HNF1A is a novel oncogene that regulates human pancreatic cancer stem cell properties.

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a human PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, decreased PCSC marker expression, and downregulation of POU5F1/OCT4 expression. Conversely, HNF1A overexpression increased PCSC marker expression and tumorsphere formation in pancreatic cancer cells and drove pancreatic ductal adenocarcinoma (PDA) cell growth. Importantly, depletion of HNF1A in xenografts impaired tumor growth and depleted PCSC marker-positive cells in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of PCSC properties and novel oncogene in PDA.

Focal adhesion kinase signaling determines the fate of lung epithelial cells in response to TGF-ß.

Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-ß1 (TGF-ß1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-ß1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-ß1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-ß1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-ß1 stimulation. TGF-ß1-induced EMP is FAK- dependent, whereas TGF-ß1-induced apoptosis is favored when FAK signaling is inhibited.

GM-CSF Mediates Mesenchymal-Epithelial Cross-talk in Pancreatic Cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDA) is characterized by a dense stroma consisting of a prevalence of activated fibroblasts whose functional contributions to pancreatic tumorigenesis remain incompletely understood. In this study, we provide the first identification and characterization of mesenchymal stem cells (MSC) within the human PDA microenvironment, highlighting the heterogeneity of the fibroblast population. Primary patient PDA samples and low-passage human pancreatic cancer-associated fibroblast cultures were found to contain a unique population of cancer-associated MSCs (CA-MSC). CA-MSCs markedly enhanced the growth, invasion, and metastatic potential of PDA cancer cells. CA-MSCs secreted the cytokine GM-CSF that was required for tumor cell proliferation, invasion, and transendothelial migration. Depletion of GM-CSF in CA-MSCs inhibited the ability of these cells to promote tumor cell growth and metastasis. Together, these data identify a population of MSCs within the tumor microenvironment that possesses a unique ability, through GM-CSF signaling, to promote PDA survival and metastasis. SIGNIFICANCE: The role of stroma in pancreatic cancer is controversial. Here, we provide the first characterization of MSCs within the human PDA microenvironment and demonstrate that CA-MSCs promote tumorigenesis through the production of GM-CSF. These data identify a novel cytokine pathway that mediates mesenchymal-epithelial cross-talk and is amenable to therapeutic intervention. Cancer Discov; 6(8); 886-99. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis.

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of ß-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.

Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFß-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that 'High-RPS8 and Low-PRELP' was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated.

Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model.

The potential utility of circulating tumor cells (CTCs) to guide clinical care in oncology patients has gained momentum with emerging micro- and nanotechnologies. Establishing the role of CTCs in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of CTCs for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. Recent attempts in the literature to culture CTCs isolated from metastatic patients using monoculture have had limited success rates of less than 20%. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully expanded CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis.

Deciphering the role of stroma in pancreatic cancer.

PURPOSE OF REVIEW: This review intends to describe recent studies on pancreatic tumor-associated stroma and potential opportunities and limitations to its targeting. RECENT FINDINGS: One of the defining features of pancreatic cancer is extensive desmoplasia, or an inflammatory, fibrotic reaction. Carcinoma cells live in this complex microenvironment which is comprised of extracellular matrix (ECM), diffusible growth factors, cytokines and a variety of nonepithelial cell types including endothelial cells, immune cells, fibroblasts, myofibroblasts and stellate cells. In addition to the heterogeneity noted in the nonneoplastic cells within the tumor microenvironment, it has also been recognized that neoplastic cancer cells themselves are heterogeneous, and include a subpopulation of stem-cell like cells within tumors termed cancer stem cells. Due to the failure of current therapeutics to improve outcomes in patients with pancreatic cancer, new therapeutic avenues targeting different components of the tumor microenvironment are being investigated. In this review article, we will focus on recent studies regarding the function of the tumor stroma in pancreatic cancer and therapeutic treatments that are being advanced to target the stroma as a critical part of tumor management. SUMMARY: Recent studies have shed new light on the contribution of the pancreatic cancer fibroinflammatory stroma to pancreatic cancer biology. Additional studies are needed to better define its full contribution to tumor behavior and how to best understand the optimal ways to develop therapies that counteract its pro-neoplastic properties.

