Maternal Embryonic Leucine Zipper Kinase (MELK) Serves As A Poor Prognosis Marker And Therapeutic Target In Osteosarcoma

Objective:To investigate the role of MELK in proliferation and metastasis of osteosarcoma.Background:Osteosarcoma is the most common primary malignancy of bones and frequently affects young children and adolescents.There are several challenges associated with treating osteosarcoma owing to the aggressiveness of the disease,as well as the risk of chemoresistance.Numerous studies are being performed with the aim of identifying improved prognostic and therapeutic markers for this malignancy.Maternal embryonic leucine zipper kinase(MELK)is an oncogene that has been studied in several types of cancer in recent years.In the present study,the expression of MELK in osteosarcoma and normal tissue samples was examined,and the effects of MELK expression on osteosarcoma cellular proliferation,metastasis,the cell cycle and apoptosis were demonstrated using CCK-8,wound healing,migration and invasion and apoptosis assays.The role of MELK in cancer progression in osteosarcoma was determined,revealing the association between MELK expression and prognosis of osteosarcoma.It was demonstrated that knockdown of MELK resulted in reduced proliferation,migration and invasion in vitro along with potentiation of apoptosis and cell cycle arrest.Furthermore,the effect of the targeted MELK inhibitor,OTSSP167,on tumor progression of osteosarcoma in vitro and in vivo was assessed.Mechanistically,it was demonstrated that MELK promoted osteosarcoma proliferation and metastasis by regulating PCNA and MMP9 expression via the PI3K/Akt/mTOR signaling pathway.Thus,the present study revealed the oncogenic role played by MELK,and established MELK as a valuable prognostic and therapeutic marker in osteosarcoma.This research has been divided into following parts:1.The expression profile of MELK in osteosarcoma and its correlation with the clinicopathological parameters.2.The effect of MELK in the biological functions of osteosarcoma and the mechanisms involved.3.The role of OTSSP167 in the tumor progression of osteosarcoma.Part 1.The expression profile of MELK in osteosarcoma and its correlation with the clinicopathological paramentersBackground:MELK expression is upregulated in several types of cancer,and its upregulated expression is associated with a less favorable prognosis in cancer.In the present study,MELK expression was revealed to be significantly upregulated in osteosarcoma tissues compared with normal tissues.Compared with the hFOB1.19 cell line,MELK expression levels were also upregulated in the MNNG/HOS osteosarcoma cell line.Analysis of the clinicopathological characteristics revealed that upregulated expression of MELK was closely associated with metastasis and chemotherapy response in patients with osteosarcoma.Furthermore,MELK expression was associated with the OS of the patients;patients with high MELK expression levels had a worse prognosis than patients with low expression.Method:The differential expression level of MELK in osteosarcoma and normal control samples was assessed.Real-time PCR was used to analyze the expression of MELK at mRNA levels and immunohistochemistry were used to quantify the protein expression levels of MELK between the cancer and the control samples.Then standard statistical methods were employed to determine the clinicopathological significance of the differential expression of MELK in osteosarcoma.Results:1.RT-PCR analysis showed a significantly higher expression of MELK mRNA in Osteosarcoma cells MNNG/HOS compared to the control group hFOB1.19 cells.(p<0.05).2.Immunohistochemical staining showed MELK was overexpressed in the osteosarcoma compared to osteoblastoma.3.There was statistically significant correlation between high expression of MELK and clinicopathological parameters like metastasis and response to chemotherapy.4.Kaplan-Meier survival curves revealed lower overall survival in the patients with high MELK expression based on data obtained from the TARGET database.Conclusion:In the present study,MELK expression was revealed to be significantly upregulated in osteosarcoma tissues compared with normal tissues.Compared with the hFOB1.19 cell line,MELK expression levels were also upregulated in the MNNG/HOS osteosarcoma cell line.Analysis of the clinicopathological characteristics revealed that upregulated expression of MELK was closely associated with metastasis and chemotherapy response in patients with osteosarcoma.Furthermore,MELK expression was associated with the OS of the patients;patients with high MELK expression levels had a worse prognosis than patients with low expression.Part 2.The effect of MELK in the biological functions of osteosarcoma and the mechanisms involvedBackground:In several studies MELK has been associated with several important processes that determine the fate of several cancers.Some of these processes are proliferation,migration,invasion,apoptosis etc.In our study we explored the functional role of MELK in culmination of the biological behaviors of osteosarcoma.Method:MELK expression was knocked down in the MNNG/HOS osteosarcoma cell line using si-MELK.Knockdown of MELK activity resulted in a significant decrease in cellular proliferation and metastatic behavior.MELK has previously been revealed to regulate