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.

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 regulation of splicing factors SRSF1, SRSF2 and hnRNP A1 in context of their alternatively spliced 3'UTRs.

SRSF1, SRSF2 and hnRNP A1 are splicing factors that regulate the expression of oncogenes and tumor suppressors. SRSF1 and SRSF2 contribute to the carcinogenesis in the kidney. Despite their importance, the mechanisms regulating their expression in cancer are not entirely understood. Here, we investigated the microRNA-mediated regulation of SRSF1, SRSF2 and hnRNP A1 in renal cancer. The expression of microRNAs predicted to target SRSF1, SRSF2 and hnRNP A1 was disturbed in renal tumors compared with controls. Using qPCR, Western blot/ICC and luciferase reporter system assays we identified microRNAs that contribute to the regulation of expression of SRSF1 (miR-10b-5p, miR-203a-3p), SRSF2 (miR-183-5p, miR-200c-3p), and hnRNP A1 (miR-135a-5p, miR-149-5p). Silencing of SRSF1 and SRSF2 enhanced the expression of their targeting microRNAs. miR-183-5p and miR-200c-3p affected the expression of SRSF2-target genes, TNFRSF1B, TNFRSF9, CRADD and TP53. 3'UTR variants of SRSF1 and SRSF2 differed by the presence of miRNA-binding sites. In conclusion, we identified a group of microRNAs that contribute to the regulation of expression of SRSF1, SRSF2 and hnRNP A1. The microRNAs targeting SRSF1 and SRSF2 are involved in a regulatory feedback loop. microRNAs miR-183-5p and miR-200c-3p that target SRSF2, affect the expression of genes involved in apoptotic regulation.

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.

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.

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.

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.

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.

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.

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.

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.

microRNAs target SRSF7 splicing factor to modulate the expression of osteopontin splice variants in renal cancer cells.

SRSF7 is a SR splicing factor involved in the regulation of splicing and mRNA export of cancer-related genes. The mechanisms regulating the expression of SRSF7 are unknown. This study shows that SRSF7 expression in cancer cells is regulated by microRNAs: short, non-coding RNAs that bind to 3'UTR of target genes and downregulate their expression. We show that microRNAs miR-30a-5p and miR-181a-5p together with SRSF7 form regulatory feedback loop in which the expression of microRNAs is recurrently regulated by its target. Finally, we demonstrate that silencing of SRSF7 affects the expression of osteopontin splice variants and decreases proliferation rate of renal cancer cells.

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.

Role of temperature in diapause response to fish kairomones in crustacean Daphnia.

The effect of non-lethal thermal conditions on the diapause response to a simulated threat of fish predation was tested in the freshwater planktonic crustacean Daphnia magna. From an early developmental stage, female Daphnia were either exposed or not exposed to fish kairomones that notified of the threat of fish predation at the benign growth temperatures of 18, 22 or 26°C. The proportion of females switching from the production of subitaneous to diapausing eggs and their rate of reproduction were recorded. Besides the faster development and more intense reproduction observed at higher temperatures, a smaller proportion of the females formed protective, diapausing eggs when exposed to fish kairomones than was the case in colder conditions. The production of diapausing eggs was not observed when the threat of fish predation was absent. These results indicate an interactive effect of fish kairomones and thermal conditions on diapause induction in D. magna. We interpret these findings in the context of strategies for the maximisation of reproductive success. The production of well protected diapausing eggs (which assures low yet stable gains under diverse thermal conditions) may be a more beneficial life history strategy at lower temperatures, where the chances of survival of active individuals until maturation and successful reproduction are low. Higher temperatures permit faster maturation and more intensive reproduction that may surpass numerical losses of active descendants due to predation, making diapause a less rewarding option.