Impact of demographic and perioperative risk factors on complication rates in skin-sparing/nipple-sparing mastectomy with implant-based reconstruction using titanized polypropylene mesh (TiLOOP® Bra).

BACKGROUND: Skin/nipple-sparing mastectomies (SSM/NSSM) have been reported to have acceptable complication rates and good aesthetic outcomes with high patient satisfaction. However, in this relatively young and rapidly expanding field of reconstructive plastic surgery, differences in perioperative management are noted between breast centers. Prospective studies of complication rates using a titanized polypropylene mesh (TiLOOP® Bra) are currently lacking. METHODS: A prospective subgroup analysis was performed based on the data set of the prospective, single-arm, multicenter observational study (PRO-BRA). Early complication rates after skin/nipple-sparing mastectomy with implant-based immediate or secondary reconstruction using a titanized polypropylene mesh (TiLOOP® Bra) subpectorally were investigated in relation to demographic factors, as well as intra-and postoperative management. The subgroup consists of 258 patients. Complications were categorised into necrosis, infection, postoperative bleeding or hematoma, seroma, wound healing delays and R1-situations. RESULTS: Early complication rates of SSM/NSSM using titanium-based meshes are comparable to complication-rates using ADM's. Logistic regression shows significantly higher risk for wound healing delays, necrosis and seroma with increasing BMI, abladat- and implant-weight (OR 1,17 -1,66, p-value < 0,001). Smokers have significantly higher necrosis rates (20.7%) compared to non-smokers (5.5%) (p-value = 0.002). Discharge with drainage results in a trend toward higher rates of wound healing complications. CONCLUSION: The use of TiLOOP® Bra meshes was shown to have acceptable complication rates. Complication rates depend on certain demographic and intraoperative risk factors and should be considered in indications and information of patients.

Patient reported outcome and cosmetic evaluation following implant-based breast-reconstruction with a titanized polypropylene mesh (TiLOOP® Bra): A prospective clinical study in 269 patients.

INTRODUCTION: Implant-based or expander-supported breast reconstruction is an established surgical method after mastectomies due to cancer or to prophylactic reasons. Patient reported outcome (PRO) and cosmetic outcome after breast reconstruction with a synthetic surgical mesh was investigated in a prospective, single-arm, multi-center study. MATERIAL AND METHODS: Primary or secondary implant-based breast reconstruction with support of TiLOOP® Bra was performed in 269 patients during the PRO-BRA study. PRO 12 months after breast reconstruction was evaluated using Breast-Q questionnaire. Cosmetic outcome was evaluated by two independent experts by means of pictures taken preoperatively and at the follow-up visits. RESULTS: Breast-Q and 12 months FU were completed by 210 women. Patients without adverse event had a significantly higher Breast-Q score for "sexual well-being" (p = 0.001); "psychosocial well-being" was negatively influenced by prior therapies (p < 0.01), and older patients had significantly lower scores at 12 months FU compared to pre-OP for "satisfaction with breasts" (p < 0.01) while the opposite was true for younger patients. Unilateral surgery resulted in reduced "satisfaction with breast" at 12 months FU (p < 0.01). Radiotherapy negatively influenced "satisfaction with breast", "sexual well-being" and "physical well-being chest". The cosmetic evaluation showed a significant difference (p < 0.001) in the evaluation by the patients and experts with the patients' assessment being worse compared to experts' assessment. CONCLUSION: Our study showed that two years after implant-based breast reconstruction with support of TiLOOP® Bra PRO is influenced by different factors. This information can be used to improve the decision-making process for women who chose implant-based breast reconstruction.

Co-staining of microRNAs and their target proteins by miRNA in situ hybridization and immunohistofluorescence on prostate cancer tissue microarrays.

