H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation.

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.

Photoinducible Oncometabolite Detection.

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.

Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation.

Cardiac hypertrophy, an adaptive process that responds to increased wall stress, is characterized by the enlargement of cardiomyocytes and structural remodeling. It is stimulated by various growth signals, of which the mTORC1 pathway is a well-recognized source. Here, we show that loss of Flcn, a novel AMPK-mTOR interacting molecule, causes severe cardiac hypertrophy with deregulated energy homeostasis leading to dilated cardiomyopathy in mice. We found that mTORC1 activity was upregulated in Flcn-deficient hearts, and that rapamycin treatment significantly reduced heart mass and ameliorated cardiac dysfunction. Phospho-AMP-activated protein kinase (AMPK)-alpha (T172) was reduced in Flcn-deficient hearts and nonresponsive to various stimulations including metformin and AICAR (5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide). ATP levels were elevated and mitochondrial function was increased in Flcn-deficient hearts, suggesting that excess energy resulting from up-regulated mitochondrial metabolism under Flcn deficiency might attenuate AMPK activation. Expression of Ppargc1a, a central molecule for mitochondrial metabolism, was increased in Flcn-deficient hearts and indeed, inactivation of Ppargc1a in Flcn-deficient hearts significantly reduced heart mass and prolonged survival. Ppargc1a inactivation restored phospho-AMPK-alpha levels and suppressed mTORC1 activity in Flcn-deficient hearts, suggesting that up-regulated Ppargc1a confers increased mitochondrial metabolism and excess energy, leading to inactivation of AMPK and activation of mTORC1. Rapamycin treatment did not affect the heart size of Flcn/Ppargc1a doubly inactivated hearts, further supporting the idea that Ppargc1a is the critical element leading to deregulation of the AMPK-mTOR-axis and resulting in cardiac hypertrophy under Flcn deficiency. These data support an important role for Flcn in cardiac homeostasis in the murine model.

Using YOLO v7 to Detect Kidney in Magnetic Resonance Imaging.

Introduction: This study explores the use of the latest You Only Look Once (YOLO V7) object detection method to enhance kidney detection in medical imaging by training and testing a modified YOLO V7 on medical image formats. Methods: Study includes 878 patients with various subtypes of renal cell carcinoma (RCC) and 206 patients with normal kidneys. A total of 5657 MRI scans for 1084 patients were retrieved. 326 patients with 1034 tumors recruited from a retrospective maintained database, and bounding boxes were drawn around their tumors. A primary model was trained on 80% of annotated cases, with 20% saved for testing (primary test set). The best primary model was then used to identify tumors in the remaining 861 patients and bounding box coordinates were generated on their scans using the model. Ten benchmark training sets were created with generated coordinates on not-segmented patients. The final model used to predict the kidney in the primary test set. We reported the positive predictive value (PPV), sensitivity, and mean average precision (mAP). Results: The primary training set showed an average PPV of 0.94 ± 0.01, sensitivity of 0.87 ± 0.04, and mAP of 0.91 ± 0.02. The best primary model yielded a PPV of 0.97, sensitivity of 0.92, and mAP of 0.95. The final model demonstrated an average PPV of 0.95 ± 0.03, sensitivity of 0.98 ± 0.004, and mAP of 0.95 ± 0.01. Conclusion: Using a semi-supervised approach with a medical image library, we developed a high-performing model for kidney detection. Further external validation is required to assess the model's generalizability.

PRDM10 RCC: A Birt-Hogg-Dubé-like Syndrome Associated With Lipoma and Highly Penetrant, Aggressive Renal Tumors Morphologically Resembling Type 2 Papillary Renal Cell Carcinoma.

OBJECTIVE: To characterize the clinical manifestations and genetic basis of a familial cancer syndrome in patients with lipomas and Birt-Hogg-Dubé-like clinical manifestations including fibrofolliculomas and trichodiscomas and kidney cancer. METHODS: Genomic analysis of blood and renal tumor DNA was performed. Inheritance pattern, phenotypic manifestations, and clinical and surgical management were documented. Cutaneous, subcutaneous, and renal tumor pathologic features were characterized. RESULTS: Affected individuals were found to be at risk for a highly penetrant and lethal form of bilateral, multifocal papillary renal cell carcinoma. Whole genome sequencing identified a germline pathogenic variant in PRDM10 (c.2029 T>C, p.Cys677Arg), which cosegregated with disease. PRDM10 loss of heterozygosity was identified in kidney tumors. PRDM10 was predicted to abrogate expression of FLCN, a transcriptional target of PRDM10, which was confirmed by tumor expression of GPNMB, a TFE3/TFEB target and downstream biomarker of FLCN loss. In addition, a sporadic papillary RCC from the TCGA cohort was identified with a somatic PRDM10 mutation. CONCLUSION: We identified a germline PRDM10 pathogenic variant in association with a highly penetrant, aggressive form of familial papillary RCC, lipomas, and fibrofolliculomas/trichodiscomas. PRDM10 loss of heterozygosity and elevated GPNMB expression in renal tumors indicate that PRDM10 alteration leads to reduced FLCN expression, driving TFE3-induced tumor formation. These findings suggest that individuals with Birt-Hogg-Dubé-like manifestations and subcutaneous lipomas, but without a germline pathogenic FLCN variant, should be screened for germline PRDM10 variants. Importantly, kidney tumors identified in patients with a pathogenic PRDM10 variant should be managed with surgical resection instead of active surveillance.

Blood and lymphatic systems are segregated by the FLCN tumor suppressor.

Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.

Assessment of multiphasic contrast-enhanced MR textures in differentiating small renal mass subtypes.

