Amplification of the 9p13.3 chromosomal region in prostate cancer.

Amplification of the 9p13.3 chromosomal region occurs in a subset of prostate cancers (PCs); however, the target gene or genes of this amplification have remained unidentified. The aim of this study was to investigate the 9p13.3 amplification in more detail to identify genes that are potentially advantageous for cancer cells. We narrowed down the minimally amplified area and assessed the frequency of the 9p13.3 amplification. Of the clinical samples from untreated PCs that were examined (n = 134), 9.7% showed high-level amplification, and 32.1% showed low-level amplification. Additionally, in clinical samples from castration-resistant PCs (n = 70), high- and low-level amplification was seen in 14.3% and 44.3% of the samples, respectively. We next analyzed the protein-coding genes in this chromosomal region for both their expression in clinical PC samples as well as their potential as growth regulators in PC cells. We found that the 9p13.3 amplification harbors several genes that are able to affect the growth of PC cells when downregulated using siRNA. Of these, UBAP2 was the most prominently upregulated gene in the clinical prostate tumor samples. © 2016 Wiley Periodicals, Inc.

Prevalence and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers.

BACKGROUND: TMPRSS2-ERG gene fusion is the most frequent genetic alteration in prostate cancer. However, information about its distribution in lymph node positive prostate cancers and the prognostic significance in these advanced tumors is unknown. METHODS: Gene fusion status was determined by fluorescence in situ hybridization on a tissue-microarray constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from the primary tumors and corresponding lymph node metastases. Data were correlated with various tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, disease-specific, and overall survival. RESULTS: TMPRSS2-ERG fusion was detected in 43.5% of the primary tumors. Conversely, only 29.9% of the metastasizing components showed the fusion. Concordance in TMPRSS2-ERG status between primary tumors and metastases was 70.9% (Kappa 0.39); 20.9% and 8.1% of the patients showed the mutation solely in their primary tumors and metastases, respectively. TMPRSS2-ERG fusion was not correlated with specific histopathological tumor features but predicted favorable biochemical recurrence-free, disease-specific and overall survival independently when present in the primary tumor (P < 0.05 each). CONCLUSION: TMPRSS2-ERG fusion is more frequent in primary prostate cancer than in corresponding metastases suggesting no selection of fusion-positive cells in the metastatic process. The gene fusion in primary tumors independently predicts favorable outcome.

The miR-15a-miR-16-1 locus is homozygously deleted in a subset of prostate cancers.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate the expression of protein coding genes. In this study, we screened highly informative prostate cancer cell lines and xenografts (n = 42) for miRNA gene copy number and expression changes. The expression profiling showed distinction between cell lines and xenografts as well as between androgen sensitive and independent models. Only a few copy number alterations that were associated with expression changes were identified. Most importantly, the miR-15a-miR-16-1 locus was found to be homozygously deleted in two samples leading to the abolishment of miR-15a, but not miR-16, expression. miR-16 is also expressed from another genomic locus. Mutation screening of the miR-15a-miR-16-1 gene in the model systems as well as clinical samples (n = 50) revealed no additional mutations. In conclusion, our data indicate that putative tumor suppressors, miR-15a and miR-16-1, are homozygously deleted in a subset of prostate cancers, further suggesting that these miRNAs could be important in the development of prostate cancer.

Methods for identifying and studying genetic alterations in hormone-dependent cancers.

Genetic alterations underlying the development of cancer include large chromosomal aberrations, such as amplifications, deletions and translocations as well as small changes in sequence, i.e. mutations. Thus, different methods are needed to reveal various types of genetic changes. Fluorescence in situ hybridisation (FISH) is a versatile technique for detecting chromosomal alterations either in cultured cells or even in formalin-fixed paraffin-embedded tissue. For screening mutations, denaturing high-performance liquid chromatography (DHPLC) provides a relatively fast, cheap and sensitive option. The only special requirement is the HPLC equipment suitable for the analysis. As a screening tool, it does not reveal the actual base pair change, which in the end needs to be done by sequencing. FISH and DHPLC can both be utilized in research as well as in clinical diagnostic laboratories.

