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

doi:10.1016/j.bbrc.2004.05.056

Biochemical and Biophysical Research Communications

Volume 319, Issue 3, 2 July 2004, Pages 795-800

https://doi.org/10.1016/j.bbrc.2004.05.056 Get rights and content

Abstract

The 14-3-3σ 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 14-3-3σ 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 14-3-3σ protein is epithelial cell-specific. Among prostate pathological specimens, >95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3σ in the cytoplasm whereas <20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3σ in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3σ mRNA level was elevated by 5-aza-2^′-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3σ reduction in DU145 and PC3 cells, as 14-3-3σ protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3σ gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3σ expression is down-regulated during the neoplastic transition of prostate epithelial cells.

Section snippets

Materials and methods

Chemicals and antibodies. A DNA methyltransferase inhibitor 5-aza-2^′-deoxycytidine (Aza-C) was purchased from Sigma (St. Louis, MO). MG132 (Z-Leu-Leu-Leu-aldehyde) was from Peptide Institute (Osaka, Japan). TRAIL was from R&D Systems (Minneapolis, MN). Rabbit polyclonal antibodies against 14-3-3ζ, and 14-3-3τ/θ, and goat polyclonal antibody against 14-3-3σ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture. Primary human prostate epithelial cells (PrECs) and prostate

Western blot analysis

We analyzed the expression of 14-3-3 proteins in PrECs and human prostate cancer cell lines (DU145, PC3, and LNCaP) by Western blot analysis. Reduced expression of 14-3-3σ was observed in DU145 and PC3 cells, whereas absence of expression in LNCaP cells (Fig. 1, top panel). In contrast, the levels of other isoforms of 14-3-3 protein including 14-3-3ζ and 14-3-3τ/θ are not basically different among these cells (Fig. 1, middle and bottom panels).

Immunocytochemistry and immunohistochemistry

We next investigated subcellular localization of

Discussion

In human cells, seven different isoforms of 14-3-3 protein regulate diverse cellular processes by binding to proteins with numerous functions [18]. The σ isoform is predominantly expressed in keratinocytes and breast epithelial cells [4], [9]. The expression of 14-3-3σ is directly induced by p53 after DNA damage [8]. Elevated levels of 14-3-3σ enforce a G2/M cell cycle arrest by sequestering cdc2/cyclinB1 complexes in the cytoplasm and are required for a stable G2/M arrest after DNA damage [6].

