Yunshan Wang

CUL4A Induces Epithelial–Mesenchymal Transition and Promotes Cancer Metastasis by Regulating ZEB1 Expression

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial-mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells.

Estrogen promotes stemness and invasiveness of ER-positive breast cancer cells through Gli1 activation

BackgroundAlthough long-term estrogen (E2) exposure is associated with increased breast cancer (BC) risk, and E2 appears to sustain growth of BC cells that express functional estrogen receptors (ERs), its role in promoting BC stem cells (CSCs) remains unclear. Considering that Gli1, part of the Sonic hedgehog (Shh) developmental pathway, has been shown to mediate CSCs, we investigated whether E2 and Gli1 could promote CSCs and epithelial-mesenchymal transition (EMT) in ER+ BC cell lines.MethodsWe knocked down Gli1 in several BC cells using a doxycycline-controlled vector, and compared Gli1-knockdown cells and Gli1+ cells in behavior and expression of ER, Gli1, ALDH1 (BC-CSC marker), Shh, Ptch1 (Shh receptor) and SOX2, Nanog and Bmi-1 (CSC-associated transcriptions factors), using PCR; tissue microarrays, western blot; chromatin immunoprecipitation q-PCR, confocal immunofluorescence microscopy; fluorescence-activated cell sorting; annexin–flow cytometry (for apoptosis); mammosphere culture; and colony formation, immunohistochemistry, Matrigel and wound-scratch assays.ResultsBoth mRNA and protein expressions of ER correlated with those of Gli1 and ALDH1. E2 induced Gli1 expression only in ER+ BC cells. E2 promoted CSC renewal, invasiveness and EMT in ER+/Gli1+ cells but not in Gli1-knockdown cells.ConclusionsOur results indicate that estrogen acts via Gli1 to promote CSC development and EMT in ER+ BC cells. These findings also imply that Gli1 mediates cancer stem cells, and thus could be a target of a novel treatment for ER+ breast cancer.

CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to Erlotinib via transcriptional regulation of EGFR

BackgroundCUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear.MethodsExpression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT in vitro and tumorigenesis in vivo, respectively.ResultsWe found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpression of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities.ConclusionsOur results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.

Rapamycin inhibits FBXW7 loss-induced epithelial-mesenchymal transition and cancer stem cell-like characteristics in colorectal cancer cells

Increased cell migration and invasion lead to cancer metastasis and are crucial to cancer prognosis. In this study, we explore whether FBXW7 plays any role in metastatic process. We show that depletion of FBXW7 induces epithelial-mesenchymal transition (EMT) in human colon cancer cells along with the increase in cell migration and invasion. Moreover, FBXW7 deficiency promotes the generation of colon cancer stem-like cells in tumor-sphere culture. mTOR inhibition by rapamycin suppresses FBXW7 loss-driven EMT, invasion and stemness. Our results define the FBXW7/mTOR axis as a novel EMT pathway that mediates cancer invasion.

Metformin exerts anticancer effects through the inhibition of the Sonic hedgehog signaling pathway in breast cancer

Metformin, a widely prescribed antidiabetic drug, has previously been shown to lower the risk of certain types of cancer, including that of breast cancer, and to improve prognosis. Its anticancer effects, which are mediated by the activation of AMP-activated protein kinase (AMPK), have become notable. The Sonic hedgehog (Shh) signaling pathway is involved in changes in mammary ducts and malignant transformation. The aim of the present study was to elucidate the role of the Shh pathway in mediating the anticancer effects of metformin and the correlation between AMPK and the Shh pathway. We investigated the effectiveness of metformin in inhibiting the proliferation, migration, invasion and stemness of breast cancer cells in vitro using RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, cell proliferation assay, scratch-wound assay (cell migration assay), cell invasion assay, mammosphere culture and flow cytometry. In in vivo experiments, a tumor xenograft model was used to detect the effects of metformin on cancer cell proliferation. The results revealed that the treatment of breast cancer cells with metformin led to the inhibition of the Shh signaling pathway. Importantly, metformin inhibited recombinant human Shh (rhShh)-induced cell migration, invasion, and stemness, and impaired cell proliferation both in vitro and in vivo. Furthermore, the small interfering RNA (siRNA)-mediated downregulation of AMPK reversed the inhibitory effects of metformin on rhShh-induced Gli-1 expression and stemness. Our findings identified a role of the Shh signaling pathway in the anticancer effects of metformin in breast cancer. Furthermore, we revealed that the metformin-mediated inhibition of the Shh signaling pathway may be dependent on AMPK.

CUL4A is overexpressed in human pituitary adenomas and regulates pituitary tumor cell proliferation.

Cullin 4A (CUL4A) encodes a core subunit of an E3 ubiquitin ligase that targets proteins for ubiquitin-mediated degradation, and aberrant expression of the CUL4A is found in many tumor types. However, its roles and clinicopathologic significance in pituitary adenomas are not clear. The aim of this study was to investigate the possible role of CUL4A in pituitary tumorigenesis. Immunohistochemistry was used to examine CUL4A expression in human normal pituitaries and pituitary tumors with respect to various clinicopathologic factors in pituitary adenomas. Cell proliferation was assessed by MTT and colony formation, and migration and invasion were analyzed by Transwell and Matrigel assays after CUL4A overexpression or knockdown in pituitary tumor cells. Overexpression of CUL4A was frequently observed in pituitary adenomas compared with normal adenohypophysial tissue and significantly associated with tumor progressiveness and invasion. CUL4A overexpression in GH3 adenoma cells increased colony numbers, cell viability and cell invasion and silencing CUL4A in AtT20 adenoma cells decreased cell proliferation, migration and invasion. Mechanistically, CUL4A could modulate the expression of p53, p21, and p27 in pituitary tumor cells. In addition, high levels of CUL4A expression also significantly inversely correlated with the p53 protein level in human pituitary adenomas. Our results indicate that CUL4A enhances pituitary cell proliferation, migration and invasion and may thus contribute to pituitary tumor development and progression.

