Binhua P. Zhou

Dual regulation of Snail by GSK-3β-mediated phosphorylation in control of epithelial–mesenchymal transition

The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial–mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. Here we find that Snail is highly unstable, with a short half-life about 25 min. We show that GSK-3β binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its β-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3β results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3β together function as a molecular switch for many signalling pathways that lead to EMT.

Cytoplasmic localization of p21 Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu -overexpressing cells

Amplification or overexpression of HER-2/neu in cancer cells confers resistance to apoptosis and promotes cell growth. The cellular localization of p21Cip1/WAF1 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Here we show that HER-2/neu-mediated cell growth requires the activation of Akt, which associates with p21Cip1/WAF1 and phosphorylates it at threonine 145, resulting in cytoplasmic localization of p21Cip1/WAF1. Furthermore, blocking the Akt pathway with a dominant-negative Akt mutant restores the nuclear localization and cell-growth-inhibiting activity of p21Cip1/WAF1. Our results indicate that HER-2/neu induces cytoplasmic localization of p21Cip1/WAF1 through activation of Akt to promote cell growth, which may have implications for the oncogenic activity of HER-2/neu and Akt.

HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.

HER-2/neu amplification or overexpression can make cancer cells resistant to apoptosis and promotes their growth. p53 is crucial in regulating cell growth and apoptosis, and is often mutated or deleted in many types of tumour. Moreover, many tumours with a wild-type gene for p53 do not have normal p53 function, suggesting that some oncogenic signals suppress the function of p53. In this study, we show that HER-2/neu-mediated resistance to DNA-damaging agents requires the activation of Akt, which enhances MDM2-mediated ubiquitination and degradation of p53. Akt physically associates with MDM2 and phosphorylates it at Ser166 and Ser186. Phosphorylation of MDM2 enhances its nuclear localization and its interaction with p300, and inhibits its interaction with p19ARF, thus increasing p53 degradation. Our study indicates that blocking the Akt pathway mediated by HER-2/neu would increase the cytotoxic effect of DNA-damaging drugs in tumour cells with wild-type p53.

Loss of FBP1 by snail-mediated repression provides metabolic advantages in basal-like breast cancer

The epithelial-mesenchymal transition (EMT) enhances cancer invasiveness and confers tumor cells with cancer stem cell (CSC)-like characteristics. We show that the Snail-G9a-Dnmt1 complex, which is critical for E-cadherin promoter silencing, is also required for the promoter methylation of fructose-1,6-biphosphatase (FBP1) in basal-like breast cancer (BLBC). Loss of FBP1 induces glycolysis and results in increased glucose uptake, macromolecule biosynthesis, formation of tetrameric PKM2, and maintenance of ATP production under hypoxia. Loss of FBP1 also inhibits oxygen consumption and reactive oxygen species production by suppressing mitochondrial complex I activity; this metabolic reprogramming results in an increased CSC-like property and tumorigenicity by enhancing the interaction of β-catenin with T-cell factor. Our study indicates that the loss of FBP1 is a critical oncogenic event in EMT and BLBC.

β-catenin interacts with and inhibits NF-κB in human colon and breast cancer

Abstract β-catenin plays an important role in development and homeostasis. Deregulated β-catenin is involved in oncogenesis. In this study, we found that β-catenin can physically complex with NF-κB, resulting in a reduction of NF-κB DNA binding, transactivation activity, and target gene expression. Repressed NF-κB activity is found in human colon cancer cells in which β-catenin is activated. Importantly, activated β-catenin was found to inhibit the expression of NF-κB target genes, including Fas and TRAF1. Furthermore, a strong inverse correlation was identified between the expression levels of β-catenin and Fas in colon and breast tumor tissues, suggesting that β-catenin regulates NF-κB and its targets in vivo. Thus, β-catenin may play an important role in oncogenesis through the crossregulation of NF-κB.

G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer

Breast cancers are highly heterogeneous but can be grouped into subtypes based on several criteria, including level of expression of certain markers. Claudin-low breast cancer (CLBC) is associated with early metastasis and resistance to chemotherapy, while gene profiling indicates it is characterized by the expression of markers of epithelial-mesenchymal transition (EMT) - a phenotypic conversion linked with metastasis. Although the epigenetic program controlling the phenotypic and cellular plasticity of EMT remains unclear, one contributor may be methylation of the E-cadherin promoter, resulting in decreased E-cadherin expression, a hallmark of EMT. Indeed, reduced E-cadherin often occurs in CLBC and may contribute to the early metastasis and poor patient survival associated with this disease. Here, we have determined that methylation of histone H3 on lysine 9 (H3K9me2) is critical for promoter DNA methylation of E-cadherin in three TGF-β-induced EMT model cell lines, as well as in CLBC cell lines. Further, Snail interacted with G9a, a major euchromatin methyltransferase responsible for H3K9me2, and recruited G9a and DNA methyltransferases to the E-cadherin promoter for DNA methylation. Knockdown of G9a restored E-cadherin expression by suppressing H3K9me2 and blocking DNA methylation. This resulted in inhibition of cell migration and invasion in vitro and suppression of tumor growth and lung colonization in in vivo models of CLBC metastasis. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also paves a way for the development of new treatment strategies for CLBC.

