Tumorigenesis Of Colorectal Cancer Cells Research Articles

BMP suppresses Wnt signaling via the Bcl11b-regulated NuRD complex to maintain intestinal stem cells.

Lgr5+ intestinal stem cells (ISCs) are crucial for the intestinal epithelium renewal and regeneration after injury. However, the mechanism underlying the interplay between Wnt and BMP signaling in this process is not fully understood. Here we report that Bcl11b, which is downregulated by BMP signaling, enhances Wnt signaling to maintain Lgr5+ ISCs and thus promotes the regeneration of the intestinal epithelium upon injury. Loss of Bcl11b function leads to a significant decrease of Lgr5+ ISCs in both intestinal crypts and cultured organoids. Mechanistically, BMP suppresses the expression of Bcl11b, which can positively regulate Wnt target genes by inhibiting the function of the Nucleosome Remodeling and Deacetylase (NuRD) complex and facilitating the β-catenin-TCF4 interaction. Bcl11b can also promote intestinal epithelium repair after injuries elicited by both irradiation and DSS-induced inflammation. Furthermore, Bcl11b deletion prevents proliferation and tumorigenesis of colorectal cancer cells. Together, our findings suggest that BMP suppresses Wnt signaling via Bcl11b regulation, thus balancing homeostasis and regeneration in the intestinal epithelium.

Dehydroepiandrosterone suppresses human colorectal cancer progression through ER stress-mediated autophagy and apoptosis in a p53-independent manner.

Colorectal cancer (CRC) is one of the primary contributors to cancer-related fatalities, with up to 80% of advanced CRC cases exhibiting mutations in the p53 gene. Unfortunately, the development of new compounds targeting mutant p53 is quite limited. The anticancer effects of Dehydroepiandrosterone (DHEA) on various cancers have been reported. However, the suppressive effect of DHEA on CRC cells harboring wild-type or mutant p53 gene remains controversial. This study emphasized revealing the suppressive mechanism and the effect of DHEA on CRC cell tumorigenesis in the presence of wild-type or mutant p53 gene. We demonstrate that DHEA causes CRC cell death and cell cycle arrest in a dose and time-dependent manner. Notably, DHEA exhibits similar inhibitory effects on CRC cells regardless of the p53 gene status. Further study reveals that DHEA induces endoplasmic reticulum (ER) stress and triggers PERK/eIF2/ATF4/CHOP UPR signaling pathway to activate autophagy followed by apoptosis, which was confirmed by suppression of 4-phenylbutyric acid (an ER stress inhibitor) or knockdown either ATF4 or CHOP. DHEA-induced apoptosis was attenuated by silencing ATG5 gene in either p53+/+ or p53-/- CRC cells, indicating autophagy regulation of apoptosis. Furthermore, DHEA treatment accompanied by bafilomycin A1 (a blocker of autophagosome degradation) leads to the accumulation of ATF4, CHOP, DR5, and p21 levels in CRC cells, implying that the degradative autophagy machinery regulates these four molecules. Consistently, DHEA demonstrates its inhibitory effect by suppressing CRC tumor formation in vivo. Altogether, we provide compelling evidence that DHEA is a potential therapeutic candidate for CRC patient treatment regardless of the p53 status through ER stress-PERK-autophagy-apoptosis axis.

TSPYL5 inhibits the tumorigenesis of colorectal cancer cells in vivo by triggering DNA damage.

