Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFβ pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFβ signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.     

Phospho-specific Smad3 signaling: Impact on breast oncogenesis

Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.     

KiSS1 gene as a novel mediator of TGFβ-mediated cell invasion in triple negative breast cancer

The invasive and metastatic phenotypes of breast cancer correlate with high recurrence rates and poor survival outcomes. Transforming growth factor-β (TGFβ) promotes tumor progression and metastasis in aggressive breast cancer. Here, we identified the kisspeptin KiSS1 as a downstream target of canonical TGFβ/Smad2 pathway in triple negative breast cancer cells. We also found KiSS1 expression to be required for TGFβ-induced cancer cell invasion. Indeed, knockdown expression of KiSS1 blocked TGFβ-mediated cancer cell invasion as well as metalloproteinase (MMP9) expression and activity. Interestingly, Kisspeptin-10 (KP-10), the smallest active form of kisspeptin also stimulates cancer cell invasive behavior through activation of MAPK/Erk pathway. We described a positive feedback loop between KiSS1 and p21 downstream of TGFβ, further contributing to TGFβ-induced cancer cell invasion. Lastly, we explored both the clinical utility of KiSS1 as a lymph node involvement predictive tool and its potential as a therapeutic target. We found KiSS1 high expression to correlate with lymph node positive status. Furthermore, blocking KiSS1 using a specific small peptide antagonist (p234) impaired TGFβ-mediated cell invasion and MMP9 induction. Together, our results define an essential role of KiSS1 in regulating TGFβ pro-invasive effects and define KiSS1 as a therapeutic new target for triple negative breast cancer.     

Transforming growth factor-β activates c-Myc to promote palatal growth

During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth, elevation and fusion of the two palatal shelves. Interestingly, the palatal mesenchyme expresses all three transforming growth factor (TGF) β isoforms (1, 2, and 3) throughout these steps of palatogenesis. However, the role of TGFβ in promoting proliferation of palatal mesenchymal cells has never been explored. The purpose of this study was to identify the effect of TGFβ on human embryonic palatal mesenchymal (HEPM) cell proliferation. Our results showed that all isoforms of TGFβ, especially TGFβ3, increased HEPM cell proliferation by up‐regulating the expression of cyclins and cyclin‐dependent kinases as well as c‐Myc oncogene. TGFβ activated both Smad‐dependent and Smad‐independent pathways to induce c‐Myc gene expression. Furthermore, TBE1 is the only functional Smad binding element (SBE) in the c‐Myc promoter and Smad4, activated by TGFβ, binds to the TBE1 to induce c‐Myc gene activity. We conclude that HEPM proliferation is manifested by the induction of c‐Myc in response to TGFβ signaling, which is essential for complete palatal confluency. Our data highlights the potential role of TGFβ as a therapeutic molecule to correct cleft palate by promoting growth. J. Cell. Biochem. 113: 3069–3085, 2012. © 2012 Wiley Periodicals, Inc.     

APC and Smad7 link TGFβ type I receptors to the microtubule system to promote cell migration

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3β (GSK-3β). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-β (TGFβ) and is known to inhibit various TGFβ-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFβ stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3β kinases to facilitate local TGFβ/p38-dependent inactivation of GSK-3β, accumulation of β-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFβ type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFβ.     

Trigenic neural crest-restricted Smad7 over-expression results in congenital craniofacial and cardiovascular defects

Smad7 is a negative regulator of TGFβ superfamily signaling. Using a three-component triple transgenic system, expression of the inhibitory Smad7 was induced via doxycycline within the NCC lineages at pre- and post-migratory stages. Consistent with its role in negatively regulating both TGFβ and BMP signaling in vitro, induction of Smad7 within the NCC significantly suppressed phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo, resulting in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion cells. At the cellular level, increased cell death was observed in pharyngeal arches. However, cell proliferation and NCC-derived smooth muscle differentiation were unaltered. NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were not affected, but subsequent colonization of the OFT was significantly reduced. Induction of Smad7 in post-migratory NCC resulted in interventricular septal chamber septation defects, suggesting that TGFβ superfamily signaling is also essential for cardiac NCC at post-migratory stages to govern normal cardiac development. Taken together, the data illustrate that tightly regulated TGFβ superfamily signaling plays an essential role during craniofacial and cardiac NCC colonization and cell survival in vivo.     

