Progression Of Colon Carcinoma Research Articles

Agrimol B inhibits colon carcinoma progression by blocking mitochondrial function through the PGC-1α/NRF1/TFAM signaling pathway.

The activation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) stimulates the transcription of the downstream target proteins, mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1), which induces mitochondrial biogenesis and promotes colorectal tumorigenesis. Agrimol B (Agr) is a constituent of Agrimonia pilosa Ledeb. that exerts anticancer effects. Herein, we aimed to investigate the antitumor activity of Agr and its mechanism of action. The interaction between Agr and PGC-1α was predicted by molecular docking. After the treatment with different concentrations of Agr (0, 144, 288, and 576 nM), the cell viability, migration rate, proliferation rate, and apoptosis rate of human colon cancer HCT116 cells were determined. Mitochondrial activity, cellular reactive oxygen species (ROS), and mitochondrial membrane potential were assessed to measure the regulatory effect of Agr on mitochondrial function. Western blotting (WB) assay was used to examine the expression of PGC-1α, NRF1, and TFAM, as well as of the pro-apoptotic proteins, Bax and Caspase-3, and the antiapoptotic protein (Bcl-2). Finally, subcutaneous tumor xenograft model mice were used to evaluate the effect of Agr on colorectal cancer (CRC) in vivo. The molecular docking results revealed a high likelihood of Agr interacting with PGC-1α. Agr inhibited the proliferation and migration of HCT116 cells, promoted ROS production and mitochondrial oxidative stress, inhibited mitochondrial activity, and decreased mitochondrial membrane potential. Agr induced cell apoptosis and, in combination with PGC-1α, impaired mitochondrial biogenesis and suppressed the expression of NRF1 and TFAM. Agr also suppressed the expression of Bcl-2 and Cleaved-Caspase-3 and increased the expression of Bax and Caspase-3. In addition, the in vivo antitumor effect and mechanism of Agr were confirmed by using a subcutaneous tumor xenograft mouse model. Our findings demonstrated that Agr regulates the expression of PGC-1α, thereby inducing mitochondrial dysfunction and promoting tumor cell apoptosis. This work highlights the potential of Agr as a promising therapeutic candidate in CRC.

Pan-cancer analysis of the angiotensin II receptor-associated protein as a prognostic and immunological gene predicting immunotherapy responses in pan-cancer.

Background: Understanding interior molecular mechanisms of tumorigenesis and cancer progression contributes to antitumor treatments. The angiotensin II receptor-associated protein (AGTRAP) has been confirmed to be related with metabolic products in metabolic diseases and can drive the progression of hepatocellular carcinoma and colon carcinoma. However, functions of AGTRAP in other kinds of cancers are unclear, and a pan-cancer analysis of AGTRAP has not been carried out. Methods and materials: We downloaded data from The Cancer Genome Atlas and Genotype-Tissue Expression dataset and The Human Protein Atlas databases and then used R software (version 4.1.1) and several bioinformatic tools to conduct the analysis. Results: In our study, we evaluated the expression of AGTRAP in cancers, such as high expression in breast cancer, lung adenocarcinoma, and glioma and low expression in kidney chromophobe. Furthermore, our study revealed that high expression of AGTRAP is significantly related with poor prognosis in glioma, liver cancer, kidney chromophobe, and so on. We also explored the putative functional mechanisms of AGTRAP across pan-cancer, such as endoplasmic reticulum pathway, endocytosis pathway, and JAK-STAT signaling pathway. In addition, the connection between AGTRAP and tumor microenvironment, tumor mutation burden, and immune-related genes was proven. Conclusion: Our study provided comprehensive evidence of the roles of AGTRAP in different kinds of cancers and supported the relationship of AGTRAP and tumorous immunity.

COL11A1 is Downregulated by miR-339-5p and Promotes Colon Carcinoma Progression.

