The role of transforming growth factor-beta-mediated tumor-stroma interactions in prostate cancer progression: an integrative approach.
ABSTRACT: We have implemented a hybrid cellular automata model based on the structure of human prostate that recapitulates key interactions in nascent tumor foci between tumor cells and adjacent stroma. Model simulations show how stochastic interactions between tumor cells and stroma may lead to a structural suppression of tumor growth, modest proliferation, or unopposed tumor growth. The model incorporates key aspects of prostate tumor progression, including transforming growth factor-beta (TGF-beta), matrix-degrading enzyme activity, and stromal activation. It also examines the importance of TGF-beta during tumor progression and the role of stromal cell density in regulating tumor growth. The validity of one of the key predictions of the model about the effect of epithelial TGF-beta production on glandular stability was tested in vivo. These experimental results confirmed the ability of the model to generate testable biological predictions in addition to providing new avenues of experimental interest. This work underscores the need for more pathologically representative models to cooperatively drive computational and biological modeling, which together could eventually lead to more accurate diagnoses and treatments of prostate cancer.
Project description:Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-? receptor II (TGF?R2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGF?R2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-?) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGF?R2 function in 50% of the stromal cell population resulted in malignant transformation of the nontumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGF?R2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (?SMA)] through elevation of TGF-?1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGF?R2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-?1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-?1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-? signaling to tumor promotion by tumor stromal cells.
Project description:Transforming growth factor (TGF)-beta is an important paracrine factor in tumorigenesis. Ligand binding of the type I and II TGF-beta receptors initiate downstream signaling. The role of stromal TGF-beta signaling in prostate cancer progression is unknown. In mice, the conditional stromal knockout of the TGF-beta type II receptor expression (Tgfbr2(fspKO)) resulted in the development of prostatic intraepithelial neoplasia and progression to adenocarcinoma within 7 months. Clinically, we observed a loss of TGF-beta receptor type II expression in 69% of human prostate cancer-associated stroma, compared to 15% of stroma associated with benign tissues (n=140, P-value <0.0001). To investigate the mechanism of paracrine TGF-beta signaling in prostate cancer progression, we compared the effect of the prostatic stromal cells from Tgfbr2(fspKO) and floxed TGF-beta type II receptor Tgfbr2(floxE2/floxE2) mice on LNCaP human prostate cancer cells in vitro and tissue recombination xenografts. Induction of LNCaP cell proliferation and tumorigenesis was observed by Tgfbr2(fspKO) prostate stroma as a result of elevated Wnt3a expression. Neutralizing antibodies to Wnt3a reversed LNCaP tumorigenesis. The TGF-beta inhibition of Wnt3a expression was in part through the suppression of Stat3 activity on the Wnt3a promoter. In conclusion, the frequent loss of stromal TGF-beta type II receptor expression in human prostate cancer can relieve the paracrine suppression of Wnt3a expression.
Project description:Prostate cancer (PCa) is a malignant tumor that seriously threatens men's health worldwide. Recently, stromal cells in the tumor microenvironment (TME) have been reported to contribute to the progression of PCa. However, the role and mechanism of how PCa cells interact with stromal cells to reshape the TME remain largely unknown. Here, using a spontaneous prostate adenocarcinoma (PRAD) model driven by the loss of Pten and Hic1, we found that M2 macrophages markedly infiltrated the stroma of Pten and Hic1 double conditional knockout (dCKO) mice compared with those in control (Ctrl) mice due to higher TGF-β levels secreted by HIC1-deleted PCa cells. Mechanistically, TGF-β in TME promoted the polarization of macrophages into "M2" status by activating the STAT3 pathway and modulating c-Myc to upregulate CXCR4 expression. Meanwhile, TGF-β activated the fibroblasts to form cancer-associated fibroblasts (CAFs) that secrete higher CXCL12 levels, which bound to its cognate receptor CXCR4 on M2 macrophages. Upon interaction with CAFs, M2 macrophages secreted more CXCL5, which promoted the epithelial-mesenchymal transition (EMT) of PCa via CXCR2. Moreover, using the TGF-β receptor I antagonist, galunisertib, significantly inhibited the tumor growth and progression of the TRAMP-C1 cell line-derived subcutaneous tumor model. Finally, we confirmed that the stromal microenvironment was shaped by TGF-β in HIC1-deficient PCa and was associated with the progression of PCa.
