Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous-cell carcinoma

Abstract

Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

Keywords: Biomedicine; In vivo labeling; Mass spectrometry; Post-translational modifications.

Figure 1

Workflow employed for characterizing tyrosine phosphorylation pattern in ESCC cell lines. A super‐SILAC mix generated by pooling lysates from ¹³C₆ lysine and ¹³C₆ arginine labeled TE2 and TE8 cell lines was spiked at 1:5 ratio into all the ESCC cell lines as well as non‐neoplastic cell line Het‐1A. Protein lysates were digested using trypsin and tyrosine phosphopeptides were enriched using anti‐phosphotyrosine antibody. Phosphopeptides were quantitated using spiked‐in internal standard.

Figure 2

EPHA2 is hyperphosphorylated in ESCC cell lines. (A) A heat map representing relative phosphorylation levels of candidate molecules across four ESCC cell lines compared to Het‐1A. (B) MS spectra showing relative difference in the abundance of EphA2 phosphopeptide (VLEDDPEATyTTSGGKIPIR) across all the cell lines used in the study. Mass‐to‐charge ratio 748.03 represents unlabeled version of phosphopeptide from cell lysates while 752.04 represents SILAC‐labeled version from super‐SILAC mix.

Figure 3

Differential phosphorylation of EPHA2 pathway mediators. (A) Schematic showing domain architecture of EPHA2. (B) EphA2 pathway and various cellular processes that it regulates. Red color marks proteins identified in this study. Phosphorylation sites identified in the study are shown in yellow.

Figure 4

EphA2 knockdown affects cell proliferation and invasion capability. (A) Immunoblotting showing p‐EphA2 and EphA2 protein levels in scrambled siRNA‐ and EphA2‐siRNA‐transfected TE8 cells. (B) Crystal violet staining of TE8 cells transfected with scrambled siRNA and EphA2‐siRNA. (C) Bar graph quantifying absorbance of dissolved dye in the wells shown in (B). (D) A representative microscopic field showing TE8 cells transfected with scrambled siRNA and EphA2‐siRNA that invaded matrigel invasion chambers. (E) Bar graph representing number of cells per field that penetrated through the matrigel.