Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells.

Simple Summary: Sperm-associated antigen 5 (SPAG5) is associated with tumour initiation, progression, and resistance to front-line therapeutics in many cancer types. However, the role of SPAG5 in triple-negative breast cancer (TNBC) remains controversial, and evidence for SPAG5-mediated chemoresistance in TNBC is lacking. Our research demonstrates that SPAG5 may be an important determinant of the efficacy of doxorubicin and docetaxel, the most widely used chemotherapy, in TNBC. We focused on the genetic removal of the SPAG5 gene by using a gene editing technique to determine if the SPAG5 deletion in the TNBC cell lines could downregulate chemoresistance. We found that the SPAG5 deletion contributed to a sensitive phenotype in the TNBC cells. Our results may help practitioners predict patients' response to doxorubicin and docetaxel, and targeting SPAG5 may represent a promising way to overcome chemoresistance in TNBC patients. Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, the SPAG5 knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines. The knockout of SPAG5 was confirmed on both gene and protein levels using genomic PCR, DNA sequencing and western blotting. The functional loss of SPAG5 was determined by colony-formation assay. SPAG5-regulated doxorubicin- and docetaxel-resistance was assessed by MTT and apoptosis assays. The results indicated that all the SPAG5 knockout MDA-MB-231 and BT549 clones were biallelic, where one allele was replaced by the donor template, and the other allele had the same "T" insertion (indel) adjacent to the cutting sites of gRNAs at the exon 1 boundary, irrespective of the gRNAs and cell lines. The locus of indel interrupted the SPAG5 transcription by damaging the GT-AG mRNA processing rule. Deletion of SPAG5 decreased clonogenicity in both MDA-MB-231 and BT549 cells. SPAG5 was able to regulate the resistance and the drug-induced apoptosis of both doxorubicin and docetaxel. In conclusion, recombinant plasmid-based CRISPR-Cas9 technology can be used to delete the SPAG5 gene in the TNBC cell lines. SPAG5 has an important role in regulating cell proliferation and doxorubicin- and docetaxel-resistance in MDA-MB-231 and BT549 cells. [ABSTRACT FROM AUTHOR]