The co-expression of miRNAs and their target proteins was studied on tissue microarrays from different prostate cancer (PCa) patients. PCa of primary Gleason pattern 4 (GP4), lymph node metastases of GP4, distant metastases, and normal tissue from the transitional and peripheral zones were co-stained by fluorescent miRNA in situ hybridization (miRisH) and protein immunohistofluorescence (IHF). The miRNAs and corresponding target proteins include the pairs miR-145/ERG, miR-143/uPAR, and miR-375/SEC23A. The fluorescence-stained and scanned tissue microarrays (TMAs) were evaluated by experienced uropathologists. The pair miR-145/ERG showed an exclusive staining for miR-145 in the nuclei of stromal cells, both in tumor and normal tissue, and for ERG in the cytoplasm with/without co-expression in the nucleus of tumor cells. The pair miR-143/uPAR revealed a clear distinction between miR-143 in the nuclei of stromal cells and uPAR staining in the cytoplasm of tumor cells. Metastases (lymph node and distant) however, showed tumor cells with cytoplasmic staining for miR-143/uPAR. In normal tissues, beside the nuclei of the stroma cells, gland cells could also express miR-143 and uPAR in the cytoplasm. miR-375 showed particular staining in the nucleoli of GP4 and metastatic samples, suggesting that nucleoli play a special role in sequestering proteins and miRNAs. Combined miRisH/IHF allows for the study of miRNA expression patterns and their target proteins at the single-cell level.

Exploring the MIR143-UPAR Axis for the Inhibition of Human Prostate Cancer Cells In Vitro and In Vivo.

MIR143 is pathologically downregulated and may function as a tumor suppressor in prostate cancer. Likewise, the urokinase plasminogen activator receptor (UPAR) is overexpressed in prostate carcinoma, representing a negative prognostic marker and putative therapeutic target gene. In this paper, we establish UPAR as a new direct target of MIR143. Luciferase reporter gene constructs identify one of the two in silico-predicted binding sites as functionally relevant for direct MIR143 binding to the 3' UTR, and, concomitantly, transfection of MIR143 reduces UPAR protein levels in prostate carcinoma cells in vitro. Inhibitory effects on cell proliferation and colony formation, spheroid growth and integrity, and cell viability are extensively analyzed, and they are compared to direct small interfering RNA (siRNA)-mediated uPAR knockdown or combined microRNA (miRNA)-siRNA treatment. Switching to a therapeutically more relevant in vivo model, we demonstrate tumor-inhibitory effects of MIR143 replacement therapy by systemic treatment of mice bearing subcutaneous PC-3 tumor xenografts with MIR143 formulated in polymeric nanoparticles. This efficient, nanoparticle-mediated delivery of intact MIR143 mediates the marked downregulation of uPAR protein, but not mRNA levels, thus indicating translational inhibition rather than mRNA degradation. In summary, we identify UPAR as a direct target gene of MIR143, and we establish the therapeutic anti-tumor potential of nanoparticle-based MIR143 replacement in prostate cancer.

Pregnancy following Unilateral Immediate Breast Reconstruction with Titanized Polypropylene Mesh (TiLOOP(R) Bra) without Compromising the Result.

Immediate breast reconstruction after mastectomy due to cancer or as a prophylactic treatment is widely preferred to avoid psychosocial distress, poor body image, and diminished sexual well-being. An increasing number of women undergoing breast reconstruction are in childbearing age; however, only limited data are available on the cosmetic outcome of patients undergoing implant-based breast reconstruction with a surgical mesh and subsequent pregnancy. This is a case report of a female patient who underwent unilateral implant-based breast reconstruction with a titanized surgical mesh implant (TiLOOP Bra). Twenty-two months after reconstruction, the woman delivered a healthy child. No adverse events occurred. The patient breastfed with the contralateral breast. The cosmetic result and patient-reported outcome was excellent. Pregnancy after breast reconstruction with a synthetic surgical mesh is not contradictory to an excellent cosmetic outcome.

Cytotoxicity of AMANTADIG - a semisynthetic digitoxigenin derivative - alone and in combination with docetaxel in human hormone-refractory prostate cancer cells and its effect on Na+/K+-ATPase inhibition.

