Browsing by Author "Blobe, Gerard C"

The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and homeostasis. BMPs have been shown to pattern the dorsoventral axis in zebrafish (Danio rerio) early during embryonic development by establishing a dorsal-to-­ventral ligand gradient. During tumorigenesis, BMPs primarily function as tumor promoters, as an increase in BMP expression is associated with an increase in invasion, migration, epithelial-­to-­mesenchymal transition (EMT), proliferation and angiogenesis.

Although it is clear that BMPs play multiple roles in these biological events, the precise mechanism by which BMPs mediate these functions is not fully understood. Canonically, BMP ligands signal through cell surface receptor complexes that phosphorylate transcription factors, Smad1, Smad5 and Smad8, which mediate BMP-­ specific gene transcription. While studying BMP signaling during cancer progression, we determined that BMPs unexpectedly signal through the canonical TGF-β-­responsive transcription factors, Smad2 and Smad3.

We determined that 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, ALK3/6, and the Smad2/3 phosphorylating receptors, ALK5/7. BMP signaling through Smad1/5/8 induces MET, while Smad1/5 and Smad2 mediate dorsoventral axis patterning in zebrafish embryos and Smad3 facilitates invasion.

Taken together, our data provides evidence that BMP-­induced Smad2 and Smad3 phosphorylation occurs through a non-­canonical signaling mechanism to mediate multiple biological events. Thus, the signaling mechanisms utilized by BMPs and TGF-­β superfamily receptors are broader than previously appreciated.

The transforming growth factor beta (TGF-beta) signaling pathway is a critical regulator of multiple biological processes that are involved in cancer progression, such as proliferation, migration, invasion and metastasis. TGF-beta ligands bind to multiple high-affinity receptors (TbetaRI, TbetaRII, TbetaRIII), whose expression on the cell surface, and subsequent ability to transduce signaling, can be modulated by ectodomain shedding.

TbetaRIII, also known as betaglycan, is the most abundantly expressed TGF-beta receptor. TbetaRIII suppresses breast cancer progression through inhibiting migration, invasion, metastasis, and angiogenesis. TbetaRIII binds TGF-beta ligands, with membrane-bound TbetaRIII presenting ligand to enhance TGF-beta signaling. However, TbetaRIII can also undergo ectodomain shedding, releasing soluble TbetaRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TbetaRIII on TGF-beta signaling and breast cancer biology, here I describe TbetaRIII mutants with impaired (Delta-Shed-TbetaRIII) or enhanced ectodomain shedding (SS-TbetaRIII). Relative to wild-type (WT)-TbetaRIII, Delta-Shed-TbetaRIII increased TGF-beta signaling and blocked TbetaRIII's ability to inhibit breast cancer cell migration and invasion. Conversely, SS-TbetaRIII, which increased soluble TbetaRIII production, decreased TGF-beta signaling and increased TbetaRIII-mediated inhibition of breast cancer cell migration and invasion.

TbetaRI is released from the cell surface by a common sheddase of the A disintegrin and metalloproteinase (ADAM) family, ADAM17. This shedding event results in a downregulation of TGF-beta signaling. Here, I present evidence that a closely related protease, ADAM10, may be a novel sheddase for TbetaRI. A specific ADAM10 inhibitor was able to increase cell surface expression of TbetaRI, and decrease levels of circulating soluble TbetaRI in vivo. Interestingly, inhibition of ADAM10 concurrently increased shedding of TbetaRIII, and was able to alter TGF-beta signaling in a TbetaRIII-dependent manner.

Together, these studies suggest that ectodomain shedding of TGF-beta receptors is an important determinant for regulation of TGF-beta-mediated signaling and biology.

Endoglin, an endothelial cell specific transforming growth factor-beta (TGF-beta) superfamily co-receptor, has an essential role in angiogenesis, with endoglin null mice having an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin resulting in the vascular disease hereditary hemorrhagic telangiectasia type I. While endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta responsive pathways, the ALK5/Smad2/3 pathway and the ALK1/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Recently, overexpression of wild type endoglin has been demonstrated to increase ALK1 signaling, supporting a role for endoglin as an important regulator of the ALK1 pathway. Here we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by TGF-beta superfamily receptors 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. This basal phosphorylation primes and is necessary for subsequent phosphorylation of endoglin by ALK1. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin mediated inhibition of Smad1/5/8 signaling and endothelial cell migration. Taken together these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the embyronal tumor neuroblastoma, where differentiation is a validated treatment strategy, remain unclear. The neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here we identify critical components of the differentiating stroma secretome and describe preclinical testing of a novel therapeutic strategy based on their mechanism of action.

