DNA methylation-regulated DDX27 promotes colorectal cancer progression through EZH2.

Anaplastic lymphoma kinase (ALK) has been described in a range of human cancers and is involved in cancer initiation and progression via activating multiple signaling pathways, such as the PI3K-AKT, CRKL-C3G, MEKK2/3-MEK5-ERK5, JAK-STAT and MAPK signal pathways. Recently ALK and LTK ligand 1 (ALKAL1) also named “augmentor-β” or “FAM150A” is identified as a potent activating ligands for human ALK that bind to the extracellular domain of ALK. However, due to its poor stability, the mechanisms of ALKAL1 underlying the tumor progression in the human cancers including colorectal cancer have not been well documented. Herein, ALKAL1 expression was evaluated by RNA sequencing datasets from The Cancer Genome Atlas (TCGA) of 625 cases colorectal cancer, immunohistochemical analysis of 377 cases colorectal cancer tissues, and Western blotting even Real-time PCR of 10 pairs of colorectal cancer tissues and adjacent normal tissues, as well as 8 colorectal cancer cell lines. Statistical analysis was performed to explore the correlation between ALKAL1 expression and clinicopathological features in colorectal cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between ALKAL1 expression and overall survival. In vitro and in vivo assays were performed to assess the biological roles of ALKAL1 in colorectal cancer. Gene set enrichment analysis (GSEA), Western blotting and luciferase assays were used to identify the underlying signal pathway involved in the tumor progression role of ALKAL1. As a result, we showed that ALKAL1 was upregulated in colorectal cancer tissues and cell lines. Upregulation of ALKAL1 correlated with tumor malignancy and poor prognosis in colorectal cancer. ALKAL1 silencing inhibited tumorigenesis, metastasis and invasion of colorectal cancer cells, and inhibited SHH signaling pathway, which is essential for ALKAL1 induced migration. Our findings reveal a new mechanism by which ALKAL1 participates in colorectal cancer migration and invasion via activating the SHH signaling pathway.

The Effect of Statins in Colorectal Cancer Is Mediated Through the Bone Morphogenetic Protein Pathway

Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the molecular mechanisms that link circRNAs with colorectal cancer (CRC) are not well understood. In the present study, we attempted to provide novel basis for targeted therapy for CRC from the aspect of circRNA-microRNA (miRNA)-mRNA interaction. We investigated the expression of circRNAs in five paired CRC tissues and adjacent non-tumor tissues by microarray analysis. Differentially expressed circRNAs were identified between CRC tissues and non-cancerous matched tissues. We focused on hsa_circ_0005100, which is located on chromosome 1 and derived from FMN2, and thus we named it as circFMN2. The expression of circFMN2 was detected in 88 CRC tissues and cell lines by quantitative real-time PCR. Functional assays were performed to evaluate the effects of circFMN2 on proliferationin vitro, and on tumorigenesisin vivo. The relationship between circFMN2 and miR-1182 was confirmed by luciferase reporter assay. circFMN2 was found to be significantly up-regulated in CRC tissues and cell lines. Moreover, knockdown of circFMN2 significantly inhibited cell proliferation and migrationin vitro. Bioinformatics analysis predicted that there is a circFMN2/miR-1182/hTERT axis in CRC progression. Dual-luciferase reporter system validated the direct interaction of circFMN2, miR-1182, hTERT. Western blot verified that inhibition of circFMN2 decreased hTERT expression. Importantly, we demonstrated that circFMN2 was up-regulated in serum exosomes from CRC patients. In conclusion, circFMN2 is a central component linking circRNAs to progression of CRC via an miR-1182/hTERT axis.