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These results suggest that 14-3-3σ stimulates both the nuclear export of c-Jun and its degradation via FBW7-dependent ubiquitin-mediated proteolysis, thus preventing the activation of the EMT program. Loss of 14-3-3σ has been observed in multiple human tumors, suggesting that it might function as a tumor suppressor (16, 18–21, 23, 78). However, the mechanisms by which it might inhibit tumor progression remain unclear.
Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ^−/− cells. 14-3-3σ^−/− cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.
Stratifin expression is a novel prognostic factor in human gliomas
2011, Pathology Research and Practice
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The study of Radhakrishnan and Martinez [23] established Stratifin as an oncogene and implicated MAPK and PI3K signaling as important for Stratifin-induced transformation. Moreover, several studies also confirm the role of Stratifin as a contributor to drug resistance in breast cancer cells and chemoresistance in hormone-refractory prostate cancer cells [24,11]. In addition, Stratifin expression was linked to the resistance to radiation therapy in lung carcinomas and resistance to 5-fluorouracil treatment in colorectal carcinoma cells [25].
Stratifin (14-3-3σ or SFN) is a member of the 14-3-3 family of proteins which play critical roles in different cellular signaling processes. Stratifin as a potential tumor suppressor gene plays an important role in carcinogenesis and metastasis. The aim of this study was to investigate the expression of Stratifin in human gliomas and to analyze its expression profile with respect to tumor development.
The expression pattern of Stratifin was analyzed by immunohistochemistry and/or Western blotting in tumor samples from 186 patients with different grades of gliomas. Prognostic significance was assessed using Kaplan–Meier survival estimates and Cox regression analyses.
The expression pattern of Stratifin was correlated with the pathological and clinical characteristics of the patients with gliomas. Western blot analysis indicated that the average optical densitometry (OD) ratio of Stratifin in high-grade gliomas (World Health Organization [WHO] grade III/IV) was lower than in low-grade tumors (WHO grade I/II, p = 0.026). In addition, statistical analysis showed that patients expressing a high level of Stratifin have favorable overall survival rates relative to those expressing a low level of this protein. Cox multi-factor analysis showed that Stratifin (p = 0.02) was an independent prognosis factor for human gliomas.
Our results provide convincing evidence that the expression of Stratifin is down-regulated in human gliomas. Its expression level is correlated with the clinicopathological parameters and prognosis in patients with gliomas. Pending validation targeting, Stratifin might also be a novel opportunity to improve the therapy of this tumor.
Regulation of 14-3-3σ expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation
2008, Cancer Letters
Citation Excerpt :
It is important to point out that this is not the only mechanism for 14-3-3σ down-regulation. In some breast cancer and prostate cancer cell lines, loss of 14-3-3σ has been shown to occur at the post-translational level and appears to be mediated by proteasome directed protein degradation [10,14]. Interestingly, in some tumor types, 14-3-3σ expression appears to be increased rather than lost.
Increased 14-3-3σ expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3σ mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpG’s in the gene’s CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2–7, which did not express 14-3-3σ. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3σ expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3σ expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3σ expression in thyroid carcinomas is regulated by CpG island hypermethylation.
14-3-3 sigma increases the transcriptional activity of the androgen receptor in the absence of androgens
2007, Cancer Letters
Citation Excerpt :
Huang et al. [39] correlated increased nuclear localization of 14-3-3 sigma with decreasing differentiation of tumours. Consistent with this, strong expression of 14-3-3 sigma was observed in the androgen-independent prostate cancer cell lines PC3, DU145, and 22Rv1, and expression of 14-3-3 sigma led to increased chemoresistance [36,38,40]. Increased expression of 14-3-3 sigma was also observed in response to treatment with androgens, and a reporter containing the 14-3-3 sigma promoter was induced within three hours of androgen treatment [39].
The androgen receptor (AR) is a ligand-activated transcription factor that regulates numerous target genes, including prostate-specific antigen (PSA). We examined the ability of each member of the 14-3-3 family to modulate transcription of PSA through the AR. Despite significant homology within the 14-3-3 family we observed differences in the ability of each isoform to alter the transcriptional activity of the AR. Significantly, 14-3-3 sigma activated PSA-luciferase reporters not only at castrate levels of androgens, but also in the complete absence of androgens. 14-3-3 sigma also increased expression of the endogenous PSA gene in the absence of androgens. Knockdown of the AR by siRNA oligonucleotides abolished activation of these reporters by 14-3-3 sigma. These findings may have greatest significance in hormone refractory prostate cancer where the AR may be activated in a ligand-independent manner.
Regulation of corneal angiogenesis in limbal stem cell deficiency
2006, Progress in Retinal and Eye Research
Corneal angiogenesis is associated with a variety of corneal diseases, and is sometimes vision threatening. In recent years, with the discovery of major pro- and anti-angiogenic factors in the cornea, details of the angiogenic process are gradually unveiled. Of note, corneal inflammation and neovascularization associated with severe limbal stem cell (LSC) deficiency is a clinically challenging issue in that the condition persists long after the initial insult, and will not improve without transplantation of LSCs. However, to date the molecular mechanism by which LSC transplantation restores corneal avascularity is not fully understood. In addition to discussing major pro-angiogenic factors involved in corneal neovascularization, this review article also focuses on possible molecular mechanisms underlying persistent inflammation and neovascularization following severe LSC deficiency, and anti-angiogenic factors expressed by human limbo-corneal epithelial cells (HLCECs).
Most of the recently discovered corneal anti-angiogenic factors belong to extracellular matrix proteins that aquire angio-inhibitory activity only after proper proteolytic processing. Our recent findings showed that the secretion of endostatin (derived from basement membrane collagen XVIII) and restin (from collagen XV) by HLCECs were enhanced when HLCECs were cultivated on amniotic membrane (AM). This adds to the advantage of transplanting ex vivo expanded HLCECs cultivated on AM in that the anti-angiogenic activity of the epithelial cells is augmented in a physiological way. Furthermore, proteomic profiling of HLCECs and human conjunctival epithelial cells (HCECs) identified a 14-3-3 protein (stratifin) preferentially expressed by HLCECs. In addition to functioning as a cell cycle controller, keratinocyte-derived stratifin induces MMPs which are involved in the generation of restin (by MMP-1) and endostatin (by MMP-3). These findings highlight the significance of delicate epithelial–matrix interactions in the maintenance of corneal avascularity.
Human astrocytes express 14-3-3 sigma in response to oxidative and DNA-damaging stresses
2006, Neuroscience Research
The 14-3-3 protein family consists of seven isoforms, most of which are expressed abundantly in neurons and glial cells, although the σ isoform, a p53 target gene originally identified as an epithelium-specific marker, has not been identified in the human central nervous system. Here, we show that human astrocytes in culture expressed 14-3-3σ under stress conditions. By Western blot, the expression of 14-3-3σ, p53 and p21 was coordinately upregulated in astrocytes following exposure to hydrogen peroxide, 4-hydroxy-2-nonenal (4-HNE) or etoposide, a topoisomerase II inhibitor. 14-3-3σ was induced by treatment with 5-aza-2′-deoxycytidine, suggesting a hypermethylated status of the gene promoter in astrocytes. In vivo, a small subset of hypertrophic reactive astrocytes, often showing a multinucleated morphology, expressed 14-3-3σ in active demyelinating lesions of multiple sclerosis (MS) and ischemic lesions of cerebral infarction, where the expression of 4-HNE and 8-hydroxy-2′-deoxyguanosine was enhanced in reactive astrocytes. Microarray analysis of etoposide-treated astrocytes verified upregulation of p53-responsive genes and concurrent downregulation of mitotic checkpoint-regulatory genes. These observations suggest that 14-3-3σ might serve as a marker of oxidative and DNA-damaging stresses inducing the mitotic checkpoint dysfunction in reactive astrocytes under pathological conditions.

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View full text

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

Abstract

Section snippets

Materials and methods

Western blot analysis

Immunocytochemistry and immunohistochemistry

Discussion

Trends Biochem. Sci.

J. Biol. Chem.

Mol. Cell

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Immunity

14-3-3 proteins: structure, function, and regulation

Annu. Rev. Pharmacol. Toxicol.

Molecular evolution of the 14-3-3 protein family

J. Mol. Evol.

Complementary DNA cloning of a novel epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells

Cell. Growth Differ.

The 14-3-3 cancer connection

Nat. Rev. Cancer

14-3-3σ is required to prevent mitotic catastrophe after DNA damage

Nature