Expression of heparan sulfate proteoglycans (HSPGs), including TβRIII, GPC1, GPC3, SDC3, and SDC4, is decreased in neuroblastoma, high in the stroma, and suppresses tumor growth. High expression of TβRIII, GPC1, and SDC3 is associated with improved patient prognosis. HSPGs signal via heparan sulfate binding to FGFR1 and FGF2, which leads to phosphorylation of FGFR1 and Erk MAPK, and upregulation of the transcription factor inhibitor of DNA binding 1 (Id1). Surface expression and treatment with soluble HSPGs promotes neuroblast differentiation via this signaling complex. Expression of individual HSPGs positively correlates with Id1 expression in neuroblastoma patient samples and multivariate regression demonstrates that expression of HSPGs as a group positively correlates with Id1 expression, underscoring the clinical relevance of this pathway. HSPGs also enhance differentiation from FGF2 released by the stroma and FGF2 is identified as a potential serum prognostic biomarker in neuroblastoma patients.

The anticoagulant heparin has similar differentiating effects to HSPGs, decreasing neuroblast proliferation and reducing tumor growth while extending survival in an orthotopic xenograft model of neuroblastoma. Dissection of individual sulfation sites identifies 2-O-, 3-O-de-sulfated heparin (ODSH) as a differentiating agent that suppresses orthotopic xenograft growth and metastasis in two models while avoiding anticoagulation. These studies form the preclinical rationale for a multicenter clinical trial currently being proposed.

In conclusion, these studies translate mechanistic insights in neuroblast HSPG function to identify heparins as differentiating agents for clinical development in neuroblastoma, while demonstrating that tumor stroma biology can inform design of targeted molecular therapeutics.

The secreted extracellular protein, transforming growth factor  induced (TGFBI or IGH3), has context dependent tumor suppressor and tumor promoting roles, similar to TGF-, in part by promoting chemotherapy sensitivity, but then increased IGH3 levels promote cancer progression, with elevated levels correlating to poorer clinical outcomes. IGH3 is regulated by the transforming growth factor-β (TGF-β) signaling pathway, which is an important regulator of breast cancer progression. However, how the breast cancer microenvironment regulates TGF-β signaling during breast cancer progression remains poorly understood. In previous studies, we identified IGH3 as a secreted protein able to promote TGF- signaling. Here, we demonstrate that IGH3 promotes breast tumor growth by promoting activation of the TGF-1 ligand from its latent form in the breast cancer tumor microenvironment. CRISPR/Cas9 silencing of IGH3 decreased tumor growth and decreased activation of latent TGF-1 in a breast cancer models. Mechanistically, IGH3 activates latent TGF-1 through its cysteine rich domain, specifically cysteine 65, and partially through its FAS1-4 domain. Increased IGH3 expression in breast cancer patients correlates with poorer clinical outcomes. These results indicate that TGF-β-mediated induction of IGH3 expression is one mechanism for sustaining elevated TGF-β signaling in the breast cancer tumor microenvironment, with IGH3 setting up positive feed forward loop by promoting activation of latent TGF-1, driving breast cancer growth. Thus, mechanisms to target IGH3 have potential utility in advanced cancers, while assessing IGH3 levels could be a biomarker for chemotherapy resistance and tumor progression.

Breast cancer remains among the most common cancers of the developed world. Despite advances in treatment modalities, deaths due to breast cancer are the second leading cause of cancer death among women. The transforming growth factor-beta (TGF-β) pathway is an important modulator of breast cancer progression, acting in a tumor suppressing fashion in early carcinogenesis but switching in a poorly understood fashion to a promoter of cancer progression in later stages. Mutations and loss of function of TGF-β components are common across a variety of cancers. In particular, the expression of the type III TGF-β receptor (TβRIII) is decreased with cancer grade and clinical progression in prostate, lung, ovarian, and pancreatic cancers. In an effort to enhance our understanding of the biology of TGF-β on carcinogenesis, this dissertation looks at the role of TβRIII in breast cancer progression.

Through an examination of clinical specimens, loss of TβRIII was seen at both the message and protein levels with increasing tumor grade. Analysis of correlated patient outcomes showed that low TβRIII expression was predictive of a shorter time to recurrence, demonstrating clinical relevance for TβRIII expression. The contribution of TβRIII to tumor progression was further examined by examining known TGF-β functions, including proliferation, apoptosis, migration, and invasion. TβRIII had no effect on proliferation or apoptosis, but had a suppressive effect on metastasis in vivo, as mammary cancer cells stably expressing TβRIII that were orthotopically injected exhibited lower metatstatic burden and local invasion. In vitro, breast cancer cells exhibited suppression of migration and invasion in transwell assays. Finally, soluble TβRIII (sTβRIII) was shown to recapitulate the suppressive effects on invasion.

To further explore other potential mechanisms by which TβRIII may be mediating its tumor suppressive effects, I examined the contribution of the cytoplasmic domain of TβRIII, which is known to be critical in the regulation of TβRIII cell surface expression and downstream signaling. In vitro, I demonstrated that abrogation of the cytoplasmic domain attenuates the TβRIII-mediated suppression of migration and invasion. TβRIII's suppressive effects are also concomitant with loss of TGF-β signaling, as abrogation of the cytoplasmic domain failed to attenuate TGF-β signaling while the full length receptor was able to do so. In vivo, I also showed that in the absence of the cytoplasmic domain, TβRIII is unable to suppress metastasis and local invasion. Finally, a closer dissection of the cytoplasmic domain revealed that abolishing the interaction of TβRIII with the scaffolding protein GIPC also attenuated TβRIII's ability to dampen TGF-β signaling and invasion.

In sum, TβRIII was established as a prognostic marker for recurrence-free survival of breast cancer patients and as a suppressor of metastasis, migration, and invasion. Furthermore, several mechanisms contribute to TβRIII's tumor suppressive effects, namely the generation of sTβRIII and the interaction of TβRIII with GIPC. Taken together, these studies further demonstrate the importance of TGF-β signaling in cancer biology, elucidate mechanisms by which TβRIII suppresses breast carcinogenesis, and expand upon our understanding of the emerging roles of TβRIII in regulating tumor biology in general.

The type III TGF-β receptor (TβRIII) is a TGF-β co-receptor that presents ligand to the type II TGF-β receptor to initiate signaling. TβRIII also undergoes ectodomain shedding to release a soluble form (sTβRIII) that can bind ligand, sequestering it away from cell surface receptors. We have previously identified a TβRIII extracellular mutant that has enhanced ectodomain shedding (“super shedding (SS)” – TβRIII-SS). Here we utilize TβRIII-SS to study the balance of cell surface and soluble TβRIII in the context of lung cancer. We demonstrate that expressing TβRIII-SS in lung cancer cell models induces epithelial-mesenchymal transition (EMT) and that these TβRIII-SS (EMT) cells are less migratory, invasive and adhesive and more resistance to gemcitabine. Moreover, TβRIII-SS (EMT) cells exhibit decreased tumorigenicity but increased tumor growth in vitro and in vivo. These studies suggest that the balance of cell surface and soluble TβRIII may regulate a dichotomous role for TβRIII during cancer progression.

We have also demonstrated that cathepsin G and neutrophil elastase, proteases stored in the azurophil granules of neutrophils, can induce cleavage of cell surface TβRIII though no functional sTβRIII was detected. Similarly, activated neutrophils could induce TβRIII shedding on cancer cells. Interestingly, neutrophils generate sTβRIII that is resistant to shedding. These studies suggest that neutrophils may modulate the balance of cell surface and soluble TβRIII in the tumor microenvironment.

Neuroblastoma is a pediatric cancer arising from undifferentiated neural crest-derived precursor cells. Treatment strategies for neuroblastoma aim to promote neuroblast differentiation, however current therapies available are only modestly effective. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that act to promote neuroblast differentiation, though the precise factors released and their mechanism of action in neuroblastoma remains unclear. Here, we identify a novel component of the differentiating stroma secretome and harness stroma biology to inform the use of a combination therapy for neuroblastoma treatment.

HBEGF expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from late-stage disease containing very little stroma or HBEGF. Addition of soluble HBEGF to neuroblastoma cell lines leads to increased neuroblast differentiation and decreased proliferation. Heparan sulfate proteoglycans (HSPGs) and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor (EGFR), leading to activation of the ERK1/2 and STAT3 pathways and upregulation of the inhibitor of DNA binding 1 transcription factor.

