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Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by a marked desmoplastic tumor stroma that is formed under the influence of transforming growth factor (TGF)-β. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-β pathway through activation of Smad4 and secretion of biglycan (Bgn). However, whether this pathway also functions in human PDAC cells has not yet been studied. Here, we show that RNA interference-mediated silencing of TAp73 in PANC-1 cells strongly reduced the stimulatory effect of TGF-β1 on BGN. TAp73-mediated regulation of BGN, and inhibition of TGF-β signaling through a (Smad-independent) ERK pathway, are reminiscent of what we previously observed for the small GTPase, RAC1b, prompting us to hypothesize that in human PDAC cells TAp73 and RAC1b are part of the same tumor-suppressive pathway. Like TAp73, RAC1b induced SMAD4 protein and mRNA expression. Moreover, siRNA-mediated knockdown of RAC1b reduced TAp73 mRNA levels, while ectopic expression of RAC1b increased them. Inhibition of BGN synthesis or depletion of secreted BGN from the culture medium reproduced the promigratory effect of RAC1b or TAp73 silencing and was associated with increased basal and TGF-β1-dependent ERK activation. BGN also phenocopied the effects of RAC1b or TAp73 on the expression of downstream effectors, like the EMT markers E-cadherin, Vimentin and SNAIL, as well as on negative regulation of the ALK2-SMAD1/5 arm of TGF-β signaling. Collectively, we showed that tumor-suppressive TAp73-Smad4-Bgn signaling also operates in human cells and that RAC1b likely acts as an upstream activator of this pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. The aim of our study was to reveal if transcriptionally active p73 (TAp73), a homolog of the well-known tumor suppressor p53, inhibits tumor progression through promoting canonical TGF-β/Smad signaling and by preventing non-canonical extracellular signal-regulated kinases (ERK)1/2-mediated TGF-β signaling. Using PDAC-derived tumor cell lines, we showed that TAp73 suppresses epithelial-mesenchymal transition by inducing the expression of epithelial markers while suppressing that of mesenchymal markers. We further demonstrated that TAp73 upregulates the expression of the TGF-β signaling intermediate SMAD4 and that SMAD4 acts a mediator of TAp73-induced inhibition of ERK activation and cell motility. Measuring the levels of TAp73 and/or SMAD4 could help to predict whether TGF-β preferentially uses an oncogenic or a tumor suppressive pathway in a given patient and at a specific time. Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-β. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-β/Smad signaling, while preventing non-canonical TGF-β signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-β1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73—but not the β isoform—interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-β-stimulated pro-invasive ERK activation as an underlying mechanism. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells' migratory activity in the absence or presence of TGFβ1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFβ1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFβ-rich tumor microenvironment. [ABSTRACT FROM AUTHOR]

Background: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2. Methods: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2-ALK5 physical interaction. Results: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2-AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 μM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2-APand TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5. [ABSTRACT FROM AUTHOR]

Background: We have previously shown in pancreatic ductal adenocarcinoma (PDAC) cells that the SRC inhibitors PP2 and PP1 effectively inhibited TGF-β1-mediated cellular responses by blocking the kinase function of the TGF-β type I receptor ALK5 rather than SRC. Here, we investigated the ability of the clinically utilised SRC/ABL inhibitor dasatinib to mimic the PP2/PP1 effect. Methods: The effect of dasatinib on TGF-β1-dependent Smad2/3 phosphorylation, general transcriptional activity, gene expression, cell motility, and the generation of tumour stem cells was measured in Panc-1 and Colo-357 cells using immunoblotting, reporter gene assays, RT-PCR, impedance-based real-time measurement of cell migration, and colony formation assays, respectively. Results: In both PDAC cell lines, dasatinib effectively blocked TGF-β1-induced Smad phosphorylation, activity of 3TPlux and pCAGA(12)-luc reporter genes, cell migration, and expression of individual TGF-β1 target genes associated with epithelial-mesenchymal transition and invasion. Moreover, dasatinib strongly interfered with the TGF-β1-induced generation of tumour stem cells as demonstrated by gene expression analysis and single cell colony formation. Dasatinib also inhibited the high constitutive migratory activity conferred on Panc-1 cells by ectopic expression of kinase-active ALK5. Conclusions: Our data suggest that the clinical efficiency of dasatinib may in part be due to cross-inhibition of tumour-promoting TGF-β signalling. Dasatinib may be useful as a dual TGF-β/SRC inhibitor in experimental and clinical therapeutics to prevent metastatic spread in late-stage PDAC and other tumours. [ABSTRACT FROM AUTHOR]