Multiple roles of mothers against decapentaplegic homolog 4 in tumorigenesis,stem cells,drug resistance,and cancer therapy

The transforming growth factor(TGF)-βsignaling pathway controls many cellular processes,including proliferation,differentiation,and apoptosis.Abnormalities in the TGF-βsignaling pathway and its components are closely related to the occurrence of many human diseases,including cancer.Mothers against decapentaplegic homolog 4(Smad4),also known as deleted in pancreatic cancer locus 4,is a typical tumor suppressor candidate gene locating at q21.1 of human chromosome 18 and the common mediator of the TGF-β/Smad and bone morphogenetic protein/Smad signaling pathways.It is believed that Smad4 inactivation correlates with the development of tumors and stem cell fate decisions.Smad4 also interacts with cytokines,miRNAs,and other signaling pathways,jointly regulating cell behavior.However,the regulatory function of Smad4 in tumorigenesis,stem cells,and drug resistance is currently controversial.In addition,Smad4 represents an attractive therapeutic target for cancer.Elucidating the specific role of Smad4 is important for understanding the mechanism of tumorigenesis and cancer treatment.Here,we review the identification and characterization of Smad4,the canonical TGF-β/Smad pathway,as well as the multiple roles of Smad4 in tumorigenesis,stem cells,and drug resistance.Furthermore,we provide novel insights into the prospects of Smad4-targeted cancer therapy and the challenges that it will face in the future.     

The role of Smad7 in oral mucositis

Oral mucositis, a severe oral ulceration, is a common toxic effect of radio- or chemoradio-therapy and a limiting factor to using the maximum dose of radiation for effective cancer treatment. Among cancer patients, at least 40% and up to 70%, of individuals treated with standard chemotherapy regimens or upper-body radiation, develop oral mucositis. To date, there is no FDA approved drug to treat oral mucositis in cancer patients. The key challenges for oral mucositis treatment are to repair and protect ulcerated oral mucosa without promoting cancer cell growth. Oral mucositis is the result of complex, multifaceted pathobiology, involving a series of signaling pathways and a chain of interactions between the epithelium and submucosa. Among those pathways and interactions, the activation of nuclear factor-kappa B (NF-κB) is critical to the inflammation process of oral mucositis. We recently found that activation of TGFβ(transforming growth factor β) signaling is associated with the development of oral mucositis. Smad7, the negative regulator of TGFβ signaling, inhibits both NF-κB and TGFβ activation and thus plays a pivotal role in the prevention and treatment of oral mucositis by attenuating growth inhibition, apoptosis, and inflammation while promoting epithelial migration. The major objective of this review is to evaluate the known functions of Smad7, with a particular focus on its molecular mechanisms and its function in blocking multiple pathological processes in oral mucositis.     

Smad7 regulates terminal maturation of chondrocytes in the growth plate

Members of the bone morphogenetic protein (BMP) superfamily, including transforming growth factor-betas (TGFβ), regulate multiple aspects of chondrogenesis. Smad7 is an intracellular inhibitor of BMP and TGFβ signaling. Studies in which Smad7 was overexpressed in chondrocytes demonstrated that Smad7 can impact chondrogenesis by inhibiting BMP signaling. However, whether Smad7 is actually required for endochondral ossification in vivo is unclear. Moreover, whether Smad7 regulates TGFβ in addition to BMP signaling in developing cartilage is unknown. In this study, we found that Smad7 is required for both axial and appendicular skeletal development. Loss of Smad7 led to impairment of the cell cycle in chondrocytes and to defects in terminal maturation. This phenotype was attributed to upregulation of both BMP and TGFβ signaling in Smad7 mutant growth plates. Moreover, Smad7−/− mice develop hypocellular cores in the medial growth plates, associated with elevated HIF1α levels, cell death, and intracellular retention of types II and X collagen. Thus, Smad7 may be required to mediate cell stress responses in the growth plate during development.     

