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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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PDGF Receptors and FGF-2 Are Expressed in Human Normal Cervix and in Cervical SCCExpression of PDGF receptors and FGF-2 was assessed in human hysterectomy samples and in human cervical SCC lesions by immunohistochemistry. Representative pictures from analysis of a total of three separate human normal cervixes and SCC lesions are shown. PDGF receptor-α was exclusively expressed by stromal cells underlying the epithelium, whereas PDGF receptor-β was expressed by stromal cells and pericytes. Similarly, FGF-2 was expressed by stromal cells. Although qualitative, the staining intensity of FGF-2 is clearly lower in all of the normal human samples than in comparable analyses of neoplastic human cervixes, and consistent with the results from the mouse (Figures 4C and 5B, and unpublished data), where FGF-2 is expressed in the normal cervical stroma at low levels and clearly up-regulated in the neoplastic cervix. As in the mouse model, PDGF-CC was predominantly expressed by the cervical epithelium. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.
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pmed-0050019-g006: PDGF Receptors and FGF-2 Are Expressed in Human Normal Cervix and in Cervical SCCExpression of PDGF receptors and FGF-2 was assessed in human hysterectomy samples and in human cervical SCC lesions by immunohistochemistry. Representative pictures from analysis of a total of three separate human normal cervixes and SCC lesions are shown. PDGF receptor-α was exclusively expressed by stromal cells underlying the epithelium, whereas PDGF receptor-β was expressed by stromal cells and pericytes. Similarly, FGF-2 was expressed by stromal cells. Although qualitative, the staining intensity of FGF-2 is clearly lower in all of the normal human samples than in comparable analyses of neoplastic human cervixes, and consistent with the results from the mouse (Figures 4C and 5B, and unpublished data), where FGF-2 is expressed in the normal cervical stroma at low levels and clearly up-regulated in the neoplastic cervix. As in the mouse model, PDGF-CC was predominantly expressed by the cervical epithelium. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.

Mentions: FGF-2 and its mRNA are reportedly elevated in cervical cancers in humans [26,45,46]. Moreover, a study of human cervical carcinomas employing in situ hybridization previously reported that FGF-2 was expressed by stromal fibroblasts [47]. Extending upon these studies, we performed immunohistochemical staining of high-grade dysplastic lesions (HSIL/CIN3) or carcinomas of the human cervix, revealing prominent expression of FGF-2 in stromal cells (Figure 6; and unpublished data). Parallel analysis of the expression pattern of PDGF receptors in human cervical lesions revealed an expression pattern in the stroma similar to that of FGF-2 (Figure 6). Similar to the HPV/E2 mice, PDGF-CC was abundantly expressed by the neoplastic epithelium, consistent with a paracrine signaling circuit operating between the tumor cells and CAFs (Figure 6). Additionally, we analyzed normal human cervical samples derived from hysterectomies. Much as in the normal mouse cervix, the expression of the PDGF receptors was detectable in stromal fibroblasts and pericytes, and PDGF-CC was expressed by the epithelial compartment (Figure 6). Recognizing the qualitative nature of the analysis of archival human samples, the expression of both PDGF receptors and FGF-2 appears to be lower in normal versus cancerous stromal fibroblasts, consistent with the clear evidence in the mouse that PDGF signaling up-regulates FGF-2 in the neoplastic and cancerous stroma. Independent validation of our immunohistochemical analyses of human cervical cancer specimens comes from recently published data on the Web site of the Human Protein Atlas project [48] (http://www.proteinatlas.org/) (Figure S7). Notably, FGF-2, PDGF receptor-α, and PDGF receptor-β were found to be expressed in the stroma of 9/11, 12/12, and 9/9 cervical cancers, respectively, whereas expression in the overt squamous cancer cells was not consistently detected, the exception being a single case that expressed PDGF receptor-β (Figure S7 and Table S2). Collectively, these results and conclusions contradict a recent study of human cervical cancer cell lines and biopsy specimens, based solely on immunohistochemical analysis using antibodies with poorly characterized specificity. That report concluded that the PDGF receptors were expressed in a series of cultured human cervical cancer cell lines and in the tumor cell compartment of approximately half of the human cervical cancer specimens analyzed [49]. In contrast, we have been unable to detect expression of either PDGF receptor by RT-PCR or western blot analysis using validated antibodies, nor growth inhibition by imatinib, during careful analysis of three of the cervical cancer cell lines used in this study [49] (Figure S8). Moreover, and consistent with our analyses, meta-analysis of the gene expression profile of human cervical cancer cell lines in the National Center for Biotechnology Information (NCBI) GEO database revealed no detectable transcripts for either of the PDGF receptors in three separate studies of HeLa cells (unpublished data), one of the cell lines stated to express PDGFR in the report in question [49]. Our data, as well as that from the Human Protein Atlas project, from analysis of human cervical cancer cell lines and human tissues are consistent with the more definitive analysis in the mouse, indicating that PDGF receptors are predominantly expressed by stromal fibroblasts and pericytes. We leave open the possibility that certain human cervical carcinomas, for example ones that have progressed from the common squamous cell carcinoma state through an epithelial-mesenchymal transition (EMT) to a spindle cell stage, might express the PDGF receptors, a possibility that deserves future analysis with well-validated reagents. Collectively, the data suggest commonality between our genetically engineered mouse model and the prevalent form of human cervical cancer, and encourage the possibility that our findings have translational relevance to mechanisms and therapeutic interventions in human cervical neoplasia and cancer.


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)

PDGF Receptors and FGF-2 Are Expressed in Human Normal Cervix and in Cervical SCCExpression of PDGF receptors and FGF-2 was assessed in human hysterectomy samples and in human cervical SCC lesions by immunohistochemistry. Representative pictures from analysis of a total of three separate human normal cervixes and SCC lesions are shown. PDGF receptor-α was exclusively expressed by stromal cells underlying the epithelium, whereas PDGF receptor-β was expressed by stromal cells and pericytes. Similarly, FGF-2 was expressed by stromal cells. Although qualitative, the staining intensity of FGF-2 is clearly lower in all of the normal human samples than in comparable analyses of neoplastic human cervixes, and consistent with the results from the mouse (Figures 4C and 5B, and unpublished data), where FGF-2 is expressed in the normal cervical stroma at low levels and clearly up-regulated in the neoplastic cervix. As in the mouse model, PDGF-CC was predominantly expressed by the cervical epithelium. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.
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getmorefigures.php?uid=PMC2214790&req=5

pmed-0050019-g006: PDGF Receptors and FGF-2 Are Expressed in Human Normal Cervix and in Cervical SCCExpression of PDGF receptors and FGF-2 was assessed in human hysterectomy samples and in human cervical SCC lesions by immunohistochemistry. Representative pictures from analysis of a total of three separate human normal cervixes and SCC lesions are shown. PDGF receptor-α was exclusively expressed by stromal cells underlying the epithelium, whereas PDGF receptor-β was expressed by stromal cells and pericytes. Similarly, FGF-2 was expressed by stromal cells. Although qualitative, the staining intensity of FGF-2 is clearly lower in all of the normal human samples than in comparable analyses of neoplastic human cervixes, and consistent with the results from the mouse (Figures 4C and 5B, and unpublished data), where FGF-2 is expressed in the normal cervical stroma at low levels and clearly up-regulated in the neoplastic cervix. As in the mouse model, PDGF-CC was predominantly expressed by the cervical epithelium. Parameters: 400× magnification; dotted line marks epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.
Mentions: FGF-2 and its mRNA are reportedly elevated in cervical cancers in humans [26,45,46]. Moreover, a study of human cervical carcinomas employing in situ hybridization previously reported that FGF-2 was expressed by stromal fibroblasts [47]. Extending upon these studies, we performed immunohistochemical staining of high-grade dysplastic lesions (HSIL/CIN3) or carcinomas of the human cervix, revealing prominent expression of FGF-2 in stromal cells (Figure 6; and unpublished data). Parallel analysis of the expression pattern of PDGF receptors in human cervical lesions revealed an expression pattern in the stroma similar to that of FGF-2 (Figure 6). Similar to the HPV/E2 mice, PDGF-CC was abundantly expressed by the neoplastic epithelium, consistent with a paracrine signaling circuit operating between the tumor cells and CAFs (Figure 6). Additionally, we analyzed normal human cervical samples derived from hysterectomies. Much as in the normal mouse cervix, the expression of the PDGF receptors was detectable in stromal fibroblasts and pericytes, and PDGF-CC was expressed by the epithelial compartment (Figure 6). Recognizing the qualitative nature of the analysis of archival human samples, the expression of both PDGF receptors and FGF-2 appears to be lower in normal versus cancerous stromal fibroblasts, consistent with the clear evidence in the mouse that PDGF signaling up-regulates FGF-2 in the neoplastic and cancerous stroma. Independent validation of our immunohistochemical analyses of human cervical cancer specimens comes from recently published data on the Web site of the Human Protein Atlas project [48] (http://www.proteinatlas.org/) (Figure S7). Notably, FGF-2, PDGF receptor-α, and PDGF receptor-β were found to be expressed in the stroma of 9/11, 12/12, and 9/9 cervical cancers, respectively, whereas expression in the overt squamous cancer cells was not consistently detected, the exception being a single case that expressed PDGF receptor-β (Figure S7 and Table S2). Collectively, these results and conclusions contradict a recent study of human cervical cancer cell lines and biopsy specimens, based solely on immunohistochemical analysis using antibodies with poorly characterized specificity. That report concluded that the PDGF receptors were expressed in a series of cultured human cervical cancer cell lines and in the tumor cell compartment of approximately half of the human cervical cancer specimens analyzed [49]. In contrast, we have been unable to detect expression of either PDGF receptor by RT-PCR or western blot analysis using validated antibodies, nor growth inhibition by imatinib, during careful analysis of three of the cervical cancer cell lines used in this study [49] (Figure S8). Moreover, and consistent with our analyses, meta-analysis of the gene expression profile of human cervical cancer cell lines in the National Center for Biotechnology Information (NCBI) GEO database revealed no detectable transcripts for either of the PDGF receptors in three separate studies of HeLa cells (unpublished data), one of the cell lines stated to express PDGFR in the report in question [49]. Our data, as well as that from the Human Protein Atlas project, from analysis of human cervical cancer cell lines and human tissues are consistent with the more definitive analysis in the mouse, indicating that PDGF receptors are predominantly expressed by stromal fibroblasts and pericytes. We leave open the possibility that certain human cervical carcinomas, for example ones that have progressed from the common squamous cell carcinoma state through an epithelial-mesenchymal transition (EMT) to a spindle cell stage, might express the PDGF receptors, a possibility that deserves future analysis with well-validated reagents. Collectively, the data suggest commonality between our genetically engineered mouse model and the prevalent form of human cervical cancer, and encourage the possibility that our findings have translational relevance to mechanisms and therapeutic interventions in human cervical neoplasia and cancer.

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