Expression of HIF­α and their association with clinicopathological parameters in clinical renal cell carcinoma.

Objectives: This study aimed to assess the cellular localization and expression levels of hypoxia-inducible factor (HIF) -α proteins (specifically HIF-1α, HIF-2α, and HIF-3α) that play a role in the hypoxia pathway and to determine their correlation with clinicopathological parameters and patient survival in renal cell carcinoma (RCC). Materials and methods: Tissue microarray (TMA) with cores from 150 clear cell RCCs and 31 non-ccRCC samples. HIF-1α, HIF-2α, and HIF-3α antibodies were used for immunohistochemistry (IHC) of TMA to evaluate the cellular localization and expression levels of HIF-α proteins, specifically in relation to the hypoxia pathway. Results: The expression levels of the HIF-α proteins were higher in the nucleus than in the cytoplasm. Furthermore, the nuclear expression levels of all HIF-α proteins were significantly higher in clear cell RCC (ccRCC) than in non-ccRCC. Cytoplasmic HIF-3α expression was also higher in ccRCC than in non-ccRCC, whereas cytoplasmic HIF-1α and HIF-2α expression levels were similar between the different RCC types. In ccRCC, nuclear HIF-1α expression levels correlated with both nuclear HIF-2α and HIF-3α levels, whereas cytoplasmic HIF-3α expression levels were associated with HIF-1α only.In non-ccRCC, there was a positive correlation observed between nuclear HIF-1α and HIF-3α expression, but no correlation was found with HIF-2α. In patients with ccRCC, the nuclear expressions of HIF-1α and HIF-3α was significantly associated with cancer-specific survival (CSS) in univariate analysis. This association was no longer evident in multivariate analysis. Notably, there was no correlation observed between nuclear HIF-2α expression and CSS in these patients. In contrast, cytoplasmic expression levels showed no association with CSS. Conclusion: The expression levels of the three primary HIF-α proteins were found to be higher in the nucleus than in the cytoplasm. Furthermore, the results indicated that HIF-3α and HIF-1α expression levels were significant univariate factors associated with CSS in patients with clear cell RCC. These results highlight the critical role that HIF-3α and HIF-1α play in the hypoxia pathway.

Sex specific associations in genome wide association analysis of renal cell carcinoma.

Renal cell carcinoma (RCC) has an undisputed genetic component and a stable 2:1 male to female sex ratio in its incidence across populations, suggesting possible sexual dimorphism in its genetic susceptibility. We conducted the first sex-specific genome-wide association analysis of RCC for men (3227 cases, 4916 controls) and women (1992 cases, 3095 controls) of European ancestry from two RCC genome-wide scans and replicated the top findings using an additional series of men (2261 cases, 5852 controls) and women (1399 cases, 1575 controls) from two independent cohorts of European origin. Our study confirmed sex-specific associations for two known RCC risk loci at 14q24.2 (DPF3) and 2p21(EPAS1). We also identified two additional suggestive male-specific loci at 6q24.3 (SAMD5, male odds ratio (ORmale) = 0.83 [95% CI = 0.78-0.89], Pmale = 1.71 × 10-8 compared with female odds ratio (ORfemale) = 0.98 [95% CI = 0.90-1.07], Pfemale = 0.68) and 12q23.3 (intergenic, ORmale = 0.75 [95% CI = 0.68-0.83], Pmale = 1.59 × 10-8 compared with ORfemale = 0.93 [95% CI = 0.82-1.06], Pfemale = 0.21) that attained genome-wide significance in the joint meta-analysis. Herein, we provide evidence of sex-specific associations in RCC genetic susceptibility and advocate the necessity of larger genetic and genomic studies to unravel the endogenous causes of sex bias in sexually dimorphic traits and diseases like RCC.

VHL status regulates transforming growth factor-ß signaling pathways in renal cell carcinoma.

