Accumulating evidence suggests that NKX6.1 (NK homeobox 1) plays a role in various types of cancer. In our previous studies, we identified NKX6.1 hypermethylation as a promising marker and demonstrated that the NKX6.1 gene functions as a metastasis suppressor through the epigenetic regulation of the epithelial-to-mesenchymal transition (EMT) in cervical cancer. More recently, we have demonstrated that NKX6.1 methylation is related to the chemotherapy response in colorectal cancer (CRC). Nevertheless, the biological function of NKX6.1 in the tumorigenesis of CRC remains unclear. In this study, we showed that NKX6.1 suppresses tumorigenic and metastatic ability both in vitro and in vivo. NKX6.1 represses cell invasion partly through the modulation of EMT. The overexpression of NKX6.1 enhances chemosensitivity in CRC cells. To further explore how NKX6.1 exerts its tumor-suppressive function, we used RNA sequencing technology for comprehensive analysis. The results showed that differentially expressed genes (DEGs) were mainly related to cell migration, response to drug, transcription factor activity, and growth factor activity, suggesting that these DEGs are involved in the function of NKX6.1 suppressing cancer invasion and metastasis. Our results demonstrated that NKX6.1 functions as a tumor suppressor partly by repressing EMT and enhancing chemosensitivity in CRC, making it a potential therapeutic target.

Colorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer-related deaths. Discrepancies in clinical outcomes are observed even among patients with same-stage CRC due to molecular heterogeneity. Thus, biomarkers for predicting prognosis in CRC patients are urgently needed. We previously demonstrated that stage II CRC patients with NKX6.1 methylation had poor 5-year overall survival. However, the methylation frequency of NKX6.1 was only 23% in 151 pairs of CRC tissues. Thus, we aimed to develop a more robust prognostic panel for CRC using NKX6.1 in combination with three genes: LIM homeobox transcription factor 1α (LMX1A), sex-determining region Y-box 1 (SOX1), and zinc finger protein 177 (ZNF177). Through quantitative methylation analysis, we found that LMX1A, SOX1, and ZNF177 were hypermethylated in CRC tissues. LMX1A methylation was significantly associated with poor 5-year overall, and disease-free survivals in stage I and II CRC patients. Sensitivity and specificity analyses of the four-gene combination revealed the best sensitivity and optimal specificity. Moreover, patients with the four-gene methylation profile exhibited poorer disease-free survival than those without methylation. A significant effect of the four-gene methylation status on overall survival and disease-free survival was observed in early stage I and II CRC patients (p = 0.0016 and p = 0.0230, respectively). Taken together, these results demonstrate that the combination of the methylation statuses of NKX6.1, LMX1A, SOX1, and ZNF177 creates a novel prognostic panel that could be considered a molecular marker for outcomes in CRC patients.