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Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis. Citation Format: Changhwan Yoon, Jun Lu, Yukyung Jun, Bang- Jin Kim, Jacob Till, Jong Hyun Kim, Sara Keshavjee, Sandra Ryeom, Sam Yoon. KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2451.

BACKGROUND: Our previous work showed that in a mouse model of gastric adenocarcinoma (GA) with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a triple conditional or Tcon mice) accelerates tumorigenesis and metastasis. The Receptor Tyrosine Kinase (RTK)-RAS signaling pathway is altered in 60% of GAs, and KRAS is amplified or mutated in 17% of GAs. There is some evidence that RTK-RAS signaling and oncogenic KRAS are important in the epithelial-to-mesenchymal transition (EMT) and maintenance of cancer stem-like cells (CSCs). We hypothesized that RTK-RAS activation in GA CSCs increases the metastatic potential of these cells. METHODS: The RTK-RAS pathway and KRAS were examined in nontransformed HFE-145 gastric epithelial cells, tumor-derived organoids derived from Tcon mice, human GA cell lines (AGS and KATOIII), AGS xenografts, and a tissue microarray of human GAs from 115 patients undergoing surgical resection. KRAS was inhibited using shRNA, and the RTK-RAS pathway was blocked using a MEK inhibitor PD0325901. RESULTS: Kras activity was much higher in GA cell lines grown as spheroids compared to GA cell lines grown monolayers. Metastasis following KRAS activation in GA cells results from stimulation of epithelial-to-mesenchymal transition (EMT) to cancer stem-like cells (CSCs). In organoids derived from our mouse model, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown in patient tumor-derived GA cell lines. KRAS inhibition in GA spheroid cells led to reduced flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. Supporting clinical relevance, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival in 115 GA patients. CONCLUSION: In this study, we show that the addition of oncogenic KRAS into gastric epithelial cells leads to EMT and acquisition of CSC phenotypes. Inhibition of KRAS in GA cell lines and in tumor derived organoids reverses EMT and inhibit CSC phenotypes. We conclude that metastasis following KRAS activation in GA cells likely results from stimulation of EMT and transition to CSCs. Citation Format: Changhwan Yoon, Jacob Till, Soo-Jeong Cho, Kevin Chang, Jian-Xian Lin, Sandra Ryeom, Sam Yoon. KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis [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 4680.

We recently generated the first model of metastatic mixed-type gastric adenocarcinoma that closely recapitulates the human disease. Our model combines conditional loss of E-cadherin (Cdh1fl/fl) and p53 (Trp53fl/fl) with oncogenic Kras (KrasLSL-G12D/+) activation and yellow fluorescence protein (Rosa26LSL-YFP/LSL-YFP) expression in cells of the gastric parietal cell lineage (Atp4b-Cre). Median survival is 76.5 days (range 64-91 days, SD 7.2) with all mice developing local lymph node metastases and lung metastases, half developing paratracheal lymph node metastases, and 20% developing liver metastases. We previously provided proof of concept for the preclinical utility of our model by demonstrating extended survival upon treatment with a MEK inhibitor (PD0325901). Here we demonstrate the utility of this model to investigate the role of the microbiome in gastric carcinogenesis and to determine the use of circulating tumor cells (CTCs) as biomarkers of metastatic disease. The specific pathogen H. pylori is known to be causal in gastric adenocarcinoma. Further, many studies have implicated the microbiome in colon carcinogenesis. However, no studies have investigated the role of the normal microbiome in gastric carcinogenesis in the absence of H. pylori infection. We treated our model of mixed-type gastric adenocarcinoma with a commonly utilized antibiotic cocktail to deplete the gut microbiota and confirmed depletion of the microbiota by qPCR in stomach and fecal contents at necropsy. Microbiome depletion resulted in a 7.5-day increase in median survival (n=6) over vehicle treated mice (n=5, p=0.04). These data suggest a role for the normal microbiota in gastric cancer progression regardless of H. pylori infection. Given the broadly metastatic phenotype of our model, we examined whether the number of CTCs correlated with metastatic burden. Because our gastric cancer model incorporated a yellow fluorescence protein (YFP) as a marker of tumor cells, we used flow cytometry to measure CTCs in cardiac blood samples from terminal mice. We detected CTCs (defined as DAPI-;CD45-;YFP+) in all terminal mice and correlated CTC counts with lung metastatic burden. Mice with macrometastatic disease in the lung had >90 CTCs per 100 μl blood (n=3) as compared to mice with micrometastatic disease of the lung which showed Citation Format: Jacob Till, Prince Addai, Neha Bhagwat, Stephanie Yee, Erica Carpenter, Sam Yoon, Sandra Ryeom. A new mouse model of gastric adenocarcinoma: A valuable tool for preclinical studies [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 4077.

