A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer

Abstract

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p<0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P<0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation.

Phase II trial of the histone deacetylase inhibitor romidepsin in patients with recurrent/metastatic head and neck cancer

“…Interestingly, the expression of HDAC6 , associated with tumor aggressiveness in SCCHN 9 , and EGFR was down-regulated in SCCHN tumors following romidepsin, the mechanism and clinical significance of which warrants further study. Of note, recent preclinical studies of the HDAC inhibitor valproic acid in combination with hydroxyurea have shown downregulation of EGFR protein expression and triggering of apoptosis in SCCHN cells and xenografts 40 .…”

Sumoylation of HDAC2 promotes NF-κB-dependent gene expression

“…Hdac2 K462R -V5 was derived thereof by QuickChange Site-Directed Mutagenesis (Agilent, Frankfurt/Main, Germany). To generate p65-GFP, the p65 coding sequence was amplified from cDNA obtained from a human head and neck tumor as described [52], inserted into the pF25 expression vector using NheI restriction sites and verified by sequencing as described [53, 54]. siRNA pools targeting p65 and HDAC2 were purchased from Santa Cruz, RSK1 (RPS6KA1) from Thermo and RelB siRNA was published previously [55].…”

Loss of the proteins Bak and Bax prevents apoptosis mediated by histone deacetylase inhibitors

“…12 Combination of an HDI with inhibitors of signaling pathways leads to increased apopotosis in several cell line models. [13][14][15] Various reports have shown that HDIs mediate apoptosis through both the intrinsic and extrinsic pathways. Romidepsin has been shown to mediate apoptosis through the tumor necrosis factor (TNF) pathway activation of caspase 8 and downregulation of cellular FLICE-inhibitory protein, but this may be modelspecific.…”