Fangzhou Song

XBP1 induces snail expression to promote epithelial- to-mesenchymal transition and invasion of breast cancer cells

Breast cancer is highly metastatic disease and the most lethal of the gynecologic malignancies. Epithelial-to-mesenchymal is a crucial process for the invasion of epithelial tumors. Recent studies revealed that breast cancer cells that have undergone EMT acquire aggressive malignant properties, but the molecular mechanisms underlying this transition are not well-understood. In this study, we report findings that human X-box binding protein1 (XBP1) acts as a novel regulator of EMT. We found that increased expression of XBP1 was associated with the progression of breast cancer and that XBP1 protein was significantly over-expressed in matched metastatic tumor. High XBP1 protein also predicts shorter overall survival of breast cancer patients. RNA interference-mediated knockdown of XBP1 expression restored E-cadherin expression and cell-cell junction formation in breast cancer cells, suppressing cell invasion, and tumor formation. In contrast, overexpression of XBP1 decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. Our finding demonstrates the upregulated expression of the key EMT regulator Snail and that it mediated EMT activation and cell invasion by XBP1.

Ubiquitin E3 Ligase CRL4(CDT2/DCAF2) as a Potential Chemotherapeutic Target for Ovarian Surface Epithelial Cancer

Background: Cullin-RING ubiquitin ligases are the largest family of E3 ligases, but their roles in ovarian cancers remain uninvestigated. Results: Inhibition of CRL4 activity induced ovarian cancer death. Conclusion: CRL4CDT2 repression and CDT1 accumulation contributed to the genotoxic effects of MLN4924 in ovarian cancer cells. Significance: Indicating CRL4CDT2 is an effective drug target in ovarian cancers. Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4CDT2 repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4CDT2 is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.

MiR-335 inhibits migration of breast cancer cells through targeting oncoprotein c-Met

Metastasis is the leading cause of death in patients with breast cancer and aberrantly expressed microRNAs (miRNAs) are highly associated with this process. A previous study has shown that miR-335 is downregulated in breast cancer and can suppress tumor invasion and metastasis. Emerging evidences indicate that c-Met is implicated in cell scattering, migration, and invasion. However, little is known about the relationship between miR-335 expression and c-Met alteration in breast cancer. In the present study, we found that miR-335 expression was downregulated and c-Met protein expression was upregulated in two human breast cell lines. MiR-335 was found to negatively regulate c-Met protein level by directly targeting its 3′ untranslated region (UTR). Forced expression of miR-335 decreased c-Met expression at protein levels and consequently diminished hepatocyte growth factor (HGF)-induced phosphorylation of c-Met and subsequently inhibited HGF promotion of breast cancer cell migration in a c-Met-dependent manner. MiR-335 expression was increased after 5-aza-2′-deoxycytidine (5-AZA-CdR) treatment, and 5-AZA-CdR treatment resulted in the same phenotype as the effect of miR-335 overexpression. Taken together, these results demonstrate that miR-335 suppresses breast cancer cell migration by negatively regulating the HGF/c-Met pathway.

Fangchinoline Inhibits Cell Proliferation Via Akt/GSK-3beta/ cyclin D1 Signaling and Induces Apoptosis in MDA-MB-231 Breast Cancer Cells

Fangchinoline (Fan) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of Fan on cell growth and proliferation in breast cancer cells remain to be elucidated. Here, we show that Fan inhibited cell proliferation in the MDA-MB-231 breast cancer cell line through suppression of the AKT/Gsk- 3beta/cyclin D1 signaling pathway. Furthermore, Fan induced apoptosis by increasing the expression of Bax (relative to Bcl-2), active caspase 3 and cytochrome-c. Fan significantly inhibited cell proliferation of MDA- MB-231 cells in a concentration and time dependent manner as determined by MTT assay. Flow cytometry analysis demonstrated that Fan treatment of MDA-MB-231 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. Further analysis demonstrated that Fan decreased the phosphorylation of AKT and GSK-3beta. In addition, Fan up-regulated active caspase3, cytochrome-c protein levels and the ratio of Bax/Bcl-2, accompanied by apoptosis. Taken together, these results suggest that Fan is a potential natural product for the treatment of breast cancer.

DAXX silencing suppresses mouse ovarian surface epithelial cell growth by inducing senescence and DNA damage

Mouse ovarian surface epithelium (OSE) is a single layer of cubodial epithelial cells that covers the ovary surface and is involved in regulating the secretion and transport of 17β-hydroxysteroid dehydrogenase. Recently, OSE cells have attracted particular interest as a major source of ovarian cancer. Death-associated protein DAXX along with PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) reportedly play roles in transcriptional regulation and apoptosis. However, little is known regarding a role for DAXX in mOSE cells. In this study, we both over-expressed DAXX and depleted DAXX in primary mOSE cells. We found that Daxx deletion accelerated senescence in a p53/p21-dependent manner and promoted DNA damage by interacting with PML bodies without affecting cell cycle progression. These results suggest that DAXX may transform mOSE cells to an ovarian oncogenic phenotype and may be an anti-cancer target.

Proteomic analysis of cervical cancer cells treated with adenovirus-mediated MDA-7.

