SW480 colorectal cancer cells that naturally express Lgr5 are more sensitive to the most common chemotherapeutic agents than Lgr5-negative SW480 cells

Abstract

Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) is a colorectal cancer (CRC) stem cell marker. The role of Lgr5-expressing stem cells in resistance to chemotherapy is controversial. The notion that Lgr5-expressing cells are more chemotherapy resistant is supported by some data; other data do not support this notion. We hypothesized that Lgr5-expressing cells would be more chemotherapy sensitive, as Lgr5 is usually a marker of dividing cells. We tested this hypothesis by exploiting two natural variants of SW480 CRC cells: the less-differentiated Lgr5-expressing floating fraction and the more-differentiated Lgr5-depleted attached fraction. We estimated chemotherapy sensitivity using an XTT Cell Proliferation Assay Kit. We confirmed that the detected chemotherapy sensitivity differences were Lgr5-driven by overexpressing Lgr5. SW480 CRC cells that naturally express Lgr5 are those that are floating, and they are more sensitive to the chemotherapeutic compounds irinotecan (maximum difference approximately two times, 0.0001<P<0.0052) and oxaliplatin (maximum difference ∼1.5 times, 0.0001<P<0.0024) than Lgr5-negative (attached) SW480 cells. At IC50, the difference in sensitivity between these two fractions of SW480 cells to the drugs was twice as prominent with irinotecan as with oxaliplatin (P<0.0001). SW480 cells that naturally express Lgr5 were slightly more sensitive to 5-fluorouracil than non-Lgr5-expressing cells (0.0002<P<0.0344). Transfected Lgr5-overexpressing attached cells showed similar results with irinotecan and oxaliplatin, confirming that the detected differences in sensitivity to these drugs were Lgr5 driven. Most likely, Lgr5 makes cells vulnerable to chemotherapy, increasing their propensity to divide through activation of the Wnt/β-catenin pathway. There is one exception, Lgr5-overexpressing cells did not show this increase in sensitivity to 5-fluorouracil. Remarkably, Lgr5 reduced the number of floating cells two-fold (instead of increasing it, as one would expect from a stemness-determining factor) and slightly increased the number of attached differentiated cells at a significantly high transfection efficiency for these SW480 cells (∼30%). These results suggest that Lgr5, although purported to be a cancer stem cell marker, is not sufficient to promote floating in these colon cancer cells. In conclusion, CRC cells that naturally express Lgr5 are more sensitive to the most commonly used CRC chemotherapeutic compounds than those that are naturally Lgr5 negative.