Inhibition of RNA Binding in SND1 Increases the Levels of miR-1-3p and Sensitizes Cancer Cells to Navitoclax

SND1 is an RNA-binding protein overexpressed in large variety of cancers. SND1 has been proposed to enhance stress tolerance in cancer cells, but the molecular mechanisms are still poorly understood. We analyzed the expression of 372 miRNAs in the colon carcinoma cell line and show that SND1 silencing increases the expression levels of several tumor suppressor miRNAs. Furthermore, SND1 knockdown showed synergetic effects with cancer drugs through MEK-ERK and Bcl-2 family-related apoptotic pathways. To explore whether the SND1-mediated RNA binding/degradation is responsible for the observed effect, we developed a screening assay to identify small molecules that inhibit the RNA-binding function of SND1. The screen identified P2X purinoreceptor antagonists as the most potent inhibitors. Validation confirmed that the best hit, suramin, inhibits the RNA binding ability of SND1. The binding characteristics and mode of suramin to SND1 were characterized biophysically and by molecular docking that identified positively charged binding cavities in Staphylococcus nuclease domains. Importantly, suramin-mediated inhibition of RNA binding increased the expression of miR-1-3p, and enhanced sensitivity of cancer cells to Bcl-2 inhibitor navitoclax treatment. Taken together, we demonstrate as proof-of-concept a mechanism and an inhibitor compound for SND1 regulation of the survival of cancer cells through tumor suppressor miRNAs

Inhibition of RNA Binding in SND1 Increases the Levels of miR-1-3p and Sensitizes Cancer Cells to Navitoclax

SND1 is an RNA-binding protein overexpressed in large variety of cancers. SND1 has been proposed to enhance stress tolerance in cancer cells, but the molecular mechanisms are still poorly understood. We analyzed the expression of 372 miRNAs in the colon carcinoma cell line and show that SND1 silencing increases the expression levels of several tumor suppressor miRNAs. Furthermore, SND1 knockdown showed synergetic effects with cancer drugs through MEK-ERK and Bcl-2 family-related apoptotic pathways. To explore whether the SND1-mediated RNA binding/degradation is responsible for the observed effect, we developed a screening assay to identify small molecules that inhibit the RNA-binding function of SND1. The screen identified P2X purinoreceptor antagonists as the most potent inhibitors. Validation confirmed that the best hit, suramin, inhibits the RNA binding ability of SND1. The binding characteristics and mode of suramin to SND1 were characterized biophysically and by molecular docking that identified positively charged binding cavities in Staphylococcus nuclease domains. Importantly, suramin-mediated inhibition of RNA binding increased the expression of miR-1-3p, and enhanced sensitivity of cancer cells to Bcl-2 inhibitor navitoclax treatment. Taken together, we demonstrate as proof-of-concept a mechanism and an inhibitor compound for SND1 regulation of the survival of cancer cells through tumor suppressor miRNAs

Probabilistic analysis of gene expression measurements from heterogeneous tissues

Motivation: Tissue heterogeneity, arising from multiple cell types, is a major confounding factor in experiments that focus on studying cell types, e.g. their expression profiles, in isolation. Although sample heterogeneity can be addressed by manual microdissection, prior to conducting experiments, computational treatment on heterogeneous measurements have become a reliable alternative to perform this microdissection in silico. Favoring computation over manual purification has its advantages, such as time consumption, measuring responses of multiple cell types simultaneously, keeping samples intact of external perturbations and unaltered yield of molecular content

Traditional and Novel Treatments for Prostate Cancer - Discoveries at the molecular level

