EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma

Abstract

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase-dependent and kinase-independent functions, both potentially involved in CCRCC progression. These results might have important implications on therapeutic approaches to CCRCC, since the disruption of the interaction between EGFR/SGLT1, mediated by anti-EGFR antibodies and/or SGLT1 inhibitors, might constitute a novel therapeutic target for CCRCC treatment, and new clinical trials should be evaluated on the basis of this therapeutic proposal.

Keywords: Clear cell renal cell carcinoma; EGFR; EGFR-variantIII; FISH analysis; SGLT1; kinase-dependent EGFR function; kinase-independent EGFR function; p-STAT3; p-p44/42 MAPK; pAKT.

Figure 1

Morphologic and immunohistochemical features of Clear Cell renal Cell Carcinoma. A. Haematoxylin & Eosin stain shows typical CCRCC morphologic features (original magnification 100×); B. Immunohistochemistry for EGFR displaying diffuse and intense membranous and membranous-cytoplasmic immunoreactivity (original magnification 100×); C. Immunohistochemistry for SGLT1 on non-neoplastic renal parenchyma showing intense immunostaining on the luminal brush border of proximal renal tubules (original magnification 400×); D. Immunohistochemistry for SGLT1 on CCRCC showing diffuse and intense, predominantly membranous immunoreactivity (original magnification 200×); E. Immunohistochemistry for p-AKT showing diffuse and intense nuclear immunoreactivity (original magnification 100×); F. Immunohistochemistry for p-p44/42 MAPK displaying focal and intense nuclear-cytoplasmic immunoreactivity (original magnification 100×); G. Immunostaining for p-STAT3 displaying moderate nuclear immunoreactivity (original magnification 100×); H. FISH analysis with centromeric probe for chromosome 7 (green hybridization signals) and locus-specific EGFR gene (orange hybridization signals) showing 4 and 4 hybridization signals, respectively, which excludes gene amplification (ratio: 1), confirming chromosome 7 polysomy (original magnification 1000×).

Figure 2

Real-time polymerase chain reaction analysis for EGFR. Box and whisker plots were used to summarize the distribution of mRNA levels in CCRCC and normal tissue controls. Statistical analysis by Mann Whitney test showed significant differences in mRNA levels between neoplastic and non-neoplastic tissues with *P-values of 0.0003.