The roles of COL11A1 in cancer have been increasingly considered, but the understandings of the effects of COL11A1 on colon carcinoma progress are much limited yet. qRT-PCR and Western blot were utilized to evaluate COL11A1 expression at mRNA and protein levels, respectively, in colon carcinoma cell lines. Afterward, the tumorigenesis biological effects of COL11A1 were examined by CCK-8, colony formation, Transwell, and wound healing methods. Moreover, upstream miRNAs containing the binding sites with COL11A1 were predicted by the bioinformatics methods. The interplay between COL11A1 and miR-339-5p was identified by a dual-luciferase assay. COL11A1 expression was prominently upregulated in colon carcinoma cell lines relative to that in normal human colon mucosal epithelial cell lines, and it was related to tumor stages. The outcomes of in-vitro experiments suggested that interfering with COL11A1 remarkably repressed the malignant behaviors of SW480 and SW620 cells. MiR-339-5p was markedly lowly expressed in colon carcinoma cell lines. Furthermore, miR-339-5p directly targeted and negatively regulated COL11A1 expression. COL11A1 upregulation promoted colon carcinoma cell functions, while overexpressing miR-339-5p evidently attenuated the promotion. These results proved the modulation of the miR-339-5p/COL11A1 axis in colon carcinoma cells, and miR-339-5p repressed colon carcinoma progression via COL11A1 downregulation. These results offer new underlying targets for the accurate therapy of colon carcinoma patients.

Silencing circular RNA-friend leukemia virus integration 1 restrained malignancy of CC cells and oxaliplatin resistance by disturbing dyskeratosis congenita 1.

Circular-RNA friend leukemia virus integration 1 (circ-FLI1; hsa_circ_0000370) is a noninvasive biomarker for the diagnosis of colon carcinoma (CC). Herein, we intended to investigate its functions and competing endogenous RNA (ceRNA) mechanisms in CC cells. In terms of expression status, circ-FLI1 was abnormally upregulated in CC patients’ tumors and cells, paralleled with DKC1 upregulation and miR-197-3p downregulation. Most strikingly, there was a direct target relationship between miR-197-3p and circ-FLI1 or DKC1 based on the starbase database, dual-luciferase reporter assay, and RNA immunoprecipitation. Functionally, the colony formation assay, MTS method, fluorescence-activated cell sorting method, cell cycle and apoptosis assays, and transwell assays were performed, and the results revealed that interfering circ-FLI1 and re-expressing miR-197-3p could restrict colony formation, cell viability, cell cycle progression, and migration/invasion of CC cells with apoptosis rate elevation; besides, they promoted oxaliplatin (L-OHP)-induced cell viability inhibition. Furthermore, there were counteractive effects between circ-FLI1 silencing and miR-197-3p depletion, miR-197-3p overexpression and DKC1 restoration on regulating CC cell functions and L-OHP resistance. With a xenograft tumor model, the anti-growth role of circ-FLI1 silencing was also found in vivo with or without L-OHP treatment. Collectively, we demonstrated that circ-FLI1 might confer L-OHP resistance and malignant progression of CC presumably through the circ-FLI1/miR-197-3p/DKC1 ceRNA axis.

Epithelial Wnt secretion drives the progression of inflammation-induced colon carcinoma in murine model

SummaryColon cancer is initiated by stem cells that escape the strict control. This process is often driven through aberrant activation of Wnt signaling by mutations in components acting downstream of the receptor complex that unfetter tumor cells from the need for Wnts. Here we describe a class of colon cancer that does not depend on mutated core components of the Wnt pathway. Genetically blocking Wnt secretion from epithelial cells of such tumors results in apoptosis, reduced expression of colon cancer markers, followed by enhanced tumor differentiation. In contrast to the normal colonic epithelium, such tumor cells autosecrete Wnts to maintain their uncontrolled proliferative behavior. In humans, we determined certain cases of colon cancers in which the Wnt pathway is hyperactive, but not through mutations in its core components. Our findings illuminate the path in therapy to find further subtypes of Wnt-dependent colon cancer that might be responsive to Wnt secretion inhibitors.

