Liyan Xue

Distinct Genomic Alterations in Prostate Cancers in Chinese and Western Populations Suggest Alternative Pathways of Prostate Carcinogenesis

Prostate cancer is significantly more common in Western men than in Asian men, but the basis for this difference remains unknown. Because genomic studies of Asian prostate cancer are very limited, we used a genome-wide approach to reveal the genomic alterations in Chinese prostate cancers. We found a significant reduction in the frequency of certain somatic genomic changes that are commonly found in Western prostate cancers, including the 21q22.2-22.3 deletion, which involves the TMPRSS2:ERG fusion gene, and 10q deletion, which causes PTEN inactivation. Array results were confirmed by PCR-based molecular copy-number counting in selected samples. The different frequencies of these genomic changes were further evaluated by fluorescent in situ hybridization and immunohistochemistry analyses of tissue microarray samples. These alterations might be key genetic changes underlying the regional/ethnic difference in clinical incidence and might be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Our findings suggest that tumors arise in Western and Chinese populations by alternative pathogenetic mechanisms.

The Association of CCND1 Overexpression and Cisplatin Resistance in Testicular Germ Cell Tumors and Other Cancers

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Absolute Quantitation of DNA Methylation of 28 Candidate Genes in Prostate Cancer Using Pyrosequencing

Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa from BPH. Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001). Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.

High-resolution genome-wide copy-number analysis suggests a monoclonal origin of multifocal prostate cancer

Many human cancers present as multifocal lesions. Understanding the clonal origin of multifocal cancers is of both etiological and clinical importance. The molecular basis of multifocal prostate cancer has previously been explored using a limited number of isolated markers and, although independent origin is widely believed, the clonal origin of multifocal prostate cancer is still debatable. We attempted to address clonal origin using a genome‐wide copy‐number analysis of individual cancer and high‐grade prostatic intraepithelial neoplasia (HGPIN) lesions. Using Affymetrix array 6.0 copy‐number analysis, we compared the genomic changes detected in 48 individual cancer and HGPIN lesions, isolated from 18 clinically localized prostate cancer cases. Identical genomic copy‐number changes, shared by all same‐case cancer foci, were detected in all 13 informative cases displaying multiple tumor foci. In addition, individual HGPIN lesions in the two multifocal‐HGPIN cases available shared identical genomic changes. Commonly known genomic alterations, including losses at 6q15, 8p21.3‐8p21.2, 10q23.2‐10q23.31, 16q22.3, 16q23.2‐16q23.3 and 21q22.2‐21q22.3 regions and gain of 8q24.3 were the most frequently detected changes in this study and each was detected in all same‐case foci in at least one case. Microarray data were confirmed by fluorescence in situ hybridization in selected foci. Our high‐resolution genome‐wide copy‐number data suggest that many multifocal cases derive from a single prostate cancer precursor clone and that this precursor may give rise to separate HGPIN foci and may further progress to multifocal invasive prostate cancer. These findings, which demonstrate the monoclonal origin of multifocal prostate cancer, should significantly enhance our understanding of prostate carcinogenesis.

Identification of ZDHHC14 as a novel human tumour suppressor gene

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase‐dependent apoptotic pathway and heterozygous knockout of ZDHHC14 decreased cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down‐regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

Abstract 4858: Identification of ZDHHC14 as a novel tumor suppressor gene commonly downregulated in human cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tumor suppressor genes (TSGs) play critical roles in preventing tumorigenesis and they are frequently inactivated in tumours. Recently developed high-density microarrays can detect subchromosomal deletions, recurrence of which usually indicates the location of TSGs within the deleted region. We analyzed testicular germ cell tumour (TGCT) clinical samples using SNP arrays and found a frequent small deletion on the region 6q25.3 containing only one known gene, ZDHHC14. While its cellular function is unknown, ZDHHC14 belongs to the recently discovered DHHC family, which are predicted to be involved in protein palmitoylation, a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Consistently, we found a dramatic under-expression of ZDHHC14 mRNA and protein in TGCTs, and this associated with chemoresistance. Oncomine database mining showed that ZDHHC14 is also under-expressed in lymphoma, liposarcoma, brain, kidney, lung and colorectal cancers. Thus, it appears that ZDHHC14 downregulation may be involved in other cancers. We studied ZDHHC14 expression in prostate cancer (PCa), detecting a decrease at mRNA and protein level. We also detected that ZDHHC14 mRNA was downregulated in a pilot study on breast cancer samples. As genomic loss of the ZDHHC14 region was only detected in a small number of PCa samples, we checked whether promoter hypermethylation was the cause for ZDHHC14 downregulation. However, no changes in methylation status were found. We then sequenced the whole genomic region surrounding ZDHHC14 by next generation sequencing in TGCTs and PCa and found several mutations in the promoter, the coding region, as well as in intronic regions. Finally, we tested the function of ZDHHC14 in cell-based studies. We generated a 293 T-REx tetracycline inducible ZDHHC14 overexpressing stable cell line, which showed that ZDHHC14 overexpression decreased cell viability. The induction of apoptosis by ZDHHC14 overexpression was detected both by FACS and caspase 7 and PARP cleavage analyses. This was confirmed by transient ZDHHC14 overexpression in the PCa cell line 22RV1. In vivo we xenografted mice using both tetracycline inducible ZDHHC14 overexpressing 293 T-REx cells and control cells transfected with the empty vector. ZDHHC14 expression was induced by tetracycline at the beginning of inoculation and we detected that ZDHHC14 overexpression blocked tumour initiation completely. In conclusion, these results implicate ZDHHC14 as a tumour suppressor gene commonly inactivated in human cancers, indicating that it might exert its tumor suppressor role through the induction of programmed cell death. This is the first study showing the involvement of ZDHHC14 in a specific pathway, the classic caspase-dependent apoptosis. 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 4858. doi:1538-7445.AM2012-4858