Cardiac glycosides (CGs) are natural compounds used to treat congestive heart failure. They have garnered attention as a potential cancer treatment option, especially because they bind to Na+/K+-ATPase as a target and activate intracellular signaling pathways leading to a variety of cellular responses. In this study we evaluated AMANTADIG, a semisynthetic cardenolide derivative, for its cytotoxic activity in two human androgen-insensitive prostate carcinoma cell lines, and the potential synergistic effects with docetaxel. AMANTADIG induced cytotoxic effects in both cell lines, and a combination with docetaxel showed a moderate and strong synergism in DU145 and PC-3 cells, respectively, at concentrations considerably lower than their IC50 values. Cell cycle analyses showed that AMANTADIG and its synergistic combination induced G2/M arrest of DU145 and PC-3 cells by modulating Cyclin B1, CDK1, p21 and, mainly, survivin expression, a promising target in cancer therapy. Furthermore, AMANTADIG presented reduced toxicity toward non-cancerous cell type (PBMC), and computational docking studies disclosed high-affinity binding to the Na+/K+-ATPase α subunit, a result that was experimentally confirmed by Na+/K+-ATPase inhibition assays. Hence, AMANTADIG inhibited Na+/K+-ATPase activity in PC-3 cells, as well as in purified pig kidney at nanomolar range. Altogether, these data highlight the potent effects of AMANTADIG in combination with docetaxel and offer important insights for the development of more effective and selective therapies against prostate cancer.

Differential prognostic value of MYC immunohistochemistry in subtypes of papillary renal cell carcinoma.

The histomorphological subtyping of papillary renal cell carcinomas (pRCCs) has improved the predictions of patients' long-term survival. Based on our previous results, we hypothesized that the MYC proto-oncogene would show differential expression in pRCC subtypes. Using a multi-institutional cohort of 204 pRCC patients we assessed the additional value of the immunohistochemical markers MYC, MINA53, and Ki67 in predicting patient's long-term survival. The clinical endpoints were overall survival (OS) and cancer-specific survival (CSS). Nomograms were constructed to predict each patient's risk of death (OS). The incorporation of the MYC staining patterns allowed the stratification of pRCC type 1 patients into better and worse prognostic groups. None of the patients with pRCC type 1 tumors and favorable MYC staining patterns died from tumor-related causes. This prognostic value was independent of the patient's age at surgery, the pathological tumor stage and presence of lymph node invasion. we could show that the immunohistochemical assessment of MYC and the histomorphological subtyping of pRCC stratifies pRCC type 1 tumors with regard to OS and CSS. The determination of the histomorphologic pRCC subtype in combination with the MYC immunohistochemical staining patterns allows a more accurate prediction of patients' individual risk of death.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na+/K+-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species Digitalis mariana ssp. heywoodii. Due to its complex structure, glucoevatromonoside cannot be obtained economically by total chemical synthesis. Here we describe two methods for glucoevatromonoside production, both using evatromonoside obtained by chemical degradation of digitoxin as the precursor. 1) Catalyst-controlled, regioselective glycosylation of evatromonoside to glucoevatromonoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as the sugar donor and 2-aminoethyldiphenylborinate as the catalyst resulted in an overall 30 % yield. 2) Biotransformation of evatromonoside using Digitalis lanata plant cell suspension cultures was less efficient and resulted only in overall 18 % pure product. Structural proof of products has been provided by extensive NMR data. Glucoevatromonoside and its non-natural 1-3 linked isomer neo-glucoevatromonoside obtained by semisynthesis were evaluated against renal cell carcinoma and prostate cancer cell lines.

Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes.

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

A new semisynthetic cardenolide analog 3ß-[2-(1-amantadine)- 1-on-ethylamine]-digitoxigenin (AMANTADIG) affects G2/M cell cycle arrest and miRNA expression profiles and enhances proapoptotic survivin-2B expression in renal cell carcinoma cell lines.

