- FGFR-4 in prostate cancer
- Inactivation of Sprouty proteins in prostate cancer
- FGF receptors as key survival factors in prostate cancer
- Cellular senescence and prostate cancer
- Cellular senescence and benign prostatic hyperplasia (BPH)
- Mouse models of prostate cancer
Recent studies in our laboratory have shown that a germline polymorphism of the FGFR-4 gene resulting in expression of arginine at codon 388 (Arg388 ) is associated with prostate cancer incidence and the occurrence of aggressive disease. The FGFR-4 genotype of men undergoing radical prostatectomy and controls of the same race was determined and the genotype correlated with clinical and pathological parameters. The presence of the FGFR-4 Arg388 allele is strongly associated with the occurrence of prostate cancer in white men and is also correlated with both increased pathological stage and the occurrence of pelvic lymph node metastasis in men undergoing radical prostatectomy. Expression of FGFR-4 Arg388 in immortalized prostatic epithelial cells results in increased cell motility and invasion through Matrigel when compared to cells expressing the FGFR-4 Gly388 allele. Thus, presence of the FGFR-4 Arg388 allele is associated with both an increased incidence and clinical aggressiveness of prostate cancer and results in increased cellular motility and invasiveness in immortalized prostate epithelial cells. Current efforts are focused on:
- Directly testing the ability of the FGFR-4 Arg388 allele to promote metastasis in vivo using transgenic and orthotopic mouse models of prostate cancer
- Determination of the mechanism by which the FGFR-4 Arg388 allele promotes increased motility and metastasis
- Confirming the role of the FGFR-4 Arg388 allele in prostate cancer incidence in a prospective trial and analysis of its impact on patient outcome in men treated with radiation therapy or watchful waiting.
Immunohistochemistry with anti-FGFR-4 antibody showing strong staining in prostate cancer cells.
Wang J, Stockton D, and Ittmann M. 2004. The FGFR-4 Arg388 allele is associated with prostate cancer initiation and progression. Clin Cancer Res. 10: 6169-6178.
Ropiquet F, Giri D, Kwabi-Addo, B, Mansukhani A, and Ittmann M. 2000. Increased expression of fibroblast growth factor 6 in human prostatic intraepithelial neoplasia and prostate cancer. Cancer Res. 60: 4245-4250.
Recently a new family of regulators of FGF activity has been identified. The Sprouty gene family negatively regulates FGF signaling in a variety of systems and could potentially limit the biological activity of FGFs in prostate cancer. Immunohistochemical analysis of normal and neoplastic prostate tissues using tissue microarrays revealed that Sprouty1 protein is down-regulated in approximately 40 percent of prostate cancers when compared with matched normal prostate. By quantitative real-time PCR analysis we found that Sprouty1 mRNA levels were significantly decreased in prostate cancers in vivo in comparison to normal prostate. In prostate cancer cell lines there is loss of the normal up-regulation of Sprouty1 mRNA and protein in response to FGFs. The decrease in Sprouty1 expression in the human prostate cancer, despite elevated levels of FGF ligands and FGF receptors, implies a loss of an important growth regulatory mechanism in prostate cancers that may potentiate the effects of increased FGF and FGFR expression in prostate cancer. Current efforts are focused on: (1) determining whether expression of other Sprouty genes are decreased in prostate cancer (2) understanding the mechanism by which Sprouty mRNAs/proteins are down-regulated (3) elucidating the biological and clinical consequences of Sprouty down-regulation.
Loss of expression of Sprouty1 protein in prostate cancer.
Kwabi-Addo B, Wang J, Erdem H, Vaid A, Castro P, Ayala G, and Ittmann M. 2004. Expression of Sprouty1, an inhibitor of fibroblast growth factor signal transduction, is decreased in human prostate cancer. Cancer Res 64: 4728-4735.
To determine the biological significance of FGF receptor (FGFR) activation in human prostate cancer, we disrupted FGF signaling by transfection of dominant negative FGFR in a vector containing a hygromycin resistance gene. The dominant negative FGFR construct inhibited colony formation after hygromycin selection by over 99 percent in all three cell lines compared to vector controls. To determine the mechanism of this profound inhibition of colony formation we constructed replication-deficient adenovirus vectors expressing dominant negative FGFR and infected LNCaP and DU145 prostate cancer cells. Infection with the dominant negative FGFR expressing adenovirus led to extensive cell death. Surprisingly, flow cytometry and cytogenetic analysis revealed that the dominant negative FGFR receptor led to arrest in the G2 phase of the cell cycle prior to cell death in both cell lines. Western blot analysis of dominant negative FGFR infected cells showed accumulation of cyclin B1 in treated cells while cdc2 kinase activity was decreased. This implies a failure to form or activate cyclin B1/cdc2 kinase complexes in such cells. These findings reveal an unexpected dependence on FGF receptor signal transduction for prostate cancer cells to traverse the G2/M checkpoint and ultimately for cell survival. We are seeking to understand the mechanism by which loss of FGF signaling leads to G2 arrest and cell death in prostate cancer. Current efforts are focused on the role of CDC25c in the observed G2 arrest. We are also using an expression microarray based approach to explore the mechanism by which G2 arrest occurs in response to loss of FGF signaling. Finally, we are testing the use of dominant negative FGFR and small molecule inhibitors of FGF signal transduction as therapeutic modalities in mouse models of prostate cancer.
