David Barron

Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma

We investigated the effects of mechanical stretch and induced stimulation of lung parenchyma on the activation of proinflammatory transcription factors in normal mice and in a mouse model of asthma. Mechanical stretching of lung parenchyma led to increased activation of NF‐κB and AP‐1 transcription factors. Incubation of lung parenchyma with methacholine increased the activation of NF‐κB, which was further augmented by stretch. Activation of NF‐κB in response to mechanical stretch was associated with the phosphorylation and degradation of IκBα and the activation of IκB kinase. Stretch‐induced activation of NF‐κB involves activation of stretch‐activated (SA) channels and the production of free radicals. Mechanical stretch and/or treatment with methacholine resulted in an increased activation of ERK1/2 and p38 MAP kinase, and the inhibition of the activity of these kinases partially blocked the stretch‐induced NF‐κB and AP‐1 activation. A greater level of NF‐κB and ERK1/2 activity was observed in the asthmatic mice, which was further increased by mechanical stretching. The level of cyclooxygenase‐2, an NF‐κB‐regulated enzyme, was also higher in lung parenchyma from asthmatic mice than in normal mice. Our data suggest that mechanical stretching of lung parenchyma activates NF‐κB and AP‐1, at least in part, through the activation of MAP kinase signaling pathways.—Kumar, A., Lnu, S., Malya, R., Barron, D., Moore, J., Corry, D. B., Boriek, A. M. Mechanical stretch activates nuclear factor‐kappaB, activator protein‐1, and mitogen‐activated protein kinases in lung parenchyma: implications in asthma. FASEB J. 17, 1800–1811 (2003)

RUNX1 is essential for mesenchymal stem cell proliferation and myofibroblast differentiation

Significance Recruitment, proliferation, and differentiation of myofibroblasts are common in many disease states. Mechanisms that regulate proliferation and differentiation are poorly understood, although TGF-β is a key inducer of differentiation. Here, we report, for the first time to our knowledge, that runt-related transcription factor 1 (RUNX1) regulates mesenchymal stem cell (MSC) biology and progenitor cell commitment to myofibroblasts. In this work, we describe the first identification, to our knowledge, of tissue-resident MSCs from adult normal human prostate gland and the role of these MSCs as myofibroblast precursors. We also pinpoint the role of RUNX1 in regulating proliferation and differentiation in both marrow-derived and tissue-resident MSCs. Perturbation of RUNX1 activity may provide insights for developing antifibrotic and anticancer therapies via targeting the reactive stroma microenvironment. Myofibroblasts are a key cell type in wound repair, cardiovascular disease, and fibrosis and in the tumor-promoting microenvironment. The high accumulation of myofibroblasts in reactive stroma is predictive of the rate of cancer progression in many different tumors, yet the cell types of origin and the mechanisms that regulate proliferation and differentiation are unknown. We report here, for the first time to our knowledge, the characterization of normal human prostate-derived mesenchymal stem cells (MSCs) and the TGF-β1–regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-β1 resulted in commitment to myofibroblasts. TGF-β1–regulated runt-related transcription factor 1 (RUNX1) was required for cell cycle progression and proliferation of progenitors. RUNX1 also inhibited, yet did not block, differentiation. Knockdown of RUNX1 in prostate or bone marrow-derived MSCs resulted in cell cycle arrest, attenuated proliferation, and constitutive differentiation to myofibroblasts. These data show that RUNX1 is a key transcription factor for MSC proliferation and cell fate commitment in myofibroblast differentiation. This work also shows that the normal human prostate gland contains tissue-derived MSCs that exhibit multilineage differentiation similar to bone marrow-derived MSCs. Targeting RUNX1 pathways may represent a therapeutic approach to affect myofibroblast proliferation and biology in multiple disease states.

TGF-β1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse.

TGF-β1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression.

Recruitment of CD34+ Fibroblasts in Tumor-Associated Reactive Stroma: The Reactive Microvasculature Hypothesis

Reactive stroma co-evolves with cancer, exhibiting tumor-promoting properties. It is also evident at sites of wound repair and fibrosis, playing a key role in tissue homeostasis. The specific cell types of origin and the spatial/temporal patterns of reactive stroma initiation are poorly understood. In this study, we evaluated human tumor tissue arrays by using multiple labeled, quantitative, spectral deconvolution microscopy. We report here a novel CD34/vimentin dual-positive reactive fibroblast that is observed in the cancer microenvironment of human breast, colon, lung, pancreas, thyroid, prostate, and astrocytoma. Recruitment of these cells occurred in xenograft tumors and Matrigel plugs in vivo and was also observed in stromal nodules associated with human benign prostatic hyperplasia. Because spatial and temporal data suggested the microvasculature as a common site of origin for these cells, we analyzed microvasculature fragments in organ culture. Interestingly, fibroblasts with identical phenotypic properties and markers expanded radially from microvasculature explants. We propose the concept of reactive microvasculature for the evolution of reactive stroma at sites of epithelial disruption common in both benign and malignant disorders. Data suggest that the reactive stroma response is conserved among tissues, in normal repair, and in different human cancers. A more clear understanding of the nature and origin of reactive stroma is needed to identify novel therapeutic targets in cancer and fibrosis.