The Molecular Networks of microRNAs and Their Targets in the Drug Resistance of Colon Carcinoma.

Simple SummaryWe systematically reviewed the recent scientific publications describing the role of microRNAs in the regulation of drug resistance in colon cancer. To clarify the intricate web of resulting genetic and biochemical interactions, we used a machine learning approach aimed at creating: (i) networks of validated miRNA/target interactions involved in drug resistances and (ii) drug-centric networks, from which we identified the major clusters of proteins affected by drugs used in the treatment of colon cancer. Finally, to facilitate a high-level interpretation of these molecular interactions, we determined the cellular pathways related with drug resistance and regulated by the miRNAs in colon cancer.Drug resistance is one of the major forces driving a poor prognosis during the treatment and progression of human colon carcinomas. The molecular mechanisms that regulate the diverse processes underlying drug resistance are still under debate. MicroRNAs (miRNAs) are a subgroup of non-coding RNAs increasingly found to be associated with the regulation of tumorigenesis and drug resistance. We performed a systematic review of the articles concerning miRNAs and drug resistance in human colon cancer published from 2013 onwards in journals with an impact factor of 5 or higher. First, we built a network with the most studied miRNAs and targets (as nodes) while the drug resistance/s are indicated by the connections (edges); then, we discussed the most relevant miRNA/targets interactions regulated by drugs according to the network topology and statistics. Finally, we considered the drugs as nodes in the network, to allow an alternative point of view that could flow through the treatment options and the associated molecular pathways. A small number of microRNAs and proteins appeared as critically involved in the most common drugs used for the treatment of patients with colon cancer. In particular, the family of miR-200, miR34a, miR-155 and miR-17 appear as the most relevant microRNAs. Thus, regulating these miRNAs could be useful for interfering with some drug resistance mechanisms in colorectal carcinoma.

Decorin deficiency promotes epithelial-mesenchymal transition and colon cancer metastasis

The tumor microenvironment encompasses a complex cellular network that includes cancer-associated fibroblasts, inflammatory cells, neo-vessels, and an extracellular matrix enriched in angiogenic growth factors. Decorin is one of the main components of the tumor stroma, but it is not expressed by cancer cells. Lack of this proteoglycan correlates with down-regulation of E-cadherin and induction of β-catenin signaling. In this study, we investigated the role of a decorin-deficient tumor microenvironment in colon carcinoma progression and metastasis. We utilized an established model of colitis-associated cancer by administering Azoxymethane/Dextran sodium sulfate to adult wild-type and Dcn−/- mice. We discovered that after 12 weeks, all the animals developed intestinal tumors independently of their genotype. However, the number of intestinal neoplasms was significantly higher in the Dcn−/− microenvironment vis-à-vis wild-type mice. Mechanistically, we found that under unchallenged basal conditions, the intestinal epithelium of the Dcn−/− mice showed a significant increase in the protein levels of epithelial-mesenchymal transition associated factors including Snail, Slug, Twist, and MMP2. In comparison, in the colitis-associated cancer evoked in the Dcn−/− mice, we found that intercellular adhesion molecule 1 (ICAM-1) was also significantly increased, in parallel with epithelial-mesenchymal transition signaling pathway-related factors. Furthermore, a combined Celecoxib/decorin treatment revealed a promising therapeutic efficacy in treating human colorectal cancer cells, in decorin-deficient animals. Collectively, our results shed light on colorectal cancer progression and provide a protein-based therapy, i.e., treatment using recombinant decorin, to target the tumor microenvironment.

P-40 A correlation between BCL-2 modifying factor, p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. These genes that can detect colon cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate the correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients.

Neutrophil Extracellular Trap-Associated CEACAM1 as a Putative Therapeutic Target to Prevent Metastatic Progression of Colon Carcinoma.