proliferation,migration and invasion.We used CCK-8 and flowcytometric cell cycle analysis to assess the proliferative potential.Wound healing assay and transwell was used to see the variance in migratory and invasive capacities and apoptotic potential was analyzed by flowcytometry.In addition to identifying the role of MELK in osteosarcoma,the underlying mechanism was also identified.PCNA is a known oncogene and has been implicated in the proliferation of cancers while MMPs are proteolytic enzymes that serve a role in extracellular matrix remodeling,affecting cell growth,differentiation,migration,invasion and angiogenesis.The PI3K/AKT/mTOR pathway is associated with a variety of cellular functions and is known to serve a distinct role in cancer progression.In this study we hypothesized that MELK affected the proliferation and migration of osteosarcoma cells by regulating PCNA and MMP9 expression via the PI3K/Akt/mTOR signaling pathway.Results:1.The results of western blot showed a significant decline in MELK expression level after transient transfection in the respective cells.2.CCK-8 assay showed a significant decrease in the proliferative potential of osteosarcoma cell lines after transient transfection3.Trasnwell assay showed a lower migration and invasion in the cell lines transfected with siRNAs compared to the respective controls.4.The change migratory potential of cells after knockdown of MELK was also studied using wound healing assay which showed a diminished potential of migration in MELK knockdown group.5.Flowcytometry showed an arrest of the cell cycle and a higher apoptotic potential in the cell lines after knockdown of MELK.6.Western blot analysis of changes in protein expression following knockdown of MELK.Cells transfected with si-MELK exhibited lower expression levels of p-PI3K p85(Tyr485),p-AKT(Ser473),p-mTOR,MMP9 and PCNA.Conclusions:MELK expression was knocked down in the MNNG/HOS osteosarcoma cell line using si-MELK.Knockdown of MELK activity resulted in a significant decrease in cellular proliferation.MELK has previously been revealed to regulate proliferation,migration and invasion.In agreement with the previous studies,suppressing MELK in the MNNG/HOS osteosarcoma cell line reduced proliferation.Part 3.The role of OTSSP167 in the tumor progression of osteosarcomaBackground:OTSSP167 is a potent MELK inhibitor,which has been demonstrated to possess antitumor properties in several types of cancer.OTSSP167 is a protein kinase inhibitor that prevents MELK phosphorylation and abrogates its downstream substrates.OTSSP167 is being developed as an anticancer drug for patients with solid tumors,which have not responded to established modes of treatment,and is emerging as a promising therapeutic for treatment of several types of solid tumors.In the present study,OTSSP167 served an inhibitory role in osteosarcoma progression through suppression of MELK.Methods:The cells were seeded into 96-well plates.The plate was incubated at 37℃for 24 h,and subsequently treated with various concentrations of OTSSP167(0,2,4,8,16,32 and 64 nM)for 48 h.For evaluation of cell viability,10 μl CCK-8 reagent was added to each well and incubated at 37℃ for 4 h.The cell viability was determined by measuring the absorbance at 450 nm using Varioskan microplate reader.For tumorigenesis assays,1x107 MNNG/HOS cells,resuspended in 200 μl PBS,were injected subcutaneously into the armpits of the mice.The tumor volume was measured daily using a Vernier caliper,with the following formula:1/2 x a x b2 where’a’ and ’b’ represent the length and width of the tumor,respectively.After 7 days,when the tumor volumes reached～100mm3,the tumor bearing mice were orally fed Vehicle or OTSSP167(10 mg/kg,daily).The tumor volumes were measured daily using Vernier calipers.After 7 days,when the maximum tumor volumes reached～1,000 mm3 the mice were euthanized using cervical dislocation method and then the growth of the tumors was examined.Results:1.OTSSP167,a MELK inhibitor,was used to assess its effects on proliferation.First,the IC50 of OTSSP167 was determined,which was determined to be 40 nM/ml.2.There was also a dose dependent decrease in MELK expression levels following treatment with OTSSP167.3.Using 40 nM OTSSP167,the proliferation of cells treated with vehicle or OTSSP167 was assessed.There was a significant decrease in the proliferation of cells treated with OTSSP167.4.The tumor volume exhibited a gradual decrease in the group fed OTSSP167 compared with the control group fed vehicle.There was a significant difference in the tumor weight between the two groups.5.To evaluate the results,the protein expression levels were assessed between the groups,and the expression of MELK was revealed to be lower in the mice fed OTSSP167.Conclusion:In the present study,OTSSP167 served an inhibitory role in osteosarcoma progression through suppression of MELK.When xenografted tumors were produced in nude mice and were orally fed OTSSP167,there was a gradual decrease in the size of tumors over time,whereas the control group,which was fed the vehicle alone,possessed tumors which grew over time.This is in agreement with the in vitro proliferation assays and strongly supports the hypothesis that MELK is responsible for tumor progression and its knockdown or inhibition by siRNA or OTSSP167,respectively,could reduce tumor progression.