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Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.

Pietras K, Pahler J, Bergers G, Hanahan D - PLoS Med. (2008)

Bottom Line: Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts.Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects.Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Diabetes Center and Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States ofAmerica. kristian.pietras@licr.ki.se

ABSTRACT

Background: Important support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.

Methods and findings: We used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.

Conclusions: This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

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FGF-2 Is Expressed by CAFs and Repressed by Specific Inhibition of PDGF Receptor Signaling(A) Analysis of cells isolated by FACS from the cervixes of 3.5-mo-old HPV/E2 mice by sorting for expression of PDGFR-α and -β or c-kit. RT-PCR was performed to assess the expression of FGF-2, the squamous epithelial marker K14, the fibroblast cell marker vimentin, and the housekeeping gene L19 as a loading control.(B) Representative immunohistochemical staining of FGF-2 in the transformation zone of the uterine cervixes of HPV/E2 mice that had or had not been treated with imatinib displaying CIN3 lesions. The expression pattern was analyzed in at least five different mice of similar histological stage from each treatment group. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. As a control for specificity, the primary antibody was pre-blocked by incubation with recombinant mouse FGF-2 . E, epithelium; S, stroma.(C) Expression of FGF-2 analyzed by quantitative RT-PCR following a long (14 d) and a short (3 d) treatment with imatinib (Student t-test, t = 3.5, p < 0.05). Expression of FGF-2 analyzed by quantitative RT-PCR following a 3-d treatment with control IgG or inhibitory antibodies against PDGFR-α and PDGFR-β (Student t-test, t = 3.1, p < 0.05). Note that for technical reasons, a different primer set was used in this experiment compared to the experiment shown in Figure 4C, yielding different absolute values of expression (for details, see Materials and Methods). Error bars indicate the standard error of the mean.
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pmed-0050019-g005: FGF-2 Is Expressed by CAFs and Repressed by Specific Inhibition of PDGF Receptor Signaling(A) Analysis of cells isolated by FACS from the cervixes of 3.5-mo-old HPV/E2 mice by sorting for expression of PDGFR-α and -β or c-kit. RT-PCR was performed to assess the expression of FGF-2, the squamous epithelial marker K14, the fibroblast cell marker vimentin, and the housekeeping gene L19 as a loading control.(B) Representative immunohistochemical staining of FGF-2 in the transformation zone of the uterine cervixes of HPV/E2 mice that had or had not been treated with imatinib displaying CIN3 lesions. The expression pattern was analyzed in at least five different mice of similar histological stage from each treatment group. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. As a control for specificity, the primary antibody was pre-blocked by incubation with recombinant mouse FGF-2 . E, epithelium; S, stroma.(C) Expression of FGF-2 analyzed by quantitative RT-PCR following a long (14 d) and a short (3 d) treatment with imatinib (Student t-test, t = 3.5, p < 0.05). Expression of FGF-2 analyzed by quantitative RT-PCR following a 3-d treatment with control IgG or inhibitory antibodies against PDGFR-α and PDGFR-β (Student t-test, t = 3.1, p < 0.05). Note that for technical reasons, a different primer set was used in this experiment compared to the experiment shown in Figure 4C, yielding different absolute values of expression (for details, see Materials and Methods). Error bars indicate the standard error of the mean.

Mentions: Having functionally implicated FGF-2 in the angiogenic phenotype, we sought to identify the cell type in cervical neoplasias and cancer that expressed FGF-2, by using FACS. Total RNA was extracted from the pools of cells labeled positively for either of the imatinib receptor tyrosine kinase targets, i.e., for PDGF receptor-α plus PDGF receptor-β, or for c-kit, and subjected to semiquantitative RT-PCR analysis to establish the identity of the cells. The pool of cells sorted for expression of c-kit was positive for expression of the basal squamous epithelial gene K14, but not for the mesenchymal gene vimentin, indicating that c-kit is expressed by a subset of cervical keratinocytes (Figure 5A). In contrast, the pool of cells sorted for expression of PDGF receptors contained the transcripts for the mesenchymal gene vimentin, but not K14, in accordance with earlier experiments showing the PDGF receptors were selectively expressed in CAFs and pericytes (Figures 1A and 5A). The transcript for FGF-2 was predominant in the pool sorted for expression of PDGF receptors, indicating that FGF-2 was largely produced by PDGF receptor-expressing cells (Figure 5A). Next, we employed immunostaining of tissue sections to visualize the cells expressing FGF-2 in the cervixes of HPV/E2 mice. FGF-2 was found to be predominantly expressed by stromal cells, in particular the subset of cells in proximity to the basal keratinocytes (Figure 5B). In accordance with the diminished FGF-2 mRNA levels seen following treatment with imatinib, the staining intensity for FGF-2 was reduced in tissue sections from imatinib-treated mice (Figure 5B). To ascertain which stromal cell type expressed FGF-2, we performed coimmunostaining of FGF-2 and markers for various prevalent cell types within the cervix. FGF-2 expression colocalized with expression of PDGF receptor-α and vimentin, indicating that FGF-2 is expressed by CAFs, but not with markers for endothelial cells, leukocytes, macrophages, mast cells, NK cells, or dendritic cells (Figure S4).


Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.

