impart E2-induced mitogenic signaling have not been fully understood. This gap in knowledge constitutes a significant impediment in effective prevention and treatment of this disease. Thus, the purpose of this study is to identify the precise signaling processes that impart E2 signaling and the molecular mechanisms underlying the resistance to hormonal therapy in breast cancer. In this study, we found that SIRT1, a type III histone deacetylase (HDAC), plays a critical role in E2-induced tumor growth as well as chemoresistance in human breast cancer cells. Functional inactivation of this gene abolished E2-induced tumor growth and made it more susceptible to hormonal- and chemotherapy-induced growth arrest and apoptosis. Additionally, our study showed that mammary stem-cells require Sirt1 for self-renewal and maintenance in the undifferentiated-state. Interestingly, mammary gland specific Sirt1-knockdown significantly reduced breast tumor growth and metastasis in syngeneic and genetically engineered mouse (GEM) models of breast cancer by limiting breast cancer stem cell (BCSC) self-renewal. RNA-seq analysis provided evidence that Sirt1 deletion is associated with significantly reduced expression of genes involved in BCSC self-renewal, tumor growth and metastasis, and drug resistance. Treatment of GEM mouse models of breast cancer with SIRT1 inhibitor reduced tumor growth with increased overall survival. Altogether, our study provides strong evidence that SIRT1 is a key regulator of E2-induced tumor growth signaling, and a potential modifier of drug resistance. Functional inactivation of this gene will effectively block mammary tumor development and circumvent drug resistance, which in turn could significantly increase the disease-free survival. Citation Format: Utkarsh Parwal, Parth Patel, Sabarish Ramachandran, Rajneesh Pathania, Allison Bridges, Pragya Rajpurohit, Puttur D. Prasad, Muthusamy Thangaraju. SIRT1 requires for mammary stem cell self-renewal and maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-040.