Neutrophils promote tumor growth and metastasis at multiple stages of cancer progression. One mechanism through which this occurs is via release of neutrophil extracellular traps (NETs). We have previously shown that NETs trap tumor cells in both the liver and the lung, increasing their adhesion and metastasis following postoperative complications. Multiple studies have since shown that NETs play a role in tumor progression and metastasis. NETs are composed of nuclear DNA-derived web-like structures decorated with neutrophil-derived proteins. However, it is unknown which, if any, of these NET-affiliated proteins is responsible for inducing the metastatic phenotype. In this study, we identify the NET-associated carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) as an essential element for this interaction. Indeed, blocking CEACAM1 on NETs, or knocking it out in a murine model, leads to a significant decrease in colon carcinoma cell adhesion, migration and metastasis. Thus, this work identifies NET-associated CEACAM1 as a putative therapeutic target to prevent the metastatic progression of colon carcinoma.

A correlation between BCL-2 modifying factor, p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.

Microcystin-LR promotes migration via the cooperation between microRNA-221/PTEN and STAT3 signal pathway in colon cancer cell line DLD-1

Previous researches have reported that microcystin-LR (MC-LR) contributes to the progression of multiple types of carcinomas including colon cancer; however, the underlying molecular mechanisms remain unclear and require in-depth investigation. Here, the colon cell line DLD-1 was arranged for the analysis by the microRNA microarray which was associated with the cancer metastasis after MC-LR exposure. 31 human microRNAs were differentially expressed, including miR-221, which targeted 3′-UTR of PTEN mRNA and PTEN level was down-regulated by MC-LR treatment. Besides, MC-LR also induced the phosphorylation of STAT3, which can be reversed by adding miR-221 inhibitor and PTEN expression plasmid. Furthermore, miR-221 inhibitor, STAT3 siRNA and PTEN expression plasmid could reverse the effects of MC-LR induced migration with the accumulation of β-catenin in nuclei. In conclusion, our study suggested that MC-LR promoted the progression of colon carcinoma, at least in part, by regulating the expression miR-221, PTEN and STAT3 phosphorylation, which offers a novel perspective to understand the connection between MC-LR and colon cancer.

XIAP facilitates breast and colon carcinoma growth via promotion of p62 depletion through ubiquitination-dependent proteasomal degradation.

X-linked inhibitor of apoptosis protein (XIAP) possesses a critical role in promotion of cell survival and maintenance of cellular homeostasis. In cancer, elevated XIAP expression has been associated with malignancy, poor prognosis, and treatment resistance. However, the underlying mechanisms of these effects remain unclear. XIAP has previously been proposed to promote tumor growth through suppression of autophagy. In this study, we examined the expression of XIAP and p62, two critical mediators of autophagy, in breast and colon cancer. We observed a negative correlation between XIAP and p62 expression in normal and cancer tissues of breast and colon, and that the ratio of XIAP and p62 expression determines the cancer phenotype. In vitro, we observed that XIAP interacted with p62 and also that XIAP depletion resulted in increased expression of p62. XIAP functioned as an ubiquitination E3 ligase towards p62 and suppressed p62 expression through ubiquitin-proteasomal degradation. Furthermore, XIAP enhanced cancer cell proliferation, viability, and colony formation in vitro via suppression of p62. In addition, we demonstrated that XIAP-enhanced tumor growth is dependent on depletion of p62 in vivo. Herein, we have therefore delineated a novel mechanism by which XIAP contributes to development and progression of breast and colon carcinoma.

MicroRNAs as biomarkers for the progression and prognosis of colon carcinoma.