Objective To study the clinicopathological significance and biological behavior of human colorectal cancer (CRC) with the expression of angiopoietin-like protein 1 (ANGPTL1). Methods The expression of ANGPTL1 protein in 98 paraffin embedded CRC specimens and paired adjacent non-cancerous tissues were detected by immunohistochemistry. The relationship between the protein expression and clinicopathological features was analyzed. Western blot and qRT-PCR were used to examine the expression of ANGPTL1 protein and mRNA levels in 24 paired fresh CRC and adjuvant non-cancerous tissues. Finally, ANGPTL1 expression in CRC cell lines with different metastatic capabilities was detected by WB. Cell invasion and migration were detected using ANGPTL1siRNA. Results ANGPTL1 expression in CRC tissues was much lower than that in non-cancerous tissues (44% vs. 76%, P<0.01). ANGPTL1 expression was negatively associated with T stage (χ2=5.766, P=0.016), lymph node metastasis (χ2=5.571, P=0.018) and TNM stage (χ2=7.773, P=0.005). Moreover, CRC patients with ANGPTL1 positive expression had a better prognosis. ANGPTL1 protein and mRNA levels in CRC tissues were higher than in non-cancerous tissues (t=3.126, P=0.005; t=2.523, P=0.019). ANGPTL1 interference promoted cell invasion and migration of HCT116 cells. Conclusion ANGPTL1 expression is closely associated with tumor T stage, lymph node metastasis, TNM stage and worse prognosis of CRC patients. ANGPTL1 inhibits cell invasion and migration of CRC cells. Key words: Colorectal neoplasms; Angiopoietin 1; Neoplasm invasioness; Prognosis

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

Background Clinical trials have shown that cancers originating from different tissues driven by the same oncogene respond differently to targeted anticancer drugs. We aimed to understand different signaling patterns in KRAS mutant cells derived from non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic cancer. Materials and methods We optimized a 50 phosphoprotein antibody-based assay on the Luminex 200 platform. We then exposed a panel of 15 KRAS mutant cell lines (5 cell lines each originating in the lung, pancreas and colon) to a DMSO control (n = 3) and clinically significant concentrations (Cmax achieved in humans adjusted for protein binding in culture medium) of a PI3K (GDC-0941), AKT (AZD5363), m-TOR (everolimus), BRAF (vemurafenib), EGFR (gefitinib), MEK (trametinib) and an HSP90 inhibitor (luminespib) for 1 hr. We quantified the change in phosphorylation of proteins for each drug compared to control. Logistic regression analysis was used to analyse differences between KRAS-driven cell lines originating from different anatomical sites. Results There were changes in phosphorylation related to the pharmacodynamic effects of the drug independent of cell line of origin; however, there were interesting differences between KRAS mutant cells originating from different anatomical sites. In NSCLC cell lines, p-EGFR levels changed significantly less when exposed to PI3K, AKT and m-TOR inhibitors (p = 0.047, 0.022 and 0.047, respectively) when compared to cells originating from CRC and pancreatic cancer. CRC cell lines, when compared to NSCLC and pancreatic cancer cell lines, showed significantly less changes in phosphorylation of key cell cycle regulators such as CHK1 when exposed to PI3K, AKT and m-TOR inhibitors, (p = 0.001, 0.047 and 0.047, respectively) and RB when exposed to an AKT and m-TOR inhibitor (p = 0.047 and 0.047, respectively). Interestingly, pancreatic cell lines showed significantly more changes in p-m-TOR compared to CRC and NSCLC cell lines following exposure to PI3K and AKT inhibitors (p = 0.0095 and 0.022, respectively). Of note, drugs not directly targeting the PI3K pathway differentially regulated different nodes in the PI3K pathway, for example, BRAF inhibitors significantly differentially changed levels of phosphorylation at different nodes in the PI3K pathway such as AKT in NSCLC cell lines, p = 0.047, p70S6K in CRC cell lines, p = 0.0472 and PRAS40 in the pancreatic cancer cell lines, p = 0.022. Conclusion These results suggest that there are significant differences in signaling patterns caused by PI3K pathway inhibitors in KRAS mutant NSCLC, CRC and pancreatic cancer cell lines. Our findings shed light on the putative use of PI3K pathway inhibitors in KRAS mutant cancers. They also question the universal application of solely using genetic mutations to stratify patients in ‘basket’ clinical studies. Citation Format: Adam Stewart, Elizabeth Coker, Anna Minchom, Parames Thavasu, Alexandros Georgiou, Anguraj Sadanandam, Timothy A. Yap, Johann S. de Bono, Bissan Al-Lazikani, Udai Banerji. KRAS and clinical context: Differential dynamic signaling output of KRAS mutant lung, colorectal and pancreatic cancer cell lines when exposed to targeted anticancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3099.

