Targeting CHK2 to reverse tamoxifen resistance by reducing BQ323636.1 expression in estrogen receptor positive breast cancer

Abstract

Breast cancer is the most prevalent cancer among women. About 75% of breast cancer patients are estrogen receptor positive (ER+ve). Tamoxifen (TAM) is a selective ER modulator (SERM) which targets the mechanism of action of ER. It is the most frequently used first-line adjuvant treatment for ER+ve breast cancer.

Even though TAM can improve the prognosis of ER+ breast cancer patients, TAM resistance remains a problem. Much effort has been spent studying TAM resistance, but the exact mechanisms remain unclear. Our group previously identified BQ323636.1 (BQ), a novel splice variant of nuclear receptor co-repressor 2 (NCOR2) was strongly associated with TAM resistance. Wild type NCOR2 is transcriptional repressor of ER, while the truncated variant BQ does not repress gene transcription initiated by ER. Overexpression of BQ could compromise the function of NCOR2, resulting in TAM resistance.

The exact cause of BQ overexpression is still poorly understood. TAM can cause DNA damage which triggers DNA damage response (DDR) driven by the ATM-CHK2 signaling cascade. Our research group previously hypothesized that activation of DDR induced by TAM may lead to increased expression of BQ which could lead to tamoxifen resistance. Preliminary data showed that knockdown of ATM resulted in downregulation of BQ expression. However, ATM knockdown resulted in cytotoxicity in both normal breast and breast cancer cell-lines.

CHK2 is a direct downstream kinase of ATM. We therefore hypothesized that inhibiting CHK2 by knockdown expression or with inhibitors might be a better strategy to reduce BQ expression and reverse tamoxifen resistance. Preliminary data showed that CHK2 knockdown is not cytotoxic. PV1019 is a small inhibitor that targets CHK2. The maximal non-lethal dosage of PV1019 on the non-cancerous breast epithelial cell-line was identified.

From the results of cell viability assays, it was confirmed that non-toxic dose of PV1019 not only reversed TAM resistance in BQ-overexpressed, TAM-resistant cell-lines LCC2 (MCF-7R) and AK47 (ZR-75R), but also sensitized TAM-response in TAM-sensitive MCF-7 and ZR-75-1 cell-lines. Treatment with PV1019 re-established TAM-induced apoptosis in LCC2 (MCF-7R) and AK47 (ZR-75R) and further sensitized MCF-7 and ZR-75-1 to apoptosis. Moreover, PV1019 could reduce the expression of BQ in BQ-overexpressing cell-lines LCC2 (MCF-7R) and AK47 (ZR-75R).

CHK2 knockdown in the LCC2 (MCF-7R) cell-line was also employed to validate the role of CHK2 inhibition in TAM resistance and BQ expression. Silencing CHK2 expression could reverse TAM resistance and reduce BQ expression, indicating CHK2 was associated with BQ expression. Nude mice experiments also showed that either PV1019 treatment or CHK2 knockdown reversed TAM resistance thus reducing tumor growth in the xenograft model. In addition, nuclear level of pCHK2 were found to positive correlate with nuclear level of phosphorylated-CHK2 in tumor samples of the patients in Tissue Microarray study.

The results of my study suggest that targeting CHK2 could be a possible strategy for reducing TAM resistance in breast cancer.