NSUN7 knockdown increases the sensitivity of tamoxifen-resistant MCF-7 cells to 4-hydroxytamoxifen

Tamoxifen is the commonly prescribed drug for estrogen receptor positive breast cancer treatment. Recently dysregulation in RNA 5-methylcytosine (m5C) modification and its regulating enzymes have been suggested as potential contributors in chemotherapeutic resistance in various cancers, however their role in determining tamoxifen resistance in breast cancer remains unknown. In this study we analyzed the expression of m5C modulating enzymes recognized as writers (NSUN2-7), readers (YBX-1 and ALYREF) and eraser (TET3), and ELISA based assay to check global methylation status in tamoxifen-resistant breast cancer cell line (MCF-7 Tam1) compared to the sensitive MCF-7 cell line. We found that global m5C status and NSUN7 writer enzyme expression was significantly higher in MCF-7 Tam1 cells. Furthermore, Small interfering RNA mediated NSUN7 depletion significantly restored the sensitivity and resulted in an approximate 50% decrease in IC50 value compared to the control. Furthermore, NSUN7 deficient cells showed a significant reduction in migration and colony formation ability. RNA sequencing and Gene set enrichment analysis showed that NSUN7 targeted genes were enriched in pathways and processes regulating cell cycle, cell migration and proliferation. Further screening of differentially expressed genes revealed that NSUN7 deficiency caused dysregulation in experimentally determined genes associated with drug resistance particularly tamoxifen resistance. The hub genes identified in NSUN7-deficient tamoxifen-resistant cell line showed significant association with the disease prognosis in terms of overall and disease-free survival. Hence, NSUN7 is a potential therapeutic target for combating tamoxifen resistance in breast cancer cells. Moreover, it has a critical role in determining metastatic characteristics in breast cancer which makes it a candidate prognostic marker. Overall design: Since NSUN7 writer enzyme expression was significantly higher in MCF-7 Tam1 cells, therefore, to investigate its role in tamoxifen resistance and cancer progression, we knocked down its expression using siRNA. We performed gene expression profiling analysis using data obtained from RNA-seq of scrambled/control-siRNA and NSUN7 targeted siRNA treated MCF-7 Tam1 cells each having two replicates. Comparative gene expression profiling of RNA-seq data of control treated MCF-7 Tam1 cells and its KD derivatives followed by gene set enrichment analysis was performed.