Prostate cancer is a heterogeneous group of diseases and there is a need for more efficient and targeted methods of treatment. In this study, the potential of gene expression data and RNA ...interference technique were combined to advance future personalized prostate cancer therapeutics. To distinguish the most promising in vivo prevalidated prostate cancer drug targets, a bioinformatic analysis was carried out using genome-wide gene expression data from 9873 human tissue samples. In total, 295 genes were selected for further functional studies in cultured prostate cancer cells due to their high mRNA expression in prostate, prostate cancer or in metastatic prostate cancer samples. Second, RNAi based cell viability assay was performed in VCaP and LNCaP prostate cancer cells. Based on the siRNA results, gene expression patterns in human tissues and novelty, endoplasmic reticulum function associated targets AIM1, ERGIC1 and TMED3, as well as mitosis regulating TPX2 were selected for further validation. AIM1, ERGIC1, and TPX2 were shown to be highly expressed especially in prostate cancer tissues, and high mRNA expression of ERGIC1 and TMED3 associated with AR and ERG oncogene expression. ERGIC1 silencing specifically regulated the proliferation of ERG oncogene positive prostate cancer cells and inhibited ERG mRNA expression in these cells, indicating that it is a potent drug target in ERG positive subgroup of prostate cancers. TPX2 expression associated with PSA failure and TPX2 silencing reduced PSA expression, indicating that TPX2 regulates androgen receptor mediated signaling. In conclusion, the combinatorial usage of microarray and RNAi techniques yielded in a large number of potential novel biomarkers and therapeutic targets, for future development of targeted and personalized approaches for prostate cancer management.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first ...conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7 , HPGD , EPHX2 , and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG -positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.
Immunohistochemical detection of prostate-specific antigen (PSA) is an aid in determining the prostatic origin of metastatic cells. However, small amounts of PSA have also been found in non-prostatic ...tissues and tumors, for example in some breast carcinomas, by highly sensitive immunofluorometric methods, but also by immunohistochemistry. Our aim was to evaluate the prevalence and prognostic value of histologically confirmed PSA immunoreactivity in breast carcinoma. Sections of formalin-fixed, paraffin-embedded samples from 171 breast carcinomas were immunostained for PSA. The staining results were compared with the mitotic activity, tumor size, histological grade, steroid receptors and follow-up data. For analysis the material was divided into subgroups according to the patients' age (pre- and postmenopausal). PSA was found by immunohistochemistry in 54 (32%) breast carcinomas. In survival analysis of the whole patient material PSA positivity did not show prognostic value. Among premenopausal patients concomitant estrogen receptor and PSA-negativity proved to be associated with high risk of breast cancer death (RR 6.2), also after adjustment for tumor size, histological grade, and axillary lymph node status. Among postmenopausal patients PSA positivity was associated with progesterone receptor positivity and high differentiation but not with age, nodal status, or mitotic activity. PSA can be detected by immunohistochemistry in a considerable number of breast carcinomas. PSA immunoreactivity alone does not seem to have any value as general prognosticator of breast carcinoma patients. However, concomitant absence of PSA and estrogen receptors was an indicator of unfavourable prognosis among premenopausal patients.
Abstract
There is an urgent need for reliable markers to identify patients whose PCa is most likely to recur after initial therapy and progress to lethal disease. While Gleason score is considered ...the most accurate predictive marker for disease-specific mortality after primary treatment of localized prostate cancer (PCa), the majority of PCas cluster into intermediate group of Gleason scores 6 and 7 where there is considerable variation in treatment response, disease recurrence and disease-specific death. In this study, we analyzed the predictive value of elevated nuclear Stat5a/b expression in PCas treated by radical prostatectomy or active surveillance. The critical role of Stat5a/b in promotion of PCa growth and progression has been well-documented in preclinical PCa models. Specifically, Stat5a/b is critical for prostate cancer cell viability in vitro and for prostate tumor growth in vivo, and Stat5a/b promotes metastatic dissemination of prostate cancer in nude mice. Here, we demonstrate that in intermediate Gleason score PCas treated by radical prostatectomy, high levels of nuclear Stat5a/b predicted both early recurrence (univariate analysis; p<0.0001, multivariate analysis; HR=0.55, p=0.017) and early PCa-specific death (univariate analysis; p=0.028). In addition, high nuclear Stat5a/b predicted early disease recurrence in both univariate (p<0.0001) and multivariate (HR=0.62; p<0.01) analysis in the entire cohort of patients treated by radical prostatectomy (n=562) regardless of the Gleason score. In the second group of patients treated by active surveillance, elevated nuclear Stat5a/b expression was associated with early PCa-specific death (p=0.0453) by univariate analysis (Cox regression analysis: 60% increased risk of death, p=0.034). If confirmed in future prospective studies, nuclear Stat5a/b may become a useful independent predictive marker of recurrence of lethal PCa after radical prostatectomy for intermediate Gleason score PCas.
Citation Format: Tuomas Mirtti, Benjamin E. Leiby, Pär Stattin, Anders Bergh, Marja T. Nevalainen, Junaid Abdulghani, Miia Pavela, Elina Aaltonen, Anita Mamtani, Kalle A. Alanen, Lars Egevad, Torvald Granfors, Andreas Josefsson. Nuclear Stat5a/b predicts early recurrence and prostate cancer-specific death in patients treated by radical prostatectomy abstract. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C41.
The nuclear DNA content of 62 pancreatic adenocarcinomas was analysed by flow cytometry from paraffin-embedded material. Radical surgery could be performed in 12 of the 24 cases with diploid ...carcinoma, but only in 3 of the 38 cases with a non-diploid tumour (P = 0.0002); the radically resected carcinomas also had a lower fraction of cells in the S-phase (P = 0.009). Non-diploid nuclear DNA content (38 cases, 61 per cent) was associated with advanced stage (P = 0.002), poor histological differentiation (grade II or III, P = 0.004), and primary tumour site in the body or the tail as compared with the head (P = 0.01). The median survival time of the patients with diploid carcinoma was 13 +/- 3 (SE) months, and that of the patients with non-diploid carcinoma 3 +/- 1 months (P = 0.0001). The DNA index with the cutoff value 1.4 was a slightly more powerful prognostic factor than DNA ploidy, and it was the most important independent prognostic factor in Cox's multivariate analysis (P less than 0.001) followed by histological grade (P less than 0.03). We conclude that diploid pancreatic carcinomas are associated with a longer survival than the non-diploid ones, and that radically operable carcinomas form a special subgroup with frequent diploidy and less aggressive biological behaviour.
To investigate whether breast carcinomas found to be DNA diploid by flow cytometry (FCM) are still diploid if reassessed by image cytometry (ICM).
In a series of 286 breast cancers analyzed by FCM ...there were 100 (35%) cancers that were classified as DNA diploid. Fourteen of the 100 diploid cases were selected for further analysis with ICM because the patient had died of breast cancer within 11-84 months after the diagnosis (a group with unfavorable outcomes), and 19 cases were selected at random from the cases who had no recurrence of cancer during follow-up of six or more years (a favorable group).
Eleven (33%) of the 33 cases turned out to be DNA nondiploid, with a DNA index > or = 1.2 when analyzed by ICM. Nine of the 11 DNA aneuploid samples by ICM were found among the 14 patients with unfavorable prognoses and only 2 among the 19 patients with favorable outcomes (P = .002). The five-year survival rate of the women with DNA diploid cancer by both methods was 86%, whereas that of patients with DNA aneuploid cancer by ICM was 36% (P = .002).
The results show that some breast carcinomas classified as DNA diploid based on FCM are not DNA diploid by ICM and that such carcinomas are associated with poorer outcomes than the ones that are DNA diploid also by ICM. The prognostic significance of DNA ploidy in breast cancer may need to be reexamined in studies where both FCM and ICM are used.
