Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 in colorectal cancer

Abstract

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a 7-transmembrane receptor reportedly expressed in stem cells of the intestinal crypts and hair follicles of mice. Overexpression of LGR5 is observed in some types of cancer; however, there has been no specific assessment in colorectal tumorigenesis. We performed quantitative RT-PCR for LGR5 expression in 37 representative cancer cell lines, and showed that LGR5 mRNA was frequently overexpressed in colon cancer cell lines. Moreover, LGR5 expression was higher in colon cancer cell lines derived from metastatic tumors compared with those from primary tumors. In clinical specimens, there was significant overexpression of LGR5 in 35 of 50 colorectal cancers (CRCs), and in seven of seven sporadic colonic adenomas, compared with matched normal mucosa. This suggests up-regulation of LGR5 from the early stage of colorectal tumorigenesis. LGR5 expression showed marked variation among CRC cases and correlated significantly with lymphatic invasion, vascular invasion, tumor depth, lymph node metastasis, and tumor stage (IIIC vs. IIIB). In addition to cancer cells, crypt base columnar cells of the small intestine and colon were shown by in situ hybridization to express LGR5. This is the first report suggesting the involvement of LGR5, not only in early events but also in late events in colorectal tumorigenesis.

Figure 1

Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) expression in a variety of cancer cell lines. LGR5 mRNA expression was evaluated by qRT‐PCR analysis in 11 colon cancer cell lines, five hepatocellular carcinoma (HCC) cell lines, 10 ovarian cancer cell lines, and 11 lung cancer cell lines. LGR5 expression was normalized to that of GAPDH. The average LGR5 expression in the 37 cell lines was set at 1 (bold line). LGR5 mRNA was frequently overexpressed in colon cancer cell lines (5/11) compared with other cancer cell lines (only Hep G2 for HCC, H889 for lung cancer, and none of the ovarian cancers). All colon cancer cell lines derived from metastatic sites showed over‐expression of LGR5.

Figure 2

Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) expression in colorectal tumor tissue and matched normal mucosa. LGR5 mRNA expression was estimated by qRT‐PCR and was normalized to that of GAPDH. (a) LGR5 expression, in 50 cases of colorectal cancer (CRC) and matched normal colorectal mucosa, was evaluated (upper column, mRNA expression in tumors; lower column, mRNA expression in normal colonic mucosa). The majority of CRCs (70%) showed a more than three times greater expression of LGR5 compared with matched normal mucosa. (b) In seven sporadic colorectal adenomas, LGR5 expression was significantly higher than in matched normal mucosa. (c) Mean (SD) mRNA expression levels for LGR5 in CRC, normal mucosa, and colorectal adenoma. LGR5 mRNA expression was approximately 13‐fold higher in CRCs as compared with normal mucosa, and LGR5 expression showed marked variation in cancers as compared with adenomas.

Figure 3

In situ hybridization of Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) mRNA in colorectal cancers (CRCs) and adenomas. De‐paraffinized sections were stained with H&E (a,d,g), and hybridized with antisense (b,e,h) or sense (c,f,i) probes for LGR5. Scale bars, 100 μm. Expression of the antisense signal coincided specifically with cancer cells (a–c, ×40; d–f, ×200) and adenoma cells (g–i, ×200). The LGR5 signal was diffusely expressed over the entire cancer tissue.

Figure 4

In situ hybridization of Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) mRNA in normal epithelium of the small intestine. De‐paraffinized serial sections were stained with H&E (a,b), hybridized with antisense (c,d) and sense (e,f) probes for LGR5, and immunolabeled for Ki‐67 (g,h) and chromogranin A (i,j). Scale bars, 25 μm. The enlarged crypt epithelium is shown on the right (b,d,f,h,j). Expression of antisense signal for LGR5 coincided specifically with four crypt base columnar (CBC) cells (arrowheads) between Paneth cells in the crypt of the small intestine (×400) (d). Nuclei of the CBC cells were lightly stained for Ki‐67 (arrowheads, h). The cells hybridized with antisense probe did not express chromogranin A (arrow, j).

Figure 5

Correlation between Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) mRNA expression and clinicopathological findings in colorectal cancers (CRCs). LGR5 expression in CRCs was divided by that in matched normal mucosa (T/N ratio). Lymph node metastasis (N)‐positive cases, those with lymphatic invasion (Ly) and vascular invasion (V), and pT4 cases, showed significantly higher LGR5 expression compared with other CRCs. LGR5 expression did not correlate with other clinicopathological findings such as age and gender of the patient, and tumor differentiation, location, and size.

Figure 6

Association between Leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5) expression and malignant potential of colorectal cancer (CRC). LGR5 expression in CRCs was divided by that in matched normal mucosa (T/N ratio). (a) Mean LGR5 expression in stages II (lymph node metastasis 0), IIIB (lymph node metastases 1–3) and IIIC (lymph node metastases ≥4). LGR5 expression increased stepwise with tumor stage progression. (b) LGR5 expression correlated positively with the number of lymph node metastases (r = 0.59, P < 0.0001).