A possible role of NUAK2 in the PERK-ATF4 unfolded protein response pathway in ovarian cancer

Abstract

NUAK2, also known as sucrose non-fermenting-like AMPK-related kinase, belongs to the AMPK serine/threonine kinase family. While little is known about the exact biological functions of NUAK2, this kinase has been reported to be overexpressed in multiple cancer types. Analysis of public cancer genomic datasets revealed that NUAK2 mRNA is remarkably elevated in ovarian cancer relative to normal ovaries. Therefore, the primary aim of this study is to investigate the functional impact of NUAK2 overexpression on ovarian carcinogenesis and the possible molecular underpinnings.

To demonstrate the clinical significance of NUAK2 overexpression in ovarian cancer, we examined NUAK2 protein expression by immunohistochemistry in a cohort of ovarian tumour tissues and correlated histoscores with various clinicopathological parameters. In concordance with the public databases, NUAK2 expression was significantly higher in ovarian cancer tissues compared to benign and borderline ovarian tumour tissues. Histoscores of nuclear NUAK2 in particular were positively correlated with high grade, and associations between nuclear NUAK2 overexpression and poor overall survival and disease-free survival rates also existed. In vitro functional assays were then performed to determine whether NUAK2 plays roles in regulating proliferation and migration of ovarian cancer cells. Cell proliferation and migration were impeded upon depletion of NUAK2 in ovarian cancer cells as determined by MTT and transwell assays respectively.

We further aimed to understand the physiological roles of NUAK2 that might be implicated in ovarian carcinogenesis by interrogating the functional groups of genes deregulated by NUAK2 in SKOV3 cells by RNA-seq. A list of differentially expressed genes was subjected to pathway enrichment analysis. Among the top-scoring pathways, several pathways where NUAK2 has no known reported function by far were identified. Notably, the A TF4-dependent unfolded protein response pathway implicated in ER homeostasis was significantly enriched. Among the many candidates that were deregulated by NUAK2, a group of ATF4 target genes was further validated. Quantitative-PCR showed downregulated ATF3, PPP1R15A and SNAI2 mRNA upon depletion of NUAK2 in SKOV3 and OVCAR3. Reactive oxygen species (ROS) quantification by flow cytometry demonstrated a reduced cytosolic ROS level after NUAK2 knockout in SKOV3. We further revealed by Western blotting and subcellular fractionation that ER stress-induced ATF4 expression and thereby its nuclear entry were impeded upon depletion of NUAK2 in SKOV3, possibly through the regulation of the upstream kinase PERK at the protein level.

Together, this study characterised NUAK2 overexpression in ovarian cancer, which is functionally required for cell growth and migration, and also pointed to the prognostic value of its nuclear form in predicting tumour aggressiveness and disease outcomes of ovarian cancer patients. This study also revealed a hitherto novel role of NUAK2 in regulating the unfolded protein response pathway as ATF4 target genes are enriched in the NUAK2 transcriptome. Functional findings suggested the potential role of NUAK2 in maintaining ER homeostasis by counteracting cytotoxic ROS accumulation in ovarian cancer cells. Mechanistically, NUAK2 acts as a facilitator in promoting the nuclear import of ATF4 via its potential regulation on PERK.