Nephron-Sparing Surgery for Adult Xp11.2 Translocation Renal Cell Carcinoma at Clinical T1 Stage: A Multicenter Study in China.

PURPOSE: To evaluate the oncologic efficacy and feasibility of nephron-sparing surgery (NSS) in adult Xp11.2 translocation renal cell carcinoma (RCC). PATIENTS AND METHODS: Seventy patients with Xp11.2 translocation RCC and 273 with conventional RCC from five institutions in Nanjing were retrospectively studied. All patients were older than 18 years and were categorized into clinical T1 (cT1) stage using preoperative imaging. Using the preoperative imaging and electronic medical records, anatomical and pathological features were collected and analyzed. RESULTS: Among patients with Xp11.2 translocation RCC, 18/36 (50.0%) with cT1a and 12/34 (35.3%) with cT1b tumors underwent NSS. The respective proportions in the conventional RCC group were 121/145 (83.4%) and 93/128 (72.7%). Among cT1a tumors, the Xp11.2 translocation RCCs tended to be adjacent to the collecting system, sinus, and axial renal midline compared with conventional RCCs. Patients with Xp11.2 translocation RCCs who underwent NSS had comparable progression-free survival (PFS) and overall survival to radical nephrectomy (RN) patients (P > 0.05). Among cT1b tumors, surgical margin positivity and pelvicalyceal, vascular, and region lymphatic involvement were more likely to occur in the Xp11.2 translocation RCCs (P < 0.05). Patients with Xp11.2 translocation RCC who underwent RN had a more favorable PFS than those who underwent NSS (P = 0.048). However, multivariate analysis of PFS did not identify surgical method as a risk factor (P = 0.089). CONCLUSIONS: Among adults with Xp11.2 translocation RCC, NSS can be an alternative for patients with cT1a tumor but should be performed with more deliberation in patients with cT1b tumors.

Estradiol increases risk of topoisomerase IIß-mediated DNA strand breaks to initiate Xp11.2 translocation renal cell carcinoma.

BACKGROUND: Xp11.2 translocation renal cell carcinoma (tRCC) is defined by translocation of the transcription factor E3 (TFE3) gene located on chromosome Xp11.2. Due to the high incidence in women, 17ß-estradiol (E2) may be a factor influencing TFE3 breaks, and the topoisomerase II (TOP2) poison is considered one of the important risk factors in mediating DNA breaks. In this study, we investigated the potential pathogenesis of Xp11.2 tRCC using the renal epithelial cell line HK-2. METHODS: Immunofluorescence assay was performed to analyze DNA breaks by quantifying phosphorylation of H2AX (γH2AX), and the micronuclei (MN) assay was designed for monitoring chromosome breaks. The chromatin immunoprecipitation (CHIP) was used to detect whether proteins bound to specific DNA site, and the co-immunoprecipitation (Co-IP) was used to confirm whether proteins bound to other proteins. In some experiments, siRNA and shRNA were transfected to knockdown target genes. RESULTS: Our results demonstrated that DNA double-strand breaks were mediated by TOP2ß in HK-2 cells, and this process could be amplified through estrogen receptor α (ERα)-dependent pathway induced by E2. After performing translocation site analysis using target region sequencing data in two Xp11.2 tRCC cell lines and ten Xp11.2 tRCC patients, we confirmed that TOP2ß and ERα could both bind to TFE3 translocation sites directly to mediate DNA breaks in a E2-dependent manner. However, TOP2ß and ERα were not observed to have direct interaction, indicating that their collaborative may be implemented in other ways. Besides, TFE3 was found to be upregulated through NRF1 with increasing E2 concentration, which could increase the risk of TFE3 breaks. CONCLUSION: Our results indicate that E2 amplifies TOP2ß-mediated TFE3 breaks by ERα-dependent pathway, and E2 upregulates TFE3 by NRF1 to increase the risk of TFE3 breaks. This suggests that E2 is an important pathogenic factor for Xp11.2 tRCC pathogenesis. Video Abstract.

Estrogen associates with female predominance in Xp11.2 translocation renal cell carcinoma.

Based on the epidemiological characteristics of susceptibility and age selectivity for women in Xp11.2 translocation renal cell carcinoma (Xp11.2 tRCC), we inferred that estrogen was to be blamed. Rad54 like 2 (Rad54l2) which might be one of key effector proteins of DNA damage mediated by estrogen was downregulated in numerous cancers, however, its role in epidemiological characteristics of Xp11.2 tRCC was needed to further study. We reviewed 1005 Xp11.2 tRCC cases and collected estrogen data and then compared the onset time of Xp11.2 tRCC cases in female with estrogen changing trend. An RNA-sequencing was performed in estrogen treated HK-2 cells and subsequently bioinformatic analysis was applied based on the Cancer Genome Atlas (TCGA) and GEO database. The male-to-female ratio of Xp11.2 tRCC was 1:1.4 and the median age of onset was 29.7 years old. The onset trend of female was similar to estrogen physiological rhythm (r = 0.67, p < 0.01). In Xp11.2 tRCC and HK-2 cells after estrogen treatment, Rad54l2 was downregulated, and GSEA showed that pathways significantly enriched in DNA damage repair and cancer related clusters after estrogen treated, as well as GO and KEGG analysis. Downregulation of Rad54l2 was in numerous cancers, including renal cell carcinoma (RCC), in which Rad54l2 expression was significantly decreased in male, age over 60 years old, T2&T3&T4 stages, pathologic SII&SIII&SIV stages as well as histologic G3&G4 grades, and cox regression analysis proved that Rad54l2 expression was a risk factor for overall survival, disease-specific survival and progression-free interval in univariate analysis. There existed female predominance in Xp11.2 tRCC and Rad54l2 might play vital role in estrogen mediating female predominance in Xp11.2 tRCC.

