Chromosomal regions showing homozygous deletion in tumors are not commonly found and are usually the site of genes involved in tumor suppression. We previously identified the homozygous deletion of a discrete region at 18q22.3 in 50% of breast tumors and 58% of prostate tumors. There are no known genes in this region. Our hypothesis for this study was the following: Encoded within this 18q region is a transcript that when lost plays a role in the development of breast and prostate cancer. The objective of this study was to determine whether sequences within the region of deletion are transcribed in primary breast and prostate epithelial cells. A microarray experiment using RNA isolated from primary mammary epithelial cells and primary prostate epithelial cells enabled us to detect an approximately 500 basepair region that is transcribed in normal breast and prostate epithelial cells. This is a novel transcribed sequence that has not been described previously. A quantitative real‐time TaqMan assay verified the existence of the transcript in breast and prostate epithelial cell RNA. The novel transcript was also detected in high abundance in RNA isolated from brain, cervix, spleen, and thymus. Through our experiments, we hope to identify a new molecular pathway in breast and prostate cancer that could provide new targets for developing therapeutics.

Maspin is a serine protease inhibitor with anti-tumor activity, including inhibition of tumor growth, angiogenesis, invasion, motility, and metastasis. Normal mammary and prostate cells express maspin at high levels. In contrast, breast and prostate cancer tissue samples and cell lines exhibit reduced or no expression of the maspin transcript. Previously we have demonstrated that introduction of an intact chromosome 18 into the bone-derived metastatic prostate cancer cell line, PC-3, resulted in reduced in vitro growth and in vivo metastatic potential. The goal of this study was to determine whether maspin is the tumor/metastasis suppressor on chromosome 18 responsible for this phenotype. To investigate whether maspin, when produced at endogenous levels, is capable of inhibiting metastasis to bone we transfected a bacterial artificial chromosome (BAC) genomic clone containing the maspin gene into PC-3 cells that aggressively metastasize to bone in an animal model. The BAC transfected PC-3 cells exhibited an in vitro phenotype consistent with maspin acting as a tumor suppressor. Analysis of the PC-3 maspin transfectants in an in vivo bone metastasis assay resulted in significant reduction of the number and severity of skeletal metastasis, compared with parental PC-3 cells. However, maspin had no effect on the ability of PC-3 cells to metastasize to extra-skeletal sites in this model. These results indicate that maspin expression likely plays a role in reducing the tumor cell's ability to seed to bone or in inhibition of growth in the bone microenvironment. However, it does not affect the ability to metastasize to distant sites.

Prostate cancer is the second leading cause of cancer deaths among American men. The loss of Y chromosome has been frequently observed in primary prostate cancer as well as other types of cancer. Earlier, we showed that introduction of the human Y chromosome suppresses the in vivo tumorigenicity of the prostate cancer cell line PC-3. To further characterize the Y chromosome, we have developed a high-density bacterial artificial chromosome (BAC) microarray containing 178 BAC clones from the human Y chromosome. BAC microarray was used for array comparative genomic hybridization on prostate cancer samples and cell lines. The most prominent observation on prostate cancer specimens was a deletion at Yp11.2 containing the TSP.Y tandem gene array. Out of 36 primary prostate tumors analyzed, 16 (44.4%) samples exhibited loss of TSPY gene copies. Notably, we observed association between the number of TSPY copies in the blood and the incidence of prostate cancer. Moreover, PC-3 hybrids with an intact Yp11.2 did not grow tumors in nude mice, whereas PC-3 hybrids with a deletion at Yp11.2 grew tumors in nude mice.

3578 Chromosomal alterations, such as amplification and deletions, are frequently observed during the progression of cancer. Although prostate cancer has a major genetic component, many of the specific genes involved in prostate cancer incidence and progression have remained elusive. Several studies revealed that loss of chromosome 18q occurs in approximately 19% to 48% of prostate cancer cases and that this loss is associated with metastasis. Allelic imbalance (AI) analysis of metastatic prostate cancer samples revealed at least two distinct regions of loss, one located at 18q21.2-q21.32 (7 cM) and the other at 18q22.3 (6 cM). Somatic cell genetic experiments have demonstrated that the introduction of chromosome 18 into prostate cancer cell lines affect both the growth and metastatic potential of these cancer cells. These data support the presence of one or more tumor suppressor genes on chromosome 18q. To localize the tumor suppressor gene(s) on 18q, we are using comparative genomic hybridization on a high-resolution genomic array (Array-CGH). The array contains 160 bacterial artificial chromosomes (BACs) which covers the two regions of 18q identified by AI analysis. This high resolution array allowed us to identify individual BACs which were amplified and deleted in prostate cancer specimens. In particular, BAC RPCI11 25L3 was found to be deleted in 50% of the human prostate tumors analyzed (10/20). Fluorescence in situ hybridization (FISH) analysis on paraffin embedded tumor samples verified the homozygous deletion of the BAC clone in the 4 prostate samples analyzed. Using flanking markers, the region of loss in the tumors is being further characterized. The identification of this tumor suppressor gene will help us better understand the molecular basis of prostate cancer.

Loss of heterozygosity and allelic imbalance data has shown that there are two distinct regions of loss on chromosome 18q associated with the progression of prostate cancer (CaP). To investigate the functional significance of chromosome 18q loci in CaP, we utilized the technique of microcell-mediated chromosome transfer to introduce an intact chromosome 18 into the human prostate cancer cell line, PC-3. Three of the resulting hybrid lines were compared to the PC-3 cells in vitro and in vivo. The hybrid cell lines, containing an intact copy of the introduced chromosome 18, exhibited a substantial reduction in anchorage-dependent and independent growth in vitro. These hybrid cell lines also made smaller tumors in nude mice following subcutaneous injection compared to PC-3 cells. Because tumor growth was not completely eliminated by introduction of chromosome 18, we assessed the ability of the hybrids to metastasize to bone after intra-cardiac inoculation in a nude mouse model. Mice inoculated with PC-3 hybrids containing intact copies of chromosome 18 had significantly fewer bone metastases and dramatically improved survival compared to PC-3 cells. In addition, the introduction of chromosome 18 significantly reduced tumor burden in extraskeletal sites. This was not because of differences in growth rates because mice bearing hybrids were monitored for metastases over twice as long as mice bearing PC-3 cells. Taken together, these data suggest that chromosome 18 has a functional role in CaP to suppress growth and metastases. Identification of the responsible gene(s) may lead to molecular targets for drug discovery.