Abstract 4588: Frequency and clinical characteristics of c-MET mutation in malignant melanoma

Purpose: C-MET is over-expressed in metastatic melanoma and have copy number gains at chromosome 7 in late stages of melanoma progression. However, the presence of C-MET mutation is not well known in melanoma. We analyzed tumor samples of patients with malignant melanoma to identify the frequency and the clinicopathology of C-MET mutations. Methods: We identified 103 patients with metastatic mutation who underwent testing for c-MET mutation and reviewed the clinical data of these patients. The sequencing analyses were performed by Sequenom Assay, and the mutations were confirmed with Sanger Sequencing analysis. Clinical characteristics were correlated with C-MET mutation status, and a survival analysis was performed to identify significant associations. Results Among the 103 patients, eleven (11%) patients had melanoma harboring a C-MET mutation. Five (4.5%) patients had N375S mutation; two (1.8%) patients had R988C mutation; two (1.8%) had T1010I; one (1%) patient had H1112R and another patient had N375/T1010 mutation. A median age at diagnosis, sex, race and the status of ulceration at the primary site and the stages at diagnosis were similar between patients with a C-MET mutation and those with wild type. There were no significant differences in the overall survival from the time of stage IV diagnosis between the two groups, but there was a trend of a shorter median duration from the time of the initial diagnosis to distant metastases among patients with a C-MET mutation. (23.6 months vs. 15.9 months, p=0.09). Conclusions We found C-MET mutations in 11% of patients with malignant melanoma. There was a tendency to have distant metastasis at an earlier time in patients with a C-MET mutation. This is the first report describing the frequency and clinical characteristics of C-MET mutations in patients with malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4588. doi:1538-7445.AM2012-4588

Abstract 1440: Mechanisms of TGF-β1-induced myofibroblast/CAF differentiation in human prostate-derived mesenchymal stem cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Reactive stroma in prostate cancer is typified by the co-evolution of myofibroblasts/CAFs. This reactive stroma is associated with most human carcinoma and is predictive of progression. TGF-β1 is a key factor in regulating reactive stroma biology. However, the origin of myofibroblasts and the mechanisms of how TGF-β1 recruits, activates and induces their differentiation are essentially unknown. We have identified mesenchymal stem cells from normal human prostate gland and evaluated their biology in a novel 3D co-culture system. Human prostate-derived mesenchymal stem cells (hpMSCs) were CD44+/CD90+, expressed other mesenchymal stem cell genes, and exhibited multipotent differentiation patterns. When co-cultured with LNCaP cells in Millicell-CM inserts or co-inoculated in nude mice, self-organizing organoids formed with a core of stromal cells and a peripheral mantel of LNCaP cells. To investigate the role of TGF-β1, the hpMSCs were co-cultured with LNCaP cells engineered to overexpress active TGF-β1. Significantly, LNCaP cells engineered to overexpress TGF-β1 induced hpMSC differentiation to prototypical reactive stroma myofibroblasts. Microarray analysis of stroma revealed 1617 gene probes with more than a 2 fold-change in the presence of TGF-β1, showing that expression of TGF-β1 in LNCaP cells drives differential gene expression in hpMSCs. Of those gene expression profiles, RUNX1 and ID1 were identified as key transcription factors in hpMSCs that mediate TGF-β1-induced myofibroblast differentiation. Knockdown of RUNX1 in hpMSCs significantly promoted differentiation to myofibroblasts. Conversely, overexpression of RUNX1 inhibited a myofibroblast gene expression signature. Together, these data implicate RUNX1 as a major regulator of the stem cell state of tissue-derived myofibroblast stem/progenitor cells and therefore a modulator of the co-evolution of reactive stroma. Furthermore, ID1 may function as a negative regulator of reactive stroma formation by promoting proliferation of transiently amplifying cells and inhibiting myofibroblast differentiation. Our data show that reactive stroma in prostate cancer initiates from activation and differentiation of CD44+/CD90+ endogenous hpMSCs through TGF-β1/RUNX1/ID1 pathway. Understanding these mechanisms is important for developing new strategies to target the microenvironment niche. Citation Format: Woosook Kim, David Barron, Rebeca San Martin, Steven Ressler, David Rowley. Mechanisms of TGF-β1-induced myofibroblast/CAF differentiation in human prostate-derived mesenchymal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1440. doi:10.1158/1538-7445.AM2013-1440

Abstract 4265: Human prostate mesenchymal stem cells in prostate cancer: Mechanisms of TGF-β1 promoted myofibroblast/CAF differentiation

