Phenotype Of Colon Cancer Cells Research Articles

Cinobufotalin regulates the USP36/c-Myc axis to suppress malignant phenotypes of colon cancer cells in vitro and in vivo.

Ubiquitin-specific protease 36 (USP36) has been reported to exhibit oncogenic effects in various malignancies, but the function of USP36 in colon cancer progression remains indefinite. Herein, we aimed to determine the role and mechanism of USP36 in malignant phenotypes of colon cancer cells and explore the potential drug targeting USP36. Bioinformatics analyses indicated that USP36 is highly expressed and significantly related to tumor stages in colon cancer. Besides, USP36 was further up-regulated in oxaliplatin (Oxa)-resistant colon cancer cells. Colony formation, Edu staining, Transwell, wound healing, sphere formation, and CCK-8 assays were conducted and showed that the proliferation, Oxa-resistance, migration, stemness, and invasion of HCT116 cells were promoted after overexpressing USP36, while suppressed by USP36 knockdown. Mechanically, USP36 enhances c-Myc protein stabilization in HCT116 cells via deubiquitination. AutoDock tool and ubiquitin-AMC hydrolysis assay identified cinobufotalin (CBF), an anti-tumor drug, maybe a USP36 inhibitor by inhibiting its deubiquitination activity. CBF significantly prohibited proliferation, migration, invasion, and stemness of HCT116 cells and reversed Oxa-resistance, whereas enforced expression of USP36 blocked these effects. Moreover, in vivo analyses confirmed the oncogenic role of USP36 and the therapeutic potential of CBF in the malignancy of colon cancer. In conclusion, CBF may be a promising therapeutic agent for colon cancer due to its regulation of the USP36/c-Myc axis.

METTL14 Suppresses Tumor Stemness and Metastasis of Colon Cancer Cells by Modulating m6A-Modified SCD1.

Colon cancer (CC) is a malignant disease of the digestive tract, and its rising prevalence poses a grave threat to people's health. N6-methyladenosine (m6A) modification is essential for various crucial life processes through modulating gene expression. Methyltransferase-like 14 (METTL14), the m6A methylation transferase core protein, and its aberrant expression is intimately correlated to tumor development. This study was conducted to probe the impacts and specific mechanisms of METTL14 on the biological process of CC. Bioinformatics data disclosed that METTL14 was significantly attenuated in CC. Functional assays were executed to ascertain how METTL14 affected CC tumorigenicity, and METTL14 overexpression caused a notable decline in viability, migration, invasion, and stemness phenotype of CC cells. Then, in-depth mechanistic studies displayed that stearoyl-CoA desaturase 1 (SCD1) was a downstream target gene of METTL14-mediated m6A modification. METTL14 overexpression substantially augmented the m6A modification of SCD1 mRNA and diminished the SCD1 mRNA level. In addition, we revealed that YTHDF2 was the m6A reader to recognize METTL14 m6A-modified SCD1 mRNA and abolish its stability. Finally, we also validated that METTL14 might impede the tumorigenic process of CC through SCD1 mediated Wnt/β-catenin signaling. Taken together, this study presented that METTL14 performed as a potential therapeutic target in CC with important implications for the prognosis amelioration of CC patients.

Potassium voltage‑gated channel subfamily E member 4 facilitates the malignant progression of colon cancer by enhancing EGF containing fibulin extracellular matrix protein 2 expression.

Colon cancer is a highly invasive and metastatic cancer with a poor prognosis. The University of Alabama at Birmingham Cancer data analysis portal (UALCAN) database indicates that potassium voltage-gated channel subfamily E member 4 (KCNE4) is highly expressed in colon cancer tissues. UALCAN data also show that KCNE4 expression is positively associated with individual cancer stages and negatively associated with patient survival. Therefore, the aim of the current study was to elucidate the functional role of KCNE4 in the biological behaviors of colon cancer cells and to investigate the underlying molecular mechanism. The gene EGF containing fibulin extracellular matrix protein 2 (EFEMP2) was found to be positively correlated with KCNE4 in colon cancer based on analysis performed using the LinkedOmics database; notably, upregulated EFEMP2 expression has been reported to be closely associated with the malignant phenotypes of colon cancer cells. The differences in the expression levels of KCNE4 and EFEMP2 between human colon cancer and normal colonic mucosa cell lines were assessed via reverse transcription-quantitative PCR and western blot assays. In addition, the proliferation, migration and invasion of colon cancer cells were determined using Cell Counting kit-8, colony formation, would healing and Transwell assays, and a co-immunoprecipitation assay was performed to confirm the interaction between KCNE4 and EFEMP2. The results of the study demonstrated that KCNE4 and EFEMP2 are markedly upregulated in colon cancer cells. In addition, KCNE4 interacted with and bound to EFEMP2. The suppressive effects of KCNE4 knockdown on the proliferation, colony formation, migration and invasion of colon cancer cells were attenuated by EFEMP2 overexpression. On the basis of these findings, it may be concluded that KCNE4 acts as an oncogene in colon cancer via the promotion of EFEMP2 expression.

