Reactivation of androgen receptor-regulated lipid biosynthesis drives the progression of castration-resistant prostate cancer.

Androgen receptor (AR) is a transcriptional activator that, in prostate cells, stimulates gene expression required for various cellular functions, including metabolisms and proliferation. AR signaling is also essential for the development of hormone-dependent prostate cancer (PCa) and its activity can be blocked by androgen-deprivation therapies (ADTs). Although PCa patients initially respond well to ADTs, the cancer inevitably relapses and progresses to lethal castration-resistant prostate cancer (CRPC). Although AR activity is generally restored in CRPC despite the castrate level of androgens, it is unclear whether AR signaling is significantly reprogrammed. In this study, we examined the AR cistrome in a PCa cell line-derived CRPC model using integrated bioinformatical analyses. Significantly, we found that the AR cistrome is largely retained in the CRPC stage. In particular, AR-mediated lipid biosynthesis is highly conserved and reactivated during the progression to CRPC, and increased level of lipid synthesis is associated with poor prognosis. The restoration of lipid biosynthetic pathways is partially due to the increased expression of AR splice variants. Blocking lipid/cholesterol synthesis in AR variants-expressing CRPC cell line and xenograft models markedly reduces tumor growth through inhibition of mTOR pathway. Silencing the expression of a fatty acid elongase, ELOVL7, also leads to the regression of CRPC xenograft tumors. These results demonstrate the importance of reactivation of AR-regulated lipid biosynthetic pathways in driving CRPC progression, and suggest that ADTs may be therapeutically enhanced by blocking lipid biosynthetic pathways.

ADAM-mediated amphiregulin shedding and EGFR transactivation.

INTRODUCTION: The ectodomain shedding of epidermal growth factor receptor (EGFR) ligands, such as amphiregulin (AREG), by ADAMs (A Disintegrin And Metalloproteases) can be stimulated by G protein-coupled receptor (GPCR) agonists. Interactions between the CXCR4 GPCR and the CXCL12 chemokine have been shown to mediate gene transcription and cellular proliferation in non-transformed and transformed prostate epithelial cells, as well as motility/invasiveness in transformed cells. OBJECTIVES: In this report, we investigated the ability of CXCL12 to stimulate amphiregulin ectodomain shedding in non-transformed and transformed prostate epithelial cells that respond proliferatively to sub-nanomolar levels of CXCL12 and amphiregulin. MATERIALS AND METHODS: Non-transformed N15C6 and transformed PC3 prostate epithelial cells were assessed for amphiregulin shedding, ADAM activation, Src phosphorylation and EGFR activation using ELISA, immunoblot, and immunoprecipitation techniques, and for proliferation using cell counting after stimulation with CXCL12 or vehicle. RESULTS: The results of these studies identify CXCL12 as a novel inducer of amphiregulin ectodomain shedding and show that both basal and CXCL12-mediated amphiregulin shedding are ADAM10- and Src kinase-dependent in non-transformed N15C6 cells. In contrast, amphiregulin shedding is not amplified subsequent to stimulation with exogenous CXCL12, and is not reduced subsequent to metalloprotease- or Src kinase-inhibition, in highly aggressive PC3 prostate cancer cells. These data also show that CXCL12-mediated cellular proliferation requires EGFR transactivation in a Src- and ADAM-dependent manner in non-transformed prostate epithelial cells. However, these same mechanisms are dysfunctional in highly transformed prostate cancer cells, which secrete amphiregulin in an autocrine manner that cannot be repressed through metalloprotease- or Src kinase inhibition. CONCLUSION: These findings show that non-transformed and transformed prostate epithelial cells may employ different mechanisms to activate EGFR ligands and thereby utilize the EGFR axis to promote cellular proliferation.

The inflammatory microenvironment of the aging prostate facilitates cellular proliferation and hypertrophy.

