Alternative splicing and its role in pathologies of the endocrine system

Różnicowe składanie pierwotnego transkryptu (splicing alternatywny pre-mRNA) jest procesem polegającym na usuwaniu rejonów, które nie kodują dojrzałej cząsteczki mRNA (intronów) i łączeniu rejonów kodujących (eksonów) w różnych konfiguracjach, dzięki czemu powstają cząsteczki mRNA o różnej sekwencji nukleotydów. Różnicowemu składaniu podlegają transkrypty prawie wszystkich kodujących białka genów człowieka, przyczyniając się do powstawania wielkiej różnorodności izoform białek kodowanych przez stosunkowo niewielką liczbę genów. Proces splicingu alternatywnego w istotny sposób przyczynia się do prawidłowego funkcjonowania komórek, również tych biorących udział w sygnalizacji hormonalnej. Zaburzenia różnicowego składania pre-mRNA prowadzą do nieprawidłowości w procesach komórkowych i w konsekwencji mogą prowadzić do zaburzeń endokrynnych. Zaburzenia różnicowego składania premRNA wynikają z mutacji w tak zwanych miejscach splicingowych bądź z nieprawidłowego działania czynników splicingowych, czyli białek zaangażowanych w regulację tego procesu. Niniejsza praca ma na celu przedstawienie roli różnicowego składania pre-mRNA w patologiach układu endokrynnego, takich jak nowotwory endokrynne (guzy przysadki, rak tarczycy, prostaty, jajnika, piersi, insulinoma), izolowany niedobór hormonu wzrostu, dysgenezja gonad. Omówione zostały także przykłady patologii niezwiązanych bezpośrednio z układem endokrynnym, w których wykryto zaburzenia alternatywnego splicingu transkryptów genów biorących udział w sygnalizacji hormonalnej. Artykuł kończy przegląd możliwości diagnostycznych i terapeutycznych w chorobach endokrynnych, wykorzystujących proces różnicowego składania pre-mRNA. (Endokrynol Pol 2011; 62 (2): 160–170)Alternative splicing of pre-mRNA is a process in which noncoding regions of primary transcript are removed and coding regions are joined in different manners to produce mRNA molecules of different sequences. Alternative splicing affects nearly all human genes and is a key source of diversity of proteins coded by a relatively small number of genes. Since alternative splicing is of crucial importance for the proper functioning of cells, including those involved in hormonal signalling, aberrations of alternative splicing can lead to disruption of cellular mechanisms and in consequence result in serious endocrine pathologies. Disturbances of alternative splicing include mutations of consensus splice regulatory sites and improprieties in the action of splicing factors, the proteins involved in regulating the process. In consequence of disturbed alternative splicing, improperly spliced mRNA and protein isoforms can be produced which can lead to disruption of function of their wild type counterparts. This review aims to discuss the role of alternative splicing in pathologies of the endocrine system and gives examples that highlight the importance of this process in the proper functioning of hormones, hormone receptors and other factors involved in hormonal regulation. The examples given include endocrine-related tumours (pituitary tumours, cancers of the thyroid, prostate, ovary and breast, and insulinoma), isolated growth hormone deficiency, and Frasier syndrome. Non-endocrine pathologies in which aberrant alternative splicing of transcripts of genes involved in hormonal signalling have been detected are also described. Finally, we discuss future perspectives on the possible usage of alternative splicing in diagnostics and therapy. (Pol J Endocrinol 2011; 62 (2): 160–170

Znaczenie TRIP11 i ścieżki sygnałowej 3,5,3’-trójjodotyroniny w progresji raka nerki oraz wpływ na czas przeżycia chorych z rakiem nerki

