microRNA-504 inhibits cancer cell proliferation via targeting CDK6 in hypopharyngeal squamous cell carcinoma
- Authors:
- Published online on: March 19, 2014 https://doi.org/10.3892/ijo.2014.2349
- Pages: 2085-2092
Abstract
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and approximately 500,000 cases are diagnosed every year (1). In spite of considerable advances in multimodality therapy, including surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients with HNSCC is only approximately 50% (2). Hypopharyngeal SCC (HSCC) comprises 20% of all HNSCC, with an incidence of approximately 10 cases per million people-years (3). HSCC has a very poor prognosis compared with other HNSCC, with 5-year survival rates ranging from 30–35% (4,5). Local tumor recurrence and distant metastasis after conventional therapy appear to be major contributing factors for restricted survival of HSCC patients. Survival rates of HSCC patients have not markedly improved despite recent advanced combination therapies (6). Therefore, understanding the molecular pathways of metastasis accompanying HSCC would help to improve diagnosis, approaches to therapy and prevention of the disease.
The discovery of non-coding RNAs (ncRNAs) in the human genome was an important conceptual breakthrough in the post-genome sequencing era (7). Improved understanding of ncRNAs is necessary for continued progress in cancer research. microRNAs (miRNAs) are endogenous small ncRNA molecules (18–25 bases in length) that regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner (8). Currently, 2,578 human mature miRNAs are registered at miRBase release 20.0 (http://www.mirbase.org/). miRNAs are unique in their ability to regulate multiple protein-coding genes. Bioinformatic predictions indicate that miRNAs regulate approximately 30–60% of the protein-coding genes in the human genome (9).
Numerous reports suggest that miRNAs are aberrantly expressed in many human cancers and that they play significant roles in the initiation, development, and metastasis of those cancers (10,11). Some highly expressed miRNAs can function as oncogenes by repressing tumor suppressors, whereas low level miRNAs can function as tumor suppressors by negatively regulating oncogenes (10,12). It is believed that normal regulatory mechanisms can be disrupted by the aberrant expression of tumor-suppressive or oncogenic miRNAs in cancer cells. Therefore, identification of aberrantly expressed miRNAs is an important first step toward elucidating miRNA-mediated oncogenic pathways.
Based on these considerations, we have constructed miRNA expression signatures using HNSCC clinical specimens and investigated the specific role of miRNAs in HNSCC oncogenesis using differentially expressed miRNAs (13,14). Our recent studies demonstrated that miR-1, miR-29s, miR-133a, miR-218, miR-489 and miR-874 functioned as tumor suppressors in HNSCC through their targeting of several types of oncogenic genes (13–23). Our HSCC and esophageal SCC miRNA expression signatures revealed that miR-504 was significantly downregulated in cancer tissues, suggesting that this miRNA is a candidate tumor suppressor in human SCC cells (13,24). However, several recent reports indicated that miR-504 functions as an oncogene (25,26). These data contradict our hypothesis that miR-504 functions as a tumor suppressor. The aim of this study was to investigate the functional significance of miR-504 in cancer cells and to identify novel targets regulated by miR-504 in HSCC cells.
We used two different sources of mature miR-504 to restore miR-504 function. Transfection of those miRNAs inhibited cancer cell proliferation. Genome-wide gene expression analysis of miR-504 transfectants and in silico database analysis showed that cyclin-dependent kinase 6 (CDK6) was a candidate target of miR-504 in HSCC cells. Tumor suppressive miR-504-regulated targets provide new insight into the potential mechanisms of HSCC oncogenesis and suggest novel therapeutic strategies for treatment of the disease.
