Abstract 4547: Treatment of human medullary thyroid carcinoma (MTC) with either proteasome (Pr) or histone deacetylase (HDAC) inhibitors leads to a fall in RET mRNA levels and, in turn, a decrease in RET protein expression providing alternate strategies to reduce RET expression in a tyrosine-kinase driven disease
Authors: Poruchynsky Marianne S. , Gramza Ann W. , Wells Samuel A. , Fojo Tito 
Published: June 2012

Abstract
Medullary thyroid carcinoma (MTC), a neuro-endocrine cancer of parafollicular C-cells accounts for 4-10% of all thyroid cancers and has low response rates to “cytotoxic chemotherapy”. The RET proto-oncogene encodes the RET (Rearranged during Transfection) transmembrane tyrosine kinase (TK) receptor, a pivotal player and a logical target for treatment of MTC. While trials with vandetanib (V) a RET TK inhibitor (TKI) in patients with MTC have been promising, we recognized a TKI is unlikely to have maximum benefit administered alone. We sought to combine V + a drug with a broad activity profile and no overlapping toxicities. Recognizing the failure of traditional “cytotoxic” agents we investigated the proteasome (Pr) inhibitor, bortezomib (B) not yet explored clinically in MTC. Initial experiments using TT MTC cells with a mutant C634W RET, found V + B non-antagonistic in cytotoxicity assays, and V showed decreased phospho-RET expression in TT cells with 0.5 or 1 µM in 1.5h. But while V did not change RET protein levels, B decreased RET protein 7 fold and secondarily phospho-RET levels. That this was secondary to Pr inhibition and not unique to B was confirmed by treating TT cells 6h or 24h with three other Pr inhibitors – epoxomycin, lactacystin or MG132. We observed 5-9 fold decreases in RET protein, which followed a time course and a dose response, with greater reduction at 24h than at 6h. To assess if the decrease in RET protein was due to decreased RET mRNA levels, we assessed RET mRNA expression and were surprised to find a 4-9 fold decrease with Pr inhibitors. Since Pr inhibitors reduced RET by reducing mRNA levels, and reasoning this would provide an alternate strategy to impact RET function, we studied the effect of three HDAC inhibitors on RET mRNA and protein expression. Treatment of TT cells with vorinostat, belinostat, or romidepsin (R) decreased RET mRNA 3-4 fold at 24h and protein levels 4 and 7-14 fold, at 24 and 48h respectively. Furthermore B + R had greater impact than either drug separately, depressing RET protein in TT cells an additional 2-3 fold to levels 4-12 fold less than that in untreated cells, suggesting mechanisms whereby Pr and HDAC inhibitors reduce mRNA levels might differ. We have pursued the latter by examining mRNA and protein levels of 3 candidate transcription factors identified in a literature search. To date we have shown TTF1 and E2F1 mRNA levels are decreased 4-58 fold by both Pr and HDAC inhibitors whereas EGR1 levels only fall with Pr inhibitors. siRNA studies that modulate transcription factor levels will clarify their possible roles in the Pr and HDAC inhibitor drug effects and help elucidate whether these inhibitors, either alone or in combination, can prove useful in MTC therapy by reducing the crucial RET TK.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4547. doi:10.1158/1538-7445.AM2011-4547