Dipak Giri

Interleukin-6 is an autocrine growth factor in human prostate cancer

Prostate cancer is the most common cancer in American men and the second leading cause of cancer deaths in this group. We have found that interleukin (IL)-6 protein concentrations are increased approximately 18-fold in clinically localized prostate cancers when compared to normal prostate tissue. Normal and neoplastic prostatic epithelial cells in culture, with the exception of LNCaP cells, secrete IL-6. Addition of exogenous IL-6 to primary epithelial cells in culture or the LNCaP prostate cancer cell line leads to phosphorylation of Stat-3 and increases in net cell proliferation. The concentration of IL-6 receptor is increased eightfold in the prostate cancer tissues and is increased in the cancer cells by immunohistochemistry. The increased expression of IL-6 receptor is correlated with increased proliferation of prostate cancer cells in vivo as assessed by Ki67 immunohistochemistry. These findings strongly support the hypothesis that IL-6 acts as a significant autocrine growth factor in vivo for primary, androgen-dependent prostate cancers.

Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression

The PTEN gene encodes a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase pathway and is inactivated in a wide variety of malignant neoplasms. High rates of loss of heterozygosity are observed at the 10q23.3 region containing the human PTEN gene in prostate cancer and other human malignancies, but the demonstrated rate of biallelic inactivation of the PTEN gene by mutation or homozygous deletion is significantly lower than the rate of loss of heterozygosity. The transgenic adenocarcinoma of mouse prostate model is a well characterized animal model of prostate cancer. Analysis of prostate cancer progression in transgenic adenocarcinoma of mouse prostate 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 the progression of prostate cancer 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.

Interleukin-8 is a paracrine inducer of fibroblast growth factor 2, a stromal and epithelial growth factor in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is an extremely common disease of older men in which there is benign overgrowth of the prostatic transition zone, leading to obstruction of urine outflow. Fibroblast growth factor (FGF) 2, a potent growth factor for prostatic stromal and epithelial cells, is increased twofold in BPH and its concentration is correlated with stromal proliferation in this condition. Immunohistochemistry of normal and hyperplastic prostate revealed that FGF2-expressing stromal cells were present in higher numbers near the epithelial acini, implying that epithelial cells may express a factor that induces FGF2 expression by stromal cells. Conditioned medium from primary cultures of prostatic epithelial cells was capable of inducing increased expression of FGF2 by primary stromal cultures. Blocking experiments with neutralizing anti-interleukin (IL)-8 antibodies and pretreatment with lipopolysaccharide, which down-regulates the IL-8 receptor, show that this inducing activity is because of the presence of IL-8 in the epithelial-conditioned medium. Analysis of normal prostatic peripheral zone and BPH tissue by enzyme-linked immunosorbent assay reveals that IL-8 is present at increased levels in hyperplastic prostate. Therefore IL-8 produced by prostatic epithelial cells can induce FGF2, a potent stromal and epithelial growth factor, and in this manner promote the abnormal proliferation of the prostatic transition zone that is critical in the pathogenesis of BPH.

Inactivation of the PTEN tumor suppressor gene is associated with increased angiogenesis in clinically localized prostate carcinoma.

The PTEN tumor suppressor gene encodes a dual-specificity protein phosphatase that may play a key role in modulating integrin-mediated signals. Inactivation of the PTEN gene has been detected in a small percentage of clinically localized prostate cancers but is common in metastatic disease. It has been shown in glioblastoma cell lines that loss of chromosome 10q, where the PTEN gene is located, is associated with increased angiogenic activity in the conditioned medium attributable to downregulation of thrombospondin-1, a negative regulator of angiogenesis. Therefore, we wished to determine whether inactivation of PTEN might be associated with increased angiogenesis in prostate cancers, because increased angiogenesis in localized cancers is associated with development of metastatic disease. Angiogenesis was assessed by counting microvessels in areas of maximal neovascularization after immunostaining with anti-factor VIII-related antigen antibodies in eight cases with proven homozygous deletion of the PTEN gene and 24 control cases. There was a statistically significant correlation between PTEN inactivation and increased microvessel counts. The microvessel density was higher at all Gleason scores in the cases with PTEN inactivation compared with control cases with the same score. To determine whether the increased angiogenesis in cases with PTEN inactivation was caused by downregulation of expression of the angiogenesis inhibitor thrombospondin-1, we analyzed a subset of the cases by immunostaining with anti-thrombospondin-1 antibody. Approximately 25% of cases showed decreased staining of prostate cancer cells, but there was no correlation with PTEN inactivation. Thus, PTEN inactivation is associated with increased angiogenesis, but the increased angiogenesis is not attributable to downregulation of thrombospondin-1 expression.

Alternative splicing of fibroblast growth factor receptors in human prostate cancer.

Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells and may promote proliferation of neoplastic prostatic epithelial cells via autocrine or paracrine mechanisms. The affinity of FGFs for FGF receptors 1–3 is critically dependent on an alternative splicing event involving the coding region for the carboxy terminal portion of the third extracellular immunoglobulin‐like domain that leads to two different isoforms of each receptor (IIIb and IIIc). We therefore sought to determine whether changes in alternative splicing of FGF receptors occur in human prostate cancer.

