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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 the PDGF Receptors during Cervical CarcinogenesisThe cervixes of estrogen-treated normal mice (N/E2), HPV/E2 mice with CIN3 lesions (at 3 mo of age), or with cervical squamous cell carcinomas, SCC (at 5 mo of age) were compared. Expression patterns were analyzed in at least five different mice of the same genotype or similar histological stage. Dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.(A) Immunostaining of PDGFR-α and -β (200× magnification; PDGFR, red; cell nuclei/DAPI, blue). Immunostaining of PDGF-CC (400× magnification).(B) Coexpression of PDGF receptors and the mesenchymal cell marker vimentin in stromal fibroblasts of the transformation zone of HPV/E2 mice (200× magnification; PDGF-α receptors, red; vimentin, green; merge; cell nuclei/DAPI, blue).(C) Coexpression of PDGFR-β and the pericyte marker NG2 in the cervical stroma of the transformation zone of HPV/E2 mice (400× magnification; PDGFR-β, red; NG2, green; cell nuclei/DAPI, blue).(D and E) Quantitative PCR analysis of the expression of PDGF receptors (D) and ligands (E) in the cervixes of estrogen-treated normal or HPV mice with progressing tumor development. Error bars indicate the standard error of the mean.
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pmed-0050019-g001: Expression of the PDGF Receptors during Cervical CarcinogenesisThe cervixes of estrogen-treated normal mice (N/E2), HPV/E2 mice with CIN3 lesions (at 3 mo of age), or with cervical squamous cell carcinomas, SCC (at 5 mo of age) were compared. Expression patterns were analyzed in at least five different mice of the same genotype or similar histological stage. Dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.(A) Immunostaining of PDGFR-α and -β (200× magnification; PDGFR, red; cell nuclei/DAPI, blue). Immunostaining of PDGF-CC (400× magnification).(B) Coexpression of PDGF receptors and the mesenchymal cell marker vimentin in stromal fibroblasts of the transformation zone of HPV/E2 mice (200× magnification; PDGF-α receptors, red; vimentin, green; merge; cell nuclei/DAPI, blue).(C) Coexpression of PDGFR-β and the pericyte marker NG2 in the cervical stroma of the transformation zone of HPV/E2 mice (400× magnification; PDGFR-β, red; NG2, green; cell nuclei/DAPI, blue).(D and E) Quantitative PCR analysis of the expression of PDGF receptors (D) and ligands (E) in the cervixes of estrogen-treated normal or HPV mice with progressing tumor development. Error bars indicate the standard error of the mean.

Mentions: The transformation zone between squamous and columnar epithelium of the uterine cervix is implicated as the site of origin of the human cancer [34]. Similarly, in K14-HPV16 female transgenic mice whose estrogen levels are maintained by time-release implants (HPV/E2 mice), incipient neoplasias first appear in the transformation zone, arising out of the HPV-16 oncogene-expressing squamous epithelium; the progressive neoplastic lesions are associated with an aberrant (“reactive”) stroma [28]. Motivated by the well-established association of PDGF signaling with regulation of fibroblast phenotypes and by our previous observations that vascular pericytes in tumors are dependent on PDGF signaling [35–37], we investigated the expression of PDGF ligands and receptors during cancer progression in HPV/E2 mice. In normal, estrogen-treated female mice (N/E2 mice), as well as in HPV/E2 mice, both PDGF receptor-α and -β were expressed by cells populating the stroma of the cervical transformation zone, as revealed by immunostaining (Figure 1A). The expression of PDGF receptors persisted in the apparently denser stroma of CIN3 and SCC lesions (Figure 1A). Immunostaining revealed that stromal cells expressing both PDGF α- and β-receptors coexpress the mesenchymal cell marker vimentin, demonstrating together with morphological (spindle-shaped cells with indented nuclei) and histological (patterns of tissue localization ) criteria that these cells are fibroblasts (Figure 1B and unpublished data). Additionally, PDGF β-receptor was also expressed by pericytes, as indicated by costaining with the mural cell marker NG2 (Figure 1C). Quantitative PCR analysis revealed that the cervical expression of both PDGF α- and β-receptors was increased concomitant with neoplastic progression; the cervix of HPV/E2 mice with SCC displayed a 2.7-fold and 1.6-fold increase in expression of PDGF α- and β-receptor, respectively, compared to N/E2 mice (Figure 1D). Expression of vimentin was found to be similarly increased (Figure 1D), indicating that the elevated expression of PDGF receptors in the tissue largely results from an increased cellularity of the stroma.


