Increased Expression of Pregnancy Up-Regulated Non-Ubiquitous Calmodulin Kinase Is Associated with Poor Prognosis in Clear Cell Renal Cell Carcinoma

Purpose

The aims of this study were to evaluate the clinical significance and potential prognostic value of pregnancy up-regulated non-ubiquitous calmodulin kinase (PNCK) in clear cell renal cell carcinoma (ccRCC) patients.

Materials and Methods

The expression of PNCK mRNA was determined in 24 paired samples of ccRCCs and adjacent normal tissues using real-time RT-PCR. The expression of PNCK was determined in 248 samples of ccRCCs and 92 paired samples of adjacent normal tissues by immunohistochemical analysis. Statistical analysis was performed to define the relationship between PNCK expression and the clinical features of ccRCC.

Results

The mRNA level of PNCK was significantly higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). An immunohistochemical analysis of 92 paired tissue specimens showed that PNCK expression was higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). Moreover, there was a significant correlation between the PNCK expression and various clinicopathological parameters such as Fuhrman grade (p = 0.011), tumor size (p<0.001), T stage (p<0.001) and N stage (p = 0.015). Patients with higher PNCK expression had shorter overall survival time than those with lower PNCK expression (p<0.001). Multivariate analysis indicated that PNCK expression was an independent predictor for poor survival of ccRCC patients.

Conclusions

To our knowledge, this is the first study that determines the relationship between PNCK and prognosis in ccRCC. We found that increased PNCK expression is associated with poor prognosis in ccRCC. PNCK may represent a novel prognostic marker for ccRCC.     

The in vitro synthesis of 7-hydroxy dehydroepiandrosterone by human mammary tissues

Normal and tumorous human mammary tissues were incubated in vitro with [7α-³H] dehydroepiandrosterone and [7α-³H] dehydroepiandrosterone sulphate. Tritium-labelled 7α-hydroxy dehydroepiandrosterone was identified as a principal metabolite from four of the five studies with normal tissue and all seven studies with tumorous tissue.     

Hexokinase 3 dysfunction promotes tumorigenesis and immune escape by upregulating monocyte/macrophage infiltration into the clear cell renal cell carcinoma microenvironment

Purpose: This study aimed to identify the potential prognostic role of HK3 and provide clues about glycolysis and the microenvironmental characteristics of ccRCC.Methods: Based on the Cancer Genome Atlas (TCGA, n = 533) and Gene expression omnibus (GEO) (n = 127) databases, real-world (n = 377) ccRCC cohorts, and approximately 15,000 cancer samples, the prognostic value and immune implications of HK3 were identified. The functional effects of HK3 in ccRCC were analyzed in silico and in vitro.Results: The large-scale findings suggested a significantly higher HK3 expression in ccRCC tissues and the predictive efficacy of HK3 for tumor progression and a poor prognosis. Next, the subgroup survival and Cox regression analyses showed that HK3 serves as a promising and independent predictive marker for the prognosis and survival of patients with ccRCC from bioinformatic databases and real-world cohorts. Subsequently, we found that HK3 could be used to modulate glycolysis and the malignant behaviors of ccRCC cells. The comprehensive results suggested that HK3 is highly correlated with the abundance of immune cells, and specifically stimulates the infiltration of monocytes/macrophages presenting surface markers, regulates the immune checkpoint molecules PD-1 and CTLA-4 of exhaustive T cells, restrains the immune escape of tumor cells, and prompts the immune-rejection microenvironment of ccRCC.Conclusion: In conclusion, the large-scale data first revealed that HK3 could affect glycolysis, promote malignant biologic processes, and predict the aggressive progression of ccRCC. HK3 may stimulate the abundance of infiltrating monocytes/macrophages presenting surface markers and regulate the key molecular subgroups of immune checkpoint molecules of exhaustive T cells, thus inducing the microenvironmental characteristics of active anti-tumor immune responses.     

Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

Background: Tumor-associated macrophages (TAMs) dominate the malignancy of cancers by perturbing the tumor microenvironment (TME). However, the clinical implications of heterogeneous subpopulations of TAMs in clear cell renal cell carcinoma (ccRCC) remain to be elucidated.Methods: We comprehensively evaluated the prognostic implications, biological behaviors, and immunogenomics features of the C-C Motif Chemokine Ligand 5 (CCL5) expression and CCL5⁺ TME in vitro and in 932 real-world ccRCC patients from testing and public validation cohorts. Flow cytometry was used to examine the functional patterns of CCL5⁺ TAMs with TME cell-infiltrating characterizations.Results: Our results identified distinct prognostic clusters with gradual changes in clinicopathological indicators based on CCL5 expression. Knockdown of CCL5 significantly restrained cell viability, migration capabilities of ccRCC cells, and the inhibits the proliferation and chemotaxis of THP1-derived TAMs. Mechanically, down-regulation of CCL5 arrested epithelial-mesenchymal transition by modulating the PI3K/AKT pathway in ccRCC cells. In ccRCC samples with CCL5 upregulation, the proportion of CCL5⁺ TAMs and PD-L1⁺ CD68⁺ TAMs were prominently increased, showing a typical suppressive tumor immune microenvironment (TIME). Besides, intra-tumoral CCL5⁺ TAMs showed distinct pro-tumorigenic TME features characterized by exhausted CD8⁺ T cells and increased expression of immune checkpoints. Furthermore, elevated CCL5⁺ TAMs infiltration was prominently associated with a dismal prognosis for patients with ccRCC.Conclusion: In conclusion, this study first revealed the predictive value of the chemokine CCL5 on the progression and TME of ccRCC. The intra-tumoral CCL5⁺ TAMs could be applied to comprehensively evaluate the prognostic patterns as well as unique TME characteristics among individuals, allowing for the identification of immunophenotypes and promotion of treatment efficiency for ccRCC.     

Sulle Cause Determinanti La Formazione Di Tumori in Ibridi Di Nicotiana

Abstract

Cause of tumor formation in NICOTIANA hybrids. Further serological studies. — Additional researches have been carried out about genetical tumors in genus Nicotiana. By means of serological procedure the affinity among pairs of species that give rise to tumorous and non-tumorous hybrids has been tested, considering also their phyletic interrelation. Using ion exchange chromatography a qualitative analysis of the total proteins of tumorous and non-tumorous tissue of hybrids and of normal leaves taken from hybrids and their parental species was carried out. Serological analysis of affinity between the species tested gave following results:

1) higher serological affinity was found between species belonging to the same subgenus than between species belonging to different subgenera.

2) serological affinity between pairs of species giving rise to tumorous hybrids is higher than the affinity observed between other pairs of species that give rise to normal hybrids in spite of the same sistematic position. The same serological responses were obtained studying many species of Nicotiana with the single apparent exception of N. langsdorffii. The results of chromatographical analysis have revealed a high constancy of protein composition of the normal and tumorous tissue of hybrids. This conferms the genetical uniformity of the whole plant.     

Hsa_circ_0003596 enhances the development of cell renal clear cell carcinoma through the miR-502-5p/IGF1/PI3K/AKT axis

Accumulating research findings have shown that circular RNAs (circRNAs) play an indispensable role in tumorigenesis and tumor progression. The current study aimed to explore the role and modulatory mechanism of hsa_circ_0003596 in clear cell renal cell carcinoma (ccRCC). Quantitative real-time polymerase chain reaction was adopted to detect the expression of hsa_circ_0003596 in ccRCC tissue and cell lines. 5-Ethynyl-2′-deoxyuridine, cell counting kit 8 and the colony formation assay were utilized to assess the proliferation potential of the ccRCC cells. Transwell along with wound healing assays were adopted to quantify infiltration coupled with the migration potential of the cells. The current research study found that the circRNA hsa_circ_0003596 was overexpressed in ccRCC tissue and cell lines. Further, result showed that hsa_circ_0003596 was associated with distant metastasis of renal cancer. Notably, the knockdown of hsa_circ_0003596 can lower the proliferation, infiltration and migration potential of ccRCC cells. The results of in vivo experiments found that the reduction of hsa_circ_0003596 significantly hampered the growth of tumors in mice. In addition, it was evident that hsa_circ_0003596 acts as a “molecular sponge” for miR-502-5p to upregulate the expression of the microRNA-502-5p (miR-502-5p) target insulin-like growth factor 1 (IGF1R). Furthermore, it was found that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling was the downstream cascade of hsa_circ_0003596/miR-502-5p/IGF1R cascade, which is partly responsible for the cancer-promoting effect. Overall, the results of the present study showed that hsa_circ_0003596 facilitated the proliferation, infiltration and migration of ccRCC through the miR-502-5p/IGF1R/PI3K/AKT axis. Therefore, it was evident that hsa_circ_0003596 can serve as a possible biomarker and therapeutic target against ccRCC.     

Urinary exosome miR‐30c‐5p as a biomarker of clear cell renal cell carcinoma that inhibits progression by targeting HSPA5

Exosome‐derived miRNAs are regarded as biomarkers for the diagnosis and prognosis of many human cancers. However, its function in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, differentially expressed miRNAs from urinal exosomes were identified using next‐generation sequencing (NGS) and verified using urine samples of ccRCC patients and healthy donors. Then, the exosomes were analysed in early‐stage ccRCC patients, healthy individuals and patients suffering from other urinary system cancers. Thereafter, the target gene of the miRNA was detected. Its biological function was investigated in vitro and in vivo. The results showed that miR‐30c‐5p could be amplified in a stable manner. Its expression pattern was significantly different only between ccRCC patients and healthy control individuals, but not compared with that of other urinary system cancers, which indicated its specificity for ccRCC. Additionally, the overexpression of miR‐30c‐5p inhibited ccRCC progression in vitro and in vivo. Heat‐shock protein 5 (HSPA5) was found to be a direct target gene of miR‐30c‐5p. The depletion of HSPA5 caused by miR‐30c‐5p inhibition reversed the promoting effect of ccRCC growth. In conclusion, urinary exosomal miR‐30c‐5p acts as a potential diagnostic biomarker of early‐stage ccRCC and may be able to modulate the expression of HSPA5, which is correlated with the progression of ccRCC.     

Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

The intracellular Ca²⁺ regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca²⁺ entry (SOCE) is a major Ca²⁺ entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.     

Semaphorin 6D as an independent predictor for better prognosis in clear cell renal cell carcinoma

IntroductionClear cell renal cell carcinoma (ccRCC) is the most common type of RCC and is associated with poor survival. However, the mechanisms underlying its development have not been thoroughly investigated. Semaphorin 6D (SEMA6D) is differentially expressed in various cancers, including lung adenocarcinoma and colorectal cancer. However, the role and mechanism of SEMA6D in ccRCC remain unexplored.Materials and methodsWe obtained 25 pairs of ccRCC tissue samples and 57 urine samples from patients with ccRCC and 52 urine samples from healthy volunteers. We performed RNA sequencing and compared the results with data from The Cancer Genome Atlas database to identify our gene of interest, SEMA6D. To verify the differential expression of SEMA6D, we used real-time quantitative polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay. Finally, we conducted in vitro proliferation, migration and invasion experiments.ResultsSEMA6D expression was significantly lower in ccRCC tissue compared to that in normal tissue. Comparative analysis of our results with data from online databases revealed that the expression level of SEMA6D in ccRCC tissue correlated with the clinical stage and pathological grade of ccRCC. Furthermore, higher SEMA6D expression was associated with improved quality of life of patients with ccRCC. In addition, the diagnostic value of SEMA6D was confirmed using data from two Gene Expression Omnibus ccRCC databases. The results showed that SEMA6D can be used as a predictor for ccRCC diagnosis, with an area under the curve of 0.9642.ConclusionSEMA6D may serve as a diagnostic and prognostic biomarker for ccRCC.     

Expression of calcium-binding protein regucalcin mRNA in hepatoma cells

Whether the gene expression of hepatic Ca²⁺-binding protein regucalcin is altered in hepatomas was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). Rat hepatoma was induced by continuous feeding of basal diet containing 0.06% 3-methyl-4-dimethylaminoazobenzene (3-Me-DAB). After 35 weeks feeding, rats were sacrificed, and the non-tumorous and tumorous tissues of the livers were removed. In individual rats, the regucalcin mRNA levels in the tumorous tissues were generally decreased in comparison with that of the non-tumorous tissues of the chemical-fed rats, although the chemical administration might decrease the mRNA expression in normal rat liver, suggesting that the chemical administration causes a suppresive effect on the mRNA expression. When the genomic DNA extracted from the liver tumorous tissues was digested with restriction enzymes (EcoRI, BamHI and HindIII) and analyzed by Southern blotting, no rear-ranged band was found in the regucalcin gene from the hepatoma. Interestingly, in the transplantable Morris hepatoma cells, the regucalcin mRNA was markedly expressed, while the albumin mRNA was expressed only slightly. The present study demonstrates that regucalcin mRNA is clearly expressed in the transformed cells (Morris hepatoma cells).     