Benign Prostatic Hypertrophy (BPH, also known as benign prostatic hyperplasia or benign prostatic enlargement), is one of the most common benign proliferative conditions associated with aging in men and is pathologically characterized by the proliferation of fibroblast/myofibroblast and epithelial cell types in the prostate. Previous studies from our laboratory have shown that the CXC-type chemokines, CXCL5 and CXCL12, are secreted by aging prostate stroma and promote both proliferative and transcriptional responses from prostate epithelial cells. Using array-based gene expression profiling and quantitative reverse-transcriptase polymerase chain reaction, we now show that the transcriptome of the aging prostate stroma is characterized by the up-regulation of several genes that encode secreted inflammatory mediators, including secreted CXC-type chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL12), interleukins (IL11, IL33), and transcripts with cytokine homology (CYTL1). At the protein level, ELISA experiments demonstrated that CXCL1, CXCL5, and CXCL6 were secreted by primary prostate stromal fibroblasts explanted from aging prostate stroma. Dose-response assays confirmed that, like CXCL5 and CXCL12, CXCL1 and CXCL6 promote low-level proliferative responses from both prostate stromal fibroblasts and epithelial cells. Taken together, these data suggest that inflammatory mediators are secreted by prostatic stroma consequent to aging, that the levels of these mediators are sufficient to promote low-level increases in the proliferative rate of both epithelial and stromal fibroblast cell types. Moreover, these processes may account for the low-level, but cumulative, proliferation of both epithelial and fibroblastic/myofibroblastic cell types that characterizes the aging-associated development of benign prostatic hypertophy.

Loss of expression of human spectrin src homology domain binding protein 1 is associated with 10p loss in human prostatic adenocarcinoma.

The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bp1, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bp1. These experiments demonstrated that 4/6 tumors (67%) with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46%) tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

Postatrophic hyperplasia of the prostate gland: neoplastic precursor or innocent bystander?

Postatrophic hyperplasia (PAH) of the prostate gland often demonstrates overlapping histological features with prostatic adenocarcinoma (PCA). These features include small acinar growth and enlarged nuclei with prominent nucleoli. Recent work has demonstrated that PAH is a proliferative, noninvoluting lesion. PAH is also histologically distinct from simple atrophy (SA), which has intermediate- to large-sized glands, minimal cytoplasm, and inconspicuous nuclei. However, despite overlapping features between PAH and PCA, high-grade prostatic intraepithelial neoplasm (HGPIN) is still considered the only direct neoplastic precursor to PCA. HGPIN resembles PCA in its topographic distribution, cytological appearance, and molecular alterations including chromosome 8p loss and chromosome 8 centromeric gain. To examine the hypothesis that PAH is the earliest histologically distinct precursor to HGPIN or PCA, the frequency, distribution, proliferative state, and chromosome 8 gain of benign prostate, SA, PAH, HGPIN, and PCA were analyzed. Forty radical prostatectomy specimens from men with clinically localized PCA were systematically analyzed. Proliferation was determined by Ki-67 immunohistochemistry (MIB-1) on formalin-fixed, paraffin-embedded tissue and quantified by digital image analysis from a total of 5,510 sample areas with benign, SA, PAH, HGPIN, and PCA. A tissue microarray was constructed to evaluate 8c gain using interphase fluorescence in situ hybridization. SA foci (n = 129) and PAH foci (n = 114) were identified in the 40 cases of which 74% (95 of 129) and 88% (100 of 114) were seen in the peripheral zone, respectively (P = 0.006). PAH and SA were identified adjacent to PCA in 28% (32 of 114) and 14% (18 of 129) of foci examined, respectively (P = 0.007). The median number of proliferating nuclei increased significantly from benign (1.20%), SA (2.67%), PAH (3.62%), HGPIN (6.14%), to PCA (12.00%) (P < 0.001). The median percentage of nuclei with more than three centromeric probe signals (chromosome 8c gain) for SA, HGPIN, PAH, and PCA were 2.1, 2.8, 4.0, and 6.0%, respectively, as compared to benign prostate with 1.3% (P = 0.006). In conclusion, the present study identified a strong topographic association between PAH and PCA. PAH is also seen often to be closely associated with chronic inflammation. Proliferation of PAH is significantly greater than benign prostatic epithelium and SA but less than HGPIN or PCA. Gain of 8c is significantly greater in PAH than benign prostate, SA, and even HGPIN. These findings demonstrate a strong association between PAH and PCA, supporting its role as a neoplastic precursor.

