Zhongyin Yang

A Novel Plant Homeodomain Finger 10-Mediated Antiapoptotic Mechanism Involving Repression of Caspase-3 in Gastric Cancer Cells

The mechanisms governing tumorigenesis of gastric cancer have been an area of intense investigation. Currently, plant homeodomain (PHD) finger (PHF) proteins have been implicated in both tumor suppression and progression. However, the function of PHF10 has not been well characterized. Here, we show that various levels of PHF10 protein were observed in gastric cancer cell lines. Alteration of PHF10 expression, which is associated with tumor cell growth, may result in apoptosis in gastric cancer cells both in vitro and in vivo. Knockdown of PHF10 expression in gastric cancer cells led to significant induction of caspase-3 expression at both the RNA and protein levels and thus induced alteration of caspase-3 substrates in a time-dependent manner. Moreover, results from luciferase assays indicated that PHF10 acted as a transcriptional repressor when the two PHD domains contained in PHF10 were intact. Combined with previous findings, our data suggest that PHF10 transcriptionally regulates the expression of caspase-3. Finally, by using systematic reporter deletion and chromatin immunoprecipitation assays, we localized a region between nucleotides −270 and −170 in the caspase-3 promoter that was required for the efficient inhibition of caspase-3 promoter activity by PHF10. Collectively, our findings show that PHF10 repressed caspase-3 expression and impaired the programmed cell death pathway in human gastric cancer at the transcriptional level. Mol Cancer Ther; 9(6); 1764–74. ©2010 AACR.

Metallopanstimulin-1 regulates invasion and migration of gastric cancer cells partially through integrin β4

MPS-1 (metallopanstimulin-1), also known as ribosomal protein S27, was overexpressed in gastric cancer cells. However, how MPS-1 contributes to gastric carcinogenesis has not been well characterized. Here, we show that high expression of MPS-1 was observed in gastric cancer tissues and associated with gastric cancer cell metastasis. Alteration of MPS-1 expression regulates invasion and migration of gastric cancer cells both in vitro and in vivo. Furthermore, by using Signal-Net and cluster analyses of microarray data we identified integrin β4 (ITGB4) as a downstream target of MPS-1 that mediates its effects on cell metastasis. Knockdown of MPS-1 expression in gastric cancer cells led to significant reduction of ITGB4 expression at both the RNA and protein levels. Mechanically, we found that overexpression of ITGB4 in MPS-1 knockdown cells largely recovers the ability of invasion and migration. Conversely, knockdown of ITGB4 partially reduced cell invading/migrating ability induced by MPS-1 overexpression. Moreover, MPS-1 and ITGB4 expressions are positively correlated in gastric cancer cell lines and tissues. Finally, the survival analyses show that the expression of MPS-1 and ITGB4 is associated with poor outcomes in gastric cancer patients. Collectively, our findings suggest that MPS-1 regulates cell invasiveness and migration partially through ITGB4 and that MPS-1/ITGB4 signaling axis may serve as therapeutic targets in the treatment of gastric cancer.

P27(Kip1), regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells.

BackgroundGastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer.MethodsBased on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA.ResultsHMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition.ConclusionsThe results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial cell differentiation.

Knockdown of metallopanstimulin-1 inhibits NF-κB signaling at different levels: The role of apoptosis induction of gastric cancer cells

The ribosomal protein S27 (metallopanstimulin‐1, MPS‐1) has been reported to be a multifunctional protein, with increased expression in a number of cancers. We reported previously that MPS‐1 was highly expressed in human gastric cancer. Knockdown of MPS‐1 led to spontaneous apoptosis and repressed proliferation of human gastric cancer cells in vitro and in vivo. However, how does MPS‐1 regulate these processes is unclear. Here we performed microarray and pathway analyses to investigate possible pathways involved in MPS‐1 knockdown‐induced apoptosis in gastric cancer cells. Our results showed that knockdown of MPS‐1 inhibited NF‐κB activity by reducing phosphorylation of p65 at Ser536 and IκBα at Ser32, inhibiting NF‐κB nuclear translocation, and down‐regulating its DNA binding activity. Furthermore, data‐mining the Gene‐Regulatory‐Network revealed that growth arrest DNA damage inducible gene 45β (Gadd45β), a direct NF‐κB target gene, played a critical role in MPS‐1 knockdown‐induced apoptosis. Over‐expression of Gadd45β inhibited MPS‐1 knockdown‐induced apoptosis via inhibition of JNK phosphorylation. Taken together, these data revealed a novel pathway, the MPS‐1/NF‐κB/Gadd45β signal pathway, played an important role in MPS‐1 knockdown‐induced apoptosis of gastric cancer cells. This study sheds new light on the role of MPS‐1/NF‐κB in apoptosis and the possible use of MPS‐1 targeting strategy in the treatment of gastric cancer.

Upregulated expression of LOX is a novel independent prognostic marker of worse outcome in gastric cancer patients after curative surgery

Lysyl oxidase (LOX) initiates the enzymatic stage of collagen and elastin cross-linking. It also has intracellular functions involved in the regulation of cell differentiation, motility/migration and gene transcription. Aberrant expression of the LOX gene has been reported in multiple tumors. However, the correlation of its expression with clinicopathological parameters and its prognostic significance in gastric cancer remains largely unknown. In order to address this problem, total RNA of paired tissue samples (n=10) and a tissue microarray containing 161 paired tissues from patients with gastric cancers at different stages were collected. Quantitative real-time PCR and immunochemistry assay were conducted to investigate the expression of LOX. Based on the results, LOX mRNA was increased in gastric cancer tissues compared with the adjacent normal mucosa. Immunohistochemical detection revealed that expression of LOX was associated with depth of tumor invasion (P<0.05), lymph node status (P<0.05), TNM stage (P<0.05) and survival (P<0.05). Cox regression analysis revealed that positive expression of LOX (P=0.026) was an independent prognostic marker for survival in patients with gastric cancer.

Integrity of the LXXLL motif in Stat6 is required for the inhibition of breast cancer cell growth and enhancement of differentiation in the context of progesterone

BackgroundProgesterone is essential for the proliferation and differentiation of mammary gland epithelium. Studies of breast cancer cells have demonstrated a biphasic progesterone response consisting of an initial proliferative burst followed by sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the effects of progesterone on mammary cell growth and differentiation remain to be determined. Recently, it was demonstrated that signal transducer and activator of transcription 6 (Stat6) is a cell growth suppressor. Similar to progesterone-bound PR, Stat6 acts by inducing the expression of the G1 cyclin-dependent kinase inhibitors p21 and p27. The possible interaction between Stat6 and progesterone pathways in mammary cells was therefore investigated in the present study.MethodsChIP and luciferase were assayed to determine whether Stat6 induces p21 and p27 expression by recruitment at the proximal Sp1-binding sites of the gene promoters. Immunoprecipitation and Western blotting were performed to investigate the interaction between Stat6 and PR-B. The cellular DNA content and cell cycle distribution in breast cancer cells were analyzed by FACS.ResultsWe found that Stat6 interacts with progesterone-activated PR in T47D cells. Stat6 synergizes with progesterone-bound PR to transactivate the p21 and p27 gene promoters at the proximal Sp1-binding sites. Moreover, Stat6 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. Stat6 knockdown also abolished the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. In addition, knockdown of Stat6 expression prevented the induction of breast cell differentiation markers, previously identified as progesterone target genes. Finally, Stat6 gene expression levels increased following progesterone treatment, indicating a positive auto-regulatory loop between PR and Stat6.ConclusionsTaken together, these data identify Stat6 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells.