In silico and in vitro analysis of the impact of single substitutions within EXO-motifs on Hsa-MiR-1246 intercellular transfer in breast cancer cell.

MiR-1246 has recently gained much attention and many studies have shown its oncogenic role in colorectal, breast, lung, and ovarian cancers. However, miR-1246 processing, stability, and mechanisms directing miR-1246 into neighbor cells remain still unclear. In this study, we aimed to determine the role of single-nucleotide substitutions within short exosome sorting motifs - so-called EXO-motifs: GGAG and GCAG present in miR-1246 sequence on its intracellular stability and extracellular transfer. We applied in silico methods such as 2D and 3D structure analysis and modeling of protein interactions. We also performed in vitro validation through the transfection of fluorescently labeled miRNA to MDA-MB-231 cells, which we analyzed by flow cytometry and fluorescent microscopy. Our results suggest that nucleotides alterations that disturbed miR-1246 EXO-motifs were able to modulate miRNA-1246 stability and its transfer level to the neighboring cells, suggesting that the molecular mechanism of RNA stability and intercellular transfer can be closely related.

Expression levels of sonic hedgehog pathway genes and their targets are upregulated in early clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU­SKI43, cyclopamine and GLI­antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786­O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription­quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma­associated zinc finger protein (GLI)1­3, BCL2 apoptosis regulator (BCL2), MYC proto­oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786­O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU­SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU­SKI43 appear to be potential anti­renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.

A Role of Inflammation and Immunity in Essential Hypertension-Modeled and Analyzed Using Petri Nets.

Recent studies have shown that the innate and adaptive immune system, together with low-grade inflammation, may play an important role in essential hypertension. In this work, to verify the importance of selected factors for the development of essential hypertension, we created a Petri net-based model and analyzed it. The analysis was based mainly on t-invariants, knockouts of selected fragments of the net and its simulations. The blockade of the renin-angiotensin (RAA) system revealed that the most significant effect on the emergence of essential hypertension has RAA activation. This blockade affects: (1) the formation of angiotensin II, (2) inflammatory process (by influencing C-reactive protein (CRP)), (3) the initiation of blood coagulation, (4) bradykinin generation via the kallikrein-kinin system, (5) activation of lymphocytes in hypertension, (6) the participation of TNF alpha in the activation of the acute phase response, and (7) activation of NADPH oxidase-a key enzyme of oxidative stress. On the other hand, we found that the blockade of the activation of the RAA system may not eliminate hypertension that can occur due to disturbances associated with the osmotically independent binding of Na in the interstitium. Moreover, we revealed that inflammation alone is not enough to trigger primary hypertension, but it can coexist with it. We believe that our research may contribute to a better understanding of the pathology of hypertension. It can help identify potential subprocesses, which blocking will allow better control of essential hypertension.

Prognostic significance of VHL, HIF1A, HIF2A, VEGFA and p53 expression in patients with clear­cell renal cell carcinoma treated with sunitinib as first­line treatment.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell cancer, characterized by the highest mortality rate among other RCC subtypes due to the occurrence of metastasis and drug resistance following surgery. The Von Hippel­Lindau tumor suppressor (VHL)­hypoxia­inducible factor 1 subunit α (HIF1A)/hypoxia­inducible factor 2α (HIF2A)­vascular endothelial growth factor A (VEGFA) protein axis is involved in the development and progression of ccRCC, whereas sunitinib, a tyrosine kinase inhibitor, blocks the binding of VEGFA to its receptor. The aim of the present study was to examine the possible association of the gene expression of VHL, HIF1A, HIF2A, VEGFA and tumor protein P53 (P53) in cancer tissue with the outcome of ccRCC patients who were treated with sunitinib as first­line therapy following nephrectomy. A total of 36 ccRCC patients were enrolled, 11 of whom were administered sunitinib post­operatively. Tumor and control samples were collected, and mRNA and protein levels were assessed by reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. High mRNA and protein expression levels of HIF2A and VEGFA were found to be associated with shorter overall survival (OS) and progression­free survival (PFS) rates, as well as with unfavorable risk factors of cancer recurrence and mortality. Resistance to sunitinib was also observed; the OS and PFS rates were shorter (median OS and PFS: 12 and 6 months, respectively, vs. undetermined). Sunitinib resistance was associated with high HIF2A and VEGFA protein levels (b=0.57 and b=0.69 for OS and PFS, respectively; P<0.001). Taken together, the findings of this study suggest that the protein levels of HIF2A and VEGFA in tumor tissue may serve as independent prognostic factors in ccRCC. ccRCC patients with increased intratumoral HIF2A and VEGFA protein levels, and unaltered VHL protein levels, are not likely to benefit from sunitinib treatment following nephrectomy; however, this hypothesis requires verification by large­scale replication studies.

Vitamin D and its low calcemic analogs modulate the anticancer properties of cisplatin and dacarbazine in the human melanoma A375 cell line.

Melanoma represents a significant challenge in cancer treatment due to the high drug resistance of melanomas and the patient mortality rate. This study presents data indicating that nanomolar concentrations of the hormonally active form of vitamin D, 1α,25­dihydroxyvitamin D3 [1α,25(OH)2D3], its non­calcemic analogues 20S­hydroxyvitamin D3 and 21­hydroxypregnacalciferol, as well as the low­calcemic synthetic analog calcipotriol, modulate the efficacy of the anticancer drugs cisplatin and dacarbazine. It was observed that vitamin D analogs sensitized melanoma A375 cells to hydrogen peroxide used as an inducer of oxidative stress. On the other hand, only 1α,25(OH)2D3 resulted in a minor, but significant effect on the proliferation of melanoma cells treated simultaneously with dacarbazine, but not cisplatin. Notably, cisplatin (300 µM) exhibited a higher overall antiproliferative activity than dacarbazine. Cisplatin treatment of melanoma cells resulted in an induction of apoptosis as demonstrated by flow cytometry (accumulation of cells at the subG1 phase of the cell cycle), whereas dacarbazine caused G1/G0 cell cycle arrest, with the effects being improved by pre­treatment with vitamin D analogs. Treatment with cisplatin resulted in an initial increase in the level of reactive oxygen species (ROS). Dacarbazine caused transient stimulation of ROS levels and the mitochondrial membrane potential (Δψm) (after 1 or 3 h of treatment, respectively), but the effect was not detectable following prolonged (24 h) incubation with the drug. Vitamin D exhibited modulatory effects on the cells treated with dacarbazine, decreasing the half maximal inhibitory concentration (IC50) for the drug, stimulating G1/G0 arrest and causing a marked decrease in Δψm. Finally, cisplatin, dacarbazine and 1α,25(OH)2D3 displayed modulatory effects on the expression of ROS and vitamin D­associated genes in the melanoma A375 cells. In conclusion, nanomolar concentrations of 1,25(OH)2D3 only had minor effects on the proliferation of melanoma cells treated with dacarbazine, decreasing the relative IC50 value. However, co­treatment with vitamin D analogs resulted in the modulation of cell cycle and ROS responses, and affected gene expression, suggesting possible crosstalk between the signaling pathways of vitamin D and the anticancer drugs used in this study.

Overexpression of the YAP1 oncogene in clear cell renal cell carcinoma is associated with poor outcome.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70-80%). Yes-associated protein 1 (YAP1) protein is a nuclear effector of the Hippo pathway and acts as a transcriptional co-activator of genes involved in the processes of growth and development of tissues. Hippo signaling, with its key kinases, MST2 and large tumor suppressor kinase 1 (LATS1), plays a significant role in the negative regulation of the amount and activity of YAP1 protein. Components of the Hippo pathway and YAP1 levels are frequently dysregulated in a variety of tumors, suggestive of their possible involvement in carcinogenesis. Our aim was to evaluate gene and protein expression profiles of YAP1, MST2 and LATS1 and the methylation status of MST2 and LATS1 promoters in ccRCC. mRNA levels of MST2, LATS1 and YAP1 genes were assessed in the tumor and matched normal kidney tissues of 86 patients, and in 12 samples of local metastases by quantitative PCR (qPCR). Proteins were semi-quantified in 58 patient samples by western blotting. Hypermethylation of LATS1 and MST2 promoters was measured by methylation­specific high­resolution-melting qPCR. We found that LATS1 promoter hypermethylation, decreased LATS1 mRNA/protein and increased YAP1 mRNA/protein levels in tumor samples were associated with higher TNM and Fuhrman's stages and patient survival. Higher YAP1 mRNA levels were associated with poor outcome (HR=4.03, p=0.036). No changes in MST2 (promoter/mRNA/protein) were found. We propose that deregulation of LATS1 and YAP1 expression is associated with ccRCC progression and poor patient survival. Measurement of YAP1 mRNA levels in paired tumor-normal kidney tissue samples may serve as a new prognostic factor in ccRCC.

