Synergistic Interaction of the Class IIa HDAC Inhibitor CHDI0039 with Bortezomib in Head and Neck Cancer Cells

Abstract

In contrast to class I/IIb/pan histone deacetylase inhibitors (HDACi), the role of class IIa HDACi as anti-cancer chemosensitizing agents is less well understood. Here, we studied the effects of HDAC4 in particular and the class IIa HDACi CHDI0039 on proliferation and chemosensitivity in Cal27 and cisplatin-resistant Cal27CisR head and neck squamous cell cancer (HNSCC). HDAC4 and HDAC5 overexpression clones were generated. HDAC4 overexpression (Cal27_HDAC4) increased proliferation significantly compared to vector control cells (Cal27_VC). Chicken chorioallantoic membrane (CAM) studies confirmed the in vitro results: Cal27_HDAC4 tumors were slightly larger than tumors from Cal27_VC, and treatment with CHDI0039 resulted in a significant decrease in tumor size and weight of Cal27_HDAC4 but not Cal27_VC. Unlike class I/pan-HDACi, treatment with CHDI0039 had only a marginal impact on cisplatin cytotoxicity irrespective of HDAC4 and HDAC5 expression. In contrast, the combination of CHDI0039 with bortezomib was synergistic (Chou–Talalay) in MTT and caspase 3/7 activation experiments. RNAseq indicated that treatment with CHDI0039 alters the expression of genes whose up- or downregulation is associated with increased survival in HNSCC patients according to Kaplan–Meier data. We conclude that the combination of class IIa HDACi with proteasome inhibitors constitutes an effective treatment option for HNSCC, particularly for platinum-resistant cancers.

Targeting histone deacetylases in head and neck squamous cell carcinoma: molecular mechanisms and therapeutic targets

“…Class I HDAC1 Expression levels: up-regulated expression in LSCC samples [36] and HNSCC samples according to TCGA, CPTAC and HPA databases Cell proliferation: regulating the expression of PCNA and affecting cell proliferation by reducing Mrna stability [37] Invasion and metastases: interacting with RAR-α to regulate OSCC EMT and invasion through RAR-β [38]; associated with lymph node metastases and advanced clinical stage in LSCC [36] Cell death: acting as a central gene in autophagy process in HNSCC [39] Therapy resistance: down-regulated expression in cisplatin-resistant HNSCC, inversely associated with adhesion loss and drug resistance [40] HDAC2 Expression levels: positive HDAC2 nuclear staining was detected in 80/93 OSCC samples and 11/20 OED samples [41] Cell proliferation: associated with transcriptional repression and tumor maintenance in SCC [47] Invasion and metastases: maintaining HIF-1α stability, promoting cell invasion and metastases of oral cancer [44]; correlated with adhesion loss and aggressiveness, promoting the occurrence and depth of invasion in OSCC [32,42] Cell death: playing a significant role in trichodermin [45] orγ-bisabolene [46] induced apoptosis in HNSCC HDAC3 Immunosuppression: acting as the target of PKM2-mediated immunosuppression in HNSCC [49] HDAC8 Expression levels: up-regulated expression in OSCC [44] and HNSCC samples according to TCGA database Cell death: knocking down HDAC8 induce apoptotic cell death through caspase activation and autophagy in OSCC [51,52] Class II HDAC4 Expression levels: up-regulated expression in NPC [55], Cal27 [56] and ESCC [57] cells, but the TCGA, CPTAC and HPA databases revealed a down-regulated expression in HNSCC, indicating that HDAC4 may possess dual roles in cancer development…”

“…Cell death: playing roles in regulating EBV lytic replication and cell death [56] HDAC7 Cell proliferation: HDAC7 knockdown resulted in growth suppression through G2/M arrest in MEC [63]; targeting of HDAC7 could potentially disrupt the miR-4465-EphA2 pathway and impede the progression of NPC [64] Invasion and metastases: target of miR-140-5p mediated invasion and migration suppression of TSCC [62] Cell death: HDAC7 knockdown in MEC promote the apoptosis and autophagy process [63] HDAC9 Expression levels: up-regulated expression in OSCC [34] Cell proliferation: promoting OSCC carcinogenesis by targeting transcription factor MEF2D and pro-apoptotic factor NR4A1/ Nur77 [34] Cell death: low expression of HDAC9 lead to G0/G1 cycle arrest in OSCC cells and increase cell apoptosis [34] HDAC6 Expression levels: down-regulated expression according to TCGA, CPTAC and HPA databases, however, Sakuma et al [69] observed HDAC6 expression increased in OSCC Cell proliferation: miR-433 downregulate HDAC6 expression by targeting its 3′UTR, leading to the inhibition of OSCC cell growth [71]; miR-206 impede proliferation of HNSCC cells through targeting HDAC6 through PTEN/AKT/Mtor signal [72] Cell death: related to autophagy and mediates the regulation between ER stress and autophagy [34] Prognostic significance: negative associations between HDAC6 expression and the OA of OSCC patients [73]; significant differences in HDAC6 expression between early stage and advanced stage OSCC samples [69] Therapy resistance: pharmacological target for overcoming chemotherapy resistance and preventing OSCC recurrence [34] HDAC10 Expression levels: TCGA database analysis showed HDAC10 expression was increased in HNSCC samples…”

“…HDAC4 was overexpressed in nasopharyngeal carcinoma (NPC) cells and promoted their proliferation and metastasis by upregulating TYK2-STAT1 phosphorylation through HDAC4/LHPP signal axis [55]. HDAC4 overexpression in Cal27 cells also led to increased proliferation compared to control cells [56]. Additionally, HDAC4 was overexpressed in esophageal squamous cell carcinoma (ESCC), where its overexpression was closely linked to increased tumor grade, advanced clinical stage and unfavorable survival outcomes [57].…”

“…The protein analysis using CPTAC and HPA databases has the potential to complement RNA data. Andrographolide could inhibit Epstein Barr virus (EBV) reactivation, linked to viral transmission and oncogenesis in HNSCC cells [56]. Andrographolide also boosted HDAC5 and HDAC9 expression in EBV-positive cells, interacting with MEF2D, specific protein (Sp) 1 and Sp3 to regulate EBV lytic replication and cell death [56].…”

“…Andrographolide could inhibit Epstein Barr virus (EBV) reactivation, linked to viral transmission and oncogenesis in HNSCC cells [56]. Andrographolide also boosted HDAC5 and HDAC9 expression in EBV-positive cells, interacting with MEF2D, specific protein (Sp) 1 and Sp3 to regulate EBV lytic replication and cell death [56]. While HDAC5 played a relatively minor role in the cytotoxicity of HDAC inhibitors.…”