Supplementary Figures and Legends 1-3

There are two supplementary figures. Figure S1 shows Western blot analysis of polyps from control and sildenafil treated mice, for cGMP signaling pathways (VASP, beta-catenin, AKT). Figure S2 shows RT-qPCR analysis of the mucosa and polyps from control and sildenafil treated animals for the expression of phosphodiesterases.

AbstractDNA 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.

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.

GPR109A is a receptor for niacin or nicotinic acid, which has been utilized as an antidyslipidemic drug for several decades without its mechanism being fully understood. Following the identification of GPR109A, it was determined that the receptor was expressed not only in adipocytes but also in spleen, immune cells, colon, retinal pigment epithelial cells, and breast tissue as well. GPR109A activation has been shown to have more than just an effect on lipids, also promoting tissue-specific regulation of metabolism and inflammation. The antiinflammatory role of GPR109A has been demonstrated in many different tissue types, potentially improving disease states from Parkinson's disease to diabetic retinopathy. In particular, its impact on colon and breast cancer suggests that GPR109A functions as a tumor suppressor. Therefore GPR109A may prove to be a potential target for future cancer prevention strategies.

Although much progress has been made in recent years in its treatment and prevention, cancer is still the second leading cause of death in the United States. Surgical removal of the tumor is not possible in all cancer types; therefore, chemotherapy and radiation therapy have become the standard course of treatment and are often the only option for more late stage and metastatic tumors. Unfortunately, chemotherapy and radiation therapy resistance are the greatest challenge for physicians trying to eradicate disease, prevent tumor recurrence, and inhibit distant metastasis. This resistance is derived from a heterogeneous population of cells within the tumor known as cancer stem cells (CSCs). CSCs are able to maintain a higher capacity for self-renewal due to a more efficient DNA repair system. CSCs are actively cycling, therefore, the repair mechanism of homologous recombination (HR) plays a significant role in repairing double-stranded DNA breaks that inevitably accumulate. Several studies have examined the link between efficient DNA repair and CSC self-renewal and found that proteins involved in HR repair are elevated in many human cancers. RAD51-associated protein 1 (RAD51AP1), which is responsible for the successful resolution of HR during DNA repair, is overexpressed in wide variety of human cancers. The present study sought to determine the functional role of RAD51AP1 in CSC self-renewal and its relevance to cancer growth and progression and also drug resistance. Our studies provide evidence that RAD51AP1 plays a critical role in CSC self-renewal and maintenance in breast, lung, and colon cancers. To determine the functional role of RAD51AP1 in cancer growth and progression, we generated genetically engineered mouse (GEM) models in breast and lung in WT (Rad51ap1+/+) and Rad51ap1-knockout (KO, Rad51ap1-/-) background and found that Rad51ap1 deletion significantly delayed the time of tumor formation and distant metastasis, which in turn increase the overall survival. Rad51ap1 inactivation in human breast and lung cancer cell lines, significantly reduced tumor growth in xenograft mouse model. This findings are also recapitulated in mouse mammary tumor cell lines (AT3 and 4T1). We also investigated the functional role of RAD51AP1 on colon cancer growth and progression using AOM/DSS and Apcmin/+ models of colon cancer and found that smaller tumor burden in KO mice compared to WT suggesting that Rad51ap1 may have a significant role in tumor growth. Taken together, these data demonstrate that RAD51AP1 plays a critical role in CSC growth and self-renewal in many human cancers and RAD51AP1 could be a novel therapeutic target for cancer prevention and treatment. Citation Format: Allison E. Bridges, Pragya Rajpurohit, Parth Patel, Nagendra Singh, Putter D. Prasad, Muthusamy Thangaraju. RAD51AP1 is a novel therapeutic target for cancer prevention and treatment [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 3681.

Deferiprone (DFP) is a hydroxypyridinone-derived iron chelator currently in clinical use for iron chelation therapy. DFP has also been known to elicit antiproliferative activities, yet the mechanism of this effect has remained elusive. We herein report that DFP chelates the Fe2+ ion at the active sites of selected iron-dependent histone lysine demethylases (KDMs), resulting in pan inhibition of a subfamily of KDMs. Specifically, DFP inhibits the demethylase activities of six KDMs - 2A, 2B, 5C, 6A, 7A and 7B - with low micromolar IC50s while considerably less active or inactive against eleven KDMs - 1A, 3A, 3B, 4A-E, 5A, 5B and 6B. The KDM that is most sensitive to DFP, KDM6A, has an IC50 that is between 7- and 70-fold lower than the iron binding equivalence concentrations at which DFP inhibits ribonucleotide reductase (RNR) activities and/or reduces the labile intracellular zinc ion pool. In breast cancer cell lines, DFP potently inhibits the demethylation of H3K4me3 and H3K27me3, two chromatin posttranslational marks that are subject to removal by several KDM subfamilies which are inhibited by DFP in cell-free assay. These data strongly suggest that DFP derives its anti-proliferative activity largely from the inhibition of a sub-set of KDMs. The docked poses adopted by DFP at the KDM active sites enabled identification of new DFP-based KDM inhibitors which are more cytotoxic to cancer cell lines. We also found that a cohort of these agents inhibited HP1-mediated gene silencing and one lead compound potently inhibited breast tumor growth in murine xenograft models. Overall, this study identified a new chemical scaffold capable of inhibiting KDM enzymes, globally changing histone modification profiles, and with specific anti-tumor activities.

