Tam How

The type III TGF-β receptor suppresses breast cancer progression

TheTGF-β�signalingpathwayhasacomplexroleinregulatingmammarycarcinogenesis.�Herewedemon- stratethatthetypeIIITGF-β�receptor�(TβRIII,�orbetaglycan),�aubiquitouslyexpressedTGF-β�coreceptor,� regulatedbreastcancerprogressionandmetastasis.�MosthumanbreastcancerslostTβRIIIexpression,�with� lossofheterozygosityoftheTGFBR3�genelocuscorrelatingwithdecreasedTβRIIIexpression.�TβRIIIexpres- siondecreasedduringbreastcancerprogression,�andlowTβRIIIlevelspredicteddecreasedrecurrence-free� survivalinbreastcancerpatients.�RestoringTβRIIIexpressioninbreastcancercellsdramaticallyinhibited� tumorinvasivenessinvitroandtumorinvasion,�angiogenesis,�andmetastasisinvivo.�TβRIIIappearedto� inhibittumorinvasionbyundergoingectodomainsheddingandproducingsolubleTβRIII,�whichbinds� andsequestersTGF-β�todecreaseTGF-β�signalingandreducebreastcancercellinvasionandtumor-induced� angiogenesis.�OurresultsindicatethatlossofTβRIIIthroughallelicimbalanceisafrequentgeneticevent� duringhumanbreastcancerdevelopmentthatincreasesmetastaticpotential.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

The Type III Transforming Growth Factor-β Receptor as a Novel Tumor Suppressor Gene in Prostate Cancer

The transforming growth factor-beta (TGF-beta) signaling pathway has an important role in regulating normal prostate epithelium, inhibiting proliferation, differentiation, and both androgen deprivation-induced and androgen-independent apoptosis. During prostate cancer formation, most prostate cancer cells become resistant to these homeostatic effects of TGF-beta. Although the loss of expression of either the type I (TbetaRI) or type II (TbetaRII) TGF-beta receptor has been documented in approximately 30% of prostate cancers, most prostate cancers become TGF-beta resistant without mutation or deletion of TbetaRI, TbetaRII, or Smads2, 3, and 4, and thus, the mechanism of resistance remains to be defined. Here, we show that type III TGF-beta receptor (TbetaRIII or betaglycan) expression is decreased or lost in the majority of human prostate cancers as compared with benign prostate tissue at both the mRNA and protein level. Loss of TbetaRIII expression correlates with advancing tumor stage and a higher probability of prostate-specific antigen (PSA) recurrence, suggesting a role in prostate cancer progression. The loss of TbetaRIII expression is mediated by the loss of heterozygosity at the TGFBR3 genomic locus and epigenetic regulation of the TbetaRIII promoter. Functionally, restoring TbetaRIII expression in prostate cancer cells potently decreases cell motility and cell invasion through Matrigel in vitro and prostate tumorigenicity in vivo. Taken together, these studies define the loss of TbetaRIII expression as a common event in human prostate cancer and suggest that this loss is important for prostate cancer progression through effects on cell motility, invasiveness, and tumorigenicity.

Loss of Betaglycan Expression in Ovarian Cancer: Role in Motility and Invasion

The transforming growth factor-beta (TGF-beta) superfamily members, TGF-beta, activin, and inhibin, all have prominent roles in regulating normal ovarian function. Betaglycan, or the type III TGF-beta receptor, is a coreceptor that regulates TGF-beta, activin, and inhibin signaling. Here, we show that betaglycan expression is frequently decreased or lost in epithelial derived ovarian cancer at both the mRNA and protein level, with the degree of loss correlating with tumor grade. Treatment of ovarian cancer cell lines with the methyltransferase inhibitor 5-aza-2-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in significant synergistic induction of betaglycan message levels and increased betaglycan protein expression, indicating that epigenetic silencing may play a role in the loss of betaglycan expression observed in ovarian cancer. Although restoring betaglycan expression in Ovca429 ovarian cancer cells is not sufficient to restore TGF-beta-mediated inhibition of proliferation, betaglycan significantly inhibits ovarian cancer cell motility and invasiveness. Furthermore, betaglycan specifically enhances the antimigratory effects of inhibin and the ability of inhibin to repress matrix metalloproteinase levels in these cells. These results show, for the first time, epigenetic regulation of betaglycan expression in ovarian cancer, and a novel role for betaglycan in regulating ovarian cancer motility and invasiveness.

TβRIII suppresses non-small cell lung cancer invasiveness and tumorigenicity

The transforming growth factor-beta (TGF-beta) superfamily has essential roles in lung development, regulating cell proliferation, branching morphogenesis, differentiation and apoptosis. Although most lung cancers become resistant to the tumor suppressor effects of TGF-beta, and loss or mutation of one of the components of the TGF-beta signaling pathway, including TbetaRII, Smad2 and Smad4 have been reported, mutations are not common in non-small cell lung cancer (NSCLC). Here we demonstrate that the TGF-beta superfamily co-receptor, the type III TGF-beta receptor (TbetaRIII or betaglycan) is lost in the majority of NSCLC specimens at the mRNA and protein levels, with loss correlating with increased tumor grade and disease progression. Loss of heterozygosity at the TGFBR3 genomic locus occurs in 38.5% of NSCLC specimens and correlates with decreased TbetaRIII expression, suggesting loss of heterozygosity as one mechanism for TbetaRIII loss. In the H460 cell model of NSCLC, restoring TbetaRIII expression decreased colony formation in soft agar. In the A549 cell model of NSCLC, restoring TbetaRIII expression significantly decreased cellular migration and invasion through Matrigel, in the presence and absence of TGF-beta1, and decreased tumorigenicity in vivo. In a reciprocal manner, shRNA-mediated silencing of endogenous TbetaRIII expression enhanced invasion through Matrigel. Mechanistically, TbetaRIII functions, at least in part, through undergoing ectodomain shedding, generating soluble TbetaRIII, which is able to inhibit cellular invasiveness. Taken together, these results support TbetaRIII as a novel tumor suppressor gene that is commonly lost in NSCLC resulting in a functional increase in cellular migration, invasion and anchorage-independent growth of lung cancer cells.

