Accumulating evidence indicates that hyperactive Wnt signalling occurs in association with the development and progression of human breast cancer. As a consequence of engaging the canonical Wnt pathway, a beta-catenin-T-cell factor (TCF) transcriptional complex is generated, which has been postulated to trigger the epithelial-mesenchymal transition (EMT) that characterizes the tissue-invasive phenotype. However, the molecular mechanisms by which the beta-catenin-TCF complex induces EMT-like programmes remain undefined. Here, we demonstrate that canonical Wnt signalling engages tumour cell dedifferentiation and tissue-invasive activity through an Axin2-dependent pathway that stabilizes the Snail1 zinc-transcription factor, a key regulator of normal and neoplastic EMT programmes. Axin2 regulates EMT by acting as a nucleocytoplasmic chaperone for GSK3beta, the dominant kinase responsible for controlling Snail1 protein turnover and activity. As dysregulated Wnt signalling marks a diverse array of cancerous tissue types, the identification of a beta-catenin-TCF-regulated Axin2-GSK3beta-Snail1 axis provides new mechanistic insights into cancer-associated EMT programmes.
Slug (Snail2) plays critical roles in regulating the epithelial-mesenchymal transition (EMT) programs operative during development and disease. However, the means by which Slug activity is controlled remain unclear. Herein we identify an unrecognized canonical Wnt/ GSK3β/β-Trcp1 axis that controls Slug activity. In the absence of Wnt signaling, Slug is phosphorylated by GSK3β and subsequently undergoes β-Trcp1-dependent ubiquitination and proteosomal degradation. Alternatively, in the presence of canonical Wnt ligands, GSK3β kinase activity is inhibited, nuclear Slug levels increase, and EMT programs are initiated. Consistent with recent studies describing correlative associations in basal-like breast cancers between Wnt signaling, increased Slug levels, and reduced expression of the tumor suppressor Breast Cancer 1, Early Onset (BRCA1), further studies demonstrate that Slug-as well as Snail-directly represses BRCA1 expression by recruiting the chromatin-demethylase, LSD1, and binding to a series of E-boxes located within the BRCA1 promoter. Consonant with these findings, nuclear Slug and Snail expression are increased in association with BRCA1 repression in a cohort of triple-negative breast cancer patients. Together, these findings establish unique functional links between canonical Wnt signaling, Slug expression, EMT, and BRCA1 regulation.lug (also termed Snail2) is a C 2 H 2 zinc-finger transcriptional repressor belonging to the three-member family of Snail proteins (Snail, Slug, and Smuc) (1, 2). First recognized for its participation in events associated with the epithelial-mesenchymal transition (EMT) programs that characterize early development, more recent studies have identified postnatal roles for Slug in a wide variety of carcinomatous states (1-7). To date, Slug expression has been linked to cancer stem cell formation, cell cycle regulation and apoptosis as well as invasion and metastasis (1-7). However, the Slug protein, like that of Snail, is rapidly turned over by the ubiquitin-proteasome system in vivo, and the key factors responsible for regulating Slug protein stability and activity remain largely undefined (2,8). Interestingly, recent studies have identified a subset of breast cancer patients (the so-called basal-like breast carcinoma phenotype) whose lesions display an EMT-like signature associated with increased Wnt signaling, upregulated Slug expression, and epigenetic silencing of the tumor suppressor BRCA1 (7, 9-13). Despite these associations, however, no molecular pathways have been established that functionally link these potentially disparate phenotypes together.Herein we demonstrate that Slug protein stability and activity are controlled by a heretofore undescribed GSK3β-dependent phosphorylation process that primes phospho-Slug for ubiquitination by the E3 ligase β-Trcp1 and its subsequent proteasomal degradation. In the presence of canonical Wnt agonists, however, GSK3β activity is suppressed and Slug phosphorylation is blocked, thereby allowing Slug protein levels and activity to...
Aberrant activation of canonical Wingless-type MMTV integration site family (Wnt) signaling is pathognomonic of colorectal cancers (CRC) harboring functional mutations in either adenomatous polyposis coli or β-catenin. Coincident with Wnt cascade activation, CRCs also up-regulate the expression of Wnt pathway feedback inhibitors, particularly the putative tumor suppressor, Axin2. Because Axin2 serves as a negative regulator of canonical Wnt signaling in normal cells, recent attention has focused on the utility of increasing Axin2 levels in CRCs as a means to slow tumor progression. However, rather than functioning as a tumor suppressor, we demonstrate that Axin2 acts as a potent promoter of carcinoma behavior by up-regulating the activity of the transcriptional repressor, Snail1, inducing a functional epithelialmesenchymal transition (EMT) program and driving metastatic activity. Silencing Axin2 expression decreases Snail1 activity, reverses EMT, and inhibits CRC invasive and metastatic activities in concert with global effects on the Wnt-regulated cancer cell transcriptome. The further identification of Axin2 and nuclear Snail1 proteins at the invasive front of human CRCs supports a revised model wherein Axin2 acts as a potent tumor promoter in vivo.invasion | E-cadherin | basement membrane | GSK3β | tankyrase
Efforts to develop unbiased screens for identifying novel functionblocking monoclonal antibodies (mAbs) in human carcinomatous states have been hampered by the limited ability to design in vitro models that recapitulate tumor cell behavior in vivo. Given that only invasive carcinoma cells gain permanent access to type I collagen-rich interstitial tissues, an experimental platform was established in which human breast cancer cells were embedded in 3D aldimine cross-linked collagen matrices and used as an immunogen to generate mAb libraries. In turn, cancer-cell-reactive antibodies were screened for their ability to block carcinoma cell proliferation within collagen hydrogels that mimic the in vivo environment. As a proof of principle, a single function-blocking mAb out of 15 identified was selected for further analysis and found to be capable of halting carcinoma cell proliferation, inducing apoptosis, and exerting global changes in gene expression in vitro. The ability of this mAb to block carcinoma cell proliferation and metastatic activity was confirmed in vivo, and the target antigen was identified by mass spectroscopy as the α 2 subunit of the α 2 β 1 integrin, one of the major type I collagen-binding receptors in mammalian cells. Validating the ability of the in vitro model to predict patterns of antigen expression in the disease setting, immunohistochemical analyses of tissues from patients with breast cancer verified markedly increased expression of the α 2 subunit in vivo. These results not only highlight the utility of this discovery platform for rapidly selecting and characterizing function-blocking, anticancer mAbs in an unbiased fashion, but also identify α 2 β 1 as a potential target in human carcinomatous states.n mammalian systems, a specialized form of extracellular matrix (ECM), termed the basement membrane, normally separates epithelial cells from the underlying type I collagen-rich interstitial matrix (1, 2). Thus, in mature animals and under physiologic conditions, the epithelium does not establish stable physical contacts with interstitial tissues (1, 2). In contrast, in neoplastic states, transformed epithelial cells (i.e., carcinomas) dissolve the intervening basement membrane barrier and establish adhesive interactions with the newly exposed type I collagen fibrillar network (1-5). As carcinoma cells begin to infiltrate the interstitial matrix, they rapidly adapt themselves to their 3D environment and initiate the proliferative phenotypes that define tumor progression at both primary and metastatic sites (2, 6, 7). Indeed, emphasizing the importance of the tumor-ECM interface, carcinoma cells do not simply use the surrounding interstitial matrix as a passive substrate, but actively promote increased type I collagen deposition within the peritumoral microenvironment as a means of further enhancing invasive activity, local growth, and cancer stem cell formation (7-12).Despite the importance of the carcinoma cell-type I collagen interface in vivo, therapeutic interventions that directly inte...
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