RNA-Seq-based analysis of differential gene expression associated with hepatitis C virus infection in a cell culture.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver diseases. Unfortunately, the mechanisms of HCV infection-induced liver injury and host-virus interactions are still not well recognized. To better understand these processes we determined the changes in the host gene expression that occur during HCV infection of Huh-7.5 cells. As a result, we identified genes that may contribute to the immune and metabolic cellular responses to infection. Pathway enrichment analysis indicated that HCV induced an increased expression of genes involved in mitogen-activated protein kinases signaling, adipocytokine signaling, cell cycle and nitrogen metabolism. In addition, the enrichment analyses of processes and molecular functions revealed that the up-regulated genes were mainly implicated in the negative regulation of phosphorylation. Construction of the pathway-gene-process network enabled exploration of a much more complex landscape of molecular interactions. Consequently, several essential processes altered by HCV infection were identified: negative regulation of cell cycle, response to endoplasmic reticulum stress, response to reactive oxygen species, toll-like receptor signaling and pattern recognition receptor signaling. The analyses of genes whose expression was decreased upon HCV infection showed that the latter were engaged in the metabolism of lipids and amino acids. Moreover, we observed disturbance in the cellular antiviral defense. Altogether, our results demonstrated that HCV infection elicits host response that includes a very wide range of cellular mechanisms. Our findings significantly broaden the understanding of complex processes that accompany HCV infection. Consequently, they may be used for developing new host-oriented therapeutic strategies.

Decreased expression of RASSF1A tumor suppressor gene is associated with worse prognosis in clear cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70-80%) and is associated with poor prognosis in 40% of cases mainly due to metastasis in the course of the disease. RASSF1, with its isoforms RASSF1A and RASSF1C, is a tumor suppressor gene which has not been fully analyzed in ccRCC yet. The epigenetic downregulation of RASSF1A is commonly associated with promoter hypermethylation. The aim of the present study was to compare the ccRCC outcomes with the expression of RASSF1A and RASSF1C. Tissues were obtained from 86 ccRCC patients. RASSF1A and RASSF1C mRNA levels were assessed in tumor and matched normal kidney tissue, and in 12 samples of local metastases by quantitative PCR (qPCR). RASSF1A and RASSF1C proteins levels were semi-quantified in 58 samples by western blot analysis and their tissue localization was assessed by immunohistochemistry. Hypermethylation of RASSF1A promoter was measured by high-resolution-melting methylation-specific qPCR. RASSF1A mRNA levels were 4 and 5 times lower in 66% of tumor and 75% metastasized samples. RASSF1A hypermethylation was found in 40% of analyzed T cases. RASSF1A protein expression was 5 or 20 times decreased in 70% tumor and 75% metastatic samples, respectively. RASSF1A hypermethylation, mRNA and protein levels were associated with TNM progression and higher Fuhrman's grading. Decreased RASSF1A expression, hypermethylation, TNM and Fuhrman's grading were associated with poorer overall survival (OS). Cox hazard ratio (HR) analysis revealed predictor role of RASSF1A mRNA levels on OS and progression-free survival (PFS) in relation to Fuhrman's grading (OS HR=2.25, PFS HR=2.93). RASSF1C levels were increased in ccRCC; no correlations with clinicopathological variables were found. We conclude that RASSF1C gene is not involved in ccRCC progression and we propose that the measurements of RASSF1A mRNA levels in paired tumor-normal kidney tissue could serve as a new prognostic factor in ccRCC.

The Hippo pathway in colorectal cancer.

Colorectal cancer (CRC) is one the most frequently diagnosed neoplastic diseases worldwide. Currently, aside from traditional chemotherapy, advanced CRCs are treated with modern drugs targeting cellular components such as epithelial growth factor receptor (EGFR). Since up to 70% of metastasized CRCs are drug resistant, the description of recent progress in cellular homeostasis regulation may shed new light on the development of new molecular targets in cancer treatment. The Hippo pathway has recently become subject of intense investigations since it plays a crucial role in cell proliferation, differentiation, apoptosis and tumourigenesis. Components of the Hippo pathway are deregulated in various human malignancies, and expression levels of its major signal transducers were proposed as prognostic factors in colorectal cancer. In this review we focused on recent data regarding Hippo pathway, its up-stream signals and down-stream effectors. Hippo negatively regulates its major effectors, YAP1 and TAZ kinases, which act as transcriptional co-activators inducing expression of genes involved not only in tissue repair and proliferation but are also oncoproteins involved in tumour development and progression. The deregulation of Hippo pathway components was found in many malignancies. The interactions between Hippo and Wnt/ß-catenin signalling, crucial in the maintenance of cell homeostasis, have been described in relation to the control of intestinal stem cell proliferation and CRC development. The recently discovered positive feedback loop between activated YAP1 and increased EGFR/KRAS signalling found in oesophageal, ovarian and hepatocellular cancer has been related to the CRC progression and resistance to EGFR inhibitors during CRC therapy.

