LGR5 Is a Gastric Cancer Stem Cell Marker Associated with Stemness and the EMT Signature Genes <i>NANOG</i>, <i>NANOGP8</i>, <i>PRRX1</i>, <i>TWIST1</i>, and <i>BMI1</i>

<div><p>Background</p><p>Accumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear.</p><p>Methods</p><p>qPCR was performed to measure the expression profiles of stemness and EMT signature genes and their association with putative CSC markers in gastric cancer tissues, cancer cell lines and sphere cells. Western blot analysis was used to confirm the results of the transcript analysis. Cell proliferation, cell migration, drug resistance and sphere cell growth assays were conducted to measure the expansion and invasion abilities of the cells. Tumor xenograft experiments were performed in NOD/SCID mice to test cell stemness <i>in vivo</i>. Flow cytometry and immunofluorescence staining were used to analyze cell subpopulations.</p><p>Results</p><p>The expression of <i>LGR5</i> was strikingly up-regulated in sphere cells but not in cancer tissues or parental adherent cells. The up-regulation of <i>LGR5</i> was also positively associated with stemness regulators (<i>NANOG</i>, <i>OCT4</i>, <i>SOX2</i>, and <i>AICDA</i>) and EMT inducers (<i>PRRX1</i>, <i>TWIST1</i>, and <i>BMI1)</i>. In addition, sphere cells exhibited up-regulated vimentin and down-regulated E-cadherin expression. Using gene-specific primers, we found that the <i>NANOG</i> expression primarily originates from the retrogene <i>NANOGP8</i>. Western blot analysis showed that the expression of both LGR5 and NANOG is significantly higher in sphere cells. <i>LGR5</i> over-expression significantly enhanced sphere cell growth, cell proliferation, cell migration and drug resistance in MGC803 cells. Tumor xenografts in nude mice showed that sphere cells are at least 10 times more efficient at tumor initiation than adherent cells. Flow cytometry analysis showed that ~20% of sphere cells are LGR5+/CD54+, but only ~3% of adherent cells are Lgr5+/CD54+. Immunofluorescence staining supports the above results.</p><p>Conclusion</p><p>The <i>LGR5</i>-expressing fraction of CD54+ cells represents gastric cancer CSCs, in which <i>LGR5</i> is closely associated with stemness and EMT core genes, and <i>NANOG</i> expression is mainly contributed by the retrogene <i>NANOGP8</i>. Sphere cells are the best starting materials for the characterization of CSCs.</p></div

Tumor initiation rate in nude mice by sphere and adherent cells.

<p>Tumor initiation rate in nude mice by sphere and adherent cells.</p

Tumor growth after subcutaneous inoculation in nude mice.

<p><b>A</b>. Tumor growth in nude mice after injection of MGC803 tumor sphere cells (2X10⁶) into the left rear flank of mice and the parental adherent cells (2X10⁶) into the right rear flank of mice. <b>B</b>. Representative xenograft tumors are shown. <b>C</b>. Size comparison of subcutaneous tumors following the injection of an equal number of MGC803 tumor sphere cells and MGC803 adherent cells (2X10⁶). *<i>p</i><0.05; **<i>p</i><0.01. <b>D</b>. H&E staining analysis of xenograft tumors derived from MGC803 sphere cells. The scale bars = 20 μm.</p

Detection of LGR5, NANOG and CD44 proteins in MGC803 sphere cells and adherent cells.

<p>Detection of LGR5, NANOG and CD44 proteins in MGC803 sphere cells and adherent cells.</p

Drug resistance analysis by MTT assay.

<p>MGC803 cells transfected with LGR5 and mock-transfected cells were compared. The black line with solid circles represents MGC803 mock-transfected cells, and the gray line with hollow circles represents LGR5-over-expressing cells. *<i>p</i><0.05; **<i>p</i><0.01.</p

Result of cell proliferation and sphere cell growth assays.

<p><b>A</b>. Cell proliferation assay results for LGR5-transfected MGC803 cells and the mock-transfected MGC803 cells. <b>B</b>. Sphere cell growth assay results for LGR5-transfected MGC803 cells and the mock-transfected MGC803 cells. <b>C</b>. Images of the sphere cell growth in LGR5-transfected and mock-transfected conditions. **<i>p</i><0.01.</p

Microscopic observation of sphere cells and adherent cells.

<p><b>A</b>. MGC803 adherent cells. <b>B</b>. MGC803 sphere cells. All the images are 400× magnified.</p

Primers used in this study.

<p>Primers used in this study.</p

Detection of gene expression profiles by qPCR.

<p><b>A</b>. Gastric cancer tissues versus adjacent normal tissues. <b>B</b>. MGC803 (a poorly differentiated cancer cell line) adherent cells versus GES-1 (a gastric epithelial cell line) adherent cells. <b>C</b>. MGC803 sphere cells versus MGC803 adherent cells. <b>D</b>. Expression of E-cadherin and vimentin in sphere cells versus parental adherent cells. <b>ECAD</b> stands for E-cadherin; <b>VIM</b> stands for vimentin. <b>E</b>. Expression of NANOG1-S and NANOG1/P8 in sphere cells versus parental adherent cells. <b>NANOG1-S</b> stands for NANOG1-specific primers and <b>NANOG1/P8</b> stands for NANOG1 and NANOGP8 shared primers.</p

Cell migration analysis by wound-healing assays.

<p>MGC803 cells transfected with LGR5 and mock-transfected cells were compared. <b>A</b>. The cell wounds were visualized at 0 h, 12 h, 24 h, and 48 h; <b>B</b>. Migration analysis at different time points. *<i>p</i><0.05; **<i>p</i><0.01.</p