Lithium chloride regulates the proliferation of stem-like cells in retinoblastoma cell lines: a potential role for the canonical Wnt signaling pathway

Abstract

Purpose: Cancer stem cells are found in many tumor types and are believed to lead to regrowth of tumor mass due to their chemoresistance and self-renewal capacity. We previously demonstrated small subpopulations of cells in retinoblastoma tissue and cell lines that display cancer stem cell-like activities, including expression of stem cell markers, Hoechst dye exclusion, slow cycling, and self-renewal ability. Identifying factors regulating stem cell proliferation will be important for selectively targeting stem cells and controlling tumor growth. Wingless and Int1 (Wnt) signaling is an essential cellular communication pathway that regulates proliferation and differentiation of non-neoplastic stem/progenitor cells in the retina and other tissues, but its role in cancer stem cells in the retinal tumor retinoblastoma is unknown. In this study, we investigated whether the Wnt pathway activator lithium chloride (LiCl) regulates proliferation of retinoblastoma cancer stem-like cells.

Methods: The number of stem-like cells in Weri and Y79 retinoblastoma cell line cultures was measured by 5-bromo-2-deoxyuridine (BrdU) pulse-chase, immunohistochemistry, and quantitative polymerase chain reaction (PCR) for stem cell marker genes. The cell lines were sorted into stem-like and non-stem-like populations by fluorescence-activated cell sorting (FACS), using an antibody against the stem cell marker ATP-binding cassette, subfamily G, member 2 (ABCG2). Activated Wnt signaling was measured in the sorted cells by western blotting and immunolocalization of the central mediator beta-catenin.

Results: LiCl increased the number of stem-like cells, measured by BrdU retention and elevated expression of the stem cell marker genes Nanog, octamer transcription factor 3 and 4 (Oct3/4), Musashi 1 (Msi1), and ABCG2. Sorted ABCG2-positive stem-like cells had higher levels of beta-catenin than ABCG2-negative non-stem cells, suggesting elevated canonical Wnt signaling. Furthermore, stem cell marker gene expression increased after small interfering RNA (siRNA) knock-down of the Wnt inhibitor secreted frizzled-related protein 2 (SFRP2).

Conclusions: These results indicate that the cancer stem-like cell population in retinoblastoma is regulated by canonical Wnt/beta-catenin signaling, which identifies the Wnt pathway as a potential mechanism for the control of stem cell renewal and tumor formation in retinoblastoma tumors in vivo.

Figure 1

LiCl expands the population of stem-like cells in the Weri and Y79 retinoblastoma cell lines. A: LiCl significantly increased the number of ABCG2 and Musashi-1 (Msi-1) immunoreactive cells. Weri and Y79 human retinoblastoma cells were treated with 0, 20, and 40 mM LiCl for 48 h and then immunostained. The number of cells expressing ABCG2 (left) or Musashi-1 (Msi-1; right) were counted. The differences between 20 mM LiCl and control, and 40 mM LiCl and control, were significant at a p-value<0.001 (*). Each experiment was repeated eight times. B: LiCl increased the expression of stem cell marker genes Nanog and Oct3/4. QPCR was performed on Weri and Y79 cells treated with 20 mM or 40 mM LiCl. LiCl increased Nanog and Oct3/4 levels, and there was significantly greater expression with 40 mM LiCl treatment compared with 20 mM LiCl at a p-value<0.05, indicated by the asterisk. Each experiment was repeated three times.

Figure 2

The number of slow cycling cells was increased by LiCl. Y79 cells were incubated with LiCl and pulse-chased with BrdU for a total of 3 weeks. The number of live cells retaining BrdU was measured by flow cytometry. The percent of gated live BrdU+ cells was calculated and normalized to untreated cells and there was significantly greater BrdU+ cells in the LiCl-treated samples at a p-value<0.05, indicated by the asterisk. Each experiment was repeated three times.

Figure 3

LiCl decreased the proliferative index of the retinoblastoma cell lines. Weri and Y79 retinoblastoma cells were treated with 0, 20, 40 mM LiCl for 48 h and then immunostained. LiCl significantly decreased the number of Ki67 immunoreactive cells (n=8).

Figure 4

Elevated canonical Wnt signaling in flow-sorted retinoblastoma cancer stem-like cells. A: Weri cells were sorted by flow cytometry using an antibody against the stem cell marker ABCG2. RNA and protein were then extracted from the ABCG2-positive and ABCG2-negative cells. Western blotting on sorted ABCG2+ stem cells showed higher β-catenin levels, indicating higher canonical Wnt signaling in the stem cells (n=3, p=0.048). β-catenin levels were normalized to β-actin, and are expressed as a ratio of the respective ABCG2-negative cells. B-F: Representative images showing the immunoreactivity of the stem cell markers ABCG2 and Msi1, and the canonical Wnt signaling mediator β-catenin in the retinoblastoma cell lines Weri and Y79. In the top right panels, ABCG2 immunoreactivity is red and β-catenin is green. In the top left panels, Msi1 immunoreactivity is green and β-catenin is red. The bottom panels show the negative controls, in which Weri and Y79 cells were treated with control rabbit IgG and FITC or TRITC secondary antibodies. The adjacent bright-field images are also shown. All images were taken at 40x. G: QPCR on canonical Wnt signaling receptors demonstrated higher expression of the receptors LRP6, Fz3, Fz4, and Fz6 in the in ABCG2-positive (cancer stem-like cells) than ABCG2-negative (non-stem-like cells). Data are from two independent experiments; each experiment had three replicates.

Figure 5

Cancer stem-like cells have active Wnt signaling in the retinoblastoma cell lines. The percent of co-localized Msi1 and nuclear β-catenin was quantified in the Weri and Y79 cell lines. Immunohistochemistry demonstrated that tumor stem cells (Msi1+) were more often positive for nuclear β-catenin than non-stem cells (Msi-), indicating active Wnt signaling. The difference in nuclear β-catenin was statistically significant at p<0.001 (*). The experiment was repeated five times.

Figure 6

The Wnt inhibitor SFRP2 regulates cancer stem-like cells. A: The Wnt inhibitor SFRP2 was decreased in Weri and Y79 cells by addition of LiCl, as measured by QPCR on unsorted cells. The difference between 20 mM and 40 mM LiCl was statistically significant at p<0.05 (*) and the difference between treated and untreated was statistically significant at p<0.01 (**). The experiment was repeated four times. B: Reducing SFRP2 levels using siRNA (average % reduction was 63% relative to scrambled siRNA) increased stem cell marker gene expression. The increase in Oct3/4 expression was statistically significant at p=0.05 (*). The experiment was repeated four times.