Cyclin E and SV40 Small t Antigen Cooperate to Bypass Quiescence and Contribute to Transformation by Activating CDK2 in Human Fibroblasts*

Abstract

Cyclin E overexpression is observed in multiple human tumors and linked to poor prognosis. We have previously shown that ectopic expression of cyclin E is sufficient to induce mitogen-independent cell cycle entry in a variety of tumor/immortal cell lines. Here we have investigated the rate-limiting step leading to cell cycle entry in quiescent normal human fibroblasts (NHF) ectopically expressing cyclin E. We found that in serumstarved NHF, cyclin E forms inactive complexes with CDK2 and fails to induce DNA synthesis. Coexpression of SV40 small t antigen (st), but not other tested oncogenes, efficiently induces mitogen-independent CDK2 phosphorylation on Thr-160, CDK2 activation, and DNA synthesis. Additionally, in contactinhibited NHF ectopically expressing cyclin E, st induces cell cycle entry, continued proliferation, and foci formation. Coexpression of cyclin E and st also bypasses G 0 /G 1 arrests induced by CDK inhibitors. Although CDK2 is dispensable for G 0 /G 1 cell cycle entry and normal proliferation in mammals, CDK2 activity is an essential rate-limiting step in NHF with deregulated cyclin E expression and altered PP2A activity, which endows primary cells with transformed features. Consequently, CDK2 could be targeted therapeutically in tumors that involve these alterations. These data also suggest that alterations prior to cyclin E deregulation facilitate proliferation of tumor cells by bypassing mitogenic requirements and negative regulation by adjacent cells.Cyclin E expression is finely regulated during the cell cycle, and its expression is dramatically reduced in quiescent cells. As cells enter G 1 from G 0 following mitogenic stimulation, D-type cyclin-CDKs 3 partially inactivate pRB/p130, which activates expression of E2F-dependent genes, including the cyclin E gene. Cyclin E binds and activates CDK2 and phosphorylates several substrates, including members of the pRB family, which further facilitates cyclin E accumulation and CDK2 activation. Cyclin E-CDK2 also phosphorylates replication factors, centrosomal proteins, and NPAT/p220, a transcription factor that controls histone synthesis. Cyclin E is subsequently degraded by the proteasome, a process that is tightly regulated and involves two separate ubiquitin ligases (reviewed in Ref. 1). The activity of the cyclin E-CDK2 complex is positively regulated by CAK, which opens the activating T loop in CDK2 via phosphorylation of threonine 160, and negatively regulated by phosphorylation of threonine 14/tyrosine 15 and binding to CDK inhibitors (CKIs), p21 and p27, of the KIP family (reviewed in Ref.2). Cyclin E is overexpressed in many tumors as a result of gene amplification, disrupted proteolysis, and/or alterations in the pRB/E2F pathway. Cyclin E overexpression has been linked to poor prognosis in breast cancer, non-small cell lung carcinoma, larynx squamous cell carcinoma, and adrenocortical tumors (reviewed in Ref.3). Two main mechanisms for cyclin E-associated tumorigenesis have been considered to date: induction of genomic instability and...

Coordinated Activation of the Origin Licensing Factor CDC6 and CDK2 in Resting Human Fibroblasts Expressing SV40 Small T Antigen and Cyclin E

“…However, we have not observed overt disruption of cyclin E/CDK2/CKI complexes, and cyclin E and st coexpression can induce cell cycle entry when CKIs are expressed at very high levels (12). This suggests that st creates a pool of cyclin E/CDK2 complexes that is protected from CKI inhibition.…”

“…In contrast, expression of cyclin E in NHF at levels found in cancer cells is not sufficient to induce mitogen-independent exit from quiescence (12). However, when cyclin E is deregulated in quiescent NHF, co-expression of SV40 st induces mitogen-independent cell cycle entry.…”

“…The bypass of quiescence induced by the expression of st and cyclin E is dependent on CDK2 activation (12). Thus, contrary to what is seen in normal murine cells (13), CDK2 activity appears essential for cell cycle progression when it is oncogenically driven by cyclin E and st expression (12).…”

“…Cyclin E deregulation in certain human tumor cell lines and immortalized rat fibroblasts is associated with mitogen-independent cell cycle entry and progression through the cell cycle (11). However, when cyclin E is ectopically expressed in quiescent normal human fibroblasts (NHF), cells remain in G0 (12).…”

“…The bypass of quiescence induced by the expression of st and cyclin E is dependent on CDK2 activation (12). Thus, contrary to what is seen in normal murine cells (13), CDK2 activity appears essential for cell cycle progression when it is oncogenically driven by cyclin E and st expression (12). Because st is known to target pathways uniquely required for the transformation of human cells (14,15), tumor cells with altered pathways that mimic st/cyclin E expression could predictably be sensitive to selective inhibition of CDK2 activity.…”