Elevated Levels Of TGF-β Research Articles

Inhibition of TGF-β Enhances the In Vivo Antitumor Efficacy of EGF Receptor–Targeted Therapy

EGF receptor (EGFR)-targeted monoclonal antibodies (mAb), such as cetuximab, execute their antitumor effect in vivo via blockade of receptor-ligand interactions and engagement of Fcγ receptors on immune effector cells that trigger antibody-dependent cell-mediated cytotoxicity (ADCC). We show that tumors counteract the in vivo antitumor activity of anti-EGFR mAbs by increasing tumor cell-autonomous expression of TGF-β. We show that TGF-β suppresses the expression of key molecular effectors of immune cell-mediated cytotoxicity, including Apo2L/TRAIL, CD95L/FasL, granzyme B, and IFN-γ. In addition to exerting an extrinsic inhibition of the cytotoxic function of immune effectors, TGF-β-mediated activation of AKT provides an intrinsic EGFR-independent survival signal that protects tumor cells from immune cell-mediated apoptosis. Treatment of mice-bearing xenografts of human head and neck squamous cell carcinoma with cetuximab resulted in emergence of resistant tumor cells that expressed relatively higher levels of TGF-β compared with untreated tumor-bearing mice. Although treatment with cetuximab alone forced the natural selection of TGF-β-overexpressing tumor cells in nonregressing tumors, combinatorial treatment with cetuximab and a TGF-β-blocking antibody prevented the emergence of such resistant tumor cells and induced complete tumor regression. Therefore, elevated levels of TGF-β in the tumor microenvironment enable tumor cells to evade ADCC and resist the antitumor activity of cetuximab in vivo. Our results show that TGF-β is a key molecular determinant of the de novo and acquired resistance of cancers to EGFR-targeted mAbs, and provide a rationale for combinatorial targeting of TGF-β to improve anti-EGFR-specific antibody therapy of EGFR-expressing cancers.

Cimetidine enhances delayed-type hypersensitivity responses and serum interleukin (IL)-2, -10, -12, and IL-17 levels after burn injury in an animal model

The immunosuppression that occurs after burn injury causes an increase in susceptibility to infection. The aim was to investigate time-related alterations in various cytokines following thermal injury and to modulate cytokines by use of an immunomodulant, cimetidine. Male Balb/c mice were anesthetized and given a 10% total body surface area full-thickness burn by submerging in 90°C water for 9 s. Time-dependent changes in delayed type hypersensitivity (DTH) and serum levels of the cytokines IL-2, IL-10, IL-12, IL-17 and TGFβ were then assessed at various post-burn day (PBD) timepoints. Effects of 10 mg cimetidine/kg on DTH responses and cytokine levels were evaluated up to PBD 14. In comparison to healthy non-burned control mice, levels of IL-2 and IL-17 significantly decreased at PBD 3, 5, 10, and 14, those of IL-10 at PBD 1, 3, 5, and 10, and those of IL-12 at PBD 1, 3, 5, 10, and 14. Administration of cimetidine significantly augmented the levels of IL-2 (at PBD 3, 5, and 10), IL-10 (at PBD 1 and 5), IL-12 (at PBD 3, 5, 10, and 14), and IL-17 (at PBD 3 and 14) as compared to those in burned counterparts who did not receive drug. In comparison to healthy mice, biphasic alterations were observed regarding TGFβ levels; values were significant decreased and increased at PBD 3 and PBD 14, respectively. Cimetidine significantly diminished the elevated TGFβ levels at PBD 14. Cimetidine also significantly augmented DTH responses at PBD 5, 10, and 14 as compared to responses in non-drug-treated burned hosts. Taken together, the results here showed significant time-dependent changes in serum cytokines levels after burn injury and that cimetidine was able to significantly augment IL-2, IL-10, IL-12, and IL-17 levels as well as DTH responses that are normally suppressed following thermal trauma.