INTRODUCTION: The Cancer Genome Atlas found that the receptor tyrosine kinase (RTK)-Ras signaling pathway is altered in half of gastric adenocarcinomas (GAs). We added oncogenic Kras to the Atp4b-Cre;Cdh1fl/fl;Trp53fl/fl mouse model of GA and found accelerated tumorigenesis and metastasis. We also found Kras activity to be higher in gastric cancer stem-like cells (CSCs). Thus we hypothesized that Kras activty is critical for maintenance of gastric CSCs and promotes CSC phenotypes. METHODS: Human and murine GA cell lines were examined. The effect of Kras pathway inhibition was examined in GA spheroid cells and monolayer cells in various in vitro assays. Kras activity and Kras pathway inhibition was examined in CSCs, primary tumors and metastases in our GA mouse model. The combination of chemotherapy and Kras pathway inhibition was tested in human GA xenografts. RESULTS: Kras inhibition with shRNA or MEK inhibitor PD0325901 decreased the ability of GA cells to form spheroids and deceased expression of the stem cell transcription factor, Sox2. CD44(+) gastric CSCs had 65.8-75.4% higher migration, 64.2-79.5% higher invasion, and 63.1-70.2% more anchorage-independent growth compared to unselected cells. These properties could all blocked by 71.1-82.7% with Kras shRNA or PD0325901. In our GA mouse model, PD0325901 starting at 6 weeks of life increased median survival from 76 days to 95 days. Primary tumors and metastases from these mice with and without treatment with MEK inhibition are being analyzed for level of CD44 CSCs, Kras activity, and extent of metastases. Gastric CSCs were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed in human GA xenografts transducing cells with Kras shRNA. CONCLUSION: Kras is more active in gastric CSCs than non-CSCs and promotes malignant phenotypes including metastasis and chemotherapy resistance. Kras pathway inhibition can block these CSC phenotypes, and thus may prove useful in various combination therapies. Note: This abstract was not presented at the meeting. Citation Format: Changhwan Yoon, Kevin K. Chang, Jacob Till, Sandra W. Ryeom, Sam S. Yoon. Oncogenic Kras activation in gastric adenocarcinoma promotes cancer stem cell phenotypes including metastasis & chemotherapy resistance [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 2897. doi:10.1158/1538-7445.AM2017-2897

Hereditary cancer syndromes represent a powerful and tractable biologic system for identifying cancer-causing genes. Though the syndromes themselves may be rare, their study can provide insights into the basis of the more common sporadic forms of the cancer. Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal dominant bone dysplasia / bone cancer syndrome. This hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, and pathologic fractures. Most notably, 35% of affected individuals develop bone MFH, a sarcoma that in its sporadic form accounts for 6% of all bone cancers and is believed to be etiologically related to osteosarcoma. Indeed, one of our affected family members developed histologically-proven osteosarcoma, thus further supporting a genetic link between these tumor types. Using a linkage based approach, we previously mapped the DMS-MFH tumor suppressor gene locus to chromosome 9p21-22 (1) and then through LOH analysis demonstrated that hereditary and sporadic forms of MFH most likely share a single underlying genetic etiology (2). We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously unrecognized terminal exons of the methylthioadenosine phosphorylase (MTAP) gene. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine/methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Intriguingly, two of the novel MTAP exons arose from early and independent retroviral integration events in primate genomes at least 40 MYR ago and since their genomic integration have gained a functional role. Six distinct retroviral-sequence containing MTAP isoforms, each of which can physically interact with archetype MTAP (i.e., exons 1-8), are identified. The disease-causing / cancer-associated mutations occur within one of these retroviral-derived exons. The mutations result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Based on these findings in a hereditary form of bone sarcoma, we then analyzed the expression of these MTAP isoforms in a sample set (n=16) of sporadic osteosarcoma samples. All tumor samples expressed similar levels of the archetype MTAP RNA sequence but the expression pattern of the splice variants varied markedly between nearly all the samples. The majority of samples did not express SV1 (n=11/16) and nearly half did not express SV6 (n=9/16). Taken together, these results identify the first gene involved in the development of bone MFH / osteosarcoma and have potential implications for the treatment of this human cancer. References: 1. Am J Hum Genet. 64:801-7; 1999. 2. Genes Chromosomes Cancer. 27:191-5; 2000. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-89. doi:1538-7445.AM2012-LB-89