Introduction: TRIP11 is a multifunctional protein localizing either to Golgi apparatus, acting as a golgin, or in the nucleus, acting as coactivator of transcription mediated by thyroid hormone receptor (THR) and hypoxia induced factor (HIF). Triiodothyronine (T3) regulates nuclear localization of TRIP11 by inducing its phosphorylation. The exact mechanism of this regulation unknown. The expressions of THR and HIF are disturbed in various cancers, including renal cell cancer (RCC). In this study we aimed to analyze: 1) the mechanism of T3-dependent subcellular localization of TRIP11; 2) the significance of TRIP11 and T3 signaling pathway in RCC progression. Material and methods: TRIP11 subcellular localization was analyzed using immunocytochemistry in RCC-derived cell line treated with T3, T3-agarose and PI3K inhibitor, wortmannin. The expressions of TRIP11 and genes involved in T3 signaling and hypoxia were investigated using qPRC in 36 pairs of RCC tumor-control samples, followed by validation/survival analysis in an independent cohort of >450 renal cancer patients. Results: Wortmannin disrupted T3-dependent nuclear transport of TRIP11. T3-agarose did not change TRIP11 localization, precluding extracellular T3-mediated mechanism. The expressions of TRIP11, HIF-1β, THRA, THRB, FURIN, VEGFA, and GLUT1 were disturbed in renal cancer. Expressions of TRIP11 and HIF-1β correlated with tumor grades. Decreased expressions of TRIP11, THRA, and THRB correlated with poor survival of RCC patients. Conclusions: 1) T3 induces nuclear TRIP11 localization via PI3K-dependent mechanism; 2) disturbed expression of T3 signaling pathway genes correlates with RCC progression. The specific mechanisms by which altered T3 signaling may contribute to RCC progression require further investigation.Wstęp: TRIP11 jest wielofunkcyjnym białkiem lokalizującym się w aparacie Golgiego lub w jądrze komórkowym, gdzie działa jako koak­tywator transkrypcji zależnej od receptorów hormonów tarczycy (THR) i czynnika indukowanego hipoksją (HIF). 3,5,3’-trójjodotyronina (T3) reguluje subkomórkową lokalizację TRIP11 wpływając na jego fosforylację. Dokładny mechanizm tej regulacji jest jednak nieznany. Ekspresja THR i HIF jest zaburzona w nowotworach, w tym w raku nerkowokomórkowym (RCC). Celem pracy było poznanie: 1. mecha­nizmu, za pośrednictwem którego T3 indukuje zmiany w lokalizacji TRIP11; 2. znaczenia TRIP11 i ścieżki sygnałowej T3 w progresji RCC. Materiały i metody: Lokalizację komórkową TRIP11 określano immunocytochemicznie w wywodzącej się z RCC linii komórkowej traktowanej T3, T3-agarozą i wortmaniną (inhibitorem kinazy PI3K). Ekspresja TRIP11 i genów zaangażowanych w odpowiedź komórki na T3 i hipoksję badano metodą qPCR w 36 parach guz RCC–kontrola nienowotworowa. Walidację wyników analizy qPCR oraz analizę czasu przeżycia prowadzono na niezależnej kohorcie chorych z RCC (n > 450). Wyniki: Wortmanina blokuje indukowany przez T3 transport TRIP11 do jądra komórkowego. T3-agaroza nie wpływa na lokalizację TRIP11, co wyklucza zewnątrzkomórkowy mechanizm działania T3. Ekspresja TRIP11, HIF-1β, THRA, THRB, FURIN, VEGFA i GLUT1 jest zaburzona w RCC. Ekspresja TRIP11 i HIF-1β koreluje ze stopniem zaawansowania nowotworu. Obniżona ekspresja TRIP11, THRA i THRB koreluje z krótszym czasem przeżycia chorych z RCC. Wnioski: 1. T3 zmienia lokalizację TRIP11 na jądrową poprzez mechanizm zależny od kinazy PI3K. 2. Zaburzona ekspresja genów ścieżki T3 koreluje z czasem przeżycia chorych z RCC. Dokładny mechanizm, za pośrednictwem którego T3 może wpływać na progresję RCC wymaga dalszych badań

Thyroid Hormones and Cancer: A Comprehensive Review of Preclinical and Clinical Studies

