Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells

Abstract

The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.J. Clin. Invest. 104:865-874 (1999). MethodsCell lines. The human cell lines used in this study are described in ref. 21. Bosc23 cells were a gift of M. Santoro (Consiglio Nazionale delle Ricerche, Naples, Italy). All cell lines were grown in DMEM containing 10% FCS. PC Cl 3 and PC-D3 cells (normal thyrocytes engineered to stably overexpress cyclin D3) were grown in Ham's F12 medium supplemented with 5% calf serum in the presence of 6 hormones (thyrotropin, hydrocortisone, insulin, transferrin, somatostatin, and glycyl-histidyllysine; Sigma Chemical Co., St. Louis, Missouri, USA).Immunoperoxidase staining. Immunohistochemistry was performed using anti-p27 monoclonal antibody k25020 (Transduction Laboratories, Lexington, Kentucky, USA) or anti-p27 polyclonal antibody C-19 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) as described previously (15,16). Antigen retrieval was performed by microwave irradiation. To define p27 expression we used cutoff values that have been defined in previous papers (15,16). Tumors were considered to be p27-positive when 50% or more of the tumor cells stained positive; if less than 50% of cells stained positive, a tumor was considered p27-negative. Counts were performed in 5 random high-power fields. At least 500 cells were counted.Western blotting, immunoprecipitation, and kinase assay. Cells were lysed in NP-40 buffer containing protease inhibitors. Proteins were separated on polyacrylamide gels and transferred to nitrocellulose membranes (Hybond-C; Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were incubated with primary and secondary antibodies and...

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27kip1

“…Since p27 kip1 phosphorylation leads to its turnover, one possibility is that, by directly or indirectly increasing the rate of p27 kip1 phosphorylation, the PI3K/Akt pathway targets it to degradation and that PTEN would decrease the rate of p27 kip1 turnover by preventing activation of this pathway. Accordingly, we have recently shown that p27 kip1 expression is lost in approximately 40% of primary thyroid carcinomas (Baldassarre et al, 1999).…”

“…The human thyroid carcinoma cell lines used in this study have been previously described (Baldassarre et al, 1999). All cell lines were grown in Dulbecco's modi®ed eagle medium (DMEM) containing 10% fetal calf serum.…”

Deregulated degradation of the cdk inhibitor p27 and malignant transformation

“…Alternatively, this could be due to an unknown mechanism for the elimination of p27. In some tumors, p27 is aberrantly localized to the cytoplasm [49,[63][64][65][66]. Sequestration of p27 in the cytoplasm may serve to block p27's inhibitory activity toward cyclin E/cdk2 complexes and allow increased proliferation.…”

Impairment of the p27kip1 function enhances thyroid carcinogenesis in TRK-T1 transgenic mice

“…It is interesting to observe that all these oncogenes identified in thyroid cancer activate a signaling pathway that results in an increased MAPK activity, according to which one specific signaling pathway that leads to MAPK activation plays a major role in the generation of PTC. However, other genetic lesions have also been reported in PTCs: decreased levels of PTEN and PTPRJ, a dual-specific phosphatase and a receptor type tyrosine phosphatase respectively , and alterations of the p27 kip protein function (Baldassarre et al 1999) represent frequent features of thyroid malignancies. p27 kip1 has classically been regarded as a cell-cycle inhibitor based on its potent inhibitory activity of cyclin E/cdk2 and the observation that its forced expression results in G1 arrest (Toyoshima & Hunter 1994).…”

“…Also in thyroid malignant neoplasias, the impairment of the p27 function is very frequent: a reduction in p27 kip1 protein levels has been previously described in 10 out of 28 papillary carcinomas, 3 out of 9 follicular carcinomas, and 6 out of 8 anaplastic carcinomas. Moreover, 80% of p27 kip1 -expressing tumors shows an uncommon cytoplasmic localization of p27 kip1 protein, associated with a high cdk2 activity (Baldassarre et al 1999). Several components account for the reduced p27 protein levels in thyroid malignant neoplasias: both PTEN and PTPRJ, whose expression is drastically reduced in thyroid malignant neoplasias , increase the stability of the p27 protein.…”