Renal cancer secretome induces migration of mesenchymal stromal cells.

BACKGROUND: Advanced renal cell carcinoma (RCC) is therapeutically challenging. RCC progression is facilitated by mesenchymal stem/stromal cells (MSCs) that exert remarkable tumor tropism. The specific mechanisms mediating MSCs' migration to RCC remain unknown. Here, we aimed to comprehensively analyze RCC secretome to identify MSCs attractants. METHODS: Conditioned media (CM) were collected from five RCC-derived cell lines (Caki-1, 786-O, A498, KIJ265T and KIJ308T) and non-tumorous control cell line (RPTEC/TERT1) and analyzed using cytokine arrays targeting 274 cytokines in addition to global CM proteomics. MSCs were isolated from bone marrow of patients undergoing standard orthopedic surgeries. RCC CM and the selected recombinant cytokines were used to analyze their influence on MSCs migration and microarray-targeted gene expression. The expression of genes encoding cytokines was evaluated in 100 matched-paired control-RCC tumor samples. RESULTS: When compared with normal cells, CM from advanced RCC cell lines (Caki-1 and KIJ265T) were the strongest stimulators of MSCs migration. Targeted analysis of 274 cytokines and global proteomics of RCC CM revealed decreased DPP4 and EGF, as well as increased AREG, FN1 and MMP1, with consistently altered gene expression in RCC cell lines and tumors. AREG and FN1 stimulated, while DPP4 attenuated MSCs migration. RCC CM induced MSCs' transcriptional reprogramming, stimulating the expression of CD44, PTX3 and RAB27B. RCC cells secreted hyaluronic acid (HA), a CD44 ligand mediating MSCs' homing to the kidney. AREG emerged as an upregulator of MSCs' transcription. CONCLUSIONS: Advanced RCC cells secrete AREG, FN1 and HA to induce MSCs migration, while DPP4 loss prevents its inhibitory effect on MSCs homing. RCC secretome induces MSCs' transcriptional reprograming to facilitate their migration. The identified components of RCC secretome represent potential therapeutic targets.

Coordinated reprogramming of renal cancer transcriptome, metabolome and secretome associates with immune tumor infiltration.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer. The molecules (proteins, metabolites) secreted by tumors affect their extracellular milieu to support cancer progression. If secreted in amounts detectable in plasma, these molecules can also serve as useful, minimal invasive biomarkers. The knowledge of ccRCC tumor microenvironment is fragmentary. In particular, the links between ccRCC transcriptome and the composition of extracellular milieu are weakly understood. In this study, we hypothesized that ccRCC transcriptome is reprogrammed to support alterations in tumor microenvironment. Therefore, we comprehensively analyzed ccRCC extracellular proteomes and metabolomes as well as transcriptomes of ccRCC cells to find molecules contributing to renal tumor microenvironment. METHODS: Proteomic and metabolomics analysis of conditioned media isolated from normal kidney cells as well as five ccRCC cell lines was performed using mass spectrometry, with the following ELISA validation. Transcriptomic analysis was done using microarray analysis and validated using real-time PCR. Independent transcriptomic and proteomic datasets of ccRCC tumors were used for the analysis of gene and protein expression as well as the level of the immune infiltration. RESULTS: Renal cancer secretome contained 85 proteins detectable in human plasma, consistently altered in all five tested ccRCC cell lines. The top upregulated extracellular proteins included SPARC, STC2, SERPINE1, TGFBI, while downregulated included transferrin and DPP7. The most affected extracellular metabolites were increased 4-hydroxy-proline, succinic acid, cysteine, lactic acid and downregulated glutamine. These changes were associated with altered expression of genes encoding the secreted proteins (SPARC, SERPINE1, STC2, DPP7), membrane transporters (SLC16A4, SLC6A20, ABCA12), and genes involved in protein trafficking and secretion (KIF20A, ANXA3, MIA2, PCSK5, SLC9A3R1, SYTL3, and WNTA7). Analogous expression changes were found in ccRCC tumors. The expression of SPARC predicted the infiltration of ccRCC tumors with endothelial cells. Analysis of the expression of the 85 secretome genes in > 12,000 tumors revealed that SPARC is a PanCancer indicator of cancer-associated fibroblasts' infiltration. CONCLUSIONS: Transcriptomic reprogramming of ccRCC supports the changes in an extracellular milieu which are associated with immune infiltration. The proteins identified in our study represent valuable cancer biomarkers detectable in plasma.