Thyroid hormones take major part in normal growth, development and metabolism. Over a century of research has supported a relationship between thyroid hormones and the pathophysiology of various cancer types. In vitro studies as well as research in animal models demonstrated an effect of the thyroid hormones T3 and T4 on cancer proliferation, apoptosis, invasiveness and angiogenesis. Thyroid hormones mediate their effects on the cancer cell through several non-genomic pathways including activation of the plasma membrane receptor integrin αvβ3. Furthermore, cancer development and progression are affected by dysregulation of local bioavailability of thyroid hormones. Case-control and population-based studies provide conflicting results regarding the association between thyroid hormones and cancer. However, a large body of evidence suggests that subclinical and clinical hyperthyroidism increase the risk of several solid malignancies while hypothyroidism may reduce aggressiveness or delay the onset of cancer. Additional support is provided from studies in which dysregulation of the thyroid hormone axis secondary to cancer treatment or thyroid hormone supplementation was shown to affect cancer outcomes. Recent preclinical and clinical studies in various cancer types have further shown promising outcomes following chemical reduction of thyroid hormones or inhibition or their binding to the integrin receptor. This review provides a comprehensive overview of the preclinical and clinical research conducted so far

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis

The Role of <i>TCOF1</i> Gene in Health and Disease: Beyond Treacher Collins Syndrome

The nucleoli are membrane-less nuclear substructures that govern ribosome biogenesis and participate in multiple other cellular processes such as cell cycle progression, stress sensing, and DNA damage response. The proper functioning of these organelles is ensured by specific proteins that maintain nucleolar structure and mediate key nucleolar activities. Among all nucleolar proteins, treacle encoded by TCOF1 gene emerges as one of the most crucial regulators of cellular processes. TCOF1 was initially discovered as a gene involved in the Treacher Collins syndrome, a rare genetic disorder characterized by severe craniofacial deformations. Later studies revealed that treacle regulates ribosome biogenesis, mitosis, proliferation, DNA damage response, and apoptosis. Importantly, several reports indicate that treacle is also involved in cancer development, progression, and response to therapies, and may contribute to other pathologies such as Hirschsprung disease. In this manuscript, we comprehensively review the structure, function, and the regulation of TCOF1/treacle in physiological and pathological processes

Ciliary Genes in Renal Cystic Diseases

Cilia are microtubule-based organelles, protruding from the apical cell surface and anchoring to the cytoskeleton. Primary (nonmotile) cilia of the kidney act as mechanosensors of nephron cells, responding to fluid movements by triggering signal transduction. The impaired functioning of primary cilia leads to formation of cysts which in turn contribute to development of diverse renal diseases, including kidney ciliopathies and renal cancer. Here, we review current knowledge on the role of ciliary genes in kidney ciliopathies and renal cell carcinoma (RCC). Special focus is given on the impact of mutations and altered expression of ciliary genes (e.g., encoding polycystins, nephrocystins, Bardet-Biedl syndrome (BBS) proteins, ALS1, Oral-facial-digital syndrome 1 (OFD1) and others) in polycystic kidney disease and nephronophthisis, as well as rare genetic disorders, including syndromes of Joubert, Meckel-Gruber, Bardet-Biedl, Senior-Loken, Alstr&ouml;m, Orofaciodigital syndrome type I and cranioectodermal dysplasia. We also show that RCC and classic kidney ciliopathies share commonly disturbed genes affecting cilia function, including VHL (von Hippel-Lindau tumor suppressor), PKD1 (polycystin 1, transient receptor potential channel interacting) and PKD2 (polycystin 2, transient receptor potential cation channel). Finally, we discuss the significance of ciliary genes as diagnostic and prognostic markers, as well as therapeutic targets in ciliopathies and cancer

