Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma.

Epithelial cancers (carcinoma) account for 80%-90% of all cancers. The development of carcinoma is associated with disrupted epithelial organization and solid ductal structures. The mechanisms underlying the morphological development of carcinoma are poorly understood, but it is thought that loss of cell polarity is an early event. Here we report the characterization of the development of human breast lesions leading to carcinoma. We identified a unique mechanism that generates solid ducts in carcinoma through progressive loss of polarity and collapse of the luminal architecture. This program initiates with asymmetric divisions of polarized cells that generate a stratified epithelium containing both polarized and depolarized cells. Stratified regions form cords that penetrate into the lumen, subdividing it into polarized secondary lumina. The secondary lumina then collapse with a concomitant decrease in RhoA and myosin II activity at the apical membrane and ultimately lose apical-basal polarity. By restoring RhoA activity in mice, ducts maintained lumen and cell polarity. Notably, disrupted tissue architecture through luminal collapse was reversible, and ducts with a lumen were re-established after oncogene suppression in vivo. This reveals a novel and common mechanism that contributes to carcinoma development by progressively disrupting cell and tissue organization.

Cell Polarity Proteins in Breast Cancer Progression.

Breast cancer, one of the leading causes of cancer related death in women worldwide, is a heterogeneous disease with diverse subtypes that have different properties and prognoses. The developing mammary gland is a highly proliferative and invasive tissue, and some of the developmental programs may be aberrantly activated to promote breast cancer progression. In the breast, luminal epithelial cells exhibit apical-basal polarity, and the failure to maintain this organizational structure, due to disruption of polarity complexes, is implicated in promoting hyperplasia and tumors. Therefore, understanding the mechanisms underlying loss of polarity will contribute to our knowledge of the early stages leading to the pathogenesis of the disease. In this review, we will discuss recent findings that support the idea that loss of apical-basal cell polarity is a crucial step in the acquisition of the malignant phenotype. Oncogene induced loss of tissue organization shares a conserved cellular mechanism with developmental process, we will further describe the role of the individual polarity complexes, the Par, Crumbs, and Scribble, to couple cell division orientation and cell growth. We will examine symmetric or asymmetric cell divisions in mammary stem cell and their contribution to the development of breast cancer subtypes and cancer stem cells. Finally, we will highlight some of the recent advances in our understanding of the molecular mechanisms by which changes in epithelial polarity programs promote invasion and metastasis through single cell and collective cell modes. J. Cell. Biochem. 117: 2215-2223, 2016. © 2016 Wiley Periodicals, Inc.

The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding.

Myostatin is a member of the transforming growth factor-beta (TGF-beta) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-beta class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.

Numb regulates cell tension required for mammary duct elongation.

The mammary gland undergoes extensive expansion of a ductal network through the stroma during puberty and is an excellent model for understanding epithelial tube morphogenesis. To investigate a role for Numb, a multifaceted adapter protein, in epithelial tube morphogenesis, we conditionally deleted it from the mammary epithelium. We report that Numb-depletion results in altered extracellular-matrix organization, reduced cell tension, altered cell shape, and increased cell packing density, which results in a 50% reduction in mammary duct elongation. Using laser ablation in vitro and geometric-based cell force inference in vivo, we determined that Numb-deficient cells have altered cortical tension. Duct elongation defects were associated with altered E-cadherin distribution, but were independent of proliferation, apoptosis in ducts or end buds. This highlights a critical role for Numb in a mechanical mechanism that is required to maintain cell packing density during epithelial tube elongation.

CTCF facilitates DNA double-strand break repair by enhancing homologous recombination repair.

The repair of DNA double-strand breaks (DSBs) is mediated via two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is recruited to DSB through its zinc finger domain independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to γ-irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing agents and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway.

Bone morphogenetic protein 2 stimulates noncanonical SMAD2/3 signaling via the BMP type 1A receptor in gonadotrope-like cells: implications for FSH synthesis.

