Protein expression profiling in the spectrum of renal cell carcinomas

Abstract

In this study, we aimed to evaluate the protein expression profile of a spectrum of renal cell carcinomas (RCC) to find potential biomarkers for disease onset and progression and therefore, prospective therapeutic targets. A 2D-gel based proteomic analysis was used to outline differences in protein levels among different subtypes of renal cell carcinomas, including clear cell carcinomas, papillary lesions, chromophobe tumors and renal oncocytomas. Spot pattern was compared to the corresponding normal kidney from the same patients and distinctive, differentially expressed proteins were characterized by mass spectrometry. Twenty-one protein spots were found differentially expressed between clear cell RCC and normal tissue and 38 spots were found expressed in chromophobe tumors. Eleven proteins were identified, with most differentially expressed -by fold change- between clear cell tumors and the corresponding normal tissue. Two of the identified proteins, Triosephosphate isomerase 1 (TPI-1) and Heat Shock protein 27 (Hsp27), were further validated in a separate set of tumors by immunohistochemistry and expression levels were correlated with clinicopathologic features of the patients. Hsp27 was highly expressed in 82% of the tumors used for validation, and all cases showed strong immunoreactivity for TPI-1. In both Hsp27 and TPI-1, protein expression positively correlated with histologic features of the disease. Our results suggest that the subjacent cytogenetic abnormalities seen in different histological types of RCC are followed by specific changes in protein expression. From these changes, Hsp27 and TPI-1 emerged as potential candidates for the differentiation and prognosis in RCC.

Keywords: Hsp27; Renal cell carcinoma; TPI-1; biomarker; protein profiling; proteomics.

Figure 1

Representative 2-D PAGE images. Full gels are shown for normal kidney (A), a clear cell carcinoma (B) and a chromophobe tumor (C). Composite images (master gels) resulting from combining all samples are shown in panels D-F also for normal, clear cell and chromophobe tumors. Differentially expressed protein spots are shown in panels G-I as indicated by arrowheads.

Figure 2

Immunohistochemical detection of Hsp27 in renal cell carcinomas. (A) Clear cell carcinoma showing strong cytoplasmic expression of Hsp27. B. A higher magnification to show the intense cytoplasmic and membrane staining. (C-D) A poorly differentiated clear cell carcinoma with sarcomatoid features also strongly expressing Hsp27. Expression in normal kidney was heterogeneous and restricted to some tubules (E). Glomeruli were consistently negative (F). A positive control RCC cell line pellet (786.O) formalin fixed and paraffin embedded demonstrating high Hsp27 expression (G). Isotype-matched negative control (H). Magnification: 5X for panels A and C; 16X for B, D, E-H.

Figure 3

Validation of TPI-1 expression in FFPE RCC tissues. TPI-1 positivity in clear cell carcinomas (A-D). The staining pattern is predominantly mixed cytoplasmic and nuclear (arrowheads). (E) Positve control (786.O cell line pellet) and isotype matched negative control (F). Magnification: 5X for A and C; 16X for B, D, E-F.

Figure 4

Venn diagram to intersect the lists of upregulated proteins generated in the present study and two others from published proteomic articles on RCC. Four proteins: Hsp27, TPI-1, Alpha-Enolase and SOD, two of them validated in the present study, were common to these three datasets.