Eukaryotic elongation factor-2 kinase expression is an independent prognostic factor in colorectal cancer

Colorectal cancer (CRC) is the second most common malignant disease and the fourth leading cause of cancer-related death worldwide, accounting for more than 650,000 deaths annually [1]. In particular, CRC incidence in many Asian countries has increased 2- to 4-fold over the last two decades [2]. Despite the recent advancements in CRC treatment, up to 50% patients whom underwent tumor resection experience cancer recurrence, among which another half subsequently developed virtually incurable metastasis. The prognostication of CRC patients remains a clinical challenge with tumor-node-metastasis (TNM) staging as the most commonly used prognostic tool in the clinical setting. However, the clinical value of this system in guiding suitable therapy has recently been questioned. For instance, adjuvant therapy is recommended for all stage III patients with CRC but it remains controversial for stage II patients as its toxicities may outweigh benefits. It is therefore pivotal to identify early-stage CRC patients with predicted gloomy outcomes pending for more aggressive treatment. In this respect, the utility of molecular predictive and prognostic markers has helped forecast clinical outcome and treatment responsiveness for deciding better interventions for CRC patients. For example, KRAS mutation has been established as a negative predictor for response to epidermal growth factor receptor-targeted therapy in patients with metastatic CRC [3]. Also, programmed death (PD)-ligand 1 (L1) expression on tumor cells predicts efficacy of PD-L1 and PD-1 inhibition-based therapies in different types of cancer [4]. Thus, the development of novel prognostic marker for clinical outcome prediction in CRC is highly warranted.

Eukaryotic elongation factor-2 kinase (EEF2K) is a calcium/calmodulin-dependent protein kinase that plays a role in regulating protein synthesis [5]. EEF2K phosphorylates its downstream target eukaryotic elongation factor-2 (EEF2), a GTPase that promotes the translocation of the nascent protein chain from A site to P site on ribosome in elongation phase during mRNA translation [6]. The phosphorylation of EEF2 at threonine 56 by EEF2K inhibits the interaction of EEF2 with ribosome and thereby inhibiting protein elongation. In our recent study, a tumor-suppressive role of EEF2K was observed in CRC, where silencing of EEF2K induced a pro-survival autophagic response through the AMPK-ULK pathway and promoted CRC growth through increasing cell size, viability and clonogenicity. In contrast, overexpressing EEF2K reduced colon cancer cell viability and potentiated the anti-tumor efficacy of the chemotherapeutic drug oxaliplatin [7]. Consistently, De Gassart and co-workers reported that nelfinavir exerted its anti-tumor effect in colon cancer cells in an EEF2K-dependent manner [8]. An in vivo study by Faller and his colleagues also revealed that EEF2K activation led to growth arrest in APC-deficient colorectal adenomas [9]. All these pieces of evidence support EEF2K as a tumor-suppressor gene in CRC. However, the clinical significance of EEF2K downregulation in CRC remains to be established.

In the current study, we investigated the expression of EEF2K in CRC and delineated its relationship with clinicopathological parameters, including survival, in patients with CRC.

EEF2K, together with other enzymes involved in protein synthesis, were traditionally known as cellular housekeepers that play a vital role in cell viability [10]. In our study, we further extended the role of EEF2K as a prognostic biomarker for CRC in which low EEF2K expression foreshadowed worse overall survival independent of other clinicopathological parameters, including age, gender and TNM staging of the patients. This observation is consistent with our previous finding that EEF2K functioned as a tumor-suppressor gene in CRC by inhibiting autophagic survival and synergized with oxaliplatin (a commonly used chemotherapeutic drug in CRC management) to induce colon cancer cell apoptosis [7]. Our findings were further validated in TCGA cohort, in which EEF2K was also downregulated in CRC without association with patients’ clinical features, CRC subtypes or mutational status as in our cohort. However, its clinical relevance with survival outcome of CRC patients was only noted in patients with stage IV CRC. It is aware that the methodologies in assessing EEF2K expression level, and also the ethnicity of patients and underlying genetic background were different and may account for the discrepancy. Also, it should be noted that multiple comparisons were not adjusted for this analysis. Additional dedicated studies are needed for further confirmation. Despite all these, our findings indicated that EEF2K expression, alongside other molecular prognostic markers such as microsatellite instability [11], BRAF mutation status [12], gene expression profiling [13] and multi-gene mutation signature [14], might supplement TNM staging in the future for more accurate prognostication and patient stratification.

The dysregulation of EEF2K and its clinical relevance have been documented in other cancer types. It is noteworthy that, contrary to its tumor-suppressive role in CRC, EEF2K was frequently found to function as an oncogene in other cancer types. Ashour and colleagues reported an overexpression of EEF2K in pancreatic cancer [15]. Meric-Bernstam and colleagues found that EEF2K was overexpressed in breast cancer and, importantly, the expression of EEF2K was positively correlated with poor prognosis in breast cancer patients [16]. A similar finding was also reported in brain cancer by Leprivier and his colleagues [17]. The tissue-specific functions of EEF2K may provide a plausible explanation for such discrepancy. While EEF2K overexpression has been shown to promote cancer cell survival in face of nutrient starvation by inhibiting protein synthesis resulted in limitation of cellular energy exhaustion [18], mTOR complex 1-mediated inhibition of EEF2K was found to be essential for the proliferation of APC-deficient but not wild-type enterocytes [9]. This distinctive synthetic lethality circuit created by APC inactivation in CRC strongly supports the intestine-specific tumor-suppressive effect of EEF2K and its association with better prognosis in CRC patients. This finding also suggests that EEF2K may function as a tumor suppressor in other cancer types, such as endometrial [19] and gastric cancers [20], in which APC inactivation plays a significant oncogenic role. The prognostic role of EEF2K expression in these cancer types also warrants further investigation to support the notion.

Taken together, we found that EEF2K downregulation is independently associated with worse overall survival in CRC patients. To the best of our knowledge, it is the first time demonstrating the clinical relevance of EEF2K expression in CRC patients. The use of EEF2K as a prognostic marker could be promising in identifying high-risk CRC patients to improve their survival with more aggressive treatment.