Nucleolar Proteins and Non-Coding RNAs: Roles in Renal Cancer

Renal cell cancer is the most frequent kidney malignancy. Most RCC cases are classified as clear cell renal cell carcinoma (ccRCC), characterized by high aggressiveness and poor prognosis for patients. ccRCC aggressiveness is defined by classification systems based on changes in morphology of nucleoli, the membraneless substructures of nuclei. The latter act as the sites of ribosome biogenesis as well as the hubs that trap and immobilize proteins, preventing their action in other cellular compartments. Thereby, nucleoli control cellular functioning and homeostasis. Nucleoli are also the sites of activity of multiple noncoding RNAs, including snoRNAs, IGS RNA, and miRNAs. Recent years have brought several remarkable discoveries regarding the role of nucleolar non-coding RNAs, in particular snoRNAs, in ccRCC. The expression of snoRNAs is largely dysregulated in ccRCC tumors. snoRNAs, such as SNHG1, SNHG4 and SNHG12, act as miRNA sponges, leading to aberrant expression of oncogenes and tumor suppressors, and directly contributing to ccRCC development and progression. snoRNAs can also act without affecting miRNA functioning, by altering the expression of key oncogenic proteins such as HIF1A. snoRNAs are also potentially useful biomarkers of ccRCC progression. Here, we comprehensively discuss the role of nucleolar proteins and non-coding RNAs in ccRCC

piRNAs and PIWI Proteins as Diagnostic and Prognostic Markers of Genitourinary Cancers

piRNAs (PIWI-interacting RNAs) are small non-coding RNAs capable of regulation of transposon and gene expression. piRNAs utilise multiple mechanisms to affect gene expression, which makes them potentially more powerful regulators than microRNAs. The mechanisms by which piRNAs regulate transposon and gene expression include DNA methylation, histone modifications, and mRNA degradation. Genitourinary cancers (GC) are a large group of neoplasms that differ by their incidence, clinical course, biology, and prognosis for patients. Regardless of the GC type, metastatic disease remains a key therapeutic challenge, largely affecting patients’ survival rates. Recent studies indicate that piRNAs could serve as potentially useful biomarkers allowing for early cancer detection and therapeutic interventions at the stage of non-advanced tumour, improving patient’s outcomes. Furthermore, studies in prostate cancer show that piRNAs contribute to cancer progression by affecting key oncogenic pathways such as PI3K/AKT. Here, we discuss recent findings on biogenesis, mechanisms of action and the role of piRNAs and the associated PIWI proteins in GC. We also present tools that may be useful for studies on the functioning of piRNAs in cancers

Classification of proteins that were differently expressed in DIO1(+) cells when compared with DIO1(-) cells.

<p>The graph shows results of analysis performed using PANTHER (<a href="http://pantherdb.org/" target="_blank">http://pantherdb.org</a>).</p

Decreased Expression of SRSF2 Splicing Factor Inhibits Apoptotic Pathways in Renal Cancer

Serine and arginine rich splicing factor 2(SRSF2) belongs to the serine/arginine (SR)-rich family of proteins that regulate alternative splicing. Previous studies suggested that SRSF2 can contribute to carcinogenic processes. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, highly aggressive and difficult to treat, mainly due to resistance to apoptosis. In this study we hypothesized that SRSF2 contributes to the regulation of apoptosis in ccRCC. Using tissue samples obtained from ccRCC patients, as well as independent validation on The Cancer Genome Atlas (TCGA) data, we demonstrate for the first time that expression of SRSF2 is decreased in ccRCC tumours when compared to non-tumorous control tissues. Furthermore, by employing a panel of ccRCC-derived cell lines with silenced SRSF2 expression and qPCR arrays we show that SRSF2 contributes not only to splicing patterns but also to expression of multiple apoptotic genes, including new SRSF2 targets: DIABLO, BIRC5/survivin, TRAIL, BIM, MCL1, TNFRSF9, TNFRSF1B, CRADD, BCL2L2, BCL2A1, and TP53. We also identified a new splice variant of CFLAR, an inhibitor of caspase activity. These changes culminate in diminished caspase-9 activity and inhibition of apoptosis. In summary, we show for the first time that decreased expression of SRSF2 in ccRCC contributes to protection of cancer cells viability