Cardiac glycosides are well known in the treatment of cardiovascular diseases; however, their application as treatment option for cancer patients is under discussion. We showed that the cardiac glycoside digitoxin and its analog AMANTADIG can inhibit the growth of renal cell carcinoma (RCC) cell lines and increase G2/M cell cycle arrest. To identify the signaling pathways and molecular basis of this G2/M arrest, microRNAs were profiled using microRNA arrays. Cardiac glycoside treatment significantly deregulated two microRNAs, miR-2278 and miR-670-5p. Pathway enrichment analysis showed that all cardiac glycoside treatments affected the MAPK and the axon guidance pathway. Within these pathways, three genes, MAPK1, NRAS and RAC2, were identified as in silico targets of the deregulated miRNAs. MAPK1 and NRAS are known regulators of G2/M cell cycle arrest. AMANTADIG treatment enhanced the expression of phosphorylated MAPK1 in 786-O cells. Secondly, we studied the expression of survivin known to be affected by cardiac glycosides and to regulate the G2/M cell phase. AMANTADIG treatment upregulated the expression of the pro-apoptotic survivin-2B variant in Caki-1 and 786-O cells. Moreover, treatment with AMANTADIG resulted in significantly lower survivin protein expression compared to 786-O control cells. Summarizing, treatment with all cardiac glycosides induced G2/M cell cycle arrest and downregulated the miR-2278 and miR-670-5p in microarray analysis. All cardiac glycosides affected the MAPK-pathway and survivin expression, both associated with the G2/M phase. Because cells in the G2/M phase are radio- and chemotherapy sensitive, cardiac glycosides like AMANTADIG could potentially improve the efficacy of radio- and/or chemotherapy in RCCs.

MiRNA-21 Expression Decreases from Primary Tumors to Liver Metastases in Colorectal Carcinoma.

OBJECTIVE: Metastasis is the major cause of death in colorectal cancer patients. Expression of certain miRNAs in the primary tumors has been shown to be associated with progression of colorectal cancer and the initiation of metastasis. In this study, we compared miRNA expression in primary colorectal cancer and corresponding liver metastases in order to get an idea of the oncogenic importance of the miRNAs in established metastases. METHODS: We analyzed the expression of miRNA-21, miRNA-31 and miRNA-373 in corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of primary colorectal cancer, liver metastasis and healthy tissues of 29 patients by quantitative real-time PCR. RESULTS: All three miRNAs were significantly up-regulated in the primary tumor tissues as compared to healthy colon mucosa of the respective patients (p < 0.01). MiRNA-21 and miRNA-31 were also higher expressed in liver metastases as compared to healthy liver tissues (p < 0.01). No significant difference of expression of miRNA-31 and miRNA-373 was observed between primary tumors and metastases. Of note, miRNA-21 expression was significantly reduced in liver metastases as compared to the primary colorectal tumors (p < 0.01). CONCLUSION: In the context of previous studies demonstrating increased miRNA-21 expression in metastatic primary tumors, our findings raise the question whether miRNA-21 might be involved in the initiation but not in the perpetuation and growth of metastases.

The New Semisynthetic Cardenolide Analog 3ß-[2-(1-Amantadine)-1-on-ethylamine]-digitoxigenin (AMANTADIG) Efficiently Suppresses Cell Growth in Human Leukemia and Urological Tumor Cell Lines.

BACKGROUND/AIM: The use of cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic cardenolide analog AMANTADIG (3ß-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the cardenolide digitoxin on leukemia and urological tumor cell lines. MATERIALS AND METHODS: The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay. RESULTS: AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both cardenolides. CONCLUSION: The new cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines.

Piwil 2 expression is correlated with disease-specific and progression-free survival of chemotherapy-treated bladder cancer patients.

