Targeting platelet-mediated CAP-1 upregulation in colorectal cancer: Mechanisms and small-molecule inhibitor development.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and vascular function, and it's role in biology are thought to be well understood. Although the effects of VEGF on angiogenesis, i.e. on endothelial cells, have been extensively studied, its effects on tumor cell function remain to be clearly elucidated. Our laboratory has demonstrated the VEGF receptors are present and active on colorectal cancer (CRC) cells. Recently we have studied the phenotypic changes in human CRC cells to determine the role of autocrine/intracrine VEGF signaling on tumor cell function. Using cell lines with somatic knockout of the VEGF gene we have been able to show that loss of VEGF expression led to significantly decreased cell growth and increased spontaneous apoptosis in CRC cells. Depletion of VEGF also increased the in vitro sensitivity of cells to the cytotoxic effects of the chemotherapeutic agent 5-fluorouracil. Importantly, these effects are not mediated in an autocrine or paracrine fashion, as neutralization of extracellular VEGF with a monoclonal antibody, or inhibition of kinase activity with small molecule inhibitors had no effect on CRC cell survival. These studies support a novel role for VEGF in cell survival as an intracrine factor. In our attempt to characterize the molecular mechanisms responsible for the intracrine pro-survival role of VEGF, we have performed gene microarray analyses on a pair of CRC cells with and without expression of VEGF. These studies show a clear difference in expression patterns of genes between cells that produce or lack VEGF, but surprisingly find no significant changes in gene expression when VEGF function is inhibited by functional antibodies (Bevacizumab). Our preliminary analyses of the gene expression profiles indicate that CRC cells lacking VEGF have increased levels of multiple receptor tyrosine kinases. In other biochemical assays, we also find that cells lacking VEGF have increased activation of survival and proliferation pathways compared to normal cells. Based on our data, we hypothesize that loss of VEGF may induce activation of other survival pathways that compensate for depletion of VEGF. Our microarray studies also indicate significant increases in a factor that has been implicated in increased metastasis in breast and lung cancer. This factor may be responsible for the increased migration and invasion of CRC cells lacking VEGF observed in vitro. Future studies will elucidate the exact role of VEGF in tumor cell survival and metastasis. Citation Format: Rajat Bhattacharya, Shaija Samuel, Fan Fan, Ganiraju Manyam, Veera Baladandayuthapani, Lee Ellis. Role of intracrine vascular endothelial growth factor (VEGF) signaling in colorectal cancer cell survival and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 375. doi:10.1158/1538-7445.AM2013-375

Sa1993a Reduced Expression of Prostaglandin Transporter Promotes Angiogenesis in Gastric Cancer

Purpose: African-American (AA) breast cancer (BC) and colorectal cancer (CRC) patients experience higher mortality rates than Non-Hispanic Caucasian (CA) patients. These disparities are thought to be due to race/ethnicity-specific tumor molecular biology. Our initial gene expression investigations have identified a Beta B2-Crystallin (CRYBB2) gene that is preferentially over-expressed in CRC of AA patients. However, the precise role of CRYBB2 in aggressiveness of CRC and BC has not been examined. Therefore, we hypothesize that biological mechanism linked to CRYBB2 could contribute to the disparate progression of CRC and BC in AA patients. Experimental Design: Gene expression studies were performed using Affymetrix Human Genome U133 Plus 2.0 arrays and mRNA samples extracted from 10 fresh frozen Stage III CRCs (5 from AA and 5 from CA) that were previously analyzed for the microsatellite instability (MS) status. Established CRC (HT29 and SW480) and BC (MBA-MD 231) cell lines were used to examine the phenotypic expression of CRYBB2. Also, paired lesions of human CRC (hCRC) and human mice CRC (hmCRC) xenograft were assessed for CRYBB2 by western blot and immunofluorescence (IF). To study the involvement of CRYBB2 in CRC and BC biology, siRNA mediated gene silencing was used to inhibit CRYBB2 expression. The apoptosis of CRC and BC cells after knockdown of CRYBB2 was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The epithelial-mesenchymal transition (EMT) of CRC and BC cells was analyzed by western blot for E-cadherin, MM9 and vimentin expression. Western blot analysis was performed on lysates of CRC and BC cells to assess the effect of CRYBB2 knockdown for expression/ activation of the AKT, and mitogen-activated protein kinase pathway proteins. Results: Our microarray analyses have suggested that several key genes involved in tumor metastasis (CRYBB2, VSNL-1and PFN2) are up-regulated in MS-CRCs of AA but down regulated in MS-CRCs of CA patients. The phenotypic expression of CRYBB2 was found to be higher in CRC tissues as compared to matching normal tissues. Over-expression of CRYBB2 correlated with metastatic CRC and N-cadherin expression and the expression of CRYBB2 was substantially increased in metastatic lesion of CRC and BC xenografts as compared to the primary tumors. Knockdown of CRYBB2 induced apoptosis in CRC and BC cells. In addition, Knockdown of CRYBB2 suppressed EMT signatures, such as decreasing the expression of MMP9, vimentin (mesenchymal markers) and increasing the expression of E-cadherin (epithelial marker). Furthermore, depletion of CRYBB2 decreased the phosphorylation of AKT (p-AKT) in CRC and BC cells. CRYBB2 was mainly localized in cytoplasm of BC and CRC cells. Conclusions: Our data suggest that CRYBB2 inhibits apoptosis, activates EMT process and AKT phosphorylation by which it contributes the aggressiveness of CRC and BC. It may serve as a promising biomarker for identifying CRC and BC patients at high risk for metastases. It may also be useful as a therapeutic target to inhibit the growth and metastasis of CRC and BC. These studies are supported by NIH/NCI funds (R21-CA171251 and U54- CA 118948). Citation Format: Harvey L. Bumpers, Venkat Katkoori, Dongquan Chen, Upender Manne. The role of BetaB2-crystallin in progression of colorectal and breast cancers in African Americans. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C56. doi:10.1158/1538-7755.DISP13-C56

CAMSAP2 has been reported to act as an oncogene in hepatocellular carcinoma. However, the expression CAMSAP2 and its potential roles in colorectal cancer remain unclear. In this study, qRT-PCR and immunoblotting analysis were used to detect the mRNA and protein levels of CAMSAP2 in colorectal cancer tissues and cell lines. Wound-healing, transwell migration and invasion assay were performed to determine whether CAMSAP2 promotes the capabilities of migration and invasion of colorectal cancer cells. The results showed that CAMSAP2 was highly elevated in colorectal cancer tissues and cell lines. Moreover, the high CAMSAP2 expression was positively correlated with tumor invasion depth, lymph node metastasis, distant metastasis, and the poor prognosis of colorectal cancer. Additionally, ectopic expression of CAMSAP2 in colorectal cancer cells promoted the migration and invasion in vitro and enhanced the lung metastasis in nude mice. Conversely, silencing CAMSAP2 resulted in an opposite phenomenon. By gain- and loss-of function experiments, we demonstrated that MMP-1 was a substantial downstream target of CAMSAP2, and it played a crucial role in regulating the migration and invasion induced by CAMSAP2 in colorectal cancer cells. Mechanistically, CAMSAP2 promoted the activation of JNK/c-Jun signaling pathway and subsequently upregulated the transcription activity of MMP-1. Taken together, our findings demonstrated that CAMSAP2 promoted colorectal cancer cell migration, invasion and metastasis through activation of JNK/c-Jun/MMP-1 signaling pathway, indicating CAMSAP2 is a promising therapeutic target for the treatment of metastatic colorectal cancer patients.

