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Loss of TGF-β type II receptor (TβRII, encoded by Tgfbr2) expression in the prostate stroma contributes to prostate cancer initiation, progression, and invasion. We evaluated whether TβRII loss also affected prostate cancer bone metastatic growth. Immunohistologic analysis revealed that TβRII expression was lost in cancer-associated fibroblasts in human prostate cancer bone metastatic tissues. We recapitulated the human situation with a conditional stromal Tgfbr2 knockout (Tgfbr2-KO) mouse model. Conditioned media from primary cultured Tgfbr2-KO or control Tgfbr2-flox prostatic fibroblasts (koPFCM or wtPFCM, respectively) were applied to C4-2B prostate cancer cells before grafting the cells tibially. We found that koPFCM promoted prostate cancer cell growth in the bone and development of early mixed osteoblastic/osteolytic bone lesions. Furthermore, the koPFCM promoted greater C4-2B adhesion to type-I collagen, the major component of bone matrix, compared to wtPFCM-treated C4-2B. Cytokine antibody array analysis revealed that koPFCM had more than two-fold elevation in granulocyte colony-stimulating factor and CXCL1, CXCL16, and CXCL5 expression relative to wtPFCM. Interestingly, neutralizing antibodies of CXCL16 or CXCL1 were able to reduce koPFCM-associated C4-2B type-I collagen adhesion to that comparable with wtPFCM-mediated adhesion. Collectively, our data indicate that loss of TGF-β responsiveness in prostatic fibroblasts results in upregulation of CXCL16 and CXCL1 and that these paracrine signals increase prostate cancer cell adhesion in the bone matrix. These microenvironment changes at the primary tumor site can mediate early establishment of prostate cancer cells in the bone and support subsequent tumor development at the metastatic site. Mol Cancer Res; 10(4); 494–503. ©2012 AACR.

Figure S1. Copy number changes in PTEN region. Figure S2. Analysis of TP53 Aberrations. Figure S3. Allelic imbalance analysis. Figure S4. Example of HOXA9 immunohistochemistry and digital analysis output images. Figure S5. Gene expression analysis of genes within the 3 regions of copy number loss in CAS.

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Supplementary Figure 3 from Tissue-Specific Consequences of Cyclin D1 Overexpression in Prostate Cancer Progression

Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-β receptor II (TGFβR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGFβR2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-β) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFβR2 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 TGFβR2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-β1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFβR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-β1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-β1 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-β signaling to tumor promotion by tumor stromal cells. Cancer Res; 71(4); 1272–81. ©2011 AACR.

Supplementary Figure 2 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

PDF - 1790K, ALCAM expression is upregulated in response to the loss of ECadherin (S1); TGFbeta-induced shedding (S2); ALCAM serum half-life in mice (S3); ALCAM shedding by prostate and breast cancer cells in vitro and in vivo (S4); Compound-32/ADAM17 inhibitor dosing in vivo (S5); TGFbeta-induced migration is abrogated by the loss of ALCAM in breast and lung cancer cells (S6); Representative TRAcP stained bone lesion created by PC3 shControl and shALCAM after intratibial injection into immunodeficient mice (S7); Immune response modulation and wound healing does not effect serum ALCAM (S8); Graph represents tumor cell proliferation over time as assessed by total cell counts over 72 hours (S9).

Supplementary Video 5 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

Supplementary Table 3 from Altered TGF-β Signaling in a Subpopulation of Human Stromal Cells Promotes Prostatic Carcinogenesis

Supplementary Table 2 from Altered TGF-β Signaling in a Subpopulation of Human Stromal Cells Promotes Prostatic Carcinogenesis

The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in normal prostate fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1–overexpressing fibroblasts (NPFcyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1NPF-cyclin D1 cells) similar to that seen with carcinoma-associated fibroblasts (CAF). Microarray analysis showed that the expression profiles between CAFs and NPFcyclin D1 cells were highly concordant including cyclin D1 up-regulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue specific. [Cancer Res 2007;67(17):8188–97]

Supplementary Figure Legends 1-7 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Table 1 from Altered TGF-β Signaling in a Subpopulation of Human Stromal Cells Promotes Prostatic Carcinogenesis

Supplementary Figure 6 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 4 from Role for Stromal Heterogeneity in Prostate Tumorigenesis

Supplementary Figure 2 from Tissue-Specific Consequences of Cyclin D1 Overexpression in Prostate Cancer Progression

