EZH2 regulates pancreatic cancer cells through E2F1, GLI1, CDK3, and Mcm4.

Pancreatic cancer (PC) is one of the most common malignant tumors in digestive tract. To explore the role of epigenetic factor EZH2 in the malignant proliferation of PC, so as to provide effective medical help in PC. Sixty paraffin sections of PC were collected and the expression of EZH2 in PC tissues was detected by immunohistochemical assay. Three normal pancreas tissue samples were used as controls. The regulation of EZH2 gene on proliferation and migration of normal pancreatic cell and PC cell were determined by MTS, colony forming, Ki-67 antibody, scratch and Transwell assays. Through differential gene annotation and differential gene signaling pathway analysis, differentially expressed genes related to cell proliferation were selected and verified by RT-qPCR. EZH2 is mainly expressed in the nuclei of pancreatic tumor cells, but not in normal pancreatic cells. The results of cell function experiments showed that EZH2 overexpression could enhance the proliferation and migration ability of PC cell BXPC-3. Cell proliferation ability increased by 38% compared to the control group. EZH2 knockdown resulted in reduced proliferation and migration ability of cells. Compared with control, proliferation ability of cells reduced by 16%-40%. The results of bioinformatics analysis of transcriptome data and RT-qPCR demonstrated that EZH2 could regulate the expression of E2F1, GLI1, CDK3 and Mcm4 in normal and PC cells. The results revealed that EZH2 might regulate the proliferation of normal pancreatic cell and PC cell through E2F1, GLI1, CDK3 and Mcm4.

An overview of CDK3 in cancer: clinical significance and pharmacological implications.

Cyclin-dependent kinase 3 (CDK3) is a major player driving retinoblastoma (Rb) phosphorylation during the G0/G1 transition and in the early G1 phase of the cell cycle, preceding the effects of CDK4/cyclin D, CDK6/cyclin D, and CDK2/cyclin E. CDK3 can also directly regulate the activity of E2 factor (E2F) by skipping the role of Rb in late G1, potentially via the phosphorylation of the E2F1 partner DP1. Beyond the cell cycle, CDK3 interacts with various transcription factors involved in cell proliferation, differentiation, and transformation driven by the epidermal growth factor receptor (EGFR)/rat sarcoma virus (Ras) signaling pathway. The expression of CDK3 is extremely low in normal human tissue but upregulated in many cancers, implying a profound role in oncogenesis. Further evaluation of this role has been hampered by the lack of selective pharmacological inhibitors. Herein, we provide a comprehensive overview about the therapeutic potential of targeting CDK3 in cancer.

Norcantharidin regulates ERα signaling and tamoxifen resistance via targeting miR-873/CDK3 in breast cancer cells.

MiR-873/CDK3 has been shown to play a critical role in ERα signaling and tamoxifen resistance. Thus, targeting this pathway may be a potential therapeutic approach for the treatment of ER positive breast cancer especially tamoxifen resistant subtype. Here we report that Norcantharidin (NCTD), currently used clinically as an ani-cancer drug in China, regulates miR-873/CDK3 axis in breast cancer cells. NCTD decreases the transcriptional activity of ERα but not ERß through the modulation of miR-873/CDK3 axis. We also found that NCTD inhibits cell proliferation and tumor growth and miR-873/CDK3 axis mediates cell proliferation suppression of NCTD. More important, we found that NCTD sensitizes resistant cells to tamoxifen. NCTD inhibits tamoxifen induced the transcriptional activity as well ERα downstream gene expressions in tamoxifen resistant breast cancer cells. In addition, we found that NCTD restores tamoxifen induced recruitments of ERα co-repressors N-CoR and SMRT. Knockdown of miR-873 and overexpression of CDK3 diminish the effect of NCTD on tamoxifen resistance. Our data shows that NCTD regulates ERα signaling and tamoxifen resistance by targeting miR-873/CDK3 axis in breast cancer cells. This study may provide an alternative therapy strategy for tamoxifen resistant breast cancer.

