The Expression of Cell Cycle-Related Genes in USP8-Mutated Corticotroph Neuroendocrine Pituitary Tumors and Their Possible Role in Cell Cycle-Targeting Treatment

Abstract

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes' expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

Keywords: Cushing’s disease; USP48; USP8; cell cycle; corticotroph PitNET; flavopiridol; p27; palbociclib; pituitary; roscovitine.

Figure 1

The expression of selected genes encoding cell cycle-related proteins in corticotroph PitNETs stratified according to deubiquitinase gene mutation status.

Figure 2

Expression of cell cycle-related proteins in corticotroph PitNETs stratified according to deubiquitinase gene mutations; (A). The results of the quantification of immunoreactivity; (B). The examples of staining results.

Figure 3

The effect of inhibitors of cyclin-dependent kinases (CDKs), palbociclib, roscovitine and flavopiridol, on AtT-20/D16v-F2 corticotroph cells. (A). Simplified scheme of cell cycle regulation indicating the specificity of each CDK inhibitor; (B). The effect of cell cycle inhibitors on viability and proliferation of AtT-20/D16v-F2 cells. Difference in viability and proliferation of treated cells was evaluated in comparison to untreated cells. ** indicates p < 0.001, *** indicates p < 0.0001; (C). The expression of genes encoding key cell cycle regulators in AtT-20/D16v-F2 cells treated with palbociclib, roscovitine and flavopiridol. Difference in the gene expression level in treated cells was evaluated in comparison to untreated cells. * indicates p < 0.05, ** indicates p < 0.001, *** indicates p < 0.0001.

Figure 4

Treatment of AtT-20/D16v-F2 cells with overexpression of USP8-mutated and wild-type variant with cyclin-dependent kinase inhibitors (CDKIs); Control—empty vector, USP8wt—wild type USP8 overexpression; USP8mut—overexpression of mutated USP8. (A). Overexpression of mutated and wild-type Usp8 in AtT-20/D16v-F2 cells (Western blot membrane and densitometry results) * indicates p < 0.05; Original western blot figures can be found at Figure S1; (B). The expression levels of Cdkn1b and Cdc25a in AtT-20/D16v-F2 cells with overexpression of Usp8-mutated and wild-type variants. Cdk6 and Ccnd2 expression levels in AtT-20/D16v-F2 cells were not determined since they were below qRT-PCR detection.; (C). Proliferation of AtT-20/D16v-F2 cells with overexpression of Usp8-mutated and wild-type variants; (D). Proliferation rate of AtT-20/D16v-F2 cells with overexpression of USP8-mutated and wild-type variants treated with CDKIs: palbociclib, roscovitine and flavopiridol, measured with the BrdU assay; (E). The viability of AtT-20/D16v-F2 cells with overexpression of Usp8-mutated and wild-type variants treated with CDKIs: palbociclib, roscovitine and flavopiridol, measured with the MTT assay.

Figure 5

Treatment of AtT-20/D16v-F2 cells with p27 knockdown using cell cycle inhibitors. (A). Transfection efficiency and lowering of p27 expression with the shRNA hairpins sh_p27.1 (targeting 3′UTR) and sh_p27.2 (targeting coding sequence). Microscopic pictures were taken by the use of Olympus CKX53 with 10 × objective. p27 expression level as determined with Western blot and quantified with densitometry; * indicates p < 0.05, *** indicates p < 0.0001. (B). Change of proliferation rate in AtT-20/D16v-F2 cells with efficient p27 downregulation.; * indicates p < 0.05. (C). The effect of the cell cycle inhibitors palbociclib, roscovitine and flavopiridol on the proliferation rate of cells with downregulated p27 and control cells (scrambled shRNA, SCR) as measured with the BrdU assay. (D). The effect of cell cycle inhibitors on the viability of cells with downregulated p27 and control cells (SCR) as measured with the MTT assay.

Figure 6

Treatment of AtT-20/D16v-F2 cells overexpressing p27 with cell cycle inhibitors. (A). Expression of p27 in AtT-20/D16v-F2 cells after transfection with expression plasmid vector with Cdkn1b coding sequence (pcDNA3.1_p27) and control cells (transfected with empty vector pcDNA3.1) as determined with Western blot and quantified with densitometry; ** indicates p < 0.001; (B). Proliferation rate in AtT-20/D16v-F2 cells with p27 overexpression and control cells; * indicates p < 0.05; (C). The effect of the cell cycle inhibitors palbociclib, roscovitine and flavopiridol on proliferation rate of cells overexpressing p27 and control cells as measured with the BrdU assay. (D). The effect of cell cycle inhibitors on viability of cells with p27 overexpression and control cells, measured with the MTT assay.