Predominance of IL-10 and TGF-β production from the mouse macrophage cell line, RAW264.7, in response to crude antigens from Clonorchis sinensis

Parasitic helminths are well-known to have the ability to modulate host immune responses. In this study, we investigated the fundamental immunoregulatory mechanism of the liver fluke Clonorchis sinensis (C. sinensis) using a murine macrophage RAW 264.7 (RAW) cell line and mouse bone marrow derived macrophages (BMDMs). We found that C. sinensis crude antigen (CA) is able to differentiate macrophage RAW cells into dendritic-like cells that can be detected by morphological observations. In addition, CA induces prominent secretion of anti-inflammatory cytokines such as IL-10 and TGF-β; however, we did not observe changes in cell surface markers that are involved in antigen recognition, antigen presentation, and T cell activation. Additionally, CA treatment induced ERK and JNK phosphorylation, and unexpectedly, elevated levels of IL-10 and TGF-β were inhibited by the addition of an ERK-specific inhibitor. Taken together, these data demonstrate that CA from C. sinensis may modulate host immune responses by upregulating anti-inflammatory cytokines via the regulation of ERK.

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log2) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7–14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.

Increased TGF‐β1‐mediated suppression of growth and motility in castrate‐resistant prostate cancer cells is consistent with Smad2/3 signaling

Elevated TGF-β levels are associated with prostate cancer progression. Although TGF-β is a tumor suppressor for normal epithelial and early-stage cancer cells, it may act paradoxically as a tumor promoter in more advanced cancers, although its effects are largely cell and context dependent. This study analyzed prostate cancer responses to TGF-β signaling in an isogenic model of androgen-sensitive and castration-resistant prostate cancer cells. Phosphorylation and nuclear translocation of Smad2 and Smad3 were analyzed using immunoblotting. Proliferation and cell cycle responses to TGF-β1 (5 ng/ml) were assessed using growth assays and flow cytometry for DNA content, as well as Western blot and immunoprecipitation of cell cycle proteins. Both androgen-sensitive (LNCaP) and castration-resistant (C4-2 and C4-2B) prostate cancer cell lines demonstrated TGF-β1-induced phosphorylation and nuclear translocation of Smad2/3 that was robust in metastatic lines. Smad phosphorylation was completely abrogated with inhibition of ALK-5 kinase activity using the kinase inhibitor, SB-431542. Increased sensitivity to TGF-β1-mediated growth inhibition was observed in C4-2 and C4-2B cells, as compared to LNCaP cells. This was paralleled with downregulation of Cyclin D and increased association of p15(Ink4b) or p27(Kip) with CDK's. Additionally, TGF-β1 inhibited motility and invasion of metastatic cell lines. TGF-β-mediated suppression of growth and motility is enhanced in metastatic, castration-resistant prostate cancer cells. Enhanced TGF-β1-induced Smad2 and -3 signaling in prostate cancer cells may correlate with tumor suppressive activity. Therefore, the direct effects of TGF-β1 on prostate cancer cells post-castration may be anti-tumorigenic and growth-suppressive.

Triggered Firing and Atrial Fibrillation in Transgenic Mice With Selective Atrial Fibrosis Induced by Overexpression of TGF-β1

Calcium transient triggered firing (CTTF) is induced by large intracellular calcium (Ca(i)) transient and short action potential duration (APD). We hypothesized that CTTF underlies the mechanisms of early afterdepolarization (EAD) and spontaneous recurrent atrial fibrillation (AF) in transgenic (Tx) mice with overexpression of transforming growth factor β1 (TGF-β1). MHC-TGFcys(33)ser Tx mice develop atrial fibrosis because of elevated levels of TGF-β1. We studied membrane potential and Ca(i)transients of isolated superfused atria from Tx and wild-type (Wt) littermates. Short APD and persistently elevated Ca(i) transients promoted spontaneous repetitive EADs, triggered activity and spontaneous AF after cessation of burst pacing in Tx but not Wt atria (39% vs. 0%, P=0.008). We were able to map optically 4 episodes of spontaneous AF re-initiation. All first and second beats of spontaneous AF originated from the right atrium (4/4, 100%), which is more severely fibrotic than the left atrium. Ryanodine and thapsigargin inhibited spontaneous re-initiation of AF in all 7 Tx atria tested. Western blotting showed no significant changes of calsequestrin or sarco/endoplasmic reticulum Ca(2+)-ATPase 2a. Spontaneous AF may occur in the Tx atrium because of CTTF, characterized by APD shortening, prolonged Ca(i) transient, EAD and triggered activity. Inhibition of Ca(2+) release from the sarcoplasmic reticulum suppressed spontaneous AF. Our results indicate that CTTF is an important arrhythmogenic mechanism in TGF-β1 Tx atria.

