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Commentary on Prostatic Neovascularization and Vascular Endothelial Growth Factor
00225347/97/1576-23.W0
Vol. 157,2040-2041, June 1997 Printed in U S A .
lME JOURNAL OF URoLoov
Copyright 8 1997 by AMERICAN U~ouxjlcALik?sc€IATION, bc.
This Month in Investigative Urology COMMENTARY ON PROSTATIC NEOVASCULARIZATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR Over the years i t has become clear from the clinical management of prostate cancer (PCa) that this disease kills patients when tumor cells progress to a stage where they grow rapidly in a n androgen-free environment. At this time, the cell's ability to differentiate has been greatly altered and some cells have gained access to extra-prostatic sites (mainly in bones), forming metastases. Several interrelated and/or independent processes must be up- or down-regulated to generate such poorly differentiated and invasive PCa cells. One of them is the formation of a capillary neovascularization system, believed to be dependent on angiogenic factors originating from the cancer cells. New blood vessels are essential to cellular survival in solid tumors, either at the primary or distant sites. Similarly to other malignancies, it has been shown that angiogenesis occurs in PCa. At first, vascular density was assessed by staining endothelial cells in prostate tissue sections with antibodies specific to factor VIII-related antigen. When normal prostate, nodules in benign prostatic hyperplasia (BPH), pre-malignant lesions (prostatic intraepithelial neoplasia or PIN), and PCa were examined, an altered pattern and a progressively increasing number of microvessels was observed. Adjacent to normal epithelium, the vascular network appears orderly, as opposed to cancer where microvessels appear more randomly distributed. I t was proposed that neovascularization begins in BPH and that it keeps progressing in a stepwise fashion in the subsequent proliferative pre-malignant and malignant stages involving epithelial cell growth. For instance, a 2-fold increase in vascular density was found between BPH and PCa. Furthermore, in PCa the number of microvessels is statistically different between clinically localized low vs high score disease, reaching, in the latter instance, levels similar to those observed in PCa with bone metastases. There is also a general agreement that increased microvessel density is predominantly associated with poorly differentiated tumors and that it correlates with invasive properties, risk of metastasis, and poor clinical outcome. Vascular density is thus seen as a valuable indicator of PCa progression. The question of whether it can be used as an independent prognostic factor remains open. More recently, as reported by two independent teams in this issue of Investigative Urology (Ferrer et al. and Jackson et al.), attempts were made to verify if the recently discovered specific mitogen for endothelial cells, the vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), was expressed in the prostate, and if i t was more specifically related to PCa. Although, the number of reports on this subject is still limited, some consensus was reached on the VEGF immunostaining in different cell types (PCa, endothelial, stromal) in prostate tissue sections. Complementary studies (in situ hybridization) may be needed to ascertain the cell types producing VEGF, but data from VEGF amplification by polymerase chain reaction using RNA from PCa tissues (Jackson et al.), coupled to VEGF expression by cultured human PCa cell lines (Ferrer et al., and Harper et al.1) support the concept that it is synthesized by PCa cells. Because of the specificity of action of VEGF, its detection on endothelial cells is believed to represent binding to surface receptors. With respect to the stromal VEGF immunoreactivity, it is consistent with the following possibilities; first that VEGF, a heparin-binding GF, binds to extracellular matrix proteins, and second, that infiltrating lymphocytes are a source of VEGF. Lastly, the presence of VEGF in non-malignant glandular epithelial cells is controversial (cf. the two articles in this issue), while previous reports agreed on such VEGF distribution.1.2 Therefore, the constitutive expression of VEGF in normal prostate cells suggests that under physiological conditions, it has an important role to play in the regulation of blood vessel function. Even though VEGF might be contributing to PCa, since tumor cells apparently stain more intensely than normal glandular cells, one cannot ascertain that the VEGF originating from PCa cells does so, given