Interplay between desmoglein2 and hypoxia controls metastasis in breast cancer.

Metastasis is the major cause of cancer death. An increased level of circulating tumor cells (CTCs), metastatic cancer cells that have intravasated into the circulatory system, is particularly associated with colonization of distant organs and poor prognosis. However, the key factors required for tumor cell dissemination and colonization remain elusive. We found that high expression of desmoglein2 (DSG2), a component of desmosome-mediated intercellular adhesion complexes, promoted tumor growth, increased the prevalence of CTC clusters, and facilitated distant organ colonization. The dynamic regulation of DSG2 by hypoxia was key to this process, as down-regulation of DSG2 in hypoxic regions of primary tumors led to elevated epithelial-mesenchymal transition (EMT) gene expression, allowing cells to detach from the primary tumor and undergo intravasation. Subsequent derepression of DSG2 after intravasation and release of hypoxic stress was associated with an increased ability to colonize distant organs. This dynamic regulation of DSG2 was mediated by Hypoxia-Induced Factor1α (HIF1α). In contrast to its more widely observed function to promote expression of hypoxia-inducible genes, HIF1α repressed DSG2 by recruitment of the polycomb repressive complex 2 components, EZH2 and SUZ12, to the DSG2 promoter in hypoxic cells. Consistent with our experimental data, DSG2 expression level correlated with poor prognosis and recurrence risk in breast cancer patients. Together, these results demonstrated the importance of DSG2 expression in metastasis and revealed a mechanism by which hypoxia drives metastasis.

Author Correction: MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells.

In the version of Supplementary Fig. 6c originally published with this Article, the immunoprecipitation (IP) and immunoblotting (IB) tags in the top panel were mislabelled. In addition, in Supplementary Fig. 6e, the blot of the IP: Numb; IB: ß-Trcp panel for HCT15 was mistakenly duplicated for HCT116. The correct versions of these figures are shown below. An independent repeat of the experiments presented in Supplementary Fig. 6c and e, showing results that are consistent with those reported in the unprocessed blots, have been deposited in figshare ( 10.6084/m9.figshare.7570685 ).

HSP60 overexpression increases the protein levels of the p110α subunit of phosphoinositide 3-kinase and c-Myc.

Heat shock protein 60 (HSP60) is a chaperone protein which plays an essential role in facilitating the folding of many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in various types of human cancers. However, proteins induced by HSP60 to mediate transformation remain largely unknown. Here we show that HSP60 overexpression increases the protein levels of the p110α subunit of phosphoinositide 3-kinase (PI3K). The amino acid domain 288-383 of HSP60 is used to increase the protein levels. Overexpression of HSP60 also induces the levels of phosphorylated Akt. In addition, the amino acid domain 288-383 of HSP60 is used to induce c-Myc expression. Finally, a mono-ubiquitinated form of ß-catenin has a higher activity to activate ß-catenin downstream targets compared to wild-type ß-catenin. These results indicate that HSP60 overexpression induces the levels or activity of multiple oncogenic proteins to mediate transformation.

TET1 regulates hypoxia-induced epithelial-mesenchymal transition by acting as a co-activator.

BACKGROUND: Hypoxia induces the epithelial-mesenchymal transition, EMT, to promote cancer metastasis. In addition to transcriptional regulation mediated by hypoxia-inducible factors, HIFs, other epigenetic mechanisms of gene regulation, such as histone modifications and DNA methylation, are utilized under hypoxia. However, whether DNA demethylation mediated by TET1, a DNA dioxygenase converting 5-methylcytosine, 5mC, into 5-hydroxymethylcytosine, 5hmC, plays a role in hypoxia-induced EMT is largely unknown. RESULTS: We show that TET1 regulates hypoxia-responsive gene expression. Hypoxia/HIF-2α regulates the expression of TET1. Knockdown of TET1 mitigates hypoxia-induced EMT. RNA sequencing and 5hmC sequencing identified the set of TET1-regulated genes. Cholesterol metabolic process genes are among the genes that showed high prevalence and statistical significance. We characterize one of the genes, INSIG1 (insulin induced gene 1), to confirm its expression and the 5hmC levels in its promoter. Knockdown of INSIG1 also mitigates hypoxia-induced EMT. Finally, TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity. TET1 acts as a co-activator to further enhance the expression of INSIG1 together with HIF-2α. We define the domain in HIF-1α that interacts with TET1 and map the domain in TET1 that confers transactivation to a 200 amino acid region that contains a CXXC domain. The TET1 catalytically inactive mutant is capable of rescuing hypoxia-induced EMT in TET1 knockdown cells. CONCLUSIONS: These findings demonstrate that TET1 serves as a transcription co-activator to regulate hypoxia-responsive gene expression and EMT, in addition to its role in demethylating 5mC.

MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells.

Asymmetrical cell division (ACD) maintains the proper number of stem cells to ensure self-renewal. In cancer cells, the deregulation of ACD disrupts the homeostasis of the stem cell pool and promotes tumour growth. However, this mechanism is unclear. Here, we show a reduction of ACD in spheroid-derived colorectal cancer stem cells (CRCSCs) compared with differentiated cancer cells. The epithelial-mesenchymal transition (EMT) inducer Snail is responsible for the ACD-to-symmetrical cell division (SCD) switch in CRCSCs. Mechanistically, Snail induces the expression of microRNA-146a (miR-146a) through the ß-catenin-TCF4 complex. miR-146a targets Numb to stabilize ß-catenin, which forms a feedback circuit to maintain Wnt activity and directs SCD. Interference with the Snail-miR-146a­ß-catenin loop by inhibiting the MEK or Wnt activity reduces the symmetrical division of CRCSCs and attenuates tumorigenicity. In colorectal cancer patients, the Snail(High)Numb(Low) profile is correlated with cetuximab resistance and a poorer prognosis. This study elucidates a unique mechanism of EMT-induced CRCSC expansion.

