Yongkun Wei

IKKβ Suppression of TSC1 Links Inflammation and Tumor Angiogenesis via the mTOR Pathway

TNFalpha has recently emerged as a regulator linking inflammation to cancer pathogenesis, but the detailed cellular and molecular mechanisms underlying this link remain to be elucidated. The tuberous sclerosis 1 (TSC1)/TSC2 tumor suppressor complex serves as a repressor of the mTOR pathway, and disruption of TSC1/TSC2 complex function may contribute to tumorigenesis. Here we show that IKKbeta, a major downstream kinase in the TNFalpha signaling pathway, physically interacts with and phosphorylates TSC1 at Ser487 and Ser511, resulting in suppression of TSC1. The IKKbeta-mediated TSC1 suppression activates the mTOR pathway, enhances angiogenesis, and results in tumor development. We further find that expression of activated IKKbeta is associated with TSC1 Ser511 phosphorylation and VEGF production in multiple tumor types and correlates with poor clinical outcome of breast cancer patients. Our findings identify a pathway that is critical for inflammation-mediated tumor angiogenesis and may provide a target for clinical intervention in human cancer.

Epidermal Growth Factor Receptor Cooperates with Signal Transducer and Activator of Transcription 3 to Induce Epithelial-Mesenchymal Transition in Cancer Cells via Up-regulation of TWIST Gene Expression

Aberrant epidermal growth factor receptor (EGFR) signaling is a major cause of tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether deregulated EGFR pathway is involved in epithelial-mesenchymal transition (EMT), an early event that occurs during metastasis of cancers of an epithelial origin. Here, we show that EGF induces EGFR-expressing cancer cells to undergo a transition from the epithelial to the spindle-like mesenchymal morphology. EGF reduced E-cadherin expression and increased that of mesenchymal proteins. In search of a downstream mediator that may account for EGF-induced EMT, we focused on transcription repressors of E-cadherin, TWIST, SLUG, and Snail and found that cancer cells express high levels of TWIST and that EGF enhances its expression. EGF significantly increases TWIST transcripts and protein in EGFR-expressing lines. Forced expression of EGFR reactivates TWIST expression in EGFR-null cells. TWIST expression is suppressed by EGFR and Janus-activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) inhibitors, but not significantly by those targeting phosphoinositide-3 kinase and MEK/ERK. Furthermore, constitutively active STAT3 significantly activates the TWIST promoter, whereas the JAK/STAT3 inhibitor and dominant-negative STAT3 suppressed TWIST promoter. Deletion/mutation studies further show that a 26-bp promoter region contains putative STAT3 elements required for the EGF-responsiveness of the TWIST promoter. Chromatin immunoprecipitation assays further show that EGF induces binding of nuclear STAT3 to the TWIST promoter. Immunohistochemical analysis of 130 primary breast carcinomas indicates positive correlations between non-nuclear EGFR and TWIST and between phosphorylated STAT3 and TWIST. Together, we report here that EGF/EGFR signaling pathways induce cancer cell EMT via STAT3-mediated TWIST gene expression.

CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. Here, we demonstrate that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupted EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibited EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promoted mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cells.

Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers.

Methylation of lysine 27 on histone H3 (H3K27) by the EZH2 complex is an epigenetic mark that mediates gene silencing. EZH2 is overexpressed in many cancers and correlates with poor prognosis in both breast and prostate cancers. However, the status of H3K27 methylation and its clinical implication in cancer patients have not been reported. We thus examined trimethylation of H3K27 (H3K27me3) by immunohistochemistry and its association with clinical variables and prognosis in breast, ovarian, and pancreatic cancers. We found that H3K27me3 expression was significantly lower in breast, ovarian and pancreatic cancers than in normal tissues (62% in breast cancer vs. 88% in normal breast tissue, P = 0.001; 38.4% in ovarian cancer vs. 83.3% in normal ovarian tissue, P < 0.05; and 26% in pancreatic cancer vs. 89% in normal pancreatic tissue, P < 0.001). H3K27me3 expression showed significant prognostic impact in breast, ovarian and pancreatic cancers in univariate survival analyses. In all three cancer types, patients with low expression of H3K27me3 had significantly shorter overall survival time when compared with those with high H3K27me3 expression. In a multivariate model, H3K27me3 expression was an independent prognostic value for overall survival in all three cancer types. These results suggest that H3K27me3 expression is a prognostic indicator for clinical outcome in patients with breast, ovarian, and pancreatic cancers.