Early detection is critical for the treatment of colon carcinoma. However, current biomarkers for its diagnosis and prognosis are insufficient and improvement is required. Aberrantly expressed microRNAs (miRNAs/miRs) in colon carcinoma have been identified to function as potential diagnostic and prognostic biomarkers. In the present study, 245 differentially expressed miRNAs between colon carcinoma and normal tissues were identified by a bioinformatics analysis of a dataset from The Cancer Genome Atlas. A six-miRNA (miR-149, miR-3189, miR-3677, miR-3917, miR-4999 and miR-6854) prognostic prediction system was established, which is able to independently and effectively predict the prognosis of colon carcinoma patients [P<0.001, area under the receiver operating characteristic curve (AUC)=0.763]. Furthermore, the six miRNAs were highly correlated with the tumor-nodes-metastasis (TNM) stage and were able to distinguish between different stages (high vs. low TNM stage, P<0.001). Of note, combination of the six-miRNA signature and TNM stage provides an improved prediction of the patient's prognosis (AUC=0.797). Functional enrichment analysis revealed the possible mechanistic involvement of these predictive miRNAs in cancer-associated biological processes and pathways. Taken together, the present study demonstrated the promising potential of the novel six-miRNA model as an independent factor for the prediction of the progression and prognosis of colon carcinoma.

MiR-299-3p functions as a tumor suppressor via targeting Sirtuin 5 in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the most common cancers in the world, with the characteristics of high morbidity and mortality. Though the levels of diagnosis and treatment of HCC have been largely improved recently, the prognosis of these patients remains unacceptable. Thus, it is urgent for us to discover the exact mechanisms and determine some new biomarkers for HCC. Previous studies revealed that microRNAs (miRNAs) played a critical role in the occurrence and progression of HCC. And miR-299-3p has been reported to be closely related to the progression of colon carcinoma, lung cancer and clear cell renal cell carcinoma and so on. However, the exact expression and functions of miR-299-3p in HCC are still uncovered. Here, we reported for the first time that miR-299-3p was downregulated in HCC. Clinically, statistical analysis showed that miR-299-3p expression was significantly associated with tumor size (P = 0.007), venous infiltration (P = 0.028), Edmondson-Steiner grading (P = 0.042) and TNM stage (P = 0.012). In addition, HCC patients with lower miR-299-3p expression had worse 3-year overall survival and disease-free survival (P = 0.0012, P = 0.0002). Functionally, Transwell assays, Wound healing assay, MTT assay and plate clone formation assay revealed that miR-299-3p inhibited the migration, invasion and proliferation of HCC cells. Furthermore, bioinformatics tools, luciferase reporter assay, real-time PCR, Pearson's correlation coefficient analysis, immunohistochemistry and Western blot showed that Sirtuin 5 (SIRT5) was a downstream target of miR-299-3p in HCC cells. In addition, rescue experiments indicated that SIRT5 mediated the effects of miR-299-3p on migration, invasion and proliferation of HCC cells. Thus, we conclude that miR-299-3p suppresses migration, invasion and proliferation of HCC cells via directly targeting SIRT5. MiR-299-3p may be a potential prognosis indicator and therapeutic target for HCC.

Let‑7i‑5p inhibits the proliferation and metastasis of colon cancer cells by targeting kallikrein‑related peptidase6.

Colorectal cancer is one of the most common types of malignancies worldwide, and as it is often diagnosed at an advanced stage, it is a serious threat to human health. MicroRNAs are important regulators of the growth and metastasis of colon cancer (CC). In the present study, the results demonstrated that kallikrein‑related peptidase6 (KLK6) plays a critical role in suppressing colon carcinoma progression. To further investigate whether microRNAs affect the impact of KLK6, a bioinformatics approach was employed, which indicated that let‑7i‑5p may directly target KLK6. Furthermore, the expression level of let‑7i‑5p was significantly negatively correlated with the expression of KLK6 at the mRNA and protein levels in CC. Functionally, overexpression of let‑7i‑5p inhibited the proliferation and invasion of CC cells, and suppressed the growth of CC invitro. The luciferase reporter assays revealed that let‑7i‑5p targeted the KLK6 3'‑untranslated region. Collectively, these results indicated that let‑7i‑5p inhibited the proliferation and metastasis of CC cells by targeting KLK6, thereby blocking the cell cycle and promoting apoptosis in colon cells. Therefore, the present study revealed that the let‑7i‑5p/KLK6 axis may be a potential target for new therapeutic strategies to treat colon tumors.