Materials and methods
Clinical specimens
Twenty-three pairs of primary HSCC and corresponding normal epithelial samples were obtained from patients with HSCC at Chiba University Hospital (Chiba, Japan) from 2005 to 2013. The samples considered normal were free of cancer cells as determined by pathologic examination. The backgrounds and clinicopathological characteristics of the patient are summarized in Table I. The patients were classified according to the 2002 Union for International Cancer Control (UICC) TNM staging criteria prior to treatment. Written consent for tissue donation for research purposes was obtained from each patient before tissue collection. The protocol was approved by the Institutional Review Board of Chiba University. The specimens were immersed in RNAlater (Qiagen, Valencia, CA, USA) and stored at −20°C until RNA was extracted.
RNA isolation
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA concentrations were determined spectrophotometrically, and molecular integrity was checked by gel electrophoresis. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Cell culture
The following human cell lines were used: FaDu (derived from a primary lesion of hypopharyngeal SCC), SAS (derived from a primary lesion of tongue SCC), HSC3 (derived from lymph node metastasis of tongue SCC), HCT116 p53+/+ (derived from a colon adenocarcinoma) and its p53−/− derivative. All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C.
Quantitative real-time RT-PCR (qPCR)
cDNA synthesis and PCR procedures were described in our previous reports (13,27). The expression levels of miR-504 (Assay ID: 002084) were analyzed by TaqMan quantitative real-time PCR (TaqMan® MicroRNA Assay; Applied Biosystems) and normalized to RNU48 (Assay ID: 001006). TaqMan® probes and primers for CDK6 (P/N: Hs01026371_m1), RB1 (P/N: Hs01078066_m1), CDC23 (P/N: 00946641_m1), CCND1 (P/N: 00765553_m1) and GUSB (P/N: Hs00939627_m1) as an internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products). The ΔΔCt method was adopted and applied to calculate the relative quantities of subject genes. All reactions were performed in triplicate and included negative control reactions that lacked cDNA.
Mature miRNA transfection
To perform gain of function studies, we utilized two different sources of mature miR-504: miR-504-A, Ambion Pre-miR miRNA Precursor, PM12429 (Applied Biosystems) and miR-504-T, miRIDIAN Mimic, MIMAT0002875 (Thermo Scientific Dharmacon, Waltham, MA, USA). Pre-miR Negative Control #2 (AM17111, Applied Biosystems) was used for negative control experiments. The miRNA transfection procedures and confirmation of miRNA transfection efficiency were described in our previous reports (13,27).
Cell proliferation assay
Cells were transfected with 10 nM miRNA by reverse transfection and plated in 96-well plates at 3×103 cells per well. After 72 h, cell proliferation was determined with the XTT assay using the Cell Proliferation Kit II (Roche Molecular Biochemicals, Mannheim, Germany) as previously reported (13,27).
Flow cytometry
Cell cycle status was examined using an APC BrdU Flow kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s protocol. Briefly, SAS and FaDu cells were transiently transfected with miR-control, miR-504-A or miR-504-T. Seventy-two hours after transfection, 10 μM BrdU was added to the medium and incubated for 6 h. The cells were then trypsinized and fixed with paraformaldehyde and permeabilized with saponin. After DNase treatment, the cells were stained with anti-BrdU antibodies and 7AAD and analyzed with a FACSCalibur flow cytometer (BD Bioscience).
Target gene search for miR-504
To identify miR-504 target genes, we used genome-wide gene expression analysis and in silico analysis. First, we performed genome-wide gene expression analysis using miR-504 transfection of SAS and FaDu. SurePrint G3 Human GE 8×60K Microarray (Agilent Technologies) was used for expression profiling of miR-504 transfectants in comparison with negative control miRNA transfectants. The genes that were downregulated in miR-504 transfectant were then categorized into Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using GeneCodis analysis (28) (http://genecodis.cnb.csic.es/). The target site for miR-504 was analyzed with TargetScan Release 6.2 (http://www.targetscan.org/).