Project description:AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-? signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-? signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-? receptor 1 specific inhibitor, blocks this TGF-? induced biology. Importantly, stromal TGF-? signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-? signaling induced AR activation in LNCaP cells, indicating that stromal TGF-? signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-? induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC.
Project description:Prostate stroma-specific TGF-beta signaling induces morphological changes in LNCaP cells. We have previously shown that stromal TGF-beta signaling regulates prostate tumor growth. To further delineate the underlying mechanisms, we generated LNCaP cells overexpressing an HA-tagged constitutively activate TGF-beta1 ligand (LNCaP-HA-TGF-β1(a)) and control LNCaP cells (LNCaP-Ctrl), and performed in vitro co-cultures of LNCaP-HA-TGF-β1(a) and LNCaP-Ctrl cells on top of the confluent HPS-19I cells, a human prostate stromal cell line. Since LNCaP cells are defective in TGF-beta receptor I (TbetaRI / ALK-5) that is essential for mediating TGF-beta signaling, only HPS19I cells are able to respond to TGF-beta ligand in these co-cultures. This provides a unique opportunity to study how prostate stromal cell-specific TGF-beta signaling regulates PCa biology. To identify the prostate epithelia-specific gene that was regulated by prostate stromal TGF-beta signaling, we also treated HPS19I cells using conditioned media collected from LNCaP- HA-TGF-β1(a) cells and LNCaP-Ctrl cells cultured in RPMI1640 supplemented with 0.2% FBS. After 6 days of treatment, we extracted total RNA from these HPS19I cells and performed microarray. Overall design: HPS-19I prostate stroma cells from LNCaP+HPS-19I co-culture, with or without TGF-beta KEYWORDS: two group comparison
Project description:The tumor-stroma crosstalk is a dynamic process fundamental in tumor development. In hepatocellular carcinoma (HCC), the progression of malignant hepatocytes frequently depends on transforming growth factor (TGF)-beta provided by stromal cells. TGF-beta induces an epithelial to mesenchymal transition (EMT) of oncogenic Ras-transformed hepatocytes and an upregulation of platelet-derived growth factor (PDGF) signaling. To analyse the influence of the hepatic tumor-stroma crosstalk onto tumor growth and progression, we co-injected malignant hepatocytes and myofibroblasts (MFBs). For this, we either used in vitro-activated p19(ARF) MFBs or in vivo-activated MFBs derived from physiologically inflamed livers of Mdr2/p19(ARF) double-null mice. We show that co-transplantation of MFBs with Ras-transformed hepatocytes strongly enhances tumor growth. Genetic interference with the PDGF signaling decreases tumor cell growth and maintains plasma membrane-located E-cadherin and beta-catenin at the tumor-host border, indicating a blockade of hepatocellular EMT. We further generated a collagen gel-based three dimensional HCC model in vitro to monitor the MFB-induced invasion of micro-organoid HCC spheroids. This invasion was diminished after inhibition of TGF-beta or PDGF signaling. These data suggest that the TGF-beta/PDGF axis is crucial during hepatic tumor-stroma crosstalk, regulating both tumor growth and cancer progression.