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.

Guanylyl cyclase-C (GC-C) agonists increase cGMP levels in the intestinal epithelium to promote secretion. This process underlies the utility of exogenous GC-C agonists such as linaclotide for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Because GC-C agonists have limited use in pediatric patients, there is a need for alternative cGMP-elevating agents that are effective in the intestine. The present study aimed to determine whether the PDE-5 inhibitor sildenafil has similar effects as linaclotide on preclinical models of constipation. Oral administration of sildenafil caused increased cGMP levels in mouse intestinal epithelium demonstrating that blocking cGMP-breakdown is an alternative approach to increase cGMP in the gut. Both linaclotide and sildenafil reduced proliferation and increased differentiation in colon mucosa, indicating common target pathways. The homeostatic effects of cGMP required gut turnover since maximal effects were observed after 3 days of treatment. Neither linaclotide nor sildenafil treatment affected intestinal transit or water content of fecal pellets in healthy mice. To test the effectiveness of cGMP elevation in a functional motility disorder model, mice were treated with dextran sulfate sodium (DSS) to induce colitis and were allowed to recover for several weeks. The recovered animals exhibited slower transit, but increased fecal water content. An acute dose of sildenafil was able to normalize transit and fecal water content in the DSS-recovery animal model, and also in loperamide-induced constipation. The higher fecal water content in the recovered animals was due to a compromised epithelial barrier, which was normalized by sildenafil treatment. Taken together our results show that sildenafil can have similar effects as linaclotide on the intestine, and may have therapeutic benefit to patients with CIC, IBS-C, and post-infectious IBS.

Abstract Intestinal cyclic guanosine monophosphate (cGMP) signaling regulates epithelial homeostasis and has been implicated in the suppression of colitis and colon cancer. In this study, we investigated the cGMP-elevating ability of the phosphodiesterase-5 (PDE5) inhibitor sildenafil to prevent disease in the azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven colorectal cancer model. Treatment of mice with sildenafil activated cGMP signaling in the colon mucosa and protected against dextran-sulfate sodium (DSS)-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared with untreated controls. Polyps that did form in sildenafil treated mice were less proliferative and more differentiated compared with polyps from untreated mice, but apoptosis was unaffected. Polyps in sildenafil treated mice were also less inflamed; they exhibited reduced myeloid-cell infiltration and reduced expression of iNOS, IFNγ, and IL6 compared with untreated controls. Most of the protection conferred by sildenafil was during the initiation stage of carcinogenesis (38% reduction in multiplicity). Administration of sildenafil during the later promotion stages did not affect multiplicity but had a similar effect on the polyp phenotype, including increased mucus production, and reduced proliferation and inflammation. In summary, the results demonstrate that oral administration of sildenafil suppresses polyp formation and inflammation in mice treated with AOM/DSS. This validation of PDE5 as a target highlights the potential therapeutic value of PDE5 inhibitors for the prevention of colitis-driven colon cancer in humans. Cancer Prev Res; 10(7); 377–88. ©2017 AACR. See related editorial by Piazza, p. 373.

Abstract The cGMP signaling pathway regulates homeostasis in the colon epithelium and has been implicated in the suppression of colorectal cancer. Exisulind is a weak phosphodiesterase-5 (PDE5) inhibitor that was previously shown to reduce polyp formation in familial adenomatous polyposis (FAP) patients. Despite this validation of PDE5 as a therapeutic target, the failure of Exisulind due to toxicity halted further trials of cGMP elevating agents. The present study tested the effect of the PDE5 inhibitor sildenafil, and the receptor guanylyl-cyclase C (GCC) agonist linaclotide on tumorigenesis in the ApcMin/+ mouse model of colon cancer. Administration of either sildenafil or linaclotide to both wild type and ApcMin/+ mice caused dramatic effects on homeostasis that included reduced proliferation and increased goblet cell differentiation. With treatment beginning at 4 weeks, sildenafil and linaclotide caused a 50% and 67% (respectively) reduction in the number of polyps per mouse. Furthermore, the polyps in sildenafil and linaclotide treated animals exhibited a reduced proliferative index and increased mucus density compared to polyps from untreated mice. The mean polyp size was not changed by treatment with either drug. In support of a tumor-suppressive role for cGMP signaling in the intestine, the ApcMin/+ mice showed reduced levels of endogenous GCC agonists in the intestinal mucosa compared to wild type animals. This suggests that part of the tumor preventative effects observed here could be due to compensation for this deficit by augmenting cGMP signaling with PDE5 inhibitors or GCC agonists. While the tumor suppressive mechanism is not fully understood, the results presented here demonstrate in a preclinical model, that increasing intestinal cGMP levels by targeting PDE5 or GCC may be a viable chemoprevention strategy for human FAP patients. Citation Format: Sarah K. Sharman, Bianca N. Islam, Yali Hou, Allison Bridges, Nagendra Singh, Subbaramiah Sridhar, Frankin G. Berger, Darren D. Browning. Sildenafil suppresses tumorigenesis in ApcMin/+ mouse model [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 2226. doi:10.1158/1538-7445.AM2017-2226

Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMP-dependent protein kinase (PKG2) to activate forkhead box 0 (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. Fox03a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. Fox03a levels were reduced in Prkg2(-/-) animals, and Fox0 target genes were unaffected by 8Br-cGMP challenge in vitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized Fox03a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors.