Gomisin A (G.A) is a dietary lignan compound from Schisandra chinensis. In this study, the effect of G.A on the proliferation and metastasis of colorectal cancer (CRC) cells was investigated using several CRC cell lines and a lung metastasis mouse model. Both oral and intraperitoneal administration of G.A (50 mg/kg) inhibited lung metastasis of CT26 cells. Various concentrations of G.A were incubated with CRC cell lines and their viability was determined using a cell counting kit-8 assay. G.A significantly decreased the viability of various CRC cell lines, whereas it did not change the proliferation of normal colon cells. G.A induced G0/G1 phase arrest and apoptosis of CT26 and HT29 cells by regulating cyclin D1/cyclin-dependent kinase 4 (CDK4) expression and apoptotic proteins such as caspases and B-cell lymphoma-2 (Bcl-2) family proteins, respectively. G.A-induced apoptosis was mediated by AMPK/p38 activation in CRC cells. A non-cytotoxic concentration of G.A inhibited epithelial–mesenchymal transition of CRC cells by modulating E-cadherin and N-cadherin expression levels. Moreover, the migration and invasion of CRC cells were reduced by G.A treatment. Especially, G.A decreased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities. G.A ameliorated lung metastasis of CRC cells by decreasing cell survival and metastatic abilities of CRC cells. Thus, G.A might be a potential novel therapeutic agent for metastatic CRC.

Background: Lung adenocarcinoma (LUAD) has a higher objective response rate to single-agent KRASG12C inhibitors (e.g., AMG 510) than colorectal cancer (CRC) patients (32.2% vs 7.1%). It is known that molecular heterogeneities exist between KRASMut LUAD and CRC tissues. We hypothesize that co-mutations and oncogenic pathways may confer resistance to KRAS inhibitors in CRC cells. Methods: Whole-exome and transcriptome profiles of KRASmut LUAD (n=168) and microsatellite stable (MSS) CRC (n=183) patients were downloaded from TCGA. Mutation profiles of KRASmut patients were also obtained from AACR Project-GENIE. Gene essentiality scores derived from the whole genome CRISPR screenings were downloaded from the DepMap to evaluate the relative oncogene addiction to KRAS in lung (n=133) and CRC (n=59) cell lines. Differential gene expressions studies were performed using EBayes, followed by gene set enrichment analysis (GSEA). CRC cell lines SW837, SW1463, SW403, and HCT15 were treated with AMG 510, a KRASG12C inhibitor, and Pictilisib, a PIK3CA inhibitor, and cell viability was measured up to 96 hours after treatment. Results: The KRASmut LUAD cohort had co-mutation clusters remarkable for (i) no co-mutation (59%, 99/168), (ii) KEAP1mut and/or STK11mut (20.2%, 34/168), and (iii) a mixed group with a frequent mutation in other genes (mean=2) but without a clear recurrent pattern (20.8%, 35/168). In CRC, KRASmut tumors formed clusters of (i) no co-mutation (59.6%, 109/183), (ii) PIK3CAmut (26.2%, 48/183), and (iii) a similar cluster with frequent co-mutation (mean=2) without a recurrent pattern (14.2%, 26/183). Similar co-mutation patterns were also observed in AACR Project-GENIE LUAD and CRC cohorts. KRAS oncogene addiction was seen in both KRASMut lung (median scores, -1.1 vs -0.4, p<0.001) and CRC (median scores, -1.3 vs -0.5, p<0.001) cell lines than KRASwt cells. However, KRASmut/PIK3CAmut co-mutated CRC cell lines (n=10) were significantly less dependent on the KRAS gene than KRASmut/PIK3CAwt cells (n=22) (median scores, -1.0 vs -1.4, p=0.03). Two KRASG12C/PIK3CAwt CRC cell lines that are highly dependent on KRAS (SW837 and SW1463 cells) also showed higher sensitivities (IC50<5uM) to AMG 510 and Pictilisib relative to KRASnon-G12C/PIK3CAmut CRC cell lines SW403 and HCT15 (IC50>10uM). GSEA revealed that the Hedgehog, spermatogenesis, and pancreas-beta cells genesets were significantly active (Normalized enrichment scores > 1, FDR<0.05) in KRAS KO-resistant CRC cells, but the same genesets were not enriched in KRAS KO-resistant lung cancer cells. Conclusion: Somatic co-mutations and transcriptomics signatures in KRASmut LUAD and CRC may predict sensitivities to KRAS inhibitors. KRASmut CRC cells with PIK3CAmut or active hedgehog pathway are resistant to KRAS gene KO, suggesting multiple drug resistance mechanisms to KRAS inhibitors exist in KRASmut CRC cells. Future studies should emphasize identifying new drug targets, as optimal drug combinations of KRAS inhibitors may not be same for all KRASmut CRC patients. Citation Format: Saikat Chowdhury, Vinay K. Pattalachinti, Valsala Haridas, Scott Kopetz, John Paul Shen. Multi-omics profiling to identify mechanisms of resistance to KRAS inhibition: A comparative study on colorectal and lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B004.

