Roles of HDACs in chromatin remodelling and response to chemotherapy in cancer

Abstract

BACKGROUND: The higher-order structure of chromatin changes in response to extracellular and environmental signals. We observed nuclear morphological changes in biopsied cancer tissue after chemotherapy. Since chromatin structure dictates gene expression, and therefore function, further investigation of this phenomenon may increase our understanding of therapeutic responses.

I hypothesised that nuclear morphological changes in cancer in response to DNA-damage by chemotherapy are mediated by histone deacetylases (de Ruijter, van Gennip et al.). Methods: Ovarian cancer cell lines PEO1/PEO4 (platinum sensitive/resistant) were selected as in vitro models, and primary ovarian cancer xenografts OV1002 and HOX424 as in vivo models. Expression levels of HDACs, heterochromatin protein 1 (HP1), and DNA damage response (DDR) proteins were profiled by Western blot analysis after treatment with cisplatin.

Immunofluorescence imaging was undertaken using confocal microscopy, and nuclear texture and γH2AX foci were measured in Image J. Cell cycle and apoptosis were detected by flow cytometry. Thirty eight different ovarian cancer biopsies and 175 xenograft samples were assessed for HDAC and HP1 expression in response to chemotherapy by quantitative immunofluorescence. HDAC2 expression was modulated by interfering RNAs (siRNA). Results: I demonstrated nuclear morphological changes in clinical tumours, xenografts, and cell lines in response to platinum chemotherapy by robust measurement of nuclear texture. Expression of HDAC2 increased in PEO1 cells treated with cisplatin at 24h, and this was accompanied by high expression of HP1s.

Expression of components of both HDACs and DDR pathways (pBRCA1, γH2AX, pATM, pATR) showed time dependent changes after cisplatin treatment.

Knockdown of HDAC2 reduced the expression of HP1, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DDR induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, I observed reversible alteration of chromatin patterns during cisplatin treatment to some degree. In clinical ovarian cancer specimens, expression of HDAC4, HDAC8 and HP1γ significantly increased after chemotherapy in sensitive patients, with enhanced heterogeneity in chromatin pattern. HDAC2, HDAC8, and HP1 expression were also increased after carboplatin treatment in carboplatin-sensitive xenografts.

CONCLUSION: These results demonstrate alterations in nuclear morphology after chemotherapy, and implicate HDACs in having a role in higher order chromatin changes and in cellular DNA damage responses in ovarian cancer both in vitro and in vivo.