A control engineering approach to understanding the TGF-β paradox in cancer

TGF-β, a key cytokine that regulates diverse cellular processes, including proliferation and apoptosis, appears to function paradoxically as a tumour suppressor in normal cells, and as a tumour promoter in cancer cells, but the mechanisms underlying such contradictory roles remain unknown. In particular, given that this cytokine is primarily a tumour suppressor, the conundrum of the unusually high level of TGF-β observed in the primary cancer tissue and blood samples of cancer patients with the worst prognosis, remains unresolved. To provide a quantitative explanation of these paradoxical observations, we present, from a control theory perspective, a mechanistic model of TGF-β-driven regulation of cell homeostasis. Analysis of the overall system model yields quantitative insight into how cell population is regulated, enabling us to propose a plausible explanation for the paradox: with the tumour suppressor role of TGF-β unchanged from normal to cancer cells, we demonstrate that the observed increased level of TGF-β is an effect of cancer cell phenotypic progression (specifically, acquired TGF-β resistance), not the cause. We are thus able to explain precisely why the clinically observed correlation between elevated TGF-β levels and poor prognosis is in fact consistent with TGF-β's original (and unchanged) role as a tumour suppressor.

Plasma TGF-β1, MMP-1 and MMP-3 Levels in Chronic Pancreatitis

Chronic pancreatitis (CP) presenting clinically with upper abdominal pain, as well as exocrine and endocrine insufficiencies, is characterized by irreversible morphological and functional alterations in the pancreas. The objective of the present study is to investigate the plasma levels of transforming growth factor-β 1 (TGF-β1), matrix metalloproteinases MMP-1 (collagenase) and MMP-3 (stromelysin) in CP. A total of 71 CP patients and 100 control subjects were considered for the study. Plasma levels of TGF-β1, MMP-1 and MMP-3 were determined by enzyme-linked immunosorbent assay in patients and control subjects. The plasma levels of TGF-β1 and MMP-1 were significantly elevated in patients compared to control group (*P=0.0301, **P<0.0001). However, there was no significant difference in the plasma levels of MMP-3 between patients and controls (P=0.3756). The elevated levels of TGF-β1 and MMP-1 may influence the inflammatory reactions by enhancing the pancreatic stellate cell activation and deposition of extracellular matrix resulting in pancreatic fibrosis. Thus, the present study highlights the role of fibrogenic cytokine marker TGF-β1 and matrix metalloproteinases in the pathogenesis of CP.

Inverse correlation between allergy markers and Helicobacter pylori infection in children is associated with elevated levels of TGF-β

We evaluated allergy/hypersensitivity clinical markers and their correlation with Helicobactor pylori infection in children and adults to analyze how early acquisition of H. pylori could modulate allergic disorder expression. H. pylori presence was assessed by the rapid urease test and histology of antrum biopsies in 165 patients. Skin tests, serum IgE, and two clinical allergy questionnaires were performed. Allergy severity was operationally defined using a combined score. Findings were correlated with H. pylori status and cytotoxin-associated gene A presence in pediatric and adult patients. Transforming growth factor β (TGF-β) levels were measured by an enzyme-linked immunosorbent assay in serum and gastric biopsies of H. pylori (+) patients. H. pylori (-) children had more positive skin tests to a higher number of antigens than H. pylori (+) children (P<0.05). Operationally defined allergy inversely correlates with H. pylori infection in children, but not in adults. The percentage of H. pylori infection was lower in children with severe allergy (32.3%) compared with children with mild allergy (43.4%) or no allergy (64.3%) (P<0.05). Colonization with virulent strains (cytotoxin-associated gene A+) showed a nonsignificant inverse correlation with severity of allergies in pediatric patients. H. pylori-infected children, but not adults, without allergy markers showed increased levels of TGF-β compared with allergic children both in serum and gastric mucosa (P<0.05). There was a strong inverse correlation between allergy markers and H. pylori infection in pediatric patients associated with elevated levels of TGF-β locally and systemically. H. pylori-associated chronic gastritis might downregulate clinical allergy expression.