Combination of Quercetin or/and siRNA-loaded DDAB-mPEG-PCL hybrid nanoparticles reverse resistance to Regorafenib in colon cancer cells

BackgroundColorectal cancer (CRC) is the second leading cause of cancer death. Although Regorafenib showed survival benefits in patients with CRC, reports imply the recurrence of malignant phenotype resulting from chemotherapy. Evidence demonstrated that a5β1 integrin plays an important role in the Regorafenib treatment, which, may be led to resistance. In this study, the effects of /siRNA or/ and Quercetin loaded DDAB-mPEG-PCLnanoparticles could reverse this resistance phenotype in colon cancer cells in vitro.MethodsRegorafenib-resistant Ls-180 colon cancer cell line was developed by long-term exposure to Regorafenib. Quercetin and Regorafenib were separately encapsulated into mPEG-PCL micelles through the nano-precipitation method and characterized by DLS. Optimized doses of Quercetin and Regorafenib were used for combination therapy of resistant cells followed cytotoxicity study using MTT. Gene expression levels of the β1 subunit of integrin were determined by the real-time method of RT-PCR.ResultsDeveloped Regorafenib resistant LS-180 showed to have Regorafenib IC50 of 38.96 ± 1.72 µM whereas IC50 in non-resistant cells were 8.51 ± 0.29 µM, which meaningful was lower statistically compared to that of a resistant one. The β1 mRNA level of whole α5β1 integrin was significantly higher in the resistant cells compared to those of non-resistant ones. Gene expression levels in each siRNA-loaded nanoparticle and Quercetin-loaded one were lower than that in mock experiments. Finally, when these two types of nanoparticles were used to treat resistant cells, gene expression decrease of integrin indicated a greater effect that could be capable of reverse resistancy.ConclusionResults of this study demonstrated another confirmation of involving integrins in cancer resistance following chemotherapy using Regorafenib. Also, it indicated how using siRNA targeting integrin could enhance the plant derivatives like Quercetin effects to reverse resistance in vitro.

Abstract 1324: IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist

Abstract Background & Aims: The IL-1 subfamily, IL-36, is increasingly being implicated as a potent cytokine family involved in chronic inflammatory conditions such as psoriasis and IBD. Recent work has also suggested IL-36 cytokines may act as a potential adjuvant in immunotherapy regimens to augment the anti-cancer immune response. Given the pluripotent nature of IL-1 cytokines and the relationship between inflammation and tumorigenesis, we investigated the effects of IL-36 signalling in colorectal cancer (CRC). Methods: IL-36 family expression in CRC progression was investigated by the NCBI GEO transcriptomic database, and then by immunohistochemistry (IHC) and qRT-PCR. Pro-tumorigenic properties such as cancer cell migration, invasion and proliferation were investigated in 2D and 3D models. In vivo models were performed using Balb-c mice which were subcutaneously inoculated with CT26 cells and treated with IP injections of recombinant IL-36R agonists (IL-36α, β, γ) or antagonist (IL-36RN). Tumour tissues were excised and studied by flow cytometry and IHC analysis. Additionally, CRISPR-Cas9 mediated IL-36R KO CT26 cells were generated and subcutaneously injected into Balb-c mice, with changes in tumour growth and immune cell infiltrate investigated. Results: IL-36R expression was shown to significantly increase with stage of disease in the adenoma-carcinoma sequence, including distant metastases. Additionally, expression of all family members was increased in tumour tissue relative to adjacent normal tissue in a separate colon cancer cohort. In vitro IL-36R signalling on cancer cells augmented a pro-tumorigenic phenotype by induction of pro-tumorigenic cytokines/chemokine production, increased cellular migration, invasion and proliferation of tumour cells. In vivo inhibition of IL-36R signalling by both administration of recombinant IL-36RN and through deletion of the IL-36R gene in CT26 cells, resulted in a significant reduction in tumour growth. This reduction was associated with a significant decrease in tumour cell proliferation, as well as an increase in CD8+ T cell infiltrate in IL-36R KO groups. Of note, administration of IL-36R agonists also resulted in reduced tumour growth, albeit to a lesser extent, associated with an increase in CD4+ and CD8+ T cell infiltration. Conclusions: This data indicates that IL-36 family members, similar to other IL-1 family members, have dual functions in colon cancer. In addition, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for CRC patients with IL-36R+ tumour cells. Citation Format: Kevin J. Baker, Charlotte O'Donnell, Micheal O'Riordain, Elizabeth Brint, Maura Bendix, Aileen Houston. IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1324.