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines.

INTRODUCTION: Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot). MATERIAL AND METHODS: HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR). RESULTS: Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs. CONCLUSIONS: We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.

Identification of a suitable qPCR reference gene in metastatic clear cell renal cell carcinoma.

There is no data on reference gene (RG) selection in metastatic clear-cell renal cell carcinoma (mccRCC) for quantitative PCR (qPCR) data normalization. We aimed at selecting the most stable RG for further determination of new prognostic markers. Thirty-five nonmetastatic and 35 mccRCC patients undergoing radical nephrectomy were included. Paired primary tumor (T, n = 70) and normal (C, n = 70) kidney fragments were collected; from 12 out of 35 mccRCC cases, we also collected metastasized regional lymph nodes and adrenal gland tissues (M, n = 12). After RNA extraction, reverse transcription and qPCR were performed. Samples were divided into four analyzed groups. Fifteen candidate RGs were tested by RefFinder tool and manual statistics. To present the importance of RG selection, TP53 gene expression levels in samples were normalized with the use of RG data. RPL13 gene was the most stable RG in analysis of 35 primary tumor nonmetastatic versus 35 mccRCC samples and matched metastasized T/C/M samples (n = 12, each group). GUSB was the most suitable RG in total 152 samples and in paired T and C (n = 140) kidney samples. Expression of GUSB, RPL13, and the RPL13 + RPLP0 pair were independent of clinical/sample variables. Normalization of TP53 expression levels showed variability of GAPDH and ACTB assays. GUSB or RPL13 assays should be used in mccRCC for qPCR data normalization whereas GAPDH and ACTB assays should be avoided. Prior RG studies should precede each qPCR gene expression study since RG selection is associated with the origin and proportion of specimens.

Sorting signal targeting mRNA into hepatic extracellular vesicles.

Intercellular communication mediated by extracellular vesicles has proved to play an important role in normal and pathological scenarios. However not too much information about the sorting mechanisms involved in loading the vesicles is available. Recently, our group has characterized the mRNA content of vesicles released by hepatic cellular systems, showing that a set of transcripts was particularly enriched in the vesicles in comparison with their intracellular abundance. In the current work, based on in silico bioinformatics tools, we have mapped a novel sequence of 12 nucleotides C[TA]G[GC][AGT]G[CT]C[AT]GG[GA], which is significantly enriched in the set of mRNAs that accumulate in extracellular vesicles. By including a 3'-UTR containing this sequence in a luciferase mRNA reporter, we have shown that in a hepatic cellular system this reporter mRNA was incorporated into extracellular vesicles. This study identifies a sorting signal in mRNAs that is involved in their enrichment in EVs, within a hepatic non-tumoral cellular model.

[Role of the Hippo pathway in cell proliferation and organ size control. Disorders of the pathway in cancer diseases]. / Rola szlaku Hippo w kontroli proliferacji komórkowej i wielkosci narzadów oraz jego zaburzenia w chorobach nowotworowych

The Hippo pathway (also known as SWH--Salvador/Warts/Hippo), discovered for the first time in Drosophila melanogaster, is responsible for cell proliferation and organ size control in mammalian systems. The components of the pathway are two kinases and their adaptor proteins which inhibit the transcription co-activator YAP by phosphorylation. When the pathway is inactive (as an effect of upstream component gene expression disorders), activated YAP is translocated to the nucleus where it cooperates with TEAD transcription factor and promotes expression of genes that regulate cell proliferation and apoptosis. YAP acts generally as an oncogene, although there are some reports describing its role as a tumor suppressor. Since all of the core components are well known, the latest reports provide mostly information about upstream components of the Hippo pathway or its interaction with other biochemical pathways. Because of the Hippo pathway's role in the cell cycle, it has become a very attractive object for studies of the genetic background of cancer. The under- or overexpression of genes involved in the Hippo pathway has been described in many different types of cancers. Moreover, it has been shown that there is a strong connection between cancer cell phenotype and highly activated YAP presence in the nucleus. This paper reviews the most important data about Hippo pathway regulation in Drosophila and mammals, including its numerous disorders and their implications for cell function.