TGF-β1 → SMAD/p53/USF2 → PAI-1 transcriptional axis in ureteral obstruction-induced renal fibrosis.

Chronic kidney disease constitutes an increasing medical burden affecting 26 million people in the United States alone. Diabetes, hypertension, ischemia, acute injury, and urological obstruction contribute to renal fibrosis, a common pathological hallmark of chronic kidney disease. Regardless of etiology, elevated TGF-β1 levels are causatively linked to the activation of profibrotic signaling pathways initiated by angiotensin, glucose, and oxidative stress. Unilateral ureteral obstruction (UUO) is a useful and accessible model to identify mechanisms underlying the progression of renal fibrosis. Plasminogen activator inhibitor-1 (PAI-1), a major effector and downstream target of TGF-β1 in the progression of several clinically important fibrotic disorders, is highly up-regulated in UUO and causatively linked to disease severity. SMAD and non-SMAD pathways (pp60(c-src), epidermal growth factor receptor [EGFR], mitogen-activated protein kinase, p53) are required for PAI-1 induction by TGF-β1. SMAD2/3, pp60(c-src), EGFR, and p53 activation are each increased in the obstructed kidney. This review summarizes the molecular basis and translational significance of TGF-β1-stimulated PAI-1 expression in the progression of kidney disease induced by ureteral obstruction. Mechanisms discussed here appear to be operative in other renal fibrotic disorders and are relevant to the global issue of tissue fibrosis, regardless of organ site.

Inhibiting TGF-β Activity Improves Respiratory Function in mdx Mice

Respiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-β play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-β signaling improves respiratory function, mdx mice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-β), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdx mice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-β antagonism may be a useful therapeutic approach for treating DMD patients.

Elevated Dickkopf-2 levels contribute to the abnormal phenotype of human osteoarthritic osteoblasts

The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/β-catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf-1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a-dependent Wnt/β-catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/β-catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor β1 (TGF-β1) in OA osteoblasts, and exogenous TGF-β1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF-β1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF-β1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF-β1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype.

Blocking cancer metastasis with TGF-beta antagonists

I of epithelial-mesechymal transition (EMT) has been shown to confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFβ is a potent inducer of EMT. We found that chemotherapeutic drug doxorubicin activates TGFβ signaling in breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced stem cell properties in the murine 4T1 breast cancer cells in vitro, which were inhibited by a TGFβ type I receptor kinase inhibitor (TβRI-KI). Th ese observations suggest that the adverse activation of TGFβ pathway by chemotherapeutics in the cancer cells together with elevated TGFβ levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. We investigated the potential synergistic anti-tumor activity of TβR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TβRI-KI enhanced the effi cacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was signifi cantly blocked when TβR1-KI was administered in combination with doxorubicin. Our results indicate that the combination treatment of doxorubicin with a TGFβ inhibitor has the potential to reduce the dose and consequently the toxic side-eff ects of doxorubicin, and improve its effi cacy in the inhibition of breast cancer growth and metastasis.