PURPOSE: This study seeks to evaluate the use of quantitative texture parameters extracted from multiphasic contrast-enhanced magnetic resonance (MR) imaging in differentiating between benign and malignant masses (oncocytoma vs. clear cell and papillary RCC) and between common subtypes of renal cell carcinoma (clear cell vs. papillary RCC) in small renal masses (< 4 cm). METHOD: One-hundred and forty-two renal lesions (90 clear cell and 22 papillary RCCs; 30 oncocytomas) were identified in a cohort of 41 patients (18 men, 23 women: mean age, 52.8 ± 14.4 years) who underwent preoperative multiphasic contrast-enhanced MR with four phases (unenhanced, arterial, venous, and delayed) between 2015 and 2016. In this study, texture features were extracted from entire cross-sectional tumoral region in three consecutive slices containing the largest cross-sectional area from each of the four phases. The change in imaging feature between precontrast imaging and each postcontrast phase was calculated. Data dimension reduction and feature selection were performed by conducting (1) pairwise Wilcoxon rank test followed by modified false discovery rate adjustment, and (2) Lasso regression. Multivariate modeling incorporating the selected features was performed using random forest classification method. RESULTS: Histogram imaging features were informative variables in differentiating between benign and malignant masses, while textures imaging features were of added value in differentiating between subtypes of RCCs. Papillary RCCs were distinguished from clear cell RCCs (sensitivity 65.5%, specificity 88%, and accuracy 77.9%), oncocytomas from clear cell RCCs (sensitivity 67.3%, specificity 88.9%, and accuracy 79.3%), and oncocytomas from papillary and clear cell RCCs (sensitivity 64.7%, specificity 85.9%, and accuracy 77.9%). CONCLUSIONS: A combination of histogram and texture imaging features on multiphasic MR can help differentiate histologic cell types in common small renal masses (< 4 cm).

Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Renal functional outcomes after robotic multiplex partial nephrectomy: the National Cancer Institute experience with robotic partial nephrectomy for 3 or more tumors in a single kidney.

OBJECTIVE: To identify renal function outcomes after robotic multiplex partial nephrectomy (RMxPNx), we reviewed our institutional database at the National Institutes of Health, National Cancer Institute. To our knowledge, we present the largest series of RMxPNx renal function outcomes to date. Robotic partial nephrectomy has been employed for oncologic control and to prevent dialysis dependence in hereditary multifocal renal cell carcinoma conditions. We have termed robotic surgery on a single kidney with three or more lesions a RMxPNx. MATERIALS AND METHODS: We evaluated patients from a prospectively maintained database at a single institution (NIH/NCI) that underwent RMxPNx from 2007 to 2013. Demographic and operative data were compiled with statistical analysis with T test performed to determine renal function outcomes. RESULTS: A total of 54 patients underwent RMxPNx. Mean number of tumors removed was 8.63 (range 3-52). Mean preoperative creatinine and eGFR were 1.02 ± 0.26 mg/dL and 85.4 ± 21.5 mL/min, respectively. Postoperatively, creatinine increased from baseline by 0.45 mg/dL (p < 0.001). Similarly, a mean decrease in eGFR by 24.6 mL/min was observed (p < 0.001). At 3-month follow-up, the creatinine increase from baseline was 0.05 mg/dL (p = 0.10) and mean decrease in eGFR was 3.01 mL/min (p = 0.21). When stratifying based on preoperative CKD stages I-III, similar results were observed. CONCLUSION: Robotic multiplex partial nephrectomy is a safe and feasible approach to patients with multifocal renal masses. These complex surgeries have a demonstrated learning curve, but this minimally invasive approach for nephron-sparing surgery allows patients to preserve renal function where they would otherwise require open surgery or a radical nephrectomy.

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.

Diagnosis and management of BHD-associated kidney cancer.

In addition to the associated cutaneous and pulmonary manifestations, individuals with the Birt-Hogg-Dubé (BHD) syndrome have an increased risk of developing kidney cancer, which is often bilateral and multifocal. The risk of developing a renal tumor in this population does not decrease with age and therefore warrants a lifelong screening approach. We recommend abdominal imaging every 36 months in individuals without renal lesions at initial screening. Once renal tumors are identified, they should be followed with interval imaging studies until the largest tumor reaches 3 cm in maximal diameter, at which point nephron-sparing surgery should be ideally pursued. While the histology of renal tumors can vary in the BHD syndrome, most tumors possess a relatively indolent natural history and do not require adjuvant therapy if resected when localized to the kidney. With this approach, the vast majority of patients will achieve a curative oncologic outcome and avoid the medical sequelae of chronic renal insufficiency that could otherwise result from total nephrectomy.

Lipid cell tumors in two women with von Hippel-Lindau syndrome.

BACKGROUND: Lipid-cell tumors are rare, functioning ovarian neoplasms. They have not been reported in women with von Hippel-Lindau syndrome, an autosomal-dominant tumor-suppressor gene mutation that is associated with renal cell carcinoma, and other vascular tumors. CASES: Two women with von Hippel-Lindau syndrome and kidney tumors were evaluated for secondary amenorrhea, hirsutism, and complex adnexal masses seen on computed tomography. The first patient had known renal cancer and bilateral adnexal masses, one with central necrosis. Because metastatic renal cell cancer could not be excluded on frozen section, bilateral salpingo-oophorectomy was performed. The second patient underwent right salpingo-oophorectomy after human chorionic gonadotropin testing confirmed that the ovarian tumor produced testosterone. Final pathology in both cases revealed testosterone-secreting lipid cell tumors. CONCLUSION: Lipid cell ovarian tumors should be considered in women with von Hippel-Lindau presenting with adnexal mass, amenorrhea, and hirsuitism.

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen-related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 h.