Testis-specific protein Y-encoded-like 5 (TSPYL5) suppresses several cancers in vivo, including colorectal cancer (CRC); however, its mechanism and role in CRC cell tumorigenesis in vivo remain unknown. To elucidate the molecular mechanisms of colorectal cancer and find new therapeutic targets to improve CRC patient outcomes. Male mice (4 weeks old, 16-22 g) were housed in sterile cages in a temperature-controlled room (20-25°C) with a 12 h light/dark cycle and ad libitum food and water. TSPYL5 overexpressing or non-overexpressing HCT116 cells were used to create a nude mouse tumor model. Tumor tissue was evaluated histologically after hematoxylin and eosin (H and E) staining. TUNEL staining assessed tumor cell apoptosis. Ki67 expression in excised tumor tissue was measured by immunohistochemistry. Western blotting examined double-stranded break (DBS)-associated protein expression in vivo. IBM SPSS Statistics for Windows, Version 21.0 was used for all analyses (IBM Corp., Armonk, NY, USA). At least three independent experiments yield a mean value ± standard deviation. Unpaired Student's t-tests compared groups. One-way analysis of variance and Dunnett's test were used to compare groups with a P value < 0.5. TSPYL5 overexpression inhibited CRC cell tumorigenicity and damaged tumor cells in vivo. TSPYL5 overexpression also significantly increased Bax and p-H2AX (early double-stranded break indicators) and decreased Ki67, Bcl-2, and peroxisome proliferator-activated receptor expression. Collectively, TSPYL5 overexpression inhibited the tumorigenicity of CRC cells in vivo by inducing DNA damage.

Oridonin promotes endoplasmic reticulum stress via TP53-repressed TCF4 transactivation in colorectal cancer

BackgroundThe incidence of colorectal cancer and cancer death rate are increasing every year, and the affected population is becoming younger. Traditional Chinese medicine therapy has a unique effect in prolonging survival time and improving the prognosis of patients with colorectal cancer. Oridonin has been reported to have anti-cancer effects in a variety of tumors, but the exact mechanism remains to be investigated.MethodsCell Counting Kit-8 assay (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) staining assay, Tranwell, and Wound healing assays were performed to measure cell proliferation, invasion, and migration capacities, respectively. The protein and mRNA expression levels of various molecules were reflected by Western blot and Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR). Transcription Factor 4 (TCF4) and its target genes were analyzed by Position Weight Matrices (PWMs) software and the Gene Expression Omnibus (GEO) database. Immunofluorescence (IF) was performed to visualize the expression and position of Endoplasmic Reticulum (ER) stress biomarkers. The morphology of the ER was demonstrated by the ER tracker-red. Reactive Oxygen Species (ROS) levels were measured using a flow cytometer (FCM) or fluorescent staining. Calcium ion (Ca2+) concentration was quantified by Fluo-3 AM staining. Athymic nude mice were modeled with subcutaneous xenografts.ResultsOridonin inhibited the proliferation, invasion, and migration of colorectal cancer, and this effect was weakened in a concentration-dependent manner by ER stress inhibitors. In addition, oridonin-induced colorectal tumor cells showed increased expression of ER stress biomarkers, loose morphology of ER, increased vesicles, and irregular shape. TCF4 was identified as a regulator of ER stress by PWMs software and GEO survival analysis. In vitro and in vivo experiments confirmed that TCF4 inhibited ER stress, reduced ROS production, and maintained Ca2+ homeostasis. In addition, oridonin also activated TP53 and inhibited TCF4 transactivation, further exacerbating the elevated ROS levels and calcium ion release in tumor cells and inhibiting tumorigenesis in colorectal cancer cells in vivo.ConclusionsOridonin upregulated TP53, inhibited TCF4 transactivation, and induced ER stress dysregulation in tumor cells, promoting colorectal cancer cell death. Therefore, TCF4 may be one of the important nodes for tumor cells to regulate ER stress and maintain protein synthesis homeostasis. And the inhibition of the TP53/TCF4 axis plays a key role in the anti-cancer effects of oridonin.

The role of KNTC1 in the regulation of proliferation, migration and tumorigenesis in colorectal cancer

BackgroundCurrent findings have revealed that kinetochore-associated protein 1 (KNTC1) plays a pivotal role in the carcinogenesis of numerous types of cancer. This study was undertaken to inspect the role and probable underlying mechanisms of KNTC1 during the genesis and progression of colorectal cancer. MethodsImmunohistochemistry was implemented to determine KNTC1 expression levels in colorectal cancer tissues and para-carcinoma tissues. The association between KNTC1 expression profiles and several clinicopathological traits of colorectal cancer cases was examined employing Mann–Whitney U, Spearman, and Kaplan–Meier analyses. To track the proliferation, apoptosis, cell cycle, migration and in vivo carcinogenesis of colorectal cancer cells, KNTC1 was knocked down in colorectal cell line via RNA interference. To investigate the potential mechanism, the expression profile alterations of associated proteins were detected using human apoptosis antibody arrays, and verified by Western blot analysis. ResultsIn colorectal cancer tissues, KNTC1 was substantially expressed, and it was associated with the pathological grade as well as overall survival rate of the disease. The knockdown of KNTC1 was able to inhibit proliferation, cell cycle, migration and in vivo tumorigenesis of colorectal cancer cells, but promote apoptosis. ConclusionsKNTC1 is a key player in the emergence of colorectal cancer and may serve as an early diagnostic indicator of precancerous lesions.