the limitations of the currently available methods. Interestingly, Jackson et al. mentioned the presence of cancer cell nests, ranging from negative to strongly positive VEGF reactivity. They also provided preliminary data on the presence of high molecular weight VEGF species, in addition to the known variants (34-46 kDa); yet there was no difference between BPH and PCa. Would VEGF expression then correlate, like the microvessel density, with the degree of tumor differentiation? Ferrer et al. addressed this question and unexpectedly, found more VEGF in well differentiated cancer cells, while Harper et al.1 rather suggested an increasing VEGF expression along with dedifferentiation. The latter observation is consistent with data obtained in PCa cell lines exhibiting different phenotypes, that is, more VEGF in poor vs. well differentiated cells. It is worth mentioning that among epithelial cell types, the neuroendocrine (NE) cells, sparsely distributed within normal glandular structures and carcinomas, highly contribute to the production of VEGF.' This finding is of significance considering that the presence of NE cells has already been associated with a poor prognosis.:$,.'The difficulty to specifically identify NE cells in tissue sections and assess their specific contribution to the VEGF "pool" may partly explain the apparent lack of correlation between VEGF and neovascularization in PCa. It is conceivable that the VEGF produced by NE cells in prostate tumors would act as a paracrine GF on endothelial cells and induce the formation of new blood vessels, which in turn favor further PCa cell growth and spreading. This is likely to require the turning on of a switch (set point t where effects of other stirnulatory c?-tclliines/nngio,nenic (;Fs :Ire ~11 cwxrts balanced with that of inhibitory signals. It is also possible that VEGF. oftcw descrihrtl ;IS n i ~ l ~ I l t i f ' ~ i n c t i o ncytokine. other regulatory functions. Collectively, the overall findings from expression studies are likely to reflect all sites where VEGF is present, irrespectiwly of where it is potentially active (endothelial cells) and whether it is being synthesized, secreted (epithelial and stromal cells, lymphocytes t , or sequestrated stroma I . Consequently, studies specifically directed on the VEGF receptor subtypes expressed in endothelial cells are needed to truly establish its contribution in neovascularization, and its association with progression of PCa. 2040
COMMENTARY ON PROSTATIC NEOVASCULARIZATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR 2041
Finally, considering the widespread distribution of VEGF in diverse cellular constituente of the prostate, it is premature to conclude on a specific role in PCa. However, direct approaches in animal models bearing different types of tumors proved beyond any doubt the crucial role of this GF in neovascularization and tumor progression. Among others are molecular strategies targeting the VEGF protein itself or its receptors, whereby full length or anti-sense cDNA are tranafected into cancer cells, and yield increased and decreased vascularity and tumor growth, respectively. Also, VEGF neutralizing antibodies were shown to be efficient in perturbing the effect of VEGF on tumor formation. These observations open novel research avenues on VEGF and on proteins involved in the VEGF signaling pathways, which may become useful diagnostic4 prognostic markers and serve as a basis for the development of antiangiogenic strategies in view of therapeutic interventions in PCa.
Simone Chevalier, Ph.D., Associate Professor, Dept. Surgery (Urology Division), McGill University Scientijk Director, Urologic Oncology Research Group, The Montreal General Hospital Research Institute, Montreal REFERENCES
1. Harper, M. E., Glynnejones, E., Goddard, L., Thurston,V. J. and Griffiths, K: Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells. Br. J. Cancer, 7 4 910, 1996. 2. Brown, L. F., Yeo, K. T., Berse, B., Morgentaler, A, Dvorak, H. F. and Rosen, S.: Vascular permeability factor (vascular endothelid
growth factor) is strongly expressed in the normal male genital tract and is present in substantial quantities in emen. J. Urol., 184: 576, 1995.
3. Aprikian, A. G., Cordon-Cardo, C., Fair, W. R., Zhang, Z.-F., Bazinet, M., Hamdy, S. M.and Reuter, V. E.: Neuroendoerine differentiation in metastatic prostatic adencarcinoma. J. Urol., 161:914,1994. 4. Weinstein, M. H., Partin, A. W., Veltri, R. W. and Epstein, J. L.: Neuroendoerine differentiation in prostate cancer: enhanced prediction of progression after radical prostatectomy. Hum. Pathol., 27: 683, 1996.
