Transcriptional signature of histone deacetylase inhibition in multiple myeloma: biological and clinical implications

Abstract

Histone deacetylases (HDACs) affect cell growth at the transcriptional level by regulating the acetylation status of nucleosomal histones. HDAC inhibition induces differentiation and/or apoptosis in transformed cells. We recently showed that HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), potently induce apoptosis of human multiple myeloma (MM) cells. In this study, we focused on MM as a model to study the transcriptional profile of HDAC inhibitor treatment on tumor cells and to address their pathophysiological implications with confirmatory mechanistic and functional assays. We found that MM cells are irreversibly committed to cell death within few hours of incubation with SAHA. The hallmark molecular profile of MM cells before their commitment to SAHA-induced cell death is a constellation of antiproliferative and/or proapoptotic molecular events, including down-regulation of transcripts for members of the insulin-like growth factor (IGF)/IGF-1 receptor (IGF-1R) and IL-6 receptor (IL-6R) signaling cascades, antiapoptotic molecules (e.g., caspase inhibitors), oncogenic kinases, DNA synthesis/repair enzymes, and transcription factors (e.g., XBP-1, E2F-1) implicated in MM pathophysiology. Importantly, SAHA treatment suppresses the activity of the proteasome and expression of its subunits, and enhances MM cell sensitivity to proteasome inhibition by bortezomib (PS-341). SAHA also enhances the anti-MM activity of other proapoptotic agents, including dexamethasone, cytotoxic chemotherapy, and thalidomide analogs. These findings highlight the pleiotropic antitumor effects of HDAC inhibition, and provide the framework for future clinical applications of SAHA to improve patient outcome in MM.

Figures

Fig. 1.

Hierarchical clustering of gene expression profiling data (obtained by oligonucleotide-microarray analysis) in SAHA-treated vs. control human MM-1S cells.

Fig. 2.

Functional clustering analysis of genes implicated in cytokine-induced proliferative/antiapoptotic signaling pathways (A) and oncogenes/tumor suppressor genes (B). SAHA down-regulates signaling pathways for MM cell proliferation and survival, including IGF/IGF-1R and IL-6R/gp130, suppresses expression of multiple oncogenes, and up-regulates several tumor suppressor genes. Color saturation is proportional to magnitude of the difference from the respective control. (C) SAHA treatment (1 μM, 0–24 h) suppresses IGF-1 autocrine production by MM-1S cells. (D) NF-κB DNA binding ELISA confirms that SAHA (1 μM, 0–24 h) suppresses NF-κB activity in MM-1S cells. (E and F) IGF/IGF-1R/Akt pathway protects against apoptosis induced by HDAC inhibition. (E) IGF-1 (200 ng/ml), but not IL-6 (200 ng/ml), reduces the percentage of specific cell death of MM-1S cells after treatment with SAHA (1 μM for 48 h) (MTT survival assay). (F) SAHA-induced cell death (quantified by MTT, mean ± SD) in MM-1S cells transfected with a vector expressing constitutively active Akt or control (neo) vector, after overnight serum starvation, and incubation with or without SAHA for additional 48 h. MM-1S cells transfected with constitutively active Akt construct have reduced sensitivity to SAHA.

Fig. 3.

Effects of SAHA on regulators of apoptosis and sensitivity to caspase-dependent drug-induced apoptosis. (A) Functional clustering analysis for changes in gene expression of regulators of apoptosis. (B) p53 DNA-binding ELISA confirms that SAHA treatment (1 μM0–24 h) induces activation of p53. (C and D) MTT assays confirm that SAHA pretreatment (50 nM for 24 h) enhances sensitivity of MM-1S cells to dexamethasone (0.1 μM for an additional 48 h) (C) or the immunomodulatory thalidomide derivative IMID-1 (CC-4047) (0.01 μM for 48 h) (D).

Fig. 4.

Effects of SAHA on DNA synthesis and repair, cell cycle regulation telomerase activity, and chemosensitivity. (A and B) Functional clustering analysis for genes involved in cell cycle regulation, DNA synthesis and repair (A), and cell cycle regulation (B). (C) Telomeric repeat amplification protocol assay for quantification of telomerase (hTERT) activity indicates that SAHA suppresses both constitutive and IGF-1 (200 ng/ml)-induced hTERT activity. (D) MTT assays confirm that SAHA pretreatment (50 nM for 24 h) enhances the sensitivity of MM-1S cells to doxorubicin (25 ng/ml for additional 48 h).

Fig. 5.

Functional impact of SAHA on the ubiquitin/proteasome pathway. (A) Functional clustering analysis for genes of the ubiquitin/proteasome pathway. (B) 20S proteasome activity assays confirm that HDAC inhibition by SAHA (1 μM, 24 h incubation) suppresses both constitutive and IGF (200 ng/ml)-induced activity of the proteasome. (C) MTT assays confirm that SAHA pretreatment (50 nM for 24 h) enhances sensitivity of MM-1S cells to proteasome inhibitor PS-341 (bortezomib) (5 nM for additional 24 h).