Epigenetic regulation of hypoxia-responsive gene expression: focusing on chromatin and DNA modifications.

Mammalian cells constantly encounter hypoxia, which is a stress condition occurring during development and physiological processes. To adapt to this inevitable condition, cells develop various mechanisms to cope with this stress and survive. In addition to the activation/stabilization of transcriptional regulators (hypoxia-inducible factors), other epigenetic mechanisms of gene regulation are used. These mechanisms are mediated by various players including transcriptional coregulators, chromatin-modifying complexes, histone modification enzymes and changes in DNA methylation status. Recent progress in all the fields mentioned above has greatly improved the knowledge of how gene regulation contributes to the hypoxic response. This review should shed light on the molecular epigenetic mechanisms of hypoxia-induced gene regulation and help understand the processes adapted by cells to cope with hypoxia.

Hypoxia-regulated target genes implicated in tumor metastasis.

Hypoxia is an important microenvironmental factor that induces cancer metastasis. Hypoxia/hypoxia-inducible factor-1α (HIF-1α) regulates many important steps of the metastatic processes, especially epithelial-mesenchymal transition (EMT) that is one of the crucial mechanisms to cause early stage of tumor metastasis. To have a better understanding of the mechanism of hypoxia-regulated metastasis, various hypoxia/HIF-1α-regulated target genes are categorized into different classes including transcription factors, histone modifiers, enzymes, receptors, kinases, small GTPases, transporters, adhesion molecules, surface molecules, membrane proteins, and microRNAs. Different roles of these target genes are described with regards to their relationship to hypoxia-induced metastasis. We hope that this review will provide a framework for further exploration of hypoxia/HIF-1α-regulated target genes and a comprehensive view of the metastatic picture induced by hypoxia.

Epigenetic reprogramming and post-transcriptional regulation during the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a developmental process that is important for organ development, metastasis, cancer stemness, and organ fibrosis. The EMT process is regulated by different signaling pathways as well as by various epigenetic and post-transcriptional mechanisms. Here, we review recent progress describing the role of different chromatin modifiers in various signaling events leading to EMT, including hypoxia, transforming growth factor (TGF)-ß, Notch, and Wnt. We also discuss post-transcriptional mechanisms, such as RNA alternative splicing and the effects of miRNAs in EMT regulation. Furthermore, we highlight on-going and future work aimed at a detailed understanding of the epigenetic and post-transcriptional mechanisms that regulate EMT. This work will shed new light on the cellular and tumorigenic processes affected by EMT misregulation.

Interplay between HDAC3 and WDR5 is essential for hypoxia-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is important for organ development, metastasis, cancer stemness, and organ fibrosis. Molecular mechanisms to coordinately regulate hypoxia-induced EMT remain elusive. Here, we show that HIF-1α-induced histone deacetylase 3 (hdac3) is essential for hypoxia-induced EMT and metastatic phenotypes. Change of specific chromatin states is associated with hypoxia-induced EMT. Under hypoxia, HDAC3 interacts with hypoxia-induced WDR5, recruits the histone methyltransferase (HMT) complex to increase histone H3 lysine 4 (H3K4)-specific HMT activity, and activates mesenchymal gene expression. HDAC3 also serves as an essential corepressor to repress epithelial gene expression. Knockdown of WDR5 abolishes mesenchymal gene activation but not epithelial gene repression during hypoxia. These results indicate that hypoxia induces different chromatin modifiers to coordinately regulate EMT through distinct mechanisms.

Interaction between HSP60 and beta-catenin promotes metastasis.

Heat shock protein 60 (HSP60) plays an essential role in assisting many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in different types of human cancers with metastasis (e.g. pancreatic cancer and large bowel carcinoma). However, the role of HSP60 in metastasis remains little known. Aberrant activation of beta-catenin plays a key role in tumorigenesis and metastasis. Here, we show that overexpression of HSP60 induces metastatic phenotypes in vitro and in vivo. HSP60 interacts with beta-catenin, increases beta-catenin protein levels through the apical domain and enhances its transcriptional activity. Short-interference RNA-mediated repression of beta-catenin reverts metastatic activity caused by HSP60 overexpression. Proteosomal activity is not required for the induction of beta-catenin by HSP60. Coexpression of HSP60 and nuclear beta-catenin predicts a worse prognosis of metastatic head and neck cancer patients. These results implicate a novel role of HSP60 in metastasis.

Direct regulation of HSP60 expression by c-MYC induces transformation.

The c-MYC proto-oncogene encodes a ubiquitous transcription factor involved in cell proliferation and tumorigenesis. Heat shock protein 60 (HSP60) plays an essential role in assisting many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in different types of human cancer. Here we show that c-MYC directly activates HSP60 transcription through an E-box (CACGTG) site located in the proximal promoter of the HSP60 gene. Overexpression of HSP60 induces transformation. Short-interference RNA (siRNA) mediated repression of HSP60 reduces transformation caused by c-MYC overexpression. These results indicate that c-MYC may promote transformation through the induction of HSP60 expression.