Introduction: Colorectal cancer (CRC) arises from the cumulative effects of genetic and epigenetic alterations. Cur- rent treatment of metastatic CRC relies on combination of chemotherapy and targeted therapies such as anti-EGFR therapies. The success of targeted therapies relies on the detection of actionable targets and predictive biomarkers of resistance. The study aims to determine mutations in common actionable targets and predictive biomarkers of re- sistance to anti-EGFR therapies in Malaysian CRC patients. Methods: Mutations in 10 CRC tissues were determined by next-generation sequencing with a panel of 7 cancer-related genes covering all exons in KRAS, BRAF, PIK3CA, PTEN, TP53, NRAS, and EGFR genes. Immunohistochemistry was used to determine mismatch repair (MMR) status. Results: Of the ten samples, 5 and 4 samples harboured two and one mutation, respectively and one had no mu- tation. All were missense mutations and were in five genes, namely, KRAS, PIK3CA, TP53, BRAF, and EGFR. They were, G12D, G12V, G12A, G13D, and V14I in KRAS, E545K, K733R, and D1056N in PIK3CA, G199V, D259Y, and R282W in TP53, V600E in BRAF and G696R in EGFR. Deficient mismatch repair (dMMR) was detected in three samples, of which two had KRAS mutation. Conclusion: Mutations in KRAS codon 12 and 13, BRAF and PIK3CA which predict resistance to anti-EGFR therapies and three TP53 mutations were found. This is the first report of EGFR mutation in Malaysian CRC patients. It is predicted to be a pathogenic variant. dMMR, one of the biomarkers for treatment with immune checkpoint inhibitor was also detected.
The objective of this study was to determine the effect of miR‑29a‑3p inhibitor on the migration and invasion of colorectal cancer cell lines (CRC) and the underlying molecular mechanisms. miR‑29a‑3p was detected using reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) in the CRC cell lines HCT11, CaCo2, HT29, SW480 and SW620. An invasive subpopulation designated SW480‑7 was derived from the parental cell line, detected by Transwell and Transwell Matrigel assays. Cytoskeleton Regulators RT2 profiler PCR array and western blot analysis were utilized to identify the alterations in expression of downstream mRNAs. siRNA against CDC42BPA was transfected into SW480‑7 and effects on cell migration and invasion were investigated. Data obtained showed that miR‑29a‑3p was detected in these five CRC cell lines. miR‑29a‑3p inhibitor had no effect on viability but stimulated cell migration and invasion of SW480‑7 cells. In contrast, miR‑29a‑3p mimic suppressed cell migration and invasion. TargetScan miRBD and DIANA were employed to identify the potential direct target genes of miR‑29a‑3p in the Cytoskeleton Regulators RT2-Profiler PCR array. Cytoskeleton Regulators RT2-Profiler PCR array data showed that 3 out of the 5 predicted targets genes, CDC42BPA (2.33-fold), BAIAP2 (1.79-fold) and TIAM1 (1.77-fold), in the array were upregulated by miR‑29a‑3p. A significant increase in expression IQGAP2, PHLDB2, SSH1 mRNAs and downregulation of PAK1 mRNA was also detected with miR‑29a‑3p inhibition. Increase in CDC42BPA, SSH1 and IQGAP2 mRNA expression correlated with increased protein level in miR‑29a‑3p transfected SW-480-7 cells. Silencing of CDC42BPA (an enhancer of cell motility) partially abolished miR‑29a‑3p inhibitor-induced stimulation of cell migration and invasion. miR‑29a‑3p expression in stage II and III CRC is relatively lower than that of stage I CRC. However, the data need to be interpreted with caution due to the small sample size. In conclusion, inhibition of miR‑29a‑3p stimulates SW480‑7 cell migration and invasion and downstream expression IQGAP2, PHLDB2, SSH1 mRNAs are upregulated whilst PAK1 mRNA is downregulated. Silencing of CDC42BPA expression partially reduces miR29a‑3p inhibitor-induced migration and invasion of SW480‑7 cells.