Src heterodimerically activates Lyn or Fyn to serve as targets for the diagnosis and treatment of esophageal squamous cell carcinoma

Although Src is one of the oldest and most investigated oncoproteins, its function in tumor malignancy remains to be defined further. In this study, we demonstrated that the inhibition of Src activity by ponatinib effectively suppressed several malignant phenotypes of esophageal squamous cell carcinoma (ESCC) both in vitro and in vivo, whereas it did not produce growth-inhibitory effects on normal esophageal epithelial cells (NEECs). Importantly, we combined phosphoproteomics and several cellular and molecular biologic strategies to identify that Src interacted with the members of Src-family kinases (SFKs), such as Fyn or Lyn, to form heterodimers. Src interactions with Fyn and Lyn phosphorylated the tyrosine sites in SH2 (Fyn Tyr¹⁸⁵ or Lyn Tyr¹⁸³) and kinase domains (Fyn Tyr⁴²⁰ or Lyn Tyr³⁹⁷), which critically contributed to ESCC development. By contrast, Src could not form heterodimers with Fyn or Lyn in NEECs. We used RNA sequencing to comprehensively demonstrate that the inhibition of Src activity effectively blocked several critical tumor-promoting pathways, such as JAK/STAT, mTOR, stemness-related, and metabolism-related pathways. Results of the real-time polymerase chain reaction (RT-PCR) assay confirmed that Lyn and Fyn were critical effectors for the Src-mediated expression of tumor growth or metastasis-related molecules. Furthermore, results of the clinical ESCC samples showed that the hyperactivation of pSrc Tyr⁴¹⁹, Fyn Tyr¹⁸⁵ or Tyr⁴²⁰, and Lyn Tyr¹⁸³ or Tyr³⁹⁷ could be biomarkers of ESCC prognosis. This study illustrates that Src/Fyn and Src/Lyn heterodimers serve as targets for the treatment of ESCC.