Involvement of CUL4A in Regulation of Multidrug Resistance to P-gp Substrate Drugs in Breast Cancer Cells

CUL4A encodes a core component of a cullin-based E3 ubiquitin ligase complex that regulates many critical processes such as cell cycle progression, DNA replication, DNA repair and chromatin remodeling by targeting a variety of proteins for ubiquitination and degradation. In the research described in this report we aimed to clarify whether CUL4A participates in multiple drug resistance (MDR) in breast cancer cells. We first transfected vectors carrying CUL4A and specific shCUL4A into breast cancer cells and corresponding Adr cells respectively. Using reverse transcription polymerase chain reactions and western blots, we found that overexpression of CUL4A in MCF7 and MDA-MB-468 cells up-regulated MDR1/P-gp expression on both the transcription and protein levels, which conferred multidrug resistance to P-gp substrate drugs, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. On the other hand, silencing CUL4A in MCF7/Adr and MDA-MB-468/Adr cells led to the opposite effect. Moreover, ERK1/2 in CUL4A-overexpressing cells was highly activated and after treatment with PD98059, an ERK1/2-specific inhibitor, CUL4A-induced expression of MDR1/P-gp was decreased significantly. Lastly, immunohistochemistry in breast cancer tissues showed that P-gp expression had a positive correlation with the expression of CUL4A and ERK1/2. Thus, these results implied that CUL4A and ERK1/2 participated in multi-drug resistance in breast cancer through regulation of MDR1/P-gp expression.

C2ORF40 suppresses breast cancer cell proliferation and invasion through modulating expression of M phase cell cycle genes

Recently, it has been suggested that C2ORF40 is a candidate tumor suppressor gene in breast cancer. However, the mechanism for reduced expression of C2ORF40 and its functional role in breast cancers remain unclear. Here we show that C2ORF40 is frequently silenced in human primary breast cancers and cell lines through promoter hypermethylation. C2ORF40 mRNA level is significantly associated with patient disease-free survival and distant cancer metastasis. Overexpression of C2ORF40 inhibits breast cancer cell proliferation, migration and invasion. By contrast, silencing C2ORF40 expression promotes these biological phenotypes. Bioinformatics and FACS analysis reveal C2ORF40 functions at G2/M phase by downregulation of mitotic genes expression, including UBE2C. Our results suggest that C2ORF40 acts as a tumor suppressor gene in breast cancer pathogenesis and progression and is a candidate prognostic marker for this disease.

Evaluating the Prognostic Significance of FBXW7 Expression Level in Human Breast Cancer by a Meta-analysis of Transcriptional Profiles

The tumor suppressor gene FBXW7 is mutated in numerous types of human cancers leading to loss of its function and/or expression. However the clinic significance of FBXW7 alterations remains largely unknown. Here, we carried out a meta-analysis of 10 gene expression microarray studies for a total 1900 patients of breast cancer with clinic information to evaluate the prognostic impact of FBXW7 mRNA expression. The FBXW7 mRNA levels significantly reduced in breast cancer compared to normal tissues. In addition, significant difference in the FBXW7 mRNA levels was found among molecular subtypes (normal-like, luminal A, luminal B, ERBB2 and basal). ERBB2 and basal tumors had significantly lower average FBXW7 mRNA level than normal-like tumors, whereas luminal A and B tumors have the lowest average FBXW7 mRNA level. The patients with higher FBXW7 mRNA level significantly increased disease-free survival, particularly in the group of patients with ER negative and basal subtype tumors. Moreover, higher FBXW7 mRNA level also significantly increased overall survival in the patients with ER negative tumors. But we strikingly found opposite effect of FBXW7 expression on overall survival in different subtypes. The patients with higher FBXW7 mRNA level significantly decreased overall survival in normal-like subtype while the patients with higher FBXW7 mRNA level significantly increased overall survival in ERBB2 and Basal subtype. Taken together, our results suggest that FBXW7 mRNA levels were a prognostic factor for disease-free and overall survival according to ER status and molecular subtypes.

α-enolase promotes tumorigenesis and metastasis via regulating AMPK/mTOR pathway in colorectal cancer.

The α‐enolase (ENO1) plays pivotal roles in several types of cancer, but its clinical significance, functional role, and possible mechanism in colorectal cancer (CRC) have remained unclear. Expression level of ENO1 in CRC tissues was examined by qRT‐PCR, Western blot, and immunohistochemistry. The effects of ENO1 on cell growth were investigated by MTT, colony formation, flow cytometry assays, and in vivo tumorigenic capacity analysis. The impacts of ENO1 on cell migration and invasion were also explored by scratch‐healing, Transwell or Matrigel chamber assays, and in vivo metastatic capacity analysis. Our results showed that the expression level of ENO1 was significantly elevated in CRC tissues. High expression level of ENO1 was associated with disease progression in CRC patients. Overexpression of ENO1 in HCT116 cell line promoted cell proliferation, migration, and invasion in vitro as well as tumorigenesis and metastasis in vivo. In other hand, ablation of ENO1 in HCT116 cells led to totally reverse effects. Mechanistically, we revealed ENO1 could regulate AMPK/mTOR signaling pathway. AMPK pathway activation or mTOR pathway suppression blocked these ENO1 induced alterations. Together, our results demonstrated that ENO1 is a potent promoter of CRC genesis and metastasis at least in part though regulating AMPK/mTOR pathway. These findings also suggested that ENO1 may be a promising therapeutic target in CRC patients.