Abstract 138: FOXO proteins mediate adaptive resistance to PI3K inhibition in mucinous colorectal cancers
Authors: Kuracha Murali R. , Thomas Peter , Loggie Brian W. , Govindarajan Venkatesh 
Published: July 2017

Abstract
Purpose: Mucinous colon adenocarcinomas (MCAs) comprise a substantial fraction (10-15%) of sporadic colorectal cancers (CRCs). MCAs show a distinct range of genetic modifications compared to nonmucinous CRCs and are more prone to peritoneal dissemination and distant metastasis. These aggressive cancers show a poorer response to chemotherapy and are often difficult to treat. A higher mutation rate in effectors of the KRAS-RAF-MEK (80% vs 42%) and PI3K-AKT-mTOR (60% vs 22%) pathways in MCAs over nonmucinous CRCs suggested potential targets for therapy. Our previous results showed that, MCAs, though initially sensitive to PI3K inhibition (PI3Ki), later develop adaptive resistance. The purpose of this study was to test whether FOXO transcription factors mediate this adaptive resistance to PI3Ki single agent treatment.
Methods: PI3K was inactivated in MCA cell lines, LS174T (KRAS G12D, PI3K H1047R) and RW7213 (KRAS G12C, PI3K WT), by treatment with GDC0941 (Pictilisib), a small molecule inhibitor of class I PI3K. Alterations in phosphorylation of effectors in the PI3K-AKT or MEK-ERK pathways were determined using western blots. FOXO nuclear localization was assessed by immunocytochemistry, confocal microscopy and nuclear fractionation studies. Expression of FOXO proteins (FOXO1, FOXO3 and FOXO4) was reduced by short inhibitory RNAs (siRNAs). FOXO1 was also inhibited in MCA cells using AS1842856, a small molecule inhibitor.
Results: Immunohistochemical analysis and nuclear fractionation studies showed significant nuclear enrichment of FOXO1 and FOXO3 in MCA cell lines in response to PI3K inhibitor treatment. Upon siRNA-mediated knockdown of FOXOs, rebound ERK phosphorylation seen in response to PI3Ki treatment was substantially reduced. Concomitant with this decrease was a reduction in expression of receptor tyrosine kinases (RTKs) IR, IGFR, HER2 and HER3. Combinatorial treatment of PI3K and FOXO1 inhibitors resulted in a more pronounced loss of cell viability compared to single agent treatment.
Conclusions: Our results suggest that resistance to PI3K single agent inhibition seen in MCA cells is mediated, at least in part, by nuclear translocation of FOXO proteins, particularly FOXO1. Our results support a model where PI3K inhibition results in increased FOXO nuclear localization which in turn, leads to transcriptional upregulation and activation of RTKs which then lead to rebound activation of the MEK-ERK pathway and increased survival and resistance. A role for FOXO proteins in mediating PI3Ki resistance in KRAS-mutant MCA cells has not been described before. Our results suggest that PI3Ki single agent therapy is unlikely to be successful in KRAS-mutant MCAs due to FOXO-mediated resistance thus providing a rationale for combination treatment strategies.
Citation Format: Murali R. Kuracha, Peter Thomas, Brian W. Loggie, Venkatesh Govindarajan. FOXO proteins mediate adaptive resistance to PI3K inhibition in mucinous colorectal cancers [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 138. doi:10.1158/1538-7445.AM2017-138