Figure 1. MELK signaling pathway
Maternal embryonic leucine zipper kinase (MELK)
As a serine/threonine kinase, Maternal embryonic leucine zipper kinase (MELK) is highly conserved across a variety of mammalian and non-mammalian species. It belongs to the AMPK/Snf1 family since there is a conserved serine/threonine kinase domain in the region of N terminus.
This catalytic domain is followed by a ubiquitin associated (UBA) domain, which is just similar to other AMPK/Snf1 family members. This UBA domain is of paramount importance for MELK’s catalytic activity. At the same time, MELK is activated by the auto-phosphorylation.
MELK was originally found to be expressed during early development of embryos and cellular processes, suggesting that it plays an important role in embryogenesis and cell cycle control. In addition, MELK is also expressed in some human cancers as well as stem cell populations where it shows unique spatial and temporal patterns of expression in specific tissues. MELK makes contributions to a wide range of cellular and biological processes including cell cycle control, cell migration, cell proliferation, apoptosis, cell renewal, cell migration, oncogenesis, embryogenesis, and cancer treatment resistance and recurrence.
Figure 2. Schematic diagram of MELK protein
MELK signaling pathway
MELK signaling cascade
As a regulator, MELK interacts a wide range of proteins and activates some proteins via phosphorylation. It binds tightly to the zinc finger-like protein 9 (ZPR9) and then phosphorylates it, which causes its nuclear accumulation. ZPR9 interacts with the transcription factor v-MYB avian myeloblastosis viral oncogene homolog 2 (B-Myb) in the nucleus. B-Myb is an important regulator of cell differentiation and proliferation. In such case, MELK indirectly enhances its transcriptional activity. At the same time, MELK also interacts with nuclear inhibitor of protein Ser/Thr phosphatase-1 (NIPP1) that is a transcription and splicing factor. MELK functions by binding a threonine-phosphorylated motif to FHA domain of NIPP1. This MELK-NIPP1 interaction is more significant during the process of mitosis, which leads to an inhibition of pre-mRNA splicing. In addition, it also phosphorylates PDK1 at threonine 354 after the interaction, which highly inhibits its activity. CDC25B, another target protein of MELK, is a protein-tyrosine phosphatase that triggers mitosis by activating protein kinase CDK1. In cancer, MELK plays an essential part. It has the interaction with p53 that is of paramount importance in cancer-related regulations. Through the phosphorylation of Ser15 in the amino-terminal transactivation domain of p53, MELK enhances the stability and activity of p53. This further enhances 53-dependent apoptosis and cell cycle arrest.
In melanoma cells, MELK is activated by E2F1 which directly binds to the MELK promoter and increases its level of transcription. E2F1 expression is stimulated by MAPK pathway. MELK can phosphorylate many proteins which are substrates of BRAF or MEK. The inhibition of MELK stops the growth of melanoma and it is resistant to vemurafenib, one of the BRAF inhibitors. In addition, NF-kB pathway can be regulated by MELK via Sequestosome 1 SQSTM1. This regulation accounts for the significant role MELK plays in promoting melanoma growth. ASK1 is phosphorylated by MELK, which increases its activity and stimulates the activation of ASK1-mediated signaling to JNK and p38 kinases. With the interaction of MELK-Smad, transforming growth factor-β (TGF-β) transcription is upregulated. This is required for TGF-β-mediated biological functions including cell growth arrest and apoptosis.
With the discovery of MELK, more and more signaling proteins and pathways that regulate and control the activity of MELK have been discovered. Early biochemical studies found that exogenously expressed murine MELK binds to the zinc-finger-like Zpr9, which leads to the activation of B-Myb, one important oncogenic transcription factor in murine cell lines. MAP kinases (AMPK) are highly associated with MELK. They regulate and affect each other. Especially, the c-Jun NH(2)-terminal kinase (JNK) which is one of the AMPK family proteins shows significant regulation with MELK in a cancer-specific manner. The JNK pathway is indispensable to the regulation of apoptosis, cell proliferation, and inflammatory responses in cancers. Many experimental results suggest that MELK directly binds to c-JUN which is the downstream oncogenic transcription factor target of JNK. This interaction of c-JUN and MELK is negatively affected when the kinase activity of MELK is diminished. This shows that the kinase activity of MELK is necessary for its interaction with c-JUN. Actually, the interaction with JNK/c-JUN is tumor-specific and usually occurs in GSC rather than normal progenitors.
The transcription factor/oncogene FOXM1, a major regulator for cell cycle progression, is usually overexpressed in a wide range of human cancers. MELK can form a protein complex with it and induce the phosphorylation of FOXM1 which facilitates FOXM1 transcriptional activity and induces the expression of various mitotic regulators including CDC25B, Aurora B and Survivin. In addition to direct interaction and phosphorylation of FOXM1 in GSCs, MELK also interacts with and regulates p53, VEGF, and Wnt/β-catenin pathways in cancers including GBM.
Relationship with diseases
Serine/threonine kinases, the kind which MELK belongs to, represent a suitable class of proteins for targeted therapies in cancer. Given that MELK is overexpressed in many human cancers, MELK is likely to take part in tumorigenesis and makes a great contribution. Especially in more aggressive forms of astrocytoma, the expression of MELK is much higher. The similar cases occur in some melanoma, breast cancer, and glioblastoma. In many preclinical studies, MELK is predicted to be a promising potential anticancer target in diverse tumor entities. Based on related studies so far, MELK plays an essential role in oncogenesis. The data have shown that MELK expression is higher in tumor-derived progenitor cells and in cancers of non-differentiated cells. For instance, in mammary tumors, MELK is associated with initiation, namely tumorigenesis. A variety of cell lines that are established from colorectal carcinoma show a much higher level of MELK expression. At the same time, MELK is also related to anti-apoptotic activities of breast cancer cells by interacting with Bcl-G. Bcl-G is one of pro-apoptotic members in the Bcl-2 family. Bcl-G can induce apoptosis which is suppressed after MELK is overexpressed. This greatly promotes mammary carcinogenesis and leads to poor patient survival in breast cancer and glioblastoma multi-forms.
As for the resistance of rectal cancer to chemoradiotherapy, MELK may also make a contribution, as the therapeutic target. In the previous studies, the knockdown of MELK decreases the transformed phenotype of multiple tumor cell lines determined by in vivo xenograft assays as well as in vitro proliferation and anchorage-independent growth. Scientists identified one potent inhibitor which inhibits the expression of MELK significantly. This inhibitor is named thiazole antibiotic siomycin A. In the mouse trails, the growth of tumor in vivo is inhibited after the treatment of glioblastoma with siomycin A. In the model of glioblastoma-derived brain tumor stem cells, siomycin A treatment also shows positive regulation in self-regeneration. It also decreases invasion and induces apoptosis after inhibiting MELK. MELK provides a growth advantage for neoplastic cells and derived tumor progression since it is aberrantly reactivated in cancer stem cells. Even though MELK also maintains a certain level of expression in normal progenitor cells, the dysregulation of MELK may cause carcinogenesis in a series of cell types. All previous studies have indicated that the overexpression of MELK promotes the development of tumors. However, the inhibition of MELK sufficiently affects proliferation and other properties of tumors, which means that MELK-based cancer therapies are promising.