FBXL6 depletion restrains clear cell renal cell carcinoma progression

BackgroundF-box proteins play important roles in cell cycle and tumorigenesis. However, its prognostic value and molecular function in clear cell renal cell carcinoma (ccRCC) remain unclear. In this study, we established a survival model to evaluate the prognosis of patients with ccRCC using the F-box gene signature and investigated the function of FBXL6 in ccRCC.MethodsComprehensive bioinformatics analyses were used to identify differentially expressed F-box and hub genes associated with ccRCC carcinogenesis. Based on the F-box gene signature, we constructed a risk model and nomogram to predict the overall survival (OS) of patients with ccRCC and assist clinicians in decision-making. Finally, we verified the function and underlying molecular mechanisms of FBXL6 in ccRCC using CCK-8 and EdU assays, flow cytometry, and subcutaneous xenografts.ResultsA risk model based on FBXO39, FBXL6, FBXO1, and FBXL16 was developed. In addition, we drew a nomogram based on the risk score and clinical features to assess the prognosis of patients with ccRCC. Subsequently, we identified FBXL6 as an independent prognostic marker that was highly expressed in ccRCC cell lines. In vivo and in vitro assays revealed that the depletion of FBXL6 inhibited cell proliferation and induced apoptosis. We also demonstrated that SP1 regulated the expression of FBXL6.ConclusionsFBXL6 was first identified as a diagnostic and prognostic marker in patients with ccRCC. Loss of FBXL6 attenuates proliferation and induces apoptosis in ccRCC cells. SP1 was also found to regulate the expression of FBXL6.     

Circ_0101692 knockdown retards the development of clear cell renal cell carcinoma through miR-384/FN1 pathway

PurposeCircular RNA_0101692 (circ_0101692) is overexpressed in clear cell renal cell carcinoma (ccRCC) by microarray analyses. However, its function and action mechanism in ccRCC tumorigenesis is still elusive.MethodsWestern blotting and qRT-PCR were executed to assess the circ_0101692, miR-384 and FN1 expression in ccRCC cells and tissues. Target relationships among them were determined via dual luciferase reporter and/or RNA immunoprecipitation assays. Cell proliferation was evaluated by CCK-8 assay. Caspase-3 activity assay was utilized to analyze cell apoptosis. To find out whether ccRCC cells might migrate, a transwell assay was performed. To assess the effects of circ_0101692 on tumor development in vivo, a mouse xenograft model was used.ResultsHigh expression of circ_0101692 and FN1, and decreased miR-384 were determined in ccRCC. Cell growth, migration and viability were decreased whereas cell apoptosis was stimulated when circ_0101692 was knockdown. miR-384 inhibitor transfection attenuated the inhibiting impacts of circ_0101692 silencing on ccRCC cell progression. FN1 deletion further inverted the cancer-promoting effect of miR-384 downregulation on cell viability and migration. In addition, circ_0101692 could sponge miR-384 to relieve the inhibition of miR-384 on FN1 in ccRCC.ConclusionsCirc_0101692 targeted miR-384/FN1 axis to facilitate cell proliferation, migration and repress apoptosis, thereby accelerating the development of ccRCC. This points out that circ_0101692/miR-384/FN1 axis might be a prospective target implemented for the future treatment of ccRCC.     

MicroRNA-153-5p promotes the proliferation and metastasis of renal cell carcinoma via direct targeting of AGO1

MicroRNAs (miRNAs) have been demonstrated to affect the biological processes of cancers and showed great potential for prognostic biomarkers. In this study, we screened differentially expressed miRNAs in ccRCC based on three dimensions of metastasis, prognosis, and differential expression compared to normal tissue using bioinformatics algorithms. MiR-153-5p was identified as a candidate miRNA to promote ccRCC occurrence and progression. Clinically, we found that miR-153-5p was significantly upregulated and related to unfavorable clinical features in ccRCC. Besides, miR-153-5p served as an independent prognostic biomarker. Functionally, miR-153-5p depletion remarkably inhibited the proliferation and metastasis of ccRCC via the phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Furthermore, AGO1 was proved to be a direct target of miR-153-5p. AGO1 is associated with favorable clinical features and exhibited independent prognostic value in ccRCC. Besides, we observed that AGO1 knockdown significantly promoted tumor proliferation and metastasis. Downregulation of AGO1 partly abolished the oncogenic effects of miR-153-5p knockdown. Furthermore, miR-153-5p combined with AGO1 showed more robust prognostic significance in ccRCC. In conclusion, we found that the newly identified miR-153-5p/AGO1 axis was responsible for tumor occurrence and progression via PI3K/Akt signaling, which may therefore provide promising therapeutic targets and prognostic biomarkers for patients with ccRCC.Subject terms: Cancer screening, miRNAs     

High Expression of KCa3.1 in Patients with Clear Cell Renal Carcinoma Predicts High Metastatic Risk and Poor Survival

Background

Ca²⁺-activated K⁺ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca²⁺-activated K⁺ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival.

Methodology/Principal Findings

We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro.

Conclusions/Significance

Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.