Targeting Mcl-1 Degradation by Bergenin Inhibits Tumorigenesis of Colorectal Cancer Cells.

Myeloid leukemia 1 (Mcl-1) is frequently overexpressed in human malignancies and emerged as a promising drug target. In this study, we verified the inhibitory effect of bergenin on colorectal cancer cells both in vivo and in vitro. In an in vitro setting, bergenin significantly reduced the viability and colony formation and promoted apoptosis of CRC cells dose-dependently. Bergenin decreased the activity of Akt/GSK3β signaling and enhanced the interaction between FBW7 and Mcl-1, which eventually induced Mcl-1 ubiquitination and degradation. Using the HA-Ub K48R mutant, we demonstrated that bergenin promotes Mcl-1 K48-linked polyubiquitination and degradation. In vivo studies showed that bergenin significantly reduced tumor size and weight without toxicity to vital organs in mice. Overall, our results support the role of bergenin in inhibiting CRC cells via inducing Mcl-1 destruction, suggesting that targeting Mcl-1 ubiquitination could be an alternative strategy for antitumor therapy.

Chemotherapeutic Risk lncRNA-PVT1 SNP Sensitizes Metastatic Colorectal Cancer to FOLFOX Regimen.

Recent studies have identified that long noncoding RNA (lncRNA) might affect the responses to anticancer drug treatment, including colorectal cancer (CRC). However, the association between single-nucleotide polymorphisms (SNPs) in PVT1 and the chemotherapy response in metastatic colorectal cancer has yet to be clarified. In this study, the PVT1 rs2278176 CT/TT genotypes were found to be associated with an increased overall survival (OS) and progression-free survival (PFS) compared with the CC genotype. Furthermore, patients harboring the rs2278176 CT/TT genotypes had a greater chance of achieving clinical benefit from 5-Fluorouracil/leucovorin combined with oxaliplatin (FOLFOX). In vivo nude mice experiments demonstrated that the CRISPR/Cas9 mediated rs2278176 C to T mutation significantly inhibited the tumorigenesis of colorectal cancer cells treated with 5-Fu, but not control DMSO treated cells. Furthermore, the apoptotic rate was significantly enhanced by treatment with 5-Fu in the CRC cells carrying with the CT/TT genotypes. Functional studies demonstrated that the PVT1 rs2278176 C to T mutation altered the binding site for hsa-miR-297, and that hsa-miR-297 downregulated Glutathione S-Transferase Alpha 2(GSTA2), a member of phase II detoxification enzyme, in an Argonaute 2(Ago2)-dependent manner. Moreover, GSTA2 levels were downregulated in the cancer tissues of patients carrying rs2278176 CT/TT genotypes. High GSTA2 expression predicted poor clinical outcome in metastatic colorectal cancer treated with FOLFOX. In conclusion, this study provided that PVT1 with rs2278176 T allele altered the binding affinity with hsa-miR-297, leading to decreased GSTA2 expression and sensitized CRC cells to FOLFOX chemotherapy, suggesting rs2278176 CT/TT genotypes might serve as a predictive biomarker to improve prognosis in patients with metastatic CRC treated with FOLFOX.

MiR-26a enhances colorectal cancer cell growth by targeting RREB1 deacetylation to activate AKT-mediated glycolysis.