The relationship between myocardial ATP content and the increase in left ventricular resting tension during ischaemia (ischaemic contracture) was studied in isolated rat hearts perfused for 15 min ...either with aerobic buffer (pO2 greater than 500 mmHg) containing non-glycolytic substrate, acetate (5 mmol X litre-1), or with hypoxic buffer (pO2 less than 10 mmHg) with glucose (10 mmol X litre-1) before making them totally ischaemic for 10 min. ATP was determined spectrophotometrically from extracts of frozen whole hearts. Left ventricular tension was recorded by intraventricular balloon catheter. Myocardial ATP content was 15.4 +/- 1.0 mumol X g-1 dry weight (mumol X g-1) after 10 min stabilising period, 14.1 +/- 0.9 mumol X g-1 after 15 min aerobic perfusion (plus acetate) and 9.0 +/- 1.3 mumol X g-1 after 15 min hypoxic perfusion (plus glucose). During 10 min of ischaemia ATP decreased in aerobic hearts to 5.4 +/- 1.1 mumol X g-1 and to 7.9 +/- 1.0 mumol X g-1 in hypoxic hearts. The left ventricular resting tension increased during ischaemia in hypoxic hearts to 9 +/- 5% of control systolic pressure (0 = diastolic pressure, 100 = systolic pressure during stabilising period), whereas in aerobic hearts the tension began to increase immediately and was 84 +/- 22% of systolic pressure at the end of the ischaemic period. In parallel control experiments, hearts were also perfused either with glucose-containing aerobic buffer or acetate-containing hypoxic buffer. ATP was well preserved during aerobic perfusion (plus glucose) but decreased markedly during hypoxic perfusion (plus acetate). There was no increase in resting tension in the aerobic hearts (plus glucose) whereas the resting tension increased considerably during hypoxic perfusion (plus acetate). The results indicate that the initiation of ischaemic contracture occurs at much higher myocardial ATP level when ATP comes from mitochondrial sources than when ATP is generated by anaerobic glycolysis.
TMPRSS2-ERG and other gene fusions involving ETS factors and genes with strong promoter elements are common in prostate cancer. Although ERG activation has been linked to invasive properties of ...prostate cancers, the precise mechanisms and pathways of ERG-mediated oncogenesis remain poorly understood. Here, we show that ERG knockdown in VCaP prostate cancer cells causes an activation of cell adhesion, resulting in strongly induced active beta(1)-integrin and E-cadherin expression as well as changes in WNT signaling. These observations were corroborated by data from ERG-overexpressing nontransformed prostate epithelial cells as well as gene expression data from clinical prostate cancer samples, which both indicated a link between ERG and epithelial-to-mesenchymal transition (EMT). Upregulation of several WNT pathway members was seen in ERG-positive prostate cancers, with frizzled-4 (FZD4) showing the strongest overexpression as verified by both reverse transcription-PCR and immunostaining. Both ERG knockin and knockdown modulated the levels of FZD4 expression. FZD4 silencing could mimic the ERG knockdown phenotype by inducing active beta(1)-integrin and E-cadherin expression, whereas FZD4 overexpression reversed the phenotypic effects seen with ERG knockdown. Taken together, our results provide mechanistic insights to ERG oncogenesis in prostate cancer, involving activation of WNT signaling through FZD4, leading to cancer-promoting phenotypic effects, including EMT and loss of cell adhesion.
There are currently no effective therapies for metastatic prostate cancer because the molecular mechanisms that underlie the metastatic spread of primary prostate cancer are unclear. Transcription ...factor Stat3 is constitutively active in malignant prostate epithelium, and its activation is associated with high histological grade and advanced cancer stage. In this work, we hypothesized that Stat3 stimulates metastatic progression of prostate cancer. We show that Stat3 is active in 77% of lymph node and 67% of bone metastases of clinical human prostate cancers. Importantly, adenoviral gene delivery of wild-type Stat3 (AdWTStat3) to DU145 human prostate cancer cells increased the number of lung metastases by 33-fold in an experimental metastasis assay compared with controls. Using various methods to inhibit Stat3, we demonstrated that Stat3 promotes human prostate cancer cell migration. Stat3 induced the formation of lamellipodia in both DU145 and PC-3 cells, further supporting the concept that Stat3 promotes a migratory phenotype of human prostate cancer cells. Moreover, Stat3 caused the rearrangement of cytoplasmic actin stress fibers and microtubules in both DU145 and PC-3 cells. Finally, inhibition of the Jak2 tyrosine kinase decreased both activation of Stat3 and prostate cancer cell motility. Collectively, these data indicate that transcription factor Stat3 is involved in metastatic behavior of human prostate cancer cells and may provide a therapeutic target to prevent metastatic spread of primary prostate cancer.
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