ResearchPad - renal-cancer https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[CRISPR-mediated ablation of overexpressed EGFR in combination with sunitinib significantly suppresses renal cell carcinoma proliferation]]> https://www.researchpad.co/article/elastic_article_14711 Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma.

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<![CDATA[Editorial Comment: Comparison of Immediate vs Deferred Cytoreductive Nephrectomy in Patients With Synchronous Metastatic Renal Cell Carcinoma Receiving Sunitinib: The SURTIME Randomized Clinical Trial]]> https://www.researchpad.co/article/N6fee5f49-c29d-4f12-bbf8-1212fbf92b2a ]]> <![CDATA[Editorial Comment: Feasibility and safety of irreversible electroporation (IRE) in patients with small renal masses: Results of a prospective study]]> https://www.researchpad.co/article/N6e2e5e60-afae-4d7f-b94c-ca4fb0935a80 ]]> <![CDATA[Editorial Comment: The Emerging Role of Stereotactic Ablative Radiotherapy for Primary Renal Cell Carcinoma: A Systematic Review and Meta-Analysis]]> https://www.researchpad.co/article/N27ee745a-c7c7-4c50-bf73-298ff7339d86 ]]> <![CDATA[GRP78 expression in tumor and perinephric adipose tissue is not an optimal risk stratification marker for clear cell renal cell carcinoma]]> https://www.researchpad.co/article/5c61b7d2d5eed0c484937fff

Objective

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, which is difficult to treat and lacks a reliable prognostic marker. A previous study showed that the endoplasmic reticulum stress marker, glucose-regulated-protein-78 (GRP78), is a potential prognostic marker for ccRCC. The present study aimed to: (1) examine whether GRP78 was upregulated in ccRCC compared with matched non-neoplastic renal tissue; and (2) investigate whether GRP78 expression in ccRCC tissue or perinephric adipose tissue has any association with ccRCC aggressiveness.

Methods

A retrospective cross-sectional study of 267 patients who underwent nephrectomy for renal tumors between June 2013 and October 2017 was conducted at Princess Alexandra Hospital, Brisbane, Australia. Software-assisted quantification of average grey value of staining intensity (staining intensity method) and proportion of positive pixels (positive pixel method) was applied to measure expression of GRP78 in archived specimens of renal tumor tissues (n = 114), adjacent non-neoplastic renal tissues (n = 68), and perinephric adipose tissues (n = 60) in participants diagnosed with ccRCC.

Results

GRP78 was not upregulated in renal tumor tissue compared with paired normal renal tissue. In tumor tissue, GRP78 expression did not show any association with ccRCC aggressiveness using either quantification method. In adipose tissue, downregulation of GRP78 demonstrated poor correlation with increased probability of metastasis, with one unit increase in average grey value of GRP78 staining weakly correlating with a 17% increase in the odds ratio of metastasis (95% confidence interval: 0.99 to 1.38, p = 0.07).

Conclusion

GRP78 is not valuable as a risk stratification marker for ccRCC.

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<![CDATA[Physical activity and sedentary behavior in relation to mortality among renal cell cancer survivors]]> https://www.researchpad.co/article/5b297b78463d7e06147f8d6c

Background

The relations of physical activity and sedentary behavior to mortality risk among patients with renal cell cancer have not yet been evaluated.

Methods

We conducted a prospective cohort study among 667 renal cell cancer survivors aged 50–71 years of the National Institutes of Health (NIH)-AARP Diet and Health Study with a median follow-up time of 7.1 years. Post-diagnosis physical activity, TV viewing, and total sitting time were assessed using self-administered questionnaires. Hazard ratios (HRs) and 95% confidence intervals (CIs) for mortality were estimated using Cox proportional hazards models.

Results

Increasing levels of moderate to vigorous physical activity were related to decreased risk of overall mortality [multivariable-adjusted HRs for <1 hr/wk (reference), 1 to 3 hrs/wk, ≥3 to <7 hrs/wk, and ≥7 hrs/wk = 1.0, 1.16, 0.94, and 0.60 (95% CI = 0.38–0.96; p-trend = 0.03)]. In contrast, television viewing was associated with increased risk of overall mortality in the age- and sex-adjusted model (HR for >4 hrs/d vs. 0 to 2 hrs/d = 1.52, 95% CI = 1.02–2.26; p-trend = 0.04), but the relation was attenuated following further control for other covariates (multivariable-adjusted HR = 1.44, 95% CI = 0.92–2.24; p-trend = 0.11). Total sitting time was unrelated to all-cause mortality.

Conclusion

Among renal cancer patients, moderate to vigorous physical activity is associated with decreased risk of overall mortality. Clinicians should consider discussing the potential benefits of physical activity for longevity among survivors of renal cell cancer.

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<![CDATA[Intratumoral Administration of Holmium-166 Acetylacetonate Microspheres: Antitumor Efficacy and Feasibility of Multimodality Imaging in Renal Cancer]]> https://www.researchpad.co/article/5989d9d4ab0ee8fa60b65446

Purpose

The increasing incidence of small renal tumors in an aging population with comorbidities has stimulated the development of minimally invasive treatments. This study aimed to assess the efficacy and demonstrate feasibility of multimodality imaging of intratumoral administration of holmium-166 microspheres (166HoAcAcMS). This new technique locally ablates renal tumors through high-energy beta particles, while the gamma rays allow for nuclear imaging and the paramagnetism of holmium allows for MRI.

Methods

166HoAcAcMS were administered intratumorally in orthotopic renal tumors (Balb/C mice). Post administration CT, SPECT and MRI was performed. At several time points (2 h, 1, 2, 3, 7 and 14 days) after MS administration, tumors were measured and histologically analyzed. Holmium accumulation in organs was measured using inductively coupled plasma mass spectrometry.

Results

166HoAcAcMS were successfully administered to tumor bearing mice. A striking near-complete tumor-control was observed in 166HoAcAcMS treated mice (0.10±0.01 cm3 vs. 4.15±0.3 cm3 for control tumors). Focal necrosis and inflammation was present from 24 h following treatment. Renal parenchyma outside the radiated region showed no histological alterations. Post administration CT, MRI and SPECT imaging revealed clear deposits of 166HoAcAcMS in the kidney.

Conclusions

Intratumorally administered 166HoAcAcMS has great potential as a new local treatment of renal tumors for surgically unfit patients. In addition to strong cancer control, it provides powerful multimodality imaging opportunities.

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<![CDATA[Resistance after Chronic Application of the HDAC-Inhibitor Valproic Acid Is Associated with Elevated Akt Activation in Renal Cell Carcinoma In Vivo]]> https://www.researchpad.co/article/5989dae6ab0ee8fa60bbd999

Targeted drugs have significantly improved the therapeutic options for advanced renal cell carcinoma (RCC). However, resistance often develops, negating the benefit of these agents. In the present study, the molecular mechanisms of acquired resistance towards the histone deacetylase (HDAC) inhibitor valproic acid (VPA) in a RCC in vivo model were investigated. NMRI:nu/nu mice were transplanted with Caki-1 RCC cells and then treated with VPA (200 mg/kg/day). Controls remained untreated. Based on tumor growth dynamics, the mice were divided into “responders” and “non-responders” to VPA. Histone H3 and H4 acetylation and expression of cell signaling and cell cycle regulating proteins in the RCC mouse tumors were evaluated by Western blotting. Tumor growth of VPA responders was significantly diminished, whereas that of VPA non-responders even exceeded control values. Cdk1, 2 and 4 proteins were strongly enhanced in the non-responders. Importantly, Akt expression and activity were massively up-regulated in the tumors of the VPA non-responders. Chronic application (12 weeks) of VPA to Caki-1 cells in vitro evoked a distinct elevation of Akt activity and cancer cells no longer responded with cell growth reduction, compared to the short 2 week treatment. We assume that chronic use of an HDAC-inhibitor is associated with (re)-activation of Akt, which may be involved in resistance development. Consequently, combined blockade of both HDAC and Akt may delay or prevent drug resistance in RCC.