Expression of the type III TGF-β receptor (TβRIII), an HSPG, is epigenetically regulated in neuroblastoma cells via direct binding of the N-Myc transcription factor to Sp-1 sites on the TβRIII promoter. Analysis of patient microarray data demonstrate that other members of the differentiating stroma secretome, including HBEGF and EGFR, are positively correlated with TβRIII expression, suggesting that these proteins may be co-regulated. Treatment with inhibitors aimed at blocking N-Myc function, including inhibitors of histone deacetylases, DNA methyltransferases (DNMTs), and aurora kinase A (AurkA) can promote neuroblast differentiation and decrease proliferation. The combination of the DNMT inhibitor decitabine with the AurkA inhibitor MLN8237 enhances differentiation and reduces proliferation compared to either agent alone. Importantly, the combination of clinically achievable doses of these targeted agents dramatically reduces tumor growth in orthotopic xenograft models of neuroblastoma, identifying a novel combination therapy that may benefit children with this disease.

In conclusion, these studies dissect the tumor microenvironment to identify an important member of the differentiating stroma secretome, while also revealing a combination therapy for clinical development that has the potential to decrease adverse side effects and increase effectiveness of neuroblastoma treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Gemcitabine is a nucleoside pyrimidine analog that has long been the backbone of chemotherapy for PDAC, both as a single agent, and more recently, in combination with nab-paclitaxel. Since gemcitabine is hydrophilic, it must be transported through the hydrophobic cell membrane by transmembrane nucleoside transporters. Human equilibrative nucleoside transporter-1 (hENT1) and human concentrative nucleoside transporter-3 (hCNT3) both have important roles in the cellular uptake of the nucleoside analog gemcitabine. While low expression of hENT1 and hCNT3 has been linked to gemcitabine resistance clinically, mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. We identified that the matricellular protein Cysteine-Rich Angiogenic Inducer 61 (CYR61) negatively regulates expression of hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 significantly increased expression of hENT1 and hCNT3 and cellular uptake of gemcitabine. CRSIPR-mediated knockout of CYR61 sensitized PDAC cells to gemcitabine-induced apoptosis. Conversely, adenovirus-mediated overexpression of CYR61 decreased hENT1 expression and reduced gemcitabine-induced apoptosis. We demonstrate that CYR61 is expressed primarily by stromal pancreatic stellate cells (PSCs) within the PDAC tumor microenvironment, with Transforming Growth Factor- β (TGF-β) inducing the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in an in vitro co-culture assay with PDAC cells. Our results identify CYR61 as a TGF-β induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

The Transforming Growth Factor-β (TGF-β) superfamily of ligands and receptors play critical roles in angiogenesis, evidenced by their essential role in physiologic angiogenesis and their perturbation in pathologic angiogenesis. In development, loss or mutation of Activin receptor-Like Kinase 1 (ALK1), an endothelium- specific TGF-β superfamily receptor kinase, leads to disordered angiogenesis and results in the hereditary vascular disease, Hereditary Hemorrhagic Telangiectasia (HHT). A co-receptor in the TGF-β superfamily, the type III Transforming Growth Factor-β Receptor (TβRIII), is known to be important for the vasculature, with genetic deletion of TβRIII resulting in embryonic lethality in mice characterized by severe defects in cardiac and hepatic vasculogenesis. Despite these observations, how TβRIII regulates the vasculature remains unknown. The aim of this study is to elucidate how TβRIII regulates ALK1 signaling and endothelial behavior.

Here, we establish that vascular endothelial cells express relatively high levels of TβRIII, alongside the canonical endothelium-restricted co-receptor, endoglin. Moreover, using biophysical and biochemical techniques, we have established that TβRIII forms stable complexes with ALK1. Through genetic knockout approaches using CRISPR, we demonstrate that loss of TβRIII impairs ALK1-induced Smad phosphorylation as well as endothelial angiogenic potential, including the ability to form capillary tubes.

We have uncovered a novel TβRIII/ALK1 interaction, the role of TβRIII in ALK1-mediated signaling and TβRIII’s role in functional endothelial biology. Understanding TβRIII’s function in the developing endothelium may lead to the development of innovative pharmacological treatments aimed at normalizing angiogenesis and improve our ability to anticipate the potential adverse effects of therapies targeting TGF-β superfamily receptors.