LIFR is a breast cancer metastasis suppressor upstream of the Hippo-YAP pathway and a prognostic marker

There is a pressing need to identify prognostic markers of metastatic disease and targets for treatment. Combining high-throughput RNA sequencing, functional characterization, mechanistic studies and clinical validation, we identify leukemia inhibitory factor receptor (LIFR) as a breast cancer metastasis suppressor downstream of the microRNA miR-9 and upstream of Hippo signaling. Restoring LIFR expression in highly malignant tumor cells suppresses metastasis by triggering a Hippo kinase cascade that leads to phosphorylation, cytoplasmic retention and functional inactivation of the transcriptional coactivator YES-associated protein (YAP). Conversely, loss of LIFR in nonmetastatic breast cancer cells induces migration, invasion and metastatic colonization through activation of YAP. LIFR is downregulated in human breast carcinomas and inversely correlates with metastasis. Notably, in approximately 1,000 nonmetastatic breast tumors, LIFR expression status correlated with metastasis-free, recurrence-free and overall survival outcomes in the patients. These findings identify LIFR as a metastasis suppressor that functions through the Hippo-YAP pathway and has significant prognostic power.

lncRNA Directs Cooperative Epigenetic Regulation Downstream of Chemokine Signals

lncRNAs are known to regulate a number of different developmental and tumorigenic processes. Here, we report a role for lncRNA BCAR4 in breast cancer metastasis that is mediated by chemokine-induced binding of BCAR4 to two transcription factors with extended regulatory consequences. BCAR4 binding of SNIP1 and PNUTS in response to CCL21 releases the SNIP1s inhibition of p300-dependent histone acetylation, which in turn enables the BCAR4-recruited PNUTS to bind H3K18ac and relieve inhibition of RNA Pol II via activation of the PP1 phosphatase. This mechanism activates a noncanonical Hedgehog/GLI2 transcriptional program that promotes cell migration. BCAR4 expression correlates with advanced breast cancers, and therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 strongly suppresses breast cancer metastasis in mouse models. The findings reveal a disease-relevant lncRNA mechanism consisting of both direct coordinated protein recruitment and indirect regulation of transcription factors.

MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation

ABSTRACT Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.

Down-regulation of Myeloid Cell Leukemia-1 through Inhibiting Erk/Pin 1 Pathway by Sorafenib Facilitates Chemosensitization in Breast Cancer

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidyl-prolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and up-regulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells.

GSK-3β Targets Cdc25A for Ubiquitin-Mediated Proteolysis, and GSK-3β Inactivation Correlates with Cdc25A Overproduction in Human Cancers

The Cdc25A phosphatase positively regulates cell-cycle transitions, is degraded by the proteosome throughout interphase and in response to stress, and is overproduced in human cancers. The kinases targeting Cdc25A for proteolysis during early cell-cycle phases have not been identified, and mechanistic insight into the cause of Cdc25A overproduction in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) phosphorylates Cdc25A to promote its proteolysis in early cell-cycle phases. Phosphorylation by GSK-3beta requires priming of Cdc25A, and this can be catalyzed by polo-like kinase 3 (Plk-3). Importantly, a strong correlation between Cdc25A overproduction and GSK-3beta inactivation was observed in human tumor tissues, indicating that GSK-3beta inactivation may account for Cdc25A overproduction in a subset of human tumors.

ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1

Epithelial–mesenchymal transition (EMT) is associated with characteristics of breast cancer stem cells, including chemoresistance and radioresistance. However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties. Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response. Radioresistant subpopulations of breast cancer cells derived from ionizing radiation exhibit hyperactivation of the kinase ATM and upregulation of ZEB1, and the latter promotes tumour cell radioresistance in vitro and in vivo. Mechanistically, ATM phosphorylates and stabilizes ZEB1 in response to DNA damage, ZEB1 in turn directly interacts with USP7 and enhances its ability to deubiquitylate and stabilize CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to radiation. These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.