iTRAQ-based analysis for the identification of MARCH8 targets in human esophageal squamous cell carcinoma

MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberrant expression in human esophageal squamous cell carcinoma. However, exact mechanisms by which it regulates cancer have been poorly understood. We applied high-throughput quantitative proteomics approach to identify downstream protein targets of MARCH8. Silencing of endogenous MARCH8 in ESCC cells followed by LC-MS/MS analysis led to identification of 1,029 unique proteins showing altered expression post MARCH8 knockdown. Several previously reported MARCH8 target proteins viz. TFR1, syntaxin-4, e-cadherin and CD44 were found to be upregulated. Furthermore, new putative targets of MARCH8, including β2M, were identified in the present study. We demonstrated that MARCH8 interacts with and ubiquitinates CDH1 and β2M. Inhibiting proteasome activity with MG132 prevented CDH1 and β2M degradation, indicating that MARCH8 might be targeting CDH1 and β2M for proteasomal degradation. Further, loss of β2M and CDH1 expression significantly and inversely correlated with MARCH8 expression in ESCC tissues (r =  -0.737 and  - 0.651, respectively; p < 0.01). In conclusion, our present study has led to identification of new targets of MARCH8 and suggests the role of MARCH8 in regulating CDH1 and β2M turnover in esophageal cancer cells. SIGNIFICANCE: The use of quantitative proteomics carried out has led to the recognition of new targets of MARCH8. The present study gives a broad understanding of the molecular remodeling arising in the ESCC after MARCH8 knockdown. The study also solidifies the idea that role of MARCH8 is not just limited to immunomodulation as silencing of MARCH8 affects various other processes such as protein processing and localization. This study might help in understanding the regulation of MARCH8 in ESCCs and the mechanism by which MARCH8 might be facilitating cancer cells to evade immune surveillance.