FGF7 and FGF2 are increased in benign prostatic hyperplasia and are associated with increased proliferation

PURPOSE To determine if overexpression of FGF7 and FGF2 occurs in benign prostatic hyperplasia (BPH) and if so, whether such overexpression is correlated with increased proliferation of epithelial and/or stromal cells. MATERIALS AND METHODS The FGF7 and FGF2 content of protein extracts of normal peripheral zone, normal transition zone and hyperplastic prostatic tissues were determined by enzyme-linked immunoabsorption assay. Proliferation of epithelial and stromal cells was assessed by immunohistochemistry with anti-Ki67 antibodies on frozen sections of the same tissues used for protein extraction. The in vitro effects of FGF7 and FGF2 on proliferation were assessed by addition of recombinant growth factor to primary cultures of prostatic epithelial and stromal cells. RESULTS We have found that both FGF7 and FGF2 are overexpressed in hyperplastic prostate in comparison to normal peripheral and transition zone tissue. FGF7 is a potent mitogen for epithelial cells in culture. Consistent with these in vitro effects, quantitative analysis of cellular proliferation by Ki67 immunohistochemistry revealed a strong correlation of epithelial proliferation with FGF7 content in BPH tissue, consistent with a key role for this growth factor in driving the abnormal epithelial proliferation in BPH. FGF2 is mitogenic for stromal cells in culture and there was a weaker correlation of FGF2 content with increased stromal proliferation. CONCLUSION Overexpression of FGF7 and FGF2 may play an important role in the abnormal cellular proliferation seen in benign prostatic hyperplasia.

FGF9 is an autocrine and paracrine prostatic growth factor expressed by prostatic stromal cells.

Polypeptide growth factors, including members of the fibroblast growth factor (FGF) family, play an important role in the growth and maintenance of the normal prostate. We have found that FGF9 is expressed at high levels in the normal peripheral and transition zone of the human prostate. Analysis of FGF9 production by primary cultures of prostatic epithelial and stromal cells has shown that FGF9 is produced and secreted by the prostatic stromal cells. Neither of these processes appears to be modulated by androgens. Production of FGF9 by stromal cells in vivo was confirmed by immunohistochemistry. FGF9 is a potent mitogen for both prostatic epithelial and stromal cells in culture and is a more potent mitogen for these cells than either FGF2 or FGF7, two other FGFs expressed in the human prostate. FGF9 is an abundant secreted growth factor that can act as both a paracrine mitogen for epithelial cells and an autocrine mitogen for stromal cells. Western blot analysis of tissue extracts from the normal and hyperplastic transition zone shows that FGF9 is present at two to threefold higher levels in the hyperplastic transition zone. Overexpression of this paracrine and autocrine growth factor may play an important role in the epithelial and stromal proliferation in benign prostatic hyperplasia. J. Cell. Physiol. 180:53–60, 1999.

Interleukin-1α Is a Paracrine Inducer of FGF7, a Key Epithelial Growth Factor in Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is an extremely common disease of older men in which there is benign overgrowth of the prostatic transition zone, leading to obstruction of urine outflow. FGF7, a potent growth factor for prostatic epithelial cells, is increased by threefold in BPH and is correlated with increased epithelial proliferation in this condition. Immunohistochemistry of normal and hyperplastic prostate revealed that FGF7-expressing fibroblastic cells were present in higher numbers near the epithelial acini, implying that epithelial cells may express a factor that induces FGF7 expression by stromal cells. Conditioned medium (CM) from primary cultures of prostatic epithelial cells was capable of inducing a two- to sixfold increase in the expression of FGF7 by primary stromal cultures. Blocking experiments with neutralizing anti-interleukin-1α (Il-1α) antibodies and IL-1Ra, an Il-1α receptor antagonist, show that this inducing activity was due to the presence of Il-1α in the epithelial CM. Analysis of normal prostatic peripheral zone and BPH tissue by enzyme-linked immunoabsorption assay reveal that Il-1α is present at increased levels in hyperplastic prostate and that levels of Il-1α correlate strongly with tissue FGF7 concentration in BPH. Therefore Il-1α is produced by prostatic epithelial cells and can induce FGF7, a potent epithelial growth factor, which can in turn lead to further epithelial growth and increased Il-1α secretion, thus establishing a double paracrine loop that is functionally equivalent to an autocrine growth loop. This double paracrine loop may play a key role in the abnormal proliferation of the transition zone, which is critical to the pathogenesis of BPH.

FGF-10 is expressed at low levels in the human prostate

Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells. FGF‐10 is a secreted growth factor that binds to FGF receptor‐2 IIIb, which is expressed in prostatic epithelial cells and thus can potentially act as a growth factor for these cells. Prior work has indicated that FGF10 may play an important role in the development of the rat prostate, but its role in the adult human prostate is unclear.