Investigating the Tumor Suppressor Role of RECQ1 Helicase in Breast Cancer

Abstract

RECQ1 is a DNA helicase essential for genomic stability in human cells. Germline mutations in RECQ1 have been linked to breast cancer susceptibility, but the mechanism by which RECQ1 increases cancer risk remains to be elucidated. RECQ1 has diverse roles in DNA repair and modulates the cellular response to DNA damaging agents. Previous studies have indicated that RECQ1 may have a role in gene regulation. Thus, we hypothesized that RECQ1 acts as a tumor suppressor by altering the expression of genes and signaling pathways important in tumorigenesis. To test this hypothesis, we performed RNA-Seq analyses of CRISPR/Cas9- derived isogenic pairs of MDA-MB-231 cell lines that either expressed wild-type RECQ1 (RECQ1-WT) or were knockouts for RECQ1 (RECQ1-KO). The RNA-Seq results were validated by RT-qPCR. We found that the long-term loss of RECQ1 expression in MDA-MB-231 cells resulted in differential expression of thousands of genes, including significantly increased expression of genes involved in cell adhesion, invasion, and migration. Consistent with this, RECQ1-KO cells displayed increased cell proliferation, migration, and invasion in cell-based assays. One of the strongest upregulated genes in RECQ1-KO cells was matrix metalloprotease 1 (MMP1), known to be important in breast cancer development and metastasis. Mechanistically, we demonstrated that RECQ1 regulates MMP1 transcription and chromatin openness through mediators at the MMP1 promoter. Remarkably, knockdowns of MMP1 restored the cell migration ability of RECQ1-KO to a level comparable to RECQ1-WT cells, indicating that the increased migration seen in RECQ1-KO is largely due to increased MMP1 expression. Xenografts of RECQ1-KO cells in nude mice resulted in significantly more tumors than RECQ1-WT cells, indicating the tumorigenic effect of RECQ1-KO cells. To gain a better understanding of how long-term loss of RECQ1 affects molecular pathways related to tumorigenesis, we performed comprehensive pathway analyses using various bioinformatic tools. This analysis revealed a significant enrichment of genes associated with the induction of the senescence-associated secretory phenotype (SASP), a pro-tumorigenic pathway in RECQ1-KO cells. We also found significantly higher reactive oxygen species (ROS) and oxidative DNA damage in RECQ1-KO cells compared to RECQ1-WT cells, indicating increased endogenous damage. Collectively, our results demonstrated that RECQ1 serves as a tumor suppressor in MDA-MB-231 breast cancer cells and chronic loss of RECQ1 expression and function drives tumorigenesis by accumulating oxidative DNA damage, inducing SASP and other transcriptomic changes.