TMPRSS2:ERG fusion identifies a subgroup of prostate cancers with a favorable prognosis.

PURPOSE: Our aim was to assess the frequency of ERG overexpression and TMPRSS2:ERG rearrangement in prostate cancer and their association with clinicopathologic variables and outcome. EXPERIMENTAL DESIGN: The presence of the TMPRSS2:ERG rearrangement was studied by reverse transcription-PCR and fluorescence in situ hybridization in 19 prostate cancer xenografts and 7 prostate cancer cell lines. The expression of ERG was studied in the xenografts and cell lines and in 49 freshly frozen clinical prostate samples by quantitative reverse transcription-PCR. The frequency of the TMPRSS2:ERG fusion in clinical prostate cancer (n = 253) on tissue microarrays was assessed by three-color fluorescence in situ hybridization. RESULTS: Seven of 19 (37%) of the xenografts overexpressed ERG and had TMPRSS2:ERG rearrangement. Two xenografts, representing small cell carcinomas, also contained the fusion but did not express ERG. In clinical tumor specimens, the overexpression of ERG was associated with the rearrangement (P = 0.0019). Fifty of 150 (33%) of the prostatectomy specimens and 28 of 76 (37%) of the hormone-refractory prostate cancers on the tissue microarrays carried the TMPRSS2:ERG rearrangement. It was associated with longer progression-free survival in patients treated by prostatectomy (P = 0.019), and according to multivariate analysis, it was an independent predictor of favorable outcome (relative risk, 0.54; 95% confidence interval, 0.30-0.98). The fusion was not associated with Gleason score, pT stage, diagnostic prostate-specific antigen, or cell proliferation activity in prostatectomy specimens nor with the AR gene amplification in hormone-refractory tumors. CONCLUSIONS: The TMPRSS2:ERG rearrangement can be found in about one third of prostate cancers. A subgroup of prostate cancer patients with a good prognosis may be identified by the rearrangement.

Clusterin is epigenetically regulated in prostate cancer.

Lack of good models has complicated investigations on the mechanisms of prostate cancer. By far, the most commonly used transgenic mouse model of prostate cancer is TRAMP, which, however, has not been fully characterized for genetic and epigenetic aberrations. Here, we screened TRAMP-derived C2 cell line for the alterations using different microarray approaches, and compared it to human prostate cancer. TRAMP-C2 had relatively few genomic copy number alterations according to array comparative genomic hybridization (aCGH). However, the gene copy number and expression were significantly correlated (p < 0.001). Screening genes for promoter hypermethylation using demethylation treatment with 5-aza-2'-deoxycytidine and subsequent expression profiling indicated 43 putatively epigenetically silenced genes. Further studies revealed that clusterin is methylated in the TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Its expression was found to be significantly reduced (p < 0.01) in untreated and hormone-refractory human prostate carcinomas. Together with known function of clusterin, the data suggest an epigenetic component in the regulation of clusterin in prostate cancer.

CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations.

Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach.Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired.These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

No amplifications of hypoxia-inducible factor-1alpha gene in invasive breast cancer: a tissue microarray study.

OBJECTIVE: Hypoxia Inducible Factor-1 (HIF-1) is an important transcription factor that stimulates tumour growth and metastases via several pathways, including angiogenesis and altered metabolism. Activation of HIF-1 depends on the levels of its alpha-subunit, which increase during hypoxia. Recent studies showed that the HIF-1alpha gene was amplified in prostate cancer, leading to overexpression of HIF-1alpha at normoxia. The aim of this study was to evaluate the presence of HIF-1alpha gene amplifications in invasive breast cancer as an explanation for HIF-1alpha protein overexpression. METHODS: Protein and gene expression of HIF-1alpha were analyzed on a tissue microarray of 94 breast cancers by immunohistochemistry and fluorescent in situ hybridization (FISH), respectively. RESULTS: Overexpression of HIF-1alpha protein was found in 58/94 (62%) of patients. No amplifications of the HIF-1alpha gene were detected. CONCLUSION: Increased protein levels of HIF-1alpha are not associated with amplification of the HIF-1alpha gene in human breast cancer. Therefore, other mechanisms than gene amplification must be responsible for HIF-alpha overexpression at normoxia.