Reactive stroma in prostate cancer is typified by the co-evolution of myofibroblasts and carcinoma-associated fibroblasts (CAFs), yet the origin and biology of myofibroblast stem/progenitor cells are not known. TGF-β1 is overexpressed in prostate cancer cells and affects myofibroblast differentiation, a key step in formation of a tumor-promoting reactive stroma. However, specific mechanisms of how TGF-β1 regulates recruitment, activation and differentiation of myofibroblast stem/progenitor cells are poorly understood. Human prostate mesenchymal stem cell (hpMSC) lines were initiated from organ cultures of normal human prostate gland and characterized by a novel 3D co-culture model. Differentially engineered LNCaP cells to overexpress active TGF-β1 were cultured as organoids on CM-inserts and hpMSCs were co-cultured with engineered LNCaP cells either in direct contact or on laminin-coated coverslips in the bottom chamber. 3D organoids grown in serum-free media for 72 hours were analyzed by IHC, ELISA and qRT-PCR. In addition, RNAi gene knockdown experiments were performed in hpMSC to identify key transcription factors involved in myofibroblast/CAF differentiation. hpMSCs were CD44+/CD90+ and showed characteristics and gene expression similar to human mesenchymal stem cells. In the 3D model, LNCaP cells and hpMSCs self-organized into a free-floating organoid. The organoids exhibited a periphery of LNCaP cells and a core of reactive stroma-appearing cells. Significantly, LNCaP cells induced myofibroblast differentiation of hpMSC and this was promoted by LNCaP expression of active TGF-β1. Gene expression analysis identified the RUNX1 and ID1 transcription factors as upregulated in hpMSC by TGF-β1. Knockdown of RUNX1 and ID1 are being used to assess the role and mechanisms of how these transcription factors regulate myofibroblast differentiation. Together, these data suggest that the genesis and co-evolution of reactive stroma in prostate cancer results from activation and differentiation of CD44+/CD90+ endogenous hpMSC via upregulation of specific transcription factors by TGF-β1. Defining key mechanisms that recruit, activate and differentiate the origin of myofibroblasts/CAFs could provide clues to develop new strategies to target the microenvironment niche. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4265. doi:1538-7445.AM2012-4265

Abstract B23: Further defining reactive stroma in prostate cancer

Quiescent fibroblasts and smooth muscle make up the mesenchymally-derived component of normal human stroma and function to maintain tissue homeostasis by forming a reactive stroma phenotype in the presence of injury. Reactive stroma initiates early during the evolution of human prostate cancer, with foci being observed during premalignant prostatic intraepithelial neoplasia (PIN). This microenvironment response is characterized by an accumulation of vimentin positive cells that co-express several reactive stroma markers. Reactive stroma co-evolves with development of cancer foci and is typified by accumulation of myofibroblasts that express tenascin-C and pro-collagen I. Our previous studies have shown that the degree of reactive stroma is a significant predictor of biochemical recurrent disease (elevation of PSA) using either archival prostatectomy specimens or pre-operative needle biopsy material. Patients with grade 3 exhibit a significantly reduced time to biochemical recurrent disease. Moreover, we have shown significant differences in gene expression profiles in human reactive stroma grade 3 relative to normal human prostate stroma. Our previous studies have shown that reactive stroma is tumor-promoting in prostate cancer models. TGF-β has emerged as a key factor in mediating the microenvironment perturbations in a variety of cancers, however its corresponding role in reactive stroma initiation has not been examined. It is our hypothesis that prostate cancer reactive stroma is composed of myofibroblasts / CAFs and is induced from tissue-fixed and/or bone marrow derived cells (BMDCs) in a TGF-β dependent manner. Our data shows that foci of reactive stroma in human prostate cancer consist of CD34+/vimentin+ progenitor cells that are juxtaposed to the sub basal lamina surface at the stromal-epithelial junction. Bone marrow transplants with CFP reporter donors into athymic mice containing LNCaP xenografts shows a time dependent increase in recruited fibroblast-like cells of marrow origin. Mechanistic studies using a transgenic mouse overexpressing TGF-β in prostatic epithelium demonstrate foci of inflammation in nerve ganglia and vessel structures. These mice also display distinct stromal proliferative lesions reminiscent of changes seen in a subtype of human prostate cancer. These histopathological changes occur in an age-dependent manner and seem to become more prevalent after a year of age in transgenic mice. Further mechanistic studies examining TGF-β signaling and the role of BMDCs are underway to examine reactive stroma induction and progression in prostate cancer. We propose a model of reactive stroma potential, affected by activation of local progenitors and subsequent recruitment of circulating progenitors to initiate and sustain a reactive microenvironment adjacent to cancer foci. We also propose that this microenvironment is selective for more aggressive cancer phenotypes and therefore is a potential target of opportunity for more advanced prognostics and novel therapeutics. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B23