MiR-17-5p Targets and Downregulates CADM2, Activating the Malignant Phenotypes of Colon Cancer Cells.

Accumulating studies have demonstrated that CADM2 modulated malignant phenotype of various cancer cells, while its regulatory function and mechanism have not yet been reported. In this study, qRT-PCR was utilized to measure CADM2 mRNA level in normal cells and colon cancer cells, also, IHC and WB were applied to detect CADM2 protein expression in colon tissues, exhibiting low mRNA and protein levels of CADM2 in colon cancer. Applying cell function experiments, the impacts of CADM2 on colon cell phenotypes were examined, and the results illustrated that upregulating CADM2 remarkably repressed proliferation, invasion, migration, cell cycle of colon cancer cells, and facilitated cell apoptosis. Thus, it could be considered that CADM2 served as a tumor repressor gene in colon cancer. Moreover, the outcomes of dual-luciferase assay displayed that miR-17-5p could target CADM2, and overexpressing miR-17-5p could notably inhibit the mRNA and protein expression levels of CADM2. We, therefore, assumed that CADM2 was a downstream target of miR-139-5p. qRT-PCR was conducted to assess miR-17-5p level in colon cancer cells and normal cells, verifying a high miR-17-5p expression in the cancer cells. The effects of miR-17-5p on colon cell phenotypes were examined as well, where we determined that miR-17-5p served as a tumor-promoting factor. Finally, the rescue experiments exhibited that miR-17-5p could activate tumor-promoting phenotypes, while such activating effects could be reversed by upregulating CADM2. In short, the study proved that miR-17-5p facilitated malignant progression of colon cancer through targeting CADM2 at a post-transcriptional level. Our findings offer new insight into molecular therapy of colon cancer patients.

Thymidylate Synthase Overexpression Drives the Invasive Phenotype in Colon Cancer Cells.

Thymidylate synthase (TYMS) is the crucial enzymatic precursor for DNA biosynthesis and, therefore, the critical target for numerous types of chemotherapy, including the most frequently applied agent in colon cancer treatment 5-fluorouracil (5-FU). TYMS also seems to be associated with cancer metastasis and acquiring mesenchymal character by tumor cells during epithelial–mesenchymal transition (EMT). Based on that knowledge, we decided to investigate the role of TYMS in the modulation of invasive ability in colon cancer cells, where its effect on cancer metastasis has not been studied in detail before. We employed colon cancer cells isolated from different stages of tumor development, cells undergoing EMT, and TYMS overexpressing cells. The elongation ratio, cell migration, invasion assay, and MMP-7 secretion were applied to analyze the cell behavior. Important epithelial and mesenchymal markers characteristic of EMT were examined at the protein level by Western blot assay. Overall, our study showed a correlation between TYMS level and invasion ability in colon cancer cells and, above all, a crucial role of TYMS in the EMT regulation. We postulate that chemotherapeutics that decrease or inhibit TYMS expression could increase the effectiveness of the therapy in patients with colon cancer, especially in the metastatic stage.

Cordycepin Suppresses The Malignant Phenotypes of Colon Cancer Cells through The GSK3ß/ß-catenin/cyclin D1 Signaling Pathway.

