Activation of β-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters

<p>Abstract</p> <p>Background</p> <p>Epithelial-mesenchymal transition (EMT) not only confers tumor cells with a distinct advantage for metastatic dissemination, but also it provides those cells with cancer stem cell-like characters for proliferation and drug resistance. However, the molecular mechanism for maintenance of these stem cell-like traits remains unclear.</p> <p>Methods</p> <p>In this study, we induced EMT in breast cancer MCF7 and cervical cancer Hela cells with expression of Twist, a key transcriptional factor of EMT. The morphological changes associated with EMT were analyzed by immunofluorescent staining and Western blotting. The stem cell-like traits associated with EMT were determined by tumorsphere-formation and expression of ALDH1 and CD44 in these cells. The activation of β-catenin and Akt pathways was examined by Western blotting and luciferase assays.</p> <p>Results</p> <p>We found that expression of Twist induced a morphological change associated with EMT. We also found that the cancer stem cell-like traits, such as tumorsphere formation, expression of ALDH1 and CD44, were significantly elevated in Twist-overexpressing cells. Interestingly, we showed that β-catenin and Akt pathways were activated in these Twist-overexpressing cells. Activation of β-catenin correlated with the expression of CD44. Knockdown of β-catenin expression and inhibition of the Akt pathway greatly suppressed the expression of CD44.</p> <p>Conclusions</p> <p>Our results indicate that activation of β-catenin and Akt pathways are required for the sustention of EMT-associated stem cell-like traits.</p

The Regulation of Snail: On the Ubiquitin Edge

Metastasis accounts for a majority of cancer death. One key feature during metastasis is epithelial-mesenchymal transition (EMT), which is regulated by transcription factors such as Snail and Twist. In non-malignant cells, Snail has a short half-life and is degraded via ubiquitination, but its stability is increased in cancer cell. However, the mechanism by which Snail escapes ubiquitination and degradation remains unknown. Recently, we found that Dub3 is a deubiquinase of Snail. Most importantly, we determined that Dub3 responded to extracellular signals such as IL-6, and that the resultant signaling prevented Snail degradation, and promoted cancer growth, invasion, and migration. In this highlight, we present a concise picture of how the transcription factor Snail is regulated by ubiquitination in cancer cells, the role of Dub3 in this process, and its potential use as a treatment target

Twist-mediated Epithelial-mesenchymal Transition Promotes Breast Tumor Cell Invasion via Inhibition of Hippo Pathway

Twist is a key transcription factor for Epithelial-mesenchymal transition (EMT), which is a cellular de-differentiation program that promotes invasion and metastasis, confers tumor cells with cancer stem cell (CSC)-like characteristics, and increases therapeutic resistance. However, the mechanisms that facilitate the functions of Twist remain unclear. Here we report that Twist overexpression increased expression of PAR1, an upstream regulator of the Hippo pathway; PAR1 promotes invasion, migration, and CSC-like properties in breast cancer by activating the transcriptional co-activator TAZ. Our study indicates that Hippo pathway inhibition is required for the increased migratory and invasiveness ability of breast cancer cells in Twist-mediated EMT

The Context-Dependent Impact of Integrin-Associated CD151 and Other Tetraspanins on Cancer Development and Progression: A Class of Versatile Mediators of Cellular Function and Signaling, Tumorigenesis and Metastasis

As a family of integral membrane proteins, tetraspanins have been functionally linked to a wide spectrum of human cancers, ranging from breast, colon, lung, ovarian, prostate, and skin carcinomas to glioblastoma. CD151 is one such prominent member of the tetraspanin family recently suggested to mediate tumor development, growth, and progression in oncogenic context- and cell lineage-dependent manners. In the current review, we summarize recent advances in mechanistic understanding of the function and signaling of integrin-associated CD151 and other tetraspanins in multiple cancer types. We also highlight emerging genetic and epigenetic evidence on the intrinsic links between tetraspanins, the epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), and the Wnt/β-catenin pathway, as well as the dynamics of exosome and cellular metabolism. Finally, we discuss the implications of the highly plastic nature and epigenetic susceptibility of CD151 expression, function, and signaling for clinical diagnosis and therapeutic intervention for human cancer

Nanoparticle Delivery of miR-34a Eradicates Long-Term-Cultured Breast Cancer Stem Cells via Targeting C22ORF28 Directly

Rationale: Cancer stem cells (CSCs) have been implicated as the seeds of therapeutic resistance and metastasis, due to their unique abilities of self-renew, wide differentiation potentials and resistance to most conventional therapies. It is a proactive strategy for cancer therapy to eradicate CSCs. Methods: Tumor tissue-derived breast CSCs (BCSC), including XM322 and XM607, were isolated by fluorescence-activated cell sorting (FACS); while cell line-derived BCSC, including MDA-MB-231.SC and MCF-7.SC, were purified by magnetic-activated cell sorting (MACS). Analyses of microRNA and mRNA expression array profiles were performed in multiple breast cell lines. The mentioned nanoparticles were constructed following the standard molecular cloning protocol. Tissue microarray analysis has been used to study 217 cases of clinical breast cancer specimens. Results: Here, we have successfully established four long-term maintenance BCSC that retain their tumor-initiating biological properties. Our analyses of microarray and qRT-PCR explored that miR-34a is the most pronounced microRNA for investigation of BCSC. We establish hTERT promoter-driven VISA delivery of miR-34a (TV-miR-34a) plasmid that can induce high throughput of miR-34a expression in BCSC. TV-miR-34a significantly inhibited the tumor-initiating properties of long-term-cultured BCSC in vitro and reduced the proliferation of BCSC in vivo by an efficient and safe way. TV-miR-34a synergizes with docetaxel, a standard therapy for invasive breast cancer, to act as a BCSC inhibitor. Further mechanistic investigation indicates that TV-miR-34a directly prevents C22ORF28 accumulation, which abrogates clonogenicity and tumor growth and correlates with low miR-34 and high C22ORF28 levels in breast cancer patients. Conclusion: Taken together, we generated four long-term maintenance BCSC derived from either clinical specimens or cell lines, which would be greatly beneficial to the research progress in breast cancer patients. We further developed the non-viral TV-miR-34a plasmid, which has a great potential to be applied as a clinical application for breast cancer therapy