We previously reported the inhibitory effects of microRNA-26a (miR-26a) on the conversion of pyruvate to acetyl coenzyme A in glucose metabolism by directly targeting pyruvate dehydrogenase protein X component in colorectal cancer (CRC) cells (Chen B et al., BMC Cancer 2014). Here, using microRNA in situ hybridization, we confirmed that miR-26a levels were elevated in 77 human CRC tissue samples and further investigated the key miR-26a-mediated metabolic regulation elements and signaling pathways in CRC cells through quantitative proteomic dissection combined with cancer cell biology and biochemical loss-of-function analysis. We found that AKT transcription signaling was a target pathway via miR-26a-mediated deacetylation modification of Ras-responsive element-binding protein 1 (RREB1) at the Lys-60 residue. miR-26a improved the deacetylation level of RREB1, thus contributing to RREB1 binding to the AKT1 promoter to activate AKT transcription and its related signaling pathway in glycolysis. Moreover, miR-26a promoted CRC tumorigenesis in CRC cells and subcutaneous xenograft mice. Thus, miR-26a is a key regulator of CRC tumorigenesis that mediates the deacetylation modification of RREB1 to enhance AKT1 transcription and downstream target gene expression in glycolysis for CRC growth.

MicroRNA-124-3p suppresses PD-L1 expression and inhibits tumorigenesis of colorectal cancer cells via modulating STAT3 signaling.

Programmed death ligand 1 (PD-L1) plays a significant role in colorectal tumorigenesis through induction of regulatory T cells (Tregs) and suppression of antitumor immunity. Furthermore, microRNAs (miRNAs) as the posttranscriptional regulators of gene expression show considerable promise as a therapeutic target for colorectal cancer (CRC) treatment. Considering this, in vitro effects of miRNA-124 (miR-124-3p) on CRC cell tumorigenesis and Tregs differentiation via targeting PD-L1were investigated in the current study. Functional analysis showed that miR-124 is significantly downregulated in CRC tissues as compared with marginal normal samples (p < .0001), and its downregulation was negatively correlated with PD-L1 expression. Moreover, a specific region in PD-L1 3'-untranslated region was predicted as the miR-124 target and validated using the luciferase assay. Further investigation showed that transfection of HT29 and SW480 cells with miR-124 mimics significantly reduced PD-L1 mRNA, protein, and cell surface expression, and inhibited Tregs in coculture models via modulating interleukin[IL]-10, IL-2, tumor necrosis factor α, transforming growth factor beta, and interferon gamma expression levels. Besides, miR-124 overexpression decreased CRC cell proliferation and arrested cell cycle at the G1 phase through downregulation of c-Myc and induced apoptosis in CRC cells via upregulation of both intrinsic and extrinsic pathways. Also, miR-124 exogenous overexpression could reduce colony and spheroid formation ability of CRC cells via downregulating CD44 mRNA expression. miR-124 also diminished MMP-9 expression and subsequently suppressed cell migration and invasion. We also illustrated that STAT3 signaling was repressed by miR-124 in CRC cells. Taken together, our findings imply that considering the involvement of miR-124 in the regulation of PD-L1 through colorectal tumorigenesis and its remarkable antitumor effects, this miRNA could be regarded as the promising target for the development of therapeutic approaches for colorectal cancer.

LncRNA-PACER upregulates COX-2 and PGE2 through the NF-κB pathway to promote the proliferation and invasion of colorectal-cancer cells.

Backgroundp50-associated cyclooxygenase-2 extragenic RNA (PACER) is a recently identified antisense long non-coding RNA (lncRNA) located on the upstream of the promoter region of cyclooxygenase-2 (COX-2). Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells. However, the role of this lncRNA in colorectal cancer (CRC) remains elusive. Here, we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.MethodsReal-time quantitative PCR (RT-qPCR) analysis was used to evaluate the expression of PACER in CRC tissues and cells. Methyl thiazolyl tetrazolium (MTT) analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation. The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays, colony-formation assay, and transwell assays. We then used fluorescence in situ hybridization (FISH) to establish the subcellular localization of PACER. COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER. Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER. Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo. Enzyme-linked immunosorbent assay (ELISA) was then used to quantify prostaglandin E2 (PGE2) production upon knock-down of PACER.ResultsRT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells, and a high PACER-expression level was associated with poor prognosis. MTT assay, wound-healing assay, colony-formation assay, and transwell assay revealed that PACER enhanced CRC-cell proliferation, invasion, and metastasis in vitro. Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus. RT-qPCR showed that PACER increased mRNA levels of COX-2. Western blot analysis demonstrated, under normal circumstances, that knock-down of PACER decreased the COX-2 protein level. In the case of p50 absence, COX-2 protein increased rapidly and remained highly expressed after knocking down PACER. Luciferase assay revealed that PACER modulated the COX-2 promoter region. Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo. ELISA revealed that PACER knock-down inhibited PGE2 production.ConclusionsPACER modulates COX-2 expression through the nuclear factor kappa B (NF-κB) pathway in CRC. An increased level of PACER enhances proliferation, migration, and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.