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<![CDATA[The Difference in Prognosis between Renal Sinus Fat and Perinephric Fat Invasion for pT3a Renal Cell Carcinoma: A Meta-Analysis]]> https://www.researchpad.co/article/5989db16ab0ee8fa60bcd1df

Background

In the current Tumour-Node-Metastasis (TNM) classification system for renal cell carcinoma (RCC), both renal sinus fat invasion (SFI) and perinephric fat invasion (PFI) are defined as T3a, suggesting that the prognosis should be similar for the two pathologic findings. Several studies, however, have reported a worse prognosis for SFI in patients with a T3a tumor. In order to compare the prognosis of these two pathologic findings (SFI versus. PFI) in a more comprehensive way, this meta-analysis was performed.

Methods

To identify relevant studies, Medline, Embase, Cochrane Library, and Scopus database were searched from the inception until October 2014. A meta-analysis was performed using Review Manager 5.2 and STATA 11. Pooled Odds ratio (OR) and/or hazard ratio (HR) with 95% confidence interval (CI) were calculated to examine the risk or hazard association.

Results

A total of 6 studies including 1031 patients qualified for analysis. T3a RCC patients with SFI were significantly associated with poor cancer specific survival(CSS) (HR: 1.47, 95% CI: 1.19–1.83; P<0.001) compared to those with PFI. In T3aNx/N0M0 subgroup, SFI patients also showed a worse prognosis than those with PFI (CSS, HR: 1.94, 95% CI: 1.21–3.12; P = 0.006). T3a RCC patients with SFI had higher Furhman grade, greater possibility of lymph node metastasis, sarcomatoid differentiation and tumour necrosis. Main limitation is the relatively small number of included studies.

Conclusion

The present meta-analysis suggested that SFI is associated with worse CSS in patients with pT3a RCC. However, due to the small number of included studies, future studies with a large sample size are required to further verify our findings.

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<![CDATA[Relevance and Therapeutic Possibility of PTEN-Long in Renal Cell Carcinoma]]> https://www.researchpad.co/article/5989da17ab0ee8fa60b7badc

PTEN-Long is a translational variant of PTEN (Phosphatase and Tensin Homolog). Like PTEN, PTEN-Long is able to antagonize the PI3K-Akt pathway and inhibits tumor growth. In this study, we investigated the role PTEN-Long plays in the development and progression of clear cell renal cell carcinoma (ccRCC) and explored the therapeutic possibility using proteinaceous PTEN-Long to treat ccRCC. We found that the protein levels of PTEN-Long were drastically reduced in ccRCC, which was correlated with increased levels of phosphorylated Akt (pAkt). Gain of function experiments showed overexpression of PTEN-Long in the ccRCC cell line 786-0 suppressed PI3K-Akt signaling, inhibited cell proliferation, migration and invasion, and eventually induced cell death. When purified PTEN-Long was added into cultured 786-0 cells, it entered cells, blocked Akt activation, and induced apoptosis involving Caspase 3 cleavage. Furthermore, PTEN-Long inhibited proliferation of 786-0 cells in xenograft mouse model. Our results implicated that understanding the roles of PTEN-Long in renal cell carcinogenesis has therapeutic significance.

