Long-term treatment with benzodiazepines and related Z-drugs exacerbates breast cancer: clinical evidence and molecular mechanisms.

Background: Benzodiazepines (Diazepam) and related Z-drugs (Zolpidem), henceforth referred to as BZDRs, are widely used for clinical treatment of insomnia and anxiety disorders. BZDRs act on GABA type A receptors to inhibit neurotransmitters. We previously demonstrated that prolonged clinical use of BZDRs exacerbates the risk of breast cancer (BRCA).

Methods: By biomedical, health informatics platform analyses and in vivo studies, we explored clinical association between BZDR usage and BRCA development and advancement. Furthermore, by retrospective studies on patient clinical data and in vitro empirical analyses of the impact of BZDR on BRCA cells, and together with ingenuity pathway analysis (IPA) analyses, we validated the signaling pathways and identified potential intermolecular crosstalk involved.

Results: Clinical data showed that BRCA patients on long term treatment with BZDRs suffered increased mortality rate (p = 0.034). Studies on patient samples indicated that among 16 GABA receptors examined, GABRA3 (a pro-tumorigenic player) was significantly upregulated by BZDRs, which advanced BRCA disease. To support our clinical findings, we examined in vivo, the impact of BZDRs on BRCA advancement using MDA-MB231 cells to mediate metastasis in mice model. Our results show that BZDRs indeed promoted cancer advancement to the lungs and localized in the tibia. Using BRCA cell lines, we revealed the molecular-cellular effects of prolonged treatment with BZDRs in vitro. We showed significant metastasis indicated by increased cancer cell migration and invasion, which correlated well with our clinical observations. We discovered that BZDR-mediated GABRA3 stimulation was associated with downregulation of anti-tumorigenic extracellular matrix (ECM) molecules (S100B, COL6A6 and VIT) and upregulation of pro-tumorigenic FBN3 in BRCA cells. Notably, GABRA3-shRNA and GABRA3-CRISPR/Cas9 disrupted the abovementioned dynamics dramatically and suppressed BRCA cell invasion induced by BZDRs. Bioinformatics analyses highlighted molecular pathways showing interplay between GABRA3 and ECMs, which presumably exacerbated BZDR-induced BRCA progression via immune modulators.

Conclusions: Long-term clinical use of BZDRs significantly increased the mortality rate of BRCA patients. We provide in vivo and in vitro evidence confirming that BZDRs promote BRCA advancement. We revealed that BZDR-mediated BRCA signaling pathways through GABRA3-ECMs, which promotes metastasis, probably through immune modulation and changes in the tumor microenvironment.