Both SUMOylation and ubiquitination of TFE3 fusion protein regulated by androgen receptor are the potential target in the therapy of Xp11.2 translocation renal cell carcinoma.

BACKGROUND: The aggressiveness of renal cell carcinoma (RCC) associated with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 translocation RCC [Xp11.2 tRCC]) is age-dependent, which is similar to the overall trend of reproductive endocrine hormones. Therefore, this study focused on the effect and potential mechanism of androgen and androgen receptor (AR) on the progression of Xp11.2 tRCC. METHODS: The effects of androgen and AR on the proliferation and migration of Xp11.2 tRCC cells were first evaluated utilising Xp11.2 tRCC cell lines and tissues. Because Transcription factor enhancer 3 (TFE3) fusion proteins play a key role in Xp11.2 tRCC, we focused on the regulatory role of AR and TFE3 expression and transcriptional activity. RESULTS: When Xp11.2 tRCC cells were treated with dihydrotestosterone, increased cell proliferation, invasion and migration were observed. Compared with clear cell RCC, the positive rate of AR in Xp11.2 tRCC tissues was higher, and its expression was negatively associated with the progression-free survival of Xp11.2 tRCC. Further studies revealed that AR could positively regulate the transcriptional activity of TFE3 fusion proteins by small ubiquitin-related modifier (SUMO)-specific protease 1, inducing the deSUMOylation of TFE3 fusion. On the other hand, UCHL1 negatively regulated by AR plays a role in the deubiquitination degradation of the PRCC-TFE3 fusion protein. Therefore, the combination of the AR inhibitor MDV3100 and the UCHL1 inhibitor 6RK73 was effective in delaying the progression of Xp11.2 tRCC, especially PRCC-TFE3 tRCC. CONCLUSIONS: Androgen and AR function as facilitators in Xp11.2 tRCC progression and may be a novel therapeutic target for Xp11.2 tRCC. The combined use of AR antagonist MDV3100 and UCHL1 inhibitor 6RK73 increased both the SUMOylation and ubiquitination of the PRCC-TFE3 fusion protein.

Low expression of TRAF3IP2-AS1 promotes progression of NONO-TFE3 translocation renal cell carcinoma by stimulating N6-methyladenosine of PARP1 mRNA and downregulating PTEN.

BACKGROUND: NONO-TFE3 translocation renal cell carcinoma (NONO-TFE3 tRCC) is one subtype of RCCs associated with Xp11.2 translocation/TFE3 gene fusions RCC (Xp11.2 tRCCs). Long non-coding RNA (lncRNA) has attracted great attention in cancer research. The function and mechanisms of TRAF3IP2 antisense RNA 1 (TRAF3IP2-AS1), a natural antisense lncRNA, in NONO-TFE3 tRCC remain poorly understood. METHODS: FISH and qRT-PCR were undertaken to study the expression, localization and clinical significance of TRAF3IP2-AS1 in Xp11.2 tRCC tissues and cells. The functions of TRAF3IP2-AS1 in tRCC were investigated by proliferation analysis, EdU staining, colony and sphere formation assay, Transwell assay and apoptosis analysis. The regulatory mechanisms among TRAF3IP2-AS1, PARP1, PTEN and miR-200a-3p/153-3p/141-3p were investigated by luciferase assay, RNA immunoprecipitation, Western blot and immunohistochemistry. RESULTS: The expression of TRAF3IP2-AS1 was suppressed by NONO-TFE3 fusion in NONO-TFE3 tRCC tissues and cells. Overexpression of TRAF3IP2-AS1 inhibited the proliferation, migration and invasion of UOK109 cells which were derived from cancer tissue of patient with NONO-TFE3 tRCC. Mechanistic studies revealed that TRAF3IP2-AS1 accelerated the decay of PARP1 mRNA by direct binding and recruitment of N6-methyladenosie methyltransferase complex. Meanwhile, TRAF3IP2-AS1 competitively bound to miR-200a-3p/153-3p/141-3p and prevented those from decreasing the level of PTEN. CONCLUSIONS: TRAF3IP2-AS1 functions as a tumor suppressor in NONO-TFE3 tRCC progression and may serve as a novel target for NONO-TFE3 tRCC therapy. TRAF3IP2-AS1 expression has the potential to serve as a novel diagnostic and prognostic biomarker for NONO-TFE3 tRCC detection.