Activated in prostate cancer: a PDZ domain-containing protein highly expressed in human primary prostate tumors.

Critical events in prostate tumorigenesis and metastasis likely include the abnormal activation and expression of specific genes. Using RNA expression profiling techniques, we have identified a transcript originating from the activated in prostate cancer (AIPC) gene, the expression of which is preferentially up-regulated in several cultured prostate tumor cell lines and human primary prostate tumors. Sequence analysis revealed that the AIPC protein encodes six PDZ domains, which are protein-protein binding domains likely involved in protein clustering and scaffolding. Immunohistochemical analysis of a tissue microarray comprising 158 tumor, 18 high-grade prostatic intraepithelial neoplasia, and 91 normal prostate specimens with an anti-AIPC antibody demonstrated abundant AIPC protein expression in 75% of tumors, 83% of prostatic intraepithelial neoplasia lesions, and 3% of normal tissues (P < 0.0001). These data suggest that the accumulation of AIPC protein may be closely associated with the initiation or early promotion of prostate tumorigenesis.

Profiling and verification of gene expression patterns in normal and malignant human prostate tissues by cDNA microarray analysis.

cDNA microarray technology allows the "profiling" of gene expression patterns for virtually any cellular material. In this study, we applied cDNA microarray technology to profile changes in gene expression associated with human prostate tumorigenesis. RNA prepared from normal and malignant prostate tissue was examined for the expression levels of 588 human genes. Four different methods for data normalization were utilized. Of these, normalization to ACTB expression proved to be the most rigorous technique with the least probability of producing spurious results. After normalization to ACTB expression, 15 of 588 (2.6%) genes examined by array analysis were differentially expressed by a factory of 2x or more in malignant compared to normal prostate tissues. The expression patterns for 8 of 15 genes have been reported previously in prostate tissues (TGFbeta3, TGFBR3, IGFII, IGFBP2, VEGF, FGF7, ERBB3, MYC), but those of seven genes are reported here for the first time (MLH1, CYP1B1, RFC4, EPHB3, MGST1, BTEB2, MLP). These genes describe at least four metabolic and signaling pathways likely disrupted in human prostate tumorigenesis. Reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analyses quantitated with reference to ACTB expression levels verified the trends in gene expression levels observed by array analysis for 14/15 and 8/8 genes, respectively. However, RT-PCR and Northern blot analyses accurately verified the "fold" differences in expression levels for only 6/15 (40%) and 7/8 (88%) of genes examined, respectively, demonstrating the need to better validate quantitative differences in gene expression revealed by array-based techniques.

Fluorescence In Situ Hybridization (FISH) to Metaphase and Interphase Chromosomes.

The unambiguous identification of human chromosomes became possible with the discovery and implementation of G-banding techniques (1). Almost immediately, investigators developed various methods to physically map specific DNA sequences to banded chromosomes. A commonly used early technique involved the hybridization in situ of radioactively labeled probes to heat-denatured human metaphase chromosomes (reviewed in 2). These techniques were efficient, yet costly, time-consuming, and technically difficult. Isotopic hybridization in situ was rapidly superseded by nonisotopic techniques-especially those utilizing fluorescently labeled probes (3-6). This chapter describes basic methodology for the accomplishment of metaphase and interphase fluorescence in situ hybridization (FISH).

Polymerase chain reaction detection of DNA sequence deletions.

Allelic loss of human chromosome sequences contributes to tumorigenesis through the inactivation of putative tumor-suppressor genes. The Knudson hypothesis proposes that deletion or mutation must affect both alleles of the gene in order to disable tumor suppression (1). As might be expected, the effect of "two hits" on tumor-suppressor gene integrity-e.g., deletion of one allele and mutation of the remaining allele-would disable the gene from encoding gene product. The von Hippel-Lindau (VHL) gene is an example of a tumorsuppressor gene that fulfills the Knudson hypothesis-e.g., one mutant allele is inherited in the germline, and the other is mutated or deleted somatically in many clear-cell renal cell carcinomas (recently reviewed in ref. 2).

Phenotypic characterization of immortalized normal and primary tumor-derived human prostate epithelial cell cultures.

BACKGROUND: Cell lines can provide powerful model systems for the study of human tumorigenesis. However, the human prostate cancer cell lines studied most intensively by investigators (PC3, DU145, and LNCaP) were established from metastatic lesions, and it is unlikely that they accurately recapitulate the genetic composition or biological behavior of primary prostate tumors. Cell lines more appropriate for the study of human prostate primary tumors would be those derived from spontaneously immortalized cells; unfortunately, explanted prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we examined whether cell lines developed through viral gene-mediated immortalization of human normal or primary tumor prostate epithelium express aspects of the normal or malignant phenotypes, and could serve as appropriate models for normal or transformed human prostatic epithelium. METHODS: To accomplish these goals, we assessed the phenotypic expression of cell cultures established through the immortalization of normal (1532N, 1535N, 1542N, and PrEC-T) or malignant (1532T, 1535T, and 1542T) human prostate epithelium with the E6 and E7 genes of HPV-16, or the large T antigen gene of SV40. RESULTS: Examination of these cell lines for their proliferative rates and their abilities to grow with or without serum or androgen stimulation, to form colonies in soft agar, or to form tumors in vivo, suggests that they may serve as valid, useful tools for the elucidation of prostate tumorigenesis. Moreover, the observation of structural alterations involving chromosome 8, including gain of 8q in 3 of the 4 cell lines expressing aspects of the malignant phenotype, implies that these cell lines accurately recapitulate the genetic composition of primary prostate tumors. CONCLUSIONS: Taken together, these data suggest that cell lines generated from immortalized normal or primary tumor epithelium may be useful for the elucidation of early transforming events in the prostate.

Genetic characterization of immortalized human prostate epithelial cell cultures. Evidence for structural rearrangements of chromosome 8 and i(8q) chromosome formation in primary tumor-derived cells.

We have utilized a combination of conventional and spectral karyotyping (SKY) techniques and allelotype analysis to assess numerical and structural chromosome alterations in two cell lines derived from normal human prostatic epithelium, and three cell lines derived from human prostate primary tumor epithelium, immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV) 16 or the large T-antigen gene of simian virus 40 (SV40). These studies revealed trisomy for chromosome 20 and rearrangements involving chromosomes 3, 4, 8, 9, 10, 16, 17, 18, 19, 21, or 22. In addition, the four HPV-immortalized cell lines exhibited extensive duplications or translocations involving the 11q chromosomal region. Interestingly, allelotyping data disclosed loss of 8p sequences in two of the three primary tumor-derived cell lines, and SKY data revealed that the loss of 8p sequences was directly due to i(8q) chromosome formation and/or other structural alterations of chromosome 8. This provides intriguing evidence that 8p loss in primary human prostate tumors may, in some cases, result from complex structural rearrangements involving chromosome 8. Moreover, the data reported here provide direct evidence that such complex structural rearrangements sometimes include i(8q) chromosome formation.

8p22 loss concurrent with 8c gain is associated with poor outcome in prostate cancer.

OBJECTIVES: A critical issue in the management of prostate cancer is the ability to distinguish patients at risk of disease recurrence. The aim of this study was to determine whether specific physical alterations of chromosome 8 may be associated with disease recurrence and poor outcome using postoperative prostate-specific antigen (PSA) values as surrogate end points. METHODS: To test this hypothesis, we examined paired normal and tumor radical prostatectomy tissues from 25 patients with prostate cancer for chromosome 8 alterations using dual fluorescence in situ hybridization with a fluorescein-labeled 8p22-specific (8p) cosmid probe and a rhodamine-labeled 8-centromere-specific (8c) probe. The probes were enumerated in 200 nuclei per tissue. RESULTS: Of the 25 tumors examined, 22 demonstrated distinct classes of genetic alterations, or nuclear types, including disomy for 8p and 8c (1 tumor), loss of 8p and disomy for 8c (10 tumors), or loss of 8p concurrent with gain of 8c (11 tumors). The presence of even a small population of tumor nuclei characterized by the loss of 8p concurrent with the gain of 8c was correlated with poor tumor grade (P = 0.009), preoperative PSA values 11 ng/mL or higher (P = 0.022), high tumor stage (P = 0.086), and detectable, rising postoperative PSA values (P = 0.086). These observations are consistent with the hypothesis that a gain of chromosome 8 is associated with poor outcome in prostate cancer. CONCLUSIONS: 8p loss concurrent with 8c gain may successfully predict disease recurrence and poor clinical outcome before the observation of detectable postoperative PSA values in patients with prostate cancer.

8pter-p23 deletion is associated with racial differences in prostate cancer outcome.

Deletions of chromosome sequences mapping to the short arm of chromosome 8 have been observed frequently in a variety of human cancers. A small number of studies have suggested that the terminal portion of the short arm of chromosome 8, 8pter-p23, may be deleted independently of other portions of 8p in human tumors, and that deletion of the 8pter-p23 region may be correlated with poor prognosis. The aim of the present study was to physically define the minimal region of 8pter-p23 deletion and to define the frequency and prognostic significance of 8pter-p23 loss in human prostate tumors. DNA was purified from normal and tumor tissues of 45 radical prostatectomy specimens and amplified for 15 highly polymorphic microsatellite sequences, 13 spanning 8pter-p23 and 2 proximal 8p markers. Allelic loss of 8p sequences was observed in 28 of 45 (62%) tumors examined. Of these, approximately half (12 of 28; 43%) demonstrated independent loss of the 8pter-p23 region, with several tumors defining a 5-cM minimal region of deletion spanning D8S264-D8S1824-D8S1781-D8S262-D8S1798. When serum prostate-specific antigen was used as a surrogate end point marker for survival, 8pter-p23 loss was significantly associated with reduced disease-free progression (log-rank P = 0.0426). Moreover, loss of the 8pter-p23 region was significantly associated with poor survival for American Caucasian (log-rank P = 0.0024) but not African-American (log-rank P = 0.5832) prostate cancer patients. These studies suggest that independent deletion of 8pter-p23 is differentially associated with disease recurrence and poor outcome in American Caucasian but not African-American prostate cancer patients.

Homozygous and frequent deletion of proximal 8p sequences in human prostate cancers: identification of a potential tumor suppressor gene site.

By using tissue microdissection and polymerase chain reaction (PCR) techniques, we examined 85 prostate tumors that were paired with normal tissues from the same patients for allelic loss at 26 highly polymorphic microsatellite sequences, 21 spanning 8p and 5 localized to 8q. Sixty-four tumors (75%) demonstrated loss of at least one 8p locus. Separate distal and proximal regions of deletion were observed as well as an intervening, staggered breakpoint. A novel region of homozygous deletion of sequences at the D8S87 locus was detected both by multiplex PCR and by fluorescence in situ hybridization within this breakpoint region. These data suggest that a tumor-suppressor gene mapping to proximal 8p is deleted frequently and is likely to be important for tumorigenesis in prostate tumors.

Allelic loss of 8p sequences in prostatic intraepithelial neoplasia and carcinoma.

OBJECTIVES: Previous work has suggested that prostatic intraepithelial neoplasia (PIN) may be a premalignant lesion important in tumorigenesis of the prostate. However, to adequately test this hypothesis at the genetic level, it is necessary to determine whether lesions in close proximity demonstrate similar genetic alterations and, hence, whether an "evolutionary" relationship might exist between PIN and tumor in the same prostate. Therefore, the purpose of this study was to examine at least two PIN lesions per prostate (one adjacent to and another distant from malignant lesions in the same prostate) for similarities or differences in the types and frequencies of genetic alterations. METHODS: To accomplish this goal, DNA was extracted from microdissected PIN, tumor, and normal epithelial tissue samples from 48 radical prostatectomies and amplified using polymerase chain reaction techniques at highly polymorphic microsatellite repeat sequences at proximal (D8S87, 8p12) and distal (NEFL, 8p21) loci on the short arm of chromosome 8. PIN specimens were either adjacent to (within one high-power microscopic field [HPF]) or distant from (separated by two or more HPFs) tumor specimens from the same patients. RESULTS: Similar fractional allelic loss frequencies were observed for informative tumor (10 [35%] of 29) and PIN (6 [21%] of 29) samples at the NEFL locus, but allelic loss at the D8S87 locus was observed only in tumors (8 [22%] of 36 informative samples). Moreover, allelic loss at the NEFL locus involved the same allele in 4 cases and different alleles in 3 cases. Interestingly, all 4 cases with the same allele loss were from adjacent PIN and tumor tissues, and all 3 with different allele loss were from distant PIN and tumor. CONCLUSIONS: These results suggest that PIN and invasive cancer share common genetic events (eg, deletion at the NEFL locus) along the same pathway of development in the prostrate.

The relationship between prostatic intraepithelial neoplasia and prostate cancer: critical issues.

PURPOSE: Prostatic intraepithelial neoplasia (PIN) is often considered to be a premalignant lesion and the main precursor of invasive carcinoma of the prostate. We evaluated the evidence for and against PIN as a premalignant lesion and determined guidelines for the clinical management of PIN. MATERIALS AND METHODS: Literature analysis of histopathological, morphometric, phenotypic and molecular genetic evidence of progression and of clinical findings regarding PIN was done. Literature searches were performed on MEDLINE with relevant key words. RESULTS: PIN, like prostate cancer, occurs most frequently in the peripheral zone of the prostate and is usually located in close proximity to prostate cancer. The relative PIN and prostate cancer volumes vary inversely. Prostate specific antigen in cases of PIN appears to be intermediate between prostate cancer and normal levels, although this elevation may be explained by concomitant prostate cancer or benign prostatic hyperplasia. Deoxyribonucleic acid ploidy in PIN follows the aneuploid proportion as in the concomitant prostate cancer. Prostate cancer and PIN show evidence of loss of putative tumor suppressor genes on chromosome 8p. The clinical relevance of PIN biopsy findings is based on the association of neoplasia and prostate cancer. High grade PIN in core biopsies without concomitant prostate cancer has a substantial risk for prostate cancer in subsequent biopsies (24 to 73%, up to 100% when the digital rectal examination is suspicious) and should cause further biopsy sampling. CONCLUSIONS: There is convincing evidence that PIN is a precursor lesion to prostate cancer, with a close association of PIN and prostate cancer in biopsy and prostatectomy specimens. A biopsy finding of high grade PIN necessitates further investigation in patients who are candidates for radical treatment for localized prostate cancer.

Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer.

