MELK is an oncogenic kinase essential for mitotic progression in basal-like breast cancer cells
- Dana-Farber Cancer Institute, United States
- Harvard Medical School, United States
- Novartis Institutes for Biomedical Research, United States
- Harvard University, United States
Figures
Figure 1 with 2 supplements
An in vivo kinome-wide screen identifies MELK as a potential oncogenic kinase.
Pools of retroviral vectors encoding 354 human kinases and kinase-related proteins (37 pools in total, each consisting of 10–12 unique open reading frames) were transduced into HMED-DD-NeuT cells. …
Figure 1—figure supplement 1
Development of an in vivo tumorigenesis model.
In telomerase-immortalized human mammary epithelial cells expressing p53DD (HMEC-DD), expression of two potent oncogenes (such as NeuT, PIK3CA [H1047R]) is required to induce these cells to form …
Figure 1—figure supplement 2
Screen hits and their gene description.
https://doi.org/10.7554/eLife.01763.005
Figure 2 with 2 supplements
MELK is highly overexpressed in breast cancer and its overexpression strongly correlates with poor prognosis.
(A) MELK expression levels are significantly higher in breast carcinoma (n = 392, red circles) than in normal breast tissues (n = 61, blue circles) in the TCGA breast cancer cohort (Cancer Genome …
Figure 2—figure supplement 1
MELK is a top-ranking overexpressed gene in breast cancer and a strong prognostic indicator.
(A) MELK expresses at a higher level in breast tumors than in normal breast tissues. (B) MELK expression is positively correlated with the histologic grade of disease. The indicated p values rank 7th…
Figure 2—figure supplement 2
Correlation of MELK expression with breast cancer subtypes and the histologic grade of disease.
(A) Data of MELK expression and the histologic grade of disease among 1556 invasive ductal breast carcinoma (Curtis et al., 2012) were analyzed. Black lines in each group indicate median with …
Figure 3 with 3 supplements
FoxM1 is overexpressed in BBC and regulates the expression of MELK.
(A) High expression of FoxM1 in BBC. Samples in the indicated data sets were grouped into subtypes based on the PAM50 gene signature (Parker et al., 2009). ns, denotes not significant. *p<0.05, …
Figure 3—figure supplement 1
Gene amplification of MELK in BBC.
Gene expression of MELK among ER-positive and ER-negative invasive ductal breast carcinoma (A, n = 389; TCGA, 2012) and breast cancer cell lines (B, n = 55; Barretina et al., 2012). Red circles …
Figure 3—figure supplement 2
FoxM1 is overexpressed in BBC and transcriptionally regulates MELK.
(A) FoxM1 expression in subtypes of breast cancer that are defined by gene expression profiling. Samples in the two indicated cohort was grouped into subtypes based on PAM50 gene signature (Parker …
Figure 3—figure supplement 3
MELK expression in different cell cycle of BBC cells.
The indicated BBC cells were left untreated (Asynchronized, As) or treated with 100 ng/ml nocodazole for 18 hr. Mitotic cells (M) were harvested by shake-off, with the attached cells harvested as …
Figure 4 with 2 supplements
Overexpression of wild-type MELK induces oncogenic transformation.
(A) Immunoblotting analysis of Rat1-DD cells expressing vector, wild-type (WT) allele of human MELK, or an oncogenic allele of PIK3CA (H1047R). Expression of PIK3CA (H1047R) enhances Akt …
Figure 4—figure supplement 1
MELK overexpression promotes tumorigenesis.
HMEC-DD-NeuT cells were transduced with empty vector, myristoylated (myr-) or wild type (WT-) MELK. Cells were transplanted into mammary fat pads of nude mice. The number of injections and tumors …
Figure 4—figure supplement 2
MELK overexpression promotes oncogenic transformation in vitro.
(A) HMEC-DD cells were seeded in 6-well plate coated with 0.6% agar (50,000 cell per well). Bright-field images were captured 4 days after seeding. Cells were also collected for trypin digestion and …
Figure 5 with 3 supplements
MELK is essential for the growth of basal-like breast cancer cells.