Two sides of the story? Smad4 loss in pancreatic cancer versus head-and-neck cancer

TGFβ signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies.     

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.     

Peroxisome proliferator-activated receptor γ (PPARγ) plays a critical role in the development of TGFβ resistance of H460 cell

The primary goal of the study was to investigate how peroxisome proliferator-activated receptor γ (PPARγ) played a critical role in the protection of H460 cell, one of the non-small cell lung cancer (NSCLC) cells with multidrug resistance, from transforming growth factor β (TGFβ)-mediated mitoinhibition. In the study, TGFβ resistance of H460 cell was first confirmed by analyses of PPARγ expression, its interaction with TGFβ-induced Smad3 and phospho-Smad3 (p-Smad3) and survival of H460. Results showed that enable to escape from G2/M phase arrest, H460 cell had higher resistance to TGFβ-mediated mitoinhibition than CH27 (a drug sensitive control). TGFβ significantly increased PPARγ expression of H460 but not of CH27 cell whereas nuclear accumulation of p-Smad3 was only limited to CH27, the latter was believed to contribute to the induction of P^{21 waf1/cip1} and cyclin B1, cell cycle arrest at G2/M phase and TGFβ-mediated mitoinhibition of CH27 cell. TGFβ-induced PPARγ of H460 cell was further demonstrated to bind to Smad3 and p-Smad3, and GW9662 (PPARγ inhibitor) or PPARγ-specific shRNA could disrupt the binding. GW9662 also increased the nuclear accumulation of p-Smad3 that eventually led to the reduction of TGFβ resistance of H460. A transient knockdown of PPARγ with shRNA revealed a similar effect as GW9662. In addition, activation of P³⁸ instead of ERK played a critical role in TGFβ-induced expression of PPARγ, which subsequently activated RhoA in H460 cell.     

Heat shock protein 90 inhibitor ameliorates pancreatic fibrosis by degradation of transforming growth factor-β receptor

Background and aimPancreatic fibrosis increases pancreatic cancer risk in chronic pancreatitis (CP). Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis by transforming growth factor-β (TGFβ) has been shown to inhibit transforming growth factor-β receptor (TGFβR)-mediated Smad and no-Smad signaling pathways. Thus, the effects of Hsp90 inhibitor on pancreatic fibrosis are evaluated in CP mice, and the association between Hsp90 and biological functions of PSCs is further investigated in vitro.MethodsThe effects of Hsp90 inhibitor 17AAG on pancreatic fibrosis were assessed in caerulein-induced CP mice, and primary PSCs were used to determine the role of Hsp90 inhibitor 17AAG in vitro.ResultsWe observed increased expression of Hsp90 in pancreatic tissues of caerulein-induced CP mice. Hsp90 inhibitor 17AAG ameliorated pancreatic inflammation and fibrosis in caerulein-induced CP mice. In vitro, Hsp90 inhibitor 17AAG inhibited TGFβ1-induced activation and extracellular matrix accumulation of PSCs by blocking TGFβR-mediated Smad2/3 and PI3K /Akt/GSK-3β signaling pathways.Hsp90 inhibitor 17AAG degraded TGFβRII by a ubiquitin-proteasome pathway, co-immunoprecipitation showed an interaction between Hsp90 and TGFβRII in PSCs.ConclusionsThe study suggests that an Hsp90 inhibitor 17AAG remarkable prevents the development of pancreatic fibrosis in caerulein-induced CP mice, and suppresses activation and extracellular matrix accumulation of PSCs in vitro. The current results provide a potential treatment strategy based on Hsp90 inhibition for pancreatic fibrosis in CP.