Improved Analysis of Prostate Cancer: VIM3, ATG7 and P53 Form a Complex and Activate miRNA 371a-3p.

BACKGROUND/AIM: It is not possible to differentiate prostate carcinomas sufficiently to ensure that every patient receives the right therapy. New molecular markers are needed. Our objective was to identify a complex consisting of vimentin variant 3 (VIM3), autophagy-related protein 7 (ATG7) and tumor protein p53 (TP53) in prostate cancer cells and its effect on microRNA (miR)-371a-3p. MATERIALS AND METHODS: Prostate cancer cell lines (PC3, DU145, LNCaP) and the benign prostatic hyperplasia cell line BPH-1 were cultured in growth medium for 24 h, then stimulated with endothelin 1 (EDN1) (50 nM) and withaferin A (2 nM) for 24 h. Cell extracts were then analyzed by western blot. The localization of VIM3, ATG7 and TP53 in the nucleus was demonstrated with immunofluorescence staining and complex formation was demonstrated by immunoprecipitation. Cancer cell migration was analyzed with a scratch assay and agarose drop analysis. The binding of the complex to the promoter of pri-miR-371a-3p was analyzed with a non-radioactive electrophoretic mobility shift assay. VIM3 knockdown using small interfering RNA and quantitative real-time polymerase chain reaction for miR-371a-3p were performed. RESULTS: The complex was present in the nucleus of prostate cancer cells and in the BPH-1 cell line. EDN1 increased the levels of the complex partners and cell migration, whereas withaferin A reduced the levels of the complex partners and migration. The complex bound to the promoter of pri-miR-371a-3p and might be involved in its transcription. Transfection with miR-371a-3p increased migration of prostate cancer cells. VIM3 knockdown reduced miR-371a-3p expression. CONCLUSION: The VIM3-ATG7-P53 complex, with its stimulatory effect on miR-371a-3p, may have the potential to be a marker for improved differentiation between prostate carcinomas, allowing tailored therapy.

Synthesis and evaluation of radioiodinated estrogens for diagnosis and therapy of male urogenital tumours.

The preparation of 24 estrogens, their estrogen receptor (ER) affinity and studies of radioiodinated estrogen binding to ER-positive male bladder tumor cells (HTB9) are described. The estrogens with the highest affinity were selected using fluorescence anisotropy assays. A 2,2,2-trifluoroethyl group at the 11ß-position caused particularly promising affinity. (Radio)iodination was performed on the 17α-vinyl group. Binding studies on HTB9 cells revealed picomolar affinities of radioconjugates 19 and 31, indicating promising ability for targeting of urogenital tumors.

Novel noninvasive marker of regression of clear cell renal cell carcinoma (ccRCC).

OBJECTIVE: Analyzing protein kinase C (PKC) alpha, iota, and zeta as well as levels of Mxi-2 and Vim3 in regressive clear cell renal carcinomas (ccRCCs) and urine samples. MATERIAL AND METHODS: Fresh samples of ccRCCs (predominantly pT1a/b) with different degrees of regression (<10%, 30%, 50%, and 70%) vs normal renal tissue and oncocytomas were studied by Western blot, using antibodies of different PKC isoforms. Urine samples from these tumors were analyzed by ELISA (PKC isoforms, Mxi-2, and Vim3). RESULTS: With increasing degree of regression beyond 10%, nuclear Mxi-2 and Vim3 were highly overexpressed in fresh tumor samples. In urine samples, Vim3 was significantly overexpressed in oncocytoma and downregulated in RCCs with 70% regression. Western blot analysis shows that PKC alpha and iota levels were significantly increased in fresh tumor tissue samples (tumors with 30% regression). PKC zeta was expressed in normal kidney and significantly increased in oncocytoma but not found in ccRCCs. In patients' urines, Mxi-2 was significantly reduced (regression > 50%), while PKC isoform alpha was significantly increased by advanced regression rate. PKC iota in patients' urine was overexpressed in oncocytoma and reduced in all ccRCC urines. CONCLUSION: Tumor regression in ccRCC tissue shows strong nuclear overexpression of Mxi-2, Vim3, and PKC alpha and iota. In respective urines, PKC alpha was overexpressed; PKC iota was decreased. Mxi-2 and Vim3 decreased with increasing regression rates. These reagents could serve as noninvasive ccRCC markers for regression.

Teratomatous Elements in Orchiectomy Specimens Are Associated with a Reduced Relapse-Free Survival in Metastasized Testicular Germ Cell Tumors.

INTRODUCTION: The impact of teratomatous elements in orchiectomy specimens of metastasized testicular germ cell tumors (TGCT) regarding oncological outcome is still unclear. METHODS: We performed a retrospective analysis including 146 patients with metastasized TGCT analysing patient characteristics. RESULTS: Twenty-six (18%) of all patients showed teratomatous elements in the orchiectomy specimens. TGCT with teratomatous elements showed a significantly higher frequency of clinical-stage 2C-3 disease (73 vs. 49%, p = 0.031), visceral metastases (58 vs. 32%, p = 0.015), and poor prognosis (p = 0.011) than TGCT without teratomatous elements. Teratoma-containing TGCT revealed a significantly higher rate of post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND, 54 vs. 32%, p = 0.041), with teratomatous elements being more often present in the PC-RPLND specimens (43 vs. 11%, p = 0.020) than nonteratoma-containing primaries. In the Kaplan-Meier estimates, the presence of teratomatous elements in orchiectomy specimens was associated with a significantly reduced relapse-free survival (RFS) (p = 0.049) during a median follow-up of 36 months (10-115.5). CONCLUSIONS: The presence of teratomatous elements in orchiectomy specimens is associated with an advanced tumor stage, worse treatment response as well as a reduced RFS in metastasized TGCT. Consequently, the presence of teratomatous elements might act as a reliable stratification tool for treatment decision in TGCT patients.

Non-invasive urine markers for the differentiation between RCCs and oncocytoma.

BACKGROUND: Recently, our group showed that Vim3 is overexpressed in tissue samples of renal oncocytomas and Mxi-2 in clear cell renal carcinoma (ccRCC). The mechanism leading to the truncation of both proteins is known and involves with two miRs, both detectable in urine. Since the analysis of miRs is time-consuming, our aim was to identify the truncated proteins in urine instead. Furthermore, urine samples from small renal masses (SRMs) (n = 45, <4 cm) were analyzed to get a pre-surgical differentiation of the cancer subtypes. METHODS: Urines were accessed from the urological biobank (n = 350). Proteins were isolated from urine samples, and Western blots were performed. Each sample was analyzed with ELISA for the expression of Vim3 and Mxi-2. A lateral flow assay was established. For the detection of SRMs, the miRs were isolated and qRT-PCR was performed. RESULTS: A significant increase of Vim3 in urines from patients with oncocytoma (n = 20) was detectable with ELISA compared to all other subtypes of RCCs (chromophobe (n = 50), papillary (n = 40), ccRCC (n = 200), and controls (n = 40) (***p < 0.0001)). Mxi-2 was predominantly overexpressed in ccRCCs (***p < 0.0001). Lateral flow assay of Vim3 and Mxi-2 shows two bands in the case of oncocytoma and ccRCC indicating the specificity of this test. For SRMs, an overexpression of miR-15a/Mxi2 was detectable in urine samples from ccRCC and chromoRCC patients. In contrast to that, miR-498/Vim3 were predominantly overexpressed in oncocytoma patients. CONCLUSION: Both proteins (Vim3 and Mxi-2) were detectable in patients' urines and can be used for the non-invasive differentiation of kidney cancers.

[Using preorchiectomy tumor marker serum concentrations for International Germ Cell Consensus Classification (IGCCCG) risk group assignment results in significant numbers of up- and downstaging]. / IGCCCG-Fehlklassifikation (International Germ Cell Consensus Classification) durch zeitlich inkorrekte Interpretation der Serumkonzentration der Tumormarker bei metastasierten testikulären Keimzelltumoren.

BACKGROUND: The prognostic classification system of the International Germ Cell Cancer Cooperative Group (IGCCCG) for testicular germ cell tumors is based on the histological subtype, location of the primary tumor, extent of metastatic spread and prechemotherapy tumor marker serum concentrations. OBJECTIVES: In this study, we aim to identify whether the use of preorchiectomy instead of prechemotherapy tumor marker serum concentration has an impact on IGCCCG risk group assignment. MATERIALS AND METHODS: We performed a retrospective analysis including 135 patients with metastasized testicular germ cell tumors. Analysis of the clinical information with a focus on the tumor marker serum concentration preorchiectomy and prechemotherapy was performed, thus leading to the grouping of patients according to IGCCCG risk group assignment. RESULTS: Using preorchiectomy instead of prechemotherapy tumor markers led to an incorrect IGCCCG risk group classification in 8% (11/135) of all patients, and consequently to a non-guideline concordant treatment. Up-staging was observed in 8 of 11 patients, representing 6% (8/135) of the total patient cohort. Three of the 11 misclassified patients showed a down-staging and thus describe 2% (3/135) of the total patient cohort. CONCLUSIONS: Using preorchiectomy tumor markers instead of prechemotherapy serum concentration might lead to an incorrect IGCCCG risk group assignment as well as non-guideline concordant treatment. Consequently, prechemotherapy tumor marker serum concentration should be applied for guideline concordant staging of patients.

Vimentin 3 Expression in Prostate Cancer Cells.

