Discipline: Biological Sciences
Subcategory: Cancer Research
Swetha Parvathaneni - Howard University
Co-Author(s): Xing Lu, Howard University, Washington DC Ritu Chaudhary, Xiao Lynn, and Ashish Lal, National Cancer Institute, Bethesda Sudha Sharma, Howard University, Washington DC
Background: The response of cancer cells to therapeutic drugs that are aimed at causing DNA damage is dependent on genes that play roles in DNA repair. Studies have shown that cancer cells upregulate DNA repair genes as a mechanism to prevent further accumulation of mutations to gain survival advantage and chemoresistance to therapeutic drugs. Single nucleotide polymorphism in RECQ1 (also known as RECQL or RECQL1), a DNA helicase involved in DNA damage and replication stress response, is reported to influence patient’s response to gemcitabine in pancreatic cancer leading to poor survival. Gemcitabine is also used as a chemotherapeutic against triple negative breast cancer, however resistance to gemcitabine is reported in breast cancer patients. The mechanism by which gemcitabine chemoresistance is obtained is poorly understood. Given the importance of RECQ1’s role in DNA repair and it being categorized as a breast cancer susceptibility gene, we hypothesize that RECQ1 is involved in regulating gemcitabine mediated cell killing in breast cancer.
Methods: To test our hypothesis, we performed CRISPR-Cas9-mediated targeted inactivation of RECQ1 in triple negative breast cancer MDA-MB-231 cell lines. Validation of RECQ1 knockout was performed by sequencing, quantitative real-time PCR and western blotting. Cell survival and cell cycle profile of RECQ1-wildtype and RECQ1-knockout MDA-MB-231 cells following gemcitabine treatment was determined by WST-1 assay and flow cytometry, respectively.
Results: We established MDA-MB-231 cell lines that are either wild-type or knockout for RECQ1. Consistent with previous findings using siRNA or shRNA mediated knockdown, RECQ1-knockout clones displayed reduced cell growth and proliferation, decreased pre-rRNA, and increased DNA damage as compared to the wild type RECQ1 clones. RECQ1 knockout MDA-MB-231 cells were sensitive to gemcitabine treatment and displayed aberrant cell cycle profile indicating defects in cell division. We identified significant up regulation of RECQ1 mRNA in MDA-MB-231 cells following gemcitabine treatment suggesting a role in gemcitabine induced DNA damage response.
Conclusion: We have developed and validated an isogenic pair of wild-type and RECQ1 knockout MDA-MB-231 cell line as a novel in vitro model to study RECQ1 functions in breast cancer. Our initial experiments suggests that RECQ1 is involved in cellular response to DNA damage caused by gemcitabine. Our ongoing experiments are investigating the detailed mechanism of RECQ1’s response to gemcitabine and a putative role in chemo resistance in triple negative breast cancer patients.
Funder Acknowledgement(s): This work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha Sharma. We also acknowledge support from the NIMHD/NIH award number G12MD007597 and NIA/NIH award number 1R25 AG047843-02.
Faculty Advisor: Sudha Sharma, email@example.com
Role: Conception of the idea, execution and data analysis.