Rebeca San Martin

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.

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.

Reactive stroma in the prostate during late life: The role of microvasculature and antiangiogenic therapy influences.

Prostate cancer is associated to a reactive stroma microenvironment characterized by angiogenic processes that are favorable for tumor progression. Senescence has been identified as a predisposing factor for prostate malignancies. In turn, the relationships between aging, reactive stroma, and the mechanisms that induce this phenotype are largely unknown. Thus, we investigated the occurrence of reactive stroma in the mouse prostate during advanced age as well as the effects of antiangiogenic and androgen ablation therapies on reactive stroma recruitment.

Tenascin-C and integrin α9 mediate interactions of prostate cancer with the bone microenvironment

Deposition of the extracellular matrix protein tenascin-C is part of the reactive stroma response, which has a critical role in prostate cancer progression. Here, we report that tenascin C is expressed in the bone endosteum and is associated with formation of prostate bone metastases. Metastatic cells cultured on osteo-mimetic surfaces coated with tenascin C exhibited enhanced adhesion and colony formation as mediated by integrin α9β1. In addition, metastatic cells preferentially migrated and colonized tenascin-C-coated trabecular bone xenografts in a novel system that employed chorioallantoic membranes of fertilized chicken eggs as host. Overall, our studies deepen knowledge about reactive stroma responses in the bone endosteum that accompany prostate cancer metastasis to trabecular bone, with potential implications to therapeutically target this process in patients. Cancer Res; 77(21); 5977-88. ©2017 AACR.

Role of Reactive Stroma in Prostate Cancer

Reactive stroma initiates at sites of epithelial damage to mediate tissue repair and restore homeostasis. Genomic instability of epithelial cells at sites of early lesions such as prostatic intraepithelial neoplasia produces a similar breach of the epithelial barrier and an initiation of reactive stroma. Reactive stromal cells, termed myofibroblasts and carcinoma-associated fibroblasts, have been shown to originate potentially from several sources including tissue fibroblasts, resident stromal stem cells, vascular cells, and marrow-derived mesenchymal stem cells. Several growth factors such as transforming growth factor-β and interleukin-8 induce reactive stroma and regulate several downstream factors expressed in reactive stroma. Reactive stroma in prostate cancer is heterogeneous, and the amount of reactive stroma is predictive of disease progression. The heterogeneity of cells in reactive stroma is possibly a key aspect of the tumor-promoting properties. It is likely that reactive stroma biology is an important aspect of tumor progression to metastasis and acquired therapeutic resistance. Targeting the tumor microenvironment reactive stroma together with direct targeting of cancer cells may represent an effective therapeutic approach for the treatment of prostate cancer.

Abstract B92: Metabolomic landscape of African American prostate cancer: Insights into the biologic basis of the racial disparity

African American (AA) men have an approximately 60% higher incidence of prostate cancer (PCa) and have about two times greater risk of dying of the disease than their European American (EA) counterparts. Despite this staggeringly unequal burden of PCa incidence and outcome between AA and EA men, there is limited insight into the molecular mechanisms associated with this racial disparity. Using state-of-the-art mass spectrometry platform, we uncovered the very first metabolic and lipidomics landscape in PCa/benign adjacent tissue pairs and paired plasma and urine containing epidemiologic meta-data from ancestry-typed AA and EA men. A total of 190 polar and mid-polar metabolites and 495 lipids were measured in tissues and 183 metabolites and lipids were examined in the plasma and urine samples. AA PCa tissue and plasma signatures had unique alterations in metabolites and key enzymes associated with the methionine-homocysteine pathway, including adenosine and inosine levels, compared to corresponding tissues and plasma from EA case controls and EA PCa tissues. Intriguingly, methionine levels in AA PCa were also portrayed by distinct dietary practices in these patients. In addition, AA PCa tissues demonstrated unique patterns of bioenergetic metabolites and accumulated lipids reflecting impaired mitochondrial activity and TCA cycle. Further characterization of these first-in-the-field findings demonstrating reprogrammed metabolism in AA PCa relative to EA tumors could reveal insights into the biologic basis of PCa disparities and novel areas for therapeutic intervention. Citation Format: Stacy M. Lloyd*, Jie H. Gohlke*, Sumanta Basu*, Vasanta Putluri,Shaiju K Vareed, Rebeca San Martin, Thekkelnaycke Rajendiran, Tiffany A. Dorsey, Bandana Prasad, Rajni Sonavane, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Harene Venghatakrishnan, Salil Bhowmik, Alexander Zaslavsky,Nilanjan Guha, Rick Kittles, Stefan Ambs, Michael Ittmann,David Rowley, Ganesh Palapattu, Nagireddy Putluri,George Michailidis, Arun Sreekumar. Metabolomic landscape of African American prostate cancer: Insights into the biologic basis of the racial disparity [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B92.

Vitamin D receptor activation reduces VCaP xenograft tumor growth and counteracts ERG activity despite induction of TMPRSS2:ERG

Whether vitamin D is chemopreventive and/or has potential therapeutically in prostate cancer is unresolved. One confounding factor is that many prostate cancers express a TMPRSS2:ERG fusion gene whose expression is increased both by androgens and by vitamin D receptor (VDR) activation. Two challenges that limit VDR agonist use clinically are hypercalcemia and the cooperation of VDR with ERG to hyper-induce the 1α,25-dihydroxyvitamin D3 metabolizing enzyme, CYP24A1, thus reducing VDR activity. Using the VCaP TMPRSS2:ERG positive cell line as a model, we found that a nonsecosteroidal CYP24A1 resistant VDR agonist, VDRM2, substantially reduces growth of xenograft tumors without inducing hypercalcemia. Utilizing next generation RNA sequencing, we found a very high overlap of 1,25D(OH)2D3 and VDRM2 regulated genes and by drawing upon previously published datasets to create an ERG signature, we found activation of VDR does not induce ERG activity above the already high basal levels present in VCaP cells. Moreover, we found VDR activation opposes 8 of the 10 most significant ERG regulated Hallmark gene set collection pathways from Gene Set Enrichment Analysis (GSEA). Thus, a CYP24A1 resistant VDR agonist may be beneficial for treatment of TMPRSS2:ERG positive prostate cancer; one negative consequence of TMPRSS2:ERG expression is inactivation of VDR signaling.