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 the PDGF Receptors during Cervical CarcinogenesisThe cervixes of estrogen-treated normal mice (N/E2), HPV/E2 mice with CIN3 lesions (at 3 mo of age), or with cervical squamous cell carcinomas, SCC (at 5 mo of age) were compared. Expression patterns were analyzed in at least five different mice of the same genotype or similar histological stage. Dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.(A) Immunostaining of PDGFR-α and -β (200× magnification; PDGFR, red; cell nuclei/DAPI, blue). Immunostaining of PDGF-CC (400× magnification).(B) Coexpression of PDGF receptors and the mesenchymal cell marker vimentin in stromal fibroblasts of the transformation zone of HPV/E2 mice (200× magnification; PDGF-α receptors, red; vimentin, green; merge; cell nuclei/DAPI, blue).(C) Coexpression of PDGFR-β and the pericyte marker NG2 in the cervical stroma of the transformation zone of HPV/E2 mice (400× magnification; PDGFR-β, red; NG2, green; cell nuclei/DAPI, blue).(D and E) Quantitative PCR analysis of the expression of PDGF receptors (D) and ligands (E) in the cervixes of estrogen-treated normal or HPV mice with progressing tumor development. Error bars indicate the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2214790&req=5

pmed-0050019-g001: Expression of the PDGF Receptors during Cervical CarcinogenesisThe cervixes of estrogen-treated normal mice (N/E2), HPV/E2 mice with CIN3 lesions (at 3 mo of age), or with cervical squamous cell carcinomas, SCC (at 5 mo of age) were compared. Expression patterns were analyzed in at least five different mice of the same genotype or similar histological stage. Dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.(A) Immunostaining of PDGFR-α and -β (200× magnification; PDGFR, red; cell nuclei/DAPI, blue). Immunostaining of PDGF-CC (400× magnification).(B) Coexpression of PDGF receptors and the mesenchymal cell marker vimentin in stromal fibroblasts of the transformation zone of HPV/E2 mice (200× magnification; PDGF-α receptors, red; vimentin, green; merge; cell nuclei/DAPI, blue).(C) Coexpression of PDGFR-β and the pericyte marker NG2 in the cervical stroma of the transformation zone of HPV/E2 mice (400× magnification; PDGFR-β, red; NG2, green; cell nuclei/DAPI, blue).(D and E) Quantitative PCR analysis of the expression of PDGF receptors (D) and ligands (E) in the cervixes of estrogen-treated normal or HPV mice with progressing tumor development. Error bars indicate the standard error of the mean.
Mentions: The transformation zone between squamous and columnar epithelium of the uterine cervix is implicated as the site of origin of the human cancer [34]. Similarly, in K14-HPV16 female transgenic mice whose estrogen levels are maintained by time-release implants (HPV/E2 mice), incipient neoplasias first appear in the transformation zone, arising out of the HPV-16 oncogene-expressing squamous epithelium; the progressive neoplastic lesions are associated with an aberrant (“reactive”) stroma [28]. Motivated by the well-established association of PDGF signaling with regulation of fibroblast phenotypes and by our previous observations that vascular pericytes in tumors are dependent on PDGF signaling [35–37], we investigated the expression of PDGF ligands and receptors during cancer progression in HPV/E2 mice. In normal, estrogen-treated female mice (N/E2 mice), as well as in HPV/E2 mice, both PDGF receptor-α and -β were expressed by cells populating the stroma of the cervical transformation zone, as revealed by immunostaining (Figure 1A). The expression of PDGF receptors persisted in the apparently denser stroma of CIN3 and SCC lesions (Figure 1A). Immunostaining revealed that stromal cells expressing both PDGF α- and β-receptors coexpress the mesenchymal cell marker vimentin, demonstrating together with morphological (spindle-shaped cells with indented nuclei) and histological (patterns of tissue localization ) criteria that these cells are fibroblasts (Figure 1B and unpublished data). Additionally, PDGF β-receptor was also expressed by pericytes, as indicated by costaining with the mural cell marker NG2 (Figure 1C). Quantitative PCR analysis revealed that the cervical expression of both PDGF α- and β-receptors was increased concomitant with neoplastic progression; the cervix of HPV/E2 mice with SCC displayed a 2.7-fold and 1.6-fold increase in expression of PDGF α- and β-receptor, respectively, compared to N/E2 mice (Figure 1D). Expression of vimentin was found to be similarly increased (Figure 1D), indicating that the elevated expression of PDGF receptors in the tissue largely results from an increased cellularity of the stroma.

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