CDK6 is activated by the atypical cyclin I to promote E2F-mediated gene expression and cancer cell proliferation.

Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.

Atypical cyclin P regulates cancer cell stemness through activation of the WNT pathway.

PURPOSE: Cancer stem cells represent a cancer cell subpopulation that has been found to be associated with metastasis and chemoresistance. Therefore, it is vital to identify mechanisms regulating cancer stemness. Previously, we have shown that the atypical cyclin P (CCNP), also known as CNTD2, is upregulated in lung and colorectal cancers and is associated with a worse clinical prognosis. Given that other cyclins have been implicated in pluripotency regulation, we hypothesized that CCNP may also play a role in cancer stemness. METHODS: Cell line-derived spheroids, ex vivo intestinal organoid cultures and induced-pluripotent stem cells (iPSCs) were used to investigate the role of CCNP in stemness. The effects of CCNP on cancer cell stemness and the expression of pluripotency markers and ATP-binding cassette (ABC) transporters were evaluated using Western blotting and RT-qPCR assays. Cell viability was assessed using a MTT assay. The effects of CCNP on WNT targets were monitored by RNA-seq analysis. Data from publicly available web-based resources were also analyzed. RESULTS: We found that CCNP increases spheroid formation in breast, lung and colorectal cancers, and upregulates the expression of stemness (CD44, CD133) and pluripotency (SOX2, OCT4, NANOG) markers. In addition, we found that CCNP promotes resistance to anticancer drugs and induces the expression of multidrug resistance ABC transporters. Our RNA-seq data indicate that CCNP activates the WNT pathway, and that inhibition of this pathway abrogates the increase in spheroid formation promoted by CCNP. Finally, we found that CCNP knockout decreases OCT4 expression in iPSCs, further supporting the notion that CCNP is involved in stemness regulation. CONCLUSION: Our results reveal CCNP as a novel player in stemness and as a potential therapeutic target in cancer.

Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.

The atypical cyclin CNTD2 promotes colon cancer cell proliferation and migration.

Colorectal cancer (CRC) is one of the most common cancers worldwide, with 8-10% of these tumours presenting a BRAF (V600E) mutation. Cyclins are known oncogenes deregulated in many cancers, but the role of the new subfamily of atypical cyclins remains elusive. Here we have performed a systematic analysis of the protein expression levels of eight atypical cyclins in human CRC tumours and several cell lines, and found that CNTD2 is significantly upregulated in CRC tissue compared to the adjacent normal one. CNTD2 overexpression in CRC cell lines increases their proliferation capacity and migration, as well as spheroid formation capacity and anchorage-independent growth. Moreover, CNTD2 increases tumour growth in vivo on xenograft models of CRC with wild-type BRAF. Accordingly, CNTD2 downregulation significantly diminished the proliferation of wild-type BRAF CRC cells, suggesting that CNTD2 may represent a new prognostic factor and a promising drug target in the management of CRC.