Functional characterization of cancer-related mutations of ERK3

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer. Importantly, ERK3 promotes cancer cell migration and invasion by phosphorylating steroid receptor activator 3 (SRC-3), hence upregulating pro-invasive matrix metalloproteinase genes. While the link between ERK3 and cancers has been recognized, little is known about ERK3 mutations in cancer progression. In this study, we have investigated ERK3 mutations on arginine 64 (arginine 64 mutated to cysteine or histidine, R64C or R64H) and leucine 290 (leucine 290 mutated to proline or valine, L290P or L290V) that are found in cancers of lung, large intestine and skin (COSMIC database). Interestingly, both R64 and L290 residues are located in the kinase domain of ERK3 and are conserved in all ERK isoforms (ERKs1-4). In order to characterize these mutations, we generated expression vectors coating plasmids of ERK3 and each ERK3 constructs containing point mutation, and then overexpressed them in HeLa cells and lung cancer cell lines with or without stable knockdown of endogenous ERK3. Notably, we found that all of these cancer-related mutations lead to reduction of ERK3 phosphorylation at S189 within the activation loop. To study the functional impact of these ERK3 mutations on cancer cell invasiveness, we expressed each of these mutants or the wild type (WT) ERK3 in HeLa cells and lung cancer cell lines with or without stable knockdown of endogenous ERK3 and performed trans-well migration and invasion assays. Interestingly, we found that in comparison with WT ERK3, both L290P and L290V mutations significantly increased ERK3’s ability in promoting cell migration and invasion, whereas R64C and R64H mutations resulted in a decrease in cell migration. Given that all these cancer-related mutations led to reduction of ERK3 phosphorylation at S189, these results suggest that S189 phosphorylation within the activation loop is not associated with ERK3’s function in promoting cancer cell motility. To elucidate the underlying mechanism by which ERK3 L290 mutants increase cancer cell invasiveness, we examined the kinase activity of WT and mutant ERK3 in vitro by assessing the phosphorylation of SRC-3, a substrate for ERK3. We found that ERK3 L290P and L290V mutants have similar kinase activity to that of WT ERK3. ERK3 protein is known to shuttle between the nucleus and the cytoplasm, which may alter its function. Interestingly, by immunofluorescent staining, we found that both L290P and L290V mutations greatly increased the cytoplasmic localization of ERK3 proteins, whereas WT ERK3 is mostly nuclear. In conclusion, this study demonstrates that cancer-related L290P and L290V mutations lead to increased ability of ERK3 to promote cancer cell migration and invasion, possibly through their increased cytoplasmic localization

Increased Prevalence of EBV Infection in Nasopharyngeal Carcinoma Patients: A Six-Year Cross-Sectional Study

Epstein Barr Virus (EBV) is implicated in the carcinogenesis of nasopharyngeal carcinoma (NPC) and currently associated with at least 1% of global cancers. The differential prognosis analysis of NPC in EBV genotypes remains to be elucidated. Medical, radiological, pathological, and laboratory reports of 146 NPC patients were collected retrospectively over a 6-year period between 2015 and 2020. From the pathology archives, DNA was extracted from tumor blocks and used for EBV nuclear antigen 3C (EBNA-3C) genotyping by nested polymerase chain reaction (PCR). We found a high prevalence of 96% of EBV infection in NPC patients with a predominance of genotype I detected in 73% of NPC samples. Histopathological examination showed that most of the NPC patients were in the advanced stages of cancer: stage III (38.4%) or stage IV-B (37.7%). Only keratinized squamous cell carcinoma was significantly higher in EBV negative NPC patients compared with those who were EBV positive (OR = 0.01, 95%CI = (0.004–0.32; p = 0.009)), whereas the majority of patients (91.8%) had undifferentiated, non-keratinizing squamous cell carcinoma, followed by differentiated, non-keratinizing squamous cell carcinoma (7.5%). Although NPC had metastasized to 16% of other body sites, it was not associated with EBV infection, except for lung metastasis. A statistically significant reverse association was observed between EBV infection and lung metastasis (OR = 0.07, 95%CI = (0.01–0.51; p = 0.008)). Although 13% of NPC patients died, the overall survival (OS) mean time was 5.59 years. Given the high prevalence of EBV-associated NPC in our population, Saudi could be considered as an area with a high incidence of EBV-associated NPC with a predominance of EBV genotype I. A future multi-center study with a larger sample size is needed to assess the true burden of EBV-associated NPC in Saudi Arabia