Leof, Edward B.

E-Cadherin–mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P 2 ) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P 2 is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P 2 pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.

Abstract A better understanding of the antagonistic interaction and cross-talk between the pivotal oncogenic and tumor suppressor signaling pathways, and the concerted cancer signaling networks that control tumorigenesis and metastasis should give rise to novel therapeutic interventions. Hyperactivated ERBB/RAS is known to drive neoplastic transformation, tumorigenesis and metastasis in a large percentage of human cancers, and thus finding novel measures to countervail activated EGFR/HER2/RAS signals to reverse tumorigenesis and metastasis remains an important goal in cancer biology. Instead of targeting an upstream signaling component, we targeted the most downstream signaling module in the ERBB/RAS pathway, the SIAH-dependent proteolytic machinery. SIAHs are evolutionarily conserved RING E3 ligases that are the critical downstream “gatekeeper” signaling modules in the RAS signal transduction pathway. We show that human SIAH E3 ligase is upregulated in proliferating tumor cells in human tumor specimens, and that proper SIAH function is required for RAS signal transduction. By inhibiting SIAH2 E3 ligase, we have blocked both tumorigenesis and metastasis in eleven of the most aggressive human cancer cell lines known in athymic nude mice. Our observations highlight the important roles that SIAH2 plays in RAS-dependent tumorigenesis and metastasis in human cancer. In this study, we aim to identify the molecular mechanism underlying SIAH2 deficiency-mediated growth inhibition in human cancer cells. We have found that SMAD2/3/4 - the essential downstream mediators of TGFβ signaling, are novel SIAH2 substrates in that SIAH2 physically interacts with, ubiquitinates and degrades SMAD2/3/4 (not SMAD1). The identification of SIAH2-mediated SMAD2/3/4 modulation may provide an additional molecular mechanism to explain the well documented, but less understood RAS-TGFβ antagonism described in normal development, tumorigenesis and metastasis. We propose that the tumor promoting activity of SIAH2 may be mediated through the degradation of SMAD2/3/4 proteins which enhancing the growth-promoting activity of the RAS pathway while suppressing the growth inhibitory action of the TGFβ pathway. Since the substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligase, our results provide new insights into the role of altered proteolysis in mediating the RAS-TGFβ antagonism and cross-talk observed in the genesis and progression of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5073.

Transforming growth factor (TGF)-β family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-β signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-β receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-β receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-β, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-β receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-β receptor recycling as well as Smad2 phosphorylation.

Abstract Engagement of the transforming growth factor-β (TGF-β) receptor complex activates multiple signaling pathways that play crucial roles in both health and disease. TGF-β is a key regulator of fibrogenesis and cancer-associated desmoplasia; however, its exact mode of action in these pathologic processes has remained poorly defined. Here, we report a novel mechanism whereby signaling via members of the ERBB or epidermal growth factor family of receptors serves as a central requirement for the biological responses of fibroblasts to TGF-β. We show that TGF-β triggers upregulation of ERBB ligands and activation of cognate receptors via the canonical SMAD pathway in fibroblasts. Interestingly, activation of ERBB is commonly observed in a subset of fibroblast but not epithelial cells from different species, indicating cell type specificity. Moreover, using genetic and pharmacologic approaches, we show that ERBB activation by TGF-β is essential for the induction of fibroblast cell morphologic transformation and anchorage-independent growth. Together, these results uncover important aspects of TGF-β signaling that highlight the role of ERBB ligands/receptors as critical mediators in fibroblast responses to this pleiotropic cytokine. Cancer Res; 70(19); 7421–30. ©2010 AACR.

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-β (TGF-β) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-β targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-β in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-β including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-β pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-β pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-β pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

Abstract Transforming growth factor-β (TGF-β) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-β signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-β has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-β signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-β. We show that TGF-β activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2–dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-β–mediated anchorage-independent growth without affecting TGF-β transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-β action. mTORC2 promotes TGF-β–induced morphologic transformation and is required for TGF-β–induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-β–mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-β and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. [Cancer Res 2009;69(1):84–93]

Transforming growth factor (TGF)-β receptors stimulate diverse signaling processes that control a wide range of biological responses. In polarized epithelia, the TGFβ type II receptor (T2R) is localized at the basolateral membranes. Sequential cytoplasmic truncations resulted in receptor missorting to apical surfaces, and they indicated an essential targeting element(s) near the receptor's C terminus. Point mutations in the full-length receptor confirmed this prediction, and a unique basolateral-targeting region was elucidated between residues 529 and 538 (LTAxxVAxxR) that was distinct, but colocalized within a clinically significant signaling domain essential for TGFβ-dependent activation of the Smad2/3 cascade. Transfer of a terminal 84 amino-acid fragment, containing the LTAxxVAxxR element, to the apically sorted influenza hemagglutinin (HA) protein was dominant and directed basolateral HA expression. Although delivery to the basolateral surfaces was direct and independent of any detectable transient apical localization, fluorescence recovery after photobleaching demonstrated similar mobility for the wild-type receptor and a missorted mutant lacking the targeting motif. This latter finding excludes the possibility that the domain acts as a cell membrane retention signal, and it supports the hypothesis that T2R sorting occurs from an intracellular compartment.

Abstract Transforming growth factor β (TGF-β) signaling via Smad proteins occurs in various cell types. However, whereas the biological response to TGF-β can be as distinct as growth promoting (i.e., mesenchymal cells) versus growth inhibiting (i.e., epithelial cells), few discernible differences in TGF-β signaling have been reported. In the current study, we examined the role of Ras in the proliferative response to TGF-β and how it might interface with Smad-dependent and Smad-independent TGF-β signaling targets. TGF-β stimulated Ras activity in a subset of mesenchymal, but not epithelial, cultures and was required for extracellular signal-regulated kinase (ERK)–dependent transcriptional responses. Although dominant negative Ras had no effect on TGF-β internalization or Smad-dependent signaling (i.e., phosphorylation, nuclear translocation, or SBE-luciferase activity), it did prevent the hyperphosphorylation of the Smad transcriptional corepressor TG-interacting factor (TGIF). This was not sufficient, however, to overcome the mitogenic response stimulated by TGF-β, which was dependent on signals downstream of p21-activated kinase 2 (PAK2). Moreover, although the initial activation of Ras and PAK2 are distinctly regulated, TGF-β–stimulated PAK2 activity is required for Ras-dependent ERK phosphorylation and Elk-1 transcription. These findings show the requirement for crosstalk between two Smad-independent pathways in regulating TGF-β proliferation and indicate that the mechanism(s) by which TGF-β stimulates growth is not simply the opposite of its growth inhibitory actions. [Cancer Res 2007;67(8):3673–82]

Abstract Transforming growth factor-β (TGF-β) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-β in essentially all cell types, it is unlikely to be the sole mediator of cell type–specific outcomes to TGF-β stimulation. In the current study, we determined that TGF-β receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-β in vitro.