The normal colon epithelium is transformed into its neoplastic counterpart through a series of genetic alterations in driver genes including activating mutations in PIK3CA. Treatment often involves surgery followed by 5-fluorouracil (5-FU) based therapy, which has limited efficiency and serious side effects. We sought to determine whether fisetin, a dietary flavonoid, alone or in combination with 5-FU affected tumorigenesis in the mammalian intestine. We first determined the effect of fisetin, 5-FU or their combination on PIK3CA-mutant and PIK3CA wild-type colon cancer cells by assessing cell viability, colony formation, apoptosis and effects on PI3K/AKT/mTOR signaling. Treatment of PIK3CA-mutant cells with fisetin and 5-FU reduced the expression of PI3K, phosphorylation of AKT, mTOR, its target proteins, constituents of mTOR signaling complex and this treatment increased the phosphorylation of AMPKα. We then determined whether fisetin and 5-FU together or singly affected tumorigenesis in Apc Min/+ mice that also express constitutively active PI3K in the distal small intestine and colon. Tumor incidence was markedly lower in fisetin-treated FC 1 3K 1 Apc Min/+ mice that also express constitutively active PI3K in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. Fisetin could be used as a preventive agent plus an adjuvant with 5-FU for the treatment of PIK3CA-mutant colorectal cancer.

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Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States, despite substantial development in its early diagnosis and treatment. The animal models of CRC have been immensely useful for understanding CRC pathogenesis, investigating the effects of genetic modifications on CRC, and for the development of new chemopreventive/chemotherapeutic drugs. Most (>80%) CRC carry mutations in the APC gene and many (15-20%) carry mutations in the PIK3CA gene, encoding the p110 catalytic subunit of the PI3K kinase. We sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. To examine the effect of mutations in APC and PIK3CA on tumorigenesis, Min mice were crossed with the FC13K1 (FC13K1ApcMin/+). This cross resulted in a murine model with the loss of one allele of Apc throughout the intestine and the expression of a dominant active PI3K (3K1) in the distal small intestine and colon due to the expression of Cre under the control of the rat fatty acid binding protein-1 promoter (FC1). There was increased tumor multiplicity, size and a more aggressive and poorly differentiated phenotype as a consequence of synergy between APC and PIK3CA mutations. Tumors form as adenomas, but quickly progress to invasive adenocarcinomas that eventually metastasize to regional lymph nodes. Using this mouse model, we have recently shown that fisetin, a dietary flavonoid could be used as a preventive agent and an adjuvant with 5-fluorouracil (FU) for the treatment of PIK3CA-mutant CRC. Tumor incidence was markedly lower in fisetin-treated FC13K1ApcMin/+ mice in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. We extended our work by demonstrating that the effect of drugs on tumorigenesis was impacted by the mutation profile of the tumor and intratumoral heterogeneity. We developed the mouse model in which intestinal tumors were composed entirely of PIK3CA wild-type cells, entirely of PIK3CA-mutant cells, or a mixture of both. We demonstrated that low dose aspirin blocked the development of heterogeneous tumors composed of PIK3CA wild-type and PIK3CA-mutant cells but not the development of homogenous tumors composed entirely of PIK3CA wild-type cells. Thus, this new model of CRC recapitulates the effect of aspirin that was observed in humans. Sustained exposure to low dose aspirin reduced the recurrence the PIK3CA-mutant CRC cancers in humans. This aggressive murine model is an exciting model of human CRC that has the potential to be instrumental in the development of targeted chemoprevention and therapeutics. Citation Format: Naghma Khan, Farah Jajeh, Emily L. Eberhardt, Devon D. Miller, Dawn M. Albrecht, Rachel Van Doorn, Melissa D. Hruby, Morgan E. Maresh, Linda Clipson, Hasan Mukhtar, Richard B. Halberg. Novel mouse model carrying APC and PIK3CA mutations in colorectal cancer [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 2733.

Colorectal cancer (CRC) is the third most common cause of cancer-related deaths in the United States and is one of the most frequent and deadliest cancers worldwide. The relative survival rate is very poor for patients with metastatic CRC, regardless of the progress in chemotherapy. 5-Fluorouracil (5-FU) is widely used chemotherapeutic drug for the treatment of colon cancer, and the efficacious tumor therapy is generally obstructed by the progression of tumor resistance mechanisms. PIK3CA is one of the most frequently mutated genes in CRC, as about 15-20% of advanced CRC harbor activating mutations in PIK3CA exon 9 and/or exon 20. Therefore, modeling the effects of this type of mutation in the mammalian colon is significant. Fisetin (3,3',4',7-tetrahydroxyflavone), a naturally occurring flavonoid, is present in several fruits and vegetables. We have earlier shown that treatment of PIK3CA-mutant colon cancer cells with fisetin and 5-FU caused induction of apoptosis, decrease in the expression of PI3K, phosphorylation of Akt, phosphorylation of mTOR and its target proteins with an increase in the phosphorylation of AMPKα. Based on these exciting results, we determined the effect of the treatment of combination of fisetin and 5-FU on the multiplicity of colonic tumors and on the regression of invasive adenocarcinomas in FC13K1ApcMin/+ mice. In this in-vivo model of colon cancer, synergy exists between the loss of the tumor suppressor APC and the presence of a dominant active PI3K. The tumors form as adenomas, progress to invasive adenocarcinomas and eventually metastasize to regional lymph nodes, as observed in human patients. The anti-tumorigenic properties of fisetin were tested by treating these mice either early when mice had no or few visible colon polyps and late when mice had developed cancers. The treatment regime was the same for both groups. The only difference was age when treatment initiated; the early group started treatment between 25 and 30 days, whereas late group started treatment between 45 and 50 days. In the early treatment groups, fewer fisetin-treated mice developed tumors than controls and fisetin-treated animals bearing tumors developed fewer colon tumors than controls. We also found that 5-FU suppressed growth of tumors and the effects of fisetin and 5-FU are additive. At the end of the experiment, as compared to control group, there was 27% decrease in tumor counts on treatment of animals with 5-FU, 36% decrease with fisetin and 49% decrease on treatment with combination of fisetin and 5-FU. There was also decrease in tumor incidence on treatment of animals with fisetin and combination of fisetin and 5-FU. Treatment with fisetin, 5-FU, or the combination did not appear to affect tumorigenesis when given late. Interestingly, the activation of PI3K appears to make 5-FU less effective at this stage. Our data suggest that fisetin could be used as a preventive agent as well as an adjuvant with 5-FU for the prevention of PIK3CA-mutant CRC. Citation Format: Naghma Khan, Farah Jajeh, Devon Miller, Rachel Van Doorn, Emily Lauren Eberhardt, Richard B. Halberg, Hasan Mukhtar. Fisetin for the management of PIK3CA-mutant colorectal 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 1251.