CDK4 expression in chordoma: A potential therapeutic target.

Chordomas are rare bone tumors and treatment is commonly based on a combination of surgery and radiotherapy. There is no standard chemotherapy treatment for chordoma. The aim of this study was to determine the expression of cyclin-dependent kinase 4 (CDK4) in chordoma and its therapeutic implications. We evaluated CDK4 expression both in chordoma cell lines and in chordoma tissues. Also, we investigated the functional roles of CDK4 in chordoma cell growth and proliferation. Furthermore, the therapeutic implications of targeting CDK4 in chordoma were evaluated. We found CDK4 highly expressed in chordoma cell lines and in a majority (97.7%) of chordoma tissues. Higher CDK4 expression correlated with metastasis and recurrence of chordoma. Treatment of chordoma cells using CDK4 inhibitor palbociclib could efficiently inhibit chordoma cells growth and proliferation. These data demonstrate that targeting CDK4 may be useful as a novel strategy in the treatment of chordoma. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1581-1589, 2018.

Two mature products of MIR-491 coordinate to suppress key cancer hallmarks in glioblastoma.

MIR-491 is commonly co-deleted with its adjacent CDKN2A on chromosome 9p21.3 in glioblastoma multiforme (GBM). However, it is not known whether deletion of MIR-491 is only a passenger event or has an important role. Small-RNA sequencing of samples from GBM patients demonstrated that both mature products of MIR-491 (miR-491-5p and -3p) are downregulated in tumors compared with the normal brain. The integration of GBM data from The Cancer Genome Atlas (TCGA), miRNA target prediction and reporter assays showed that miR-491-5p directly targets EGFR, CDK6 and Bcl-xL, whereas miR-491-3p targets IGFBP2 and CDK6. Functionally, miR-491-3p inhibited glioma cell invasion; overexpression of both miR-491-5p and -3p inhibited proliferation of glioma cell lines and impaired the propagation of glioma stem cells (GSCs), thereby prolonging survival of xenograft mice. Moreover, knockdown of miR-491-5p in primary Ink4a-Arf-null mouse glial progenitor cells exacerbated cell proliferation and invasion. Therefore, MIR-491 is a tumor suppressor gene that, by utilizing both mature forms, coordinately controls the key cancer hallmarks: proliferation, invasion and stem cell propagation.

Cyclin-dependent kinase 6 is a chromatin-bound cofactor for NF-κB-dependent gene expression.

Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.

Activation of adenosine A2A receptors restores the altered cell-cycle machinery during impaired wound healing in genetically diabetic mice.

BACKGROUND: Cyclins drive cell-cycle progression by associating with their kinase partners, cyclin-dependent kinases (CDK). We investigated cyclin D1/CDK6, cyclin E/CDK2 complexes, and the cell-cycle negative regulators p15 and p27 in an incisional skin wound model. METHODS: Wounds were produced on the back of female diabetic mice and their normoglycemic littermates. Animals were treated with polydeoxyribonucleotide (PDRN, 8 mg/kg/i.p.), an agonist of adenosine A2(A) receptors, or its vehicle daily. Granulation tissue proliferation by Ki67 immunostaining, cyclin D/CDK6 and cyclin E/CDK2 complexes, and p21 and p16 proteins (Western blot analysis), and the histologic changes were assessed at different days (3, 6, and 12 days after injury). RESULTS: Numerous Ki67 positive cells were observed at day 3 and day 6 in the granulation tissue of normoglycemic mice. Ki67 positive cells were fewer in diabetic than in normoglycemic mice. PDRN increased Ki67 positive cells in diabetic mice. Normoglycemic mice showed the greatest upregulation of cyclin D1, CDK6, cyclin E, and CDK2 at day 6. Diabetic mice had a markedly lower expression of cyclin D1, CDK6, cyclin E, and CDK2 at day 6. They also showed a greater expression of p15 and p27 at day 6. PDRN administration in diabetic mice increased cyclin D1/CDK6 and cyclin E/CDK2 expression and reduced p15 and p27 inhibitors at day 6 after injury; moreover, it improved the impaired wound healing at day 12. CONCLUSION: Our results suggest that adenosine A2(A) receptor activation by PDRN might represent a therapeutic strategy to overcome the diabetes-impaired cell-cycle machinery.

Distinct subcellular distribution of cyclin dependent kinase 6.