RNA N6-Methyladenosine Methyltransferase METTL3 Facilitates Colorectal Cancer by Activating the m6A-GLUT1-mTORC1 Axis and Is a Therapeutic Target

RNA N6-methyladenosine (m6A) modification has recently emerged as a new regulatory mechanism in cancer progression. We aimed to explore the role of the m6A regulatory enzyme METTL3 in colorectal cancer (CRC) pathogenesis and its potential as a therapeutic target. The expression and clinical implication of METTL3 were investigated in multiple human CRC cohorts. The underlying mechanisms of METTL3 in CRC were investigated by integrative m6A sequencing, RNA sequencing, and ribosome profiling analyses. The efficacy of targeting METTL3 in CRC treatment was elucidated in CRC cell lines, patient-derived CRC organoids, and Mettl3-knockout mouse models. Using targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 dropout screening, we identified METTL3 as the top essential m6A regulatory enzyme in CRC. METTL3 was overexpressed in 62.2% (79/127) and 88.0% (44/50) of primary CRCs from 2 independent cohorts. High METTL3 expression predicted poor survival in patients with CRC (n= 374, P < .01). Functionally, silencing METTL3 suppressed tumorigenesis in CRC cells, human-derived primary CRC organoids, and Mettl3-knockout mouse models. We discovered the novel functional m6A methyltransferase domain of METTL3 in CRC cells by domain-focused CRISPR screening and mutagenesis assays. Mechanistically, METTL3 directly induced the m6A-GLUT1-mTORC1 axis as identified by integrated m6A sequencing, RNA sequencing, ribosome sequencing, and functional validation. METTL3 induced GLUT1 translation in an m6A-dependent manner, which subsequently promoted glucose uptake and lactate production, leading to the activation of mTORC1 signaling and CRC development. Furthermore, inhibition of mTORC1 potentiated the anticancer effect of METTL3 silencing in CRC patient-derived organoids and METTL3 transgenic mouse models. METTL3 promotes CRC by activating the m6A-GLUT1-mTORC1 axis. METTL3 is a promising therapeutic target for the treatment ofCRC.

Downregulation of long non-coding RNA MAFG-AS1 represses tumorigenesis of colorectal cancer cells through the microRNA-149-3p-dependent inhibition of HOXB8

BackgroundColorectal cancer (CRC) is considered as the second common death-induced cancer. More recently, association of long non-coding RNAs (lncRNAs) with CRC has been extensively investigated. Therefore, the present study was performed to determine whether lncRNA MAF BZIP Transcription Factor G Antisense RNA 1 (MAFG-AS1) could regulate biological activities of CRC cells and unravel the underlying mechanisms.MethodsCRC and corresponding adjacent tissues were collected to determine the expression of lncRNA MAFG-AS1, microRNA-149-3p (miR-149-3p) and homeobox B8 (HOXB8) by RT-qPCR. Dual luciferase reporter gene assay was used to explore the targeting relationship between miR-149-3p and lncRNA MAFG-AS1 and between miR-149-3p and HOXB8, followed by RNA immunoprecipitation for verification. Migration, proliferation, invasion, and apoptosis of HCT116 and LoVo cells were examined when lncRNA MAFG-AS1 was silenced or miR-149-3p was overexpressed. Furthermore, tumorigenicity of HCT116 and LoVo cells was measured in vivo by tumor xenograft in nude mice.ResultsLncRNA MAFG-AS1 and HOXB8 were found to be highly expressed in CRC tissues and cells, while miR-149-3p was under-expressed. LncRNA MAFG-AS1 negatively regulated miR-149-3p while miR-149-3p downregulated HOXB8. In addition, lncRNA MAFG-AS1 silencing by shRNA or miR-149-3p upregulation by mimic suppressed the migration, proliferation, invasion and tumorigenesis but promoted the apoptosis of HCT116 and LoVo cells.ConclusionTaken together, lncRNA MAFG-AS1 downregulation inhibits the malignant behaviors of CRC cells by upregulating miR-149-3p and downregulating HOXB8, providing a potential therapeutic target for CRC treatment.