Vol. 157,2040-2041, June 1997 Printed in U S A .
lME JOURNAL OF URoLoov
Copyright 8 1997 by AMERICAN U~ouxjlcALik?sc€IATION, bc.
This Month in Investigative Urology COMMENTARY ON PROSTATIC NEOVASCULARIZATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR Over the years i t has become clear from the clinical management of prostate cancer (PCa) that this disease kills patients when tumor cells progress to a stage where they grow rapidly in a n androgen-free environment. At this time, the cell's ability to differentiate has been greatly altered and some cells have gained access to extra-prostatic sites (mainly in bones), forming metastases. Several interrelated and/or independent processes must be up- or down-regulated to generate such poorly differentiated and invasive PCa cells. One of them is the formation of a capillary neovascularization system, believed to be dependent on angiogenic factors originating from the cancer cells. New blood vessels are essential to cellular survival in solid tumors, either at the primary or distant sites. Similarly to other malignancies, it has been shown that angiogenesis occurs in PCa. At first, vascular density was assessed by staining endothelial cells in prostate tissue sections with antibodies specific to factor VIII-related antigen. When normal prostate, nodules in benign prostatic hyperplasia (BPH), pre-malignant lesions (prostatic intraepithelial neoplasia or PIN), and PCa were examined, an altered pattern and a progressively increasing number of microvessels was observed. Adjacent to normal epithelium, the vascular network appears orderly, as opposed to cancer where microvessels appear more randomly distributed. I t was proposed that neovascularization begins in BPH and that it keeps progressing in a stepwise fashion in the subsequent proliferative pre-malignant and malignant stages involving epithelial cell growth. For instance, a 2-fold increase in vascular density was found between BPH and PCa. Furthermore, in PCa the number of microvessels is statistically different between clinically localized low vs high score disease, reaching, in the latter instance, levels similar to those observed in PCa with bone metastases. There is also a general agreement that increased microvessel density is predominantly associated with poorly differentiated tumors and that it correlates with invasive properties, risk of metastasis, and poor clinical outcome. Vascular density is thus seen as a valuable indicator of PCa progression. The question of whether it can be used as an independent prognostic factor remains open. More recently, as reported by two independent teams in this issue of Investigative Urology (Ferrer et al. and Jackson et al.), attempts were made to verify if the recently discovered specific mitogen for endothelial cells, the vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), was expressed in the prostate, and if i t was more specifically related to PCa. Although, the number of reports on this subject is still limited, some consensus was reached on the VEGF immunostaining in different cell types (PCa, endothelial, stromal) in prostate tissue sections. Complementary studies (in situ hybridization) may be needed to ascertain the cell types producing VEGF, but data from VEGF amplification by polymerase chain reaction using RNA from PCa tissues (Jackson et al.), coupled to VEGF expression by cultured human PCa cell lines (Ferrer et al., and Harper et al.1) support the concept that it is synthesized by PCa cells. Because of the specificity of action of VEGF, its detection on endothelial cells is believed to represent binding to surface receptors. With respect to the stromal VEGF immunoreactivity, it is consistent with the following possibilities; first that VEGF, a heparin-binding GF, binds to extracellular matrix proteins, and second, that infiltrating lymphocytes are a source of VEGF. Lastly, the presence of VEGF in non-malignant glandular epithelial cells is controversial (cf. the two articles in this issue), while previous reports agreed on such VEGF distribution.1.2 Therefore, the constitutive expression of VEGF in normal prostate cells suggests that under physiological conditions, it has an important role to play in the regulation of blood vessel function. Even though VEGF might be contributing to PCa, since tumor cells apparently stain more intensely than normal glandular cells, one cannot ascertain that the VEGF originating from PCa cells does so, given the limitations of the currently available methods. Interestingly, Jackson et