Objective Cordycepin, also known as 3ˊ-deoxyadenosine, is the main bioactive ingredient of Cordyceps militaris and possesses various pharmacological effects. This study was performed to investigate the role of cordycepin in regulating the biological behaviors of colon cancer cells and the potential mechanism behind it. Materials and Methods In this experimental study, after treatment of colon cancer cells with different concentrations of cordycepin, inhibition of proliferation was detected by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Colon cancer cell migration and invasion abilities were analyzed by wound healing and Transwell assays. Flow cytometry was performed to detect cell apoptosis. A lung metastasis model in nude mice was utilized to examine the effect of cordycepin on the metastasis of colon cancer cells in vivo. Western blot was used to quantify GSK3β, β-catenin and cyclin D1 expression levels. Results Cordycepin inhibited colon cancer cell proliferation, migration and invasion, induced apoptosis in vitro, and inhibited lung metastasis of colon cancer cells in vivo. GSK-3β inhibitor (CHIR99021) treatment abolished the effects of cordycepin on cell viability, migration, invasion and apoptosis. Additionally, cordycepin promoted the expressions of GSK3β, and inhibited β-catenin and cyclin D1 in colon cancer cells, while co-treatment with CHIR99021 reversed the above effects. ConclusionCordycepin suppresses the malignant phenotypes of colon cancer through the GSK3β/β-catenin/cyclin D1 signaling pathway.

IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist.

The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines, shown to play a role in the pathogenesis of intestinal diseases such as Inflammatory Bowel Disease (IBD). Given the link between IBD and colitis –associated cancer, as well as the involvement of other IL-1 family members in intestinal tumorigenesis, the aim of this work was to investigate whether IL-36 cytokines play a role in the pathogenesis of colon cancer. Whilst research to date has focused on the role of IL-36 family members in augmenting the immune response to induce tumour rejection, very little remains known about IL-36R signalling in tumour cells in this context. In this study we demonstrate that expression of IL-36 family member mRNA and protein are significantly increased in colorectal cancer tissue compared to adjacent non-tumour. In vitro assays showed stimulation of colon cancer cell lines with IL-36R agonists resulted in the activation of the pro-tumorigenic phenotypes of increased cellular migration, invasion and proliferation in both 2D and 3D models. In addition, the IL-36 cytokines induced strong expression of pro-inflammatory chemokines in both human and murine cell lines. Intraperitoneal injection of IL-36Ra significantly reduced tumour burden using the subcutaneous CT26 tumour model in syngeneic Balb/mice, and this was associated with a decrease in Ki-67 expression by tumour cells in the IL-36Ra- treated group relative to untreated, suggesting the inhibition of the pro-proliferative signalling of IL-36 agonists resulted in the decreased tumour size. Moreover, colon cancer cells lacking the IL-36R also showed reduced tumour growth and reduced Ki-67 expression in vivo. Taken together, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for colorectal cancer patients with IL-36R+ tumour cells.

Enterococcus faecalis promotes a migratory and invasive phenotype in colon cancer cells.

Much about the role of intestinal microbes at the site of colon cancer development and tumor progression following curative resection remains to be understood. We have recently shown that collagenolytic bacteria such as Enterococcus faecalis predominate within the colon postoperatively, particularly at the site of the colon reconnection (i.e. anastomosis) in the early period of post-surgical recovery. The presence of collagenolytic bacteria at this site correlates with the tumor progression in a mouse model of post-surgical tumor development. In the present study we hypothesized, that collagenolytic bacteria, such as E. faecalis, play an important yet to be discovered role in tumor formation and progression. Therefore the aims of this study were to assess the role of collagenolytic E. faecalis on the migration and invasion of a murine colon cancer cell line. Results demonstrated that both migration and invasion were induced by E. faecalis with collagenolytic activity being required for only invasion. Bidirectional signaling in the E. faecalis-cancer cell interaction was observed by the discovering that the expression of gelE in E. faecalis, the gene required for collagenase production, is expressed in response to exposure to CT26 cells. The mechanism by which migration enhancement via E. faecalis occurs appears to be dependent on its ability to activate pro-uPA, a key element of the urokinase-plasminogen system, a pathway that is well – known to be important in cancer cell invasion and migration. Finally, we demonstrated that collagenase producing microbes preferentially colonize human colon cancer specimens.