The aim of this study was to explore the role of microRNA-802 (miRNA-802) in the progression of colorectal cancer (CRC) and the underlying mechanism. The relative expression levels of miRNA-802 and FOXE1 in 40 paired CRC tissues and adjacent normal tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between miRNA-802 expression and the pathological indexes of CRC patients was assessed. Meanwhile, the prognostic potentials of miRNA-802 and FOXE1 in CRC patients were identified through the Kaplan-Meier method. After overexpression of miRNA-802, the changes in the proliferative, migratory, and invasive capacities of HT29 and HCT-8 cells were evaluated in vitro. The Dual-Luciferase Reporter Gene Assay was applied to investigate the binding relationship between miRNA-802 and FOXE1. Finally, the rescue experiments were carried out to uncover the role of the miRNA-802/FOXE1 axis in regulating the cellular behaviors of CRC. MiRNA-802 was significantly downregulated in CRC tissues and cell lines. CRC patients with a low level of miRNA-802 had significantly higher rates of lymphatic metastasis and distant metastasis, as well as worse overall survival. The transfection of miRNA-802 mimics remarkably attenuated the proliferation, migration, and invasion of HT29 and HCT-8 cells. FOXE1 expression was significantly upregulated in CRC tissues and cell lines. Meanwhile, the expression of FOXE1 was negatively correlated with miRNA-802 in CRC tissues. A higher level of FOXE1 indicated the worse prognosis of CRC patients. The Dual-Luciferase Reporter Gene Assay further verified the binding relationship between FOXE1 and miRNA-802. Importantly, the overexpression of FOXE1 could reverse the regulatory effects of miRNA-802 on the cellular behaviors of CRC. MiRNA-802 is significantly downregulated in CRC, and is closely related to lymphatic and distant metastasis of CRC. Furthermore, miRNA-802 alleviates the malignant progression of CRC via negatively regulating FOXE1.

Upregulation of long noncoding RNA ZFAS1 predicts poor prognosis and prompts invasion and metastasis in colorectal cancer

Retinoic acid receptor-related orphan receptor α (RORA) is a tumor-specific differentially methylated region. RORA mRNA expression is frequently downregulated in colorectal cancer (CRC) due to promoter methylation, and this methylation is correlated with the development of CRC. Here we investigated the correlation between the methylation status of the RORA promoter region and clinical CRC stages. The methylation status of RORA isoform 1 (RORA1) and isoform 4 (RORA4) promoters was investigated in 43 paired CRC specimens and adjacent normal tissues by quantitative DNA methylation analysis using the Sequenom MassARRAY system and bisulfite sequencing. The relationship between the methylation status of the RORA1 promoter and the CRC pathological stage was analyzed. RORA1 expression was evaluated using quantitative PCR. Sixteen of 43 CRC specimens (37%) and three CRC cell lines (Caco2, HT29, and HCT116) showed increased levels of methylation in the RORA1 promoter region compared with adjacent normal tissues, whereas no methylation was observed in the RORA4 promoter. Quantitative PCR showed downregulation of RORA1 expression both in CRC samples and cell lines. Furthermore, the RORA1 promoter hypomethylation status showed a significant correlation with unfavorable CRC stages (stages III and IV) compared with favorable stages (stages I and II, p = 0.014). Hypomethylation of the RORA1 promoter may have important clinical implications in unfavorable CRC development, and therefore, the methylation status of the RORA1 promoter may constitute a useful biomarker to determine an indication for postoperative therapy such as adjuvant chemotherapy in highly advanced CRC patients.

The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl-CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation-specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.

