Activation of HIF-1α/ACLY signaling axis promotes progression of clear cell renal cell carcinoma with VHL inactivation mutation / 中华病理学杂志

Objective: To explore the potential pathogenesis of clear cell renal cell carcinoma (ccRCC) based on the HIF-1α/ACLY signaling pathway, as well as to provide new ideas for the treatment of ccRCC. Methods: Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China were collected. The VHL mutation was examined using exon sequencing. The expression of HIF-1α/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines (786-O, A498, UM-RC-2, SNU-333, and Caki-2) using Western blot. The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1α in ccRCC cell lines. HeLa cells were treated with CoCl2 and hypoxia (1%O2) to activate HIF-1α and then subject to the detection of the ACLY mRNA and protein levels. The potential molecular mechanism of HIF-1α-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay (ChIP) and luciferase reporter gene assay. The effect of HIF-1α/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques. The expression of ACLY was compared between patients with ccRCC and those with benign lesions, and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis. Results: Exon sequencing revealed that 55 (70.5%) of the 78 ccRCC patients harbored a VHL inactivation mutation, and HIF-1α expression was associated with ACLY protein levels. The protein levels of ACLY and HIF-1α in ccRCC cell lines carrying VHL mutation were also correlated to various degrees. Overexpression of HIF-1α in A498 cells increased the mRNA and protein levels of ACLY, and knockdown of HIF-1α in Caki-2 cells inhibited the mRNA and protein levels of ACLY (P<0.001 for all). CoCl2 and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1α (P<0.001 for all). The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1α could directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level (P<0.001 for all). The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1α and ACLY. The depletion of HIF-1α could effectively reduce the accumulation of lipid in cells, while the overexpression of ACLY could reverse this process. At the same time, cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1α knockdown was significantly decreased, and overexpression of ACLY could restore proliferation of these tumor cells (P<0.001). Survival analysis further showed that compared with the ccRCC patients with low ACLY expression, the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival (P<0.001). Conclusions: VHL mutation-mediated HIF-1α overexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY. Targeting the HIF-1α/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.

Ginsenoside 20(S)-Rg3 upregulates tumor suppressor VHL gene expression by suppressing DNMT3A-mediated promoter methylation in ovarian cancer cells / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells.@*METHODS@#Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice.@*RESULTS@#Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels (@*CONCLUSIONS@#Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.

Germline gene testing of the RET, VHL, SDHD and SDHB genes in patients with pheochromocytoma/paraganglioma / 北京大学学报(医学版)

OBJECTIVE@#To analyze the germline variations of genes RET, VHL, SDHD and SDHB in patients with pheochromocytoma and/or paraganglioma and to evaluate variations of these genes in Chinese patients.@*METHODS@#Patients who were treated in Peking University First Hospital from September 2012 to March 2014 and diagnosed with pheochromocytoma and/or paraganglioma by pathologists were included in this study. Twelve patients were included in total, of whom 11 had pheochromocytoma, and 1 had paraganglioma. Deoxyribonucleic acid (DNA) was extracted from the leukocytes of peripheral blood of the patients. The exons 10, 11, 13-16 of the RET gene, and all exons of VHL, SDHB and SDHD genes and their nearby introns (±20 bp) were amplified with polymerase chain reactions, and the products were sent to a biotechnology company for sequencing. The sequencing results were compared with wildtype sequences of these genes to identify variations. One of the patients was diagnosed with multiple endocrine neoplasia type 2A. A family analysis was performed in his kindred, and his family members received genetic tests for the related variations.@*RESULTS@#Three patients were found to have germline gene variations. A c.136C>T (p.R46X) variation of the SDHB gene was found in a patient with malignant pheochromocytoma. A c.1901G>A (C634Y) variation, as well as c.2071G>A (p.G691S) and c.2712C>G (p.S904S) variations of the RET gene were found in a patient with multiple endocrine neoplasia type 2A. After a family analysis, five family members of this patient were found to have the same variations. c.2071G>A (p.G691S) and c.2712C>G (p.S904S) variations of the RET gene were also found in a clinical sporadic patient without evidence of malignancy. A patient with congenital single ventricle malformation and pheochromocytoma was included in this study, and no variation with clinical significance was found in the four genes of this patient.@*CONCLUSION@#25% (3/12) patients with pheochromocytoma or paraganglioma were found to have missense or nonsense germline gene variations in this study, including the c.136C>T (p.R46X) variation of the SDHB gene, the c.1901G>A (C634Y) variation of the RET gene, and c.2071G>A (p.G691S) and c.2712C>G (p.S904S) variations of the RET gene. The former two variations have already been confirmed to be pathogenic. The existence of these variations in Chinese patients with pheochromocytoma and/or paraganglioma was validated in this study, which supports the conclusion that genetic testing is necessary to be generally performed in patients with pheochromocytoma and/or paraganglioma.