To evaluate the role of pVHL in the regulation of TGF-ß signaling pathways in clear cell renal cell carcinoma (ccRCC) as well as in non-ccRCC; the expression of pVHL, and the TGF-ß pathway components and their association with clinicopathological parameters and patient's survival were explored. Tissue samples from 143 ccRCC and 58 non-ccRCC patients were examined by immunoblot. ccRCC cell lines were utilized for mechanistic in-vitro studies. Expression levels of pVHL were significantly lower in ccRCC compared with non-ccRCC. Non-ccRCC and ccRCC pVHL-High expressed similar levels of pVHL. Expression of the TGF-ß type I receptor (ALK5) and intra-cellular domain were significantly higher in ccRCC compared with non-ccRCC. In non-ccRCC, expressions of ALK5-FL, ALK5-ICD, pSMAD2/3, and PAI-1 had no association with clinicopathological parameters and survival. In ccRCC pVHL-Low, ALK5-FL, ALK5-ICD, pSMAD2/3, and PAI-1 were significantly related with tumor stage, size, and survival. In ccRCC pVHL-High, the expression of PAI-1 was associated with stage and survival. In-vitro studies revealed that pVHL interacted with ALK5 to downregulate its expression through K48-linked poly-ubiquitination and proteasomal degradation, thus negatively controlling TGF-ß induced cancer cell invasiveness. The pVHL status controls the ALK5 and can thereby regulate the TGF-ß pathway, aggressiveness of tumors, and survival of the ccRCC and non-ccRCC patients.

Transforming growth factor-ß promotes aggressiveness and invasion of clear cell renal cell carcinoma.

The molecular mechanisms whereby transforming growth factor-ß (TGF-ß) promotes clear cell renal cell carcinoma (ccRCC) progression is elusive. The cell membrane bound TGF-ß type I receptor (ALK5), was recently found to undergo proteolytic cleavage in aggressive prostate cancer cells, resulting in liberation and subsequent nuclear translocation of its intracellular domain (ICD), suggesting that ALK5-ICD might be a useful cancer biomarker. Herein, the possible correlation between ALK5 full length (ALK5-FL) and ALK5-ICD protein, phosphorylated Smad2/3 (pSmad2/3), and expression of TGF-ß target gene PAI-1, was investigated in a clinical ccRCC material, in relation to tumor grade, stage, size and cancer specific survival. Expression of ALK5-FL, ALK5-ICD, pSmad2/3 and PAI-1 protein levels were significantly higher in higher stage and associated with adverse survival. ALK5-ICD, pSmad2/3 and PAI-1 correlated with higher grade, and ALK5-FL, pSmad2/3 and PAI-1 protein levels were significantly correlated with larger tumor size. Moreover, the functional role of the TGF-ß - ALK5-ICD pathway were investigated in two ccRCC cell lines by treatment with ADAM/MMP2 inhibitor TAPI-2, which prevented TGF-ß-induced ALK5-ICD generation, nuclear translocation, as well as cell invasion. The present study demonstrated that canonical TGF-ß Smad2/3 pathway and generation of ALK5-ICD correlates with poor survival and invasion of ccRCC in vitro.

TERT promoter mutations in clear cell renal cell carcinoma.

We screened promoter region of the telomerase reverse transcriptase (TERT) for activating somatic mutations in 188 tumors from patients with clear cell renal cell carcinoma (ccRCC). Twelve tumors (6.4%) carried a mutation within the core promoter region of the gene. The mutations were less frequent in high grade tumors compared to low grade tumors [odds ratio (OR) = 0.15, 95% confidence interval (CI) = 0.03-0.72, p = 0.02]. Multivariate analysis for cause specific survival showed statistically significant poor outcome in patients with TERT promoter mutations [hazard ratio (HR) = 2.90, 95% CI = 1.13-7.39, p = 0.03]. A common polymorphism (rs2853669) within the locus seemed to act as a modifier of the effect of the mutations on patient survival as the noncarriers of the variant allele with the TERT promoter mutations showed worst survival (HR = 3.34, 95% CI = 1.24-8.98, p = 0.02). We also measured relative telomere length (RTL) in tumors and difference between tumors with and without the TERT promoter mutations was not statistically significant. Similarly, no difference in patient survival based on RTL in tumors was observed. Our study showed a relatively low frequency of TERT promoter mutations in ccRCC. Nevertheless, patients with the mutations, particularly in the absence of the rs2853669 variant showed the worst disease-specific survival. Thus, it is possible that the TERT promoter mutations define a small subset of tumors with an aggressive behavior.

Single nucleotide polymorphisms in the Wilms' tumour gene 1 in clear cell renal cell carcinoma.

