Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in gastric cancer

Abstract

Maternal embryonic leucine zipper kinase (MELK) is upregulated in a variety of human tumors, and is considered an attractive molecular target for cancer treatment. We characterized the expression of MELK in gastric cancer (GC) and measured the effects of reducing MELK mRNA levels and protein activity on GC growth. MELK was frequently overexpressed in primary GCs, and higher MELK levels correlated with worse clinical outcomes. Reducing MELK expression or inhibiting kinase activity resulted in growth inhibition, G2/M arrest, apoptosis and suppression of invasive capability of GC cells in vitro and in vivo. MELK knockdown led to alteration of epithelial mesenchymal transition (EMT)-associated proteins. Furthermore, targeting treatment with OTSSP167 in GC patient-derived xenograft (PDX) models had anticancer effects. Thus, MELK promotes cell growth and invasiveness by inhibiting apoptosis and promoting G2/M transition and EMT in GC. These results suggest that MELK may be a promising target for GC treatment.

Keywords: MELK; PDX; gastric cancer; metastasis; prognosis.

Figure 1. MELK expression in cultured GC cells and primary GC tissues, and survival in patients with GC

A. Western blotting analysis of MELK in GC cell lines. B. MELK expression in primary GC (T) and distal surgical margin normal mucosa (N) were examined by Western blotting. C. Expression of MELK by immunohistochemical staining. Original magnification: 200x. NS: normal stomach; IM: intestinal metaplasia; T: gastric cancer. D. Kaplan-Meier survival curves of overall survival for all patients with MELK-negative vs. -positive GC tissue.

Figure 2. MELK knockdown attenuated migration and invasion of GC cells in vitro and in vivo

A. MELK protein expression. Lane 1, wild type; Lane 2, scrambled shControl; Lane 3, shMELK. B. Morphologic changes in GC cells in response to MELK knockdown. Original magnification: ×100. C-F. BGC823 and SGC7901 cells were assayed for their invasive capability on Matrigel or Boyden chambers. Bars represent the mean ± SD of three independent experiments. G. The effect of MELK on metastatic colonization through blood circulation. *p< 0.05, **p< 0.01, _***p< 0.001.

Figure 3. Effects of MELK knockdown on GC cell growth in vitro and in vivo

A. Cell proliferation assay using a Cell Counting Kit-8. B. Photograph showing tumor formation in nude mice injected with BGC823-shContral and shMELK, as well as tumor growth curve. The data are shown as mean ± SD. *p< 0.05, **p <0.01, ***p< 0.001.

Figure 4. Targeting MELK expression or MELK kinase activity results in G2/M arrest and apoptosis

A. Cell cycle analysis by Flow cytometry. B. Apoptosis assessed by Annexin V and PI staining in BGC823 and SGC7901 cells treated with different concentrations of OTSSP167. C. Histogram of cell cycle distribution. Bars, mean ± SD of three independent experiments. D. Histogram of cell apoptosis. Bars, mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. MELK regulates the expression of EMT-associated proteins and AKT activity

A and B. Expression profiles of EMT components in response to MELK knockdown determined using Western blotting. Lane 1, wild type; Lane 2, scrambled shControl; Lane 3, shMELK. © Altered expression of EMT-associated proteins and Akt activity in BGC823 cells treated with LY294002.

Figure 6. OTSSP167 reduces growth of MELK-positive GC-PDX TumorGraft

A, B and C. IHC for MELK in patients' tumors. A. MELK-positive staining in the case #1 GC patient-derived TumorGraft. B. MELK-positive staining in the case #3 GC patient-derived TumorGraft. C. MELK-negative staining in the case #2 GC patient-derived TumorGraft. F0, primary GC tissue; F1, 1^st generation of TumorGraft. Original magnification: 200x. D, E and F. NOD/SCID mice bearing TumorGraft of the F3 generation were intravenously treated with OTSSP167 or vehicle control once every two days for two weeks. Tumor volume was measured every two days (right panel), and MELK expression in TumorGraft tissue after treatment was detected by IHC (middle panel). Tumor volume is shown as mean ± SD.