A proteomic study of human Merkel Cell Carcinoma

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies

Additional file 1: of Regulation of Locomotor activity in fed, fasted, and food-restricted mice lacking tissue-type plasminogen activator

Activity profiles and body weight change in mice subjected to restricted feeding in a skeleton photoperiod. (PDF 603 kb

Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway

<div><p>Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.</p></div

List of proteins found to be significantly altered in RET 9 and RET 51 lines compared to control lines.

<p>Fold change ≤1.5 is considered increased expression and between 0.2 and 0.6 is decreased expression.</p><p>List of proteins found to be significantly altered in RET 9 and RET 51 lines compared to control lines.</p

Components of an active RET signaling pathway are observed in MTC and correlate to DNA-PKcs.

<p>Immunohistochemistry of MTC array samples for phosphorylated ERK 1/2 and phosphorylated AKT indicated the presence of both of these components of an active RET signaling pathway in MTC similar to staining observed for ps2056 DNA-PKcs. 10x images of whole tissue array sample are shown.</p

Phosphorylation of DNA-PKcs at s2056 is elevated in RET 9 and RET 51 cells.

<p>A) Western blot analysis shows phospho-s2056 (ps2056) (460 Kda) to be elevated in RET9 and RET 51 cell lysates. Total DNA-PKcs was used as loading control. B) Immunocytochemistry of cells plated in chamber slides and stained for ps2056 (red) and Dapi as counterstain (blue). C) ICC revealed a significant increase in ps2056 located in the nuclei of RET 9 and RET 51 cells compared to EV. * p ≤ 0.05, error bars = s.d.</p

Inhibition of RET reduces phosphorylation of DNA-PKcs s2056 and blocking DNA-PKcs activity increases chemosensitivity.

<p>A) TT cells were treated with RPI-1 (5 μM) to inhibit RET for 24 hrs prior to nuclei isolation. Western blot analysis indicated that RET inhibition reduces DNA-PKcs s2056 phosphorylation. Total DNA-PKcs and Lamin B1 were used as loading controls. B) TT cells treated with Nu7441 (NU) (1 μM) to inhibit DNA-PKcs had a greater decrease in cell viability than DOX alone. *p ≤ 0.05, error bars = s.d.</p

RET inhibition reduces phosphorylation of DNA-PKcs.

<p>EV, RET 9 and RET 51 cells were treated with the RET inhibitor RPI-1 for 24 hours before cells were harvested and analyzed by western blot. RPI-1 treatment significantly reduced phosphorylation of DNA-PKcs at site s2056 according to Image J analysis. GAPDH was used as loading control. **p ≤ 0.005, error bars = s.d.</p

Increased expression of RET isoforms increases resistance to chemotherapy.

<p>A) Western blot analysis of RET lines show expression and phosphorylation of either RET 9 or RET 51 isoforms only in overexpressing lines and not in the control EV cells. B) RET expression causes cells to be resistant to doxorubicin treatment (DOX). *p≤ 0.01, error bars = s.d.</p