Bmi1 enhances tumorigenicity and cancer stem cell function in pancreatic adenocarcinoma.

BACKGROUND: Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma. METHODS: We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing. RESULTS: Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue. CONCLUSIONS: Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.

Inflammation and Gli2 suppress gastrin gene expression in a murine model of antral hyperplasia.

Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (Gast⁻/⁻) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1ß expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. LacZ reporter mice for Sonic hedgehog (Shh), Gli1, and Gli2 expression bred onto the Gast⁻/⁻ background revealed reduced Shh and Gli1 expression in the antra compared to wild type controls (WT). Gli2 expression in the Gast⁻/⁻ corpus was unchanged. However in the hyperplastic Gast⁻/⁻ antra, Gli2 expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that Gli2 is differentially regulated in the hyperplastic Gast⁻/⁻ antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1ß and Il-11, which promote gastric epithelial proliferation, were increased in the Gast⁻/⁻ stomach along with Infγ. To test if inflammation could account for elevated epithelial Gli2 expression in the Gast⁻/⁻ antra, the human gastric cell line AGS was treated with IL-1ß and was found to increase GLI2 but decrease GLI1 levels. IL-1ß also repressed human GAST gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ∼50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the GAST promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed Gast expression but induced Il-1ß gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate Il-1ß expression, repress Gast gene expression and increase proliferation, leading to antral hyperplasia.

Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.

BACKGROUND & AIMS: Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation. METHODS: We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-ß-cholera-toxin-overexpressing mice. RESULTS: Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-ß-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-α, -ß, and -δ (but not PKC-ϵ) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. CONCLUSIONS: Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca(2+)(i)-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin.

Histamine 3 receptor activation mediates inhibition of acid secretion during Helicobacter-induced gastritis.

AIM: To test the hypothesis that histamine 3 receptor (H3R) activation during Helicobacter infection inhibits gastric acid secretion in vivo and in vitro. METHODS: Helicobacter felis (H. felis) infected and uninfected C57Bl/6 mice were infused with either PBS or the H3 receptor antagonist thioperamide (THIO) for 12 wk. After treatment, mice were analyzed for morphological changes and gastric acid content. Total RNA was prepared from the stomachs of each group and analyzed for changes in somatostatin and gastrin mRNA abundance by real time-polymerase chain reaction (RT-PCR). Location of H3 receptors in the stomach was analyzed by co-localization using antibodies specific for the H3 receptor and parietal cell marker H(+), K(+)-ATPase ß subunit. RESULTS: Inflammation and parietal cell atrophy was observed after 12 wk of H. felis infection. Interestingly, treatment with the H3R antagonist thioperamide (THIO) prior to and during infection prevented H. felis-induced inflammation and atrophy. Compared to the uninfected controls, infected mice also had significantly decreased gastric acid. After eradication of H. felis with THIO treatment, gastric acidity was restored. Compared to the control mice, somatostatin mRNA abundance was decreased while gastrin gene expression was elevated during infection. Despite elevated gastric acid levels, after eradication of H. felis with THIO, somatostatin mRNA was elevated whereas gastrin mRNA was suppressed. Immunofluorescence revealed the presence of H3 receptors on the parietal cells, somatostatin-secreting D-cells as well as the inflammatory cells. CONCLUSION: This study shows that during H. felis infection, gastric acidity is suppressed as a consequence of an inhibitory effect on the parietal cell by H3R activation. The stimulation of gastric mucosal H3Rs increases gastrin expression and release by inhibiting release of somatostatin.

Interleukin-1beta promotes gastric atrophy through suppression of Sonic Hedgehog.

