GPNMB expression identifies TSC1/2/mTOR-associated and MiT family translocation-driven renal neoplasms

Abstract

GPNMB (glycoprotein nonmetastatic B) and other TFE3/TFEB transcriptional targets have been proposed as markers for microphthalmia (MiT) translocation renal cell carcinomas (tRCCs). We recently demonstrated that constitutive mTORC1 activation via TSC1/2 loss leads to increased activity of TFE3/TFEB, suggesting that the pathogenesis and molecular markers for tRCCs and TSC1/2-associated tumors may be overlapping. We examined GPNMB expression in human kidney and angiomyolipoma (AML) cell lines with TSC2 and/or TFE3/TFEB loss produced using CRISPR-Cas9 genome editing as well as in a mouse model of Tsc2 inactivation-driven renal tumorigenesis. Using an automated immunohistochemistry (IHC) assay for GPNMB, digital image analysis was employed to quantitatively score expression in clear cell RCC (ccRCC, n = 87), papillary RCC (papRCC, n = 53), chromophobe RCC (chRCC, n = 34), oncocytoma (n = 4), TFE3- or TFEB-driven tRCC (n = 56), eosinophilic solid and cystic RCC (ESC, n = 6), eosinophilic vacuolated tumor (EVT, n = 4), and low-grade oncocytic tumor (LOT, n = 3), as well as AML (n = 29) and perivascular epithelioid cell tumors (PEComas, n = 8). In cell lines, GPNMB was upregulated following TSC2 loss in a MiT/TFE- and mTORC1-dependent fashion. Renal tumors in Tsc2^+/- A/J mice showed upregulation of GPNMB compared with normal kidney. Mean GPNMB expression was significantly higher in tRCC than in ccRCC (p < 0.0001), papRCC (p < 0.0001), and chRCC (p < 0.0001). GPNMB expression in TSC1/2/MTOR alteration-associated renal tumors (including ESC, LOT, AML, and PEComa) was comparable to that in tRCC. The immunophenotype of tRCC and TSC1/2/MTOR alteration-associated renal tumors is highly overlapping, likely due to the increased activity of TFE3/TFEB in both, revealing an important caveat regarding the use of TFE3/TFEB-transcriptional targets as diagnostic markers. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: GPNMB; TFE3; TFEB; TSC1/2; angiomyolipoma; renal cell carcinoma; translocation renal cell carcinoma.

Figure 1

GPNMB is upregulated following TSC2 loss in a TFE3/TFEB‐ and mTORC1‐dependent fashion. (A) Immunoblotting of whole‐cell lysates from parental and TSC2 KO HEK293T cells for TSC2 and mTORC1 activation markers. (B) Immunoblotting of whole‐cell lysates from parental and TSC2 KO HEK293T cells for GPNMB. A non‐specific band (*) based on the siRNA experiment in supplementary material, Figure S2 is present in this cell line by immunoblotting. (C) Immunoblotting of cell lysates enriched for lysosomal content from parental and TSC2 KO HEK293T cells. (D) Immunoblotting of whole‐cell lysates from TSC2 KO HEK293T cells with or without 72‐h treatment with mTOR inhibitor rapamycin (200 nm). (E) RT‐qPCR for relative GPNMB gene expression in TSC2 KO HEK293T cells with or without genomic deletion of TFE3, TFEB, or TFEB and TFE3 (r = 3; ***p < 0.0001 by one‐way ANOVA). (F) Immunoblotting of whole‐cell lysates from TSC2 KO HEK293T cells with or without genomic deletion of TFE3, TFEB, or TFEB and TFE3. (G) Immunoblotting of whole‐cell lysates from TRI‐102 parental cells (TSC2 ^−/−) and TRI‐103 cells (TSC2 ^−/− with stable transfection of wild‐type TSC2) for TSC2 and mTORC1 activation markers. (H) RT‐qPCR for relative GPNMB gene expression in TRI‐102 and TRI‐103 cells. (I) Immunoblotting of cell lysates from TRI102 and TRI103 cells enriched for lysosomal content. (J) Immunoblotting of whole‐cell lysates from TRI‐102 cells with or without 72‐h treatment with mTOR kinase inhibitor Torin 1 (1 μm).

Figure 2

Renal tumors from A/J Tsc2 ^+/− mice show increased GPNMB expression. (A) Hematoxylin and eosin (H&E) and TSC2, p‐S6, and p‐4EBP1 IHC staining of cystadenoma lesions occurring in 14‐month‐old A/J Tsc2 ^+/− mice. Scale bar: 500 μm. (B) Laser capture microdissection (LCM) of renal cystadenomas from 14‐month‐old A/J Tsc2 ^+/− mice guided by p‐S6 immunostaining for mTORC1 activation. Images reduced from 100× magnification. Scale bar: 250 μm. (C) RT‐qPCR for relative GPNMB gene expression in normal kidney parenchyma and LCM‐captured renal cystadenoma tissue. (D) H&E and GPNMB IHC staining of oncocytic vacuolated carcinomas occurring in 14‐month‐old A/J Tsc2 ^+/− mice. Scale bar: 500 μm. Right panels are higher‐magnification views of the outlined area in each image.

Figure 3

Representative GPNMB immunohistochemical (IHC) staining in common renal cell carcinoma (RCC) subtypes. (A) Representative H&E and GPNMB IHC images for normal renal parenchyma, clear cell RCC (ccRCC), and papillary RCC (papRCC). All images were reduced from 200× magnification. (B) Representative H&E and GPNMB IHC images for oncocytoma and chromophobe RCC (chRCC). Note variable staining in two different chRCC cases. All images were reduced from 200× magnification.

Figure 4

Representative GPNMB staining in translocation RCC (tRCC) cases. Representative H&E and GPNMB IHC images for TFE3‐driven tRCC [PRCC–TFE3 and SFPQ–TFE3 cases confirmed by fluorescence in situ hybridization (FISH)] as well as a tRCC negative for cathepsin K immunostaining and a TFEB‐driven (FISH‐confirmed) tRCC case. All images were reduced from 200× magnification.

Figure 5

Representative GPNMB immunohistochemical (IHC) staining in TSC1/2/mTOR‐related tumors. (A) Representative H&E and GPNMB IHC images for eosinophilic solid and cystic carcinoma (ESC), eosinophilic vacuolated tumor (EVT), and low‐grade oncocytic tumor (LOT). All images were reduced from 200× magnification. (B) Representative H&E and GPNMB IHC images for conventional angiomyolipoma (AML) and perivascular epithelioid cell tumor (PEComa). All images were reduced from 200× magnification.

Figure 6

Digitally quantified GPNMB expression by renal tumor diagnosis. The point represents the median H‐score, and bars represent the interquartile range for each diagnostic group.