Decreased Levels of miR-224 and the Passenger Strand of miR-221 Increase MBD2, Suppressing Maspin and Promoting Colorectal Tumor Growth and Metastasis in Mice

868 Direct Autocrine Regulation of Colon Cancer Cell Growth by VEGF Is Mediated via Activation of VEGF-R1/2 and Transactivation of EGF-R: Direct Effect of VEGF on CRC Cell Proliferation and Reduced Apoptosis

Introduction: Vascular endothelial growth factor receptor-1 (VEGFR-1) is highly expressed in endothelial cells, macrophages and cancer cells in the tumor microenvironment. VEGFR-1 plays an important role in tumor angiogenesis, inflammation and metastasis. In colorectal cancer (CRC) cells with hyperactive Wnt signaling, its function has been linked to cell motility, anchorage-independent growth, and cell survival. To better understand the role of VEGFR-1 in CRC, we performed studies to define the subcellular location and post-translational modifications of VEGFR-1 in human CRC cells. Methods/Results: Utilizing IHC analysis, GFP-tagged imaging and cell surface receptor assays, we observed that VEGFR-1 locates mostly in intracellular compartments in CRC cells, which is distinct from its membrane receptor status in endothelial cells. Moreover, spontaneous apoptosis was increased with endogenous VEGF knockout whereas treatment with the VEGF neutralizing antibody bevacizumab had no effect on apoptosis (Samuel et al. Oncogene, in press); these studies suggest that CRC cells require VEGF/VEGFR-1 intracrine signaling for suppression of apoptosis. In order to understand the means by which VEGFR-1 cellular localization is determined, we analyzed the glycosylation of VEGFR-1 in CRC cells, endothelial cells and clinical specimens of metastatic CRC from the liver. We found that VEGFR-1 is partially N-glycosylated by high mannose oligosaccharides in the CRC cells and human tumors, in contrast to complex, mature N-glycosylation of VEGFR-1 in the endothelial cells and normal liver tissue. Conclusion: VEGFR-1 is differentially post-translationally modified and intracellularly localized in CRC cells and metastatic tumors. Because N-glycosylation is critical for the membrane receptor's intracellular trafficking and ligand interaction on the cell surface, hypo-glycosylation of VEGFR-1 in CRC cells likely contributes to its cytoplasmic localization and intracrine activation through a non-RTK pathway. The implications of intracrine function of VEGFR-1 in CRC warrant further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5132. doi:10.1158/1538-7445.AM2011-5132

RBP-J promotes cell growth and metastasis through regulating miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis in colorectal cancer