Abstract Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon but the mechanisms are poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling inhibits proliferation in the colon epithelium and that AKT may have an important role. Type-2 cGMP-dependent protein kinase (PKG2) is a central effector of cGMP in the colon epithelium and likely mediates the suppressive signaling. An important growth-inhibitory effect of AKT is the inactivation of forkhead transcription factors (FoxO). FoxO proteins are tumor suppressors that regulate growth-inhibitory and pro-apoptotic target gene expression but these proteins have not previously been examined in the intestinal tract. The present study sought to determine whether PKG2 can activate FoxO in colon cancer cells and in the colon epithelium. Activation of PKG2 in LS174T colon cancer cells inhibited cell proliferation but did not significantly affect apoptosis. Suggestive of a mechanism, it was found that PKG2 inhibited the activity of AKT but not ERK in these cells. Activation of PKG2 also significantly increased luciferase reporter activity driven by FoxO-responsive elements in LS174T cells. In support of FoxO activation, PKG2 also increased the expression of several FoxO target genes, including catalase, GADD45, and p27kip. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and this was associated with reduced redox stress. The regulation of FoxO by cGMP in vivo most likely requires PKG2, because target gene expression was reduced in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Since FoxO has not previously been studied in the colon, we first examined expression using IHC. These data showed that FoxO3a is the most prominent isoform, and was concentrated almost exclusively at the lumenal border. While FoxO1 showed negligible staining, FoxO4 was observed deeper in the crypt. In order to determine whether cGMP could activate FoxO in vivo, the PDE-5 inhibitor vardenafil (Levitra™) was used. Mice treated with vardenafil showed a dramatic mobilization of FoxO3a to the nucleus of lumenal epithelial cells. Analysis of the colon mucosa from treated animals showed increased levels of FoxO target genes and reduced redox stress. This was likely to be mediated by FoxO3a since vardenafil did not significantly affect the localization of FoxO1 or FoxO4. Taken together these data define a novel signaling pathway in the colon epithelium, where PKG2 leads to activation of FoxO. The established tumor suppressor role of FoxO proteins highlights the potential therapeutic role for cGMP signaling in colon cancer prevention. Citation Format: Allison Bridges, Bianca Islam, Sarah Sharman, Rui Wang, Subbaramiah Sridhar, Darren Douglas Browning. Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon. [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 1918. doi:10.1158/1538-7445.AM2015-1918

with miR-31-3p inhibitor, a miR-31-3p mimic or their respective controls.After 24 and 48 hours, cell lysates were collected for western blot analysis.Mouse colon tissues were collected 7 days (acute colitis) or 6 weeks (chronic colitis) after TNBS administration or 5 days after 5% DSS administration (acute colitis).Results: SP stimulated miR-31-3p expression after 0.5 and 6 hrs in NCM460-NK-1R cells, while pretreatment with the C-Jun N-terminal Kinase (JNK) inhibitor SP600125, but not with a NF-kB or ERK1/2 or PKC inhibitor (known signaling pathways activated by SP), normalized SP-induced miR-31-3p expression (p<0.05).Furthermore, IL6, IL8, TNFa, and IFN-g stimulation (alone or in combination) for 6 hrs (n=3) increased miR-31-3p expression (>2-fold, p<0.05).Bioinformatic analysis revealed that RhoA can be a potential miR-31-3p downstream target.Indeed, in NCM460 cells miR-31-3p silencing increased RhoA protein expression 24 and 48 hours post-transfection.Conversely, transfection with a miR-31-3p mimic decreased RhoA protein expression.Colonic expression of miR-31-5p was increased in the acute (7 days) TNBS (by 10.6 fold, n=8, p<0.05), and DSS (by 3.6 fold, n=7, p<0.05) colitis models, in the chronic (6 weeks) TNBS model (by 2.1-fold, n=5, p<0.05), and in colon biopsies (Origene) from active UC (by 4.2-fold, n=14, p<0.05), but not CD (n=15) patients compared to controls (n=10).Conclusions: 1) Expression of miR-31-3p in human colonic epithelial cells is increased by SP-NK-1R interactions that involve JNK activation as well as exposure of cells to proinflammatory mediators.2) RhoA is a downstream target of this miR.3) MiR-31-3p may represent a novel regulator and/or biomarker for colitis and IBD.