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors.

Simple SummaryColorectal cancer (CRC) is one of the most common malignancies in the digestive system. We have previously shown that the proprotein convertase Furin is involved in calcium regulation in cancer cells. In this study, we revealed that the malignant phenotype of colon cancer stem cells is repressed by Furin inhibition that is associated with reduced expression of LGR5 and Nanog and dysregulated expression of several calcium regulators involved in colon cancer. Our data support the idea that targeting Furin in colorectal cancer stem cells may constitute a potential therapeutic approach.Proprotein convertases or PCs are known to regulate the malignant phenotype of colon cancer cells by different mechanisms, but their effects on cancer stem cells (CSCs) have been less widely investigated. Here, we report that PCs expression is altered in colon CSCs, and the inhibition of their activity reduced colon CSCs growth, survival, and invasion in three-dimensional spheroid cultures. In vivo, repression of PCs activity by the general PC inhibitors α1-PDX, Spn4A, or decanoyl-RVKR-chloromethylketone (CMK) significantly reduced tumor expression levels of the stem cell markers LGR5 and NANOG that are associated with reduced tumor xenografts. Further analysis revealed that reduced tumor growth mediated by specific silencing of the convertase Furin in KRAS or BRAF mutated-induced colon tumors was associated with reduced expression of LGR5 and NANOG compared to wild-type KRAS and BRAF tumors. Analysis of various calcium regulator molecules revealed that while the calcium-transporting ATPase 4 (ATP2B4) is downregulated in all the Furin-silenced colon cancer cells, the Ca2+-mobilizing P2Y receptors, was specifically repressed in BRAF mutated cells and ORAI1 and CACNA1H in KRAS mutated cells. Taken together, our findings indicate that PCs play an important role in the malignant phenotype of colon CSCs and stem cell markers’ expression and highlight PCs repression, particularly of Furin, to target colon tumors with KRAS or BRAF mutation.

GABAB receptor inhibits tumor progression and epithelial-mesenchymal transition via the regulation of Hippo/YAP1 pathway in colorectal cancer.

Gamma-Aminobutyric Acid Type B Receptor (GABABR) plays essential roles in tumor progression. However, the function of GABABR in colorectal cancer (CRC) needs further clarification. As the main part of GABABR, GABABR1 expression was identified significantly lower in tumor tissues than those in non-tumor normal tissues and that CRC patients with high GABABR1 expression lived longer. Further studies indicated that knockdown of GABABR1 elevated CRC cell proliferation, migration, and invasion. Furthermore, knockdown of GABABR1 activated the expression of the epithelial-mesenchymal transition (EMT)-related proteins N-cadherin and Vimentin, whereas decrease the protein level of E-cadherin. In addition, activation of Hippo/YAP1 signaling contributes to the GABABR1 down-regulation promoted proliferation, migration, invasion and EMT in CRC cells. At last, we verified the contribution of Hippo/YAP1 signaling in the GABABR1 down-regulation impaired biological phenotype of colon cancer cells in vivo. In summary, these data indicate that GABABR1 impairs the migration and invasion of CRC cells by inhibiting EMT and the Hippo/YAP1 pathway, suggesting that GABABR1 could be a potential therapeutic target for CRC.

Proprotein convertases blockage up-regulates specifically metallothioneins coding genes in human colon cancer stem cells

Despite continuous exertion made, colon cancer still represents a major health problem and its incidence continues being high worldwide. There is growing evidence in support of the cancer stem cells (CSCs) being central in the initiation of this cancer, and CSCs have been the focus of various studies for the identification of new ways of treatment. Lately, the proprotein convertases (PCs) were reported to regulate the maturation and expression of various molecules involved in the malignant phenotype of colon cancer cells, however, the identity of the molecules regulated by these serine proteases in CSCs is unknown. In this study, we used the general PCs inhibitor, the Decanoyl-RVKR-chloromethylketone (Decanoyl-RVKR-CMK) that inhibits all the PCs found in the secretory pathway, and analyzed its effect on CSCs using RNA-seq analysis. Remarkably, from the only 9 up-regulated genes in the human SW620-derived sphere-forming cells, we identified 7 of the 11 human metallothioneins, all of them localized on chromosome 16, and zinc related proteins as downstream effectors of the PCs. The importance of these molecules in the regulation of cell proliferation, differentiation and chemoresistance, and their reported potential tumor suppressor role and loss in colon cancer patients associated with worse prognosis, suggests that targeting PCs in the control of the malignant phenotype of CSCs is a new potential therapeutic strategy in colon cancer.