Cell cycle analysis of prostate cancer cells expressing
dominant negative FGF receptor.
Ozen M, Giri D, Ropiquet F, Mansukhani A, and Ittmann M. 2001. Role of fibroblast growth factor receptor signaling in prostate cancer cell survival. J. Natl. Canc. Instit. 93: 1783-1790.
Giri D, Ropiquet F, and Ittmann M. 1999. Alterations in expression of FGF2 and its receptor FGFR-1 in human prostate cancer. Clin. Canc. Res. 5: 1063-1071
Prostate cancer is a disease characterized by a markedly increased incidence with age. Research in our laboratory has established that fibroblast growth factors act as important growth and survival factors for prostate cancer cells. Our fundamental hypothesis is that increased expression of FGFs by normal tissue in the peripheral zone of the prostate acts as stimulus to prostate cancer progression by providing critical growth and survival signals to adjacent prostate cancer cells. A corollary of this hypothesis is that individual variation in the control of expression of these progression factors may lead to differences in the incidence of prostate cancer. Recent work in our laboratory has shown cellular senescence of prostatic epithelial cells leads to expression of cytokines which in turn induce expression of FGFs by stromal cells that then act as growth factors for prostate cancer cells and in this manner promote prostate cancer progression. If so, variation in the extent of cellular senescence may explain differences in disease incidence. Our current data strongly supports this hypothesis and indicates that oxidative DNA damage is a major determinant of cellular senescence in prostatic epithelium.
Ben ign prostatic hyperplasia (BPH) is an extremely common disease of older men, occurring in more than 70 percent of men over the age of 60. BPH results in substantial morbidity in this patient population, including complications such as urinary retention, renal impairment and infection. A major factor in the pathogenesis of prostatic hyperplasia is the continuing growth of the transition zone of the prostate due to both epithelial and stromal proliferation. FGF7 is expressed in normal and hyperplastic prostate by stromal fibroblasts and acts as a potent epithelial growth factor. We have demonstrated that expression of FGF7 is increased in BPH and tissue levels of FGF7 are strongly correlated with epithelial proliferation in this condition. Using primary cultures of prostatic epithelial and stromal cells we have also shown that Il-1α is secreted by prostatic epithelial cells and can act as a paracrine inducer of FGF7 secretion by the stromal cells. Analysis of normal prostatic peripheral zone and BPH tissue reveals that Il-1α is present at increased levels in hyperplastic prostate and levels of Il-1α correlate strongly with tissue FGF7 concentration. Thus, the increased epithelial proliferation seen in BPH is driven by increased expression of Il-1α by prostatic epithelial cells acting on adjacent stromal cells to increase FGF7 expression. Our recent work indicates that cellular senescence is a major factor driving expression of IL-1α in vitro and in vivo . A similar relationship holds for epithelial derived IL-8 and stromal FGF2. Thus there is a linkage between cellular senescence and one of the most common pathologies of older men. Current efforts seek to determine if senescence increases expression of other cytokines and growth factor and the mechanism of cellular senescence in prostatic epithelial cells.
Castro P, Chen X, Gomez L, Lamb D, and Ittmann M. 2004. Interleukin-8 expression is increased in senescent prostatic epithelial cells and promotes the development of benign prostatic hyperplasia. The Prostate 60:153-159
Castro P, Giri D, Lamb D, and Ittmann M. 2003. Cellular senescence in the pathogenesis of benign prostatic hyperplasia. The Prostate 55: 30-38.
Giri D, and Ittmann M. 2001. IL-8 is a paracrine inducer of FGF2, a stromal and epithelial growth factor in benign prostatic hyperplasia. Am. J. of Path. 159: 139-147.
Giri D and Ittmann M. 2000. Il-1α is a paracrine inducer of FGF-7, a key epithelial growth factor in benign prostatic hyperplasia. Am. J. Pathol. 157: 249-255.
Ropiquet F, Giri D, Lamb D, and Ittmann M. 1999. FGF7 and FGF2 are increased in benign prostatic hyperplasia and are associated with increased proliferation. J. Urol. 162: 595-599.
