Cell Cycle Regulation by Alternative Polyadenylation of CCND1.
ABSTRACT: Global shortening of 3'UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.
Project description:Prostate cancer is one of the most common types of cancer in men and the leading cause of death in developed countries. With the aid of molecular and genetic profiling of cancers, cancer molecular subtypes are paving the way for tailored cancer therapy. FOXA1 has been identified as one of the seven molecular subtypes of prostate cancer. FOXA1 is involved in a variety of metabolic process such as glucose homeostasis and deregulation of its expression is crucial in prostate cancer progression. In this study, we investigated the effects of FOXA1 gene knock-out on the expression levels of various cancer cell metabolism and cell cycle-related protein expressions. FOXA1 gene was knocked-out by using CRISPR/Cas9 technique. While FOXA1 gene knock-out significantly altered Casp-9, Bax, CCND1, CDK4, and fibronectin protein expressions (P?<?0.05, fold change: ?40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, ?-catenin, c-Myc, and GSK-3B. Knocking-out FOXA1 gene in androgen-dependent LNCaP prostate cancer cells inhibited CCND1 protein expression. Our pre-clinical results demonstrate the importance of FOXA1 as a drug target in the treatment of prostate cancer. Impact statement Knock-out studies offer a unique way of studying the function of genes especially for developmentally lethal genes. FOXA1 has prominent roles both in breast and prostate cancer pathogenesis due to its role in ER receptor signaling pathway. FOXA1 has also been identified as one of the seven molecular subtypes of primary prostate cancer. In the present study, we used an efficient gene knock-out method, CRISPR/Cas9, in order to investigate FOXA1 function on LNCaP prostate cancer cells in vitro. FOXA1 knock-out altered cell-cycle regulator CCND1 protein expression levels. Therefore, our results suggest that FOXA1 might be a plausible drug target for prostate cancer treatment.
Project description:Aurora kinase B (AURKB) triggers the phosphorylation of serine 10 on histone H3 (H3S10ph), which is important for chromosome condensation and cytokinesis during mitosis in mammals. However, how exactly AURKB controls cell cycle and contributes to tumorigenesis as an oncoprotein under pathological conditions remains largely unknown. Here, we report that AURKB promotes gastric cancer cell proliferation in vitro and in vivo. Silencing AURKB expression inhibits gastric cell proliferation and arrests the cell cycle in G2/M phase. We demonstrate that cyclin D1 (CCND1) is a direct downstream target of AURKB that plays a key role in gastric cancer cell proliferation. AURKB is able to activate the expression of CCND1 through mediating H3S10ph in the promoter of the CCND1 gene. Furthermore, we show that AZD1152, a specific inhibitor of AURKB, can suppress the expression of CCND1 in the gastric cancer cells and inhibit cell proliferation in vitro and in vivo. Importantly, we found that high AURKB and CCND1 expression levels are correlated with shorter overall survival of gastric cancer patients. This study demonstrates that AURKB promotes gastric tumorigenesis potentially through epigenetically activating CCND1 expression, suggesting AURKB as a promising therapeutic target in gastric cancer.
Project description:BACKGROUND:Non-small cell lung cancer (NSCLC) is a life-threatening disease that has a poor prognosis and low survival rate. Cleavage factor Im 25 (CFIm25) is a RNA-binding protein that if down-regulated causes 3'UTR shortening and thus promotes the transcript stability of target genes. It is not clear whether CFIm25 and alternative polyadenylation (APA) play a role during cancer development. The purpose of this study is to explore the role of CFIm25 in lung cancer cell proliferation. METHODS:CFIm25 was knocked down in A549?cells. Western blots were carried out to determine the protein expression of CFIm25, insulin growth factor 1 receptor (IGF1R), CyclinD1 (CCND1) and TP53. Real-time qRT PCR was performed to determine the total transcript levels of CFIm25 targets and the normalized fold changes in their distal PAS (dPAS) usage. Immunofluorescence was carried out to check the expression of CFIm25, IGF1R and CCND1. Cell proliferation over time was determined using the WST-1 reagent. RESULTS:The transcript levels of CCND1 and GSK3? were significantly increased and the dPAS usage of several oncogenes (IGF1R, CCND1 and GSK3?) were decreased after CFIm25 knockdown. The protein level of IGF1R was increased, and we detected increased percentage of CCND1 positive cells and cell proliferation over time in CFIm25 knockdown cells. In addition, the mRNA and APA analysis of IGF1R using patient RNA-seq data from the Cancer Genome Atlas indicated that IGF1R is shortened in both lung adenocarcinoma and lung squamous cell carcinoma compared to normal controls. CONCLUSIONS:Our findings suggest that CFIm25 plays an important role in lung cancer cell proliferation through regulating the APA of oncogenes, including IGF1R, and promoting their protein expression.