BACKGROUND & AIMS: In both human subjects and rodent models, Helicobacter infection leads to a decrease in Shh expression in the stomach. Sonic Hedgehog (Shh) is highly expressed in the gastric corpus and its loss correlates with gastric atrophy. Therefore, we tested the hypothesis that proinflammatory cytokines induce gastric atrophy by inhibiting Shh expression. METHODS: Shh-LacZ reporter mice were infected with Helicobacter felis for 3 and 8 weeks. Changes in Shh expression were monitored using beta-galactosidase staining and immunohistochemistry. Gastric acidity was measured after infection, and interleukin (IL)-1beta was quantified by quantitative reverse-transcription polymerase chain reaction. Mice were injected with either IL-1beta or omeprazole before measuring Shh mRNA expression and acid secretion. Organ cultures of gastric glands from wild-type or IL-1R1 null mice were treated with IL-1beta then Shh expression was measured. Primary canine parietal or mucous cells were treated with IL-1beta. Shh protein was determined by immunoblot analysis. Changes in intracellular calcium were measured by Fura-2. RESULTS: All major cell lineages of the corpus including surface pit, mucous neck, zymogenic, and parietal cells expressed Shh. Helicobacter infection reduced gastric acidity and inhibited Shh expression in parietal cells by 3 weeks. IL-1beta produced during Helicobacter infection inhibited gastric acid, intracellular calcium, and Shh expression through the IL-1 receptor. Suppression of parietal cell Shh expression by IL-1beta and omeprazole was additive. IL-1beta did not suppress Shh expression in primary gastric mucous cells. CONCLUSIONS: IL-1beta suppresses Shh gene expression in parietal cells by inhibiting acid secretion and subsequently the release of intracellular calcium.

Paracrine Hedgehog signaling in stomach and intestine: new roles for hedgehog in gastrointestinal patterning.

BACKGROUND & AIMS: Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon. METHODS: Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed. RESULTS: Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle. CONCLUSIONS: These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.

Reduced pepsin A processing of sonic hedgehog in parietal cells precedes gastric atrophy and transformation.

Sonic hedgehog (Shh) is not only essential to the development of the gastrointestinal tract, but is also necessary to maintain the characteristic acid-secreting phenotype of the adult stomach. Gastrin is the only hormone capable of stimulating gastric acid and is thus required to maintain functional parietal cells. We have shown previously that gastrin-null mice display gastric atrophy and metaplasia prior to progression to distal, intestinal-type gastric cancer. Because reduced levels of Shh peptide correlate with gastric atrophy, we examined whether gastrin regulates Shh expression in parietal cells. We show here that gastrin stimulates Shh gene expression and acid-dependent processing of the 45-kDa Shh precursor to the 19-kDa secreted peptide in primary parietal cell cultures. This cleavage was blocked by the proton pump inhibitor omeprazole and mediated by the acid-activated protease pepsin A. Pepsin A was also the protease responsible for processing Shh in tissue extracts from human stomach. By contrast, extracts prepared from neoplastic gastric mucosa had reduced levels of pepsin A and did not process Shh. Therefore processing of Shh in the normal stomach is hormonally regulated, acid-dependent, and mediated by the aspartic protease pepsin A. Moreover parietal cell atrophy, a known pre-neoplastic lesion, correlates with loss of Shh processing.

Hydrogen peroxide is a diffusible paracrine signal for the induction of epithelial cell death by activated myofibroblasts.

Cell-cell signaling roles for reactive oxygen species (ROS) generated in response to growth factors/cytokines in nonphagocytic cells are not well defined. In this study, we show that fibroblasts isolated from lungs of patients with idiopathic pulmonary fibrosis (IPF) generate extracellular hydrogen peroxide (H2O2) in response to the multifunctional cytokine, transforming growth factor-beta1 (TGF-beta1). In contrast, TGF-beta1 stimulation of small airway epithelial cells (SAECs) does not result in detectable levels of extracellular H2O2. IPF fibroblasts independently stimulated with TGF-beta1 induce loss of viability and death of overlying SAECs when cocultured in a compartmentalized Transwell system. These effects on SAECs are inhibited by the addition of catalase to the coculture system or by the selective enzymatic blockade of H2O2 production by IPF fibroblasts. IPF fibroblasts heterogeneously express alpha-smooth muscle actin stress fibers, a marker of myofibroblast differentiation. Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast phenotype. Thus, myofibroblast secretion of H2O2 functions as a diffusible death signal for lung epithelial cells. This novel mechanism for intercellular ROS signaling may be important in physiological/pathophysiological processes characterized by regenerating epithelial cells and activated myofibroblasts.

Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-beta1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-beta1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-beta1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-beta receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-beta1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-beta1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-beta1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-beta1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-beta1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-beta1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.