Several studies have recently reported that the cyclin dependent kinase (cdk) 6 oncogene plays a role in differentiation of a variety of cell types. This novel function expands the previously understood function of cdk6 as a regulator of G(1) phase of the cell cycle. The proposed mechanisms of these functions both require nuclear localization. That is, cdk6 phosphorylation of the retinoblastoma protein (pRb) to regulate cell cycle, and the recently proposed transcriptional regulation to block differentiation, are both nuclear functions that predict nuclear localization of the kinase. This report provides a thorough analysis of cdk6 localization and compares the localization of a commonly used mutant cdk6 to the corrected wildtype sequence as recorded in GenBank. The widely shared mutant of cdk6 contains a tyrosine residue at amino acid 224 (instead of an aspartic acid) introducing a potential phosphorylation site to the cdk6 sequence. Results indicate a majority of cdk6 is localized to the cytoplasm with concentrations of cdk6 in the edges of the cytoplasm and in the cytoplasmic extensions of cells. The results of this study may help to better understand the emerging roles of cdk6 in cell cycle control, differentiation and cancer.

Clinicopathologic features of CDK6 translocation-associated B-cell lymphoproliferative disorders.

Cyclin-dependent protein kinase 6 (CDK6), in cooperation with cyclin Ds, drives cell cycle progression from G1 to S phase through phosphorylation and subsequent inactivation of retinoblastoma 1 protein. Alteration of this pathway results in both nonhematologic and hematologic malignancies, which include a small subset of B-cell lymphoproliferative disorders (BLPDs). We identified 5 cases of BLPD that carried CDK6 chromosomal translocations and characterized their clinical, pathologic, immunophenotypic, and genetic features. Common clinical characteristics included marked neoplastic lymphocytosis, systemic lymphadenopathy, splenomegaly, and bone marrow involvement. Three patients were diagnosed with low-grade B-cell lymphoma and had an indolent clinical course, and 2 patients (one who transformed to large B-cell lymphoma, and the other who was initially diagnosed with a high-grade B-cell lymphoma) had an aggressive clinical course. Immunophenotypically, the neoplastic B cells expressed CD5, CDK6, and cytoplasmic retinoblastoma 1 protein in all cases, expressed phospho-RB, p27kip1, and cyclin D2 in most cases, and uniformly lacked expression of all other cyclins. In 4 cases, the CDK6 translocation partner was kappa immunoglobulin light-chain gene; and in the fifth case, the CDK6 translocation partner was unknown. These distinct clinicopathologic and cytogenetic features distinguish the CDK6 translocation-associated BLPDs (CDK6-BLPDs) from other mature B-cell lymphomas.

Retinoic acid and sodium butyrate as cell cycle regulators in the treatment of oral squamous carcinoma cells.

All-trans retinoic acid (ATRA) and sodium butyrate (SB) have shown growth-inhibitory and differentiation-inducing properties to tumor cells when used as single agents or in combination, but the exact molecular mechanism still remains to be determined. In order to determine the mechanism of the synergy in treatment with RA and SB, we evaluated the growth inhibition capability of ATRA and SB, alone or in combination, in human oral squamous carcinoma cell lines SCC-1 and SCC-9, and identified the expression of cell cycle-related genes. ATRA and SB inhibited cell growth and induced cell cycle G1 arrest. The inhibition effect was more pronounced with SB than with ATRA (p = 0.000). There were interactions between ATRA and SB (p = 0.000). Consistent with the inhibition effect and G1 arrest, ATRA and SB, alone or in combination, induced the expression of G1 phase markers cyclin-dependent kinase (CDK) 6, p21, and p27; inhibited the expression of S-G2 phase proteins CDK2; and decreased Rb phosphorylation. Cyclin D1 expression was increased in the SB- and ATRA + SB-treated groups, but inhibited in the ATRA-treated group. Cyclin B1 and cyclin E expression was slightly decreased in the SB- and ATRA + SB-treated groups, but did not change in the ATRA-treated group. These results indicate that the growth inhibition and G1 arrest of oral squamous carcinoma cells in response to ATRA and/or SB correlates with the induction of G1 phase cell cycle regulatory proteins CDK6, p21, and p27 and the inhibition of S-G2 phase cell cycle regulatory protein CDK2.

Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels.

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22-q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin-dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto-oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barrett's dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high-resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.