Project description:p27 plays critical roles in cell proliferation, differentiation, and apoptosis, which have been well studied in mammals and Drosophila. However, the mechanisms underlying p27 regulation of the cell cycle have not been thoroughly researched. In this study, Genevestigator, Kaplan-Meier Plotter, and the Human Protein Atlas databases were used to analyze the expression of p27, cell division protein kinase 6 (CDK6), and cyclin D1 (CCND1), as well as its prognostic value in different tumor tissues and corresponding normal tissues. Quantitative PCR and immunohistochemistry were used to detect the expression of p27, CDK6, and CCND1 in the tissues of cancer patients. The effects of p27, CDK6, and CCND1 on the proliferation of lung cancer cells were examined by the MTT assay, and flow cytometry was used to investigate the mechanism by which p27 affected cell proliferation. Immunofluorescence, co-immunoprecipitation, and Western blotting were used to determine if p27 interacted with CDK and CCND1 to regulate the cell cycle. The results showed that p27, CDK6, and CCND1 played different roles in tumorigenesis and development, which are in accordance with CDK6 and CCND1 in affecting the cell cycle and cell proliferation. p27 regulated the cell cycle and inhibited cell proliferation by affecting formation of the cell cycle-dependent complex CDK6/CCND1, but did not directly affect the expression of CDK6 and CCND1. Moreover, CCND1 did not regulate the cell cycle alone, but rather, functioned together with CDK6. This study provides insights into the effects of p27 on tumor formation and development, and the underlying regulatory mechanisms.
Project description:Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER) positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In the present study we used 32k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2- mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1. Genomic profiling of 94 CCND1-amplified breast tumors using tiling BAC aCGH. A number of cases were hybridized as replicates or replicate as dye-swaps.
Project description:BACKGROUND: OCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized. METHODS: By manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated. RESULTS: Increasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice. CONCLUSION: OCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment.
Project description:Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1.
Project description:microRNA-374a (miR-374a) exhibits oncogenic functions in various tumor types. Here we report that miR-374a suppresses proliferation, invasion, migration and intrahepatic metastasis in colon adenocarcinoma cell lines HCT116 and SW620. Notably, we detected that PI3K/AKT signaling and its downstream cell cycle factors including c-Myc, cyclin D1 (CCND1), CDK4 and epithelial-mesenchymal transition (EMT)-related genes including ZEB1, N-cadherin, Vimentin, Slug, and Snail were all significantly downregulated after miR-374a overexpression. Conversely, cell cycle inhibitors p21 and p27 were upregulated. Expression of E-cadherin was only decreased in HCT116, without any obvious differences observed in SW620 cells. Furthermore, luciferase reporter assays confirmed that miR-374a could directly reduce CCND1. Interestingly, when CCND1 was silenced or overexpressed, levels of pPI3K, pAkt as well as cell cycle and EMT genes were respectively downregulated or upregulated. We examined miR-374a levels by in situ hybridization and its correlation with CCND1 expression in CRC tumor tissues. High miR-374a expression with low level of CCND1 was protective factor in CRC. Together these findings indicate that miR-374a inactivates the PI3K/AKT axis by inhibiting CCND1, suppressing of colon cancer progression.
Project description:Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Here we report that the integrin receptor CD49b-encoding <i>ITGA2</i>, a direct target of miR-206, promotes breast cancer stemness and metastasis. <i>ITGA2</i> knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. <i>ITGA2</i> overexpression reversed miR-206-caused cell cycle arrest in G1. RNA sequencing analyses revealed that <i>ITGA2</i> knockdown inhibits genes related to cell cycle regulation and lipid metabolism, including <i>CCND1</i> and <i>ACLY</i> as representative targets, respectively. Knockdown of <i>CCND1</i> or <i>ACLY</i> inhibits mammosphere formation of breast cancer cells. Overexpression of <i>CCND1</i> rescues the phenotype of <i>ITGA2</i> knockdown-induced cell cycle arrest. <i>ACLY</i>-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels. <i>CCND1</i> knockdown further mimics <i>ITGA2</i> knockdown in abolishing lung colonization of breast cancer cells. We identified that the low levels of <i>miR-206</i> as well as high expression levels of <i>ITGA2</i>, <i>ACLY</i> and <i>CCND1</i> are associated with an unfavorable relapse-free survival of the patients with estrogen receptor-negative or high grade breast cancer, especially basal-like or TNBC, possibly serving as potential biomarkers of cancer stemness and therapeutic targets of breast cancer metastasis.
Project description:Background: Lung cancer is the leading cause of cancer deaths. The main risk factor is smoking but the risk is also associated with various genetic and epigenetic components in addition to environmental factors. Increases in the gene copy numbers due to chromosomal amplifications constitute a common mechanism for oncogene activation. A gene-dense region on chromosome 11q13 which harbors four core regions that are frequently amplified, has been associated with various types of cancer. The important cell cycle regulatory protein cyclin D1 (CCND1) is an essential driver of the first core region of the Chr11q13 amplicon. Deregulation of CCND1 has been associated with different kinds of human malignancies including lung cancer. The EMSY (c11orf30) gene has been proposed as the possible driver of the fourth core of the 11q13 amplicon and its amplification has been associated with breast and ovarian cancers. There is no report in the literature investigating the EMSY gene in lung cancer. Methods: In this study, expression levels of the EMSY and CCND1 genes were investigated in 85 patients with non small cell lung cancer by Real Time PCR. Results: Expression of the EMSY and CCND1 genes were increased in 56 (65.8%) and 50 (58.8%) of the patients, respectively. Both genes showed a higher expression in the tumors when compared to normal tissues. A strong correlation was present between the expression rates of both genes (p<0.001). Patients with adenocarcinoma had higher expression levels of both genes (p=0.02). Conclusion: We conclude that EMSY and CCND1 work in collaboration and contribute to the pathogenesis of lung cancer.