Chaperone Hsp47 Drives Malignant Growth and Invasion by Modulating an ECM Gene Network

The extracellular matrix (ECM) is a determining factor in the tumor microenvironment that restrains or promotes malignant growth. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. A transcription network analysis showed that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or noninvasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Coexpression network analysis also showed that levels of microRNA(miR)-29b and -29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and -29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins

Epithelial Neoplasia Coincides with Exacerbated Injury and Fibrotic Response in the Lungs of \u3cem\u3eGprc5a\u3c/em\u3e-Knockout Mice Following Silica Exposure

Exposure to crystalline silica is suggested to increase the risk for a variety of lung diseases, including fibrosis and lung cancer. However, epidemiological evidences for the exposure-risk relationship are ambiguous and conflicting, and experimental study from a reliable animal model to explore the relationship is lacking. We reasoned that a mouse model that is sensitive to both lung injury and tumorigenesis would be appropriate to evaluate the exposure-risk relationship. Previously, we showed that, Gprc5a-/- mice are susceptible to both lung tumorigenesis and endotoxin-induced acute lung injury. In this study, we investigated the biological consequences in Gprc5a-/- mouse model following silica exposure. Intra-tracheal administration of fine silica particles in Gprc5a-/- mice resulted in more severe lung injury and pulmonary inflammation than in wild-type mice. Moreover, an enhanced fibrogenic response, including EMT-like characteristics, was induced in the lungs of Gprc5a-/- mice compared to those from wild-type ones. Importantly, increased hyperplasia or neoplasia coincided with silica-induced tissue injury and fibrogenic response in lungs from Gprc5a-/- mice. Consistently, expression of MMP9, TGFβ1 and EGFR was significantly increased in lungs from silica-treated Gprc5a-/- mice compared to those untreated or wild-type ones. These results suggest that, the process of tissue repair coincides with tissue damages; whereas persistent tissue damages leads to abnormal repair or neoplasia. Thus, silica-induced pulmonary inflammation and injury contribute to increased neoplasia development in lungs from Gprc5a-/- mouse model

ITCH Nuclear Translocation and H1.2 Polyubiquitination Negatively Regulate the DNA Damage Response

The downregulation of the DNA damage response (DDR) enables aggressive tumors to achieve uncontrolled proliferation against replication stress, but the mechanisms underlying this process in tumors are relatively complex. Here, we demonstrate a mechanism through which a distinct E3 ubiquitin ligase, ITCH, modulates DDR machinery in triple-negative breast cancer (TNBC). We found that expression of a nuclear form of ITCH was significantly increased in human TNBC cell lines and tumor specimens. Phosphorylation of ITCH at Ser257 by AKT led to the nuclear localization of ITCH and ubiquitination of H1.2. The ITCH-mediated polyubiquitination of H1.2 suppressed RNF8/RNF168-dependent formation of 53BP1 foci, which plays important roles in DDR. Consistent with these findings, impaired ITCH nuclear translocation and H1.2 polyubiquitination sensitized cells to replication stress and limited cell growth and migration. AKT activation of ITCH-H1.2 axis may confer TNBC cells with a DDR repression to counteract the replication stress and increase cancer cell survivorship and growth potential

Hsp47 Promotes Cancer Metastasis by Enhancing Collagen-Dependent Cancer Cell-Platelet Interaction

Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell–platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell–platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell–platelet interaction and cancer colonization in secondary organs

Stabilization of the Transcription Factors Slug and Twist by the Deubiquitinase Dub3 is a Key Requirement for Tumor Metastasis

The Epithelial-mesenchymal transition (EMT) represents a cellular de-differentiation process that provides cells with the increased plasticity required during embryonic development, tissue remodeling, wound healing and metastasis. Slug and Twist are two key EMT transcription factors (EMT-TFs) that are tightly regulated via ubiquitination and degradation. How Slug and Twist escape degradation and become stabilized in cancer cells remains unclear. One plausible mechanism of Slug and Twist stabilization involves removal of ubiquitin by deubiquitinases (DUBs). In this study, we identified Dub3 as a novel DUB for both Slug and Twist. We further found that Dub3 overexpression increased Slug and Twist protein levels in a dose-dependent manner, whereas Dub3-knockdown decreased their protein levels. Of importance, Dub3 interacted with Slug and Twist and prevented them from degradation, thereby promoting migration, invasion, and cancer stem cell (CSC)-like properties of breast cancer cells. Intriguingly, Dub3 was identified as an early response gene that was upregulated after exposure to inflammatory cytokines such as IL-6, which plays a critical role in the growth and metastasis of breast cancer cells, as well as the maintenance of breast CSCs. We found that Dub3 played an essential role in IL-6 induced EMT through stabilization of Slug and Twist. Our study has uncovered an IL-6-Dub3-Slug/Twist signaling axis during EMT and suggests potential approaches that could target Dub3 to prevent metastatic breast tumor