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<![CDATA[Analysis of SNPs and Haplotypes in Vitamin D Pathway Genes and Renal Cancer Risk]]> https://www.researchpad.co/article/5989db0aab0ee8fa60bc9cd0

In the kidney vitamin D is converted to its active form. Since vitamin D exerts its activity through binding to the nuclear vitamin D receptor (VDR), most genetic studies have primarily focused on variation within this gene. Therefore, analysis of genetic variation in VDR and other vitamin D pathway genes may provide insight into the role of vitamin D in renal cell carcinoma (RCC) etiology. RCC cases (N = 777) and controls (N = 1,035) were genotyped to investigate the relationship between RCC risk and variation in eight target genes. Minimum-p-value permutation (Min-P) tests were used to identify genes associated with risk. A three single nucleotide polymorphism (SNP) sliding window was used to identify chromosomal regions with a False Discovery Rate of <10%, where subsequently, haplotype relative risks were computed in Haplostats. Min-P values showed that VDR (p-value = 0.02) and retinoid-X-receptor-alpha (RXRA) (p-value = 0.10) were associated with RCC risk. Within VDR, three haplotypes across two chromosomal regions of interest were identified. The first region, located within intron 2, contained two haplotypes that increased RCC risk by approximately 25%. The second region included a haplotype (rs2239179, rs12717991) across intron 4 that increased risk among participants with the TC (OR = 1.31, 95% CI = 1.09–1.57) haplotype compared to participants with the common haplotype, TT. Across RXRA, one haplotype located 3′ of the coding sequence (rs748964, rs3118523), increased RCC risk 35% among individuals with the variant haplotype compared to those with the most common haplotype. This study comprehensively evaluated genetic variation across eight vitamin D pathway genes in relation to RCC risk. We found increased risk associated with VDR and RXRA. Replication studies are warranted to confirm these findings.

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<![CDATA[The Silencing of CCND2 by Promoter Aberrant Methylation in Renal Cell Cancer and Analysis of the Correlation between CCND2 Methylation Status and Clinical Features]]> https://www.researchpad.co/article/5989da0cab0ee8fa60b77fed

Cyclin D2 (CCND2) is a member of the D-type cyclins, which plays a pivotal role in cell cycle regulation, differentiation and malignant transformation. However, its expression status and relative regulation mechanism remains unclear in renal cell cancer (RCC). In our study, the mRNA expression level of CCND2 is down-regulated in 22/23 paired RCC tissues (p<0.05). In addition, its protein expression level is also decreased in 43/43 RCC tumor tissues compared with its corresponding non-malignant tissues (p<0.001). We further detected that CCND2 was down-regulated or silenced in 6/7 RCC cell lines, but expressed in “normal” human proximal tubular (HK-2) cell line. Subsequently, MSP and BGS results showed that the methylation status in CCND2 promoter region is closely associated with its expression level in RCC cell lines. Treatment with 5-Aza with or without TSA restored CCND2 expression in several methylated RCC cell lines. Among the 102 RCC tumors, methylation of CCND2 was detected in 29/102 (28%) cases. Only 2/23 (8.7%) adjacent non-malignant tissues showed methylation. We then analyzed the correlation of clinical features and its promoter methylation. Collectively, our data suggested that loss of CCND2 expression is closely associated with the promoter aberrant methylation.

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<![CDATA[Angiomyolipoma Have Common Mutations in TSC2 but No Other Common Genetic Events]]> https://www.researchpad.co/article/5989da51ab0ee8fa60b8ddd6

Renal angiomyolipoma are part of the PEComa family of neoplasms, and occur both in association with Tuberous Sclerosis Complex (TSC) and independent of that disorder. Previous studies on the molecular genetic alterations that occur in angiomyolipoma are very limited. We evaluated 9 angiomyolipoma for which frozen tissue was available from a consecutive surgical series. Seven of 8 samples subjected to RT-PCR-cDNA sequencing showed mutations in TSC2; none showed mutations in TSC1 or RHEB. Six of the seven mutations were deletions. We searched for 983 activating and inactivating mutations in 115 genes, and found none in these tumors. Similarly analysis for genomic regions of loss or gain, assessed by Affymetrix SNP6.0 analysis, showed no abnormalities. Loss of heterozygosity in the TSC2 region was commonly seen, except in patients with low frequency TSC2 mutations. We conclude that sporadic renal angiomyolipoma usually have mutations in TSC2, but not TSC1 or RHEB, and have no other common genomic events, among those we searched for. However, chromosomal translocations and gene fusion events were not assessed here. TSC2 inactivation by mutation is a consistent and likely necessary genetic event in the pathogenesis of most angiomyolipoma.