The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine‐specific demethylase 1

Epithelial–mesenchymal transition (EMT) is a transdifferentiation programme. The mechanism underlying the epigenetic regulation of EMT remains unclear. In this study, we identified that Snail1 interacted with histone lysine‐specific demethylase 1 (LSD1). We demonstrated that the SNAG domain of Snail1 and the amine oxidase domain of LSD1 were required for their mutual interaction. Interestingly, the sequence of the SNAG domain is similar to that of the histone H3 tail, and the interaction of Snail1 with LSD1 can be blocked by LSD1 enzymatic inhibitors and a histone H3 peptide. We found that the formation of a Snail1–LSD1–CoREST ternary complex was critical for the stability and function of these proteins. The co‐expression of these molecules was found in cancer cell lines and breast tumour specimens. Furthermore, we showed that the SNAG domain of Snail1 was critical for recruiting LSD1 to its target gene promoters and resulted in suppression of cell migration and invasion. Our study suggests that the SNAG domain of Snail1 resembles a histone H3‐like structure and functions as a molecular hook for recruiting LSD1 to repress gene expression in metastasis.

The Role of Snail in EMT and Tumorigenesis

Epithelial-mesenchymal transition (EMT) is a highly conserved process in which polarized, immobile epithelial cells lose tight junctions, associated adherence, and become migratory mesenchymal cells. Several transcription factors, including the Snail/Slug family, Twist, δEF1/ZEB1, SIP1/ZEB2 and E12/E47 respond to microenvironmental stimuli and function as molecular switches for the EMT program. Snail is a zinc-finger transcriptional repressor controlling EMT during embryogenesis and tumor progression. Through its N-terminal SNAG domain, Snail interacts with several corepressors and epigenetic remodeling complexes to repress specific target genes, such as the E-cadherin gene (CDH1). An integrated and complex signaling network, including the RTKs, TGF-β, Notch, Wnt, TNF-α, and BMPs pathways, activates Snail, thereby inducing EMT. Snail expression correlates with the tumor grade, nodal metastasis of many types of tumor and predicts a poor outcome in patients with metastatic cancer. Emerging evidences indicate that Snail causes a metabolic reprogramming, bestows tumor cells with cancer stem cell-like traits, and additionally, promotes drug resistance, tumor recurrence and metastasis. Despite many new and exciting developments, several challenges remain to be addressed in order to understand more thoroughly the role of Snail in metastasis. Additional investigations are required to disclose the contribution of microenvironmental factors on tumor progression. This information will lead to a comprehensive understanding of Snail in cancer and will provide us with novel approaches for preventing and treating metastatic cancers.

Inflammation: A driving force speeds cancer metastasis

It has been increasingly recognized that tumor microenvironment plays an important role in carcinogenesis. Inflammatory component is present and contributes to tumor proliferation, angiogenesis, metastasis, and resistance to hormonal and chemotherapy. This review highlights the role of inflammation in the tumor metastasis. We focus on the function of proinflammatory factors, particularly cytokines during tumor metastasis. Understanding of the mechanisms by which inflammation contributes to metastasis will lead to innovative approach for treating cancer. How tumor spreads remains an enigma and has received great attention in recent years, as metastasis is the major cause of cancer mortality. The complex and highly selective metastatic cascade not only depends on the intrinsic properties of tumor cells but also the microenvironment that they derive from. An inflammatory milieu consisting of infiltrated immune cells and their secretory cytokines, chemokines, and growth factors contribute significantly to the invasive and metastatic traits of cancer cells. Here, we review new insights into the molecular pathways that link inflammation in the tumor microenvironment to metastasis.

Phosphorylation/Cytoplasmic Localization of p21Cip1/WAF1 Is Associated with HER2/neu Overexpression and Provides a Novel Combination Predictor for Poor Prognosis in Breast Cancer Patients

Purpose: The diversity of biological functions makes p21Cip1/WAF1 (p21) a controversial marker in predicting the prognosis of breast cancer patients. Recent laboratory studies revealed that the regulation of p21 function could be related to different subcellular localizations of p21 by Akt-induced phosphorylation at threonine 145 in HER2/neu-overexpressing breast cancer cells. The purpose of this study was to verify these findings in clinical settings. Experimental Design: The expression status of the key biological markers in the HER2/neu-Akt-p21 pathway in 130 breast cancer specimens was evaluated by immunohistochemical staining and correlated with patients’ clinical parameters and survival. In addition, an antibody against phospho-p21 at threonine 145 [phospho-p21 (T145)] was also used for better validation of these findings. Results: Cytoplasmic localization of p21 is highly correlated with overexpression of phospho-p21 (T145). Both cytoplasmic p21 and overexpression of phospho-p21 (T145) are associated with high expression of HER2/neu and phospho-Akt. Cytoplasmic localization of p21 and overexpression of phospho-p21 (T145), HER2/neu, and phospho-Akt are all associated with worse overall survival. Multivariate analysis of the Cox proportional hazard regression model revealed that cytoplasmic p21 and overexpression of HER2/neu are independently associated with increased risk of death. Combining these two factors stratified patients’ survival into four distinct groups, with a 5-year survival rate of 79% in low HER2/neu and negative/nuclear p21 patients, 60% in high HER2/neu and negative/nuclear p21 patients, 29% in low HER2/neu and cytoplasmic p21 patients, and 16% in high HER2/neu and cytoplasmic p21 patients. Conclusions: The present study, in addition to supporting the mechanisms of p21 regulation derived from laboratory investigation, demonstrates the prognostic importance of phospho-p21 (T145) for the first time and also provides a novel combination of p21 and HER2/neu for better stratification of patients’ survival than any single clinicopathological or biological marker that may play important diagnostic and therapeutic roles for breast cancer patients.