HDAC6 Inhibitors Sensitize Claudin-high Breast Cancer Cells to Cysteine Depletion via Activation of PKCγ Signaling

Abstract

IntroductionTriple-negative breast cancer (TNBC) is a highly malignant breast cancer type with poor prognosis and lacks effective therapy. TNBC is not responsive to targeted therapy for hormone receptors and often exhibit resistance to current chemotherapeutic agents. Targeting tumor metabolism is an emergent strategy to treat cancer. Therefore, identification of tumor metabolic deregulations and development of effective targeted therapies are urgently needed.MethodsWe performed the epigenetic compound library screening in claudin-high breast tumor cells and identified therapeutical sensitizers to overcome the drug resistance of targeted cysteine-dependence therapy. Gene expression profiling were generated to analyze signaling pathways induced by the combined tubacin and cysteine deprivation treatment. Specific inhibitors, shRNA, and CRISPR/Cas9 gene editing approaches were used to target cellular proteins HDAC6 and PKCγ and examine their roles in cell death. Cell viability, RT-qPCR, and Western blotting assays were performed in cysteine-independent tumor cells to examine the anticancer effects of combined tubacin and cysteine deprivation treatment. ResultsWe found that TNBC has differential death responses to cysteine deprivation and the cysteine-dependence of TNBC corelates with the expression levels of claudin genes in addition to the classical EMT markers. To overcome drug resistance in claudin-high/cysteine-independent breast tumor cells, HDAC6 inhibitors were identified by the epigenetic compound library screening as potent sensitizers that synergize with cysteine deprivation to eradicate cysteine-independent tumor cells. Unexpectedly, HDAC6 knockout did not recapitulate the HDAC6 inhibitors-mediated synthetic lethality, indicating that HDAC6 is not the actual target of HDAC6 inhibitors in this context. Transcriptomic profiling revealed that HDAC6 inhibitors synergizes with cysteine depletion to trigger a profound gene transcriptional program. Notably, a zinc-related gene response was observed to accompany with a prominent increase of labile zinc in cells during cell death. We further showed that activation of PKCγ signaling is required to interfere cellular zinc homeostasis and drive HDAC6 inhibitors-mediated cell death.ConclusionOur study demonstrated that HDAC6 inhibitors function as potent sensitizers to overcome the resistance of cysteine deprivation in claudin-high breast tumor cells. Identification of such sensitizers would make the targeted cysteine-dependence therapy applicable in various subtypes of breast cancer.