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. 2010 Nov 1;70(21):8863-73.
doi: 10.1158/0008-5472.CAN-10-1295. Epub 2010 Sep 22.

Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo

Lionel W Hebbard  1 , Jochen Maurer, Amber Miller, Jacqueline Lesperance, John Hassell, Robert G Oshima, Alexey V Terskikh
Affiliations

Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo

Lionel W Hebbard et al. Cancer Res. .

Abstract

Maternal embryonic leucine zipper kinase (MELK) is expressed in several developing tissues, in the adult germ line, and in adult neural progenitors. MELK expression is elevated in aggressive undifferentiated tumors, correlating with poor patient outcome in human breast cancer. To investigate the role of MELK in mammary tumorigenesis in vivo, we used a MELK-green fluorescent protein (GFP) reporter mouse, which allows prospective isolation of MELK-expressing cells based on GFP fluorescence. We found that in the normal mammary gland, cells expressing high levels of MELK were enriched in proliferating cells that express markers of mammary progenitors. The isolation of cells with high levels of MELK in mammary tumors from MMTV-Wnt1/MELK-GFP bitransgenic mice resulted in a significant enrichment of tumorsphere formation in culture and tumor initiation after transplantation into mammary fat pads of syngeneic mice. Furthermore, using lentiviral delivery of MELK-specific shRNA and limiting dilution cell transplantations, we showed that MELK function is required for mammary tumorigenesis in vivo. Our findings identify MELK as a potential target in breast tumor-initiating cells.

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Figures

Figure 1
Figure 1. Characterization of MELK expression in normal mammary glands
A, whole mount mammary fat pad from a 6 weeks-old virgin mouse (Scale bar 100 μm). B, 9.5 days pregnant MELK-GFP female. Note the increased GFP expression in the proliferative alveoli (arrows). Scale bar 50μm. C and D, immunofluorescence of a 6 weeks-old virgin MELK-GFP transgenic mouse mammary gland demonstrating colocalization of GFP expression (red) and CD44v6 (green) in a terminal end bud (C) and predominant GFP expression in epithelial cells as demarcated by CD44v6 in a cross section of a mammary duct (D). Scale bar 30μm. E and F, detection of endogenous MELK protein by immunofluorescence in FACS isolated GFPlow (bottom 10%) and GFPhigh (top 10%) cells. Scale bar 10μm. G. Fluorescent histograms of GFPlow (blue line, median=1.1×103) and GFPhigh (green line median=5.5×103) populations. H. Q-PCR detection of MELK mRNA in GFPlow and GFPhigh populations (normalized to 18s expression). I. Quantification of endogenous MELK protein shown in E, F normalized to DAPI).
Figure 2
Figure 2. Levels of MELK expression correlate with proliferation in normal mammary cells
A, colocalization of PCNA (green) and GFP (red) in the luminal cells of the ducts in 6 weeks-old virgin mammary. DAPI staining is in blue. Scale bar 40μm. B, the cell cycle analysis of various GFP populations from freshly dissociated 6 weeks-old virgin mammary glands.
Figure 3
Figure 3. Cells with highest levels of MELK expression include mammary progenitors
A, flow cytometry analysis of dissociated normal mammary gland revealed up-regulation of GFP signal in CD24+CD29low cells compared to CD24+CD29high cells. B, the GFPhigh cells (top 10-15%) are enriched for cells with progenitor markers. Dot plot and histogram of live Lin− cells (CD45−CD31−TER119−PI−) are shown in the top panels. Cells in the lower panels are color-coded and gated as indicated. C, immunostaining for K8 and K14 demonstrated that GFPhigh cells are enriched for K8 cells, a luminal marker, while GFPlow cells are enriched for K14, a basal marker. Scale bar 50μm. D, quantification of K8 and K14 expression in GFPHigh and GFPLow fractions from MELK-GFP virgin mammary glands. GFPLow and GFPHigh cells express significantly different levels of K8 and K14, p=0.002 and p=0.0164, respectively, n = 3.
Figure 4
Figure 4. Heterogeneous expression of GFP in MMTV-Wnt1/MELK-GFP bitransgenic tumors
A, immunostaining for K8 and K14 in a wild-type MMTV-Wnt1 tumor. Scale bar 50μm. B, whole mount bright field and green fluorescent images of a bitransgenic tumor. Scale bar 50μm. C and D, immunostaining (scale bar 50μm) and quantification of K8 and K14 expression in GFPHigh and GFPLow fractions of GFP Wnt1 tumors. GFPLow and GFPHigh cells express significantly different levels of K8 and K14, p=0.003 and p=0.0215, respectively, n=3. E, flow cytometry analysis of stem/progenitor marker expression in GFPnegative(grey area), GFPlow(blue line), and GFPhigh (green line) cells from a MMTV-Wnt1/MELK-GFP bitransgenic tumor. Note GFPhigh cells express elevated CD24, CD49f, CD61 as compared to GFPlow cells. F, flow cytometry analysis (colored dot plot 5% probability with outliers) of CD61 vs GFP expression in MMTV-Wnt1/MELK-GFP bitransgenic tumor. Semi-transparent shaded areas correspond to GFPhigh (green), GFPlow (blue), and GFPnegative (grey) populations. Horizontal line denotes the level of background fluorescence (CD61 negative cells).
Figure 5
Figure 5. The GFPhigh population in MMTV-Wnt1/MELK-GFP bitransgenic tumors is enriched for tumor initiating cells
A, GFPHigh population is greatly enriched in spheroid forming activity using a sphere forming culture assay under all conditions tested; p<0.05 for all GFPhigh, one-way ANOVA. Proliferating tumorspheres (B, bright field) retain GFP expression (C). Scale bar 100μm. The matrigel cultures of GFPhigh cells have an increased total number of cells (D) but a decreased frequency of sphere forming cells (E). The mammosphere assay is linear for all cell densities and at all passages tested (F). Colony formation efficiency decreased with the first three passages. 1st passage, red, linear regression y = 0.02x + 4□R2 = 1; 2nd passage, green, y = 0.0138x - 0.4401□R2 = 0.9962; 3d passage, blue, y = 0.0084x - 1.9418□R2 = 0.9385). G. Whole-mount GFP fluorescence of a 20 days outgrowth after transplantation of 10 GFPhigh cells derived from WMG49 primary tumor. Scale bar 1mm. H&E (H) and K8, K14 and DAPI (I) staining of a solid tumor developed from transplanted GFPhigh cells clonally derived from WMG300 primary tumor. Scale bar 50μm.
Figure 6
Figure 6. MELK function is required for Wnt1-induced tumorigenesis in vitro and in vivo
Black bars = MELK shRNA, white bars = control shRNA, grey bar = MELK cDNA rescue. A and B, MELK shRNA reduced the frequency of tumorsphere-initiating cells in Wnt1 tumors. B, co-infection with virus expressing the MELK cDNA rescues tumorsphere proliferation; p < 0.05, n = 3. C, the percentage (%) of mCherry-positive cells in tumors (from D) after transduction with MELK shRNA is reduced compared to control shRNA. Unpaired t-test, p=0.0072.
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