Abstract 1564: Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation

The objective of this study is to assess the regulatory roles of reactive endosteum associated with foci of bone metastatic prostate cancer cells. Previous studies evaluated tissue arrays of human prostate cancer bone metastases and we identified a “reactive endosteum” spatially associated with foci of prostate cancer cells on trabecular bone. Reactive endosteum was characterized by elevated expression of tenascin-C, a glycoprotein deposited in the extracellular matrix of reactive stroma at sites of wound repair and in the primary tumor microenvironment in adult tissues. In general, tenascin-C is stromal derived in adult tissues. Of interest, tenascin-C is also deposited at sites of fracture repair and at sites of Brodie abscess osteomyelitis-associated inflammation in human bone. Tenascin-C has many diverse functions: it regulates cell adhesion, migration and proliferation in different pathological states while playing an important role in neuropatterning and osteogenesis during development. Moreover, tenascin-C has been shown to regulate several signal transduction pathways. To evaluate prostate cancer-bone interactions, we developed an in vitro, 3D, osteogenic organoid model composed of human mesenchymal stem cells induced to osteogenesis that were combined with human prostate VCaP cells in organ culture. In this model, foci of VCaP cells associated preferentially to regions of high tenascin-C deposition. VCaP cells also preferentially bound to purified human tenascin-C deposited on culture plates in a dose-dependent manner. VCaP cells also exhibited an elevated growth rate on osteo-mimetic plates coated with human tenascin-C as compared to control. Moreover, VCaP cells preferentially bound to human tenascin-C coated bovine trabecular bone cubes in vitro and initiated colony formation. Evaluation of potential mediators identified integrin alpha 9 beta 1 as the key mediator of attachment to human tenascin-C. Neutralization of this integrin inhibited adhesion. Evaluation of potential signaling pathways have implicated activation of EGF receptor (EGFR), Wnk1, and STAT6 pathways. Human tenascin-C exhibits EGF-like repeats and fibronectin type III domains that may mediate activation of these pathways. Of interest, adhesion and growth of VCaP cells on tenascin-C also induced elevated tenascin-C expression in these cells, a novel finding. Additional 3D organoid and in vivo xenograft studies with other cell types support the finding of induced expression of tenascin-C in epithelial cells associated with reactive stroma or matrix. In summary, our studies characterize elevated tenascin-C at sites of a reactive endosteum associated with metastatic prostate cancer foci. Data suggests that tenascin-C mediates adhesion and proliferation of cancer cells via activation of several pathways. These data may aid in developing novel therapeutic approaches to treat metastatic disease. Citation Format: Rebeca San Martin, Kenneth Pienta, David R. Rowley. Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1564.

Abstract 2268: Tenascin C as an effector of prostate cancer derived bone metastasis

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX The purpose of this study is to characterize the microenvironment changes in prostate cancer bone metastasis in the context of a reactive stroma response, and to evaluate how these changes affect metastatic colonization and proliferation. The stroma plays an important role in the maintenance of tissue homeostasis. In particular, stroma associated with secretory epithelium, like prostate acini, must be able to initiate an efficient wound repair response in the event of a breach in the epithelial layer in order to prevent infection or further damage. This reactive stroma response is characterized by the accumulation of myofibroblasts, also known as carcinoma associated fibroblasts (CAFs) followed by remodeling of extracellular matrix at the site of injury. The reactive stroma response initiates early in prostate cancer, co-evolves with disease progression, and is predictive of recurrence. One way reactive stroma remodels the microenvironment is by deposition of tenascin-C. Tenascin-C is an extracellular matrix protein that is expressed during development and is critical for neuronal patterning and osteogenesis. In contrast, expression of tenascin C in adult tissues is limited and is restricted to regions of wound repair, tissue remodeling and pathological conditions, such as cancer. To assess whether the bone exhibits a reactive tissue phenotype in the context of metastasis, human prostate cancer metastasis tissue arrays were evaluated using immunohistochemistry and spectral deconvolution. This work identified a tenascin-C expression pattern at trabeculae-associated metastatic sites, suggesting the evolution of a reactive endosteum. In order to evaluate the mechanisms involved, we developed an in vitro 3D osteogenic organoid, using human mesenchymal stem cells induced to osteoblastic differentiation, which exhibits a reactive endosteum phenotype. Co-culture with the metastatic prostate cancer cell line VCaP showed preferential binding at sites high in tenascin C deposition. Also, metastatic cells were capable of adherence to purified tenascin C in vitro, forming 3D colonies. We have identified α9β1 integrin as a mediator of prostate cancer cell adhesion to tenascin C-rich surfaces. Preliminary data has also identified signaling through WNK1 and STAT6 as additional candidate mechanisms that mediate tenascin-C induced biology in prostate cancer cells that are metastatic to bone surfaces. These studies characterize a reactive endosteum phenotype at sites of metastatic prostate cancer foci and suggest that elevated tenascin-C at these sites mediates adhesion and other biological properties of cancer cells. This study will help provide data from which to develop novel therapeutic approaches to treat metastatic disease. Citation Format: Rebeca San Martin, Kenneth Pienta, David R. Rowley. Tenascin C as an effector of prostate cancer derived bone metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A62.

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