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. 2007;9(1):R17.
doi: 10.1186/bcr1650.

Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family

Meng-Lay Lin  1 , Jae-Hyun Park, Toshihiko Nishidate, Yusuke Nakamura, Toyomasa Katagiri
Affiliations

Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family

Meng-Lay Lin et al. Breast Cancer Res. 2007.

Abstract

Introduction: Cancer therapies directed at specific molecular targets in signaling pathways of cancer cells, such as tamoxifen, aromatase inhibitors and trastuzumab, have proven useful for treatment of advanced breast cancers. However, increased risk of endometrial cancer with long-term tamoxifen administration and of bone fracture due to osteoporosis in postmenopausal women undergoing aromatase inhibitor therapy are recognized side effects. These side effects as well as drug resistance make it necessary to search for novel molecular targets for drugs on the basis of well-characterized mechanisms of action.

Methods: Using accurate genome-wide expression profiles of breast cancers, we found maternal embryonic leucine-zipper kinase (MELK) to be significantly overexpressed in the great majority of breast cancer cells. To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK. Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.

Results: Northern blot analyses on multiple human tissues and cancer cell lines demonstrated that MELK was overexpressed at a significantly high level in a great majority of breast cancers and cell lines, but was not expressed in normal vital organs (heart, liver, lung and kidney). Suppression of MELK expression by small interfering RNA significantly inhibited growth of human breast cancer cells. We also found that MELK physically interacted with Bcl-GL through its amino-terminal region. Immunocomplex kinase assay showed that Bcl-GL was specifically phosphorylated by MELK in vitro. TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.

Conclusion: Our findings suggest that the kinase activity of MELK is likely to affect mammary carcinogenesis through inhibition of the pro-apoptotic function of Bcl-GL. The kinase activity of MELK could be a promising molecular target for development of therapy for patients with breast cancers.

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Figures

Figure 1
Figure 1
Expression and distribution of MELK in human normal tissues and breast cancer cell lines. (a) Expression of MELK in 12 breast cancer specimens (case number; 42, 102, 247, 252, 302, 473, 478, 502, 552, 646, 769 and 779) by semi-quantitative RT-PCR. GAPDH served as a quantitative internal control. (b) Multiple tissue Northern blot analysis demonstrated that an approximately 2.7 kb MELK transcript was detected in the testis, thymus and small intestine. PBL, peripheral blood leukocytes. (c) Breast cancer cell line Northern blot analysis revealed that approximately 2.4 to 2.7 kb MELK variants were specifically expressed in breast cancer cell lines, but not in normal vital organs. (d) Schematic representation of three variant transcripts identified by cDNA library screening (see Materials and methods). White boxes indicate a coding region and black boxes indicate a non-coding region. Black and grey triangles indicate initiation codons, and white triangles indicate stop codons. Exon numbers are shown above each box. (e) In vitro translation assay of each variant isolated from cDNA library screening. The number within parentheses represents the predicted molecular weight (kDa) of each variant protein. (f) Expression of MELK proteins in eight breast cancer cell lines as well as human mammary epithelial cells (HMECs) shown by western blot analysis with an anti-MELK antibody. β-Actin served as a control. (g) Schematic representation of the V1, V2 and V3 forms of MELK. The shaded boxes indicate the catalytic domain (amino acids 11 to 263 of the V1 protein). The KA1 domain is the kinase-associated domain in the carboxy-terminal region.
Figure 2
Figure 2
Effect of knockdown of MELK by small-interfering RNA (siRNA) on cell viability and proliferation. Four psiH1 promoter-based siRNA constructs (si-#1, si-#2, si-#3 and si-#4) were introduced into (a) T47D and (b) MCF-7 cell lines. SC refers to scramble used as a control for siRNA experiments. Gene silencing was evaluated by semi-quantitative RT-PCR and western blot analyses at four and five days after neomycin selection, respectively. β2-microglobulin (β2MG) was used as a control for normalization of semi-quantitative RT-PCR, and β-actin was used as a control in western blot analysis. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed to evaluate cell viability at 10 days after neomycin selection, and graphed after standardization using the scramble control (SC) as 1.0 (T47D, P = 0.0003, P = 0.0013; MCF-7, P = 0.0001, P = 0.0001; unpaired t-test). Colony formation assays were carried out three weeks after neomycin selection (see Materials and methods). Two siRNA constructs (si-#3 and -#4) showed significant knockdown effects against internal MELK expression and inhibited cell growth in both T47D (a) and MCF-7 (b) cell lines. Values represent the average from triplicate experiments. Error bars indicate standard deviation.
Figure 3
Figure 3
Identification of Bcl-GL as an interacting protein for MELK. (a) Silver staining of SDS-PAGE gels that contained the pulled-down cell lysates. The enlarged area covering Bcl-G shows the differential interaction between it and wild-type MELK (WT-MELK) and kinase-dead MELK (D150A-MELK). (b) Expression of Bcl-GL in eight breast cancer cell lines as well as human mammary epithelial cells (HMECs) by western blot analysis with an anti-Bcl-GL antibody. β-Actin was used as a control. (c) Interaction of MELK with Bcl-GL. Extracts from HeLa-cells transfected with HA (hemagglutinin)-tagged WT-MELK (HA-WT-MELK) or Flag-tagged Bcl-GL (Flag-Bcl-GL), or a combination of these, were harvested 36 hours after transfection. The cell lysates were immunoprecipitated with anti-Flag M2 antibody. Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with an anti-HA antibody. (d) Direct interaction of the MELK and Bcl-GL proteins. The upper panel indicates the amount of input of WT-MELK and D150A-MELK. His-tagged WT-MELK bound to Bcl-GL, but His-tagged D150A-MELK did not. (e) Schematic representation of the amino- and carboxy-terminal deletion constructs of Bcl-GL. The C-1 and C-2 constructs have the BH2 domain deleted, and the C-3 construct has both the BH2 and BH3 domains deleted. (f) Determination of the WT-MELK binding regions of Bcl-GL by immunoprecipitation. The HA-tagged WT-MELK and various peptide sequences of Flag-tagged Bcl-GL (Figure 3e) were pulled down by immunoprecipitation with Flag-M2 antibody and then immunoblotted with rabbit anti-Flag antibody. The expression of HA-tagged WT-MELK in total cell lysates was confirmed by western blotting analysis. As a control, immunoprecipitation was performed from cells co-transfected with pCAGGSn3FC (Mock) and HA-tagged WT-MELK (HA-WT-MELK) through all steps. Arrowheads indicate expression of each Bcl-GL peptide.
Figure 4
Figure 4
MELK phosphorylates Bcl-GL in vitro. (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-GL, and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-GL fusion recombinant protein (GST-Bcl-GL) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-GL protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-GL (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-GL (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-GL proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.
Figure 5
Figure 5
MELK involvement in the apoptosis cascade through Bcl-GL. HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-GL proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t-test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).
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