14-3-3sigma is down-regulated in human prostate cancer

The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.     

14-3-3sigma mediation of cell cycle progression is p53-independent in response to insulin-like growth factor-I receptor activation

We investigated the role of 14-3-3sigma protein in insulin-like growth factor-I (IGF-I) receptor signaling. It has been previously shown that 14-3-3sigma negatively regulates cell cycle especially in response to p53-sensitive DNA damage. In this study we demonstrated that 14-3-3sigma is a positive mediator of IGF-I receptor-induced cell proliferation. Treatment with IGF-I increased 14-3-3sigma mRNA and protein levels about 4-fold, in a time-dependent manner in MCF-7 breast cancer cells. Preincubation with the phosphoinositide 3'-kinase inhibitor LY294002 significantly reduced the effects of IGF-I on 14-3-3sigma gene expression in these cells, suggesting that this effect of IGF-I occurs via the phosphoinositide 3'-kinase pathway. 14-3-3sigma is induced by IGF-I in MCF-7 cells, which express wild-type p53, as well as in MCF-7 cells transfected with a small interference RNA targeting duplex that reduced p53 expression levels. These results suggest that IGF-I induces 14-3-3sigma expression in a manner that is independent of p53. Using the small interference RNA strategy, we demonstrated that a 70-75% reduction of 14-3-3sigma mRNA levels resulted in a similar decrease in the effects of IGF-I on cell cycle progression and proliferation in MCF-7 cells. This effect was also associated with a reduction in IGF-I-induced cyclin D1 expression. Taken together, these results suggest that 14-3-3sigma positively mediates IGF-I-induced cell cycle progression.     

MicroRNA-590-3P suppresses cell survival and triggers breast cancer cell apoptosis via targeting sirtuin-1 and deacetylation of p53

Downregulation of microRNA-590-3p (miR-590-3p) is a frequently occurring, nonphysiological event which is observed in several human cancers, especially breast cancer. However, the significance of miR-590-3p still remain unclear in the progression of this disease. This study explored the role of miR-590-3p in apoptosis of breast cancer cells. Gene expression of miR-590-3p, Sirtuin-1 (SIRT1), Bcl-2 associated X protein (BAX), and p21 was evaluated with real-time polymerase chain reaction (PCR) and SIRT1 protein expression was assessed by Western blot analysis in breast cancer cell lines. Bioinformatics analysis and luciferase reporter assay were used to evaluate targeting of SIRT1 messenger RNA (mRNA) by miR-590-3p. Cells were transfected with miR-590-3p mimic and inhibitor and their effects on the expression and activity of SIRT1 were evaluated. The effects of miR-590-3p upregulation on the acetylation of p53 as well as cell viability and apoptosis were assessed by Western blot analysis, WST-1 assay, and flow cytometry, respectively. miR-590-3p expression was considerably downregulated in breast cancer cells which was accompanied by upregulation of SIRT1 expression. SIRT1 was recognized as a direct target for miR-590-3p in breast cancer cells and its protein expression and activity was dramatically inhibited by the miR-590-3p. In addition, there was an increase in p53 and its acetylated form that ultimately led to upregulation of BAX and p21 expression, suppression of cell survival, and considerable induction of apoptosis in breast cancer cells. These findings suggest that miR-590-3p exerts tumor-suppressing effects through targeting SIRT1 in breast cancer cells, which makes it a potential therapeutic target for developing more efficient treatments for breast cancer.     

Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression

14-3-3 sigma, implicated in cell cycle arrest by p53, was cloned by expression cloning through cyclin-dependent kinase 2 (CDK2) association. 14-3-3 sigma shares cyclin-CDK2 binding motifs with different cell cycle regulators, including p107, p130, p21(CIP1), p27(KIP1), and p57(KIP2), and is associated with cyclin.CDK complexes in vitro and in vivo. Overexpression of 14-3-3 sigma obstructs cell cycle entry by inhibiting cyclin-CDK activity in many breast cancer cell lines. Overexpression of 14-3-3 sigma can also inhibit cell proliferation and prevent anchorage-independent growth of these cell lines. These findings define 14-3-3 sigma as a negative regulator of the cell cycle progression and suggest that it has an important function in preventing breast tumor cell growth.     

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3if-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.     

Downregulation of wild-type p53 protein by HER-2／neu mediated PI3K pathway activation in human breast cancer cells： its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy.Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.     

ik3-2, a relative to ik3-1/Cables, is involved in both p53-mediated and p53-independent apoptotic pathways

ik3-2 is a close relative to ik3-1/Cables, an associator with cdk3 and cdk5. ik3-1/Cables has been identified to be a candidate tumor suppressor for colon and head/neck cancers. In agreement, it has been pointed out that ik3-1/Cables is a regulator for both p53- and p73-induced apoptosis [J. Biol. Chem. 277 (2002) 2951] although ectopic expression of ik3-1/Cables does not induce apoptosis. Here we show that adenovirus-mediated overexpression of ik3-2 results in apoptosis of p53-intact U2OS cells. ik3-2 binds to p53 in vivo and ectopic coexpression of ik3-2 enhances apoptosis induced by adenovirus-mediated expression of p53. Furthermore, ectopic expression of ik3-2 results in apoptosis of primary p53/Mdm2- and p53/ARF-null mouse embryo fibroblasts, indicating that ik3-2-induced apoptosis is partially p53-independent. Both the highly conserved C-terminal cyclin box-homologous domain (ik3-2-C) and the N-terminal region consisting of 70 amino acids (ik3-2-N) are responsible for ik3-2-mediated enhancement of p53-induced apoptosis. In contrast, ik3-2-induced p53-independent apoptosis is mediated through ik3-2-N. We thus identified ik3-2 as a proapoptotic factor involved in both p53-mediated and p53-independent apoptotic pathways.     