Piwi-like 2 (Piwil 2) belongs to the family of Argonaute genes/proteins. The expression of Piwil 2 is associated with stem cells. A role in tumorigenesis and/or tumor progression is proposed for different cancers but not yet for bladder cancer (BCa). We investigated the Piwil 2 expression by immunohistochemistry in a cohort of 202 BCa patients treated by cystectomy and adjuvant chemotherapy. The association between Piwil 2 expression and disease-specific (DSS) or progression-free survival (PFS) was calculated using Kaplan Meier analyses and univariate/multivariate Cox's regression hazard models.In a multivariate Cox's regression, Piwil 2 expression, either in the cytoplasm or the nucleus, was significantly associated with DSS and PFS. A weak cytoplasmic staining pattern was associated with poor DSS and tumor progression (RR=2.7; P=0.004 and RR=2.4; P=0.027). Likewise,, absent nuclear Piwil 2 immunoreactivity was associated with poor DSS and tumor progression (RR=2.3; P=0.023 and RR=2.2; P=0.022). BCa patients whose tumors exhibited a combination of weak cytoplasmic and absent nuclear immunoreactivity had a 6-fold increased risk of tumor-related death (P=0.005) compared to patients with strong expression. Considering only patients with high grade G3 tumors, a 7.8-fold risk of tumor-associated death and a 3.6-fold risk of tumor progression were detected independently of the histologic tumor subtype or the chemotherapy regimen. In summary, a combination of weak cytoplasmic and absent nuclear expression of Piwil 2 is significantly associated with an increased risk of DSS and tumor progression. This implicates that Piwil 2 could be a valuable prognostic marker for high-risk BCa patients.

The combined serum levels of miR-375 and urokinase plasminogen activator receptor are suggested as diagnostic and prognostic biomarkers in prostate cancer.

This study aimed to assess the applicability of miR-375 in combination with the soluble urokinase plasminogen activator receptor (suPAR) protein as a diagnostic and/or prognostic biomarker for prostate cancer (PCa) patients. miR-375 levels by qRT-PCR and suPAR levels by ELISA were evaluated in serum samples from 146 PCa patients, 35 benign prostate hyperplasia (BPH) patients and 18 healthy controls. Antigen levels of suPAR differed between healthy controls and PCa or BPH patients, whereas miR-375 levels differed between PCa and BPH patients or healthy controls (p < 0.001). Additionally, suPAR levels differed between the Gleason sum groups GS = 7 versus GS > 7, with higher levels in the latter group (p = 0.011), and miR-375 levels were higher in the tumor stage group T3-T4 compared with the T1-T2 group (p = 0.039). A high concentration of suPAR was associated with a poor disease-specific survival (DSS; p = 0.039). The combination of suPAR and miR-375 levels identified a patient group possessing high levels for both parameters. This was associated with a poorer 10-year overall survival (OS) and DSS, with a 6.38-fold increased risk of death and a 7.68-fold increased risk of tumor-related death (p = 0.00026 and p = 0.014; univariate Cox's regression analysis). In a multivariate Cox's regression analysis PCa patients with high levels of suPAR and miR-375 showed a 5.72-fold increased risk of death in OS (p = 0.006). In summary, the differences between the PCa/BPH/healthy control cohorts for either suPAR and miR-375 levels in conjunction with the association of combined high suPAR/miR-375 levels with a poor prognosis suggest a diagnostic and prognostic impact for PCa patients.

High coexpression of CCL2 and CX3CL1 is gender-specifically associated with good prognosis in soft tissue sarcoma patients.

Chemokines are involved in both the negative and positive regulation of inflammatory processes, angiogenesis and cancer/cancer stem cell proliferation as well as the chemoattraction of tumor cells to metastatic sites. The aim of this study was to measure the mRNA expression levels of three chemokines, CCL2, CCL7 and CX3CL1, in soft tissue sarcomas (STSs) and to assess the correlations between these levels as well as their correlations with clinicopathological data and the disease-specific survival of STS patients. The mRNA levels of CCL2, CCL7 and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death as compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) as compared to those with high expression of both parameters in female patients. In conclusion, this is the first study to show a significant correlation between combined low expression of CCL2 and CX3CL1 and a poor prognosis for STS patients, particularly in female patients.

Piwi-like 1 and 4 gene transcript levels are associated with clinicopathological parameters in renal cell carcinomas.

