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

PLoS One. 2015 Jun 11;10(6):e0127943. doi: 10.1371/journal.pone.0127943. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Neuroendocrine / metabolism*
  • Cell Line, Tumor
  • DNA-Activated Protein Kinase / metabolism*
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Indoles / pharmacology
  • Nuclear Proteins / metabolism*
  • Phosphorylation / drug effects
  • Proteomics / methods*
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins c-ret / metabolism*
  • Signal Transduction
  • Thyroid Neoplasms / metabolism*

Substances

  • Indoles
  • MAS1 protein, human
  • Nuclear Proteins
  • Proto-Oncogene Mas
  • RPI-1 compound
  • Proto-Oncogene Proteins c-ret
  • RET protein, human
  • DNA-Activated Protein Kinase
  • PRKDC protein, human

Supplementary concepts

  • Thyroid cancer, medullary

Grants and funding

This work was funded by the UAMS Department of Surgery.