Upregulation of LINC00319 indicates a poor prognosis and promotes cell proliferation and invasion in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (CSCC), an epidermal keratinocyte-derived skin tumor, is one of the most leading causes of cancer-associated morbidity and mortality worldwide. Long noncoding RNAs have emerged as key regulators of tumor development and progression. Recent studies have identified LINC00319, a long intergenic noncoding RNA, as an oncogene in lung cancer. However, the biological role of LINC00319 in CSCC remains largely unknown. The current study aimed to explore the role of LINC00319 in CSCC and uncover the molecular mechanisms. In current study, we found that LINC00319 was significantly upregulated in both CSCC tissues and cell lines. Besides, the χ2 test showed that increased expression of LINC00319 was associated with larger tumor size, advanced TNM stage, and lymphovascular invasion. Gain-of-function and loss-of-function approaches were applied to investigate the effects of LINC00319 on CSCC cells. Functional studies demonstrated that LINC00319 promoted CSCC cell proliferation, accelerated cell cycle progression, facilitated cell migration and invasion, and inhibited cell apoptosis. Mechanistic studies revealed that LINC00319 exerts its oncogenic functions in CSCC via miR-1207-5p-mediated regulation of cyclin-dependent kinase 3. Taken together, upregulation of LINC00319 implies a potential link with poor prognosis and reflects CSCC progression. Collectively, this study may provide some evidence for LINC00319 as a candidate target in CSCC treatment.

miR-125a-3p inhibits ERα transactivation and overrides tamoxifen resistance by targeting CDK3 in estrogen receptor-positive breast cancer.

Tamoxifen (TAM) is a major adjuvant therapy for patients who are diagnosed with estrogen receptor-α (ER)-positive breast cancer; however, TAM resistance occurs often during treatment and the underlying mechanism is unclear. Here, we report that miR-125a-3p inhibits ERα transcriptional activity and, thus, ER+ breast cancer cell proliferation, which causes cell-cycle arrest at the G1/S stage, inducing apoptosis and suppressing tumor growth by targeting cyclin-dependent kinase 3 (CDK3) in vitro and in vivo. In addition, CDK3 and miR-125a-3p expression levels were measured in 37 cancerous tissues paired with noncancerous samples, and their expression levels were negatively associated with miR-125a-3p level. Of interest, miR-125a-3p level is down-regulated in MCF-7 TAM-resistant (TamR) cells. Of more importance, up-regulation of miR-125a-3p resensitizes MCF-7 TamR cells to TAM, which is dependent on CDK3 expression. These results suggest that miR-125a-3p can function as a novel tumor suppressor in ER+ breast cancer by targeting CDK3, which may be a potential therapeutic approach for TamR breast cancer therapy.-Zheng, L., Meng, X., Li, X., Zhang, Y., Li, C., Xiang, C., Xing, Y., Xia, Y., Xi, T. miR-125a-3p inhibits ERα transactivation and overrides tamoxifen resistance by targeting CDK3 in estrogen receptor-positive breast cancer.

HuR promotes breast cancer cell proliferation and survival via binding to CDK3 mRNA.

HuR, a ubiquitously expressed RNA-binding protein, stabilizes mRNA and regulates its translation. HuR expression was increased at all stages of breast cancer and correlated with poor clinical outcome. However, the detailed mechanisms remain unclear. Here we reported that overexpression of HuR increased CDK3 mRNA stability and thus its protein expression in MDA-MB-231 and MCF-7 cells. Mechanistically, CDK3 mRNA was identified as a target of HuR via bioinformatics and RNA binding protein immunoprecipitation (RIP) assays. Furthermore, treatment with HuR shRNA decreased CDK3 expression, inhibited cell proliferation and promoted cell apoptosis in breast cancer. More importantly, overexpression of CDK3 reversed the suppressive effects of HuR knockdown on cell growth in both MDA-MB-231 and MCF-7 cells. Finally, HuR and CDK3 expression levels were positively correlated and significantly up-regulated in breast cancer samples. And overexpression of HuR attenuated the chemotherapeutical efficiency of breast cancer. Therefore, our results indicate that ectopic expression of HuR promotes breast cancer cell proliferation and survival by directly binding to and stabilizing CDK3 mRNA.

CDK3 is a major target of miR-150 in cell proliferation and anti-cancer effect.