3 Clonal dynamics of colon carcinoma progression

IntroductionCancer formation is initiated by a single cell that acquires sequential mutations, eventually resulting in a metastasizing carcinoma. Throughout this process numerous clones arise, each with their own unique genetic composition. All these clones have slightly different fitnesses and compete with each other; this process of competition (clonal dynamics) plays an important role in cancer progression and determines the genetic composition of the tumour as a whole.Material and methodsTo understand these clonal dynamics we have developed a viral lineage tracing system, which allows us to simultaneously track the clonal dynamics of thousands of unique clones. We applied this system to an organoid model of colon carcinoma progression, which consists of WT, APC-/- (A), APC-/- P53-/- (AP), APC-/- P53-/- KRASG12D (APK) and APC-/- P53-/- KRASG12D SMAD4-/- (APKS) organoid lines, which mimics colon carcinoma progression in patients.Results and discussionsWe found that clonal dynamics change dramatically throughout progression from A/AP to APK/APKS, where there is fast and stringent selection in A/AP organoid lines and slower selection in APK/APKS organoids lines. Furthermore Whole Genome Sequencing (WGS) of the organoids revealed that Copy Number Variation (CNV) patterns are deterministic in the A/AP organoids, with a strong preference for losses of chromosome 4 and chromosome 18, and much more varied in APK/APKS organoids. Finally, through single cell whole genome sequencing it was possible to identify the sequence of CNV events through which unique genetic clones arise in the APKS mutant organoids.ConclusionIn this study, we find reproducible differences in clonal dynamics between adenoma and carcinoma like organoids. These differences in clonal dynamics are in line with current views on colon carcinoma progression, where early in the progression of the tumour several large-scale clonal expansions occur, while late in progression clonal dynamics are less extreme.

Suppression of YAP by DDP disrupts colon tumor progression.

Colon cancer is a commonly diagnosed cancer that often has a poor prognosis. Combined with the development of drug resistance to cancer treatment agents the treatment efficacy of colon cancer can be limited. Activation of the oncogene YAP has been shown to be related to colon cancer progression and is associated with poor prognosis, drug resistance and metastasis, even under treatment. Cisplatin (DDP) is a commonly used drug that can control carcinoma progression, although its mechanisms are poorly understood. In the present study, we examined whether DDP specifically suppressed YAP in order to inhibit colon carcinoma progression. Our data revealed that Mst/Yap signaling was unusually activated in colon cancers, promoting cell proliferation and invasion. DDP treatment decreased the expression of YAP at both the transcriptional and post-translational levels, leading to cell cycle arrest, apoptosis and senescence in cancer cells, in addition to decreasing epithelial-to-mesenchymal transition, cell motility and in vitro cell invasion and migration. Ultimately, DDP increased the expression of E-cadherin and decreased the expression of vimentin. The present study also revealed that post-translational regulation of YAP phosphorylation controlled the subcellular distribution between the nucleus and the cytoplasm. In conclusion, the findings of the present study revealed that DDP was a suitable therapeutic candidate for colon cancer that specifically targets the Mst/Yap signaling pathway.

MicroRNA-299-3p suppresses proliferation and invasion by targeting VEGFA in human colon carcinoma

