Phosphoproteome Profiling of Isogenic Cancer Cell‐Derived Exosome Reveals HSP90 as a Potential Marker for Human Cholangiocarcinoma

Abstract

The northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma (CCA). Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Exosomes are stable toward circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression. Here an in vitro metastatic model of isogenic CCA cells is used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel-based and gel-free proteomics approaches are used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty-three phosphoproteins are identified with a significant change in phosphorylation level. Phos-tag western blotting and immunohistochemistry staining are then employed to validate the candidate phosphoproteins. Heat shock protein 90 is successfully confirmed as being differentially phosphorylated in relation to tumor malignancy.

Tumorigenesis, diagnosis, and therapeutic potential of exosomes in liver cancer

“…Studies have also shown that the expression of HSP90 in serum exosomes of cancer patients with highly metastatic CCA is significantly higher than that in patients with common CCA, suggesting that the level of HSP90 in serum exosomes is related to the malignant degree of the tumor, probably due to the fact that HSP90 contributes to tumor metastasis and invasion by activating related proteins in the extracellular microenvironment. Further studies have also found that the phosphorylation of HSP90, especially a decrease in the phosphorylation of HSP90B protein at the Ser255 and Ser581 amino acid sites, is inversely correlated to the metastatic ability of cholangiocarcinoma cells [159]. Therefore, exosomal HPS90 and its phosphorylation state may be used as markers for the progression of hepatobiliary tumors.…”

Molecular Profile Study of Extracellular Vesicles for the Identification of Useful Small “Hit” in Cancer Diagnosis

“…Differential exosomal phosphoproteome analysis of invasive M213 and M213D5 CCA cells has shown its potential as a biomarker in cancer. Reduced exosomal fetuin-A phosphorylation and high HSP90B phosphorylation has been detected in tissues from CCA patients with a low TNM stage, compared to those with a single high-stage TNM [100] (Table 1).…”

The role of extracellular vesicles in cholangiocarcinoma

“…Similarly, using proteomic methods, Weeraphan et al studied the exosomal phosphoproteome of M213 and M213D5 in CCA cells, and showed that Ser255 of HSP90B was highly phosphorylated in tissues of CCA patients with a low TNM stage (I and II) compared to those with a TNM stage of III or IV. ROC analysis showed that HSP90B-S255 was a new potential biomarker for metastatic CCA with an AUC value of 0.936 (sensitivity 87.27%, specificity 97.62%) [105]. Moreover, Shen et al studied exosomal miRNA in peripheral blood samples from CCA patients and healthy controls.…”