Exploring the Role of MEK1/2 Kinases in Cellular Functions and Cancer Treatment

探討MEK1/2激酶在細胞功能和腫瘤治療中的作用

Student thesis: Doctoral Thesis

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Award date3 May 2024

Abstract

The MAPK/ERK pathway is a fundamental signaling pathway that regulates cell homeostasis. It is commonly depicted as a linear RAS–RAF–MEK–ERK signaling cascade, which transduces extracellular signals into cells to regulate cell proliferation, differentiation, and survival. Dysregulation of the MAPK/ERK cascade is associated with cancer and other human diseases. Extensive research has focused on this pathway, leading to the development of drug inhibitors for cancer therapy.

Cisplatin, the first and one of the most effective platinum-based chemotherapeutic drugs, is utilized in the treatment of diverse cancer types. However, drug resistance and multiple undesirable side effects limit its curative potential. A promising approach to overcome drug resistance and mitigate toxicity is the combination of cisplatin with other therapeutic drugs. Cisplatin has been shown to induce apoptosis and intersect with the MEK/ERK signaling, but the underlying mechanism remains elusive. MEK1 and MEK2 are core transducers of the MEK/ERK cascade, playing a critical role as "gatekeeper" kinases that activate ERK1 and ERK2 by phosphorylating them at conserved threonine and tyrosine residues. They regulate cell proliferation, survival, and differentiation. Although MEK1 and MEK2 share high homology in size and structure, studies have suggested that they have nonredundant roles. However, it is still unclear how MEK1 and MEK2 function distinctively within broader signaling networks. My thesis work focuses on MEK signaling and contributes in the following two aspects:

In the first part, we investigated the MEK signaling pathway to develop synergistic strategies to improve the anti-cancer efficacy of cisplatin. Cell viability analysis showed that cisplatin was not efficient against several lung cancer cell lines. Biochemical assays indicated that low-dosage cisplatin induced the dissociation of CRAF from MEK1 and decreased the phosphorylation levels of MEK1/2. However, at high cisplatin dosages that effectively killed cells, the phosphorylated MEK1/2 levels increased. This raised our interest in exploring whether manipulating the MEK/ERK signaling could improve the cell's sensitivity to cisplatin. Dabrafenib is an approved drug that inhibits the RAF kinase upstream of MEK. We found that low dosages of dabrafenib and cisplatin exhibited synergistic effects. Mechanistically, dabrafenib obstructed the cisplatin-induced disassociation of CRAF from MEK1, leading to the accumulation of CRAF in the CRAF-BRAF-MEK complex and a high level of phosphorylated MEK. We also explored whether trametinib, an approved MEK inhibitor, could further enhance the synergistic effects of cisplatin/dabrafenib. The combinatorial dabrafenib/trametinib treatment substantially reduced the concentration of cisplatin needed to achieve effective killing of lung cancer cells. Overall, my work demonstrated that steering the MEK/ERK pathway could effectively reduce the dosage of cisplatin and achieve synergistic killing.

In the second part, we developed a proximity-based phospho-interactome (Prob-PhI) platform to dissect the interactome and substrates of MEK1 and MEK2. Briefly, the MEK1 and MEK2 kinases were fused with the engineered biotin ligase, named as BASU, followed by a self-cleaving T2A peptide and an enhanced green fluorescent protein (EGFP) to monitor their expression in living cells. Upon biotin addition, BASU empowers the proximity labeling that captures stable and transient interactors, of MEK1/2, including their substrates. To pinpoint the substrates of MEK1 and MEK2 kinases, we applied trametinib to block the MEK1 and MEK2 kinase activity and cross-validate the reduction of substrates phosphorylation. The biotinylated proteins were then captured employing streptavidin pull-down. After trypsin digestion and phosphor-peptides enrichment, the total interactome and substrates of MEK1 and MEK2 were analyzed by LC-MS/MS. During the study, a comprehensive analysis identified 182 proteins as potential interactors of MEK1, while 253 proteins were identified as potential interactors of MEK2. The phosphoproteome analysis revealed a rich landscape, encompassing 4,059 phosphosites distributed among 1,133 parental proteins associated with MEK1, and 4,286 phosphosites distributed among 1,173 parental proteins associated with MEK2. Among these phosphoproteins, 23 proteins were identified as candidate substrates of MEK1, and 26 proteins were identified as candidate substrates of MEK2. Further comparing the substrates between MEK1 and MEK2, 17 proteins were identified as unique substrates of MEK1, and 20 proteins were identified as unique substrates of MEK2. Additionally, 6 proteins were found to be substrates of both MEK1 and MEK2. Further exploration through gene ontology (GO) enrichment analyses revealed that the candidate substrates of MEK1 were involved in diverse cellular processes, including gene transcription, and metabolic processes. On the other hand, the candidate substrates of MEK2 were associated with chromatin remodeling and histone post-translational modifications, such as acetylation and methylation. Notably, the lysosome-associated membrane glycoprotein 3 (LAMP3 or DC-LAMP) was validated as a unique interacting protein of MEK2 but not MEK1. Functional validation revealed a novel MEK2-specific activity in regulating the increase of LAMP3-induced lysosomes. In brief, MEK2 phosphorylates LAMP3 at the Thr201 site to maintain the protein stability of LAMP1 and promote the functional increase of lysosomes. Taken together, the new Prob-PhI method discerns the signaling activities of MEK1 and MEK2 by delineating the distinct interactome and substrates of MEK1 and MEK2. The robust performance of Prob-PhI could also be extended to dissect other kinases.

In conclusion, my thesis work revealed a strategy to target MEK signaling to sensitize NSCLC cells for cisplatin treatment. In addition, the Prob-PhI platform discerned the signaling activities of MEK1 and MEK2 and revealed a specific activity of MEK2. Overall, my studies provided a novel target-based approach for cancer treatment and developed a new method to dissect kinases in signaling pathways.