TGF­ß1 affects the renal cancer miRNome and regulates tumor cells proliferation.

TGF­ß1 is a pleiotropic cytokine that can either promote or inhibit cancer development and progression. It was previously found that TGF­ß1 can regulate the expression of several microRNAs (miR or miRNA) involved in the progression of renal cell carcinoma (RCC). Therefore, the present study aimed to analyze the effects of TGF­ß1 on the global RCC miRNome. It was found that TGF­ß1 can regulate a complex network consisting of miRNAs and mRNAs involved in RCC transformation. In particular, TGF­ß1 was revealed to regulate the proliferation of RCC cells while concomitantly modifying the expression of oncogenic regulators, including avian erythroblastosis virus E26 (V­Ets) oncogene homolog­1 (ETS1). In addition, TGF­ß1 was demonstrated to regulate the expression of a number of miRNAs including miR­30c­5p, miR­155­5p, miR­181a­5p and miR­181b­5p. By contrast, TGF­ß1 reciprocally modified the expression of genes encoding TGF­ß1 receptors and SMADs, indicating a novel regulatory feedback mechanism mediated through the miRNAs. These data suggested that ETS1 served different roles in different subtypes of RCC tumors, specifically by functioning as an oncogene in clear cell RCC while as a tumor suppressor in papillary RCC.

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.

Correction: Kedzierska, H., et al. Decreased Expression of SRSF2 Splicing Factor Inhibits Apoptotic Pathways in Renal Cancer. Int. J. Mol. Sci. 2016, 17, 1598.

The authors wish to make the following corrections to this paper [1]: in Figure 4 the same gelscans were mistakenly pasted to illustrate splicing changes of: i) BIM in KIJ-265T and KIJ308T cells,and ii) MCL-1 in UOK171 and KIJ-265T [...].

MicroRNA-Mediated Metabolic Reprograming in Renal Cancer.

Metabolic reprogramming is one of the hallmarks of renal cell cancer (RCC). We hypothesized that altered metabolism of RCC cells results from dysregulation of microRNAs targeting metabolically relevant genes. Combined large-scale transcriptomic and metabolic analysis of RCC patients tissue samples revealed a group of microRNAs that contribute to metabolic reprogramming in RCC. miRNAs expressions correlated with their predicted target genes and with gas chromatography-mass spectrometry (GC-MS) metabolome profiles of RCC tumors. Assays performed in RCC-derived cell lines showed that miR-146a-5p and miR-155-5p targeted genes of PPP (the pentose phosphate pathway) (G6PD and TKT), the TCA (tricarboxylic acid cycle) cycle (SUCLG2), and arginine metabolism (GATM), respectively. miR-106b-5p and miR-122-5p regulated the NFAT5 osmoregulatory transcription factor. Altered expressions of G6PD, TKT, SUCLG2, GATM, miR-106b-5p, miR-155-5p, and miR-342-3p correlated with poor survival of RCC patients. miR-106b-5p, miR-146a-5p, and miR-342-3p stimulated proliferation of RCC cells. The analysis involving >6000 patients revealed that miR-34a-5p, miR-106b-5p, miR-146a-5p, and miR-155-5p are PanCancer metabomiRs possibly involved in global regulation of cancer metabolism. In conclusion, we found that microRNAs upregulated in renal cancer contribute to disturbed expression of key genes involved in the regulation of RCC metabolome. miR-146a-5p and miR-155-5p emerge as a key "metabomiRs" that target genes of crucial metabolic pathways (PPP (the pentose phosphate pathway), TCA cycle, and arginine metabolism).

TGF-ß1 targets a microRNA network that regulates cellular adhesion and migration in renal cancer.