MiR-150, a member of small non-coding RNAs, has been proven to dysregulate in different types of tumor and bear on carcinogenesis and cancer prognosis by regulating the expression of a series of gene including utrophin. Given that utrophin can compensate for dystrophin's absence and be regarded as a promising therapeutic target for Duchenne Muscular Dystrophy (DMD), we further detected the deep role of miR-150 in dystrophic muscle. Using a range of bioinformatic, molecular and cell biology techniques, we declared that miR-150 directly targets cyclin-dependent kinase 3 (CDK3) and leads to the regulation of CDK3 gene expression in both muscle-derived and non-muscle cells. The results indicated the expression of miR-150 was upregulated in mdx muscle and closely related to the lower level of CDK3. Transient transfection of miR-150 into cultured C2C12 cells led to significant decrease in cell proliferation, which is partly mediated via the 3'-UTRs of CDK3 mRNA. Targeting of CDK3 could also play a role, at least in part, in the anti-cancer activity suggested for miR-150 in previous studies. Consistently, the analysis of tumor and matched normal lung tissues indicates that miR-150 downregulation in lung tumors correlates with higher CDK3 levels. In addition, miR-150 transfection experiments with cancer-derived cell lines reveal that miR-150-mediated CDK3 suppression directly induces to growth inhibition. Collectively, our results highlight a novel activity for CDK3 in myoblast cell proliferation and confirm CDK3 as a key target that further enhances the tumor suppressor function proposed for miR-150.

Phosphorylation of NFAT3 by CDK3 induces cell transformation and promotes tumor growth in skin cancer.

The nuclear factor of activated T cells (NFAT) family proteins are transcription factors that regulate the expression of pro-inflammatory cytokines and other genes during the immune response. Although the NFAT proteins have been extensively investigated in the immune system, their role in cancer progression remains controversial. Here, we report that NFAT3 is highly expressed in various skin cancer cell lines and tumor tissues. Knockdown of endogenous NFAT3 expression by short hairpin RNA (shRNA) significantly inhibited tumor cell proliferation, colony formation and anchorage-independent cell growth. Furthermore, results of the mammalian two-hybrid assay showed that cyclin-dependent kinase 3 (CDK3) directly interacted with NFAT3 and phosphorylated NFAT3 at serine 259 (Ser259), which enhanced the transactivation and transcriptional activity of NFAT3. The phosphorylation site of NFAT3 was critical for epidermal growth factor (EGF)-stimulated cell transformation of the HaCaT immortalized skin cell line and mutation of NFAT3 at Ser259 led to a reduction of colony formation in soft agar. We also found that overexpressing wildtype NFAT3, but not mutant NFAT3-S259A, promoted A431 xenograft tumor growth. Importantly, we showed that CDK3, NFAT3 and phosphorylated NFAT3-Ser259 were highly expressed in skin cancer compared with normal skin tissues. These results provided evidence supporting the oncogenic potential of NFAT3 and suggested that CDK3-mediated phosphorylation of NFAT3 has an important role in skin tumorigenesis.

Cdk3-promoted epithelial-mesenchymal transition through activating AP-1 is involved in colorectal cancer metastasis.

Cyclin dependent kinase-3 (Cdk3) is a positive regulator of the G1 mammalian cell cycle phase. Cdk3 is involved in cancer progression, but very little is known about its mechanism in cancer development and progression. Herein, we found that Cdk3 increased colorectal cancer metastasis through promoting epithelial-mesenchymal transition (EMT) shift. Cdk3 was found to highly express in metastatic cancer and induce cell motility and invasion. Cdk3 was shown to phosphorylate c-Jun at Ser 63 and Ser 73 in vitro and ex vivo. Cdk3-phosphorylated c-Jun at Ser 63 and Ser 73 resulted in an increased AP-1 activity. Ectopic expression of Cdk3 promoted colorectal cancer from epithelial to mesenchymal transition conjugating AP-1 activation, while AP-1 inhibition dramatically decreased Cdk3-increased EMT shift. These results showed that the Cdk3/c-Jun signaling axis mediating epithelial-mesenchymal transition plays an important role in colorectal cancer metastasis.

miR-214/199a/199a* cluster levels predict poor survival in hepatocellular carcinoma through interference with cell-cycle regulators.