Mapping of the chromosomal amplification 1p21-22 in bladder cancer.

BACKGROUND: The aim of the study was to characterize a recurrent amplification at chromosomal region 1p21-22 in bladder cancer. METHODS: ArrayCGH (aCGH) was performed to identify DNA copy number variations in 7 clinical samples and 6 bladder cancer cell lines. FISH was used to map the amplicon at 1p21-22 in the cell lines. Gene expression microarrays and qRT-PCR were used to study the expression of putative target genes in the region. RESULTS: aCGH identified an amplification at 1p21-22 in 10/13 (77%) samples. The minimal region of the amplification was mapped to a region of about 1 Mb in size, containing a total of 11 known genes. The highest amplification was found in SCaBER squamous cell carcinoma cell line. Four genes, TMED5, DR1, RPL5 and EVI5, showed significant overexpression in the SCaBER cell line compared to all the other samples tested. Oncomine database analysis revealed upregulation of DR1 in superficial and infiltrating bladder cancer samples, compared to normal bladder. CONCLUSIONS: In conclusions, we have identified and mapped chromosomal amplification at 1p21-22 in bladder cancer as well as studied the expression of the genes in the region. DR1 was found to be significantly overexpressed in the SCaBER, which is a model of squamous cell carcinoma. However, the overexpression was found also in a published clinical sample cohort of superficial and infiltrating bladder cancers. Further studies with more clinical material are needed to investigate the role of the amplification at 1p21-22.

Loss of PTEN is associated with aggressive behavior in ERG-positive prostate cancer.

BACKGROUND: The associations of ERG overexpression with clinical behavior and molecular pathways of prostate cancer are incompletely known. We assessed the association of ERG expression with AR, PTEN, SPINK1, Ki-67, and EZH2 expression levels, deletion, and mutations of chromosomal region 3p14 and TP53, and clinicopathologic variables. METHODS: The material consisted of 326 prostatectomies, 166 needle biopsies from men treated primarily with endocrine therapy, 177 transurethral resections of castration-resistant prostate cancers (CRPC), and 114 CRPC metastases obtained from 32 men. Immunohistochemistry, FISH, and sequencing was used for the measurements. RESULTS: ERG expression was found in about 45% of all patient cohorts. In a multivariate analysis, ERG expression showed independent value of favorable prognosis (P = 0.019). ERG positivity was significantly associated with loss of PTEN expression in prostatectomy (P = 0.0348), and locally recurrent CRPCs (P = 0.0042). Loss of PTEN expression was associated (P = 0.0085) with shorter progression-free survival in ERG-positive, but not in negative cases. When metastases in each subject were compared, consistent ERG, PTEN, and AR expression as well as TP53 mutations were found in a majority of subjects. CONCLUSIONS: A similar frequency of ERG positivity from early to late stage of the disease suggests lack of selection of ERG expression during disease progression. The prognostic significance of PTEN loss solely in ERG-positive cases indicates interaction of these pathways. The finding of consistent genetic alterations in different metastases suggests that the major genetic alterations take place in the primary tumor. IMPACT: Interaction of PTEN and ERG pathways warrants further studies.

Contribution of ARLTS1 Cys148Arg (T442C) variant with prostate cancer risk and ARLTS1 function in prostate cancer cells.