Colorectal cancer (CRC) is one of the most aggressive malignancies of the digestive tract, characterized by aberrant post-transcriptional RNA modifications, including pseudouridine (Ψ). TruB pseudouridine synthase family member 1 (TRUB1) is a key pseudouridine synthase but its role in CRC progression remains unclear. Public databases and CRC cell lines were analysed to assess TRUB1 expression in CRC. Receiver-operating characteristic (ROC) curve analysis and survival analysis were performed to evaluate the diagnostic and prognostic significance of TRUB1. The impact of TRUB1 on tumor proliferation and Ψ modification was examined in TRUB1-knock-down HCT116 cell lines. Mechanistically, RNA sequencing of control and TRUB1-knock-down HCT116 cells was conducted to identify potential pathways, which were validated by using real-time polymerase chain reaction (PCR), Western blot, and immunofluorescence assays. TRUB1 was significantly upregulated in CRC tumor tissues and cell lines. ROC analysis showed that TRUB1 had strong diagnostic potential and its overexpression was associated with poorer overall survival in CRC patients. In TRUB1-knock-down HCT116 cells, apoptosis increased and tumor growth slowed in nude mice, with a corresponding increase in apoptosis-related proteins and decreased Ψ modification. Mechanistically, RNA sequencing indicated that tumor necrosis factor α signaling via the nuclear factor kappa B (NFκB) pathway was activated in TRUB1-knock-down HCT116 cells. Further analysis identified Baculoviral inhibitor of apoptosis proteins repeat-containing 3 (BIRC3) as a potential downstream target gene that was regulated by TRUB1 in the NFκB pathway. TRUB1 serves as a potential biomarker for CRC diagnosis and prognosis, and it can inhibit apoptosis in CRC cells via BIRC3-mediated NFκB signaling.

This study aims to investigate the function of long non-coding RNA ZEB1-AS1, reveal its molecular mechanism in colorectal cancer cell growth, and evaluate its clinical significance in colorectal cancer patients. ZEB1-AS1 has reported in the development of several cancers, but the biological role of it in colorectal cancer has not been discussed. In this report, ZEB1-AS1 expression level was measured with quantitative real-time polymerase chain reaction in 63 pairs of colorectal cancer tissues and paired adjacent non-tumor colorectal tissues. The relationship between ZEB1-AS1 expression and overall survival was analyzed by virtue of Kaplan-Meier analysis. Subsequently, small interfering RNA or lentivirus vector-mediated lncRNA ZEB1-AS1 was transfected into colorectal cancer cell lines. Cell viability and apoptosis were examined. Later, nude mouse transplantation experiment was conducted to evaluate the effect of ZEB1-AS1 on colorectal cancer development in vivo. It turns out that ZEB1-AS1 is upregulated in colorectal cancer tissues and its expression is significantly associated with overall survival rate and recurrence-free survival. Upregulation of ZEB1-AS1 colorectal cancer promotes cell proliferation and inhibits cell apoptosis. In addition, cell cycle inhibitory protein p15 participates in the oncogenic function of ZEB1-AS1. Collectively, ZEB1-AS1 has asignificant effect on colorectal cancer pathological process and serves as a valuable prognostic biomarker for colorectal cancer.

Inactivation of the UNC5C Netrin-1 Receptor Is Associated With Tumor Progression in Colorectal Malignancies

To clarify the promotive effect of LINC00346 on the malignant progression of colorectal cancer (CRC) by mediating miRNA-101-5p/MMP9 axis. Expression pattern of LINC00346 in 46 paired CRC tissues and adjacent normal tissues was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Correlation between LINC00346 level and prognosis of CRC patients was analyzed, and the LINC00346 level in CRC cell lines was examined as well. Subsequently, potential influences of LINC00346 on cellular behaviors of CRC cells were evaluated through cell counting kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Finally, Dual-Luciferase reporter gene assay was conducted to verify the binding relationship between LINC00346 and miRNA-101-5p/MMP9. LINC00346 was upregulated in CRC tissues and cell lines. Compared with CRC patients with low level of LINC00346, those with high level suffered a poorer prognosis, and higher metastatic rates (lymph node metastasis and distant metastasis). Transfection of sh-LINC00346 attenuated proliferative, migratory, and invasive abilities of CRC cells. In addition, LINC00346 was confirmed to bind to miRNA-101-5p, and the latter was binding to MMP9. Moreover, the overexpression of miRNA-101-5p decreased colony number, viability, and numbers of migratory and invasive cells. LINC00346 is upregulated in CRC and correlated with metastasis and poor prognosis of CRC. LINC00346 accelerates the malignant progression of CRC via targeting miRNA-101-5p/MMP9.

Loss of the Ras Regulator RASAL1: Another Route to Ras Activation in Colorectal Cancer