Abstract 3878: The different genetic alterations between Western and Chinese samples indicate alternate pathways of prostate cancer development

Prostate cancer is the most common male cancer in Western countries, but much less frequent in Asian countries. We systematically investigated genomic changes in prostate cancers from UK and China to determine genetic similarity and difference and potential alternative mechanisms of prostate carcinogenesis in these high and low cancer incidence populations. Using Affymetrix array 6.0 microarrays, we analyzed genome-wide genomic alterations in 32 UK and 39 Chinese prostate cancer samples. Distinct difference in genomic alterations between Western and Chinese prostate cancers were found, including loss of 21q22 and PTEN deletion, which are the most common genomic changes in Western prostate cancer but rarely detected in the Chinese samples. To further evaluate the difference between Western and Chinese samples, FISH analysis for PTEN deletion and ERG rearrangements and immunohistochemistry analysis for PTEN and ERG expressions were applied to UK (n=160) and Chinese (n=143) prostate cancer tissue microarrays (TMAs). PTEN deletion and ERG rearrangements were found at a significantly higher frequency in samples from UK (42.3% and 37.4%) than China (14.3% and 7.5% respectively) (P In conclusion, we compared genetic alterations in prostate cancer samples from UK and China and found there are significant differences between the two groups, commencing at pre-invasive, HGPIN, stage. Our findings suggest that tumours arise in Western and Chinese populations by alternative pathogenetic mechanisms. These alterations, differentially presented in these two populations may be key genetic changes underlying the regional/ethnic difference in clinical incidence and may be induced by specific environmental and/or genetic risk factors that Western men are exposed to. 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 3878. doi:10.1158/1538-7445.AM2011-3878

Abstract 4831: Monoclonal origin of multifocal cancer and the step-wise genetic events of prostate carcinogenesis

Prostate cancer is commonly detected as multiple foci in a single prostate gland and it is of etiological and clinical importance to identify the origin of these multifocal cancer lesions. Due to the limited number of markers analyzed, previous studies to address this issue are inconclusive. A high-resolution genome-wide study should address this issue better. Therefore, we have performed a genome-wide analysis using Affymetrix Array 6.0 of individually laser-capture microdissected foci to compare the genetic similarities and/or differences of these foci within the same prostate gland. Fluorescence in situ hybridization was used to confirm SNP array results. A total of 43 cancer and high-grade prostatic intraepithelial neoplasia (PIN) foci from 16 prostate cancer cases were analyzed for genomic copy number changes. Copy number changes from same-case foci were compared to determine the genetic relationship between the foci. In many cases, we found extensive genetic differences between different foci isolated from the same case. However, omniclonal copy number gains and losses were detected in 11/11 informative case-matched multifocal tumor cases and 8/8 informative case-matched PIN and tumor cases, suggesting that multiple cancer foci arise from a common prostate cancer precursor clone. In addition, a higher degree of similarity was observed between PIN and adjacent cancer foci, as compared to distant lesions. The strong genetic similarities between PIN and prostate cancer foci from the same region suggest clonal expansion of separate PIN lesions to invasive cancer. Based on the monoclonal model, we examined the step-wise accumulation of these genomic copy number changes. Loss of 10q23.2-10q23.31 and 16q were commonly shared in same-case tumor foci and, in many cases, also matched PIN foci, suggesting that they are early events in prostate carcinogenesis. Other common genomic changes, such as loss of 6q, 8p and 21q22.2-21q22.3, were found less frequently in all same-case foci and rarely in PIN lesions, suggesting these genetic events occur at a relatively later stage. In conclusion, using a high-density SNP array, we reveal a monoclonal origin for the majority of multifocal prostate cancer samples analyzed. From these SNP array data, we have also begun to elucidate the step-wise genetic events in prostate carcinogenesis, including early events such as loss of 10q23.2-10q23.31 and 16q Taken together, these findings should significantly enhance our understanding of prostate carcinogenesis and potentially affect the clinical management of this disease. 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 4831. doi:10.1158/1538-7445.AM2011-4831