Our laboratory is interested in the use of mouse models of prostate cancer to explore the pathogenesis of prostate cancer and its response to treatment. We have used the SV-40 based TRAMP model of prostate cancer to explore the role of FGF2 and the PTEN tumor suppressor gene in prostate cancer progression. To accomplish this we crossed TRAMP mice to the appropriate knockout mice and follow tumor progression. Analysis of survival and the molecular phenotype and genotype of the tumors in these mice has provided insight into the role of these genes in prostate cancer progression. In particular , analysis of prostate cancer progression in TRAMP mice bred to PTEN+/- heterozygous mice, coupled with analysis of the PTEN gene and protein in the resulting tumors, reveals that haploinsufficiency of the PTEN gene promotes prostate cancer progression in this model system. This observation provides a potential explanation for the discordance in rates of loss of heterozygosity at 10q23 and biallelic PTEN inactivation observed in prostate cancer and many human malignancies. In contrast, inactivation of even one FGF2 allele resulted in increased survival, a decrease in metastasis and inhibition of progression to the poorly differentiated phenotype in primary prostatic tumors. When compared to wild-type mice, p P oorly differentiated tumors arising in FGF+/-and FGF-/-mice expressed higher levels of VEGF, and in some cases, increased levels of acidic FGF intracellular binding protein, a nuclear FGF1 binding protein , than in wild-type mice . These findings suggest that both FGF2 mediated angiogenesis and intranuclear FGF2 activities may both promote tumor progression and support the hypothesis that FGF2 plays a significant role in prostate cancer progression in vivo . We have also established a number of transgenic mouse lines expressing FGFR-4 and Il-1α and are evaluating there phenotype and potential utility as mouse models of cancer and BPH.
Prostatic neoplasia in the TRAMP mouse model of prostate cancer
Schematic of PTEN function
Survival of TRAMP mice as a function of tumor PTEN genotype
Microarray analysis of differential gene expression in wild-type and FGF2 knockout TRAMP tumors
Polnaszek N, Kwabi-Addo B, Peterson L, Ozen M, Greenberg N, Ortega S, Basilico C, and Ittmann M. 2003. Fibroblast growth factor 2 promotes tumor progression in an autochthonous mouse model of prostate cancer. Cancer Res 63: 5724-5760.
Kwabi-Addo B, Giri D, Schmidt K, Podsypanina K, Parsons R, Greenberg N, and Ittmann M. 2001. Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression. Proc Natl Acad Sci 98: 11563-11568.
Han G, Buchanan G, Ittmann M, Harris J, Yu X, DeMayo F, Tilley W, and Greenberg N. 2005. Mutation of the androgen receptor causes oncogenic transformation of the prostate. Proc. Natl. Acad. Sci. 102: 1151-1156.
Shappell S, Thomas G, Roberts R, Herbert R, Ittmann M, Rubin M, Humphrey P, Sundberg J, Rozengurt N, Barrios R, Ward J, and Cardiff R. 2004. Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor Meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee. Cancer Res 64 : 2270-2305.
Kaplan-Lefko P, Chen T, Ittmann M, Barrios R, Ayala G, Huss W, Maddison L, Foster B, and Greenberg N. 2003. Pathobiology of Autochthonous Prostate Cancer in a Pre-clinical Transgenic Mouse Model. The Prostate 55: 219-237.
Freeman K, Gangula R, Welm B, Ozen M, Foster B, Rosen J, Ittmann M, Greenberg N, and Spencer D. 2003. Conditional activation of fibroblast growth factor (FGFR) 1, but not FGFR2, in prostate cancer cells, leads to increased osteopontin induction, increased extracellular signal-regulated kinase activation and in vivo proliferation. Cancer Res 63: 6237-6243.
Freeman K, Welm B, Gangula R, Rosen J, Ittmann M, Greenberg N and Spencer D. 2003. Inducible prostate intraepithelial neoplasia with reversible hyperplasia in conditional FGFR1-expressing mice. Cancer Res 63: 8256-8263.
Jin C, McKeehan K, Guo W, Jauma S, Ittmann M, Foster B, Greenberg NM, McKeehan WL and Wang F. 2003. Cooperation between Ectopic FGFR1 and Depression of FGFR2 in Induction of Prostatic Intraepithelial Neoplasia in the Mouse Prostate. Cancer Res 63: 8784-8790.
Wang F, McKeehan K, Yu C, Ittmann M, and McKeehan WL. 2004. Chronic activity of ectopic type 1 fibroblast growth factor receptor tyrosine kinase in prostate epithelium results in hyperplasia accompanied by intraepithelial neoplasia. The Prostate 58: 1-12.