Targeting p53 for enhanced radio- and chemo-sensitivity

p53 acts as a central mediator of the cellular response to stressful stimuli. The growth-suppressive function of p53 is lost with mutation and this occurs commonly in human cancer. In addition to suppressing cancer development and progression, wild-type p53 further confers chemo-sensitivity and radio-sensitivity upon tumor cells. Accumulated evidence over the last two decades that wild-type p53 activity is required for the efficacy of radiation and chemotherapy has led to considerable interest in development of strategies to restore normal p53 function in tumors with defective p53-dependent signaling. A number of promising discoveries, based on the knowledge of structural and functional basis of p53 mutation, p53 degradation by MDM2 and p53 family proteins, provide a foundation for future drug design. Here we review the role of p53 in enhancing the sensitivity from radiation and chemotherapy and discuss current progress on therapies targeting p53.     

Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer

We hypothesize that 14-3-3 sigma gene expression and its regulation by methylation can characterize histological types of primary human epithelial ovarian cancer. To test this hypothesis, ovarian cancer cell lines and 54 ovarian cancer tissue samples were analyzed for expression and methylation of 14-3-3 sigma gene using methylation specific PCR. The results of our experiments demonstrate that 14-3-3 sigma gene was methylated and inactivated in ES-2 ovarian cell line, which was derived from clear cell adenocarcinoma. Treatment of this cell line with demethylating agent 5-aza-2'-deoxycytidine restored the expression of 14-3-3 sigma gene. In human ovarian cancer tissues, the expression of 14-3-3 sigma protein was inactivated in most of the ovarian clear cell carcinoma tissues. Interestingly, 14-3-3 sigma protein expression was positive in significantly higher percentages of serous (89.5%), endometrioid (90%), and mucinous (81.8%) ovarian adenocarcinoma tissues. The ovarian clear cell carcinoma samples with inactivated 14-3-3 sigma protein were highly methylated, suggesting that inactivation of 14-3-3 sigma gene is through DNA methylation. Using direct DNA sequencing, 14-3-3 sigma gene methylation on all the 17 CpG sites was significantly higher in ovarian clear cell carcinoma as compared to other histological types of ovarian cancer (serous, endometrioid, and mucinous). This is the first report suggesting that 14-3-3 sigma gene expression and methylation status can characterize histological features of different types of ovarian cancer.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

p73 and MDM2 confer the resistance of epidermoid carcinoma to cisplatin by blocking p53

p73 responds to DNA damage and exerts its pro-apoptotic function. However, p73 might contribute to the development of drug-resistance in certain tumor cells. In this study, we found that p73 and MDM2 correlate with cisplatin-resistant phenotype of human epidermoid carcinoma-derived cells. p73 and MDM2 were kept at low levels in the cisplatin-sensitive KB-3-1 cells, whereas p53 was induced to be phosphorylated at Ser-15 in response to cisplatin. In contrast, p73 and MDM2 were expressed at higher levels, and cisplatin-mediated p53 phosphorylation was undetectable in the cisplatin-resistant KCP-4 cells. Enforced expression of p73 in KB-3-1 cells caused an accumulation of unphosphorylated form of p53 and MDM2, and conferred the cisplatin resistance. Collectively, our results suggest that a loss of the cisplatin sensitivity is at least in part due to a lack of cisplatin-induced p53 phosphorylation, and p73 might cooperate with MDM2 to be involved in this process.     

The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax

In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.     

Structure of the p53 C-terminus bound to 14-3-3: Implications for stabilization of the p53 tetramer

The adaptor protein 14-3-3 binds to and stabilizes the tumor suppressor p53 and enhances its anti-tumour activity. In the regulatory C-terminal domain of p53 several 14-3-3 binding motifs have been identified. Here, we report the crystal structure of the extreme C-terminus (residues 385-393, p53pT387) of p53 in complex with 14-3-3σ at a resolution of 1.28 Å. p53pT387 is accommodated by 14-3-3 in a yet unrecognized fashion implying a rationale for 14-3-3 binding to the active p53 tetramer. The structure exhibits a potential binding site for small molecules that could stabilize the p53/14-3-3 protein complex suggesting the possibility for therapeutic intervention.

Structured summary

MINT-7711943: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by X-ray crystallography (MI:0114)MINT-7711931: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by isothermal titration calorimetry (MI:0065)     

Hypoxia activates tumor suppressor p53 by inducing ATR-Chk1 kinase cascade-mediated phosphorylation and consequent 14-3-3γ inactivation of MDMX protein

It has been known that p53 can be induced and activated by hypoxia, an abnormal condition that often occurs in rapidly growing solid tumors or when normal tissues undergo ischemia. Although the ATR-Chk1 kinase cascade was associated with hypoxia-induced p53 activation, molecules that directly link this hypoxia-ATR-Chk1 pathway to p53 activation have been elusive. Here, we showed that hypoxia could induce phosphorylation of MDMX at Ser-367 and enhance the binding of this phosphorylated MDMX to 14-3-3γ, consequently leading to p53 activation. A Chk1 inhibitor or knockdown of ATR and Chk1 inhibited the phosphorylation of MDMX at Ser-367 and impaired the binding of MDMX to 14-3-3γ in addition to p53 activation in response to hypoxia. In primary mouse embryonic fibroblast cells that harbor a mutant MDMX, including the S367A mutation, hypoxia also failed to induce the binding of this mutant MDMX to 14-3-3γ and to activate p53 and its direct targets. These results demonstrate that hypoxia can activate p53 through inactivation of MDMX by the ATR-Chk1-MDMX-14-3-3γ pathway.