PTTG oncogene protein stability is regulated by post-translational modifications

TEDESCO, L.; BONFIGLIO, J.; SAPOCHNIK, M.; ATTORRESI, A.; FUERTES, M.; SENIN, S.; ARZT, E.

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Conferencia; FASEB Science Research Conferences, Ubiquitin and Cellular Regulation; 2016

FASEB

Pituitary tumor transforming gene (PTTG) is an oncogene that regulates cell cycle progression, proliferation, differentiation, repair, malignant transformation, and angiogenesis. It was identified as a member of the securin family, critical regulators of sister chromatid separation in late stage mitosis. PTTG is expressed in tissue/organ with high proliferation activities. PTTG is highly expressed in tumors derived from a variety of organs, including pituitary, colon, thyroid, breast, ovary, uterine, lung and esophagus, but no mutations have been described. Isolated from highly tumorigenic and angiogenic cells, RWD domain-containing protein SUMO Enhancer (RSUME), increases protein sumoylation. It is expressed in several normal tissues, in many tumor types and is induced by heat shock and hypoxia. The aim of the present study was to determine if high PTTG levels are due to increased stability of PTTG induced by post-translational modifications and regulated by RSUME, and if this might explain PTTG tumor abundance and pathogenic action. First, we found by western blot that RSUME increases PTTG protein stability, both in transfected COS-7 cells that do not express PTTG and in the pituitary tumoral cell lines AtT-20 and GH4 that do express PTTG endogenously. RSUME also increases PTTG stability in tumor cell lines from different origin (HEK293T, HeLa and HepG2) and in primary cultures of mouse fetal testis, a tissue that expresses high levels of PTTG. Consequently, silencing RSUME with a specific small interfering RNA (siRNA) has the opposite effect. In transfected COS-7 cells treated for different times with cycloheximide, PTTG protein levels decline, and RSUME slows the decay. We found a diminished stability of PTTG in the presence of Senp-1 (a SUMO isopeptidase) or Gam1 (a viral enzyme that inhibits sumoylation), even in the presence of RSUME. The RSUME mutant that lacks of activity as sumoylation enhancer showed a decreased action on PTTG stability. We determined by WB and immunoprecipitation that PTTG is target of sumoylation by SUMO-1, 2 and 3, and RSUME enhanced this conjugation. RSUME and SUMO-1 decreased PTTG ubiquitination. Gam1 by preventing sumoylation, restored the levels of PTTG conjugated to ubiquitin, even in the presence of RSUME. RSUME and PTTG are co-expressed in explants of different types of human pituitary tumors and correlate significantly. In stable clones of AtT-20 and GH4 cell lines expressing interference RNA specific to RSUME (shRSUME), that silencing RSUME and consequently decreased the levels of PTTG protein, we found a significant inhibition of cell proliferation with respect to shSCRAMBLE clones, and disminished tumorigenic proprieties on the athymic nude mice model. Accordingly, in shRSUME clones the decrease in proliferation was reversed by over expression of PTTG. We conclude that sumoylation of PTTG accounts for the higher protein level in cancer cells.