757 - A NOVEL ROLE OF HNF4A IN MICROSATELLITE STABILIZED COLORECTAL CANCER: PROMOTING METASTASIS OF MICROSATELLITE STABILIZED COLORECTAL CANCER BY UPREGULATING CXCL12 TO RECRUIT MDSC CELLS

Obesity epidemic is associated with increased colon cancer (CC) risk and progression. One of the mechanisms of obesity mediated CC progression may include Lipid Droplets (LDs). They are dynamic organelles storing intracellular triacylglycerols (TG) and providing fuel for diverse cell functions. LDs initiation and growth is mediated by LDs- coat acyltransferases, DGAT1/2, that catalyze the final step in TG synthesis. We/others demonstrated elevated LDs in colonic tumors (vs normal). Further, we identified a self-reinforcing negative regulatory loop involving LDs and FOXO3 that drives metabolic and tumorigenic remodeling in CC. Here, we aim to elucidate the mechanisms of DGATs mediated LDs dynamics that facilitate CC in obesity. Methods. Human CC: Tissue array, Local CC patients; TCGA and NCBI’s GEO databases, Human colon cells (CCC): HT29, HCT116, SW620, SW480; NCM460 (non-transformed), Colonospheres; Mouse: APC^min/+ colon cancer model, AOM/DSS, High Fat Diet (HFD)- obese; Oleic acid (OA, 300mM); Enzymatic inhibitors: DGAT1- A922500, DGAT2- PF-06424439; Differentially expressed genes (DEGs), RNA/cDNA, CUT&RUN, RNAseq (Illumina HiSeq 2500), RSEM/EBseq DEGs analysis, Ingenuity Pathway Analysis (IPA), Kaplan-Meier survival estimates; IHS; IFS; IB; qPCR; ChIP assay; Colony formation assay; FACS; Luciferase-Reporter Assay. Results. We found significantly higher DGATs levels in human CC tissue (vs normal) that is augmented by both the severity of the disease and obesity. Similarly, their levels are increased in CCC (vs non-transformed cells) and augmented by obesity mediator (OA). This increase in DGATs expression is mediated by MYC activation and binding to DGAT2 promoter (Fig 1A-I). Moreover, blockade of DGATs shut down the self-reinforcing regulatory loop between LDs and FOXO3. Specifically, it protected FOXO3 by attenuating PI3K and lowered LDs accumulation by reducing MYC dependent DGATs expression. Further, blockade of DGATs lowered OA facilitated growth of CCC and colonosphere (cell cycle G0/G1 arrest via FOXO3/p27kip1 protection). Moreover, treatment with DGATs inhibitors reduced colonic tumor burden in APC^min/+ mouse, on regular diet and HFD (obese). Further, this inhibition attenuated DEGs linked to metabolic and tumorigenic remodeling in CCC, colonospheres and colon of HFD-obese APC^min/+ mouse (IPA, FDR<0.05). It additionally targeted pathways that are activated in human CC and colon cancer crypts (IPA, FDR<0.05) (Fig 2A-R). Selected DEGs were validated in CCC, colonospheres, and APC^min/+ mouse colon (qPCR, p<0.05); their similarly altered transcriptional levels were found in human CC. Conclusions. We identified a novel mechanism of DGATs dependent metabolic and tumorigenic remodeling mediated by LDs and FOXO3 regulatory loop in obesity facilitated CC. This establishes a platform for new diagnostics and for development of effective treatments.

Background: Colorectal cancer (CRC) is highly heterogeneous at the genetic and molecular level, where immunotherapy with checkpoint inhibition has shown potent antitumor activity in microsatellite instability (MSI) metastatic cancer, while microsatellite stable (MSS) CRC has long been considered resistant to immunotherapy. Given that metastatic sites may predict the response or resistance to checkpoint blockade, additional efforts are required to study the mechanism of MSS CRC metastasis and to develop novel therapeutic strategies to overcome its immune resistance. Here we report a novel role of HNF4A in MSS CRC progression.
Methods: HNF4A/CXCL12/TGFBI expression was examined by qRT-PCR, Western blot and IHC staining in MSS CRC tissues and cells. The clinical significance of HNF4A/CXCL12/TGFBI in two independent cohorts of CRC was assessed by KM analysis and the Multivariate Cox hazards model. The biological roles of HNF4A in tumor growth and metastasis were detected both in vivo and in vitro. The Inflammatory Cytokines & Receptors Profiler was used to explore the targets of HNF4A in MSS CRC. Serial deletion, site-directed mutagenesis luciferase report assays and chromatin immunoprecipitation were used to determine transcriptional regulation of CXCL12 promoters by HNF4A. MDSCs were isolated by flow cytometry and the Gr-1 monoclonal antibody was utilized to block the recruitment of MDSCs into tumors.
Results: HNF4A expression was upregulated in MSS CRC and negatively correlated with PDL1 expression. Upregulation of HNF4A promoted the proliferation and metastasis of MSS CRC cells and indicated poor prognosis. Meanwhile, HNF4A overexpression facilitates immunosuppression, with accumulated M0 and Tregs while reduced CD8+T, aNK, aDendritic, Monocytes and Neutrophils. The levels of HNF4A are positively correlated with the numbers of M0, Tregs and memory B cells, whereas negatively correlated with CD8+T, aNK, aDendritic, Neutrophils, Eosinophils and M1. Furthermore, CXCL12 was found to be the most upregulated cytokine upon HNF4A overexpression in MSS CRC cells. The proportion of MDSCs was remarkably enhanced in HNF4A-high CRC tissues of mice. While the use of Gr-1 antibody or CXCL12 knockdown rescued HNF4A-mediated CRC metastases. In addition, TGFBI/PI3K-AKT signaling was hyperactive in MSS CRC. TGFBI-induced HNF4A expression was blocked by PI3K inhibitor. The combination of the Gr-1 antibody and PI3K inhibitor attenuated HNF4A-mediated MSS CRC metastasis.
Conclusions: TGFBI-induced HNF4A overexpression promotes MSS CRC metastasis by transactivating the cytokine CXCL12 and thereby recruiting MDSC cells to suppress anti-tumor immunity in MSS CRC. The combination of Gr-1 antibody and PI3K inhibitor attenuated HNF4A-mediated MSS CRC metastasis, suggesting that HNF4A and CXCL12 as promising prognostic biomarkers and novel therapeutic targets for MSS CRC.