In our previous study we found altered expression of 19 adhesion-related genes in renal tumors. In this study we hypothesized that disturbed expression of adhesion-related genes could be caused by microRNAs: short, non-coding RNAs that regulate gene expression. Here, we found that expression of 24 microRNAs predicted to target adhesion-related genes was disturbed in renal tumors and correlated with expression of their predicted targets. miR-25-3p, miR-30a-5p, miR-328 and miR-363-3p directly targeted adhesion-related genes, including COL5A1, COL11A1, ITGA5, MMP16 and THBS2. miR-363-3p and miR-328 inhibited proliferation of renal cancer cells, while miR-25-3p inhibited adhesion, promoted proliferation and migration of renal cancer cells. TGF-ß1 influenced the expression of miR-25-3p, miR-30a-5p, and miR-328. The analyzed microRNAs, their target genes and TGF-ß1 formed a network of strong correlations in tissue samples from renal cancer patients. The expression signature of microRNAs linked with TGF-ß1 levels correlated with poor survival of renal cancer patients. The results of our study suggest that TGF-ß1 coordinates the expression of microRNA network that regulates cellular adhesion in cancer.

Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3'-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes in local availability of thyroid hormones might favor a shift from a differentiated to a more proliferation-prone state of cancer tissues and cell lines.

The significance of TRIP11 and T3 signalling pathway in renal cancer progression and survival of patients.

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-1ß, THRA, THRB, FURIN, VEGFA, and GLUT1 were disturbed in renal cancer. Expressions of TRIP11 and HIF-1ß 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.

Integrated transcriptomic and metabolomic analysis shows that disturbances in metabolism of tumor cells contribute to poor survival of RCC patients.

PURPOSE: Cellular metabolism of renal cell carcinoma (RCC) tumors is disturbed. The clinical significance of these alterations is weakly understood. We aimed to find if changes in metabolic pathways contribute to survival of RCC patients. MATERIAL AND METHODS: 35 RCC tumors and matched controls were used for metabolite profiling using gas chromatography-mass spectrometry and transcriptomic analysis with qPCR-arrays targeting the expression of 93 metabolic genes. The clinical significance of obtained data was validated on independent cohort of 468 RCC patients with median follow-up of 43.22months. RESULTS: The levels of 31 metabolites were statistically significantly changed in RCC tumors compared with controls. The top altered metabolites included beta-alanine (+4.2-fold), glucose (+3.4-fold), succinate (-11.0-fold), myo-inositol (-4.6-fold), adenine (-4.2-fold), uracil (-3.7-fold), and hypoxanthine (-3.0-fold). These disturbances were associated with altered expression of 53 metabolic genes. ROC curve analysis revealed that the top metabolites discriminating between tumor and control samples included succinate (AUC=0.91), adenine (AUC=0.89), myo-inositol (AUC=0.87), hypoxanthine (AUC=0.85), urea (AUC=0.85), and beta-alanine (AUC=0.85). Poor survival of RCC patients correlated (p<0.0001) with altered expression of genes involved in metabolism of succinate (HR=2.7), purines (HR=2.4), glucose (HR=2.4), beta-alanine (HR=2.5), and myo-inositol (HR=1.9). CONCLUSIONS: We found that changes in metabolism of succinate, beta-alanine, purines, glucose and myo-inositol correlate with poor survival of RCC patients.

Induction of type 1 iodothyronine deiodinase expression inhibits proliferation and migration of renal cancer cells.

Type 1 iodothyronine deiodinase (DIO1) regulates peripheral metabolism of thyroid hormones that control cellular proliferation, differentiation and metabolism. The significance of DIO1 in cancer is unknown. In this study we hypothesized that diminished expression of DIO1, observed in renal cancer, contributes to the carcinogenic process in the kidney. Here, we demonstrate that ectopic expression of DIO1 in renal cancer cells changes the expression of genes controlling cell cycle, including cyclin E1 and E2F5, and results in inhibition of proliferation. The expression of genes encoding collagens (COL1A1, COL4A2, COL5A1), integrins (ITGA4, ITGA5, ITGB3) and transforming growth factor-ß-induced (TGFBI) is significantly altered in renal cancer cells with induced expression of DIO1. Finally, we show that overexpression of DIO1 inhibits migration of renal cancer cells. In conclusion, we demonstrate for the first time that loss of DIO1 contributes to renal carcinogenesis and that its induced expression protects cells against cancerous proliferation and migration.

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.

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5'UTRs of Selected Tumor Suppressors.

BACKGROUND: Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. FINDINGS: We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5'-untranslated region (5'UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5'UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRß1 5'UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5'UTR has higher translational regulatory potential when compared to the weakly folded TRß1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5'UTRs of complex structure. SIGNIFICANCE: This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5'UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors.

