Maternal Embryonic Leucine Zipper Kinase (MELK) as a Novel Mediator and Biomarker of Radioresistance in Human Breast Cancer

Abstract

Purpose: While effective targeted therapies exist for estrogen receptor-positive and HER2-positive breast cancer, no such effective therapies exist for triple-negative breast cancer (TNBC); thus, it is clear that additional targets for radiosensitization and treatment are critically needed.

Experimental design: Expression microarrays, qRT-PCR, and Western blotting were used to assess MELK RNA and protein expression levels. Clonogenic survival assays were used to quantitate the radiosensitivity of cell lines at baseline and after MELK inhibition. The effect of MELK knockdown on DNA damage repair kinetics was determined using γH2AX staining. The in vivo effect of MELK knockdown on radiosensitivity was performed using mouse xenograft models. Kaplan-Meier analysis was used to estimate local control and survival information, and a Cox proportional hazards model was constructed to identify potential factors impacting local recurrence-free survival.

Results: MELK expression is significantly elevated in breast cancer tissues compared with normal tissue as well as in TNBC compared with non-TNBC. MELK RNA and protein expression is significantly correlated with radioresistance in breast cancer cell lines. Inhibition of MELK (genetically and pharmacologically) induces radiation sensitivity in vitro and significantly delayed tumor growth in vivo in multiple models. Kaplan-Meier survival and multivariable analyses identify increasing MELK expression as being the strongest predictor of radioresistance and increased local recurrence in multiple independent datasets.

Conclusions: Here, we identify MELK as a potential biomarker of radioresistance and target for radiosensitization in TNBC. Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target in TNBC. Clin Cancer Res; 22(23); 5864-75. ©2016 AACR.

Figure 1.

MELK is more highly expressed in cancerous tissue and TNBC. Analysis of TCGA breast dataset demonstrates MELK expression is significantly higher in breast tumors (in red) than in normal breast tissue (in green) with FPKM values on the y-axis and individual tumor samples from cancer versus normal on the x-axis (A). Error bars represent ±SD. MELK expression is also significantly elevated in basal-like and TNBCs in the TCGA dataset (B and C). Error bars represent ±SEM. The expression of MELK in 180 breast normal and tumor samples (22 reduction mammoplasty normal and 158 tumors) was measured using RNA-sequencing analysis from the University of Michigan Translational Pathology databank (D). Data are depicted as absolute RPKM values. Differential expression between ER-negative and ER-positive human breast tumors was also confirmed in an institutional breast tumor database using qRT-PCR analysis (E). Expression is depicted normalized to control with error bars representing ±SEM.

Figure 2.

MELK expression is associated with radioresistance. Intrinsic radiosensitivity of 21 breast cancer cell lines (as measured by clonogenic survival assay area under the survival curve, AUC) was assessed and correlated to MELK RNA expression using Pearson correlation. Each dot represents an individual cell line with colors corresponding to intrinsic subtype. Mean centered log₂ RNA expression is depicted on the x-axis and the survival AUC from clonogenic survival assays are depicted on the y-axis (A). Intrinsic radiosensitivity was also correlated to MELK protein expression in 15 breast cancer cell lines with MELK expression relative to the expression in MCF-7 cells as determined by Western blotting (B). Using siRNA knockdown of MELK expression in a radiation-resistant breast cancer cell lines with high baseline MELK expression (MDA-MB-231 and BT549), radiation sensitivity was assessed using clonogenic survival assays. Knockdown of MELK expression confers radiation sensitivity with limited toxicity with an enhancement ratio of 1.55 to 1.62 in MDA-MD-231 cells (C) and 1.48 to 1.52 in BT549 cells (D). MELK knockdown was confirmed in both experiments using Western blotting for MELK expression (E and F). MELK overexpression in the radiosensitive ER-positive breast cancer cell line MCF-7, with low baseline MELK expression and high radiosensitivity confers radioresistance (G). MELK overexpression was confirmed at the protein and RNA level (H and I). All experiments were repeated in triplicate with error bars ±SEM.

Figure 3.

MELK knockdown significantly delays repair of dsDNA breaks after ionizing radiation. Representative images of γH2AX foci at 16 hours are depicted in A. Using siRNA directed against MELK, the effect of MELK knockdown on γH2AX foci formation (B) or fluorescence staining by flow cytometry (D) was evaluated at various times (30 minutes, 4 hours, 16 hours, 24 hours) after 2 Gy of ionizing radiation. The effect on dsDNA break repair caused by inhibition of MELK kinase function using the MELK inhibitor OTSSP167 was also assessed in time course by foci formation (C) or flow cytometry (E). Each experiment was run in triplicate three independent times. Similar results were found using the cell line BT549 (data not shown). Error bars represent SD.

Figure 4.

MELK inhibition significantly reduces xenograft tumor doubling time compared with radiation alone. Knockdown of MELK alone or radiation alone resulted in decreased tumor volume growth in the xenograft model (A), but the combination of radiation and MELK knockdown resulted in a synergistic and statistically significant (P < 0.01) reduction in tumor growth compared with radiation alone, and a tumor volume doubling time nearly four times as long as treatment with radiation alone (B). A similar experiment was performed using wild-type MDA-MB-231 cells injected as above, but this time treatment was with the MELK inhibitor OTSSP167 treated at 10 mg/kg daily by oral gavage. While radiation and MELK inhibitor alone did delay tumor doubling time slightly, combination therapy was significantly more effective at delay tumor growth and doubling time (C and D). MELK expression was assessed by qRT-PCR from xenograft tumors harvested during the fourth week of the experiment (E). A depiction of the experimental design is shown (F). Error bars represent ±SEM.

Figure 5.

MELK expression is associated with increased risk of local recurrence. Kaplan–Meier local RFS analysis in the Wang dataset demonstrates that patients whose tumors have higher than median expression of MELK have significantly higher rates of local recurrence after radiation and an overall poorer prognosis than patients with lower than median expression of MELK (HR for local recurrence, 2.22; P < 0.001; A). Even when divided into quartiles, increasing levels of MELK expression are associated with increased risk of locoregional (B). Similarly, in the Servant dataset, Kaplan–Meier local RFS analysis demonstrates that patients whose tumors have high expression of MELK have significantly higher rates of locoregional (C). Again, quartile expression of MELK demonstrates increasing rates of locoregional with increasing MELK expression (D).