Identification of the genes involved in prostate cancer (PCa) progression to a virulent and androgen-independent (AI) form is a major focus in the field. cDNA microarray was used to compare the gene ...expression profile of the indolent, androgen sensitive (AS) LNCaP PCa cell line to the aggressively metastatic, AI C4-2. Thirty-eight unique sequences from a 6388 cDNA array were found differentially expressed (≥2-fold, 95% CI). The expression of 14 genes was lower in C4-2 than in LNCaP cells, while the reverse was true for 24 genes. Twelve genes were validated using Q-PCR, Western blotting and immunohistochemistry (IHC) of LNCaP and C4-2 xenograft. Q-PCR showed that 10 of 12 (83.3%) genes had similar patterns of expression to the array (LNCaP>C4-2: TMEFF2, ATP1B1, IL-8, BTG1, BChE, NKX3.1; LNCaP<C4-2: BNIP3, TM4SF1, AMACR, UCH-L1). By Western blot, 4/5 genes examined: TMEFF2, NKX3.1, AMACR, and UCH-L1, not IL-8, were consistent with RNA profiling. Protein expression levels were confirmed in human tumor xenografts using IHC. A large proportion of the markers found in this expression profile is consistent with those recently identified in human PCa tissues along with several novel genes that remain to be examined. These data further demonstrate the utility of the LNCaP human PCa progression model as a tool to investigate the phenotypic changes required for the progression to AI and metastasis.
Abstract
Prostate carcinoma is the most common malignancy and the second leading cause of cancer mortality in men. In order to understand the genetic composition of lethal and advanced prostate ...cancer, we used exome sequencing to identify protein-altering mutations within a panel of 23 prostate cancer xenografts that represent a wide spectrum of advanced disease phenotypes. Although corresponding normal tissue was not available for most tumors, we were able to take advantage of increasingly deep catalogs of human genetic variation to remove most germline variants. On average, each tumor genome contains ∼200 novel nonsynonymous variants, of which the vast majority was specific to individual carcinomas. Based on the prevalence of rare nonsynonymous variants, we identified a subset of 30 genes including TP53, DLK2, GPC6 and SDF4 as putative candidates involved in tumor progression. To ascertain the frequency of mutations in these candidate genes across a large panel of tumors, we developed a molecular inversion probe (MIP) based protocol that enables efficient targeting and sequencing of tens of genes across hundreds to thousands of samples. The method requires as little as 50 nanograms of DNA as input, and is compatible with DNA isolated from both frozen and formalin formalin-fixed paraffin embedded (FFPE) tissues. We have applied this method to capture and deeply sequence the nearly ∼1,000 exons of the 30 candidate genes across 150 prostate cancer metastases on a single lane of an Illumina HiSeq2000 platform. Unexpectedly, our exome survey identified three tumors with substantially higher mutation frequencies, with 2,000-4,000 novel coding variants per exome. We used whole genome sequencing to characterize the global patterns of mutation in one of these tumors, and found a high prevalence of transition mutations within CpG islands. Experiments to determine whether hypermutation is the result of loss of DNA repair pathway function are ongoing. Collectively, our results indicate that exome sequencing combined with efficient screening of candidate genes is a powerful method to understand the genetics of advanced prostate cancer. We also report a previously undescribed subtype of prostate cancers exhibiting “hypermutated” genomes, with potential implications for resistance to cancer therapeutics.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5059. doi:1538-7445.AM2012-5059
Abstract
Prostate Cancer (PCa) metastasizes to the bone in approximately 90% of patients who die of PCa. Growth of tumor cells in the bone can lead to replacement of bone marrow, spinal cord ...compression, severe bone pain, cachexia and death. The bone response to a metastasis can manifest as a spectrum of osteoblastic to osteoblastic/osteolytic (mixed) or osteolytic responses in the same patient at different biopsy sites. A number of proteins have been proposed to promote the osteoblastic and osteolytic responses in PCa bone metastasis. Our objective was to determine whether known bone forming and degrading factors are associated with the osteoblastic or osteolytic response in pre-clinical models and clinical specimens of PCa bone metastasis. Additionally, we wanted to identify novel proteins that may be associated with the osteoblastic and osteolytic response in PCa bone metastases. Using specimens obtained at rapid autopsy at the University of Washington, we compared the gene expression profile of osteoblastic and osteolytic metastases (n=14) from 13 patients and validated our findings by qRT-PCR. We also interrogated the same factors at the protein level in 18 osteoblastic and 18 osteolytic PCa bone metastases from 27 patients by immunohistochemistry (IHC). Additionally, we are currently interrogating