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<![CDATA[Comparison of Trifecta and Pentafecta Outcomes between T1a and T1b Renal Masses following Robot-Assisted Partial Nephrectomy (RAPN) with Minimum One Year Follow Up: Can RAPN for T1b Renal Masses Be Feasible?]]> https://www.researchpad.co/article/5989da10ab0ee8fa60b79783

Purpose/Objectives

To investigate the feasibility of RAPN on T1b renal mass by assessment of Trifecta and Pentafecta rate between T1a and T1b renal mass.

Materials/Methods

We retrospectively reviewed the medical records of 277 cases of RPN performed from 2006 to 2015. Sixty patients with clinically T1b renal masses (> 4cm and ≤ 7 cm) were identified, and from 180 patients with clinically T1a renal mass, 60 patients were matched with T1b renal mass by propensity score. Tumor complexity was investigated according to R.E.N.A.L nephrometry score. “Pentafecta” was defined as achievement of Trifecta (negative surgical margin, no postoperative complications and warm ischemia time of ≤ 25 minutes) with addition of over 90% estimated GFR preservation and no chronic kidney disease stage upgrading at 1 year postoperative period. Propensity score matching was performed by OneToManyMTCH. Logistic regression models were used to identify the variables which predict the Trifecta, and Pentafecta ac.

Results

Preoperative variables (age, sex, body mass index, ASA score) were similar between T1a and T1b after propensity score matching. The median R.E.N.A.L. nephrometry score was 8 vs 9 for T1a and T1b respectively (p<0.001). The median warm ischemia time was 20.1 min vs 26.2 min (p<0.001). Positive surgical margin rate was 5% vs 6.6% (p = 0.729) and overall complication rate of 13.3%. vs 15% (p = 0.793). The rate of achievement of Trifecta rate were 65.3% vs 43.3% (p = 0.017) and Pentafecta rate were 38.3% vs 26.7% (p = 0.172). For achievement of Pentafecta, R.E.N.A.L nephrometry score (HR 0.80; 95% CI (0.67–0.97); p = 0.031) was significant predictor of achieving Pentafecta. Subanalyis to assess the component of R.E.N.A.L nephrometry score, L component (location relative to the polar lines, HR 0.63; 95% CI (0.38–1.03); P = 0.064) was relatively important component for Pentafecta achievement.

Conclusions

The rate of Pentafecta after RAPN was comparable between T1a and T1b renal masses. RAPN is a feasible modality with excellent long term outcome for patients with larger renal mass (cT1b).

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<![CDATA[Reduced Expression of Fumarate Hydratase in Clear Cell Renal Cancer Mediates HIF-2α Accumulation and Promotes Migration and Invasion]]> https://www.researchpad.co/article/5989da99ab0ee8fa60ba3026

Germline mutations of FH, the gene that encodes for the tricarboxylic acid TCA (TCA) cycle enzyme fumarate hydratase, are associated with an inherited form of cancer referred to as Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). Individuals with HLRCC are predisposed to the development of highly malignant and lethal renal cell carcinoma (RCC). The mechanisms of tumorigenesis proposed have largely focused on the biochemical consequences of loss of FH enzymatic activity. While loss of the tumor suppressor gene von Hippel Lindau (VHL) is thought to be an initiating event for the majority of RCCs, a role for FH in sporadic renal cancer has not been explored. Here we report that FH mRNA and protein expression are reduced in clear cell renal cancer, the most common histologic variant of kidney cancer. Moreover, we demonstrate that reduced FH leads to the accumulation of hypoxia inducible factor- 2α (HIF-2α), a transcription factor known to promote renal carcinogenesis. Finally, we demonstrate that overexpression of FH in renal cancer cells inhibits cellular migration and invasion. These data provide novel insights into the tumor suppressor functions of FH in sporadic kidney cancer.