Piwi-like gene family members (Piwil 1-4) are considered stem cell-associated genes/proteins. These are expressed predominantly in germline cells, but are re-expressed in different tumors. Piwil 1-4 gene expression has not previously been studied and correlated with clinicopathological parameters in renal cell carcinomas (RCC). The Piwil 1-4 transcript levels were analyzed by quantitative real-time PCR in 73 clear cell RCC (ccRCC) tissues and corresponding normal tissues. The transcript levels of Piwil 1, 2 and 4 were strongly and significantly correlated with each other, in both the tumor tissues and the normal tissues (P<0.001; Spearman's rank test). Piwil 4 gene expression was significantly higher in the ccRCC tissues than that in the corresponding normal renal tissues (P<0.001; Wilcoxon signed-rank test). When the ccRCC patient cohort was divided according to the median Piwil 1-4 expression into low- and high-expression groups and according to age into younger (≤64years) and older patient groups (>64years), the younger patients displayed significantly higher levels of Piwil 1 mRNA in comparison to the older patients (P=0.010; Fisher's exact test). Interestingly, Piwil 1 expression was left-right polarized in the normal tissues but not in the tumor tissues (P=0.004; Fisher's exact test). Altogether, associations were determined between the Piwi-like family member expression levels and clinicopathological parameters of ccRCC, suggesting a potential role for these genes/proteins in ccRCC diagnostics and tumorigenesis as well as in renal tissue embryology.

Comparative microRNA profiling of prostate carcinomas with increasing tumor stage by deep sequencing.

UNLABELLED: MicroRNAs (miRNA) posttranscriptionally regulate gene expression and are important in tumorigenesis. Previous deep sequencing identified the miRNA profile of prostate carcinoma versus nonmalignant prostate tissue. Here, we generated miRNA expression profiles of prostate carcinoma by deep sequencing, with increasing tumor stage relative to corresponding nonmalignant and healthy prostate tissue, and detected clearly changed miRNA expression patterns. The miRNA profiles of the healthy and nonmalignant tissues were consistent with our previous findings, indicating a high fidelity of the method employed. In the tumors, quantitative real-time PCR (qRT-PCR) analysis of 40 paired samples of prostate carcinoma versus normal tissue revealed significant upregulation of miR-20a, miR-148a, miR-200b, and miR-375 and downregulation of miR-143 and miR-145. Hereby, miR-375 increased from normal to organ-confined tumors (pT2 pN0), slightly decreased in tumors with extracapsular growth (pT3 pN0), but was then expressed again at higher levels in lymph node metastasizing (pN1) tumors. The sequencing data for miR-375 were confirmed by Northern blotting and qRT-PCR. The regulation for other selected miRNAs could, however, not be confirmed by qRT-PCR in individual tumor stages. MiR-200b, in addition to miR-200c and miR-375 reduced the expression of SEC23A. Interestingly, miR-375, found by sequencing in pT2 upregulated by us and others in tumor versus normal tissue, and miR-15a, found by sequencing in pT2 and pT3 and in the metastasizing tumors, target the phosphatases PHLPP1 and PHLPP2, respectively. PHLPP1 and PHLPP2 dephosphorylate members of the AKT family of signal transducers, thereby inhibiting cell growth. Coexpression of miR-15a and miR-375 resulted in downregulation of PHLPP1/2 and strongly increased prostate carcinoma cell growth. IMPLICATIONS: These genomic data reveal relevant miRNAs in prostate cancer that may have biomarker and therapeutic potential.

Role of two single nucleotide polymorphisms in secreted frizzled related protein 1 and bladder cancer risk.