Western blotting
Cells were harvested 72 h after transfection and lysates were prepared. Protein (50 μg) from each lysate was separated on a Mini-Protean TGX gel (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Immunoblotting was performed with mouse CDK6 antibody (1:500, #3136, Cell Signaling Technology, Danvers, MA, USA) with GAPDH antibody (1:1,000, ab8245, Abcam, Cambridge, UK) used as an internal control. The membrane was washed and incubated with anti-mouse IgG HRP-linked antibody (#7076, Cell Signaling Technology). Complexes were visualized with an Immun-Star™ Western Chemiluminescence kit (Bio-Rad), and the expression levels of these genes were evaluated by ImageJ software (ver.1.44; http://rsbweb.nih.gov/ij/).
Statistical analysis
The relationships between two groups and the numerical values obtained by qPCR were analysed using the paired t-test. Spearman’s rank test was used to evaluate the correlation between the expression of miR-504 and target genes. The relationships among more than three variables and numerical values were analysed using the Bonferroni adjusted Mann-Whitney U test. All analyses were performed using Expert StatView (version 4, SAS Institute Inc., Cary, NC, USA).
Results
Expression of miR-504 in HSCC clinical specimens and cell lines
To validate our past miRNA profiling results, we evaluated miR-504 expression in 23 clinical HSCC specimens. The expression levels of miR-504 were significantly lower in tumor tissues than in corresponding adjacent normal epithelia (miR-504 expression normalized to RNU48: normal, 0.0026±0.0011; tumor, 0.00070±0.00052, P<0.0001, Fig. 1). The expression levels of miR-504 in HNSCC cell lines were also lower than those in normal epithelia (miR-504 expression normalized to RNU48: FaDu, 0.000026; SAS, 0.00016; HSC3, 0.000025; Fig. 1).
Effects of miR-504 restoration on the proliferation of HNSCC cell lines
To investigate the role of miR-504, we performed gain-of-function studies using mature miRNA transfection of three HNSCC cell lines (FaDu, SAS and HSC3). We utilized two sources of mature miR-504 (miR-504-A, Ambion; miR-504-T, Thermo Scientific Dharmacon) to ensure reproducibility of the data.
The XTT assay demonstrated that cell proliferation was significantly inhibited in miR-504 transfectants in comparison with the mock or miR-control transfectant cells. Specifically, we observed the following growth, expressed as a percentage of the mock: i) FaDu-mock, 100.0±1.0; miR control, 92.6±2.7; miR-504-A, 45.5±2.3; miR-504-T, 76.4±1.9; ii) SAS-mock, 100.0±6.8; miR control, 92.0±3.9; miR-504-A, 34.0±2.4; miR-504-T, 45.1±3.3; iii) HSC3-mock, 100.0±3.7; miR control, 103.4±3.9; miR-504-A, 81.6±2.7; miR-504-T, 89.6±4.9, with P<0.0083 (Fig. 2).
Because miR-504 has been reported to promote tumorigenicity by regulating TP53, we evaluated functional effects of miR-504 in HCT116 p53+/+ cells and its p53−/− derivative cell line. The XTT assay showed that cell proliferation was significantly inhibited by miR-504 transfection in both cell lines, suggesting that miR-504 functioned as a tumor suppressor regardless of p53 status. We observed the following growth, expressed as a percentage of the mock: i) HCT116 p53+/+-mock, 100.0±3.7; miR control, 94.5±3.8; miR-504-A, 68.7±2.2; miR-504-T, 74.9±3.8; ii) HCT116 p53−/−-mock, 100.0±4.4; miR control, 82.8±2.9; miR-504-A, 63.8±2.1; miR-504-T, 65.9±2.1, with P<0.0083 (Fig. 2).
Effects of miR-504 restoration on cell cycle status in HNSCC cell lines
To study whether miR-504 affected the cell cycle status of cancer cells, we performed flow cytometric analysis of cells stained with anti-BrdU and 7AAD allowing the discrimination of cell fractions that resided in G0/G1, S or G2/M phases of the cell cycle (Fig. 3A). The fraction of FaDu cells in the G0/G1 phase was significantly larger in miR-504 transfectants in comparison with the miR control transfectants, whereas the fraction of SAS cells in G2/M phase was significantly larger in miR-504 transfectants (Fig. 3B).