(A) MELK expression levels are significantly higher in BBC cell lines than in luminal breast cancer cell lines. The MELK mRNA data in 23 established BBC and 24 luminal breast cancer cell lines were …
Figure 5—figure supplement 1
MELK knockdown in basal and luminal breast cancer cells.
(A) Conditionally knocking down MELK in five basal and five luminal breast cancer cell lines. Cells were exposed to doxcycyclin (100 ng/ml) for 3 days. Cell lysates were subjected to immunoblotting …
Figure 5—figure supplement 2
MELK knockdown does not affect the proliferation of HMECs.
(A) Immunoblotting analysis of MELK protein levels in HMECs in the presence and absence of doxycycline. (B) Quantification of MELK knockdown by q-PCR. (C) Quantification of the growth of HMECs. …
Figure 5—figure supplement 3
Generation of shMELK-resistant MELK cDNA (MELK-R).
Top, the 21-mer sequence targeted by shMELK2 is marked in bold. The silent mutations are indicated by the arrows. Bottom, the indicated shRNA (scramble or shMELK) were co-transfected with plasmid …
Figure 6 with 3 supplements
MELK downregulation induces apoptosis and impairs mitosis in BBC cells.
(A) Immunoblotting analysis of MDA-MB-468 cells carrying a scrambled control or tet-shMELK in the presence and absence of doxycycline. Both cleaved PARP and Caspase-3 levels increased upon MELK …
Figure 6—figure supplement 1
MELK inhibition induces cell death in MDA-MB-468 cells.
(A) Bright-field images of indicated cells untreated or treated with doxycycline for the induction of MELK silencing. (B) DAPI staining of MDA-MB-468 cells with tet-shMELK. The cells were untreated …
Figure 6—figure supplement 2
MELK inhibition induces cell death selectively in basal-like breast cancer cells.
(A) The indicated cells were treated with vehicle or MELK inhibitor (OTSSP167, 100 nM) for 2 days. Bright-field images of cells were captured. (B) Cell viability were treated as in (A) and assayed …
Figure 6—figure supplement 3
MELK inhibition in BBC cells induces cell death and defective mitosis.
(A) Conditionally knocking down MELK in BT549 cells induces the accumulation of cells with 4n DNA content and G2/M arrest. BT549 cells with tet-shMELK were either treated without or with doxycycline …
Figure 7 with 1 supplement
MELK is essential to sustain the oncogenic growth of BBC Cells.
(A) Effects of MELK knockdown on anchorage-independent growth of BBC cells in soft agar. The left and middle panels show crystal violet staining, and bright-field images of the colonies …
Figure 7—figure supplement 1
Efficient conditional MELK knockdown in vivo.
Mice with mammary tumors derived from the indicated cells with stable tet-shMELK were untreated or treated with doxycycline-supplemented water for 4 days. Tumors lysates were used for …
Videos
Video 1
This representative time-lapse video, related to Figure 6, shows a MDA-MB-468/tet-shMELK cell in the presence of doxycycline fails to undergo cytokinesis.
The large frame indicates the initial position of the cell, and small frame its final position. Note that the cell progresses into mitosis, which ends with a double-nuclei cell following failed …
Video 2
This representative time-lapse video, related to Figure 6, shows that in the presence of doxycycline, MDA-MB-468/tet-shMELK/GFP-H2B cells with double nuclei undergo cell death.
The two frames indicate two such cells. Frame rate is five frames per second. Time is given in hours:minutes.
Video 3
This representative time-lapse video, related to Figure 6, shows that in the presence of doxycycline, MDA-MB-468/tet-shMELK/GFP-H2B cells undergo mitosis but ending with asymmetric cell division (in the top frame) or cell death (in the bottom frame).
Frame rate is five frames per second. Time is given in hours:minutes.
Video 4
This representative time-lapse video, related to Figure 6, shows that in the absence of doxycycline, MDA-MB-468/tet-shMELK/GFP-H2B cells demonstrate efficient mitosis.
Frame rate is five frames per second. Time is given in hours: minutes.
Additional files
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Supplementary file 1
Breast cancer gene expression datasets used in this study.
- https://doi.org/10.7554/eLife.01763.030
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Supplementary file 2
Oligonucleotides used in this study.
- https://doi.org/10.7554/eLife.01763.031