Project description:Prostate stroma-specific TGF-beta signaling induces morphological changes in LNCaP cells. We have previously shown that stromal TGF-beta signaling regulates prostate tumor growth. To further delineate the underlying mechanisms, we generated LNCaP cells overexpressing an HA-tagged constitutively activate TGF-beta1 ligand (LNCaP-HA-TGF-β1(a)) and control LNCaP cells (LNCaP-Ctrl), and performed in vitro co-cultures of LNCaP-HA-TGF-β1(a) and LNCaP-Ctrl cells on top of the confluent HPS-19I cells, a human prostate stromal cell line. Since LNCaP cells are defective in TGF-beta receptor I (TbetaRI / ALK-5) that is essential for mediating TGF-beta signaling, only HPS19I cells are able to respond to TGF-beta ligand in these co-cultures. This provides a unique opportunity to study how prostate stromal cell-specific TGF-beta signaling regulates PCa biology. Overall design: LNCaP + HPS19I co-cultures, with or without TGF-beta and with or without SD208 (a TBetaRI (ALK-5) protein kinase-specific inhibitor) KEYWORDS: multiple group comparison
Project description:Aberrant transforming growth factor-? (TGF-?) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-? signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-? signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-?-induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.
Project description:This study was designed to investigate the different involvements of prostatic stromal cells from the normal transitional zone (TZ) or peripheral zone (PZ) in the carcinogenesis of prostate cancer (PCa) epithelial cells (PC-3) in vitro and in vivo co-culture models. Ultra-structures and gene expression profiles of primary cultures of human prostatic stromal cells from the normal TZ or PZ were analyzed by electron microscopy and microarray analysis. In vitro and in vivo co-culture models composed of normal TZ or PZ stromal cells and human PCa PC-3 cells were established. We assessed tumor growth and weight in the in vivo nude mice model. There are morphological and ultra-structural differences in stromal cells from TZ and PZ of the normal prostate. In all, 514 differentially expressed genes were selected by microarray analysis; 483 genes were more highly expressed in stromal cells from TZ and 31 were more highly expressed in those from PZ. Co-culture with PZ stromal cells and transforming growth factor-beta1 (TGF-beta1) increased the tumor growth of PC-3 cells in vitro and in vivo, as well as Bcl-2 expression. On the other hand, stromal cells of TZ suppressed PC-3 cell tumor growth in the mouse model. We conclude that ultra-structures and gene expression differ between the stromal cells from TZ or PZ of the normal prostate, and stroma-epithelium interactions from TZ or PZ might be responsible for the distinct zonal localization of prostate tumor formation.
Project description:Stromal-epithelial interactions dictate prostate tumorigenesis and response to castration. Hydrogen peroxide-inducible clone 5 (Hic-5/ARA55) is a transforming growth factor-beta (TGF-?)-induced coactivator of androgen receptor (AR) expressed in the prostate stroma. Interestingly, following castration, we identified epithelial expression of Hic-5/ARA55 in mouse and human prostate tissues. To determine the role of epithelial Hic-5 in prostate cancer progression and castration responsiveness, we compared LNCaP cells having Hic-5 stably expressed with the parental LNCaP cells following tissue recombination xenografts with mouse prostate stromal cells. We previously identified knocking out prostate stromal TGF-? signaling potentiated castrate-resistant prostate tumors, in a Wnt-dependent manner. The LNCaP chimeric tumors containing prostate fibroblasts conditionally knocked out for the TGF-? type II receptor (Tgfbr2-KO) resulted in larger, more invasive, and castration-resistant tumors compared those with floxed (control) stromal cells. However, the LNCaP-Hic5 associated with Tgfbr2-KO fibroblasts generated chimeric tumors with reduced tumor volume, lack of invasion and restored castration dependence. Neutralization of canonical Wnt signaling is shown to reduce prostate tumor size and restore regression following castration. Thus, we hypothesized that epithelial Hic-5/ARA55 expression negatively regulated Wnt signaling. The mechanism of the Hic-5/ARA55 effects on castration was determined by analysis of the c-myc promoter. C-myc luciferase reporter activity suggested Hic-5/ARA55 expression inhibited c-myc activity by ?-catenin. Sequential ChIP analysis indicated ?-catenin and T-cell-specific 4 (TCF4) bound the endogenous c-myc promoter in the absence of Hic-5 expression. However, the formation of a TCF4/Hic-5 repressor complex inhibited c-myc promoter activity, by excluding ?-catenin binding with TCF4 on the promoter. The data indicate Hic-5/ARA55 expression in response to castration-enabled epithelial regression through the repression of c-myc gene at the chromatin level.