Identification of candidate target genes regulated by miR-504 in HNSCC cells
To identify miR-504 target genes, we used genome-wide gene expression analysis and in silico analysis. First, we performed genome-wide gene expression analysis using two cancer cell lines (FaDu and SAS) and selected genes downregulated by miR-504 transfection compared with miR control transfection. In this analysis, 810 genes and 1,145 genes were recognized as downregulated genes (log2 ratio <−0.5) in FaDu and SAS, respectively. Entries from the microarray data were approved by the Gene Expression Omnibus (GEO) and were assigned GEO accession no. GSE37119.
Next, genes downregulated in miR-504-transfectants were categorized into KEGG pathways using GeneCodis analysis and 24 pathways were identified as significantly enriched in both lines (Tables II and III). Among these pathways, we focused on the ‘cell cycle’ pathway because this pathway has been implicated in cancer cell proliferation. A total of 19 genes were identified in this pathway and four genes (RB1, CDK6, CDC23 and CCND1) had putative miR-504 target sites predicted by the TargetScan database (Table IV).
 | Table II.Significantly enriched annotations among downregulated genes by miR-504 transfection in FaDu. |
| Table III.Significantly enriched annotations among downregulated genes by miR-504 transfection in SAS. |
CDK6 is a candidate of miR-504 regulation in HNSCC cells
We investigated the expression levels of four candidate genes in HSCC clinical specimens. CDK6 was significantly upregulated in cancer tissues (P=0.0004, Fig. 4A). Furthermore, the expression of CDK6 was inversely correlated with that of miR-504 in HSCC specimens (r=−0.43, P=0.0039, Fig. 4B).
We performed qPCR and western blotting in FaDu and SAS to investigate whether CDK6 expression was downregulated by restoration of miR-504. CDK6 mRNA expression was significantly repressed by miR-504 transfection of FaDu cells, while no changes were observed in SAS cells (Fig. 5A). The expression levels of CDK6 protein were repressed in miR-504 transfectants in comparison with mock or miR-control transfectants in both FaDu and SAS cells (Fig. 5B).
Discussion
Aberrant expression of miRNAs can disrupt the tightly regulated system by which miRNA regulates protein-coding RNA networks in cancer cells (10,12). Therefore, studies of differentially expressed miRNAs in cancer cells provide important information regarding the molecular mechanisms underlying oncogenesis and metastasis. To elucidate the molecular mechanisms underlying HNSCC, we have identified tumor-suppressive miRNAs, focusing on their regulated molecular targets and novel cancer pathways based on HNSCC expression signatures (13–24,27).
Our recent studies of miRNA expression signatures of HSCC and esophageal SCC showed that miR-504 was significantly reduced in cancer tissues compared to normal tissues (13,24). Those results suggested that miR-504 was a candidate tumor suppressor. In glioblastoma, miR-504 expression was reported to be downregulated and functioned as a tumor suppressor by regulating mesenchymal genes (29). This finding is consistent with our results. However, miR-504 has also been reported to have oncogenic functions. For example, a recent study showed that miR-504 was a negative regulator of human TP53 and directly bound to its 3′-UTR region (25). Overexpression of miR-504 induced TP53 silencing and caused inhibition of p53-mediated apoptosis and cell cycle arrest in response to stress (25). Another report showed that ectopic expression of miR-504 increased migration and invasion in an oral cancer cell line by targeting FOXP1, a member of forkhead transcriptional factors (26).
We conducted two analyses to test the conclusions of the above reports. First, we restored function using two different sources of mature miR-504 in several cancer cell lines. In HNSCC cell lines, restoration of both types of miR-504 significantly inhibited cancer cell proliferation. Similar results were observed in HPV16- and HPV18-positive cervical-SCC cell lines (data not shown). Furthermore, we investigated the anti-proliferative effects of miR-504 and p53 status using HCT116 p53+/+ and HCT116 p53−/− cells. Our data demonstrated that the anti-proliferative effect was not affected by the p53 status. In this study, our data indicated that miR-504 had a tumor-suppressive function, particularly promotion of cell cycle arrest.
A unique aspect of miRNAs is that one miRNA regulates many protein-coding genes. Thus, it is important to elucidate the molecular targets and pathways regulated by a single tumor suppressive molecule, miR-504, in cancer cells. To solve this problem, we performed genome-wide gene expression analysis using miR-504-transfectants. We categorized differentially expressed genes of miR-504-transfectants into KEGG pathways. Several pathways were enriched in this analysis and we focused on ‘cell cycle’ pathways because miR-504-transfectants underwent cell cycle arrest. Finally, CDK6 was chosen as a miR-504 target oncogenic gene that met several conditions. These included the following: i) mRNA sequence contained a putative miR-504 binding site, ii) inhibition of its expression in miR-504 transfects, and iii) overexpression in HSCC clinical specimens.
It is well known that CDK-cyclin complexes are deregulated in cancer cells, resulting in either continued proliferation or unscheduled re-entry into the cell cycle (30). Several CDK4/CDK6 inhibitors have been shown to induce G1 arrest and inhibit proliferation of tumor cells (31,32). Our data indicated that restoration of miR-504 repressed CDK6 and induced G1 arrest in FaDu cell. On the other hand, in SAS cells, restoration of miR-504 induced G2 arrest. We have no reasonable data to explain this phenomenon but it is possible that some G2 phase related genes were affected by miR-504 in SAS cells. Thus, further study is needed.
In conclusion, downregulation of miR-504 was frequently observed in HSCC clinical specimens. Restoration of miRNA significantly inhibited cancer cell proliferation, suggesting that miR-504 functioned as a tumor suppressor in HSCC cells. To the best of our knowledge, this is the first report demonstrating that tumor-suppressive miR-504 regulated ‘cell cycle’ pathways and that CDK6 was a putative target. The identification of target oncogenes regulated by miR-504 might lead to a better understanding of HSCC oncogenesis and the development of new therapeutic strategies to treat this disease.
Acknowledgements
This study was supported by JSPS KAKENHI Grant nos. 23592505, 24592590, 25462676 and 25861528.
References
|
Jemal A, Siegel R, Xu J and Ward E: Cancer statistics, 2010. CA Cancer J Clin. 60:277–300. 2010. View Article : Google Scholar | |
|
Leemans CR, Braakhuis BJ and Brakenhoff RH: The molecular biology of head and neck cancer. Nat Rev Cancer. 11:9–22. 2011. View Article : Google Scholar | |
|
Davies L and Welch HG: Epidemiology of head and neck cancer in the United States. Otolaryngol Head Neck Surg. 135:451–457. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Hoffman HT, Karnell LH, Shah JP, et al: Hypopharyngeal cancer patient care evaluation. Laryngoscope. 107:1005–1017. 1997. View Article : Google Scholar : PubMed/NCBI | |
|
Bova R, Goh R, Poulson M and Coman WB: Total pharyngolaryngectomy for squamous cell carcinoma of the hypopharynx: a review. Laryngoscope. 115:864–869. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Godballe C, Jorgensen K, Hansen O and Bastholt L: Hypopharyngeal cancer: results of treatment based on radiation therapy and salvage surgery. Laryngoscope. 112:834–838. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Carthew RW and Sontheimer EJ: Origins and mechanisms of miRNAs and siRNAs. Cell. 136:642–655. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Filipowicz W, Bhattacharyya SN and Sonenberg N: Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 9:102–114. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Esquela-Kerscher A and Slack FJ: Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer. 6:259–269. 2006. View Article : Google Scholar | |
|
Tran N, O’Brien CJ, Clark J and Rose B: Potential role of micro-RNAs in head and neck tumorigenesis. Head Neck. 32:1099–1111. 2010. View Article : Google Scholar | |
|
Caldas C and Brenton JD: Sizing up miRNAs as cancer genes. Nat Med. 11:712–714. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Kikkawa N, Hanazawa T, Fujimura L, et al: miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC). Br J Cancer. 103:877–84. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Nohata N, Hanazawa T, Kikkawa N, et al: Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma. Br J Cancer. 105:833–841. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Kinoshita T, Hanazawa T, Nohata N, et al: Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion through targeting laminin-332 in head and neck squamous cell carcinoma. Oncotarget. 3:1386–1400. 2012.PubMed/NCBI | |
|
Kinoshita T, Nohata N, Fuse M, et al: Tumor suppressive microRNA-133a regulates novel targets: moesin contributes to cancer cell proliferation and invasion in head and neck squamous cell carcinoma. Biochem Biophys Res Commun. 418:378–383. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Kinoshita T, Nohata N, Watanabe-Takano H, et al: Actin-related protein 2/3 complex subunit 5 (ARPC5) contributes to cell migration and invasion and is directly regulated by tumor-suppressive microRNA-133a in head and neck squamous cell carcinoma. Int J Oncol. 40:1770–1778. 2012.PubMed/NCBI | |
|
Kinoshita T, Nohata N, Yoshino H, et al: Tumor suppressive microRNA-375 regulates lactate dehydrogenase B in maxillary sinus squamous cell carcinoma. Int J Oncol. 40:185–193. 2012.PubMed/NCBI | |
|
Mutallip M, Nohata N, Hanazawa T, et al: Glutathione S-transferase P1 (GSTP1) suppresses cell apoptosis and its regulation by miR-133alpha in head and neck squamous cell carcinoma (HNSCC). Int J Mol Med. 27:345–352. 2011.PubMed/NCBI | |
|
Nohata N, Hanazawa T, Kikkawa N, et al: Caveolin-1 mediates tumor cell migration and invasion and its regulation by miR-133a in head and neck squamous cell carcinoma. Int J Oncol. 38:209–217. 2011.PubMed/NCBI | |
|
Nohata N, Hanazawa T, Kikkawa N, et al: Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma. Int J Oncol. 39:1099–1107. 2011.PubMed/NCBI | |
|
Nohata N, Hanazawa T, Kinoshita T, et al: Tumour-suppressive microRNA-874 contributes to cell proliferation through targeting of histone deacetylase 1 in head and neck squamous cell carcinoma. Br J Cancer. 108:1648–1658. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Nohata N, Sone Y, Hanazawa T, et al: miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma. Oncotarget. 2:29–42. 2011.PubMed/NCBI | |
|
Kano M, Seki N, Kikkawa N, et al: miR-145, miR-133a and miR-133b: tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma. Int J Cancer. 127:2804–2814. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Hu W, Chan CS, Wu R, et al: Negative regulation of tumor suppressor p53 by microRNA miR-504. Mol Cell. 38:689–699. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Yang MH, Lin BR, Chang CH, et al: Connective tissue growth factor modulates oral squamous cell carcinoma invasion by activating a miR-504/FOXP1 signalling. Oncogene. 31:2401–2411. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Ichimi T, Enokida H, Okuno Y, et al: Identification of novel microRNA targets based on microRNA signatures in bladder cancer. Int J Cancer. 125:345–352. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Tabas-Madrid D, Nogales-Cadenas R and Pascual-Montano A: GeneCodis3: a non-redundant and modular enrichment analysis tool for functional genomics. Nucleic Acids Res. 40:W478–W483. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Ma X, Yoshimoto K, Guan Y, et al: Associations between microRNA expression and mesenchymal marker gene expression in glioblastoma. Neuro Oncol. 14:1153–1162. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Malumbres M and Barbacid M: Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer. 9:153–166. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Musgrove EA, Caldon CE, Barraclough J, Stone A and Sutherland RL: Cyclin D as a therapeutic target in cancer. Nat Rev Cancer. 11:558–572. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Shapiro GI: Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol. 24:1770–1783. 2006. View Article : Google Scholar : PubMed/NCBI |