FSH is an essential regulator of mammalian reproduction. Its synthesis by pituitary gonadotrope cells is regulated by multiple endocrine and paracrine factors, including TGFß superfamily ligands, such as the activins and inhibins. Activins stimulate FSH synthesis via transcriptional regulation of its ß-subunit gene (Fshb). More recently, bone morphogenetic proteins (BMPs) were shown to stimulate murine Fshb transcription alone and in synergy with activins. BMP2 signals via its canonical type I receptor, BMPR1A (or activin receptor-like kinase 3 [ALK3]), and SMAD1 and SMAD5 to stimulate transcription of inhibitor of DNA binding proteins. Inhibitor of DNA binding proteins then potentiate the actions of activin-stimulated SMAD3 to regulate the Fshb gene in the gonadotrope-like LßT2 cell line. Here, we report the unexpected observation that BMP2 also stimulates the SMAD2/3 pathway in these cells and that it does so directly via ALK3. Indeed, this novel, noncanonical ALK3 activity is completely independent of ALK4, ALK5, and ALK7, the type I receptors most often associated with SMAD2/3 pathway activation. Induction of the SMAD2/3 pathway by ALK3 is dependent upon its own previous activation by associated type II receptors, which phosphorylate conserved serine and threonine residues in the ALK3 juxtamembrane glycine-serine-rich domain. ALK3 signaling via SMAD3 is necessary for the receptor to stimulate Fshb transcription, whereas its activation of the SMAD1/5/8 pathway alone is insufficient. These data challenge current dogma that ALK3 and other BMP type I receptors signal via SMAD1, SMAD5, and SMAD8 and not SMAD2 or SMAD3. Moreover, they suggest that BMPs and activins may use similar intracellular signaling mechanisms to activate the murine Fshb promoter in immortalized gonadotrope-like cells.

Activins bind and signal via bone morphogenetic protein receptor type II (BMPR2) in immortalized gonadotrope-like cells.

TGFß superfamily ligands greatly outnumber their receptors. Thus, receptors are shared between ligands and individual ligands can bind multiple receptors. Bone morphogenetic proteins (BMPs) bind and signal via both BMP type II (BMPR2) and activin type II (ACVR2) receptors. We hypothesized that, in addition to its canonical receptor ACVR2, activin A might similarly bind and signal via BMPR2. First, using surface plasmon resonance, we showed that activin A binds to the BMPR2 extracellular domain (ECD), though with lower affinity compared to the ACVR2-ECD. We confirmed these results in cells, where radiolabeled activin A bound to ACVR2 and BMPR2, but not to other type II receptors (AMHR2 or TGFBR2). Using homology modeling and site-directed mutagenesis, we identified key residues in BMPR2 that mediate its interaction with activin A. The soluble ECDs of ACVR2 or BMPR2 dose-dependently inhibited activin A-, but not TGFß-induced signaling in cells, suggesting that activin binding to BMPR2 could have functional consequences. To address this idea, we altered BMPR2 expression levels in immortalized murine gonadotrope-like cells, LßT2, in which activins potently stimulate follicle-stimulating hormone ß (Fshb) subunit transcription. BMPR2 expression potentiated activin A responses whereas depletion of endogenous BMPR2 with short interfering RNAs attenuated activin A-stimulated Fshb transcription. Additional data suggest, for the first time, that BMPR2 may form functional complexes with the canonical activin type I receptor, activin receptor-like kinase 4. Collectively, our data show that BMPR2, along with ACVR2, functions as a bona fide activin type II receptor in gonadotrope-like cells, thereby broadening our understanding of mechanisms of activin action.

Cycloheximide inhibits follicle-stimulating hormone ß subunit transcription by blocking de novo synthesis of the labile activin type II receptor in gonadotrope cells.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play essential roles in the regulation of vertebrate reproduction. Activins and inhibins have opposing actions on FSH (but not LH) synthesis, either inducing or inhibiting transcription of the FSHß subunit gene (Fshb). The translational inhibitor cycloheximide (CHX) produces inhibin-like effects in cultured pituitary cells, selectively suppressing FSH production. Using the murine gonadotrope-like cell line, LßT2, as a model, we tested the hypothesis that a component of the activin pathway is highly labile in gonadotrope cells and that its rapid loss following CHX treatment impairs activin-stimulated Fshb transcription. Treatment of cells with CHX for 6h, but not 1h, blocked activin A-stimulated Fshb transcription. Pre-treatment of LßT2 cells with CHX for as few as 2-3h inhibited activin A-stimulated SMAD2/3 phosphorylation without altering total SMAD2/3 protein levels. These data indicated that CHX affects activin signalling upstream of SMAD proteins, most likely at the receptor level. Indeed, CHX rapidly reduced activin A binding to LßT2 cells. We went on to show that activin A signals via the type II receptor ACVR2, rather than ACVR2B, to regulate Fshb transcription and that the receptor has a half life of ~2h in LßT2 cells. The mechanism of ACVR2 turnover remains undefined, but appears to be ligand-, proteasome-, and lysosome-independent. Collectively, these data indicate that CHX produces inhibin-like effects in gonadotropes by preventing de novo synthesis of the highly labile ACVR2, thereby blocking activin signaling to the Fshb promoter.