Accumulating evidences have shown that microRNAs (miRNAs) are vital regulators and possess huge capabilities in post-transcriptional control. Although a large number of miRNAs have been identified to be dysregulated in human cancers especially in colon cancer, our understandings of the function of most miRNAs are still largely limited. In this study, we have demonstrated that miR-299-3p plays a critical role in suppressing colon carcinoma progression by targeting Vascular Endothelial Growth Factor A (VEGFA). We observed that miR-299-3p was down-regulated in colon carcinoma tissues and colon cancer cell lines. The level of miR-299-3p was significantly negatively correlated with that of VEGFA mRNA level in colon carcinoma. More importantly, the low level of miR-299-3p predicted poor prognosis of colon cancer patients. Functionally, overexpression of miR-299-3p inhibited the proliferation and invasion of colon carcinoma cells and suppressed the growth of colon cancer xenografts in nude mice. Luciferase reporter assays showed that miR-299-3p could target VEGFA 3′ UTR. In addition, up-regulation of miR-299-3p decreased VEGFA expression both in vitro and in vivo, showing that miR-299-3p plays a suppressive effect on VEGFA via post-transcriptional control. However, ectopical expression of VEGFA could abrogate this effect and also abolish miR-299-3p-induced inhibition of cell proliferation and invasion. Taken together, our study provides evidences showing that miR-299-3p functions as a suppressor in colon cancer by targeting VEGFA, suggesting that miR-299-3p might serve as a novel target for colon cancer therapy.

Syndecan-1 deficiency promotes tumor growth in a murine model of colitis-induced colon carcinoma.

Syndecan-1 (Sdc1) is an important member of the cell surface heparan sulfate proteoglycan family, highly expressed by epithelial cells in adult organisms. Sdc1 is involved in the regulation of cell migration, cell-cell and cell-matrix interactions, growth-factor, chemokine and integrin activity, and implicated in inflammatory responses and tumorigenesis. Gastrointestinal tract represents an important anatomic site where loss of Sdc1 expression was reported both in inflammation and malignancy. However, the biological significance of Sdc1 in chronic colitis-associated tumorigenesis has not been elucidated. To the best of our knowledge, this study is the first to test the effects of Sdc1 loss on colorectal tumor development in inflammation-driven colon tumorigenesis. Utilizing a mouse model of colitis-related colon carcinoma induced by the carcinogen azoxymethane (AOM), followed by the inflammatory agent dextran sodium sulfate (DSS), we found that Sdc1 deficiency results in increased susceptibility to colitis-associated tumorigenesis. Importantly, colitis-associated tumors developed in Sdc1-defficient mice were characterized by increased local production of IL-6, activation of STAT3, as well as induction of several STAT3 target genes that act as important effectors of colonic tumorigenesis. Altogether, our results highlight a previously unknown effect of Sdc1 loss in progression of inflammation-associated cancer and suggest that decreased levels of Sdc1 may serve as an indicator of colon carcinoma progression in the setting of chronic inflammation.

Abstract A28: Mechanistic study involving the combined antiproliferative effect of Etoricoxib- cyclooxygenase-2 inhibitor and cholecystokinin-2 receptor antagonist in human pancreatic cancer cells