AIMS: To identify the clinical and functional association of miR-214/199a/199a* cluster in human hepatocellular carcinoma (HCC) and to clarify the mechanism of miR-214. METHODS: Kaplan-Meier and Cox proportional regression analyses were used to determine the association of miR-214/199a/199a* cluster levels with the survival of HCC patients. The role of miR-214 in regulating HCC cell proliferation was studied with miR-214 mimics/inhibitor-treated cells. Furthermore, the inhibition effect of miR-214 on E2F2, cyclin-dependent kinase (CDK) 3 and CDK6 expression was assessed in HCC cell lines with miR-214 mimics/inhibitors to increase/decrease miR-214 expression. Direct binding of miR-214 to the 3'-untranslated regions of E2F2, CDK3, and CDK6 was verified by dual-luciferase reporter assay. RESULTS: In analyzing HCC clinical specimens and cell lines, we discovered a uniform decrease in miR-214/199a/199a* expression in comparison with noncancerous tissue or normal liver epithelial cell lines. Higher miR-214 levels were related with improved patient survival. Overexpression of miR-214 in HCC cells inhibited proliferation by inducing G1-S checkpoint arrest. Conversely, RNA interference-mediated silencing of miR-214 promoted cell-cycle progression and accelerated the proliferation of HCC cells. E2F2, CDK3 and CDK6 were each directly targeted for inhibition by miR-214, and restoring their expression reversed miR-214 inhibition of cell-cycle progression. The relationship between expression of miR-214 and its targets was confirmed in HCC tumor xenografts and clinical specimens. CONCLUSIONS: Our results demonstrate that miR-214 has tumor-suppressive activity in HCC through inhibition of E2F2, CDK3 and CDK6.

MiR-873 regulates ERα transcriptional activity and tamoxifen resistance via targeting CDK3 in breast cancer cells.

miRNAs (microRNAs) are frequently and aberrantly expressed in many cancers. MiR-873 has been revealed to be downregulated in colorectal cancer and glioblastoma. However, its function remains unclear. Here we report that miR-873 is downregulated in breast tumor compared with normal tissue. Enforced expression of miR-873 decreases the transcriptional activity of ER (estrogen receptor)-α but not ERß through the modulation of ERα phosphorylation in ER-positive breast cancer cells. We also found that miR-873 inhibits breast cancer cell proliferation and tumor growth in nude mice. Reporter gene assays revealed cyclin-dependent kinase 3 (CDK3) as a direct target of miR-873. CDK3 was shown to be overexpressed in breast cancer and phosphorylate ERα at Ser104/116 and Ser118. Furthermore, we found that Mir-873 inhibits ER activity and cell growth via targeting CDK3. Interestingly, miR-873 was observed to be downregulated in tamoxifen-resistant MCF-7/TamR cells, while CDK3 is overexpressed in these cells. More importantly, re-expression of miR-873 reversed tamoxifen resistance in MCF-7/TamR cells. Our data demonstrate that miR-873 is a novel tumor suppressor in ER-positive breast cancer and a potential therapeutic approach for treatment of tamoxifen-resistant breast cancer.

Cyclin C is a haploinsufficient tumour suppressor.

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

CDK3 expression and its clinical significance in human nasopharyngeal carcinoma.

The aim of the current study was to investigate the expression of cyclin-dependent kinase 3 (CDK3) in human nasopharyngeal carcinoma (NPC) and to evaluate its association with the clinicopathological characteristics of patients with NPC. CDK3 expression was examined in three NPC cell lines and one nasopharyngeal epithelial cell line by western blot analysis and in 94 specimens of NPC and 40 specimens of inflamed nasopharyngeal tissue by immunohistochemistry staining. CDK3 was overexpressed in the three NPC cell lines, 5-8F, CNE1 and CNE2, compared with the NP-69 nasopharyngeal epithelial cell line, and was primarily expressed in the cytoplasm. The frequency of CDK3 expression was significantly higher in NPC specimens (67%) compared with inflamed nasopharyngeal tissue specimens (12.5%; P<0.001). CDK3 expression was associated with the degree of infiltration, lymph node metastasis and tumor node metastasis clinical staging, respectively, (P<0.001) in patients with NPC. These results revealed that the expression of CDK3 is associated with the progression of NPC, and may be a potential biomarker for prediction of the prognosis of patients with NPC.