Supplementary Figure 1 from Role for Stromal Heterogeneity in Prostate Tumorigenesis

Supplementary Figure 2 from Altered TGF-β Signaling in a Subpopulation of Human Stromal Cells Promotes Prostatic Carcinogenesis

Supplementary Figure 1 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 4 from Tissue-Specific Consequences of Cyclin D1 Overexpression in Prostate Cancer Progression

Supplementary Information and Figures 1-7 from Transforming Growth Factor-β Promotes Invasion in Tumorigenic but not in Nontumorigenic Human Prostatic Epithelial Cells

Supplementary Figure 3 from Role for Stromal Heterogeneity in Prostate Tumorigenesis

Free Article from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β. Not only is this posttranslational modification of ALCAM a marker of prostate cancer progression, the molecule is also required for effective metastasis to bone. Biochemical analysis of prostate cancer cell lines reveals that ALCAM expression and shedding is elevated in response to TGF-β signaling. Both in vitro and in vivo shedding is mediated by ADAM17. Longitudinal analysis of circulating ALCAM in tumor-bearing mice revealed that shedding of tumor, but not host-derived ALCAM is elevated during growth of the cancer. Gene-specific knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished both skeletal dissemination and tumor growth in bone. The reduced growth of ALCAM knockdown cells corresponded to an increase in apoptosis (caspase-3) and decreased proliferation (Ki67). Together, these data demonstrate that the ALCAM is both a functional regulator as well as marker of prostate cancer progression. Cancer Res; 74(5); 1404–15. ©2014 AACR.

Mechanisms of androgen dependence of the prostate are critical to understanding prostate cancer progression to androgen independence associated with disease mortality. Transient elevation of transforming growth factor-β (TGF-β) occurs after androgen ablation. To determine the role of TGF-β on prostate response to androgen ablation, conditional TGF-β type II receptor knockout mouse models of the epithelia (Tgfbr2NKX3.1KO) and stromal fibroblasts (Tgfbr2fspKO) were used. After castration, the prostates of Tgfbr2NKX3.1KO mice had apoptosis levels similar to those expected for control Tgfbr2floxE2/floxE2 mice. Prostates of Tgfbr2fspKO mice, however, had reduced regression and high levels of proliferation associated with canonical Wnt activity throughout the glandular epithelia regardless of androgen status. In contrast, Tgfbr2floxE2/floxE2 prostates had epithelial canonical Wnt activity only in the surviving proximal ducts after castration. In vitro studies showed that androgen antagonist, bicalutamide, transiently elevated both Tgfbr2floxE2/floxE2 and Tgfbr2fspKO stromal expression of Wnt-2, Wnt-3a, and Wnt-5a. The neutralization of Wnt signaling by the expression of secreted frizzled related protein-2 (SFRP-2) resulted in decreased LNCaP prostate epithelial cell proliferation in stromal conditioned media transfer experiments. In vivo tissue recombination studies using Tgfbr2fspKO prostatic stromal cells in combination with wild-type or SV40 large T antigen expressing epithelia resulted in prostates that were refractile to androgen ablation. The expression of SFRP-2 restored the Tgfbr2fspKO-associated prostate responsiveness to androgen ablation. These studies reveal a novel TGF-β, androgen, and Wnt paracrine signaling axis that enables prostatic regression of the distal ducts after androgen ablation while supporting proximal duct survival. [Cancer Res 2008;68(12):4709–18]

Supplementary Figure 1 from Altered TGF-β Signaling in a Subpopulation of Human Stromal Cells Promotes Prostatic Carcinogenesis

Supplementary Video 3 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

Supplementary Figure 3 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

Supplementary Materials and Methods, Legends for Figures and Tables from Altered TGF-β Signaling in a Subpopulation of Human Stromal Cells Promotes Prostatic Carcinogenesis

Supplementary Figure 4 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

Supplementary Figure 4 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Table S2 from Identification of SFRP1 as a Candidate Mediator of Stromal-to-Epithelial Signaling in Prostate Cancer

PDF - 96K, Legend for Supplemental Figures S1-S9.

Supplementary Video 2 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

Supplementary Video 4 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

PDF - 82K, Description of additional methods and procedures used in the study.

Supplementary Figure 2 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 1 from The Role of Transforming Growth Factor-β–Mediated Tumor-Stroma Interactions in Prostate Cancer Progression: An Integrative Approach

Supplementary Figure 3 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity