miR-32 promotes MYC-driven prostate cancer

miR-32 is an androgen receptor (AR)-regulated microRNA, expression of which is increased in castration-resistant prostate cancer (PC). We have previously shown that overexpression of miR-32 in the prostate of transgenic mice potentiates proliferation in prostate epithelium. Here, we set out to determine whether increased expression of miR-32 influences growth or phenotype in prostate adenocarcinoma in vivo. We studied transgenic mice expressing MYC oncogene (hiMYC mice) to induce tumorigenesis in the mouse prostate and discovered that transgenic overexpression of miR-32 resulted in increased tumor burden as well as a more aggressive tumor phenotype in this model. Elevated expression of miR-32 increased proliferation as assessed by Ki-67 immunohistochemistry, increased nuclear density, and higher mitotic index in the tumors. By gene expression analysis of the tumorous prostate tissue, we confirmed earlier findings that miR-32 expression regulates prostate secretome by modulating expression levels of several PC-related target genes such as Spink1, Spink5, and Msmb. Further, we identified Pdk4 as a tumor-associated miR-32 target in the mouse prostate. Expression analysis of PDK4 in human PC reveals an inverse correlation with miR-32 expression and Gleason score, a decrease in castration-resistant and metastatic tumors compared to untreated primary PC, and an association of low PDK4 expression with a shorter recurrence-free survival of patients. Although decreased PDK4 expression induces the higher metabolic activity of PC cells, induced expression of PDK4 reduces both mitotic respiration and glycolysis rates as well as inhibits cell growth. In conclusion, we show that miR-32 promotes MYC-induced prostate adenocarcinoma and identifies PDK4 as a PC-relevant metabolic target of miR-32-3p.publishedVersionPeer reviewe

miR-32 promotes MYC-driven prostate cancer

miR-32 is an androgen receptor (AR)-regulated microRNA, expression of which is increased in castration-resistant prostate cancer (PC). We have previously shown that overexpression of miR-32 in the prostate of transgenic mice potentiates proliferation in prostate epithelium. Here, we set out to determine whether increased expression of miR-32 influences growth or phenotype in prostate adenocarcinoma in vivo. We studied transgenic mice expressing MYC oncogene (hiMYC mice) to induce tumorigenesis in the mouse prostate and discovered that transgenic overexpression of miR-32 resulted in increased tumor burden as well as a more aggressive tumor phenotype in this model. Elevated expression of miR-32 increased proliferation as assessed by Ki-67 immunohistochemistry, increased nuclear density, and higher mitotic index in the tumors. By gene expression analysis of the tumorous prostate tissue, we confirmed earlier findings that miR-32 expression regulates prostate secretome by modulating expression levels of several PC-related target genes such as Spink1, Spink5, and Msmb. Further, we identified Pdk4 as a tumor-associated miR-32 target in the mouse prostate. Expression analysis of PDK4 in human PC reveals an inverse correlation with miR-32 expression and Gleason score, a decrease in castration-resistant and metastatic tumors compared to untreated primary PC, and an association of low PDK4 expression with a shorter recurrence-free survival of patients. Although decreased PDK4 expression induces the higher metabolic activity of PC cells, induced expression of PDK4 reduces both mitotic respiration and glycolysis rates as well as inhibits cell growth. In conclusion, we show that miR-32 promotes MYC-induced prostate adenocarcinoma and identifies PDK4 as a PC-relevant metabolic target of miR-32-3p.</p

Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion

Background Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. Methods We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. Results Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. Conclusions Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer.Peer reviewe

Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion

Background Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. Methods We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. Results Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. Conclusions Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p &lt; 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p &lt; 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p &lt; 0.0001) or urgent (20.4% vs. 38.5%; p &lt; 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p &lt; 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p &lt; 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Kromosomin 1p21-22 monistuman löytäminen rakkosyövässä ja ei-koodaavien RNA:iten ilmentyminen eturauhassyövässä

Eturauhas- ja rakkosyövän molekyylimekanismit Syöpä on monimutkainen sairaus, jonka aiheuttavat normaaliin soluun kertyvät geneettiset muutokset. Tällaiset geneettiset muutokset häiritsevät normaalien solujen homeostaasia ja johtavat hallitsemattomaan solujen lisääntymiseen. Näiden muutosten tunteminen on tärkeää, jotta voitaisiin kehittää entistä tehokkaampia syövän hoitomuotoja ja diagnostisia menetelmiä tappavan tautimuodon tunnistamiseksi. Virtsarakon syöpä on yleisin virtsateiden maligniteetti. Useimmat virtsarakon syövät syntyvät välimuotoisesta epiteelistä (uroteliaaliset karsinoomat). Alle 5 % virtsarakon syöpätapauksista on levyepiteeliperäisiä. Virtsarakon syöpä on heterogeeninen sairaus, jolle ovat ominaisia erilaiset geneettiset muutokset, jotka johtavat eri polkuja syövän kehittymiseen ja etenemiseen. Monet näistä geneettisistä muutoksista vaikuttavat geenien kopiolukumäärään johtaen onkogeenien monistumaan ja kasvurajoitegeenien häviämään. Tässä tutkimuksessa käytettiin sirupohjaista, vertailevaa genomista hybridisaatiomenetelmää (aCGH) geenikopiolukumäärän analysoimiseksi kliinisissä rakkosyöpänäytteissä sekä solulinjoissa. Yhdeksi uudeksi monistuma-alueeksi tunnistettiin kromosomialue 1p21-22. Työssä kartoitettiin ko. aluetta tarkemmin ja osoitettiin, että ns. minimaalinen monistuma-alue käsitti yhden miljoonan emäsparin DNA-jakson sisältäen yhteensä 11 tunnettua geeniä. Korkein monistuma-aste löytyi SCaBER-levyepiteelisyöpäsolulinjasta. Näistä neljä geeniä, TMED5, DR1, RPL5 ja EVI5, yli-ilmeni SCaBER-solulinjassa, DR1 kaikkein eniten. Julkisten tietokantojen perusteella DR1 yli-ilmenee myös kliinisissä näytteissä, jotka edustavat yleistä virtsarakkosyövän histologista tyyppiä. Eturauhassyöpä on miesten toiseksi yleisin diagnosoitu syöpä maailmassa ja yleisin kehittyneissä maissa. Taudin histologisen ja kliinisen käyttäytymisen monimuotoisuuden taustalla olevia mekanismeja tunnetaan huonosti. Eturauhassyövän molekyylitason mekanismien tunteminen mahdollistaisi uusien taudin aggressiivisuutta kuvastavien biomarkkerien löytämisen. Viime aikoina on yhä enemmän kiinnitetty huomiota mikroRNA:iden (miRNA) merkitykseen syövän kehittymisessä. Useat tutkimukset ovat osoittaneet, että miRNA-ilmentymistasot ovat muuntuneita eturauhassyövässä. Onkin mahdollista, että niitä voitaisiin käyttää ennusteellisina biomarkkereina. Kuten muissa syövissä, on epäselvää, koostuuko eturauhassyöpä eri solupopulaatioista, joilla on erilainen jakaantumiskyky, kuten syövän kantasolumallit ennustavat. Useissa tutkimuksissa on käytetty virtaussytometriaa eturauhasen kantasolujen eristämiseen. Tässä tutkimuksessa tehtiin genominlaajuinen miRNA-ilmentymisen analyysi potilaista peräisin olevista kantasolun kaltaisista soluista (SC), välivaiheen soluista ja sitoutuneista (CB) soluista. Jokainen solualapopulaatio poikkesi toisistaan miRNA-ilmentymisprofiililtaan riippumatta siitä, oliko kyseessä syöpänäyte vai ei. MiR-548c-3p:n havaittiin yli-ilmentyvän noin viisinkertaisesti SC-soluissa verrattuna CB-soluihin. Toiminnallisissa tutkimuksissa miR-548c-3p:n yli-ilmentyminen CB-soluissa vähensi erilaistumista kohti kantasoluilmiasua. MiR-548c-3p oli myös merkittävästi yli-ilmentynyt kastraatioresistentistä eturauhassyövästä (CRPC) eristetyissä soluissa verrattuna eturauhasen hyvänlaatuisesta liikakasvusta (BPH) eristettyihin epiteelisoluihin. Tämä viitaa siihen, että miR-548c-3p voisi olla eturauhassyövän aggressiivisuuden biomarkkeri. Tunnistaaksemme uusia, eri tavoin ilmentyviä miRNA:ita, syväsekvensoimme joukon kliinisiä eturauhassyöpänäytteitä. MiR-1247-5p, miR-1249, miR-1269a, miR1271-5p, miR-1290, miR-1291 ja miR-1299 ilmentyivät eri lailla syövässä ja normaalissa eturauhasessa. Niinpä näitä tutkittiin laajemmassa materiaalissa qRT-PCR-menetelmällä. MiR-1247-5p ilmentyi merkittävästi enemmän CRPC-näytteissä verrattuna ei-maligniin eturauhaseen. Ennusteohjelmien perusteella miR-1247-5p:n yksi kohdegeeni voisi olla MYCBP2 (myc:iä sitova proteiini 2). miR-1247-5p:n yli-ilmentyminen PC-3- ja LNCaP-syöpäsolulinjoissa johti MYCBP2:n ilmentymisen laskuun sekä mRNA- että proteiinitasolla. Lusiferaasireportterikoe vahvisti, että MYCBP2 on miR-1247-5p:n kohde. Useissa uuden sukupolven sekvensointitutkimuksissa on syövissä löydetty uusia ryhmiä ei-koodaavia RNA:ita. Eturauhassyöpäsolulinjoissa miRNA:iden jälkeen toiseksi yleisin ryhmä ei-koodaavia RNA:ita ovat tRNA:ista peräisin olevat fragmentit (tRFs). Niiden ominaisuudet, runsas ilmentyminen ja tarkka sekvenssi viittaavat siihen, että nämä molekyylit eivät ole satunnaisia tuotteita tRNA:n hajoamisesta. tRF:ien tarkka rooli on kuitenkin edelleen epäselvä. Tässä tutkimuksessa tRF:ien ilmentymistä analysoitiin potilaan kudosnäytteistä. Tutkimuksessa tunnistettiin yhteensä 598 erilaista tRF:ää, jotka näyttivät ilmentyvän eri lailla syövässä kuin normaalissa kudoksessa. Suurin osa tunnistetuista tRF:ista on peräisin kypsän sytosolisen tRNA:n 5’-ja 3’-päistä, mutta myös muita fragmentteja löytyi. 5’-päästä peräisin olevat tRF:t olivat eniten yli-ilmentyneitä ja 3’-päästä peräsin olevat ali-ilmentyneitä syövässä. Kolmen tRF:n ilmentymiserot varmennettiin qRT-PCR:llä. Normalisoitu tRNALys:stä ja tRNAPhe:stä peräisin olevien tRF-315:n ja tRF-544:n ilmentyvyyssuhde ennusti hyvin taudin etenemistä. Yhteenvetona tutkimuksessa löydettiin uusi monistuma-alue, jossa saattaa sijaita rakkosyövän kehityksen kannalta tärkeitä onkogeenejä. Lisäksi tunnistettiin useita eturauhassyövässä poikkeavasti ilmentyviä ns. ei-koodaavia RNA:ita. Nämä saattavat olla mekanistisesti tärkeitä eturauhassyövän kehityksen kannalta ja mahdollisia syövän aggressiivisuuden markkereita.Cancer is a complex disease, caused by accumulation of genetic alterations in normal cells. The consequence of these genetic alterations is the disruption of normal cell number homeostasis and uncontrolled cell proliferation. Understanding the molecular mechanisms behind tumorigenesis is essential in order to identify aggressive and possibly lethal form of the disease as well as to plan effective cancer therapeutic strategies. Urinary bladder cancer is the most common malignancy of the urinary tract. Most of the tumors arise from the epithelium lining the inside of the urinary bladder (urothelial carcinomas). Squamous cell carcinomas represent less than 5% of bladder cancer cases. Bladder cancer is a heterogeneous disease, characterized by different genetic alterations, leading to diverse pathways of cancer development and progression. Many of these genetic alterations consist of region-specific gains and losses of DNA copy number. Regions of DNA copy number gain or amplification commonly harbor oncogenes, whereas deleted regions harbor tumor suppressor genes. In this study, array-comparative genomic hybridization (aCGH) was performed in bladder cancer clinical samples and cell line models, revealing a common amplification at chromosomal region 1p21-22. The minimal region of the amplification was mapped to a region of about one 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. DR1 was found to be the most significantly overexpressed in the SCaBER cell line. According to published clinical sample cohorts, DR1 is overexpressed also in superficial and infiltrating bladder cancers. Prostate cancer (PC) is the second most commonly diagnosed cancer among males worldwide and the most frequently diagnosed malignancy in developed countries. The heterogeneity of histologic and clinical features of PC is well known, but the mechanisms underlying the heterogeneity are not understood. Deeper understanding of the molecular mechanisms of PC tumorigenesis is needed to discover more specific biomarkers of aggressive form of the disease. Recently, there has been increasing attention on the role of microRNAs (miRNAs) in cancer development. Several expression-profiling studies have provided evidence of aberrant expression of miRNAs in prostate cancer and have highlighted the potential use of specific miRNA expression signatures as prognostic or predictive markers. Similarly to other solid tumors, it is at present unclear whether prostate cancer is organized hierarchically into populations of cells with different proliferative potentials, as cancer stem cell (CSC) model suggests. Several studies have used flow cytometry-based approaches to isolate putative prostate stem cells. Here, genome-wide miRNA expression analysis was performed on patient-derived, stem-like cells (SC), transit-amplifying cells and committed basal (CB) cells, enriched from briefly cultured primary prostate epithelial cells. Each cell subpopulation showed a distinct miRNA expression profile, regardless of its pathologic status. MiR-548c-3p was found to be overexpressed approximately fivefold in SCs, compared with CBs. Functional studies of miR-548c-3p overexpression in CBs showed increased dedifferentiation to a more stem-like phenotype. MiR-548c-3p was also found to be significantly upregulated in CRPC-derived epithelial cells, compared with BPH-derived epithelial cells, suggesting that miR-548c-3p is a functional biomarker for PC aggressiveness. In order to identify new, differentially expressed miRNAs, the expression data obtained from recent deep-sequencing experiments on pools of clinical specimens were analyzed. MiR-1247-5p, miR-1249, miR-1269a, miR1271-5p, miR-1290, miR-1291 and miR-1299 showed differential expression in malignant samples, compared to benign ones and were selected for validation by qRT-PCR. Significant up-regulation of miR-1247-5p was found in castration-resistant prostate cancer (CRPC), compared to non-malignant prostate. The expression of miR-1247-5p was subsequently studied in PC cell lines where an up-regulation of miR-1247-5p was observed in the androgen-independent PC-3 model. According to on-line target prediction tools MYCBP2 (myc-binding protein 2) is a high-scoring potential target of miR-1247-5p. Down-regulation of MYCBP2 at both mRNA and protein levels was demonstrated by overexpression of miR-1247-5p in PC-3 and LNCaP models. Next, MYCBP2 was confirmed as a target of miR-1247-5p by using luciferase reporter assay. Several high-throughput sequencing studies in human cancers have recently led to the discovery of additional groups of non-coding RNAs. Next to miRNAs, the most abundant non-coding RNAs in prostate cancer cell lines were found to be fragments derived from tRNAs that are termed tRNA-derived RNA fragments (tRFs). The characteristic and abundant expression of the fragments, as well as their precise sequence, indicate that these molecules are not random products of tRNA degradation. However, the precise role of tRFs is still unclear. In this study, the expression of tRFs was analyzed in normal adjacent prostate and different stages of PC by RNA-sequencing. A total of 598 unique tRFs were identified, many of which appear to be deregulated in cancer samples, when compared to controls. Most of the identified tRFs are derived from the 5’ and 3’ end of mature cytosolic tRNAs, but tRFs produced from pre-tRNA trailers and leaders were also found, as well as tRFs from mitochondrial tRNAs. The 5’-derived tRFs comprised the most abundant class of tRFs and represented the major class among upregulated tRFs, while 3’-derived tRFs types were dominant among downregulated tRFs in PC. The expression of three tRFs (tRF-544, tRF-315 and tRF-562) was validated in PC using qRT-PCR. Interestingly, the normalized expression ratio of tRF-315 and tRF-544, derived from tRNALys and tRNAPhe respectively, emerged as a good indicator of progression-free survival and as a candidate prognostic marker. In conclusion, a novel amplification, which may harbour important oncogenes, was identified in bladder cancer. In addition, several differentially expressed non-coding RNAs were discovered in prostate cancer. They may be important drivers of prostate tumorigenesis and putative biomarkers of aggressive form of the disease

Kromosomin 1p21-22 monistuman löytäminen rakkosyövässä ja ei-koodaavien RNA:iten ilmentyminen eturauhassyövässä

Eturauhas- ja rakkosyövän molekyylimekanismit Syöpä on monimutkainen sairaus, jonka aiheuttavat normaaliin soluun kertyvät geneettiset muutokset. Tällaiset geneettiset muutokset häiritsevät normaalien solujen homeostaasia ja johtavat hallitsemattomaan solujen lisääntymiseen. Näiden muutosten tunteminen on tärkeää, jotta voitaisiin kehittää entistä tehokkaampia syövän hoitomuotoja ja diagnostisia menetelmiä tappavan tautimuodon tunnistamiseksi. Virtsarakon syöpä on yleisin virtsateiden maligniteetti. Useimmat virtsarakon syövät syntyvät välimuotoisesta epiteelistä (uroteliaaliset karsinoomat). Alle 5 % virtsarakon syöpätapauksista on levyepiteeliperäisiä. Virtsarakon syöpä on heterogeeninen sairaus, jolle ovat ominaisia erilaiset geneettiset muutokset, jotka johtavat eri polkuja syövän kehittymiseen ja etenemiseen. Monet näistä geneettisistä muutoksista vaikuttavat geenien kopiolukumäärään johtaen onkogeenien monistumaan ja kasvurajoitegeenien häviämään. Tässä tutkimuksessa käytettiin sirupohjaista, vertailevaa genomista hybridisaatiomenetelmää (aCGH) geenikopiolukumäärän analysoimiseksi kliinisissä rakkosyöpänäytteissä sekä solulinjoissa. Yhdeksi uudeksi monistuma-alueeksi tunnistettiin kromosomialue 1p21-22. Työssä kartoitettiin ko. aluetta tarkemmin ja osoitettiin, että ns. minimaalinen monistuma-alue käsitti yhden miljoonan emäsparin DNA-jakson sisältäen yhteensä 11 tunnettua geeniä. Korkein monistuma-aste löytyi SCaBER-levyepiteelisyöpäsolulinjasta. Näistä neljä geeniä, TMED5, DR1, RPL5 ja EVI5, yli-ilmeni SCaBER-solulinjassa, DR1 kaikkein eniten. Julkisten tietokantojen perusteella DR1 yli-ilmenee myös kliinisissä näytteissä, jotka edustavat yleistä virtsarakkosyövän histologista tyyppiä. Eturauhassyöpä on miesten toiseksi yleisin diagnosoitu syöpä maailmassa ja yleisin kehittyneissä maissa. Taudin histologisen ja kliinisen käyttäytymisen monimuotoisuuden taustalla olevia mekanismeja tunnetaan huonosti. Eturauhassyövän molekyylitason mekanismien tunteminen mahdollistaisi uusien taudin aggressiivisuutta kuvastavien biomarkkerien löytämisen. Viime aikoina on yhä enemmän kiinnitetty huomiota mikroRNA:iden (miRNA) merkitykseen syövän kehittymisessä. Useat tutkimukset ovat osoittaneet, että miRNA-ilmentymistasot ovat muuntuneita eturauhassyövässä. Onkin mahdollista, että niitä voitaisiin käyttää ennusteellisina biomarkkereina. Kuten muissa syövissä, on epäselvää, koostuuko eturauhassyöpä eri solupopulaatioista, joilla on erilainen jakaantumiskyky, kuten syövän kantasolumallit ennustavat. Useissa tutkimuksissa on käytetty virtaussytometriaa eturauhasen kantasolujen eristämiseen. Tässä tutkimuksessa tehtiin genominlaajuinen miRNA-ilmentymisen analyysi potilaista peräisin olevista kantasolun kaltaisista soluista (SC), välivaiheen soluista ja sitoutuneista (CB) soluista. Jokainen solualapopulaatio poikkesi toisistaan miRNA-ilmentymisprofiililtaan riippumatta siitä, oliko kyseessä syöpänäyte vai ei. MiR-548c-3p:n havaittiin yli-ilmentyvän noin viisinkertaisesti SC-soluissa verrattuna CB-soluihin. Toiminnallisissa tutkimuksissa miR-548c-3p:n yli-ilmentyminen CB-soluissa vähensi erilaistumista kohti kantasoluilmiasua. MiR-548c-3p oli myös merkittävästi yli-ilmentynyt kastraatioresistentistä eturauhassyövästä (CRPC) eristetyissä soluissa verrattuna eturauhasen hyvänlaatuisesta liikakasvusta (BPH) eristettyihin epiteelisoluihin. Tämä viitaa siihen, että miR-548c-3p voisi olla eturauhassyövän aggressiivisuuden biomarkkeri. Tunnistaaksemme uusia, eri tavoin ilmentyviä miRNA:ita, syväsekvensoimme joukon kliinisiä eturauhassyöpänäytteitä. MiR-1247-5p, miR-1249, miR-1269a, miR1271-5p, miR-1290, miR-1291 ja miR-1299 ilmentyivät eri lailla syövässä ja normaalissa eturauhasessa. Niinpä näitä tutkittiin laajemmassa materiaalissa qRT-PCR-menetelmällä. MiR-1247-5p ilmentyi merkittävästi enemmän CRPC-näytteissä verrattuna ei-maligniin eturauhaseen. Ennusteohjelmien perusteella miR-1247-5p:n yksi kohdegeeni voisi olla MYCBP2 (myc:iä sitova proteiini 2). miR-1247-5p:n yli-ilmentyminen PC-3- ja LNCaP-syöpäsolulinjoissa johti MYCBP2:n ilmentymisen laskuun sekä mRNA- että proteiinitasolla. Lusiferaasireportterikoe vahvisti, että MYCBP2 on miR-1247-5p:n kohde. Useissa uuden sukupolven sekvensointitutkimuksissa on syövissä löydetty uusia ryhmiä ei-koodaavia RNA:ita. Eturauhassyöpäsolulinjoissa miRNA:iden jälkeen toiseksi yleisin ryhmä ei-koodaavia RNA:ita ovat tRNA:ista peräisin olevat fragmentit (tRFs). Niiden ominaisuudet, runsas ilmentyminen ja tarkka sekvenssi viittaavat siihen, että nämä molekyylit eivät ole satunnaisia tuotteita tRNA:n hajoamisesta. tRF:ien tarkka rooli on kuitenkin edelleen epäselvä. Tässä tutkimuksessa tRF:ien ilmentymistä analysoitiin potilaan kudosnäytteistä. Tutkimuksessa tunnistettiin yhteensä 598 erilaista tRF:ää, jotka näyttivät ilmentyvän eri lailla syövässä kuin normaalissa kudoksessa. Suurin osa tunnistetuista tRF:ista on peräisin kypsän sytosolisen tRNA:n 5’-ja 3’-päistä, mutta myös muita fragmentteja löytyi. 5’-päästä peräisin olevat tRF:t olivat eniten yli-ilmentyneitä ja 3’-päästä peräsin olevat ali-ilmentyneitä syövässä. Kolmen tRF:n ilmentymiserot varmennettiin qRT-PCR:llä. Normalisoitu tRNALys:stä ja tRNAPhe:stä peräisin olevien tRF-315:n ja tRF-544:n ilmentyvyyssuhde ennusti hyvin taudin etenemistä. Yhteenvetona tutkimuksessa löydettiin uusi monistuma-alue, jossa saattaa sijaita rakkosyövän kehityksen kannalta tärkeitä onkogeenejä. Lisäksi tunnistettiin useita eturauhassyövässä poikkeavasti ilmentyviä ns. ei-koodaavia RNA:ita. Nämä saattavat olla mekanistisesti tärkeitä eturauhassyövän kehityksen kannalta ja mahdollisia syövän aggressiivisuuden markkereita.Cancer is a complex disease, caused by accumulation of genetic alterations in normal cells. The consequence of these genetic alterations is the disruption of normal cell number homeostasis and uncontrolled cell proliferation. Understanding the molecular mechanisms behind tumorigenesis is essential in order to identify aggressive and possibly lethal form of the disease as well as to plan effective cancer therapeutic strategies. Urinary bladder cancer is the most common malignancy of the urinary tract. Most of the tumors arise from the epithelium lining the inside of the urinary bladder (urothelial carcinomas). Squamous cell carcinomas represent less than 5% of bladder cancer cases. Bladder cancer is a heterogeneous disease, characterized by different genetic alterations, leading to diverse pathways of cancer development and progression. Many of these genetic alterations consist of region-specific gains and losses of DNA copy number. Regions of DNA copy number gain or amplification commonly harbor oncogenes, whereas deleted regions harbor tumor suppressor genes. In this study, array-comparative genomic hybridization (aCGH) was performed in bladder cancer clinical samples and cell line models, revealing a common amplification at chromosomal region 1p21-22. The minimal region of the amplification was mapped to a region of about one 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. DR1 was found to be the most significantly overexpressed in the SCaBER cell line. According to published clinical sample cohorts, DR1 is overexpressed also in superficial and infiltrating bladder cancers. Prostate cancer (PC) is the second most commonly diagnosed cancer among males worldwide and the most frequently diagnosed malignancy in developed countries. The heterogeneity of histologic and clinical features of PC is well known, but the mechanisms underlying the heterogeneity are not understood. Deeper understanding of the molecular mechanisms of PC tumorigenesis is needed to discover more specific biomarkers of aggressive form of the disease. Recently, there has been increasing attention on the role of microRNAs (miRNAs) in cancer development. Several expression-profiling studies have provided evidence of aberrant expression of miRNAs in prostate cancer and have highlighted the potential use of specific miRNA expression signatures as prognostic or predictive markers. Similarly to other solid tumors, it is at present unclear whether prostate cancer is organized hierarchically into populations of cells with different proliferative potentials, as cancer stem cell (CSC) model suggests. Several studies have used flow cytometry-based approaches to isolate putative prostate stem cells. Here, genome-wide miRNA expression analysis was performed on patient-derived, stem-like cells (SC), transit-amplifying cells and committed basal (CB) cells, enriched from briefly cultured primary prostate epithelial cells. Each cell subpopulation showed a distinct miRNA expression profile, regardless of its pathologic status. MiR-548c-3p was found to be overexpressed approximately fivefold in SCs, compared with CBs. Functional studies of miR-548c-3p overexpression in CBs showed increased dedifferentiation to a more stem-like phenotype. MiR-548c-3p was also found to be significantly upregulated in CRPC-derived epithelial cells, compared with BPH-derived epithelial cells, suggesting that miR-548c-3p is a functional biomarker for PC aggressiveness. In order to identify new, differentially expressed miRNAs, the expression data obtained from recent deep-sequencing experiments on pools of clinical specimens were analyzed. MiR-1247-5p, miR-1249, miR-1269a, miR1271-5p, miR-1290, miR-1291 and miR-1299 showed differential expression in malignant samples, compared to benign ones and were selected for validation by qRT-PCR. Significant up-regulation of miR-1247-5p was found in castration-resistant prostate cancer (CRPC), compared to non-malignant prostate. The expression of miR-1247-5p was subsequently studied in PC cell lines where an up-regulation of miR-1247-5p was observed in the androgen-independent PC-3 model. According to on-line target prediction tools MYCBP2 (myc-binding protein 2) is a high-scoring potential target of miR-1247-5p. Down-regulation of MYCBP2 at both mRNA and protein levels was demonstrated by overexpression of miR-1247-5p in PC-3 and LNCaP models. Next, MYCBP2 was confirmed as a target of miR-1247-5p by using luciferase reporter assay. Several high-throughput sequencing studies in human cancers have recently led to the discovery of additional groups of non-coding RNAs. Next to miRNAs, the most abundant non-coding RNAs in prostate cancer cell lines were found to be fragments derived from tRNAs that are termed tRNA-derived RNA fragments (tRFs). The characteristic and abundant expression of the fragments, as well as their precise sequence, indicate that these molecules are not random products of tRNA degradation. However, the precise role of tRFs is still unclear. In this study, the expression of tRFs was analyzed in normal adjacent prostate and different stages of PC by RNA-sequencing. A total of 598 unique tRFs were identified, many of which appear to be deregulated in cancer samples, when compared to controls. Most of the identified tRFs are derived from the 5’ and 3’ end of mature cytosolic tRNAs, but tRFs produced from pre-tRNA trailers and leaders were also found, as well as tRFs from mitochondrial tRNAs. The 5’-derived tRFs comprised the most abundant class of tRFs and represented the major class among upregulated tRFs, while 3’-derived tRFs types were dominant among downregulated tRFs in PC. The expression of three tRFs (tRF-544, tRF-315 and tRF-562) was validated in PC using qRT-PCR. Interestingly, the normalized expression ratio of tRF-315 and tRF-544, derived from tRNALys and tRNAPhe respectively, emerged as a good indicator of progression-free survival and as a candidate prognostic marker. In conclusion, a novel amplification, which may harbour important oncogenes, was identified in bladder cancer. In addition, several differentially expressed non-coding RNAs were discovered in prostate cancer. They may be important drivers of prostate tumorigenesis and putative biomarkers of aggressive form of the disease

Proteomic Landscape of Prostate Cancer: The View Provided by Quantitative Proteomics, Integrative Analyses, and Protein Interactomes

Prostate cancer is the second most frequent cancer of men worldwide. While the genetic landscapes and heterogeneity of prostate cancer are relatively well-known already, methodological developments now allow for studying basic and dynamic proteomes on a large scale and in a quantitative fashion. This aids in revealing the functional output of cancer genomes. It has become evident that not all aberrations at the genetic and transcriptional level are translated to the proteome. In addition, the proteomic level contains heterogeneity, which increases as the cancer progresses from primary prostate cancer (PCa) to metastatic and castration-resistant prostate cancer (CRPC). While multiple aspects of prostate adenocarcinoma proteomes have been studied, less is known about proteomes of neuroendocrine prostate cancer (NEPC). In this review, we summarize recent developments in prostate cancer proteomics, concentrating on the proteomic landscapes of clinical prostate cancer, cell line and mouse model proteomes interrogating prostate cancer-relevant signaling and alterations, and key prostate cancer regulator interactomes, such as those of the androgen receptor (AR). Compared to genomic and transcriptomic analyses, the view provided by proteomics brings forward changes in prostate cancer metabolism, post-transcriptional RNA regulation, and post-translational protein regulatory pathways, requiring the full attention of studies in the future