PTPN18 promotes colorectal cancer progression by regulating the c-MYC-CDK4 axis

Protein tyrosine phosphatase non-receptor type 18 (PTPN18) is often highly expressed in colorectal cancer (CRC), but its role in this disease remains unclear. We demonstrated that PTPN18 overexpression promotes growth and tumorigenesis in CRC cells and that PTPN18 deficiency yields the opposite results in vitro. Moreover, a xenograft assay showed that PTPN18 deficiency significantly inhibited tumorigenesis in vivo. PTPN18 activated the MYC signaling pathway and enhanced CDK4 expression, which is tightly associated with the cell cycle and proliferation in cancer cells. Finally, we found that MYC interacted with PTPN18 and increased the protein level of MYC. In conclusion, our results suggest that PTPN18 promotes CRC development by stabilizing the MYC protein level, which in turn activates the MYC-CDK4 axis. Thus, PTPN18 could be a novel therapeutic target in the future.

Hypoxia-induced PGC-1α Regulates Mitochondrial Function and Tumorigenesis of Colorectal Cancer Cells.

Hypoxia promotes tumor proliferation and metastasis in colorectal cancer (CRC). Since the tumor microenvironment is generally characterized by hypoxia, its understanding is important for cancer therapy. We hypothesized that hypoxia promotes the mitochondrial function, mobility, and proliferation of CRC by up-regulating peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). To assess the effects of PGC-1α under hypoxia, we investigated the mitochondrial function, cell motility, and sphere formation as well as proliferation and apoptosis of CRC. Under hypoxia, we confirmed the increased expression of PGC-1α and reduced production of reactive oxygen species (ROS) by activating anti-oxidant enzymes. Also, up-regulation of PGC-1α enhanced the motility, sphere formation, and proliferation of CRC. Under the presence of the anti-cancer drug 5-fluorouracil (5FU), up-regulation of PGC-1α under hypoxia promoted resistance of CRC against 5FU-induced apoptosis. Targeting PGC-1α could to be a powerful strategy for CRC therapy.

Effect of Galectin-3 on biological behavior of colorectal cancer cells

Objective To investigate the effects of Galectin-3 on the proliferation, migration and tumorigenesis of colorectal cancer cells. Methods The stable cell line of Galectin-3 knockout (KO) was established by CRISPR-Cas9 technique in SW480 cells and the cells without knockout of Galectin-3 were used as control group. The expression level of Galectin-3 in control group and Galectin-3 KO group were analyzed by Western blotting. The proliferation ability in control group and Galectin-3 KO group were analyzed by Edu method. The migration ability in control group and Galectin-3 KO group were analyzed by scratch test and Transwell experiment. and was used to analyze The growth of tumors cell in control group and Galectin-3 KO group were analyzed by in vivo tumorigenesis experiment. Results Two single guide RNA (sgRNA) targeting knockout Galectin-3 proteins were selected and Galectin-3 KO1 and Galectin-3 KO2 were obtained respectively. Compared with the control group [(52.19±12.09)%], the positive rate of EDU in the Galectin-3 KO1 group and Galectin-3 KO2 groups [(11.35±9.27)% and (14.39±7.88)%] decreased significantly (t=3.912, P<0.05; t=3.209, P<0.05). Compared with the control group [(81.32±9.81)%], the percentage of healing distance of scratches in Galectin-3 KO1 group and Galectin-3 KO2 group [(33.28±8.77)% and (30.19±9.10)%] decreased significantly (t=5.197, P<0.05; t=5.273, P<0.05). Compared with the control group [(201.33±19.81) cells], the average number of cell migration per visual field in Galectin-3 KO1 group and Galectin-3 KO2 group [(109.4±12.87) cells and (125.10±15.91) cells] decreased significantly (t=9.255, P<0.05; t=10.364, P<0.05). The growth rate of cells in Galectin-3 KO1 group and Galectin-3 KO2 group in nude mice was significantly lower than that in control group (P<0.05). Conclusion Detection of Galectin-3 protein level is helpful to judge the proliferation and migration of colorectal cells. Key words: Galectin-3; Colorectal cancer; Proliferation; Migration; Tumor formation