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<![CDATA[Stage-Related Alterations in Renal Cell Carcinoma – Comprehensive Quantitative Analysis by 2D-DIGE and Protein Network Analysis]]> https://www.researchpad.co/article/5989daf7ab0ee8fa60bc330e

Renal cell carcinoma accounts for about 3% of adult malignancies and 85% of neoplasms arising from the kidney. To identify potential progression markers for kidney cancer we examined non-neoplastic and neoplastic kidney tissue from three groups of patients, which represent different tumor stages (pT1, pT2, pT3) by a fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) approach combined with MALDI-ToF-MS/MS. Delta2D software package was used for gel image based quantification and statistical analysis. Thereby, a comprehensive Principal Component Analysis (PCA) could be performed and allowed a robust quality control of the experiment as well as a classification of the analyzed samples, which correlated with the predicted stages from the pathological examination. Additionally for selected candidate proteins we detected a correlation to the tumor grading as revealed by immunohistochemistry. On the 2D protein map 176 spots out of 989 were detected as at least 2-fold differentially expressed. These spots were analyzed by MALDI-ToF-MS/MS and 187 different proteins were identified. The functional clustering of the identified proteins revealed ten groups. Within these groups we found 86 enzymes, 63 proteins of unknown function, 14 transporter, 8 peptidases and 7 kinases. From the systems biology approach we could map many of these proteins in major pathways involved in remodelling of cytoskeleton, mitochondrial dysfunctions and changes in lipid metabolism. Due to complexity of the highly interconnected pathway network, further expression and functional validation of these proteins might provide new insights in kidney cancer progression to design novel diagnostic and therapeutic strategies.

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<![CDATA[Sex Differences in the Development of Malignancies among End-Stage Renal Disease Patients: A Nationwide Population-Based Follow-Up Study in Taiwan]]> https://www.researchpad.co/article/5989db37ab0ee8fa60bd36e3

Increasing evidence indicates that end-stage renal disease (ESRD) is associated with the morbidity of cancer. However, whether different dialysis modality and sex effect modify the cancer risks in ESRD patients remains unclear. A total of 3,570 newly diagnosed ESRD patients and 14,280 controls matched for age, sex, index month, and index year were recruited from the National Health Insurance Research Database in Taiwan. The ESRD status was ascertained from the registry of catastrophic illness patients. The incidence of cancer was identified through cross-referencing with the National Cancer Registry System. The Cox proportional hazards model and the Kaplan–Meier method were used for analyses. A similar twofold increase in cancer risk was observed among ESRD patients undergoing hemodialysis (HD) or peritoneal dialysis (PD) after adjusting for other potential risk factors. Patients with the highest cancer risk, approximately fourfold increased risk, were those received renal transplants. Urothelial carcinoma (UC) had the highest incidence in HD and PD patients. However, renal cell carcinoma (RCC) had the highest incidence in the renal transplantation (RT) group. In addition, female patients undergoing RT or PD had a higher incidence of RCC and UC, respectively. Male patients under HD had both higher incidence of RCC and UC. In conclusion, different dialysis modality could modify the cancer risks in ESRD patients. We also found sex effect on genitourinary malignancy when they are under different dialysis modality.

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<![CDATA[VHL Type 2B Mutations Retain VBC Complex Form and Function]]> https://www.researchpad.co/article/5989dac1ab0ee8fa60bb0da7

Background

von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1α and HIF-2α), resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease.

Methodology/Principal Findings

This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1α.

Conclusions

Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition for cancer development in VHL disease.

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<![CDATA[A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors]]> https://www.researchpad.co/article/5989da85ab0ee8fa60b9c212

Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.

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<![CDATA[High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer]]> https://www.researchpad.co/article/5989db24ab0ee8fa60bcffc4

Background

Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas.

Methodology/Principal Findings

We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1–2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3–5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines.

Conclusions/Significance

This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.

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