BACKGROUND/AIM: Vimentin3 (Vim3) was recently described as a tumour marker for the direct discrimination between benign and malignant kidney tumours. Here, we examined its expression in prostate cancer (PCa) cell lines and the regulation of its expression by endothelin receptors. MATERIALS AND METHODS: Prostate cancer cell lines (PC3, DU145, LNCap) were incubated with endothelin 1 (ET-1), BQ123 [endothelin A receptor (ETAR) antagonist], BQ788 [endothelin B receptor (ETBR) antagonist], BQ123+ET-1, BQ788+ET-1 for 24 h and a scratch assay was performed. Cell extracts were analysed by western blotting and qRT-PCR. RESULTS: ET-1 induced Vim3 overexpression. Blocking the ETBR in the different prostate cancer cell lines yielded a higher migration rate, whereby Vim3 expression was significantly increased. CONCLUSION: Vim3 concentration increases in cell lines without a functional ETBR and may be used as a marker for PCas where ETBR is frequently methylated.

EBV induced loss of sperm quality.

OBJECTIVE: To analyze the presence of Epstein-Barr-virus (EBV) in sperm samples from patients diagnosed with some impairment of the fertility parameters evaluated using seminogram and to observe if there is any difference with the normozoospermic samples. We hypothesize that an EBV infection is responsible for the upregulation of the miRNA 199-3p, which binds to the 3'UTR of endothelin-1 (ET-1). ET-1 is a key factor to produce Vimentin (Vim3), and therefore, it influences the expression of Vim3. Since Vim3 is predominantly detectable in sperms without any structural defects, the newly identified regulation mechanism can be responsible for the loss of sperm quality. MATERIAL AND METHODS: This study was performed from January 2017 to December 2020 and included 27 patients who provided ejaculated samples obtained by masturbation. Ejaculates were evaluated according to the Word Health Organization's criteria. Posteriorly, the samples were sorted according to the seminogram diagnosis and further analyzed using different enzyme-linked absorbed immune assays to determine the level or concentration of Epstein-Barr nuclear antigen (EBNA), ET-1, and Vim3. RESULTS: All sperm samples with the impairment of fertility parameters contained the EBNA and presented a downregulation of ET-1 and Vim3. In addition, sperms located in the swim ups are also partially positive for the EBV virus in different clinical aspects. CONCLUSION: Based on the regulation mechanism here presented, it seems that the EBV induces changes at the miRNA level, which are responsible for the decreasing of sperm quality.

Mxi-2 Dependent Regulation of p53 in Prostate Cancer.

BACKGROUND/AIM: Endothelin-1 (ET-1) is overexpressed in many types of cancer, inhibiting the release of the microRNA 15a (miR-15a) and inducing the production of Mxi-2. Our aim was to identify a molecular complex regulating p53 activity in prostate cancer (PCa). MATERIALS AND METHODS: DU145 cells were treated with ET-1, MAPK p38 inhibitor, Endothelin A receptor inhibitor (ETAR inhibitor) and Endothelin B receptor inhibitor (ETBR inhibitor). Extracts were analysed using Western Blot, immunoprecipitation and qRT-PCR. Furthermore, prostate cancer patient samples were analysed using qRT-PCR and ELISA. RESULTS: The hypothesised molecular complex was identified, with miR-15a, microRNA 1285 (miR-1285) and Mxi-2 levels up-regulated in patients in relation to increasing aggressiveness of PCa. CONCLUSION: A complex composed of Argonaut 2 (Ago2)/Mxi-2/miR-1285 is involved in PCa. The expression of Mxi-2 correlates with increasing PCa aggressiveness and might be used as a non-invasive marker for the diagnosis and progression of PCa.

Vimentin 3 Allows Differentiation between Normozoospermia and Oligoasthenoteratozoospermia.

Vimentin is a structural protein predominantly located in the head of sperms. The function and localization of the previously identified truncated version, Vimentin 3 (Vim3), are still unknown. To investigate whether the expression of Vim3 can be used as a reliable marker for the differentiation of sperm quality, we analyzed ejaculates from patients with oligoasthenoteratozoospermia (OAT) syndrome and normozoospermia. We identified sperms with head, neck, and tail changes, which were less positive for Vim3 in OAT syndrome compared to normozoospermia. The expression of Vim3 was significantly downregulated in patients with OAT syndrome compared to sperms from patients with normozoospermia (∗∗ p < 0.01). The ELISA analysis showed similar results as ejaculates from normozoospermic patients showed a significantly higher Vim3 concentration than patients with OAT syndrome (∗∗∗ p < 0.001). This study demonstrates that Vim3 is more highly expressed in ejaculates from patients with normozoospermia compared to ejaculates from patients with OAT syndrome. Therefore, we postulate that Vim3 can be used to determine ejaculate quality. Furthermore, we identified the marker, Vim3, to differentiate between mature sperms with no morphological changes and sperms with head, neck, and tail changes. A lateral flow assay that allows quick analysis is currently under development.