Lei Du

PURPOSE: Both CD44 and CD133 were reported as putative markers for isolating colorectal cancer stem cells (CSC). It remains to be resolved if both of these markers are of functional importance for colorectal CSC. EXPERIMENTAL DESIGN: The expression of CD44 and CD133 in normal colonic tissues and primary colorectal cancer was assessed by immunohistochemistry in a series of 60 patients on tissue microarray sections. Both in vitro clonogenic and in vivo tumorigenic assay were applied to measure CSC activities from the cells isolated from patients. Lentiviral RNA interference was used to stably knock down CD44 or CD133 in colorectal cancer cells from patients. RESULTS: We found that CD44(+) cells displayed clustered growth and they did not colocalize with CD133(+) cells within colorectal cancer. As few as 100 CD44(+) cells from a patients' tumor initiated a xenograft tumor in vivo. A single CD44(+) cell from a tumor could form a sphere in vitro which has characteristic stem cell properties and was able to generate a xenograft tumor resembling the properties of the primary tumor. Knockdown of CD44, but not CD133, strongly prevented clonal formation and inhibited tumorigenicity in xenograft model. CONCLUSIONS: These results indicate that CD44 is a robust marker and is of functional importance for colorectal CSC for cancer initiation.

PURPOSE: CD44 is of functional importance for tumor initiation and progression in colorectal cancer, but how this molecule benefits cancer cells from the tumor microenvironment, especially tumor-associated macrophages (TAM), remains poorly defined. EXPERIMENTAL DESIGN: In vivo tumorigenic assays were conducted to assess the role of murine TAMs in the tumorigenesis of human colorectal cancer cells. Both in vitro and in vivo osteopontin (OPN) expression levels in TAMs were examined by immunohistochemistry, quantitative PCR, and Western blotting. Soft agar colony formation assays were used to estimate the clonogenicity of colorectal cancer cells that had received different treatments. The relationships between the expression levels of OPN, CD44v6, and CD68 and clinical prognosis were evaluated by tissue microarray analysis. RESULTS: We found that macrophages, when coinjected or cocultured with CD44-positive colorectal cancer cells, were able to produce higher levels of OPN, which in turn facilitated the tumorigenicity and clonogenicity of the colorectal cancer cells. The knockdown of CD44 or treatment with blocking antibodies to CD44 attenuated OPN secretion. OPN, through binding to its receptor CD44, activated c-jun-NH(2)-kinase signaling and promoted the clonogenicity of colorectal cancer cells. Moreover, tissue microarray data have shown that OPN expression, in combination with CD44v6, has a negative correlation with colorectal cancer patient survival. CONCLUSIONS: These results suggest that the OPN-CD44 interaction is important for colorectal cancer progression and could serve as a potential therapeutic target for the treatment of colorectal cancer.

Cancer stem cells are implicated in tumor progression, metastasis, and recurrence, although the exact mechanisms remain poorly understood. Here, we show that the expression of cellular prion protein (PrPc, PRNP) is positively correlated with an increased risk of metastasis in colorectal cancer. PrPc defines a subpopulation of CD44-positive cancer stem cells that contributes to metastatic capacity. PrPc(+)CD44(+) colorectal cancer stem cells displayed high liver metastatic capability, unlike PrPc(-)CD44(+) stem cells, that was inhibited by RNAi-mediated attenuation of PrPc. Notably, administration of PrPc monoclonal antibodies significantly inhibited tumorigenicity and metastasis of colorectal cancer stem cells in mouse models of orthotopic metastasis. PrPc promoted epithelial to mesenchymal transition (EMT) via the ERK2 (MAPK1) pathway, thereby conferring high metastatic capacity. Our findings reveal the function of PrPc in regulating EMT in cancer stem cells, and they identify PrPc as candidate therapeutic target in metastatic colorectal cancer.