Endokriinistä hoitoa käytetään rutiinisti pitkälle edenneen eturauhassyövän hoidossa, vaikka sen solunsisäisiä vaikutuksia tunnetaan huonosti. Tässä väitöskirjatutkimuksessa hyödynsimme Tampereen yliopistollisessa sairaalassa kerättyä harvinaista potilasmateriaalia. Materiaali oli kerätty 28 potilaalta, jotka saivat endokriinistä hoitoa kolmen kuukauden ajan ennen leikkausta. Potilasmateriaalin ansiosta pääsimme tutkimaan syöpäkudosnäytteistä ilmiöitä, joita aiemmin on tutkittu pääasiassa soluviljelyolosuhteissa tai koe-eläinmalleissa. Näistä näytteistä määritimme ihmisen kaikkien tunnettujen geenien aktiivisuustasot sekä tutkimme hormonihoidon vaikutuksia niiden aktiivisuuksiin. Kliinisissä kokeissa kahden yleisimmin eturauhassyövän hoidossa käytetyn endokriinisen lääkkeen, GnRH-agonistin ja antiandrogeenin, tehokkuudessa ei ole havaittu eroja paikallisissa syövissä. Tässä tutkimuksessa saimme kuitenkin selville, että hoitojen vaikutukset eroavat toisistaan solutasolla oletettua enemmän. Eturauhassyövän kasvun tiedetään tapahtuvan lähes aina androgeenireseptorigeenin (AR-geenin) säätelyn kautta. AR:n kohdegeenejä syöpäkudoksessa ei kuitenkaan tunneta täysin tarkasti. Endokriinihoidon teho perustuu AR:n aktiivisuuden hiljentämiseen syöpäkudoksessa. Voimme siis olettaa, että osa hoidon ansiosta hiljentyneistä geeneistä ovat AR:n kohdegeenejä. Yhdistämällä oman ryhmämme sekä muiden tutkijoiden soluviljelyolosuhteissa keräämää tietoa mm. AR:n sitoutumiskohdista genomissa pystyimme osoittamaan mahdollisia AR:n kohdegeenejä näytemateriaalistamme. Lisäksi löysimme uusia potentiaalisia indikaattorigeenejä, joiden aktiivisuuden muutokset voivat osoittaa endokriinihoidon vaikutuksen vähentymistä ja syövän aggressiivisuuden lisääntymistä. Näitä indikaattorigeenejä seuraamalla voitaisiin kemoterapiahoidon aloitus ajoittaa mahdollisimman optimaaliseksi ja näin parantaa hoidon tehoa sekä lisätä potilaiden elinaikaa. Määritimme näytteistä myös äskettäin löydetyn kahden geenin fuusion yleisyyttä ja tutkimme, vaikuttaako endokriininen hoito eri tavalla niihin potilaisiin, joilla tämä geneettinen muutos on tapahtunut. Geenifuusio todettiin 60 %:lla aineiston potilaista. Huomasimme, että potilailla, joilta geenifuusio löydettiin, useat solujen kasvuun ja jakaantumiseen vaikuttavat geenit olivat aktiivisempia kuin potilailla, joilla geenifuusiota ei ollut. Havaitsimme myös endokriinisen hoidon alentavan tehokkaasti näiden samojen geenien aktiivisuutta. Voimme siis todeta, että endokriininen hoito on tehokas myös potilaille, joilta tämä geenifuusio löytyy. Eturauhassyöpä aiheuttaa Suomessa ja muissa länsimaissa toiseksi eniten syöpäkuolemia miehillä heti keuhkosyövän jälkeen.The efficient treatment of prostate cancer faces several challenges. Although an overwhelming majority of all prostate cancers can be efficiently treated by radical prostatectomy or radiation therapy, prostate cancer remains one of the main cancer killers in the Western world. The mortality rate is high for two reasons: prostate cancer has the most frequent occurrence of all cancers affecting men, and effective treatments against metastatic prostate cancer do not exist. The lethal form of prostate cancer is aggressive and metastasizes to other tissues. For this form, the most efficient treatment is endocrine therapy. However, this therapy is not curative. Resistance against treatment develops in an average of 18-24 months, and after resistance has developed, the mean overall survival time of patients with metastatic prostate cancer is only 20 months. Despite intensive studies, the molecular mechanisms leading to resistance to endocrine therapy remain obscure. The understanding of these molecular mechanisms will enhance the development of more efficient treatment methods and will improve the survival of prostate cancer patients. This study aimed to determine the molecular consequences of the two most commonly used endocrine therapies for prostate cancer. We utilized rare clinical material from 28 prostate cancer patients who had undergone neoadjuvant endocrine therapy and analyzed the expression levels of all known protein-coding genes and over 700 miRNAs from the prostate cancer samples by microarray. Furthermore, we determined the cancer-specific gene expression levels from heterogeneous prostate tissue samples using an in silico Bayesian modeling tool. The rare clinical material enabled us to study events that have previously been studied using mainly in vitro or animal models. We detected great differences in transcriptome levels between the two endocrine therapies, GnRH agonist and antiandrogens, despite their similar clinical outcomes. In addition, we determined the frequency of the most common fusion gene in prostate cancer, TMPRSS2:ERG, from the samples and determined how the endocrine treatment affected the ERG-regulated genes. In non-treated patients, the TMPRSS2:ERG fusion enhanced the expression of proliferation-related genes. Interestingly, the endocrine therapies reduced the expression of these genes and diminished the differences between fusion-positive and fusion-negative samples. In addition, we characterized possible androgen receptor dependent regulation and cancer specificity of the most differently expressed genes after endocrine therapy. Several miRNAs and two protein-coding genes (NEDD4L and TPD52) showed their potential as prognostic biomarkers for the formation of treatment resistance. However, more studies are needed to explore their potency fully. The last part of the study explored the capacity of novel polycationic peptides to enhance the transduction of viral gene transfer vectors into prostate and other cancer cells. With optimized transduction efficiency, viral gene therapy could be used as a novel treatment method for prostate and other cancers. However, our study revealed that polycationic peptides were not more efficient than polybrene and protamine sulphate, which are the small cationic compounds traditionally used in in vitro cell culture models. Thus, more studies with different approaches are needed to obtain clinically sufficient gene transfer efficiency with the current viral vectors. This study provides valuable, novel information regarding the transcriptional events in prostate cancer and can assist in the development of more effective treatment methods for prostate cancer