HOTAIR/miR-326/FUT6 axis facilitates colorectal cancer progression through regulating fucosylation of CD44 via PI3K/AKT/mTOR pathway

Metastasis is the main cause of death in colorectal cancer (CRC) patients. Aberrant fucosylation, catalyzed by the specific fucosyltransferases (FUTs), is associated with malignant behaviors. Non-conding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), emerge as key molecules in cancer malignancy. The aim of this study was to investigate HOTAIR/miR-326/FUT6 axis modified fucosylation on sLeX-CD44 (HCELL), which served as E-selectin ligand during CRC progression. Higher levels of HOTAIR and FUT6 were verified in CRC tissues and cell lines, with a positive correlation. HOTAIR was associated with poor clinical prognosis of CRC. Altered HOTAIR levels influenced proliferation, aggressiveness, apoptosis and tumorigenesis of CRC cells. HOTAIR directly harbored miR-326 binding sites and regulated FUT6 expression. Further results corroborated that HOTAIR/miR-326/FUT6 axis modified α1, 3-fucosylation of CD44, which mediated CRC malignancy. Co-modulation of HOTAIR, miR-326 and FUT6 impacted α1, 3-fucosylated CD44, which further triggered PI3K/AKT/mTOR pathway. HOTAIR also mediated CRC tumorigenesis and liver metastasis in vivo. Thus, our findings indicated that HOTAIR/miR-326/FUT6 axis mediated CRC procession through α1, 3-fucosylated CD44 via PI3K/AKT/mTOR pathway. This work rendered new therapeutic targets for CRC.

ErbB4/KITENIN-Mediated Signaling is Activated in Cetuximab-Resistant Colorectal Cancer Cells.

ErbB4/KITENIN signaling plays a role in epidermal growth factor receptor (EGFR)-independent EGF pathways mediating the invasiveness and tumorigenesis of colorectal cancer cells. However, whether alterations in ErbB4/KITENIN signaling play a role in the resistance to anti-EGFR therapy remains unclear. Here, we established cetuximab-resistant DLD1 and HT29 cells, and analyzed changes in ErbB4/KITENIN signaling. c-Jun, a final effector in ErbB4/KITENIN-mediated signaling, was upregulated, whereas KITENIN levels remained constant in both cetuximab-resistant cell lines. The phosphorylation of EGFR and ErbB4 was increased in cetuximab-resistant cells, suggesting that ErbB4/KITENIN signaling contributed to the acquisition of cetuximab resistance in the cells. Silencing of KITENIN and/or ErbB4 increased cetuximab sensitivity in cetuximab-resistant cells. This study is the first to report the activation of ErbB4/KITENIN-mediated signaling in cetuximab-resistant colorectal cancer cells and the potential clinical application of ErbB4/KITENIN-targeting therapy for overcoming anti-EGFR resistance.

O-GlcNAcylation of YY1 stimulates tumorigenesis in colorectal cancer cells by targeting SLC22A15 and AANAT.

Emerging studies have revealed that O-GlcNAcylation plays pivotal roles in the tumorigenesis of colorectal cancers. However, the underlying mechanism still remains largely unknown. Here, we demonstrated that YY1 was O-GlcNAcylated by OGT and O-GlcNAcylation of YY1 could increase the protein expression by enhancing its stability. O-GlcNAcylation facilitated transformative phenotypes of CRC cell in a YY1-dependent manner. Also, O-GlcNAcylation stimulates YY1-dependent transcriptional activity. Besides, we also identified the oncoproteins, SLC22A15 and AANAT, which were regulated by YY1 directly, are responsible for the YY1 stimulated tumorigenesis. Furthermore, we identified the main putative O-GlcNAc site of YY1 at Thr236, and mutating of this site decreased the pro-tumorigenic capacities of YY1. We concluded that O-GlcNAcylation of YY1 stimulates tumorigenesis in CRC cells by targeting SLC22A15 and AANAT, suggesting that YY1 O-GlcNAcylation might be a potential effective therapeutic target for treating CRC.