Abstract Introduction: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in United States. Due to poor prognosis and increasing incidences, over 80% of the patient are given palliative chemotherapy with combination drugs. Despite decades of efforts, the survival rate still remains 3-6%. The most common cause of pancreatic cancer are genetic alterations, cigarette smoking, obesity and lack of exercise. Patients with Type-2 Diabetes Mellitus are also on higher risk of pancreatic cancer. Although numerous chemotherapeutic drugs have been tested in this malignancy, survival of advanced pancreatic cancer has not improved over the past several decades. Because of the lack of effective treatment options available for this disease, identification of novel targets and approaches has been made. Recent studies have shown an association between the gastrointestinal hormone gastrin, the expression of Cholecystokinin -B/ gastrin receptor and an increased generation of COX-2 in colon-carcinoma cells and progression of gastric cancer, suggesting that gastrin has direct stimulating potencies. However, it remains unknown whether the combination of Cholecystokinin-2 (CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic antitumor effects on human gastric cancer. Therefore, for a better treatment and understanding the underlying mechanism that leads to pancreatic adenocarcinoma initiation, chemoresistance and progression, we further investigated the combined effect of Cyclooxygenase (COX-2) inhibitor and Cholecystokinin receptor-2 (CCK-2) antagonist on pancreatic cancer cells. Thus it is essential to recognize more effective targets/ receptors for treatment of pancreatic cancer. Materials &amp; Methods: Antiproliferative effects were carried out using Cell Proliferation Kinetics, Trypan blue staining technique, Morphological analysis and MTT Assay. Apoptotic activity was confirmed by DNA Fragmentation, Western Blot analysis and Cell cycle analysis Result and Discussion: The present study demonstrate that COX-2 enzyme is a modulator of carcinogenesis, apoptosis and angiogenesis in pancreatic cancer and is such rational target for drug development. Similarly Cholecystokinin antagonists is also a potential target for developing novel strategies for pancreatic cancer and further defines the pathway affected by CCK-BR. To study the combinatorial effect of COX-2 inhibitor and Cholecystokinin antagonist we successfully purified Etoricoxib using HPLC and further characterized by spectroscopic techniques using FT-IR, NMR and Mass spectroscopy. The anti-proliferative activity of Etoricoxib was checked on various cell lines and it induced proliferation arrest in Miapaca-2, Panc-I, BXPC-3 and ASPC-1 both in time and concentration dependent manner. The IC50 values were found to be 280 μM (Miapaca-2), 400μM (Panc-1), 300μM (BXPC-3) and 450 μM (ASPC-1) cells. The IC50 of procured Cholecystokinin antagonists YM-022, LY 288513 and L-365-260 was found out to be 0.002μM, 0.001μM and 0.01μM on Panc-1 respectively. Similarly, combinatorial studies was carried out on Panc-1, BXPC-3, ASPC-1 and Miapaca-2 respectively using cytotoxicity and apoptotic techniques. It is thus, a probable insight in stating that it has lesser side effects and thus have selectivity for cancer cell toxicity. Conclusion: The combined effect of Cyclooxygenase-2 inhibitor and Cholecystokinin-2 Receptor antagonists was calculated using Chaou Tatlay method. A combination index was calculated. As a class NSAIDs possess analgesic, antipyretic, anti-inflammatory, and there is persuasive evidence that COX-2 inhibitor suppress cancer cell proliferation owing to their role in apoptosis, compelling evidence suggest that COX-2 over-expression promotes whereas COX-2 inhibition prevents tumor initiation and promotions. NSAIDs and COX-2 selective inhibitors may have different effects on cancer may be stage dependent therefore a better understanding of the critical COX related mechanisms of carcinogenesis, proliferation, apoptosis, inflammation, angiogenesis and tumor invasion will help to define the potential of COX-2 blockage in cancer therapy. Previous studies have shown that NSAIDs and specific COX-2 inhibitors are able to enhance the effect of certain cystostaic agents, a specific COX-2 inhibitor Celecoxib enhances the cystostaic effect of doxorubicin on tumor cell lines (Wijnggaarden J.V. et.al. 2007). Results in our lab showed that combination of doxorubicin and etoricoxib synergistically inhibited the proliferation of Hela and Miapaca cell lines. These results has indicated that the combination therapy of COX-2 selective inhibitors may be the potential chemotherapeutic strategy for cancer prevention. In the present study, human pancreatic cancer cell lines, in which the CCK-2 receptor and COX-2 were expressed, was applied to examine whether blockade of the CCK-2 receptor and COX-2 exerts synergistic anti-tumor effects on human pancreatic cancer in vitro. The detailed studies are under process. In conclusion, both Etoricoxib and YM-022 had growth inhibitory and apoptosis inductive effects on the pancreatic cancer cells through down regulation of Bcl-2 with simultaneously up-regulation of BAX expression which suggest that COX-2 inhibition is new molecular targets for effective therapy against pancreatic cancer. Citation Format: Manisha Sikka. Mechanistic study involving the combined antiproliferative effect of Etoricoxib- cyclooxygenase-2 inhibitor and cholecystokinin-2 receptor antagonist in human pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A28.