Background:The molecular basis for the development of appendiceal mucinous tumours, which can be a cause of pseudomyxoma peritonei, remains largely unknown.Methods:Thirty-five appendiceal mucinous neoplasms were analysed for GNAS and KRAS mutations. A functional analysis of mutant GNAS was performed using a colorectal cancer cell line.Results:A mutational analysis identified activating GNAS mutations in 16 of 32 low-grade appendiceal mucinous neoplasms (LAMNs) but in none of three mucinous adenocarcinomas (MACs). KRAS mutations were found in 30 LAMNs and in all MACs. We additionally analysed a total of 186 extra-appendiceal mucinous tumours and found that GNAS mutations were highly prevalent in intraductal papillary mucinous tumours of the pancreas (88%) but were rare or absent in mucinous tumours of the colorectum, ovary, lung and breast (0–9%). The prevalence of KRAS mutations was quite variable among the tumours. The introduction of the mutant GNAS into a colorectal cancer cell line markedly induced MUC2 and MUC5AC expression, but did not promote cell growth either in vitro or in vivo.Conclusion:Activating GNAS mutations are a frequent and characteristic genetic abnormality of LAMN. Mutant GNAS might play a direct role in the prominent mucin production that is a hallmark of LAMN.

Purpose: SMAD4 is a key transcriptional factor of TGFb signaling and acts as a tumor suppressor in colorectal cancer. In the present study, we explored the immunologic effect of SMAD4 on the tumor microenvironment. Experimental Design: Using 99 clinical specimens and human colorectal cancer 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 patients with colorectal cancer. Results: SMAD4 knockdown from human colorectal cancer cells upregulated the expression of CXCL1 and CXCL8, which recruited neutrophils to colorectal cancer tumor via CXCR2. In turn, when neutrophils were exposed to the supernatant of SMAD4-negative colorectal cancer 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 colorectal cancer compared with that in SMAD4-positive colorectal cancer, and that both CXCL1 and CXCL8 were abundantly expressed in the tumor-infiltrating neutrophils. Neutrophils isolated from primary colorectal cancer 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 colorectal cancer patients, especially those at stage II/III, and statistical analysis indicated a 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 colorectal cancer.

Mesenchymal stem cells (MSCs) are recruited from BM to the stroma of developing tumors, where they serve as critical components of the tumor microenvironment by secreting growth factors, cytokines, and chemokines. The role of MSCs in colorectal cancer (CRC) progression was controversial. In this study, we found that C-C chemokine receptor type 5 (CCR5) ligands (i.e., C-C motif chemokine ligand 3 (CCL3), CCL4, and CCL5) were highly produced from MSCs using a chemokine array screening with conditioned media from the cultured human MSCs. A relatively strong CCR5 expression could be detected within the cytoplasm of several CRC cell lines. Regarding the effect of MSC, we found that the xenografts in which CCR5-overexpressing HCT116 cells were inoculated into immunocompromised mice were highly promoted in vivo by a mixture with MSCs. Notably, the CCR5 inhibitor, maraviroc, significantly abolished the MSC-induced tumor growth in vivo. In human clinical specimens (n = 89), 20 cases (29%) were high for CCR5, whereas 69 cases (71%) were low. Statistical analyses indicated that CCR5 expression in primary CRC was associated with CRC patients’ prognosis. Especially, stage III/IV patients with CCR5-high CRCs exhibited a significantly poorer prognosis than those with CCR5-low CRCs. Furthermore, we investigated the effects of preoperative serum CCR5 ligands on patients’ prognosis (n = 114), and found that CRC patients with high serum levels of CCL3 and CCL4 exhibited a poorer prognosis compared to those with low levels of CCL3 and CCL4, while there was no association between CCL5 and prognosis. These results suggest that the inhibition of MSC–CRC interaction by a CCR5 inhibitor could provide the possibility of a novel therapeutic strategy for CRC, and that serum levels of CCL3 and CCL4 could be predictive biomarkers for the prognosis of CRC patients.

Mutation in the KRAS gene induces prominent metabolic changes. We have recently reported that KRAS mutations in colorectal cancer (CRC) cause alterations in amino acid metabolism. However, it remains to be investigated which amino acid transporter can be regulated by mutated KRAS in CRC. Here, we performed a screening of amino acid transporters using quantitative reverse-transcription polymerase chain reaction (RT-PCR) and then identified that ASCT2 (SLC1A5) was up-regulated through KRAS signaling. Next, immunohistochemical analysis of 93 primary CRC specimens revealed that there was a significant correlation between KRAS mutational status and ASCT2 expression. In addition, the expression level of ASCT2 was significantly associated with tumor depth and vascular invasion in KRAS-mutant CRC. Notably, significant growth suppression and elevated apoptosis were observed in KRAS-mutant CRC cells upon SLC1A5-knockdown. ASCT2 is generally known to be a glutamine transporter. Interestingly, SLC1A5-knockdown exhibited a more suppressive effect on cell growth than glutamine depletion. Furthermore, SLC1A5-knockdown also resulted in the suppression of cell migration. These results indicated that ASCT2 (SLC1A5) could be a novel therapeutic target against KRAS-mutant CRC.

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