In this study, we determined the genotype distribution of two single nucleotide polymorphisms (SNPs) in secreted frizzled related protein 1 (SFRP1), rs3242 and rs921142, in a Caucasian bladder cancer case-control study. Allelic variants of the SNPs were determined using restriction fragment length polymorphism (RFLP) analysis and partly verified by sequencing analysis. Overall, DNA from 188 consecutive and 215 early-onset bladder cancer patients (≤45 years) as well as from 332 controls was investigated. Potential microRNA binding sites were determined for rs3242, and microRNA expression was analysed in cell lines and tumour specimens. We observed a remarkable distribution difference in rs3242 between bladder cancer patients and healthy controls (p=0.05). Additionally, we found a significant difference in genotype distribution (p=0.032), resulting from the difference of early-onset patients and the control group (p=0.007). The risk allele T showed increased frequency in the early-onset patient group (p=0.002). Genotype-dependent differences of microRNA binding capacity were predicted in SFRP1 mRNA for two microRNAs. Hsa-miR-3646 showed strong expression in cell lines and tumour tissue, whereas hsa-miR-603 exhibited weak expression. The rs921142 SNP showed no significant association with bladder cancer risk. This is the first study to describe an association of the SFRP1 SNP rs3242 and bladder cancer risk as well as the influence of rs3242 on genotype-dependent microRNA capacity on SFRP1 mRNA. The onset of bladder seems to be associated with the increased occurrence of the T-allele in rs3242.

The proto-oncogene ERG is a target of microRNA miR-145 in prostate cancer.

Prostate cancer is a leading cause of cancer mortality in men. One of the distinct characteristics of prostate cancer is over-expression of the ERG proto-oncogene. The TMPRSS2-ERG gene fusion, the most common gene fusion, is found in approximately 50% of prostate cancer cases. We show that certain microRNAs are extensively deregulated in prostate cancer cell lines and primary clinical cancer samples. MicroRNAs are capable of modulating post-transcriptional gene expression via inhibition of protein synthesis. Independent target prediction methods have indicated that the 3' untranslated region of the ERG mRNA is a potential target of miR-145. miR-145 is consistently down-regulated in prostate cancer. Here we show that the ERG 3' untranslated region is a regulative target of miR-145 in vitro. Ectopic expression of miR-145 led to a reduction in expression of the ERG protein. We analyzed 26 prostate cancer samples and corresponding normal tissue. ERG protein expression was found to be elevated in the tumor samples, together with increased expression of several ERG isoforms. We identified ERG proteins of 35 and 24 kDa, which may represent unknown ERG splice variants. Analyses of miR-145 and ERG mRNA expression revealed a general down-regulation of miR-145 irrespective of the presence or absence of translocations involving ERG. This observation indicates that down-regulation of miR-145 may contribute to the increased expression of most ERG splice variants sharing the miR-145 target sequence in their 3' untranslated region.

Identification of ZNF217, hnRNP-K, VEGF-A and IPO7 as targets for microRNAs that are downregulated in prostate carcinoma.

In primary prostate cancer (PCa), a major cause of cancer-related death in men, the expression of various microRNAs (miRNAs) is deregulated. We previously detected several miRNAs, for example, miR-24 and miR-22, as significantly downregulated in PCa (Szczyrba et al., Mol Cancer Res 2010;8:529-38). An in silico search predicted that zinc finger protein 217 (ZNF217) and importin 7 (IPO7) were potential target genes of these miRNAs. Additionally, for two genes that are deregulated in PCa (heterogeneous nuclear ribonucleoprotein K, hnRNP-K, and vascular endothelial growth factor A, VEGF-A), we identified two regulatory miRNAs, miR-205 and miR-29b. The regulation of the 3'-untranslated regions of the four genes by their respective miRNAs was confirmed by luciferase assays. As expected, the upregulation of ZNF217, hnRNP-K, VEGF-A and IPO7 could be verified at the protein level in the PCa cell lines LNCaP and DU145. ZNF217 and IPO7, which had not yet been studied in PCa, were analyzed in more detail. ZNF217 mRNA is overexpressed in primary PCa samples, and this overexpression translates to an elevated protein level. However, IPO7 was upregulated at the protein level alone. The inhibition of ZNF217 and IPO7 by siRNA resulted in reduced proliferation of the PCa cell lines. ZNF217 could thus be identified as an oncogene that is overexpressed in PCa and affects the growth of PCa cell lines, whereas the function of IPO7 remains to be elucidated in greater detail.