Utilizing tissue microdissection and PCR techniques, we have examined 35 prostate tumors paired with normal tissues from the same patients for allelic loss at 24 polymorphic loci spanning chromosome 10. Twenty-five tumors (71%) were deleted for at least one chromosome 10 locus. Of the total 35 tumors, 6 (17%) were deleted for 10p loci only, 5 (14%) for 10q loci only, and 14 (40%) were deleted for both 10p and 10q loci. The common region of deletion on 10p included loci D10S211-D10S89-D10S111. Fluorescence in situ hybridization of yeast artificial chromosome probes encompassing these loci demonstrated that the 10p region of deletion maps to 10p11.2. Losses involving 10p loci alone were most common in localized (5/14, 36%) and least common in metastatic (0/8) tumors. The common region of deletion on 10q included loci D10S219-D10S215, consistent with the major region of deletion recently defined for prostate tumors on 10q. Losses involving 10q loci alone were lowest in localized and locally invasive tumors (1/14 and 2/12, respectively) and highest in tumors metastatic to regional lymph nodes (2/8). These results suggest that 10p losses may define less invasive tumors, whereas 10q losses may play a role in the progression to more advanced tumor states in the prostate. Furthermore, this is the first report of allelic loss of a defined region on 10p potentially harboring tumor suppressor gene loci in human prostate cancer.

Distinct regions of allelic loss on 13q in prostate cancer.

Loss of heterozygosity (LOH) involving the long arm of chromosome 13 has been reported to occur in as many as one third of primary prostate cancers. Candidate tumor suppressor genes on 13q that may be important in the development of prostate cancer include the retinoblastoma susceptibility gene (RBI) and a gene associated with inherited breast cancer (BRCA2). To define the pattern of allelic loss of 13q in prostate cancer, LOH analysis was performed using nine mapped polymorphic markers spanning the entire chromosomal arm. Nineteen (48%) of 40 prostate cancer cases obtained following radical prostatectomy demonstrated atllelic loss with at least one marker. Furthermore, 13 (33%) of 40 cases had evidence of allelic loss involving a region of 13q14 containing RB1. To test the hypothesis that RB1 is the targeted tumor suppressor gene in this region, 37 of 40 cases were assessed for expression of pRB, the protein product of RB1 using immunohistochemical techniques. By this analysis, 8 (22%) of 37 prostate tumors demonstrated no pRB expression. However, allelic loss at RB1, assessed with an intragenic marker, did not correlate with absent pRB expression (Fisher's exact test, P=0.375). Taken together, these data confirm that allelic loss of a common region of 13q14 occurs in approximately one third of prostate cancers. Lack of correlation of LOH at RB1 with absent pRB expression suggests the existence of another tumor suppressor gene in this region important in prostate cancer.

Evidence for three tumor suppressor gene loci on chromosome 8p in human prostate cancer.

Allelic loss of human chromosome sequences is often equated with inactivation of putative tumor suppressor genes. Loss of sequences on the short arm of chromosome 8 (8p) has been observed in human cancers, especially of 8p22 in prostate tumors. By using PCR analysis of highly polymorphic microsatellite repeat markers at nine 8p loci in 135 tumors, we observed deletion of sequences at 8p22 and at two other proximal deletion domains. These novel deletion domains encompass the NEFL locus and D8S87-ANK1 loci, respectively. These data suggest that three 8p tumor suppressor gene loci may be independently deleted in human prostate cancers.

Comparative analysis of epidermal growth factor receptor gene expression and protein product in benign, premalignant, and malignant prostate tissue.

In order to more clearly define the status of epidermal growth factor receptor (EGFR) in prostate cancer, expression of EGFR transcript and protein was analyzed in paired samples of benign and malignant tissues from 30 radical prostatectomy specimens. Prostate tumors and high grade prostatic intraepithelial neoplasias (PINs) expressed significantly less EGFR protein than benign tissues or low grade PINs (P < 0.001). Expression of EGFR mRNA was analyzed in a subset of the same samples, and was higher in more prostate tumors than benign specimens (P < 0.05). However, differences in mean mRNA expression between malignant and benign tissues were not significant. EGFR mRNA was expressed at moderate or low levels in equivalent numbers of PIN lesions. These results suggest that, although EGFR mRNA expression is somewhat elevated in prostate tumors, EGFR protein expression may be down-regulated in the same malignant tissues. Furthermore, our data demonstrate phenotypic similarity between prostate tumors and high grade PIN at the level of EGFR protein expression.