Loss of SMAD4 promotes colorectal cancer progression by recruiting tumor-associated neutrophils via CXCL1/8-CXCR2 axis

Purpose:SMAD4 is a key transcriptional factor of TGF-b signaling, and acts as a tumor suppressor in colorectal cancer (CRC). In the present study, we explored the immunological effect of SMAD4 on the tumor microenvironment. Experimental Design: Using 99 clinical specimens and human CRC cell lines, we investigate the relationship between SMAD4 expression and neutrophil accumulation. We immunohistochemically analyzed expression of SMAD4, CXCL1, CXCL8, CXCR2 and other proteins with clinical specimens. Finally, we determined the serum levels of CXCL1 and CXCL8 in 125 CRC patients. Results: SMAD4 knockdown from human CRC cells up-regulated the expression of CXCL1 and CXCL8, which recruited neutrophils to CRC tumor via CXCR2. In turn, when neutrophils were exposed to the supernatant of SMAD4-negative CRC cells, they produced a large amount of CXCL1 and CXCL8 by themselves in vitro. In human clinical specimens, we found that neutrophil infiltration into the peritumoral stroma was more marked in SMAD4-negative CRC compared to that in SMAD4-positive CRC, and that both CXCL1 and CXCL8 were abundantly expressed in the tumor-infiltrating neutrophils. Neutrophils isolated from primary CRC expressed significantly higher levels of CXCL1 and CXCL8 than did those isolated from peripheral blood. Furthermore, tumor-infiltrating neutrophils expressed MMP2 and MMP9 in addition to ARG1 and IDO. Serum CXCL8 level was significantly higher in CRC patients, especially those at stage II/III, and statistical analysis indicated high CXCL8 level was associated with a shorter overall survival and relapse-free survival. Conclusions:Blockade of the CXCL1/8-CXCR2 axis could be a novel therapeutic approach against SMAD4-negative CRC.

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