Abstract LB-090: Activation of KITENIN/ErbB4-mediated signaling contributes to acquisition of cetuximab resistance in colorectal cancer cells

Abstract ErbB4/KITENIN signaling plays a role in epidermal growth factor receptor (EGFR)-independent EGF pathways mediating the invasiveness and tumorigenesis of colorectal cancer cells. However, whether alterations in ErbB4/KITENIN signaling play a role in the resistance to anti-EGFR therapy remains unclear. Here, we established cetuximab-resistant DLD1 and HT29 cells, and analyzed changes in ErbB4/KITENIN signaling. c-Jun, a final effector in ErbB4/KITENIN-mediated signaling, was upregulated, whereas KITENIN levels remained constant in both cetuximab-resistant cell lines. The phosphorylation of EGFR and ErbB4 was increased in cetuximab-resistant cells, suggesting that ErbB4/KITENIN signaling contributed to the acquisition of cetuximab resistance in the cells. Silencing of KITENIN and/or ErbB4 increased cetuximab sensitivity in cetuximab-resistant cells. This study is the first to report the activation of ErbB4/KITENIN-mediated signaling in cetuximab-resistant colorectal cancer cells and the potential clinical application of ErbB4/KITENIN-targeting therapy for overcoming anti-EGFR resistance. Citation Format: So-Yeon Park, Yi Yang, Rui Zhou, Woo Kyun Bae, Ik Joo Chung, Kyung Keun Kim, Hangun Kim. Activation of KITENIN/ErbB4-mediated signaling contributes to acquisition of cetuximab resistance in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-090.

Role of TXNDC5 in tumorigenesis of colorectal cancer cells: In vivo and in vitro evidence.

Thioredoxin domain-containing 5 (TXNDC5) is reportedly overexpressed in colorectal cancer (CRC) and is therefore considered an oncogene. However, the role of TXNDC5 in CRC tumorigenesis remains unclear. The present study aimed to explore the role of TXNDC5 in CRC tumorigenesis in vitro and in vivo under hypoxic and normoxic conditions. Analyses of patient tissue samples revealed a positive association between the expression of hypoxia-inducible factor-1α (HIF-1α) or TXNDC5 and the TNM stage of CRC. In addition, a positive correlation between the expression levels of HIF-1α and TXNDC5 was observed in CRC tissues. Furthermore, culturing RKO and HCT-116 human CRC cell lines under hypoxic conditions significantly increased the expression levels of HIF-1α and TXNDC5, whereas knockdown of HIF-1α abolished the hypoxia-induced expression of TXNDC5. Knockdown of TXNDC5 significantly decreased cell proliferation and colony formation, and increased apoptosis of both cell lines. Furthermore, knockdown of TXNDC5 markedly increased hypoxia-induced reactive oxygen species (ROS) generation, and the expression of hypoxia-induced endoplasmic reticulum stress (ER) markers (CCAAT-enhancer-binding protein homologous protein, glucose-regulated protein 78 and activating transcription factor 4) and apoptotic markers (B-cell lymphoma 2-associated X protein and cleaved caspase-8). In addition, the expression levels of TXNDC5 were significantly increased in tumor tissues compared with in adenoma and normal tissues in a mouse model of CRC tumorigenesis. In conclusion, the in vivo data demonstrated that TXNDC5 is overexpressed in CRC tissues, and this overexpression may be associated with unfavorable clinicopathological features. The in vitro data indicated that hypoxia may induce TXNDC5 expression via upregulating HIF-1α; this effect promoted CRC cell proliferation and survival under hypoxic conditions, likely via inhibiting hypoxia-induced ROS/ER stress signaling. These findings suggested that TXNDC5 functions as an important stress survival factor to maintain tumorigenesis of CRC cells under hypoxia by regulating hypoxia-induced ROS/ER stress signaling. The present study provided novel insights into the role of TXNDC5 in the tumorigenesis of CRC.