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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.

Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T - Breast Cancer Res. (2007)

Bottom Line: 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).We also found that MELK physically interacted with Bcl-GL through its amino-terminal region.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

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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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.
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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.

Mentions: To assess a possible role of MELK in mammary carcinogenesis, we knocked down the expression of endogenous MELK in the breast cancer cell lines T47D and MCF7 (Figure 2a,b), in which MELK was overexpressed at a high level, by means of the mammalian vector-based RNAi technique (see Materials and methods). We examined expression levels of MELK by semi-quantitative RT-PCR and western blot analyses, and found that the MELK-siRNAs si-#3 and si-#4 significantly reduced its expression at the transcriptional and protein levels compared with scramble-siRNA (SC), si-#1 and si-#2 (Figure 2). Colony formation and MTT assays revealed that both si-#3 and si-#4 significantlysuppressed growth of T47D and MCF7 cells (MTT assay in T47D, P = 0.0003, P = 0.0013; in MCF-7, P = 0.0001, P = 0.0001; unpaired t-test), in concordance with the results showing the knockdown effect of this gene. These results suggest that MELK has a critical role in the growth of breast cancer cells.


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.

Lin ML, Park JH, Nishidate T, Nakamura Y, Katagiri T - Breast Cancer Res. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1851384&req=5

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.
Mentions: To assess a possible role of MELK in mammary carcinogenesis, we knocked down the expression of endogenous MELK in the breast cancer cell lines T47D and MCF7 (Figure 2a,b), in which MELK was overexpressed at a high level, by means of the mammalian vector-based RNAi technique (see Materials and methods). We examined expression levels of MELK by semi-quantitative RT-PCR and western blot analyses, and found that the MELK-siRNAs si-#3 and si-#4 significantly reduced its expression at the transcriptional and protein levels compared with scramble-siRNA (SC), si-#1 and si-#2 (Figure 2). Colony formation and MTT assays revealed that both si-#3 and si-#4 significantlysuppressed growth of T47D and MCF7 cells (MTT assay in T47D, P = 0.0003, P = 0.0013; in MCF-7, P = 0.0001, P = 0.0001; unpaired t-test), in concordance with the results showing the knockdown effect of this gene. These results suggest that MELK has a critical role in the growth of breast cancer cells.

Bottom Line: 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).We also found that MELK physically interacted with Bcl-GL through its amino-terminal region.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus