WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

Aspirin inhibits proliferation and promotes differentiation of neuroblastoma cells via p21Waf1 protein up‐regulation and Rb1 pathway modulation

Several clinical and experimental studies have demonstrated that regular use of aspirin (acetylsalicylic acid, ASA) correlates with a reduced risk of cancer and that the drug exerts direct anti‐tumour effects. We have previously reported that ASA inhibits proliferation of human glioblastoma multiforme‐derived cancer stem cells. In the present study, we analysed the effects of ASA on nervous system‐derived cancer cells, using the SK‐N‐SH (N) human neuroblastoma cell line as an experimental model. ASA treatment of SK‐N‐SH (N) dramatically reduced cell proliferation and motility, and induced neuronal‐like differentiation, indicated by the appearance of the neuronal differentiation marker tyrosine hydroxylase (TH) after 5 days. ASA did not affect cell viability, but caused a time‐dependent accumulation of cells in the G₀/G₁ phase of the cell cycle, with a concomitant decrease in the percentage of cells in the G₂ phase. These effects appear to be mediated by a COX‐independent mechanism involving an increase in p21^Waf1 and underphosphorylated retinoblastoma (hypo‐pRb1) protein levels. These findings may support a potential role of ASA as adjunctive therapeutic agent in the clinical management of neuroblastoma.     

TRIM16 inhibits neuroblastoma cell proliferation through cell cycle regulation and dynamic nuclear localization

Neuroblastoma is the most common solid tumor in childhood and represents 15% of all children’s cancer deaths. We have previously demonstrated that tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite totif (TRIM) protein family, has significant effects on neuroblastoma proliferation and migration in vitro and tumorigenicity in vivo. However, the mechanism by which this putative tumor suppressor influences cell proliferation and tumorigenicity was undetermined. Here we show, for the first time, TRIM16’s striking pattern of expression and dynamic localization during cell cycle progression and neuroblastoma tumor development. In a tyrosine hydroxylase MYCN (TH-MYCN) neuroblastoma mouse model, immunohistochemical staining revealed strong nuclear TRIM16 expression in differentiating ganglia cells but not in the tumor-initiating cells. Furthermore in vitro studies clearly demonstrated that during G₁ cell cycle phase, TRIM16 protein expression is upregulated and shifts to the nucleus of cells. TRIM16 also plays a role in cell cycle progression through changes in Cyclin D1 and p27 expression. Importantly, using TRIM16 deletion mutants, an uncharacterized protein domain of TRIM16 was found to be required for both TRIM16’s growth inhibitory effects and its nuclear localization. Taken together, our data suggest that TRIM16 acts as a novel regulator of both neuroblastoma G₁/S progression and cell differentiation.     

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.     

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.     

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

Retinoblastoma protein family in cell cycle and cancer: A review

Two genes, p107 and Rb2/p130, are strictly related to RB, the most investigated tumor suppressor gene, responsible for susceptibility to retinoblastoma. The products of these three genes, namely pRb, p107, and pRb2/p130 are characterized by a peculiar steric confirmation, called “pocket,” responsible for most of the functional interactions characterizing the activity of these proteins in the homeostasis of the cell cycle. The interest in these genes and proteins springs from their ability to regulate cell cycle processes negatively, being able, for example, to dramatically slow down neoplastic growth. So far, among these genes, only RB is firmly established to act as a tumor suppressor, because its lack-of-function is clearly involved in tumor onset and progression. It has been found deleted or mutated in most retinoblastomas and sarcomas, but its inactivation is likely to play a crucial role in other types of human cancers. The two other members of the family have been discovered more recently and are currently under extensive investigation. We review analogies and differences among the pocket protein family members, in an attempt to understand their functions in normal and cancer cells. © 1996 Wiley-Liss, Inc.     

The convergence of hormone regulation and cell cycle in prostate physiology and prostate tumorigenesis

Despite the intense research focused on prostate cancer, it remains the most frequently diagnosed malignancy in men over 40-yr-of-age, and the second most frequent cause of cancer-related deaths in men in the United States (1). In 1990, the National Cancer Institute convened 50 experts and leaders from various disciplines in the prostate cancer field to discuss research directions that would help elucidate the molecular basis of this disease and reduce the incidence and mortality of prostate cancer (2). Critical issues identified at this meeting included the role of androgens and the regulation of cell cycle in prostate tumorigenesis and its progression to androgen-independence. Hormones and cell cycle clearly play important roles in normal and cancerous prostate physiology; however, little information has emerged that clearly delineates their function in the etiology of prostate cancer. Some of the mutational events that occur during prostate tumorigenesis and its progression to androgen-independence involve alterations to normal growth, developmental and apoptotic programs regulated by androgen, and the cell cycle. As such, the delineation of events by which prostate cancer cells circumvent these regulatory mechanisms will be central to our understanding of prostate tumorigenesis and to the development of new modalities to treat this disease. This article is then intended to summarize the functional convergence of androgen regulation and cell cycle in normal prostate physiology and prostate tumorigenesis.     

miR-377-5p inhibits lung cancer cell proliferation,invasion, and cell cycle progression by targeting AKT1 signaling

Lung carcinoma is the most common type of malignant tumors globally, and its molecular mechanisms remained unclear. With the aim to investigate the effects of microRNA (miR)-377-5p on the cell development, invasion, metastasis, and cycle of lung carcinoma, this study was performed. We evaluated miR-377-5p expression levels in lung cancer tissues and cell models. Cell viability, proliferation, migration, invasion abilities, and cell cycle distribution were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, crystal violet, transwell, and flow cytometry assay. Furthermore, expression levels of protein kinase B α subunit (AKT1) and proteins related to cell cycle and epithelial-mesenchymal transition (EMT) were assessed using Western blot analysis and quantitative real-time polymerase chain reaction. These results suggested that miR-377-5p was downregulated in vivo and in cell models, and miR-377-5p overexpression inhibited cell viability, proliferation, migration, invasion, and induced cell-cycle arrest. In addition, as a target of miR-377-5p, AKT1 alleviated the decreases of cell viability, proliferation, migration, invasion, the S-phase cells, the expression of cyclin D1, fibronectin, and vimentin, as well as the increases of the G0/G1-phase cells, the expression of Foxo1, p27 ^kip1, p21 ^Cip1 and E-cadherin when miR-377-5p overexpressed. In conclusion, miR-377-5p inhibited cell development and regulated cell cycle distribution and EMT by targeting AKT1, which provided a theoretical basis for further study of lung carcinoma therapeutics.     

Smad7 induces G0/G1 cell cycle arrest in mesenchymal cells by inhibiting the expression of G1 cyclins

The major Smad pathways serve in regulating the expression of genes downstream of TGFbeta signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone-marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7-induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7-inducing stimuli in a cell-type-dependent fashion.