TGF-β1 As Possible Link between Loss of Bone Mineral Density and Chronic Inflammation

BackgroundThe TGF family plays a key role in bone homeostasis. Systemic or topic application of proteins of this family apparently positively affects bone healing in vivo. However, patients with chronic inflammation, having increased TGF-β1 serum-levels, often show reduced bone mineral content and disturbed bone healing. Therefore, we wanted to identify intracellular mechanisms induced by chronic presence of TGF-β1 and their possible role in bone homeostasis in primary human osteoblasts.Methodology/Principal FindingsOsteoblasts were isolated from femur heads of patients undergoing total hip replacement. Adenoviral reporter assays showed that in primary human osteoblasts TGF-β1 mediates its signal via Smad2/3 and not Smad1/5/8. It induces proliferation as an intermediate response but decreases AP-activity and inorganic matrix production as a late response. In addition, expression levels of osteoblastic markers were strongly regulated (AP↓; Osteocalcin↓; Osteopontin↑; MGP↓; BMP 2↓; BSP2↓; OSF2↓; Osteoprotegerin↓; RANKL↑) towards an osteoclast recruiting phenotype. All effects were blocked by inhibition of Smad2/3 signaling with the Alk5-Inhibitor (SB431542). Interestingly, a rescue experiment showed that reduced AP-activities did not recover to base line levels, even 8 days after stopping the TGF-β1 application.Conclusions/SignificanceIn spite of the initial positive effects on cell proliferation, it is questionable if continuous Smad2/3 phosphorylation is beneficial for bone healing, because decreased AP-activity and BMP2 levels indicate a loss of function of the osteoblasts. Thus, inhibition of Smad2/3 phosphorylation might positively influence functional activity of osteoblasts in patients with chronically elevated TGF-β1 levels and thus, could lead to an improved bone healing in vivo.

Transforming growth factor‐β regulation of proteoglycan synthesis in vascular smooth muscle: Contribution to lipid binding and accelerated atherosclerosis in diabetes

Atherosclerosis is accelerated in the setting of diabetes, but the factors driving this phenomenon remain elusive. Hyperglycemia leads to elevated levels of transforming growth factor (TGF)-β and TGF-β has been implicated as a factor in atherosclerosis. Given the established association between hyperglycemia and elevated TGF-β, it is plausible that elevated TGF-β levels in diabetes play a pathogenic role in the development of accelerated atherosclerosis. TGF-β is a potent regulator of extracellular matrix synthesis, including many actions on proteoglycan synthesis that lead to increased binding to low-density lipoprotein and therefore potentially increased lipid retention in the vessel wall and accelerated atherosclerosis. TGF-β signals through the canonical TGF-β receptor I-mediated phosphorylation of Smad transcription factors and TGF-β signaling is also known to involve, positively and negatively, interactions with the mitogen-activated protein kinase pathways. The focus of the present review is on the effects of TGF-β on proteoglycan synthesis in vascular smooth muscle and particularly the signaling pathways through which TGF-β exerts its effects, because those pathways may be therapeutic targets for the prevention of pathological modifications in the proteoglycan component of the vessel wall in the vascular diseases of diabetes.

Elevated TGF‐β1 levels might protect HCV/ HIV‐coinfected patients from liver fibrosis

HIV accelerates hepatitis C virus (HCV)-induced liver fibrosis by mechanisms not well understood. As HIV dysregulates transforming growth factor-β1 (TGF-β1) and T regulatory (Treg) cells, both of which are involved in hepatic fibrogenesis, herein we describe their influence on liver fibrosis staging in patients with chronic hepatitis C with and without HIV coinfection. Eighty-eight subjects (42 HIV/HCV co-infected patients, 20 HCV-monoinfected patients, and 26 healthy controls) were examined. Treg cells (CD4+Foxp3+) were measured in peripheral blood using flow cytometry. An enzyme immunoassay was used to measure TGF-β1 in plasma. Liver fibrosis staging was estimated using elastometry and advanced liver fibrosis was considered for ≥ 9·5 kPa (F3-F4 Metavir estimates). Treg cells were increased in HIV/HCV-coinfected patients compared with HCV-monoinfected patients (P = 0·004), whereas TGF-β1 levels were similar in both groups of patients. While Treg cells levels were similar in both null-mild and advanced liver fibrosis patients, a high level of TGF-β1 was found in patients with low levels of liver fibrosis compared with those with advanced liver fibrosis [14·9 ng mL(-1) (5·6-37·9) vs. 5·5 ng mL(-1) (1·9-7·9) respectively P = 0·007]. In a multivariate logistic regression model, elevated TGF-β1 levels were significantly associated with not having advanced liver fibrosis [OR: 0·13 (95% CI: 0·02-0·71), P = 0·019]. While Treg cells do not influence liver fibrosis staging, elevated TGF-β1, probably through its anti-inflammatory effects, might protect HCV/HIV-coinfected patients from liver fibrosis.