869 Discovery and Therapeutic Potential of FGFR2 Gene Amplification in Diffuse Gastric Cancer

872 HSP70 Is Required for Loss of Heterozygosity in the ApcMin/+ Model of Colorectal Cancer

Rationale: Colorectal cancer (CRC) is a leading cause of cancer-related mortality. Epigenetic modifications play a significant role in the progression of CRC. KAT7, a histone acetyltransferase, has an unclear role in CRC.Methods: In this research, we analyzed the expression of KAT7 in CRC patients and its correlation with prognosis using the GEO database, western blot, and immunohistochemistry. We assessed the impact of KAT7 on CRC cell functions through cell viability, colony formation, flow cytometry, scratch, and transwell assays. Mechanistic insights were obtained via RNA sequencing and ChIP-qPCR. Additionally, we evaluated the effects of KAT7 on CRC growth and metastasis in vivo using mouse subcutaneous tumor and lung metastasis models.Results: In this study, we discovered an upregulated KAT7 signaling pathway in CRC and its association with poor patient survival. Knockdown of KAT7 promotes apoptosis and inhibits proliferation, migration, and invasion of CRC cells. Conversely, KAT7 overexpression enhanced these cellular processes. In vivo assays confirmed that knockdown of KAT7 can inhibit CRC proliferation and lung metastasis. Mechanistically, KAT7 acetylated histone H3 at lysine 14 (H3K14) to enhance MRAS transcription, which activated the MAPK/ERK pathway and promoted tumorigenesis. The enzymatic function of KAT7 as an acetyltransferase is crucial for the advancement of colorectal cancer. In KAT7 knockdown CRC cells, re-expression of KAT7, but not an acetyltransferase-deficient mutant, rescued MRAS expression, ERK phosphorylation, and CRC tumorigenesis.Conclusion: We found that KAT7 is highly expressed in CRC patients, and those with high KAT7 expression have a worse prognosis. KAT7 enhances MRAS gene transcription by promoting H3K14 acetylation, thereby activating the MAPK/ERK pathway and promoting malignant phenotypes of CRC. In summary, KAT7 represents a promising target for CRC therapy.

Background: Cancer stem cells (CSC), which possess the properties of self-renewal, multi-potential differentiation and tumor initiation, can arise either from normal stem cells or due to the influence of the tumor micro-environment. This study was done to determine the potential role of endothelial cell (EC) derived paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells. Methods: RFP-labeled human CRC cells HCT116 were co-cultured with RF24 ECs (immortalized HUVECs) under low serum conditions. After FAC-sorting for the CRC cells, the CSC population was analyzed by 1) the Aldefluor assay, 2) sphere forming ability, and 3) Western blot analysis for the CSC markers CD133 and CD44. In addition, low serum condition media obtained from either ECs or parental CRC cells (control), was added to CRC cells to determine its effect on the CSC phenotype. In order to characterize the apoptotic effect of EC derived factors on CRC cells, we performed Annexin V staining by FACS, PARP cleavage, Caspase 3 cleavage, and Bcl2 levels by Western blotting. Using MTT assay, we determined the effect of EC derived factors on chemo-sensitivity of CRC cells exposed to 5-FU. Results: Co-culturing of CRC cells with ECs markedly increased the ALDH-positive population from 5% to 18%, and the sphere forming ability by more than 4-fold in CRC cells. In addition, CRC cells also displayed increased CD133 (6-fold) and CD44 (4-fold) protein levels. Furthermore, this effect could be mimicked simply by co-culturing CRC cells with conditioned media obtained from ECs. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive population from 4% to 8%, sphere forming ability >3-fold, and the expression of CD133 (7-fold) and CD44 (6-fold). Conditioned medium from ECs, concomitantly decreased spontaneous apoptosis in CRC cells as demonstrated by a 3-fold decrease in Annexin V-positive population (21% to 7%), down-regulation of cleaved PARP and Caspase 3, and up-regulation of Bcl2. CRC cells exposed to EC conditioned medium also displayed decreased sensitivity to 5-FU [>10-fold increase of the IC50 from 2.0 μg/ml to 22.3 μg/ml (p<0.05)]. Conclusions: Exposure of CRCs to ECs increased CSC properties, and decreased spontaneous apoptosis. Since, this effect can also be mimicked by the conditioned media, additional experiments to identify and validate the soluble factors that mediate this effect are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1281.

NT5DC2 knockdown inhibits colorectal carcinoma progression by repressing metastasis, angiogenesis and tumor-associated macrophage recruitment: A mechanism involving VEGF signaling