The RNA-binding protein Snd1/Tudor-SN regulates hypoxia-responsive gene expression

Snd1 is an evolutionarily conserved RNA-binding protein implicated in several regulatory processes in gene expression including activation of transcription, mRNA splicing, and microRNA decay. Here, we have investigated the outcome of Snd1 gene deletion in the mouse. The knockout mice are viable showing no gross abnormalities apart from decreased fertility, organ and body size, and decreased number of myeloid cells concomitant with decreased expression of granule protein genes. Deletion of Snd1 affected the expression of relatively small number of genes in spleen and liver. However, mRNA expression changes in the knockout mouse liver showed high similarity to expression profile in adaptation to hypoxia. MicroRNA expression in liver showed upregulation of the hypoxia-induced microRNAs miR-96 and -182. Similar to Snd1 deletion, mimics of miR-96/182 enhanced hypoxia-responsive reporter activity. To further elucidate the function of SND1, BioID biotin proximity ligation assay was performed in HEK-293T cells to identify interacting proteins. Over 50% of the identified interactors were RNA-binding proteins, including stress granule proteins. Taken together, our results show that in normal growth conditions, Snd1 is not a critical factor for mRNA transcription in the mouse, and describe a function for Snd1 in hypoxia adaptation through negatively regulating hypoxia-related miRNAs and hypoxia-induced transcription consistent with a role as stress response regulator.Peer reviewe

The RNA-binding protein Snd1/Tudor-SN regulates hypoxia-responsive gene expression

Abstract Snd1 is an evolutionarily conserved RNA-binding protein implicated in several regulatory processes in gene expression including activation of transcription, mRNA splicing, and microRNA decay. Here, we have investigated the outcome of Snd1 gene deletion in the mouse. The knockout mice are viable showing no gross abnormalities apart from decreased fertility, organ and body size, and decreased number of myeloid cells concomitant with decreased expression of granule protein genes. Deletion of Snd1 affected the expression of relatively small number of genes in spleen and liver. However, mRNA expression changes in the knockout mouse liver showed high similarity to expression profile in adaptation to hypoxia. MicroRNA expression in liver showed upregulation of the hypoxia-induced microRNAs miR-96 and -182. Similar to Snd1 deletion, mimics of miR-96/182 enhanced hypoxia-responsive reporter activity. To further elucidate the function of SND1, BioID biotin proximity ligation assay was performed in HEK-293T cells to identify interacting proteins. Over 50% of the identified interactors were RNA-binding proteins, including stress granule proteins. Taken together, our results show that in normal growth conditions, Snd1 is not a critical factor for mRNA transcription in the mouse, and describe a function for Snd1 in hypoxia adaptation through negatively regulating hypoxia-related miRNAs and hypoxia-induced transcription consistent with a role as stress response regulator