Nadine Hempel

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.

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.

Manganese Superoxide Dismutase Enhances the Invasive and Migratory Activity of Tumor Cells

Clinically significant elevations in the expression of manganese superoxide dismutase (Sod2) are associated with an increased frequency of tumor invasion and metastasis in certain cancers. The aim of this study was to examine whether increases in Sod2 activity modulate the migratory potential of tumor cells, contributing to their enhanced metastatic behavior. Overexpression of Sod2 in HT-1080 fibrosarcoma cells significantly enhanced their migration 2-fold in a wound healing assay and their invasive potential 3-fold in a transwell invasion assay. Severity of invasion was directly correlated to Sod2 expression levels and this invasive phenotype was similarly observed in 253J bladder tumor cells, in which Sod expression resulted in a 3-fold increase in invasion compared with controls. Further, migration and invasion of the Sod2-expressing cells was inhibited following overexpression of catalase, indicating that the promigratory/invasive phenotype of Sod2-expressing cells is H(2)O(2) dependent. Sod2 overexpression was associated with a loss of vinculin-positive focal adhesions that were recovered in cells coexpressing catalase. Tail vein injections of Sod2-GFP-expressing HT-1080 cells in NCR nude mice led to the development of pulmonary metastatic nodules displaying high Sod2-GFP expression. Isolated tumors were shown to retain high Sod2 activity in culture and elevated levels of the matrix degrading protein matrix metalloproteinase-1, and a promigratory phenotype was observed in a population of cells growing out from the tumor nodule. These findings suggest that the association between increased Sod2 activity and poor prognosis in cancer can be attributed to alterations in their migratory and invasive capacity.

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.

Manganese Superoxide Dismutase (Sod2) and Redox-Control of Signaling Events That Drive Metastasis

Manganese superoxide dismutase (Sod2) has emerged as a key enzyme with a dual role in tumorigenic progression. Early studies were primarily directed at defining the tumor suppressive function of Sod2 based on its low level expression in many tumor types. It is now commonly held that loss of Sod2 expression is likely an early event in tumor progression allowing for further propagation of the tumorigenic phenotype resulting from steady state increases in free radical production. Increases in free radical load have also been linked to defects in mitochondrial function and metastatic disease progression. It was initially believed that Sod2 loss may propagate metastatic disease progression, in reality both epidemiologic and experimental evidence indicate that Sod2 levels increase in many tumor types as they progress from early stage non-invasive disease to late stage metastatic disease. Sod2 overexpression in many instances enhances the metastatic phenotype that is reversed by efficient H(2)O(2) scavenging. This review evaluates the many sequelae associated with increases in Sod2 that impinge on the metastatic phenotype. The ability to use Sod2 to modulate the cellular redox-environment has allowed for the identification of redox-responsive signaling events that drive malignancy, such as invasion, migration and prolonged tumor cell survival. Further studies of these redox-driven events will help in the development of targeted therapeutic strategies to efficiently restrict redox-signaling essential for malignant progression.

Altered Redox Status Accompanies Progression to Metastatic Human Bladder Cancer

The role of reactive oxygen species (ROS) in bladder cancer progression remains an unexplored field. Expression levels of enzymes regulating ROS levels are often altered in cancer. A search of publicly available microarray data reveals that expression of mitochondrial manganese superoxide dismutase (Sod2), responsible for the conversion of superoxide (O(2)(-)) to hydrogen peroxide (H(2)O(2)), is consistently increased in high-grade and advanced-stage bladder tumors. We aimed to identify the role of Sod2 expression and ROS in bladder cancer. Using an in vitro human bladder tumor model we monitored the redox state of both nonmetastatic (253J) and highly metastatic (253J B-V) bladder tumor cell lines. 253J B-V cells displayed significantly higher Sod2 protein and activity levels compared to their parental 253J cell line. The increase in Sod2 expression was accompanied by a significant decrease in catalase activity, resulting in a net increase in H(2)O(2) production in the 253J B-V cell line. Expression of the prometastatic and proangiogenic factors matrix metalloproteinase 9 (MMP-9) and vascular endothelial-derived growth factor (VEGF), respectively, was upregulated in the metastatic line. Expression of both MMP-9 and VEGF was shown to be H(2)O(2)-dependent, as removal of H(2)O(2) by overexpression of catalase attenuated their expression. Similarly, expression of catalase effectively reduced the clonogenic activity of 253J B-V cells. These findings indicate that metastatic bladder cancer cells display an altered antioxidant expression profile, resulting in a net increase in ROS production, which leads to the induction of redox-sensitive protumorigenic and prometastatic genes such as VEGF and MMP-9.

Recent advances in intracellular and in vivo ROS sensing: focus on nanoparticle and nanotube applications.