Purpose: We have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines, the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524, ). In this report, the ability of PP2 and PP1, the more specific Src inhibitor SU6656, and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1, Colo 357) and primary NSCLC (Tu459) cell lines were investigated. Methods: The effects of PP2, PP1, SU6656, and SB431542 on TGF-β1-dependent cell scattering/EMT, cell migration/invasion, and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR, respectively. Results: In all three cell lines tested, PP1, PP2, and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT, migration, and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast, SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1, and to a greater extent PP2, also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant. Conclusions: These data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Polycystic ovary syndrome (PCOS) is associated with the metabolic syndrome. Decreased omentin-1 levels are associated with obesity and diabetes. To study the effects of metformin treatment on omentin-1 levels in PCOS subjects and effects of omentin-1 on in vitro migration and angiogenesis. RESEARCH DESIGN AND METHODS--Serum omentin-1 was measured by ELISA. Angiogenesis was assessed by studying capillary tube formation in human microvascular endothelial cells (HMEC-1) on growth factor reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. Nuclear factor-κB (NF-κB) was studied by stably transfecting HMEC-1 cells with a cis-reporter plasmid containing luciferase reporter gene linked to five repeats of NF-κB binding sites. Akt phosphorylation was assessed by Western blotting. RESULTS--Serum omentin-1 was significantly lower in PCOS women (P < 0.05). After 6 months of metformin treatment, there was a significant increase in serum omentin-1 (P < 0.01). Importantly, changes in hs-CRP were significantly negatively correlated with changes in serum omentin-1 (P = 0.036). In vitro migration and angiogenesis were significantly increased in serum from PCOS women (P < 0.01) compared with matched control subjects; these effects were significantly attenuated by metformin treatment (P < 0.01) plausibly through the regulation of omentin-1 levels via NF-κB and Akt pathways. CRP and VEGF induced in vitro migration, and angiogenesis was significantly decreased by omentin-1. CONCLUSIONS--Increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin-treated PCOS women. Diabetes 59:3023-3031, 2010 [ABSTRACT FROM AUTHOR]

Simple Summary: Transforming growth factor (TGF)β secreted by cancer cells and acting in an autostimulatory manner has been implicated in epithelial-mesenchymal transition and cellular invasion. However, the upstream inducers/downstream effectors, or the effects on cell migration, of endogenously produced TGFβ1 remain poorly characterized. Here, we studied whether autocrine TGFβ is regulated by the small GTPase, RAC1B, and how it impacts cell motility. We found that RAC1B induced an increase in the levels of TGFβ1 mRNA and the secreted bioactive protein in pancreatic carcinoma Panc1 and breast cancer MDA-MB-231 cells. Antibody-mediated neutralization of secreted TGFβ or inhibition of its synthesis increased cell migration and the activities of promigratory genes, but downregulated those of antimigratory genes, i.e., SMAD3. Restoration of SMAD3 protein expression in these cells was able to prevent the TGFβ1 inhibition-induced rise in migratory activity, suggesting that a RAC1B-autocrine TGFβ1-SMAD3 pathway can suppress mesenchymal transdifferentiation and cell motility. Autocrine transforming growth factor (TGF)β has been implicated in epithelial-mesenchymal transition (EMT) and invasion of several cancers including pancreatic ductal adenocarcinoma (PDAC) as well as triple-negative breast cancer (TNBC). However, the precise mechanism and the upstream inducers or downstream effectors of endogenous TGFB1 remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGFβ1 via downregulation of the TGFβ type I receptor, ALK5, but whether RAC1B also impacts autocrine TGFβ signaling has not yet been studied. Intriguingly, RNA interference-mediated knockdown (RNAi-KD) or CRISPR/Cas-mediated knockout of RAC1B in TGFβ1-secreting PDAC-derived Panc1 cells resulted in a dramatic decrease in secreted bioactive TGFβ1 in the culture supernatants and TGFB1 mRNA expression, while the reverse was true for TNBC-derived MDA-MB-231 cells ectopically expressing RAC1B. Surprisingly, the antibody-mediated neutralization of secreted bioactive TGFβ or RNAi-KD of the endogenous TGFB1 gene, was associated with increased rather than decreased migratory activities of Panc1 and MDA-MB-231 cells, upregulation of the promigratory genes SNAI1, SNAI2 and RAC1, and downregulation of the invasion suppressor genes CDH1 (encoding E-cadherin) and SMAD3. Intriguingly, ectopic re-expression of SMAD3 was able to rescue Panc1 and MDA-MB-231 cells from the TGFB1 KD-induced rise in migratory activity. Together, these data suggest that RAC1B favors synthesis and secretion of autocrine TGFβ1 which in a SMAD3-dependent manner blocks EMT-associated gene expression and cell motility. [ABSTRACT FROM AUTHOR]

