Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA Signaling as Prognostic Biomarker and Therapeutic Target for Colon Cancer Invasion

Metastasis
is one of the major causes of treatment failure in the
patients with colon cancer. The aim of our study is to find key proteins
and pathways that drive invasion and metastasis in colon cancer. Eight
rounds of selection of cancer cells invading through matrigel-coated
chamber were performed to obtain highly invasive colon cancer sublines
HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell
Culture technology was used to identify the differently expressed
proteins, and the proteomics data were analyzed by ingenuity pathway
analysis. PAK1-PBD immunoprecipitation combined with Western blot
were carried out to determine Cdc42 activity, and qRT-PCR and Western
blot were used to determine gene expression. The functional role of
Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by
loss-of-function experiments including pharmacological blockade, siRNA
knockdown, chamber invasion, and WST-1 assays. Human colon cancer
tissue microarray was analyzed by immunohistochemistry for overexpression
of Cdc42BPA and its correlation with clinicopathological parameters
and patient survival outcomes. HCT116-I8 and RKO-I8 cells showed significantly
stronger invasive potential as well as decreased E-cadherin and increased
vimentin expressions compared with parental cells. The differently
expressed proteins in I8 cells compared with parental cells were identified.
Bioinformatics analysis of proteomics data suggested that Cdc42BPA
protein and Cdc42 signaling pathway are important for colon cancer
invasion, which was confirmed by experimental data showing upregulation
of Cdc42BPA and higher expression of active GTP-bound form of Cdc42
in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic
blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon
cancer cell invasion and reversed epithelial mesenchymal transition
process. Furthermore, compared with adjacent normal tissues, Cdc42BPA
expression was significantly higher in colon cancer tissues and further
upregulated in metastatic tumors in lymph nodes. More importantly,
Cdc42BPA expression was correlated with metastasis and poor survival
of the patients with colon cancer. This study provides the first evidence
that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion,
and Cdc42BPA has potential implications for colon cancer prognosis
and treatment.