Association between MLK4 expression and colon cancer patient survival

Background: Mixed Lineage Kinase 4 (MLK4) is a protein kinase from the serine/threonine kinase family functioning through the MAPK pathway. The association of MLK4 with colon cancer oncologic outcomes has not been well described. We aimed to investigate the molecular expression of MLK4 at both the protein and mRNA levels, its differential expression across stages, and the association with survival in cases of increased expression using the TCGA databases. Methods: An in-silico analysis of MLK4 in colorectal cancers was performed using the TCGA database for stage-wise expression and overall survival analysis in 288 patients. A TMA of colon cancer and matched adjacent normal colon tissue, annotated with survival data and clinical stage (AJCC 8th edition), was used to investigate differential expressions of MLK4 in colon cancer. Laser Capture microdissection was performed with FFPE samples consisting of normal tissue and tumor. MLK4 RNA was quantified using organic extraction and RT-PCR techniques. Results: IHC of MLK4 in 74 matched sample pairs revealed a significant (p < 0.05) increase in MLK4 expression (mean difference = 0.405, 95% CI = 0.2028 to 0.6080) in colon cancer compared to normal tissue. The correlation coefficient (r = 0.4721, p < 0.0001) suggested a moderate correlation between MLK4 levels in normal and tumor tissues. RT-PCR analysis in colon cancer specimens revealed a significant (p<0.05) increase in mRNA levels of MLK4 (mean difference = 2.139, 95% CI = 0.8789 to 3.400). High MLK4 expression showed a trend toward reduced overall survival compared with low expression (HR = 1.25, log-rank p = 0.079). Similarly, higher MLK4 methylation demonstrated a trend toward poorer overall survival in colon adenocarcinoma patients (HR = 1.54, 95% CI 0.96-2.47; log-rank p = 0.07). Conclusion: Increased expression of MLK4, both transcriptionally and translationally, in colon cancer suggests involvement in colon tumor pathology. Cellular-level expressions and correlations of MLK4 kinase with survival can help elucidate the complex nature of colon cancer development and identify unknown prognostic markers.