The small GTPase RAC1B has been shown to act as a powerful inhibitor of the transforming growth factor (TGF)β type I receptor ALK5 and TGFβ1/ALK5-induced epithelial–mesenchymal transition and cell motility. However, the precise mechanism has remained elusive. RNAi-mediated knockdown of RAC1B in the pancreatic ductal adenocarcinoma (PDAC)-derived cell line Panc1 failed to alter transcriptional activity from a transfected ALK5 promoter–reporter construct. In contrast, pharmacological inhibition of the proteasome decreased the abundance of ALK5 protein in cell lines of the mesenchymal subtype (Panc1, IMIM-PC-1, and breast cancer MDA-MB-231), but not in a PDAC cell line of the epithelial subtype (Colo357). Here, we focused on the inhibitory Smad protein, SMAD7, as a potential candidate for RAC1B-mediated inhibition of cell migration. In Panc1 cells devoid of RAC1B, SMAD7 protein was dramatically reduced and these cells were refractory to TGFβ1-induced upregulation of SMAD7 protein but not mRNA expression. Intriguingly, RNAi-mediated knockdown or ectopic overexpression of SMAD7 in Panc1 cells up- or downregulated, respectively, ALK5 protein expression and mimicked the suppressive effect of RAC1B on TGFβ/SMAD3-dependent transcriptional activity, target gene expression and cell migration. Transfection of SMAD7 was further able to partially rescue cells from the RAC1B knockdown-mediated increase in migratory properties. Conversely, knockdown of SMAD7 was able to partially rescue Panc1 and MDA-MB-231 cells from the antimigratory effect of ectopically expressed RAC1B. Finally, we demonstrate that RAC1B upregulation of SMAD7 protein requires intermittent transcriptional induction of the deubiquitinating enzyme USP26. Our data suggest that RAC1B induces SMAD7 by promoting its deubiquitination and establishes this Smad as one of RAC1B's downstream effectors in negative regulation of ALK5 and TGFβ1-induced cell migration in mesenchymal-type carcinoma cells. [ABSTRACT FROM AUTHOR]

The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-β type I receptor ALK5, but the molecular details of this process have remained unclear. Here, we hypothesized that RAC1B-mediated suppression of activin receptor-like kinase 5 (ALK5) involves proteinase-activated receptor 2 (PAR2), a G protein-coupled receptor encoded by F2RL1 that is crucial for sustaining ALK5 expression. We found in pancreatic carcinoma Panc1 cells that PAR2 is upregulated by TGF-β1 in an ALK5-dependent manner and that siRNA-mediated knockdown of RAC1B increased both basal and TGF-β1-induced expression of PAR2. Further, the simultaneous knockdown of PAR2 and RAC1B rescued Panc1 cells from a RAC1B knockdown-induced increase in ALK5 abundance and the ALK5-mediated increase in TGF-β1-induced migratory activity. Conversely, Panc1 cells with stable ectopic expression of RAC1B displayed reduced ALK5 expression, an impaired upregulation of PAR2, and a reduced migratory responsiveness to TGF-β1 stimulation. However, these effects could be reversed by ectopic overexpression of PAR2. Moreover, the knockdown of PAR2 alone in Panc1 cells and HaCaT keratinocytes phenocopied RAC1B's ability to suppress ALK5 abundance and TGF-β1-induced chemokinesis and growth inhibition. Lastly, we found that the RAC1B knockdown-induced increase in TGF-β1-induced PAR2 mRNA expression was sensitive to pharmacological inhibition of MEK-ERK signaling. Our data show that in pancreatic and skin epithelial cells, downregulation of ALK5 activity by RAC1B is secondary to suppression of F2RL1/PAR2 expression. Since F2RL1 itself is a TGF-β target gene and its upregulation by TGF-β1 is mediated by ALK5 and MEK-ERK signaling, we suggest the existence of a feed-forward signaling loop involving ALK5 and PAR2 that is efficiently suppressed by RAC1B to restrict TGF-β-driven cell motility and growth inhibition. [ABSTRACT FROM AUTHOR]

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-β type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of TGFBR1 to induction by TGF-β1 stimulation. RAC1B KO also increased TGF-β1-induced C-terminal SMAD3 phosphorylation, SMAD3 transcriptional activity, growth inhibition, and cell migration. The KD of ALK5 expression by RNA interference or inactivation of the ALK5 kinase activity by dominant-negative interference or ATP-competitive inhibition rescued the cells from the RAC1B KD/KO-mediated increase in TGF-β1-induced cell migration, whereas the ectopic expression of kinase-active ALK5 mimicked this RAC1B KD/KO effect. We conclude that RAC1B downregulates the abundance of ALK5 and SMAD3 signaling, thereby attenuating TGF-β/SMAD3-driven cellular responses, such as growth inhibition and cell motility. [ABSTRACT FROM AUTHOR]

Abstract Recent advances in tumor biology have revealed that a detailed analysis of the complex interactions of tumor cells with their adjacent microenvironment (tumor stroma) is mandatory in order to understand the various mechanisms involved in tumor growth and the development of metastasis. The mutual interactions between tumor cells and cellular and non-cellular components (extracellular matrix = ECM) of the tumor microenvironment will eventually lead to a loss of tissue homeostasis and promote tumor development and progression. Thus, interactions of genetically altered tumor cells and the ECM on the one hand and reactive non-neoplastic cells on the other hand essentially control most aspects of tumorigenesis such as epithelial-mesenchymal-transition (EMT), migration, invasion (i.e. migration through connective tissue), metastasis formation, neovascularisation, apoptosis and chemotherapeutic drug resistance. In this mini-review we will focus on these issues that were recently raised by two review articles in CCS.