Reversing the Malignant Proliferation and Doxorubicin Resistance in Human Triple Negative Breast Cancer Cell Line MDA-MB-231 by CRISPR/Cas9-Mediated Deletion of the KISS1R Gene

Abstract

Triple negative breast cancer (TNBC) is a treatment resistant strain of breast cancer (Aysola et al., 2012). Women with TNBC do not benefit from endocrine therapy or Human Epithelial Growth Factor Receptor 2 (HER2) targeted therapies as the relevant drug targets are absent, thus the overall survival (OS) rates of patients diagnosed with TNBC are poor (Aysola et al., 2012). Fayaz and colleagues (2019) suggest the 10-year OS rate of patients with non-metastatic TNBC is 66%, 16% lower than the average breast cancer OS rate, and the 10-year OS rate of patients with advanced TNBC is 0%. These survival rates are likely the result of the lack of effective systemic treatments.

Chemotherapy is one of the only effective systemic treatments available for the treatment of TNBC, with neoadjuvant chemotherapy (NAC) producing a pathological complete response (pCR) in approximately 40-50% of patients with TNBC (van der Ende et al., 2023). Unfortunately, studies show that if timely clearance is not achieved, long-term use often results in the acquisition of broad-spectrum drug resistance.

The use of genetic medicine to overcome chemotherapeutic drug resistance has been posited as a solution. By modifying genes that govern traits such as angiogenesis, immune evasion, replication or the expression of drug efflux channels, all of which contribute to multidrug resistance (MDR), it is possible to improve clinical outcomes in TNBC.

A known modulator of neoplastic behaviours in TNBC cells is the KISS1 receptor (KISS1R), a galanin-like G protein coupled receptor associated with a family of signal peptides, called kisspeptins. The KISS1/KISS1R mechanism is believed to influence several pathways, which regulate cell proliferation and invasiveness. A notable body of research associates KISS1R overexpression with increased proliferation in oestrogen receptor alpha (ERα) negative breast cancer cell lines (Blake et al. 2017).

qRT PCR analysis of KISS1R overexpressing cell lines suggests overexpression of KISS1R directly correlates with Breast Cancer Resistance Protein (BCRP) expression levels. BCRP, also known as MXR/ABCG2, is a key contributor to the acquisition of multidrug resistance in TNBC (Mao and Unadkat, 2014). Conversely, analysis of cell proliferation rates in MDA-MB-231 breast cancer cell cultures treated with KISS1R siRNA showed reduced proliferation, invasiveness and cell viability in screened cell lines, further evincing KISS1Rs impact on TNBCs. This suggests KISS1R is an ideal target for therapeutic intervention.

The CRISPR-Cas9 gene editing system can be used to delete or remove a gene of interest in an in vivo cell model. We hypothesized this system can be used to halt KISS1R expression in an in vitro cell model, resulting in a reversal of multidrug resistance, and a reduction in proliferation and invasiveness in MDA-MB-231 cancer cell cultures.

Two different MDA-MB-231 cell cultures were transformed by the delivery of CRISPR Cas9 ribonucleoprotein complexes, using the Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent. The efficiency of the KISS1R knockout was assessed using a T7 endonuclease cleavage assay and a Western Blot Analysis. An MTT assay was performed to compare the differences of Doxorubicin sensitivity between KISS1R knockout, and the wildtype MDA-MB-231 cells and sequence analysis was performed. The results demonstrate the feasibility of the CRISPR-Cas9 system in the targeted deletion of the KISS1R gene. The genomic cleavage detection assay indicated a cleavage fraction of 30.18 ± 2.2% (95% CI) and the cleavage efficiency of 54.94% in cultures transformed using the CRISPR Cas9 RNP complexed with sgRNA KISS1R C3. TIDE sequence trace decomposition analysis suggests the transformation efficiency was lower, at approximately 31.2%. The MTT assay results support the results of prior studies that KISS1R inhibition also affects cell viability, cancer cell migration and invasiveness, with cell viability reduced by a factor of 3.25. MTT results also suggest a reversion in chemosensitivity in KISS1R knockout cells, with a 6-fold reduction in IC50 in the KISS1R knockout cell line D1-3 when compared to wild type MDA-MB-231 with wild type IC50 = 54 nM (95% CI; 33.83 to 86.11) and D1-3 IC50 = 9 nM (95% CI; 4.420 to 18.14) This supports the targeting of the KISS1 receptor for therapeutic purposes in TNBC as well as validates the utility of the CRISPRCas9 ribonucleoprotein in inducing KO mutations for therapeutic purposes.