ARLTS1 is a recently characterized tumor suppressor gene at 13q14.3, a region frequently deleted in both sporadic and hereditary prostate cancer (PCa). ARLTS1 variants, especially Cys148Arg (T442C), increase susceptibility to different cancers, including PCa. In this study the role of Cys148Arg substitution was investigated as a risk factor for PCa using both genetic and functional analysis. Cys148Arg genotypes and expression of the ARLTS1 were explored in a large set of familial and unselected PCa cases, clinical tumor samples, xenografts, prostate cancer cell lines and benign prostatic hyperplasia (BPH) samples. The frequency of the variant genotype CC was significantly higher in familial (OR = 1.67, 95% CI = 1.08-2.56, P = 0.019) and unselected patients (OR = 1.52, 95% CI = 1.18-1.97, P = 0.001) and the overall risk was increased (OR = 1.54, 95% CI = 1.20-1.98, P = 0.0007). Additional analysis with clinicopathological data revealed an association with an aggressive disease (OR = 1.28, 95% CI = 1.05-∞, P = 0.02). The CC genotype of the Cys148Arg variant was also contributing to the lowered ARLTS1 expression status in lymphoblastoid cells from familial patients. In addition significantly lowered ARLTS1 expression was observed in clinical tumor samples compared to BPH samples (P = 0.01). The ARLTS1 co-expression signature based on previously published microarray data was generated from 1587 cancer samples confirming the low expression of ARLTS1 in PCa and showed that ARLTS1 expression was strongly associated with immune processes. This study provides strong confirmation of the important role of ARLTS1 Cys148Arg variant as a contributor in PCa predisposition and a potential marker for aggressive disease outcome.

Association of SPINK1 expression and TMPRSS2:ERG fusion with prognosis in endocrine-treated prostate cancer.

PURPOSE: The aim of the study was to examine whether TMPRSS2:ERG fusion or SPINK1 protein expression is associated with hormone responsiveness of prostate cancer and can thus be used as a biomarker. EXPERIMENTAL DESIGN: Diagnostic needle biopsies from prostate cancer patients primarily treated by endocrine therapy were evaluated for TMPRSS2:ERG fusion with fluorescence in situ hybridization and SPINK1 protein expression with immunohistochemistry. RESULTS: The frequency of TMPRSS2:ERG fusion in 178 biopsies of hormonally treated patients was 34%. Of the fusion-positive cases, 71% showed deletion between the two genes, and 23% showed gain of the fusion. The fusion was associated with high Ki-67 staining (P=0.001), age at diagnosis (P=0.024), and tumor area (P=0.006), but not with Gleason score, T stage, M stage, prostate-specific antigen (PSA), or progression-free survival. Strong positive SPINK1 expression was found in 11% (21 of 186) of the biopsies. SPINK1-positive cases had significantly shorter progression-free survival compared with SPINK1-negative cases (P=0.001). The expression was not associated with any other clinicopathologic variables studied. In a multivariate analysis, SPINK1 expression showed independent prognostic value, with a relative risk of 2.3 (95% confidence interval, 1.1-4.6). SPINK1 expression and the fusion were not associated with each other. CONCLUSIONS: There was no association between TMPRSS2:ERG fusion and prognosis, suggesting that TMPRSS2:ERG rearrangement does not implicate hormone dependence of the cancer. SPINK1 expression, found in approximately 10% of prostate cancers, was associated with aggressive form of the disease and could serve as a biomarker in endocrine-treated prostate cancer.

Genetic aberrations in prostate cancer by microarray analysis.

The aim of this study was to screen genetic as well as expression alterations in prostate cancer. Array comparative genomic hybridization (aCGH) to a 16K cDNA microarray was performed to analyze DNA sequence copy number alterations in 5 prostate cancer cell lines and 13 xenografts. The aCGH confirmed the previously implicated common gains and losses, such as gains at 1q, 7, 8q, 16p and 17q and losses at 2q, 4p/q, 6q, 8p, 13q, 16q, 17p and 18q, which have previously been identified by chromosomal CGH (cCGH). Because of the higher resolution of aCGH, the minimal commonly altered regions were significantly narrowed-down. For example, the gain of 8q was mapped to three independent regions, 8q13.3-q21.11, 8q22.2 and 8q24.13-q24.3. In addition, a novel recurrent gain at 9p13-q21 was identified. The concomitant expression analysis indicated that genome-wide DNA sequence copy number (gene dosage) was significantly associated with the expression level (p < 0.0001). The analyses indicated several individual genes whose expression was associated with the gene copy number. For example, gains of PTK2 and FZD6, were associated with the increased expression, whereas losses of TNFRSF10B (alias DR5) and ITGA4 with decreased expression. In conclusion, the aCGH mapping data will aid in the identification of genes altered in prostate cancer. The combined expression and copy number analysis suggested that even a low-level copy number change may have significant effect on gene expression, and thus on the development of prostate cancer.