Molecular characterization of TGFβ-induced epithelial-mesenchymal transition in normal finite lifespan human mammary epithelial cells

Epithelial-mesenchymal transition (EMT) is a morphogenetic program essential for embryonic development and wound healing, but can adversely cause fibrosis and metastatic cancer progression when deregulated. Here, we established a model of efficient EMT induction in normal finite lifespan human mammary epithelial cells (HMEC) using transforming growth factor beta (TGFβ). We demonstrate that EMT in HMEC occurs in three distinctive phases that are governed by a hierarchy of EMT-activating transcription factors (TFs). Loss of epithelial cell polarity (ZO-1), and acquisition of mesenchymal marker (Vimentin, Fibronectin) expression are immediate-early events, whereas switching from E-cadherin to N-cadherin protein expression occurs only after EMT-like morphological changes become apparent. The kinetics of TF induction suggests that ZEB1 and SNAIL mediate early EMT induction reinforced by ZEB2, while GOOSECOID and FOXC2 may play a role in EMT maintenance. TWIST and SLUG were not significantly induced in this system. Furthermore, we show for the first time that normal HMEC acquire a CD44 +/CD24 −/low stem cell phenotype during a third phase of EMT that is characterized by maximum TF expression levels. Our results may have important implications for understanding potential changes that might occur in normal breast epithelium under pathological conditions triggering elevated TGFβ levels.

Prolyl isomerase Pin1 controls TGF-β-Smad signaling and pulmonary fibrosis (142.12)

Abstract Normal pulmonary function requires precise control over the content and composition of the extracellular matrix (ECM). During the pathogenesis of lung fibrosis, a net increase in ECM content displaces epithelial cells and causes severe reductions of pulmonary function. This process is often associated with elevated levels of TGF-β1 which enhances ECM production by fibroblasts. However, the signaling mechanisms that coordinate ECM production remain unclear. Here, we show that Pin1, a peptidyl-prolyl isomerase which isomerizes pSer/pThr-Pro peptide bonds, reciprocally controls the production of pro- and anti-fibrogenic ECM. Pin1 knockout mice treated with bleomycin, which induces pulmonary fibrosis by elevating TGF-β1, showed significantly attenuated expression of collagens (I, III and V), TIMPs (TIMP1 and 4) and fibrogenic cytokines (TGF-β1, IL-1β and CTGF) but increased MMPs (MMP2 and 3). In primary lung fibroblasts, Pin1 was required for TGF-β1-induced phosphorylation and subsequent nuclear transactivation of Smad3. These functions were mediated by TGF-β1 dependent interactions of Pin1 with inhibitory Smad6. In Pin1 knockout cells, Smad6 remained predominantly cytoplasmic, likely attenuating Smad3 phosphorylation and transcriptional activity. These data support and extend our previous observations that Pin1 blockade in vivo significantly reduced total collagen deposition in the lungs of animal models of asthma and lung transplantation.

