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Downregulation of RING ligase praja2 boosts receptor tyrosine kinases in kidney cancer

ABSTRACT: Clear cell renal cell carcinoma (ccRCC) is the most common variant of kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and the mechanisms underlying ccRCC growth and progression will provide clues to treat this aggressive malignant tumor. Here, we report that the RING ligase praja2 is a novel component of the endocytic system that supports clathrin-mediated receptor endocytosis. At the molecular level, we identify the adaptor protein AP2 as a binding partner and substrate of praja2. Functionally, we demonstrate that praja2 is required for AP2-mediated receptor endocytosis and clearance. Downregulation of praja2 in RCC cells and tissues is associated with a marked upregulation of membrane receptors, as EGFR, VEGFR and TfR. A negative feedback loop links EGF signaling to proteolysis of praja2 and sustains downstream mitogenic and proliferative pathways. Restoring praja2 expression in RCC cells remarkably decreases EGFR levels, rewires cancer cell metabolism and inhibits RCC growth and metastatic diffusion. In praja2 knockout mice, upregulation of RTKs levels associates with profound histopathological renal alterations. Our findings identify praja2 as a component of the endocytic pathway that supports receptor endocytosis and clearance. Downregulation of praja2 in RCC cells, thus, sustains RTK signaling and promotes kidney cancer growth and diffusion.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: CHIARA D'AMBROSIO

LAB HEAD: Andrea Scaloni

PROVIDER: PXD034966 | Pride | 2024-05-21

REPOSITORIES: Pride

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Downregulation of praja2 restrains endocytosis and boosts tyrosine kinase receptors in kidney cancer.

Rinaldi Laura L Chiuso Francesco F Senatore Emanuela E Borzacchiello Domenica D Lignitto Luca L Iannucci Rosa R Donne Rossella Delle RD Fuggi Mariano M Reale Carla C Russo Filomena F Russo Nicola Antonino NA Giurato Giorgio G Rizzo Francesca F Sellitto Assunta A Santangelo Michele M De Biase Davide D Paciello Orlando O D'Ambrosio Chiara C Amente Stefano S Garbi Corrado C Dalla Emiliano E Scaloni Andrea A Weisz Alessandro A Ambrosino Concetta C Insabato Luigi L Feliciello Antonio A

Communications biology 20240220 1

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates ...[more]

PMID: 38379085

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Project description:Background: Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC growth and progression will provide clues to treat this aggressive malignant tumor. Method: Expression studies of RING ligase praja2 and tyrosine kinase receptors (RTKs) in renal cell carcinoma was evaluated by immunoblot and immunohistochemistry. Gene transcription profiling in RCC cells was evaluated by RNA sequencing and Ingenuity Pathway Analysis. Imunofluorescence assays were used to evaluate the intracellular distribution and clearance of membrane receptors in cells devoid of praja2. Ubiquitin modifications and protein-protein interaction networks were monitored by IP, western blot, mass spectrometry and proteomic analysis. Experiments in vitro and in vivo were performed to define the role of praja2 in RTKs turnover, metabolism, renal cell carcinoma growth and metastatic diffusion. Results: We report here that the ubiquitin ligase praja2 controls endocytosis and the signal strength of different types of receptors (EGFR, VEGFR, TfR). Praja2 forms a complex with- and ubiquitinates the adaptor protein AP2m required for the activity of ATPase and acidification of endosomes and lysosomes necessary for receptor endocytosis and clearance. Downregulation of praja2 blocks the endocytosis of several membrane receptors, including EGFR, VEGFR and TfR and amplifies mitogenic and proliferative signaling as shown in the clear cell renal cell carcinoma (ccRCC). Restoring praja2 expression in RCC cells reduces EGFR levels, rewires cancer cell metabolism and inhibits growth and metastatic diffusion. In vivo, praja2 knockout mice show upregulation of tyrosine kinase receptors (RTKs) and pronounced histopathological renal alterations. Conclusion: Our findings identify praja2 as an important regulator of tyrosine kinase receptor(s) turnover and can be considered a bona fide oncosuppressor because it finely regulates signaling of several types of receptors and its loss supports kidney cancer growth and diffusion.

Project description:Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Currently, there are no biomarkers for the diagnostic, prognostic, or predictive applications in RCC. MicroRNAs (miRNAs) are short non protein-coding RNAs that negatively regulate gene expression and have been shown to be involved in cancer. We analyzed a total of 70 matched pairs of clear cell RCC (ccRCC) and normal kidney tissues from the same patients by microarray analysis and validated our results by quantitative real time PCR. We identified 166 miRNAs significantly dysregulated in ccRCC. MiR-122, miR-155 and miR-210 had the highest fold changes of overexpression while miR-200c, miR-335, and miR-218 were the most downregulated. We performed extensive bioinformatics analysis including a combinatorial analysis of previously reported miRNAs dysregulated in RCC and extensive target prediction analysis. Many miRNAs were predicted to target a number of genes involved in RCC pathogenesis. Our results showed that miRNA dysregulation in RCC can be attributed in part, to chromosomal aberrations, the co-regulation of miRNA clusters, and co-expression with host genes. We also correlated miRNA expression with clinical characteristics and found miR-155 expression was correlated with ccRCC tumor size. In conclusion, our analysis showed that a number of miRNAs are dysregulated in ccRCC and may contribute to kidney cancer pathogenesis by targeting more than one key molecule. We identified mechanisms that may contribute to miRNA dysregulation in ccRCC. Dysregulated miRNAs represent potential biomarkers for kidney cancer. We preformed a miRNA microarray on 20 pairs of matched primary clear cell renal cell carcinoma (ccRCC) and normal kidney tissue from the same patient (St. Michael's Hospital, Toronto, Canada). One matched pair was used per array for a total of 20 arrays. Microarray results were validated by quantitative real time PCR using an independent set of 50 matched pairs of ccRCC and normal kidney tissue from the same patient.

			Action	DRS
	BAT_FLAG_PRAJA2.msf	Msf
	BAT_FLAG_PRAJA2_1.raw	Raw
	BAT_FLAG_PRAJA2_10.raw	Raw
	BAT_FLAG_PRAJA2_11.raw	Raw
	BAT_FLAG_PRAJA2_12.raw	Raw