The gene for polycomb group protein enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate cancer.

Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) has been found in several malignancies, including prostate cancer, with an aggressive phenotype. Amplification of the gene has previously been demonstrated in several malignancies, but not in prostate cancer. Our goal was to evaluate the gene copy number and expression alterations of EZH2 in prostate cancer. The copy number of EZH2 in cell lines (LNCaP, DU145, PC-3, 22Rv1), xenografts (n = 10), and clinical tumors (n = 191) was studied with fluorescence in situ hybridization. All cell lines had a gain of EZH2. Eight of the ten xenografts showed an increased copy number of the gene, including one case of high-level amplification (>or=5 copies of the gene and EZH2/centromere ratio >or=2). 34/125 (27%) of untreated prostate carcinomas showed increased copy number, but only one case of low-level amplification (>or=5 copies of the gene and EZH2/centromere ratio <2), whereas half (25/46) of the hormone-refractory carcinomas showed increased copy number, including seven cases of low-level amplification and three cases of high-level amplification (P < 0.0001). Expression of EZH2 was significantly (P = 0.0009) higher in hormone-refractory prostate cancer compared with that in benign prostatic hyperplasia or untreated cancer, according to quantitative real-time RT-PCR assay. Also, the expression of EZH2 protein was found to be higher in hormone-refractory tumors than in hormone-naïve tumors by immunohistochemistry. The EZH2 gene amplification was significantly (P < 0.05) associated with increased EZH2 protein expression. The data show that amplification of the EZH2 gene is rare in early prostate cancer, whereas a fraction of late-stage tumors contains the gene amplification leading to the overexpression of the gene, thus indicating the importance of EZH2 in the progression of prostate cancer.

Expression and gene copy number analysis of ERBB2 oncogene in prostate cancer.

An anti-ERBB2 antibody, trastuzumab, has been shown to be highly efficient in the treatment of metastatic breast cancers overexpressing the ERBB2 gene. It has been suggested that overexpression and even amplification of ERBB2 may play a role in the development of prostate cancer. Here, we have analyzed gene copy number and expression of the ERBB2 gene in both androgen-dependent primary and metastatic tumors, as well as recurrent hormone-refractory tumors. The expression levels were compared to the expression of ERBB2 in breast cancers with or without ERBB2 gene amplification. Of 126 prostate tumors, chromogenic in situ hybridization (CISH) revealed only 1 case containing borderline (six to eight copies) amplifications of ERBB2. This hormone-refractory tumor, however, did not express ERBB2 protein. Immunohistochemical staining of ERBB2 protein was negative (0 or 1+ intensity) in all prostate samples (n = 124) analyzed. To quantitate the level of ERBB2 mRNA expression in prostate tumors (n = 34) and cell lines (n = 3), as well as in breast tumors (n = 30) and cell lines (n = 16), real-time reverse transcriptase-polymerase chain reaction (LightCycler) methodology was used. The expression level was similar in all prostate tumor types and corresponded to the level of expression in breast carcinomas without ERBB2 amplification. Breast tumors with ERBB2 amplification expressed, on average, approximately 20 times (P < 0.001) higher mRNA levels than prostate tumors or breast carcinomas without the gene amplification. In conclusion, the expression of ERBB2 in prostate cancer is relatively low, and is not altered during disease progression. Thus, it is unlikely that treatment modalities relying on the overexpression of ERBB2 gene will be useful in treating prostate cancer.