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Gap junction-mediated intercellular communication (GJIC) is essential for the proper function of many organs, including the lens. GJIC in lens epithelial cells is increased by FGF in a concentration-dependent process that has been linked to the intralenticular gradient of GJIC required for lens transparency. Unlike FGF, elevated levels of TGF-beta are associated with lens dysfunction. We show that TGF-beta1 or -2 up-regulates dye coupling in serum-free primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) via a mechanism distinct from that utilized by other growth factors. Remarkably, the ability of TGF-beta and of FGF to up-regulate GJIC is abolished if DCDMLs are simultaneously exposed to both factors despite undiminished cell-cell contact. This reduction in dye coupling is attributable to an inhibition of gap junction assembly. Connexin 45.6, 43, and 56-containing gap junctions are restored, and intercellular dye coupling is increased, if the activity of p38 kinase is blocked. Our data reveal a new type of cross-talk between the FGF and TGF-beta pathways, as well as a novel role for TGF-beta and p38 kinase in the regulation of GJIC. They also provide an explanation for how pathologically increased TGF-beta signaling could contribute to cataract formation.

Soluble Factors and Circulating Tumor Cells in Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is an insidious form of breast cancer. Cytokines and chemokines direct the metastatic potential of disease and serve as biomarkers for disease progression. Circulating tumor cells (CTC) are an independent prognostic factor in metastatic disease. Further, TGF-β is involved in the induction of the epithelial-mesenchymal transition (EMT) which regulates the metastatic ability of IBC. As prognostic factors of aggressive disease, we measured serum cytokines and leukocyte phenotypes, and correlated the values with known CTC results of IBC and non-IBC patients.Methods: Peripheral blood (PB) from 35 IBC patients (18 non-metastatic and 17 metastatic) and 19 non-IBC patients (7 LABC, 12 MBC) was collected prior to starting a new therapy to measure a panel of 33 cytokines, chemokines, and growth factors in serum by Luminex; CTC by CellSearch™; and PB leukocyte immunophenotype by FACS. The Kruskal-Wallis and Mann-Whitney U tests determined the differences in cytokine levels between IBC, non-IBC and healthy donors (HD) and presence of CTCs.Results: There were no differences in serum cytokine, chemokine or growth factor levels between IBC and non-IBC patients. EGF, IP-10, MIG, Eotaxin, MCP-1 and TNF-RI were significantly elevated in breast cancer patients compared to HD. TNF-RI, EGF, HGF, IP-10, MIG, Eotaxin, MCP-1 and interleukin (IL)-10 were higher in IBC patients than in HD. Compared to IBC patients, non-IBC patients had fewer dysregulated cytokines relative to HD including higher EGF, Eotaxin, MCP-1 and IL-8.Perhaps counter-intuitively, non-metastatic IBC patients had higher plasma levels of IL-2, -2R, -4, -5, -10, -12p70, -15, -17, FGF-b, IFN-γ, GM-CSF, and MIP1-α than metastatic IBC patients. Moreover, these differences were not observed between LABC and MBC patients. Furthermore, compared to HD, metastatic IBC had lower IL-4, -7, -17, -12p70, IFN-γ, RANTES, but higher levels of IP-10, Eotaxin, MCP-1, and TNF-RI. These data suggest that non-metastatic IBC patients are more immune competent than metastatic IBC patients. Finally, there were differences in the immunophenotype as well as cytokine levels between IBC patients with and without CTCs. IBC patients with CTC had a lower %T-cells (p=0.003) and higher %B-cells (p=0.008) and TNF-RI (p=0.01) than IBC patients without CTCs which may lead to a decrease in cellular immunity. Cell-mediated immunity may be further compromised by the elevated levels of serum TGF-β (p= 0.064) that can also promote EMT and metastatic progression.Conclusion: We report a comprehensive analysis of the serum cytokine and chemokine profiles in IBC patients. More importantly, this is the first report of potential interactions between soluble factors, CTC, and immune parameters in IBC patients. Non-metastatic IBC patients are more immune competent than metastatic IBC patients as evidenced by the high levels of pro- and anti-inflammatory factors; however, the presence of CTC in IBC tends to shift the immune response to a TH2 polarization with a decrease in T-cells, and a concomitant increase in B-cells and serum TGF-β and TNF-RI levels. Additional studies are needed to determine the role of soluble factors in the pathogenesis and progression of IBC and the impact on clinical outcome. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2135.