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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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Expression of FGF-2 Is Repressed by Imatinib in Preclinical Trials, and Treatment with FGF-Trap Impairs Angiogenesis in the Neoplastic Cervix(A) Quantitative RT-PCR analysis evaluating expression of a set of growth and/or angiogenic regulatory factors in the neoplastic cervixes of 4-mo-old HPV/E2 mice treated with imatinib for 2 wk. The results are expressed as the ratio of expression (as percentage of the ribosomal protein gene L19) of imatinib-treated mice versus vehicle-treated mice. Bar colors indicate relative expression levels during the neoplastic progression (green = down-regulated expression compared with normal estrogen-treated cervix; red = up-regulated expression compared with normal estrogen-treated cervix; and white = unchanged expression compared with normal estrogen-treated cervix).(B and C) Quantitative RT-PCR analysis of FGF-7 (Student t-test, t = 15.9, p < 0.001) (B) and FGF-2 (Student t-test, t = 13.7, p < 0.001) (C) expression in the cervixes of: estrogen-treated normal mice (N/E2); HPV/E2 mice with CIN3 lesions (3 mo) or SCC (5 mo) and HPV/E2 mice treated from 3.5 mo to 4 mo of age with vehicle or imatinib.(D) Western blot (WB) analysis of FGF-2 expression following immunoprecipitation (IP) of FGF-2 from tissue lysates of neoplastic cervixes of mice untreated or treated with imatinib for 2 wk. Two individual tissue lysates are shown for each treatment, and every lysate for each treatment group consisted of the combined cervixes from five mice. Omission of the immunoprecipitating antibody was used as a negative control (No Ab-lane), and 50 ng of recombinant mouse FGF-2 was used as a positive control. Densitometric quantification is shown normalized to lane 1.(E) Immunostaining for the mitogenic signaling receptor for FGF-2, i.e., FGF receptor-1 (FGFR-1), in CIN3 lesions of the uterine cervix from HPV/E2 mice. Expression of FGFR-1 (green) was predominantly detected in the stroma and colocalized with a marker for endothelial cells (red, CD31). The expression pattern was analyzed in at least five different mice of similar histological stage. Parameters: 200× magnification; cell nuclei/DAPI, blue; dotted line marks epithelium–stroma boundary. Similar results were seen in analysis of cervical carcinoma lesions (unpublished data). Note that the scattered punctate shapes distal from the vasculature are non–cell-associated debris derived from the secondary antibody, as revealed by evaluation at high magnification and analysis of tissue sections in which the primary antibody was omitted. E, epithelium; S, stroma.(F) Quantification of vessel density in the cervical transformation zone of HPV/E2 mice at 4 mo of age following a 2-wk treatment with imatinib or 2 wk after a single treatment with adenoviral delivery of FGF-trap or control GFP. Student t-test, t = 5.8, p < 0.001.Error bars indicate the standard error of the mean.
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pmed-0050019-g004: Expression of FGF-2 Is Repressed by Imatinib in Preclinical Trials, and Treatment with FGF-Trap Impairs Angiogenesis in the Neoplastic Cervix(A) Quantitative RT-PCR analysis evaluating expression of a set of growth and/or angiogenic regulatory factors in the neoplastic cervixes of 4-mo-old HPV/E2 mice treated with imatinib for 2 wk. The results are expressed as the ratio of expression (as percentage of the ribosomal protein gene L19) of imatinib-treated mice versus vehicle-treated mice. Bar colors indicate relative expression levels during the neoplastic progression (green = down-regulated expression compared with normal estrogen-treated cervix; red = up-regulated expression compared with normal estrogen-treated cervix; and white = unchanged expression compared with normal estrogen-treated cervix).(B and C) Quantitative RT-PCR analysis of FGF-7 (Student t-test, t = 15.9, p < 0.001) (B) and FGF-2 (Student t-test, t = 13.7, p < 0.001) (C) expression in the cervixes of: estrogen-treated normal mice (N/E2); HPV/E2 mice with CIN3 lesions (3 mo) or SCC (5 mo) and HPV/E2 mice treated from 3.5 mo to 4 mo of age with vehicle or imatinib.(D) Western blot (WB) analysis of FGF-2 expression following immunoprecipitation (IP) of FGF-2 from tissue lysates of neoplastic cervixes of mice untreated or treated with imatinib for 2 wk. Two individual tissue lysates are shown for each treatment, and every lysate for each treatment group consisted of the combined cervixes from five mice. Omission of the immunoprecipitating antibody was used as a negative control (No Ab-lane), and 50 ng of recombinant mouse FGF-2 was used as a positive control. Densitometric quantification is shown normalized to lane 1.(E) Immunostaining for the mitogenic signaling receptor for FGF-2, i.e., FGF receptor-1 (FGFR-1), in CIN3 lesions of the uterine cervix from HPV/E2 mice. Expression of FGFR-1 (green) was predominantly detected in the stroma and colocalized with a marker for endothelial cells (red, CD31). The expression pattern was analyzed in at least five different mice of similar histological stage. Parameters: 200× magnification; cell nuclei/DAPI, blue; dotted line marks epithelium–stroma boundary. Similar results were seen in analysis of cervical carcinoma lesions (unpublished data). Note that the scattered punctate shapes distal from the vasculature are non–cell-associated debris derived from the secondary antibody, as revealed by evaluation at high magnification and analysis of tissue sections in which the primary antibody was omitted. E, epithelium; S, stroma.(F) Quantification of vessel density in the cervical transformation zone of HPV/E2 mice at 4 mo of age following a 2-wk treatment with imatinib or 2 wk after a single treatment with adenoviral delivery of FGF-trap or control GFP. Student t-test, t = 5.8, p < 0.001.Error bars indicate the standard error of the mean.

Mentions: The evidently paracrine impairment, both of the cancer cell growth rate and of the angiogenic phenotype, in the neoplastic cervix following inhibition of PDGF signaling, prompted us to investigate possible changes in growth and angiogenic regulatory signals. The expression of a panel of candidate genes encoding proliferative and antiapoptotic signaling molecules, based on a literature search for factors known to be of importance for cervical cancer growth, as well as prototypical angiogenic factors, was analyzed by quantitative RT-PCR in normal mouse cervix, and in CIN3 and SCC lesions, as depicted in Figure 4A. The genes coded with white bars indicate there was no change in expression during tumor progression, whereas the red bars symbolize the genes up-regulated during tumor progression, and green bars those down-regulated during tumor progression. The data are presented in more depth in Table S1. To assess changes in gene expression effected by treatment with imatinib, cervical tissues from HPV/E2 mice treated for 2 wk with vehicle control or with imatinib were examined. The analysis revealed that expression of fibroblast growth factor (FGF) type-7 (FGF-7; keratinocyte growth factor) was elevated throughout the progression of cervical neoplasias in HPV/E2 mice (Figure 4B; Student t-test, t = 15.9, p < 0.001), and its expression was decreased by 35% in imatinib-treated versus control tumors (Figure 4A and 4B; Student t-test, t = 10.9, p < 0.001); down-regulation of FGF-7 may therefore contribute to the observed antiproliferative effect of imatinib. The small mass of the mouse cervix and a lack of reagents with high sensitivity and specificity precluded biochemical quantiation of the protein levels of FGF-7 during the course of cervical tumor progression. Conspiciously, a large proportion of human cervical SCC lesions reportedly express FGF receptor-2IIIB, the receptor for FGF-7, in contrast to normal cervical epithelium [44]. Increased expression of insulin-like growth factors −1 and −2, and hepatocyte growth factor, was observed during neoplastic progression; notably, however, no substantive alterations in the expression of these factors, or of other candidate tumor growth factors and receptors, resulted from the treatment with imatinib (Figure 4A and Table S1).


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)

Expression of FGF-2 Is Repressed by Imatinib in Preclinical Trials, and Treatment with FGF-Trap Impairs Angiogenesis in the Neoplastic Cervix(A) Quantitative RT-PCR analysis evaluating expression of a set of growth and/or angiogenic regulatory factors in the neoplastic cervixes of 4-mo-old HPV/E2 mice treated with imatinib for 2 wk. The results are expressed as the ratio of expression (as percentage of the ribosomal protein gene L19) of imatinib-treated mice versus vehicle-treated mice. Bar colors indicate relative expression levels during the neoplastic progression (green = down-regulated expression compared with normal estrogen-treated cervix; red = up-regulated expression compared with normal estrogen-treated cervix; and white = unchanged expression compared with normal estrogen-treated cervix).(B and C) Quantitative RT-PCR analysis of FGF-7 (Student t-test, t = 15.9, p < 0.001) (B) and FGF-2 (Student t-test, t = 13.7, p < 0.001) (C) expression in the cervixes of: estrogen-treated normal mice (N/E2); HPV/E2 mice with CIN3 lesions (3 mo) or SCC (5 mo) and HPV/E2 mice treated from 3.5 mo to 4 mo of age with vehicle or imatinib.(D) Western blot (WB) analysis of FGF-2 expression following immunoprecipitation (IP) of FGF-2 from tissue lysates of neoplastic cervixes of mice untreated or treated with imatinib for 2 wk. Two individual tissue lysates are shown for each treatment, and every lysate for each treatment group consisted of the combined cervixes from five mice. Omission of the immunoprecipitating antibody was used as a negative control (No Ab-lane), and 50 ng of recombinant mouse FGF-2 was used as a positive control. Densitometric quantification is shown normalized to lane 1.(E) Immunostaining for the mitogenic signaling receptor for FGF-2, i.e., FGF receptor-1 (FGFR-1), in CIN3 lesions of the uterine cervix from HPV/E2 mice. Expression of FGFR-1 (green) was predominantly detected in the stroma and colocalized with a marker for endothelial cells (red, CD31). The expression pattern was analyzed in at least five different mice of similar histological stage. Parameters: 200× magnification; cell nuclei/DAPI, blue; dotted line marks epithelium–stroma boundary. Similar results were seen in analysis of cervical carcinoma lesions (unpublished data). Note that the scattered punctate shapes distal from the vasculature are non–cell-associated debris derived from the secondary antibody, as revealed by evaluation at high magnification and analysis of tissue sections in which the primary antibody was omitted. E, epithelium; S, stroma.(F) Quantification of vessel density in the cervical transformation zone of HPV/E2 mice at 4 mo of age following a 2-wk treatment with imatinib or 2 wk after a single treatment with adenoviral delivery of FGF-trap or control GFP. Student t-test, t = 5.8, p < 0.001.Error bars indicate the standard error of the mean.
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pmed-0050019-g004: Expression of FGF-2 Is Repressed by Imatinib in Preclinical Trials, and Treatment with FGF-Trap Impairs Angiogenesis in the Neoplastic Cervix(A) Quantitative RT-PCR analysis evaluating expression of a set of growth and/or angiogenic regulatory factors in the neoplastic cervixes of 4-mo-old HPV/E2 mice treated with imatinib for 2 wk. The results are expressed as the ratio of expression (as percentage of the ribosomal protein gene L19) of imatinib-treated mice versus vehicle-treated mice. Bar colors indicate relative expression levels during the neoplastic progression (green = down-regulated expression compared with normal estrogen-treated cervix; red = up-regulated expression compared with normal estrogen-treated cervix; and white = unchanged expression compared with normal estrogen-treated cervix).(B and C) Quantitative RT-PCR analysis of FGF-7 (Student t-test, t = 15.9, p < 0.001) (B) and FGF-2 (Student t-test, t = 13.7, p < 0.001) (C) expression in the cervixes of: estrogen-treated normal mice (N/E2); HPV/E2 mice with CIN3 lesions (3 mo) or SCC (5 mo) and HPV/E2 mice treated from 3.5 mo to 4 mo of age with vehicle or imatinib.(D) Western blot (WB) analysis of FGF-2 expression following immunoprecipitation (IP) of FGF-2 from tissue lysates of neoplastic cervixes of mice untreated or treated with imatinib for 2 wk. Two individual tissue lysates are shown for each treatment, and every lysate for each treatment group consisted of the combined cervixes from five mice. Omission of the immunoprecipitating antibody was used as a negative control (No Ab-lane), and 50 ng of recombinant mouse FGF-2 was used as a positive control. Densitometric quantification is shown normalized to lane 1.(E) Immunostaining for the mitogenic signaling receptor for FGF-2, i.e., FGF receptor-1 (FGFR-1), in CIN3 lesions of the uterine cervix from HPV/E2 mice. Expression of FGFR-1 (green) was predominantly detected in the stroma and colocalized with a marker for endothelial cells (red, CD31). The expression pattern was analyzed in at least five different mice of similar histological stage. Parameters: 200× magnification; cell nuclei/DAPI, blue; dotted line marks epithelium–stroma boundary. Similar results were seen in analysis of cervical carcinoma lesions (unpublished data). Note that the scattered punctate shapes distal from the vasculature are non–cell-associated debris derived from the secondary antibody, as revealed by evaluation at high magnification and analysis of tissue sections in which the primary antibody was omitted. E, epithelium; S, stroma.(F) Quantification of vessel density in the cervical transformation zone of HPV/E2 mice at 4 mo of age following a 2-wk treatment with imatinib or 2 wk after a single treatment with adenoviral delivery of FGF-trap or control GFP. Student t-test, t = 5.8, p < 0.001.Error bars indicate the standard error of the mean.
Mentions: The evidently paracrine impairment, both of the cancer cell growth rate and of the angiogenic phenotype, in the neoplastic cervix following inhibition of PDGF signaling, prompted us to investigate possible changes in growth and angiogenic regulatory signals. The expression of a panel of candidate genes encoding proliferative and antiapoptotic signaling molecules, based on a literature search for factors known to be of importance for cervical cancer growth, as well as prototypical angiogenic factors, was analyzed by quantitative RT-PCR in normal mouse cervix, and in CIN3 and SCC lesions, as depicted in Figure 4A. The genes coded with white bars indicate there was no change in expression during tumor progression, whereas the red bars symbolize the genes up-regulated during tumor progression, and green bars those down-regulated during tumor progression. The data are presented in more depth in Table S1. To assess changes in gene expression effected by treatment with imatinib, cervical tissues from HPV/E2 mice treated for 2 wk with vehicle control or with imatinib were examined. The analysis revealed that expression of fibroblast growth factor (FGF) type-7 (FGF-7; keratinocyte growth factor) was elevated throughout the progression of cervical neoplasias in HPV/E2 mice (Figure 4B; Student t-test, t = 15.9, p < 0.001), and its expression was decreased by 35% in imatinib-treated versus control tumors (Figure 4A and 4B; Student t-test, t = 10.9, p < 0.001); down-regulation of FGF-7 may therefore contribute to the observed antiproliferative effect of imatinib. The small mass of the mouse cervix and a lack of reagents with high sensitivity and specificity precluded biochemical quantiation of the protein levels of FGF-7 during the course of cervical tumor progression. Conspiciously, a large proportion of human cervical SCC lesions reportedly express FGF receptor-2IIIB, the receptor for FGF-7, in contrast to normal cervical epithelium [44]. Increased expression of insulin-like growth factors −1 and −2, and hepatocyte growth factor, was observed during neoplastic progression; notably, however, no substantive alterations in the expression of these factors, or of other candidate tumor growth factors and receptors, resulted from the treatment with imatinib (Figure 4A and Table S1).

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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Related in: MedlinePlus