al. mentioned the presence of cancer cell nests, ranging from negative to strongly positive VEGF reactivity. They also provided preliminary data on the presence of high molecular weight VEGF species, in addition to the known variants (34-46 kDa); yet there was no difference between BPH and PCa. Would VEGF expression then correlate, like the microvessel density, with the degree of tumor differentiation? Ferrer et al. addressed this question and unexpectedly, found more VEGF in well differentiated cancer cells, while Harper et al.1 rather suggested an increasing VEGF expression along with dedifferentiation. The latter observation is consistent with data obtained in PCa cell lines exhibiting different phenotypes, that is, more VEGF in poor vs. well differentiated cells. It is worth mentioning that among epithelial cell types, the neuroendocrine (NE) cells, sparsely distributed within normal glandular structures and carcinomas, highly contribute to the production of VEGF.' This finding is of significance considering that the presence of NE cells has already been associated with a poor prognosis.:$,.'The difficulty to specifically identify NE cells in tissue sections and assess their specific contribution to the VEGF "pool" may partly explain the apparent lack of correlation between VEGF and neovascularization in PCa. It is conceivable that the VEGF produced by NE cells in prostate tumors would act as a paracrine GF on endothelial cells and induce the formation of new blood vessels, which in turn favor further PCa cell growth and spreading. This is likely to require the turning on of a switch (set point t where effects of other stirnulatory c?-tclliines/nngio,nenic (;Fs :Ire ~11 cwxrts balanced with that of inhibitory signals. It is also possible that VEGF. oftcw descrihrtl ;IS n i ~ l ~ I l t i f ' ~ i n c t i o ncytokine. other regulatory functions. Collectively, the overall findings from expression studies are likely to reflect all sites where VEGF is present, irrespectiwly of where it is potentially active (endothelial cells) and whether it is being synthesized, secreted (epithelial and stromal cells, lymphocytes t , or sequestrated stroma I . Consequently, studies specifically directed on the VEGF receptor subtypes expressed in endothelial cells are needed to truly establish its contribution in neovascularization, and its association with progression of PCa. 2040
COMMENTARY ON PROSTATIC NEOVASCULARIZATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR 2041
Finally, considering the widespread distribution of VEGF in diverse cellular constituente of the prostate, it is premature to conclude on a specific role in PCa. However, direct approaches in animal models bearing different types of tumors proved beyond any doubt the crucial role of this GF in neovascularization and tumor progression. Among others are molecular strategies targeting the VEGF protein itself or its receptors, whereby full length or anti-sense cDNA are tranafected into cancer cells, and yield increased and decreased vascularity and tumor growth, respectively. Also, VEGF neutralizing antibodies were shown to be efficient in perturbing the effect of VEGF on tumor formation. These observations open novel research avenues on VEGF and on proteins involved in the VEGF signaling pathways, which may become useful diagnostic4 prognostic markers and serve as a basis for the development of antiangiogenic strategies in view of therapeutic interventions in PCa.
Simone Chevalier, Ph.D., Associate Professor, Dept. Surgery (Urology Division), McGill University Scientijk Director, Urologic Oncology Research Group, The Montreal General Hospital Research Institute, Montreal REFERENCES
1. Harper, M. E., Glynnejones, E., Goddard, L., Thurston,V. J. and Griffiths, K: Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells. Br. J. Cancer, 7 4 910, 1996. 2. Brown, L. F., Yeo, K. T., Berse, B., Morgentaler, A, Dvorak, H. F. and Rosen, S.: Vascular permeability factor (vascular endothelid
growth factor) is strongly expressed in the normal male genital tract and is present in substantial quantities in emen. J. Urol., 184: 576, 1995.
3. Aprikian, A. G., Cordon-Cardo, C., Fair, W. R., Zhang, Z.-F., Bazinet, M., Hamdy, S. M.and Reuter, V. E.: Neuroendoerine differentiation in metastatic prostatic adencarcinoma. J. Urol., 161:914,1994. 4. Weinstein, M. H., Partin, A. W., Veltri, R. W. and Epstein, J. L.: Neuroendoerine differentiation in prostate cancer: enhanced prediction of progression after radical prostatectomy. Hum. Pathol., 27: 683, 1996.