Ad.mda-7 inhibited growth and decreased survival in a broad array of human tumor cells, without eliciting detrimental effects in normal cells. This study demonstrates that Ad.mda-7 can effectively impede the proliferation and induce apoptosis of human cervical carcinoma cells, but the underlying mechanisms inducing cell death at protein level are unknown. Using proteome analysis, an investigation aimed at a better understanding of the antiproliferative mechanisms by Ad.mda-7 was carried out in CaSki cervical cancer cells. A total of 43 differentially expressed proteins were visualized by 2-DE and silver stain., 29 proteins of which were identified via matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF-MS) analysis, 15 were upregulated (eg, Tumor suppressor p53、Apoptosis regulator BAX、Adenylate kinase isoenzyme 1(AK1)、Growth arrest and DNA-damage-inducible protein GADD45 gamma (GADD45γ)) and 14 were downregulated (eg, Eukaryotic translation initiation factor 5A(eIF-5A)、Protein DJ-1、Annexin V、Transcription elongation factor B polypeptide 2 (TCEB2)、TRAF family member-associated NF-kappa-B activator (TRAF2)、c-Myc-responsive protein Rcl (RCL)). Among the identified proteins, the protein and mRNA alterations of six proteins were further confirmed by Western blot and semi-quantitative RT-PCR. Together, at both the mRNA and protein levels, p53, BAX, AK1, GADD45γ, and BCCIP were upregulated, while eIF-5A was downregulated following Ad.mda-7 treatment. Our findings may offer new insights into the antiproliferative mechanisms by Ad.mda-7 and its mode of action in cervical carcinoma cells.

Cytotoxic T Lymphocytes Elicited by Dendritic Cell-Targeted Delivery of Human Papillomavirus Type-16 E6/E7 Fusion Gene Exert Lethal Effects on CaSki Cells

Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

MCPH1 Protein Expression in Normal and Neoplastic Lung Tissues.

Lung cancer is the most common cause of cancer-related death in the world. The main types are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), the latter including squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma. NSCLCs account for about 80% of all lung cancer cases. Microcephalin (MCPH1), also called BRIT1 (BRCT-repeat inhibitor of hTERT expression), plays an important role in the maintenance of genomic stability. Recently, several studies have provided evidence that the expression of MCPH1 gene is decreased in several different types of human cancers. We evaluated the expression of protein MCPH1 in 188 lung cancer and 20 normal lung tissues by immunohistochemistry. Positive MCPH1 staining was found in all normal lung samples and only some cancerous tissues. MCPH1-positive cells were significantly lower in lung carcinoma compared with normal tissues. Furthermore, we firstly found that MCPH1 expression in lung adenocarcinoma is higher than its expression in squamous cell carcinoma. Change in MCPH1 protein expression may be associated with lung tumorigenesis and may be a useful biomarker for identification of pathological types of lung cancer.

ALEX1 Regulates Proliferation and Apoptosis in Breast Cancer Cells

BACKGROUND Arm protein lost in epithelial cancers, on chromosome X (ALEX) is a novel subgroup within the armadillo (ARM) family, which has one or two ARM repeat domains as opposed to more than six-thirteen repeats in the classical Armadillo family members. MATERIALS AND METHODS In the study, we explore the biological functions of ALEX1 in breast cancer cells. Overexpression of ALEX1 and silencing of ALEX1 were performed with SK-BR3 and MCF-7 cell lines. Cell proliferation and colony formation assays, along with flow cytometry, were carried out to evaluate the roles of ALEX1. RESULTS ALEX1 overexpression in SK-BR3 breast cancer cells inhibited proliferation and induced apoptosis. Furthermore, depletion of ALEX1 in MCF-7 breast cancer cells increased proliferation and inhibited apoptosis. Additional analyses demonstrated that the overexpression of ALEX1 activated the intrinsic apoptosis cascades through up-regulating the expression of Bax, cytosol cytochrome c, active caspase-9 and active caspase-3 and down-regulating the levels of Bcl-2 and mitochondria cytochrome c. Simultaneouly, silencing of ALEX1 inhibited intrinsic apoptosis cascades through down-regulating the expression of Bax, cytosol cytochrome c, active caspase-9, and active caspase-3 and up-regulating the level of Bcl-2 and mitochondria cytochrome c. CONCLUSIONS Our data suggest that ALEX1 as a crucial tumor suppressor gene has been involved in cell proliferation and apoptosis in breast cancer, which may serve as a novel candidate therapeutic target.

Association Between the Pre-mir-218 Polymorphism and Cancer Risk in the Chinese Population: a Meta-Analysis

BACKGROUND Several recent studies have explored associations between pre-mir-218 polymorphism (rs11134527) and cancer risk. However, published data are still inconclusive. To obtain a more precise estimation of the relationship in the Chinese population, we carried out a meta-analysis for the first time. MATERIALS AND METHODS Through retrieval from the PubMed, Medline, Embase, Web of Science databases, China National Knowledge Infrastructure and the Chinese BioMedical Literature Database, a total of four studies were analyzed with 3,561 cases and 3,628 controls for SNP pre-mir-218 rs11134527. We calculated odds ratios (ORs) and 95% confidence intervals (95%CIs) to explore the strength of associations. RESULTS The results showed that the rs11134527 polymorphism was associated with decreased cancer risk in GG versus AA and GG versus AA+AG models tested ( GG vs AA: OR=0.82, 95%CI: 0.71-0.94; GG vs AA+AG: OR=0.84, 95%CI: 0.74-0.96), and significantly decreased cervical cancer risk was observed in GG versus AA and GG versus AA+AG models (GG vs AA: OR=0.79, 95%CI: 0.66-0.94; GG vs AA+AG: OR=0.80, 95%CI: 0.68-0.94). However, no significant association between the rs11134527 polymorphism and hepatocellular carcinoma risk was observed in all comparison models tested (AG vs AA: OR=0.94, 95%CI: 0.79-1.11; GG vs AA: OR=0.88, 95%CI: 0.70-1.10; GG+AG vs AA: OR=0.92, 95%CI: 0.79-1.08; GG vs AA+AG: OR=0.91, 95%CI: 0.75-1.11). CONCLUSION The findings suggest that pre-miR-218 rs11134527 polymorphism may have some relation to cancer development in Chinese. However, well-designed studies with larger sample size and more detailed data are needed to confirm these conclusions.