Paraganglioma of seminal vesicle and chromophobe renal cell carcinoma: a case report and literature review / Paraganglioma da vesícula seminal e carcinoma de células renais cromófobo: um relato de caso e revisão da literatura

CONTEXT: Extra-adrenal paragangliomas are rare tumors that have been reported in many locations, including the kidney, urethra, urinary bladder, prostate, spermatic cord, gallbladder, uterus and vagina. CASE REPORT: This report describes, for the first time to the best of our knowledge, a primary paraganglioma of the seminal vesicle occurring in a 61-year-old male. The patient presented persistent arterial hypertension and a previous diagnosis of chromophobe renal cell carcinoma. It was hypothesized that the seminal vesicle tumor could be a metastasis from the chromophobe renal cell carcinoma. Immunohistochemical characterization revealed expression of synaptophysin and chromogranin in tumor cell nests and peripheral S100 protein expression in sustentacular cells. Succinate dehydrogenase A and B-related (SDHA and SDHB) expression was present in both tumors. CONCLUSIONS: No genetic alterations to the VHL and SDHB genes were detected in either the tumor tissue or tissues adjacent to the tumor, which led us to rule out a hereditary syndrome that could explain the association between paraganglioma and chromophobe renal cell carcinoma in a patient with arterial hypertension.

CONTEXTO: Paragangliomas extra-adrenais são tumores raros que têm sido relatados em muitas localizações, incluindo rim, uretra, bexiga, próstata, cordão espermático, vesícula biliar, útero e vagina. RELATO DE CASO: Este relato descreve, pela primeira vez em nosso conhecimento, um paraganglioma primário da vesícula seminal ocorrendo em um paciente do sexo masculino de 61 anos de idade. O paciente apresentou hipertensão arterial persistente e um diagnóstico prévio de carcinoma de células renais cromófobo (CCRC). Foi pensado que o tumor de vesícula seminal poderia ser uma metástase do CCRC. A caracterização imunoistoquímica revelou expressão de sinaptofisina e cromogranina nos ninhos de células tumorais e expressão de proteína S100 nas células sustentaculares. Expressão de succinato de-hidrogenase A e B relacionada (SDHA e SDHB) estiveram presentes em ambos os tumores CONCLUSÕES: Nenhuma alteração genética dos genes VHL e SDHB foi detectada nos tecidos tumorais e adjacentes ao tumor, o que nos levou a afastar uma síndrome hereditária que poderia explicar a associação entre o paraganglioma e o CCRC em um paciente com hipertensão arterial.

STAT3 inhibits the degradation of HIF-1alpha by pVHL-mediated ubiquitination

Hypoxia-inducible factor 1alpha (HIF-1alpha) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1alpha is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1alpha protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1alpha stability. We found that STAT3 interacts with C-terminal domain of HIF-1alpha and stabilizes HIF-1alpha by inhibition of pVHL binding to HIF-1alpha. The binding between HIF-1alpha and pVHL, negative regulator of HIF-1alpha stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1alpha protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1alpha protein via inhibition of interaction between pVHL and HIF-1alpha. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1alpha through competition with pVHL for binding to HIF-1alpha, and then stabilizes HIF-1alpha protein levels.

Molecular analysis of the Von Hippel-Lindau (VHL) gene in a family with non-syndromic pheochromocytoma: the importance of genetic testing

The two index patients of the family analyzed in this study had undergone bilateral adrenalectomy for pheochromocytomas. This prompted genetic analyses of the probands and seven first-degree relatives. The two pheochromocytoma patients and two additional asymptomatic family members were found to harbor a mutation c496G>T in exon 3 of the VHL gene. The family was then lost to systematic follow-up. Three years after performing the initial genetic evaluation, the sister of the probands, who was known to carry the same VHL germline mutation, was referred to our service after a pregnancy that was complicated by preeclampsia. She reported paroxysms suggestive for pheochromocytoma, but her urinary metanephrines were negative. However, computerized tomography of the abdomen showed an adrenal mass that was also positive on metaiodobenzylguanidine (MIBG) scintigraphy. This study illustrates that molecular analysis of the index patient(s) can lead to the identification of presymptomatic relatives carrying the mutation. Moreover, despite negative urinary metanephrines, the identification of a specific mutation has led to an increased suspicion and detection of a pheochromocytoma in the sister of the probands.

Dois pacientes índices da família analisada neste estudo foram submetidos a adrenalectomia bilateral devido a feocromocitoma. Foi, então, realizado o estudo genético dos pacientes e de sete parentes de primeiro grau. Os dois pacientes com feocromocitoma e dois outros membros assintomáticos da família apresentaram a mutação c496G>T no exon 3 do gene VHL. A família perdeu seguimento médico. Três anos após a realização da avaliação genética, a irmã dos pacientes, portadora da mutação, foi encaminhada para o nosso serviço após uma gestação complicada por pré-eclampsia. Ela referia paroxismos sugestivos de feocromocitoma, mas as metanefrinas urinárias eram negativas. Entretanto, a tomografia computadorizada de abdômen evidenciou uma massa adrenal que também se contrastou na cintilografia com metaiodobenzilguanidina (MIBG). Esse estudo mostra que a análise molecular do paciente índice pode levar à identificação de parentes assintomáticos portadores da mutação. Além disso, mesmo com as metanefrinas urinárias negativas, a identificação de uma mutação específica levou a um aumento da suspeita e detecção de feocromocitoma na irmã dos afetados pela doença.