The Wilms' tumour gene 1 (WT1) single nucleotide polymorphism (SNP) rs16754 has recently been described as an independent prognostic factor in acute myeloid leukaemia (AML) patients. It is of great interest to test whether WT1 SNPs can be used as a molecular marker in other cancer types in order to improve risk and treatment stratification. We performed sequencing analysis on all 10 exons of the WT1 gene in a total of 182 patients with clear cell renal cell carcinoma (ccRCC). Six different SNPs were identified, in descending order for minor allele frequency: rs2234582, rs16754, rs1799925, rs5030315, rs2234583, and rs2234581. At least one minor allele for WT1 SNP was identified in 61% of ccRCC patients. In the entire study population, only 6% carried two copies of the minor allele. The genotypes of WT1 SNPs in 78 tumour-free kidney tissue specimens were found to be in 95% concordance with corresponding tumour samples. No correlation was observed between WT1 SNP genotypes and RNA expression level. WT1 SNP genotypes did not associate with clinical and pathological characteristics. We found favourable outcomes associated with the homozygous minor allele for WT1 SNP. However, SNP genotypes did not show to be of prognostic significance when comparing wild-type versus homozygous or heterozygous for the minor allele in the entire cohort. None of the previously reported WT1 mutations in AML was found in the present study. A novel WT1 missense mutation was identified in only one patient. Our data suggest that common WT1 mutations are not involved in ccRCC. Due to too few cases harbouring the homozygous minor allele, the prognostic impact needs to be verified in larger study populations.

Telomere length in relation to immunological parameters in patients with renal cell carcinoma.

Over the last decade, telomere length (TL) has gained attention as a potential biomarker in cancer disease. We previously reported that long blood TL was associated with a poorer outcome in patients with breast cancer and renal cell carcinoma. Based on these findings, we hypothesized that certain immunological components may have an impact on TL dynamics in cancer patients. One aim of the present study was to investigate a possible association between serum cytokines and TL of peripheral blood cells, tumors and corresponding kidney cortex, in patients with clear cell renal cell carcinoma. For this purpose, a multiplex cytokine assay was used. Correlation analysis revealed significant positive correlations between tumor TL and peripheral levels of three cytokines (IL-7, IL-8 and IL-10). In a parallel patient group with various kidney tumors, TL was investigated in whole blood and in immune cell subsets in relation to peripheral levels of regulatory T cells (Tregs). A significant positive association was found between whole blood TL and Treg levels. However, the strongest correlation was found between Tregs and TL of the T lymphocyte fraction. Thus, patients with higher Treg levels displayed longer T cell telomeres, which might reflect a suppressed immune system with fewer cell divisions and hence less telomere shortening. These results are in line with our earlier observation that long blood TL is an unfavorable prognostic factor for cancer-specific survival. In summary, we here show that immunological components are associated with TL in patients with renal cell carcinoma, providing further insight into the field of telomere biology in cancer.

The PTEN regulator DJ-1 is associated with hTERT expression in clear cell renal cell carcinoma.

DJ-1 is as a novel regulator of the tumor suppressor PTEN with stimulatory effects on PI3K-AKT/PKB signaling, one possible target of which is cMyc. The catalytic unit of the telomerase complex, hTERT, can be activated at different levels, including transcriptionally by cMyc and through phosphorylation by AKT/PKB. The aim of the study was to analyze the putative signaling pathway encompassing DJ-1, cMyc and hTERT in a series of 176 renal cell carcinomas (RCC) and experimentally in cell lines. DJ-1 mRNA expression was significantly elevated in clear cell RCC (ccRCC) compared with in papillary RCC (pRCC; p = 0.005) and kidney cortex tissue (p < 0.001). ccRCC and pRCC demonstrated higher cMyc RNA levels than in kidney cortex (p < 0.001 for both) as well as increased levels of hTERT RNA (p < 0.001 and p = 0.011, respectively). DJ-1 was positively correlated to cMyc and hTERT in ccRCC (p < 0.001 and p = 0.019, respectively), but not in pRCC, indicating that this pathway could have a functional significance in ccRCC. siRNA knock down of DJ-1 induced downregulation of cMyc and hTERT mRNA associated with decreased expression of pAKT and cMyc protein levels. hTERT promoter activity was upregulated after DJ-1 transfection and this upregulation was inhibited after mutation of the cMyc binding sites. These experimental data support the functional link among DJ-1, cMyc and hTERT expression as indicated in the tumor material. Neither DJ-1, cMyc nor hTERT mRNA levels were associated with proliferation (S-phase fraction), telomere length or prognosis in ccRCC.