Pietras K, Pahler J, Bergers G, Hanahan D - PLoS Med. (2008)

FGF-2 Is Expressed by CAFs and Repressed by Specific Inhibition of PDGF Receptor Signaling(A) Analysis of cells isolated by FACS from the cervixes of 3.5-mo-old HPV/E2 mice by sorting for expression of PDGFR-α and -β or c-kit. RT-PCR was performed to assess the expression of FGF-2, the squamous epithelial marker K14, the fibroblast cell marker vimentin, and the housekeeping gene L19 as a loading control.(B) Representative immunohistochemical staining of FGF-2 in the transformation zone of the uterine cervixes of HPV/E2 mice that had or had not been treated with imatinib displaying CIN3 lesions. The expression pattern was analyzed in at least five different mice of similar histological stage from each treatment group. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. As a control for specificity, the primary antibody was pre-blocked by incubation with recombinant mouse FGF-2 . E, epithelium; S, stroma.(C) Expression of FGF-2 analyzed by quantitative RT-PCR following a long (14 d) and a short (3 d) treatment with imatinib (Student t-test, t = 3.5, p < 0.05). Expression of FGF-2 analyzed by quantitative RT-PCR following a 3-d treatment with control IgG or inhibitory antibodies against PDGFR-α and PDGFR-β (Student t-test, t = 3.1, p < 0.05). Note that for technical reasons, a different primer set was used in this experiment compared to the experiment shown in Figure 4C, yielding different absolute values of expression (for details, see Materials and Methods). Error bars indicate the standard error of the mean.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2214790&req=5

pmed-0050019-g005: FGF-2 Is Expressed by CAFs and Repressed by Specific Inhibition of PDGF Receptor Signaling(A) Analysis of cells isolated by FACS from the cervixes of 3.5-mo-old HPV/E2 mice by sorting for expression of PDGFR-α and -β or c-kit. RT-PCR was performed to assess the expression of FGF-2, the squamous epithelial marker K14, the fibroblast cell marker vimentin, and the housekeeping gene L19 as a loading control.(B) Representative immunohistochemical staining of FGF-2 in the transformation zone of the uterine cervixes of HPV/E2 mice that had or had not been treated with imatinib displaying CIN3 lesions. The expression pattern was analyzed in at least five different mice of similar histological stage from each treatment group. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. As a control for specificity, the primary antibody was pre-blocked by incubation with recombinant mouse FGF-2 . E, epithelium; S, stroma.(C) Expression of FGF-2 analyzed by quantitative RT-PCR following a long (14 d) and a short (3 d) treatment with imatinib (Student t-test, t = 3.5, p < 0.05). Expression of FGF-2 analyzed by quantitative RT-PCR following a 3-d treatment with control IgG or inhibitory antibodies against PDGFR-α and PDGFR-β (Student t-test, t = 3.1, p < 0.05). Note that for technical reasons, a different primer set was used in this experiment compared to the experiment shown in Figure 4C, yielding different absolute values of expression (for details, see Materials and Methods). Error bars indicate the standard error of the mean.
Mentions: Having functionally implicated FGF-2 in the angiogenic phenotype, we sought to identify the cell type in cervical neoplasias and cancer that expressed FGF-2, by using FACS. Total RNA was extracted from the pools of cells labeled positively for either of the imatinib receptor tyrosine kinase targets, i.e., for PDGF receptor-α plus PDGF receptor-β, or for c-kit, and subjected to semiquantitative RT-PCR analysis to establish the identity of the cells. The pool of cells sorted for expression of c-kit was positive for expression of the basal squamous epithelial gene K14, but not for the mesenchymal gene vimentin, indicating that c-kit is expressed by a subset of cervical keratinocytes (Figure 5A). In contrast, the pool of cells sorted for expression of PDGF receptors contained the transcripts for the mesenchymal gene vimentin, but not K14, in accordance with earlier experiments showing the PDGF receptors were selectively expressed in CAFs and pericytes (Figures 1A and 5A). The transcript for FGF-2 was predominant in the pool sorted for expression of PDGF receptors, indicating that FGF-2 was largely produced by PDGF receptor-expressing cells (Figure 5A). Next, we employed immunostaining of tissue sections to visualize the cells expressing FGF-2 in the cervixes of HPV/E2 mice. FGF-2 was found to be predominantly expressed by stromal cells, in particular the subset of cells in proximity to the basal keratinocytes (Figure 5B). In accordance with the diminished FGF-2 mRNA levels seen following treatment with imatinib, the staining intensity for FGF-2 was reduced in tissue sections from imatinib-treated mice (Figure 5B). To ascertain which stromal cell type expressed FGF-2, we performed coimmunostaining of FGF-2 and markers for various prevalent cell types within the cervix. FGF-2 expression colocalized with expression of PDGF receptor-α and vimentin, indicating that FGF-2 is expressed by CAFs, but not with markers for endothelial cells, leukocytes, macrophages, mast cells, NK cells, or dendritic cells (Figure S4).

Bottom Line: Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts.Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects.Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Diabetes Center and Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States ofAmerica. kristian.pietras@licr.ki.se

ABSTRACT

Background: Important support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.

Methods and findings: We used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.

Conclusions: This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

Show MeSH
Related in: MedlinePlus