HOXA13 promotes colon cancer progression through β-catenin-dependent WNT pathway

Human class I homeobox A13 (HOXA13) was initially identified as a transcription factor and has an important role in embryonic development and malignant transformation. However, the clinical significance and the molecular mechanisms of HOXA13 in colon cancer development and progression are still unknown. In this study, we found that HOXA13 was highly expressed in colon cancer tissues, and its expression was associated with histological grade, T stage, N stage and tumour size. In vitro studies showed that HOXA13 promoted colon cancer cell proliferation, migration and invasion. Bioinformatics analysis revealed that HOXA13 expression was positively correlated with the WNT signalling pathway. In vitro studies showed that HOXA13 promoted the malignant phenotype of colon cancer cells by facilitating the nuclear translocation of β-Catenin. Moreover, XAV939, an inhibitor of β-Catenin, reversed the HOXA13-mediated effects on invasion and proliferation of colon cancer cells. In vivo studies further verified that HOXA13 promoted tumour formation through the Wnt/β-Catenin pathway. Collectively, these results suggest that HOXA13 is a potential oncogene that functions by promoting the nuclear translocation of β-Catenin, thereby maintaining the proliferation and metastasis of colon cancer.

Abstract 851: MicroRNA-124 modulated collagen-prolyl hydroxylase P4HA1 expression regulates colon cancer progression

Abstract In the US, colon adenocarcinoma is the third most common cause of cancer-related deaths. Since various genetic and epigenetic alterations contribute to progression of this disease, it is necessary to evaluate these molecular events as markers of progression and as potential druggable targets to develop therapeutics. Analysis of publicly available TCGA transcriptome sequencing data using UALCAN, a bioinformatics web portal (http://ualcan.path.uab.edu/), identified in colon adenocarcinoma, overexpression of collagen prolyl-4-hydroxylase α subunit 1 (P4HA1), an enzyme involved in collagen metabolism. P4HA1 changes the composition of the extra cellular matrix by hydroxylating proline residues in collagen. We validated this in silico finding by measuring P4HA1 RNA expression levels in paired normal (n=105) and tumor (n=105) frozen samples by qRT-PCR. Our investigations showed overexpression of P4HA1 in colon cancers compared to their paired normal colonic epithelial tissues. Expression of P4HA1 was independent of pathologic stage; tumor histologic type; and the race/ethnicity, age, and gender of patients. Furthermore, Western blotting and immunohistochemical analyses showed elevated expression of P4HA1 protein in colon tumor tissues as compared to normal colonic epithelial specimens. To assess the functional role of P4HA1 in colon cancer cells, we performed in vitro and in vivo analyses using RNA interference. The results showed that depletion of P4HA1 led to reduced cell growth and cell invasion, and to less tumor growth in xenograft models. Bioinformatics analysis indicated that microRNA miR-124, a tumor suppressor, could be targeting P4HA1. Forced overexpression of miR-124 in colon cancer cells reduced P4HA1 expression and diminished the malignant phenotypes of colon cancer cells, confirming that, in these cells, miR-124 targeted P4HA1. Further, treatment of the cells with diethyl-pythiDC (100 uM), a small molecule inhibitor of P4HA1, reduced their malignant phenotypes. In sum, the present investigations show that overexpression of P4HA1 is involved in progression of colon adenocarcinomas and that it is a potential therapeutic target that is amenable to small molecule inhibition. This work was partially supported by a NIH grant (3U54CA118948) and by the ELKUS foundation grant to Dr. Manne. Citation Format: Sumit Agarwal, Balabhadrapatruni V. Chakravarthi, Michael Behring, Hyung-Gyoon Kim, Kevin Hale, Abduljalil M. Alsubaie, Sameer Al Diffalha, Shajan P. Sugandha, Sooryanarayana Varambally, Upender Manne. MicroRNA-124 modulated collagen-prolyl hydroxylase P4HA1 expression regulates colon cancer progression [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 851.

Cathepsin B Is Upregulated and Mediates ECM Degradation in Colon Adenocarcinoma HT29 Cells Overexpressing Snail.

During tumor development and ongoing metastasis the acquisition of mesenchymal cell traits by epithelial carcinoma cells is achieved through a programmed phenotypic shift called the epithelial-to-mesenchymal transition, EMT. EMT contributes to increased cancer cell motility and invasiveness mainly through invadosomes, the adhesion structures that accompany the mesenchymal migration. The invadosomes and their associated proteases restrict protease activity to areas of the cell in direct contact with the ECM, thus precisely controlling cell invasion. Our data prove that Snail-overexpressing HT-29 cells that imitate the phenotype of colon cancer cells in the early stage of the EMT showed an increase in the expression and pericellular activity of cathepsin B. It appears that the pericellular localization of cathepsin B, also observed in colon and rectum adenocarcinoma tissue samples, plays a key role in its function.

RBP7 is a clinically prognostic biomarker and linked to tumor invasion and EMT in colon cancer.

RBP7 is a member of the cellular retinol-binding protein (CRBP) family and previous data suggested a link between CRBPs and the malignant transformation of colon cancer cells. Here, we investigated the potential of RBP7 as a predictive biomarker for patients with colon cancer and determined its functional relevance for tumor progression. We analyzed RBP7 protein and mRNA expression in independent tissue collections of colon cancers with recorded follow-up data, including data from TCGA. We used gene set enrichment analyses to characterize its functional role. Effects of RBP7 on migration and invasion were determined in transwell assays. High expression of RBP7 was an independent biomarker of poor cancer specific survival in early and late stage colon cancer, and linked to colon cancer progression. Gene set enrichment analysis revealed a strong association of RBP7, colon cancer invasion and epithelial mesenchymal transition (EMT). Ectopic expression of RBP7 increased migration and invasion of colon cancer cells. Our findings demonstrate that RBP7 is a strong prognostic biomarker in colon cancer that functionally contributes to the malignant phenotype of colon cancer cells. This may aid in risk stratification for the therapeutic management of patients with colorectal cancer.

Effects of Beclin 1 overexpression on aggressive phenotypes of colon cancer cells.

Beclin 1 is involved in autophagy, differentiation, apoptosis and cancer progression, and functions as a haploinsufficient tumor suppressor gene. The aim of the present study was to elucidate the function of Beclin 1 in colon cancer. A Beclin 1-expressing plasmid was transfected into HCT-15 and HCT-116 cells, and the phenotypes and associated molecules were determined. Beclin 1 transfectants were subcutaneously injected into nude mice to determine tumor growth, and proliferation and apoptosis levels using Ki-67 immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Beclin 1 overexpression inhibited viability as determined using a Cell Counting Kit-8 assay, inhibited migration and invasion as determined using a wound healing assay or Transwell assay, and lamellipodia formation by filamentous actin staining, induced autophagy as determined using electron microscopy, and light chain 3B (LC-3B) expression, and apoptosis as determined using Annexin V staining in the two cell lines (P<0.05). Beclin 1 induced G2 arrest of HCT-15 transfectants as determined using propidium iodide staining (P<0.05), whereas HCT-116 transfectants were arrested in G1 phase (P<0.05). The two transfectants exhibited increased expression of c-Myc, cyclin D1, β-catenin, insulin-response element 1 and 78 kDa glucose-regulated protein compared with the control and mock cells as determined using the reverse transcription-quantitative polymerase chain reaction (P<0.05). Beclin 1 overexpression upregulated LC-3B and cyclin-dependent kinase 4 expression, but downregulated cyclin E expression of the cancer cell lines as determined using western blot analysis (P<0.05). Beclin 1 expression in vivo significantly suppressed the proliferation of colon cancer cells in xenograft models via inducing apoptosis by TUNEL, and inhibiting proliferation by Ki-67 expression (P<0.05). Beclin 1 overexpression may reverse aggressive phenotypes and suppress colon cancer tumor growth, and be employed as a target molecule for gene therapy of patients with colon cancer.

PBX3 Is Part of an EMT Regulatory Network and Indicates Poor Outcome in Colorectal Cancer.

Purpose: Colorectal cancers are composed of phenotypically different tumor cell subpopulations within the same core genetic background. Here, we identify high expression of the TALE transcription factor PBX3 in tumor cells undergoing epithelial-mesenchymal transition (EMT), analyze PBX3 regulation, and determine clinical associations in colorectal cancer.Experimental design: We used transcriptomic and in situ analyses to identify PBX3 expression in colorectal cancer and cell biology approaches to determine its regulation and function. Clinical associations were analyzed in independent tissue collections and gene expression datasets of colorectal cancers with recorded follow-up data.Results: PBX3 was expressed in tumor cells with high WNT activity undergoing EMT at the leading tumor edge of colorectal cancers, whereas stromal cells were PBX3 negative. PBX3 expression was induced by WNT activation and by the EMT transcription factors SNAIL and ZEB1, whereas these effects were mediated indirectly through microRNA miR-200. PBX3 was required for a full EMT phenotype in colon cancer cells. On the protein level, PBX3 expression indicated poor cancer-specific and disease-free survival in a cohort of 244 UICC stage II colorectal cancers, and was associated with metastasis in a case-control collection consisting of 90 cases with or without distant metastasis. On the mRNA level, high PBX3 expression was strongly linked to poor disease-free survival.Conclusions: PBX3 is a novel indicator of EMT in colorectal cancer, part of an EMT regulatory network, and a promising prognostic predictor that may aid in therapeutic decision making for patients with colorectal cancer. Clin Cancer Res; 24(8); 1974-86. ©2018 AACR.

Alpha‐2c Adrenergic Receptor Promotes the Malignant Phenotype of Colon Cancer Cells

Cancer remains one of the leading causes of death worldwide, with colorectal cancer (CRC) being the third most common type. Accumulating evidence shows that stress plays a pivotal role in the malignant phenotype of CRC, by promoting hallmarks of CRC including cellular proliferation, migration, invasion, and angiogenesis. During stressful situations, the sympathetic nervous system is activated, causing an elevation in levels of catecholamines, including epinephrine which elicits its effects mainly by activating adrenoceptors. Interestingly, however, the role of alpha2C adrenergic receptor (a2c‐AR) in CRC malignancy remains obscure. Here, we proposed that activation of a2c‐AR potentiates the malignant phenotype of human CRC cells. Treatment of human colon cancer cell lines, SW480 and HT‐29, with increasing concentrations of a2‐AR specific agonist (UK 14,304; 10, 30 or 100 nM) for different time points (24, 48, or 72 hours) did not cause a significant change in proliferation. However, using monolayer scratch assay, a significant increase in migration was observed after treatment with UK 14,304 (100 nM) for 8 and 12 hours in both cell lines. This was further supported by a transwell migration assay where UK 14,304 increased cell migration. Pretreatment with a specific a2c‐AR antagonist (JP 1302; 100 nM) abolished the UK 14,304‐induced cell migration. This increase in migratory capacity was concomitant with increased Rho activation evident by translocation of Rho from cytoplasm to the membrane as well as via a Rhotekin‐binding activation assay. Moreover, treatment with UK 14,304 evoked actin reorganization, an element known to be a prerequisite for cell migration. The induced Rho activation and actin cytoskeletal rearrangement could be mediated by increased oxidative stress since treatment with UK 14,304 increased ROS production. Importantly, ROS is known to induce migration of these cells. Furthermore, adhesion of colon cancer cells to TNF‐alpha‐activated HUVECs was promoted by UK 14,304. This UK14,304‐induced adhesion was followed by a dramatic increase in transendothelial migration of SW480 through HUVECs, clearly indicating that a2c‐AR activation may promote intra‐ or extravasation. Because breaking the endothelial cell barrier requires matrix digestion, we measured the levels of matrix metalloproteinases (MMPs). Importantly, stimulation of a2c‐ARs by UK14,304 increased the production of MMP2 and MMP9, and JP 1302 abolished this UK14,304‐induced production. Taken together, our data suggests that a2c‐AR potentiates the migratory and invasive capacities and hence the malignant phenotype of human colon cancer cells. Therefore, a2c‐AR may represent a novel target in the treatment of CRC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.