Bone morphogenetic proteins induce pancreatic cancer cell invasiveness through a Smad1-dependent mechanism that involves matrix metalloproteinase-2

Bone morphogenetic proteins (BMPs) have an emerging role in human cancers. Here we demonstrate that the BMP-signaling pathway is intact and functional in human pancreatic cancer cells, with several BMP signaling components and transcriptional targets upregulated in human pancreatic cancer specimens compared with normal pancreatic tissue. Functionally, multiple BMP family members, including BMP-2, BMP-4 and BMP-7, induce an epithelial to mesenchymal transition (EMT) in the human pancreatic cancer cell line Panc-1, as demonstrated by morphological alterations and loss of E-cadherin expression. BMP-mediated EMT results in an increase in invasiveness of Panc-1 cells, in part through increased expression and activity of matrix metalloproteinase (MMP)-2, a known mediator of pancreatic cancer cell invasiveness. Accompanying EMT, BMP reduces expression of the transforming growth factor (TGF)-beta superfamily receptor, transforming growth factor-beta type III receptor (TbetaRIII), for which we have previously demonstrated loss of expression during pancreatic cancer progression. Maintaining TbetaRIII expression inhibits BMP-mediated invasion and suppresses Smad1 activation. Further, Smad1 is required for BMP-induced invasiveness and partially responsible for BMP-mediated increases in MMP-2 activity. These data suggest that BMP signaling, through Smad1 induction and upregulation of MMP-2, is an important mediator of pancreatic cancer invasiveness and a potential therapeutic target for treating this deadly disease.

Endoglin Promotes Transforming Growth Factor β-mediated Smad 1/5/8 Signaling and Inhibits Endothelial Cell Migration through Its Association with GIPC

Transforming growth factor β (TGF-β) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFβ-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-β signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFβ-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-β1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-β signaling.

ALK5 phosphorylation of the endoglin cytoplasmic domain regulates Smad1/5/8 signaling and endothelial cell migration

Endoglin, an endothelial cell-specific transforming growth factor-beta (TGF-beta) superfamily coreceptor, has an essential role in angiogenesis. Endoglin-null mice have an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin result in the vascular disease hereditary hemorrhagic telangiectasia type I. Increased endoglin expression in the proliferating endothelium of tumors has been correlated with metastasis, tumor grade and decreased survival. Although endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta-responsive pathways, the activin receptor-like kinase 5 (ALK5)/Smad2/3 pathway and the activin receptor-like kinase 1 (ALK1)/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Here, we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by ALK5 in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to TGF-beta and endothelial cell migration, whereas loss of phosphorylation at both serines 646 and 649 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to bone morphogenetic protein-9. Taken together, these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.

The type III transforming growth factor-β receptor negatively regulates nuclear factor kappa B signaling through its interaction with β-arrestin2

Transforming growth factor-beta (TGF-beta) increases or decreases nuclear factor kappa B (NFkappaB) signaling in a context-dependent manner through mechanisms that remain to be defined. The type III transforming growth factor-beta receptor (TbetaRIII) is a TGF-beta superfamily co-receptor with emerging roles in both mediating and regulating TGF-beta superfamily signaling. We have previously reported a novel interaction of TbetaRIII with the scaffolding protein, beta-arrestin2, which results in TbetaRIII internalization and downregulation of TGF-beta signaling. beta-arrestin2 also scaffolds interacting receptors with the mitogen-activated protein kinase and NFkappaB-signaling pathways. Here, we demonstrate that TbetaRIII, through its interaction with beta-arrestin2, negatively regulates NFkappaB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TbetaRIII expression reduced NFkappaB-mediated transcriptional activation and IkappaBalpha degradation, whereas a TbetaRIII mutant unable to interact with beta-arrestin2, TbetaRIII-T841A, had no effect. In a reciprocal manner, short hairpin RNA-mediated silencing of either TbetaRIII expression or beta-arrestin2 expression increased NFkappaB-mediated transcriptional activation and IkappaBalpha degradation. Functionally, TbetaRIII-mediated repression of NFkappaB signaling is important for TbetaRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TbetaRIII regulates NFkappaB signaling and expand the roles of this TGF-beta superfamily co-receptor in regulating epithelial cell homeostasis.

Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development

The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and cellular homeostasis, with aberrant BMP signaling resulting in a broad spectrum of human disease. We report that BMPs unexpectedly signal through the canonical transforming growth factor β (TGF‐β)‐responsive Smad2 and Smad3. BMP‐induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP‐binding TGF‐β superfamily receptors, activin receptor‐like kinase (ALK)3/6, and the Smad2/3 phosphorylating receptors ALK5/7. BMP signaling through Smad2 mediates, in part, dorsoventral axis patterning in zebrafish embryos, whereas BMP signaling through Smad3 facilitates cancer cell invasion. Consistent with increased BMP‐mediated Smad2/3 signaling during cancer progression, Smad1/5 and Smad 2/3 signaling converge in human cancer specimens. Thus, the signaling mechanisms used by BMPs and TGF‐β superfamily receptors are broader than previously appreciated.—Holtzhausen, A., Golzio, C., How, T., Lee, Y.‐H., Schiemann, W. P., Katsanis, N., Blobe, G. C. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development. FASEB J. 28, 1248–1267 (2014). www.fasebj.org