Introduction: The human Pituitary Tumor Transforming Gene (hPTTG) is a phosphorylated proto-oncogene induced in multiple tumour types. hPTTG phosphorylation is mediated by cyclin-dependent kinase 2 (CDC2) and expression is regulated by specificity protein 1 (SP1). In thyroid cancer, hPTTG induces genetic instability and propagates growth through induction of growth factors (GFs).
Methods: The interplay between hPTTG phosphorylation, SP1 regulation and GF induction was evaluated in thyroid cells in vitro and in mouse models of PTTG over- and underexpression in vivo.
Results: EGF(5nM), TGF-α(5 nM) and IGF1(10 ng/l) induced hPTTG protein in K1(twofold, 3.6-fold and 2.3-fold, P=<0.01) and TPC-1 papillary thyroid carcinoma cells(twofold, 4-fold and 2.6-fold, P=<0.01) and in human primary thyrocytes(threefold, 2.4-fold and 2-fold, P=<0.01). These effects were associated with activation of mitogen activated protein kinase (MAPK) and phosphoinositide three-kinase (PI3K), but not with increased cellular proliferation rates. GF treatment of TPC-1 cells following siRNA knockdown of CDC2 or SP1 confirmed that GFs induce hPTTG independently of these hPTTG regulators. CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. Transient transfection with hPTTG induced EGF (1.7-fold, n=4, P=0.004), IGF1 (1.6-fold, n=5, P=0.002) and TGF-α mRNA (1.6-fold, n=3, P=0.024) expression. Treatment of human primary thyrocytes with conditioned media from hPTTG transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by GF depletion or GF receptor inhibitors. In vivo evaluation of our transgenic mouse model with thyroid-targeted hPTTG overexpression confirmed increased mEGF (2.7-fold, n=3, P=0.012) and mIGF1 (2.0-fold, n=3, P=0.02) mRNA, compared with WT mice. Further, mEGF mRNA expression was downregulated in the thyroids of PTTG−/− knockout mice (0.4-fold, n=4, P=0.001), consistent with reduced EGF mRNA expression in TPC-1 cells transfected with hPTTG siRNA (0.68-fold, n=4, P<0.05, compared to scrambled controls).
Conclusion: Together, our results indicate that hPTTG is involved in autocrine signalling mechanisms with GFs in the thyroid, independently of CDC2 and SP1, and that aberrant control of these pathways may enhance transformed cell growth.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details unavailable.
05 - 09 May 2012
European Society of Endocrinology