A new risk-scoring system to predict Xp11.2 translocation renal cell carcinoma in adults.

OBJECTIVE: The objective was to derive and validate a practical scoring system for preoperative diagnosis of Xp11.2 translocation renal cell carcinoma (RCC) in adults. METHODS: Epidemiology, symptomatology, and imaging methods were correlated between patients with common RCC and those with Xp11.2 translocation RCC using a derivation study (N = 6352) and a validation study (N = 127). Univariate analysis of risk factors was performed to derive a scoring system to predict the occurrence of Xp11.2 translocation RCC in adults. The Hosmer-Lemeshow goodness-of-fit test and receiver operating characteristic (ROC) curve were used to validate the scoring system. RESULTS: Based on odd ratios, three low-risk factors (sex, gross haematuria, and intratumoural calcification) and three high-risk factors (age, unenhanced computed tomography density, and enhancement pattern) were given weighted scores of 1 and 2, respectively. Patients who scored 3 to 5 points underwent an additional magnetic resonance imaging examination. The final scoring system had a sensitivity of 81.0% and a specificity of 98.0%. CONCLUSION: We established a practical scoring system for the preoperative diagnosis of Xp11.2 translocation RCC in adults, which can be optimised through further clinical findings in the future.

NONO-TFE3 Fusion Promotes Aerobic Glycolysis and Angiogenesis by Targeting HIF1A in NONO-TFE3 Translocation Renal Cell Carcinoma.

BACKGROUND: NONO-TFE3 translocation renal cell carcinoma (tRCC), one of the RCCs that are associated with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 tRCCs), involves an X chromosome inversion between NONO and TFE3 with the characteristics of endonuclear aggregation of NONO-TFE3 fusion protein. The oncogenic mechanisms of NONO-TFE3 fusion have not yet been fully elucidated. OBJECTIVE: This study aimed at investigating the mechanism of NONO-TFE3 fusion regulating HIF1A as well as the role of HIF-1α in the progression of NONO-TFE3 tRCC under hypoxia. METHODS: Immunohistochemistry and Western Blotting assays were performed to profile HIF-1α expression in renal clear cell carcinoma (ccRCC) or in Xp11.2 tRCC. Chromatin immunoprecipitation (ChIP), a luciferase reporter assay, and real-time quantitative PCR (RT-qPCR) were used to evaluate the regulation of HIF1A expression by NONO-TFE3 fusion. Then, the flow cytometry analysis, tube formation assays, and cell migration assays were used as well as glucose or lactic acid levels were measured to establish the impact of HIF-1α on the progression of NONO-TFE3 tRCC. Besides, the effect of HIF-1α inhibitor (PX-478) on UOK109 cells was analyzed. RESULTS: We found that HIF1A was the target gene of NONO-TFE3 fusion. In UOK109 cells, which were isolated from NONO-TFE3 tRCC samples, NONO-TFE3 fusion promoted aerobic glycolysis and angiogenesis by up-regulating the expression of HIF-1α under hypoxia. Furthermore, the inhibition of HIF-1α mediated by PX-478 suppressed the development of NONO-TFE3 tRCC under hypoxia. CONCLUSION: HIF-1α is a potential target for therapy of NONO-TFE3 tRCC under hypoxia.

The suitability of NONO-TFE3 dual-fusion FISH assay as a diagnostic tool for NONO-TFE3 renal cell carcinoma.

NONO-TFE3 RCC is a subtype of Xp11.2 translocation renal cell carcinoma (RCC). So far, only a small amount of NONO-TFE3 RCC have been reported owing to lack of effective diagnosis methods. Utilizing the novel dual-fusion fluorescence in situ hybridization (FISH) probe reported here, 5 cases of NONO-TFE3 RCC were identified and were ultimately confirmed by RT-PCR. Histopathology, all 5 cases were consisted by sheets of epithelial cells and papillary architecture. The cytoplasm was abundantly clear, and nucleoli was not prominent. Besides, the nuclear palisading, subnuclear vacuoles and psammoma bodies were identified. The most distinctive features were strong positive TFE3 staining but equivocal split signals of the TFE3 probe, which might lead to the misdiagnosis of Xp11.2 translocation RCC. The median age and median tumor size of the five patients were 41.2 years and 3.6 cm, respectively. A median following follow-up of 27 months showed moderate disease progression and prognosis in NONO-TFE3 RCC patients. In conclusion, the present study demonstrates the effectiveness and reliability of the NONO-TFE3 dual-fusion FISH probe for diagnosing NONO-TFE3 RCC. Suspected cases of Xp11.2 translocation RCC showing biphasic pattern, strong positive TFE3 staining, and equivocal split signals in the TFE3 FISH assay indicated a possibility of NONO-TFE3 RCC.