Expression of Genes Involved in Cellular Adhesion and Extracellular Matrix Remodeling Correlates with Poor Survival of Patients with Renal Cancer.

PURPOSE: Renal cell carcinoma is the most common highly metastatic kidney malignancy. Adhesion has a crucial role in the metastatic process. TGF (transforming growth factor)-ß1 is a pleiotropic cytokine that influences cancerous transformation. We hypothesized that 1) changes in the expression of adhesion related genes may influence survival rate of patients with renal cell carcinoma and 2) TGF-ß1 may contribute to changed expression of adhesion related genes. MATERIALS AND METHODS: Two-step quantitative real-time polymerase chain reaction arrays were used to analyze the expression of adhesion related genes in 77 tumors and matched pair controls. The prognostic significance of genes was evaluated in TCGA (The Cancer Genome Atlas) data on 468 patients with renal cell carcinoma. Quantitative real-time polymerase chain reaction and Western blot were applied for TGF-ß1 analysis. TGF-ß1 mediated regulation of gene expression was analyzed by TGF-ß1 supplementation of Caki-2 cells and quantitative real-time polymerase chain reaction. RESULTS: The expression of 19 genes related to adhesion and extracellular matrix remodeling was statistically significantly disturbed in renal cell carcinoma compared with controls. The 10-gene expression signature (COL1A1, COL5A1, COL11A1, FN1, ICAM1, ITGAL, ITGAM, ITGB2, THBS2 and TIMP1) correlated with poor survival (HR 2.85, p = 5.7e-10). TGF-ß1 expression was 22 times higher in renal cell carcinoma than in controls (p <0.0001). TGF-ß1 induced expression of TGFBI, COL1A1, COL5A1, COL8A1, FN1, ITGA5, ITGAM and TIMP1 in a renal cell carcinoma derived cell line. CONCLUSIONS: Disturbed expression of genes involved in adhesion and extracellular matrix remodeling develops early during renal cell carcinoma carcinogenesis and correlates with poor survival. TGF-ß1 contributes to changed expression of extracellular matrix and adhesion related genes. Bioinformatic analysis performed on a broad panel of cancers of nonkidney origin suggests that disturbed expression of genes related to extracellular matrix and adhesion may be a universal feature of cancerous progression.

Epigenetic regulation of thyroid hormone receptor beta in renal cancer.

Thyroid hormone receptor beta (THRB) gene is commonly deregulated in cancers and, as strengthened by animal models, postulated to play a tumor-suppressive role. Our previous studies revealed downregulation of THRB in clear cell renal cell carcinoma (ccRCC), but the culpable mechanisms have not been fully elucidated. Since epigenetic regulation is a common mechanism influencing the expression of tumor suppressors, we hypothesized that downregulation of THRB in renal cancer results from epigenetic aberrances, including CpG methylation and microRNA-dependent silencing. Our study revealed that ccRCC tumors exhibited a 56% decrease in THRB and a 37% increase in DNA methyltransferase 1 (DNMT1) expression when compared with paired non-neoplastic control samples. However, THRB CpG methylation analysis performed using BSP, SNaPshot and MSP-PCR consistently revealed no changes in methylation patterns between matched tumor and control samples. In silico analysis resulted in identification of four microRNAs (miR-155, miR-425, miR-592, and miR-599) as potentially targeting THRB transcript. Luciferase assay showed direct binding of miR-155 and miR-425 to 3'UTR of THRB, and subsequent in vivo analyses revealed that transfection of UOK171 cell line with synthetic miR-155 or miR-425 resulted in decreased expression of endogenous TRHB by 22% and 64%, respectively. Finally, real-time PCR analysis showed significant upregulation of miR-155 (354%) and miR-425 (162%) in ccRCC when compared with matched controls. Moreover, microRNA levels were negatively correlated with the amount of THRB transcript in tissue samples. We conclude that CpG methylation is not the major mechanism contributing to decreased THRB expression in ccRCC. In contrast, THRB is targeted by microRNAs miR-155 and miR-425, whose increased expression may be responsible for downregulation of THRB in ccRCC tumors.

Alternative splicing of iodothyronine deiodinases in pituitary adenomas. Regulation by oncoprotein SF2/ASF.

Pituitary tumors belong to the group of most common neoplasms of the sellar region. Iodothyronine deiodinase types 1 (DIO1) and 2 (DIO2) are enzymes contributing to the levels of locally synthesized T3, a hormone regulating key physiological processes in the pituitary, including its development, cellular proliferation, and hormone secretion. Previous studies revealed that the expression of deiodinases in pituitary tumors is variable and, moreover, there is no correlation between mRNA and protein products of the particular gene, suggesting the potential role of posttranscriptional regulatory mechanisms. In this work we hypothesized that one of such mechanisms could be the alternative splicing. Therefore, we analyzed expression and sequences of DIO1 and DIO2 splicing variants in 30 pituitary adenomas and 9 non-tumorous pituitary samples. DIO2 mRNA was expressed as only two mRNA isoforms. In contrast, nine splice variants of DIO1 were identified. Among them, five were devoid of exon 3. In silico sequence analysis of DIO1 revealed multiple putative binding sites for splicing factor SF2/ASF, of which the top-ranked sites were located in exon 3. Silencing of SF2/ASF in pituitary tumor GH3 cells resulted in change of ratio between DIO1 isoforms with or without exon 3, favoring the expression of variants without exon 3. The expression of SF2/ASF mRNA in pituitary tumors was increased when compared with non-neoplastic control samples. In conclusion, we provide a new mechanism of posttranscriptional regulation of DIO1 and show deregulation of DIO1 expression in pituitary adenoma, possibly resulting from disturbed expression of SF2/ASF.

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.

Untranslated regions of thyroid hormone receptor beta 1 mRNA are impaired in human clear cell renal cell carcinoma.

Thyroid hormone receptor ß1 (TRß1) is a hormone-dependent transcription factor activated by 3,5,3'-l-triiodothyronine (T3). TRß1 functions as a tumor suppressor and disturbances of the THRB gene are frequent findings in cancer. Translational control mediated by untranslated regions (UTRs) regulates cell proliferation, metabolism and responses to cellular stress, processes that are involved in carcinogenesis. We hypothesized that reduced TRß1 expression in clear cell renal cell cancer (ccRCC) results from regulatory effects of TRß1 5' and 3'UTRs on protein translation. We determined TRß1 expression and alternative splicing of TRß1 5' and 3'UTRs in ccRCC and control tissue together with expression of the type 1 deiodinase enzyme (coded by DIO1, a TRß1 target gene). Tissue concentrations of T3 (which are generated in part by D1) and expression of miRNA-204 (an mRNA inhibitor for which a putative interaction site was identified in the TRß1 3'UTR) were also determined. TRß1 mRNA and protein levels were reduced by 70% and 91% in ccRCC and accompanied by absent D1 protein, a 58% reduction in tissue T3 concentration and 2-fold increase in miRNA-204. Structural analysis of TRß1 UTR variants indicated that reduced TRß1 expression may be maintained in ccRCC by posttranscriptional mechanisms involving 5'UTRs and miRNA-204. The tumor suppressor activity of TRß1 indicates that reduced TRß1 expression and tissue hypothyroidism in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage to malignant cells.

Thyroid hormone - triiodothyronine - has contrary effect on proliferation of human proximal tubules cell line (HK2) and renal cancer cell lines (Caki-2, Caki-1) - role of E2F4, E2F5 and p107, p130.

BACKGROUND: Triiodothyronine regulates proliferation acting as stimulator or inhibitor. E2F4 and E2F5 in complexes with pocket proteins p107 or p130 stop cells in G1, repressing transcription of genes important for cell cycle progression. p107 and p130 inhibits activity of cyclin/cdk2 complexes. Expression of all those proteins could be regulated by triiodothyronine. In clear cell renal cell carcinoma many disturbances in T3 signaling pathway was described, in that type of cancer also expression of some key G1 to S phase progression regulators was shown. METHODS: We investigated role of T3 and its receptors in regulation of proliferation of HK2, Caki-2, Caki-1 cell lines (cell counting, cytometric analysis of DNA content) and expression of thyroid hormone receptors, E2F4, E2F5, p107 and p130 (western blot and semi-quantitative real time PCR). Statistical analysis was performed using one-way ANOVA. RESULTS AND CONCLUSION: We show that T3 inhibits proliferation of HK2, and stimulates it in Caki lines. Those differences are result of disturbed expression of TR causing improper regulation of E2F4, E2F5, p107 and p130 in cancer cells.