Reactive oxygen species (ROS) are increasingly being implicated in the regulation of cellular signaling cascades. Intracellular ROS fluxes are associated with cellular function ranging from proliferation to cell death. Moreover, the importance of subtle, spatio-temporal shifts in ROS during localized cellular signaling events is being realized. Understanding the biochemical nature of the ROS involved will enhance our knowledge of redox-signaling. An ideal intracellular sensor should therefore resolve real-time, localized ROS changes, be highly sensitive to physiologically relevant shifts in ROS and provide specificity towards a particular molecule. For in vivo applications issues such as bioavailability of the probe, tissue penetrance of the signal and signal-to-noise ratio also need to be considered. In the past researchers have heavily relied on the use of ROS-sensitive fluorescent probes and, more recently, genetically engineered ROS sensors. However, there is a great need to improve on current methods to address the above issues. Recently, the field of molecular sensing and imaging has begun to take advantage of the unique physico-chemical properties of nanoparticles and nanotubes. Here we discuss the recent advances in the use of these nanostructures as alternative platforms for ROS sensing, with particular emphasis on intracellular and in vivo ROS detection and quantification.

The type III TGF-β receptor suppresses breast cancer progression through GIPC-mediated inhibition of TGF-β signaling

Loss of expression of the type III transforming growth factor-beta receptor (TbetaRIII or betaglycan), a transforming growth factor-beta (TGF-beta) superfamily co-receptor, is common in human breast cancers. TbetaRIII suppresses cancer progression in vivo by reducing cancer cell migration and invasion by largely unknown mechanisms. Here, we demonstrate that the cytoplasmic domain of TbetaRIII is essential for TbetaRIII-mediated downregulation of migration and invasion in vitro and TbetaRIII-mediated inhibition of breast cancer progression in vivo. Functionally, the cytoplasmic domain of TbetaRIII is required to attenuate TGF-beta signaling, whereas TbetaRIII-mediated attenuation of TGF-beta signaling is required for TbetaRIII-mediated inhibition of migration and invasion. Mechanistically, both TbetaRIII-mediated inhibition of TGF-beta signaling and TbetaRIII-mediated inhibition of invasion occur through the interaction of the cytoplasmic domain of TbetaRIII with the scaffolding protein GAIP-interacting protein C-terminus (GIPC). Taken together, these studies support a functional role for the TbetaRIII cytoplasmic domain interacting with GIPC to suppress breast cancer progression.

Expression of the type III TGF-β receptor is negatively regulated by TGF-β

The type III transforming growth factor-beta receptor (TbetaRIII or betaglycan) is a ubiquitously expressed transforming growth factor-beta (TGF-beta) superfamily coreceptor with essential roles in embryonic development. Recent studies have defined a role for TbetaRIII in the pathogenesis of human cancers, with frequent loss of TbetaRIII expression at the message and protein level. Mechanisms for the loss of TbetaRIII expression remain to be fully defined. Advanced human cancers often have elevated circulating levels of TGF-beta1. Here, we define a specific role for TGF-beta1 in negatively regulating TbetaRIII at the message level in breast and ovarian cancer models. TGF-beta1 decreased TbetaRIII message and protein levels in ovarian (Ovca420) and breast cancer (MDA-MB-231) cell lines in both a dose- and time-dependent manner. TGF-beta1-mediated TbetaRIII repression is mediated by the type I TGF-beta receptor/Smad2/3 pathway as the activin receptor-like kinase 5 (ALK5) inhibitor, SB431542, abrogated this effect, while the expression of constitutively active ALK5 was sufficient to repress TbetaRIII expression. Mechanistically, TGF-beta1 does not affect TbetaRIII messenger RNA (mRNA) stability, but instead directly regulates the TbetaRIII promoter. We define alternative promoters for the TGFBR3 gene, a distal and proximal promoter. Although both promoters are active, only the proximal promoter was responsive and negatively regulated by TGF-beta1 and constitutively active ALK5. Taken together, these studies define TGF-beta1-mediated downregulation of TbetaRIII mRNA expression through effects on the ALK5/Smad2/3 pathway on the TGFBR3 gene proximal promoter as a potential mechanism for decreased TbetaRIII expression in human cancers.

Loss of type III transforming growth factor-β receptor expression is due to methylation silencing of the transcription factor GATA3 in renal cell carcinoma

Loss of transforming growth factor-β receptor III (TβRIII) correlates with loss of transforming growth factor-β (TGF-β) responsiveness and suggests a role for dysregulated TGF-β signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TβRIII expression is downregulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TβRIII expression in human cells. We localize GATA3′s binding to a 10-bp region of the TβRIII proximal promoter. We demonstrate that GATA3 mRNA is downregulated in all stages, of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines, and that inhibiting GATA3 expression in normal renal epithelial cells downregulates TβRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression through epigenetic silencing decreases TβRIII expression during ccRCC progression.