[CDK3 expression and its clinical significance in human nasopharyngeal carcinoma].

OBJECTIVE: To investigate the expression profile and the clinical significance of CDK3 in the tissues of nasopharyngeal carcinoma. METHOD: Immunohistochemistry was utilized to detect the expression of CDK3 in 94 cases of NPC and 40 cases of nasopharyngeal inflammation. RESULT: CDK3 was highly expressed in NPC cell lines. Immunohistochemistry showed that CDK3 was mostly expressed in the cytoplasm. The positive expression rate of NPC was 67% and that of nasopharyngeal inflammation was only 12. 5%. The difference between these two groups was highly statistically significant. The CDK3 expression in NPC was related to the TNM clinical staging of NPC (P < 0.01). CONCLUSION: The expression levels of CDK3 were obviously higher in NPC tissues. The CDK3 expression in NPC was related to the TNM clinical staging of NPC. It suggests that CDK3 expression may play an important role in the occurrence and development of nasopharyngeal carcinoma.

The retroviral cyclin of walleye dermal sarcoma virus binds cyclin-dependent kinases 3 and 8.

Walleye dermal sarcoma virus encodes a retroviral cyclin (rv-cyclin) with a cyclin box fold and transcription activation domain (AD). Co-immune precipitation (co-IP) identified an association of rv-cyclin with cyclin-dependent kinase 8 (cdk8). Cdk8 is dependent upon cyclin C and regulates transcription with the Mediator complex, a co-activator of transcription. Mutation of cyclin residues, required for cdk binding, disrupts rv-cyclin-cdk8 co-IP. Mutation or removal of the AD has no effect on cdk8 interaction. Direct rv-cyclin-cdk8 binding is demonstrated by pulldown of active cdk8 and by GST-rv-cyclin binding to recombinant cdk8. Cdk3 is also activated by cyclin C and phosphorylates retinoblastoma protein to initiate entry into the cell division cycle. Co-IP and pulldowns demonstrate direct rv-cyclin binding to cdk3 as well. The rv-cyclin functions as a structural ortholog of cyclin C in spite of its limited amino acid sequence identity with C cyclins or with any known cyclins.

Overall Cdk activity modulates the DNA damage response in mammalian cells.

In response to DNA damage, cells activate a phosphorylation-based signaling cascade known as the DNA damage response (DDR). One of the main outcomes of DDR activation is inhibition of cyclin-dependent kinase (Cdk) activity to restrain cell cycle progression until lesions are healed. Recent studies have revealed a reverse connection by which Cdk activity modulates processing of DNA break ends and DDR activation. However, the specific contribution of individual Cdks to this process remains poorly understood. To address this issue, we have examined the DDR in murine cells carrying a defined set of Cdks. Our results reveal that genome maintenance programs of postreplicative cells, including DDR, are regulated by the overall level of Cdk activity and not by specific Cdks.

Novel Hsp90 partners discovered using complementary proteomic approaches.

Hsp90 is an essential eukaryotic molecular chaperone that stabilizes a large set of client proteins, many of which are involved in various cellular signaling pathways. The current list of Hsp90 interactors comprises about 200 proteins and this number is growing steadily. In this paper, we report on the application of three complementary proteomic approaches directed towards identification of novel proteins that interact with Hsp90. These methods are coimmunoprecipitation, pull down with biotinylated geldanamycin, and immobilization of Hsp90beta on sepharose. In all, this study led to the identification of 42 proteins, including 18 proteins that had not been previously characterized as Hsp90 interactors. These novel Hsp90 partners not only represent abundant protein species, but several proteins were identified at low levels, among which signaling kinase Cdk3 and putative transcription factor tripartite motif-containing protein 29. Identification of tetratricopeptide-repeat-containing mitochondrial import receptor protein Tom34 suggests the involvement of Hsp90 in the early steps of translocation of mitochondrial preproteins. Taken together, our data expand the knowledge of the Hsp90 interactome and provide a further step in our understanding of the Hsp90 chaperone system.