PROTEIN ADDUCTION REGULATE DNA DAMAGE RESPONSE IN PRENEOPLASTIC CONDITIONS OF ESOPHAGEAL ADENOCARCINOMA

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.

Introduction
Tuft cells represent a rare population of epithelial cells in the gastrointestinal tract to sense diverse inflammatory conditions to trigger immune responses. Tuft cells have also been shown to expand in response to oncogenic Kras activation in the pancreas and influence tumor progression. However, few studies have examined tuft cells in the gastric pathologies. We have previously generated Lrig1-Kras and Mist1-Kras mouse models to recapitulate gastric metaplasia to dysplasia progression. Here, we used these models to investigate changes in tuft cell population upon Kras activation in the stomach. In addition, we examined tuft cells in various human gastric lesions including gastritis, intestinal metaplasia (IM) and gastric tumors.
Method
Lrig1-CreERT2;LSL-Kras(G12D) (Lrig1-Kras) mice were treated with 2 mg of tamoxifen and sacrificed 2 months after tamoxifen injection. Mist1-CreERT2;LSL-Kras(G12D) (Mist1-Kras) mice were treated with 5 mg of tamoxifen and sacrificed at 1, 3, and 4 months after tamoxifen injection. Gastric organoids were generated from Mist1-Kras mice at 3 and 4 months after tamoxifen treatment. Tumor formation assay was performed with subcutaneous flank injection in the nude mice. Immunostaining using antibodies against Dclk1, POU2F3, ChAT was performed to examine changes in tuft cell populations.
Result
Two months after induction of active Kras in Lrig1+ cells, foveolar hyperplasia developed and tuft cells increased in number compared to control mice, whereas neuroendocrine cells decreased (Fig 1). Mist1-Kras mice displayed progression from SPEM to IM and dysplasia and tuft cells substantially increased in the metaplastic mucosa. Organoids derived from dysplastic glands were implanted into nude mice and engraftments at 7 and 13 weeks after injection were harvested. Interestingly, tuft cells were mostly observed in the cystic or well-differentiated tubular glands with mucin expression and low Ki-67, while rarely seen in the poorly-differentiated glands. This pattern was consistently observed in tumors formed by re-implanting tumor cells in the nude mice. With POU2F3 and ChAT staining, we found that tuft cells are normally absent in human antrum and corpus (Fig 2). Tuft cells began to appear in the mucosa with inflammation and IM. Compared to tuft cells in the normal intestine and colon, which are usually POU2F3 and ChAT double-positive, tuft cells in the gastric mucosa with gastritis or IM were often POU2F3 single-positive. Tuft cells were frequently observed in gastric adenomas, however they become very rare in gastric cancers.
Conclusion
Our study demonstrates that tuft cells expand upon Kras activation during gastric metaplasia and dysplasia in mice. In the human stomach, tuft cells also increased in association with inflammation, metaplasia and benign tumors, but are then markedly decreased in gastric cancers.

Background: Gastroesophageal Reflux Disease (GERD) is a common digestive disorder that affects approximately 20% of the adult population in North America and is considered to be one of the strongest risk factors for esophageal adenocarcinoma. The exposure of esophageal cells to chronic gastroesophageal reflux induces extensive DNA damage through the formation of reactive oxygen species (ROS) causing genomic instability and cancer. A surveillance mechanism known as DNA damage response (DDR) ensures proper repair of the DNA lesions. In our previous study, we found a unique way of protein adduction by isolevuglandins (isoLGs) in precancerous conditions of the esophagus. One of the most adducted proteins is p53 that controls a critical branch of the DDR. In this study, we investigated how the protein adduction affects DNA damage response in conditions of esophageal reflux.
Methods and Results: For the first time, we found the adduction of multiple proteins by isoLGs in the esophagus of GERD patients and esophagojejunostomy mice that recapitulates human GERD conditions using immunohistochemistry. IsoLGs are formed through lipid peroxidation caused by ROS and enzymatic cyclooxygenation of polyunsaturated fatty acids. IsoLGs are highly reactive with free amines on lysine residues forming LG-lysine lactam protein adducts. Despite significant DNA damage caused by treatment with acidic bile salts (ABS), the p53-associated DDR was not activated in normal and precancerous esophageal cells. In contrast to p53, DNA damage induced by ABS led to a strong upregulation of p73 protein that shares some similar p53 targets, in the same cells, confirming a selective inhibitory effect of ABS on p53. Investigating the consequence of p53 protein adduction using focus array (84 key genes of human p53 signaling pathway), qPCR, cell cycle analysis and Chromatin immunoprecipitation sequencing (ChIP-seq) showed that the modification of p53 protein with isoLGs diminishes the genome-wide activation of p53 target genes that are critical for cell cycle arrest, apoptosis and DNA repair. Using dot blot, double-immunofluorescence, native gel, proteostat aggregation assay and amyloid staining, we found the accumulation of adducted p53 protein in intracellular amyloid-like aggregates both in vitro and in vivo. We also tested the isoLG scavengers, such as 2-hydroxybenzylamine (2-HOBA), and found that these compounds are able to restore p53-associated DDR in esophageal cells.
Conclusions: Combined, our studies revealed, for the first time how isoLGs regulate DDR in esophageal cells exposed to reflux by inactivating p53 that may play a previously unrecognized role in human tumorigenesis. Our work also helps to explain how chronic reflux induce tumorigenic process in the esophagus and opens new opportunities for the development of novel cancer chemopreventive agents.