the levels of these factors in 6 novel LuCaP xenograft models of PCa that promote an osteoblastic response and 4 that promote an osteolytic response in the tibia of immune compromised mice by IHC. Using gene expression arrays, validated by qRTPCR, we identified a putative osteoblastic factor EMID1 (p=0.01) and two putative osteolytic factors, MMP12 (p=0.03) and sFRP1 (p=0.02). By IHC, MMP12 expression approached significance (p=0.07), whereas sFRP1 was significantly higher in the osteolytic samples (p=0.02). Interestingly, we did not observed significant differences in transcript levels between osteoblastic and osteolytic metastases for a number of proposed osteoblastic and osteolytic factors including BMP2 and 7, tachykinin 1, endothelin 1, osteoprotegerin, and sclerostin. Furthermore, none of the proposed osteoblastic and osteolytic factors we interrogated by IHC showed a significant difference in protein expression between osteoblastic and osteolytic PCa bone metastases. This is the first detailed analysis of PCa osteoblastic and osteolytic factors in human specimens and animal models of PCa in the bone. Our data suggest that many of the factors important in bone remodeling may not be central to the bone response in PCa bone metastases. Additionally we have identified three factors that may have a role in bone remodeling in PCa bone metastases.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1532. doi:1538-7445.AM2012-1532
TMPRSS2 is an androgen-regulated cell surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority ...of human prostate cancer metastasis. Through the generation of mouse models with a targeted deletion of
Tmprss2
, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-Met receptor tyrosine kinase signaling, and initiated a pro-invasive EMT phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis
in vivo
. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment.
Abstract
There have been considerable advances in the detection, isolation and characterization of circulating tumor cells (CTC) and disseminated tumor cells (DTC) in prostate cancer (PCa) patients. ...However, the ability to interrogate these cells is restricted by the small number detected and isolated (typically <10), and current technologies available to characterize individual cells. We set out to determine the limit of commercially available technologies to obtain a transcriptomic profile of a single PCa cell.
To do this we isolated clonally selected Aphidicolin synchronized C4-2B PCa cells. Ten sets of C4-2B cells were isolated individually or in pools of 5 or 10 cells. All thirty samples were amplified using WT-Ovation™ One-Direct (NuGEN), hybridized on a 44K Whole Human Gene Expression Microarray (Agilent Technologies) normalized for batch effects, and normalized within and between arrays. The amplified material was also assessed by realtime-PCR.
Using a high stringency cut off (mean signal intensity of 300) on the oligonucleotide arrays 22,365 probes were Cy3 positive for 10 cells, 19,393 for 5 cells and 15,441 for 1 cell. Furthermore, using the same high stringency cut off, the sensitivity and specificity between 1 and 10 cells was 0.640 and 0.946 respectively, and between 1 and 5 cells was 0.713 and 0.932, demonstrating a very low false positive rate. Ten-thousand randomly selected pairs of commonly expressed genes had a Pearson correlation coefficient of 0.862 and 0.908 for 1 vs. 10 cells and 1 vs. 5 cells respectively, indicating that the vast majority of detected probes maintained consistent relative abundance. The cycle threshold values for single cell samples by realtime-PCR were well within the detectable range for abundant transcripts (e.g. AR and KLK3). However, other transcripts (e.g. FKBP5 and TMPRSS2) were not detected in the single cell samples and had limited detection in 5 cell samples when compared to 10 cell samples.
These data suggest that currently commercially available technologies allow for the detection of a limited but significant number of genes from a single PCa cell. We are using the methodology described herein, to determine the heterogeneity of single DTC isolated from the bone marrow of patients with PCa.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5242. doi:10.1158/1538-7445.AM2011-5242
PURPOSE: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the ...presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens. Experimental Methods: The castrate-resistant human xenograft LuCaP 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. RESULTS: In castrate mice, the tumors were delayed in G(2) with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G(1) or G(2) delay. Transforming growth factor-beta-stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. To further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 showed an increase in apoptosis and a delay in G(1). When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. CONCLUSIONS: These data indicate that IGF-I receptor inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22. (Clin Cancer Res 2009;15(24):7634-41).
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