LSR promotes epithelial ovarian cancer cell survival under energy stress through the LKB1-AMPK pathway
Introduction
Lipolysis-stimulated lipoprotein receptor (LSR), a type I transmembrane protein, was originally identified as a lipoprotein receptor in the liver [1]. LSR is also known as a tricellular tight junction protein in epithelial cells [2]. Our group reported that LSR was overexpressed in the tissues of epithelial ovarian cancer (EOC), and high LSR expression was associated with a poor prognosis in EOC [3]. However, the biological significance of LSR expression in cancer cells remains unclear.
Previously, Czulkies and colleagues illustrated that LSR-knockout CaCo-2 cells, a colorectal cancer cell line, formed much smaller tumors than their wild-type counterparts in vivo [4]. Additionally, our group demonstrated that anti-LSR monoclonal antibody for EOC and gastric cancer cell lines inhibited tumor growth in vivo by blocking lipid uptake [3,5]. These reports suggest that LSR plays an indispensable role in the tumor growth of several cancers in vivo. However, wild-type CaCo-2 cells exhibited lower proliferative potential than LSR-knockout CaCo-2 cells in growth medium in vitro [4], which seem to be a paradox in LSR expressing cancer biology. In general, tumor cells are exposed to energy stress, including glucose deprivation, low amino acid levels, and hypoxia because of insufficient vascularization in vivo [6]. Taken together, LSR expression may be a growth advantage for tumor cells especially under energy stress. Energy stresses increase intracellular AMP levels corresponding to the depletion of intracellular ATP.
AMP-activated protein kinase (AMPK) functions as a regulator of metabolism under energy stress. The active form of AMPK, phospho-AMPKα (Thr172), suppresses ATP consumption and promotes ATP production through acetyl-CoA carboxylase (ACC) phosphorylation, which protects cancer cells from oxidative stress [7]. Shaw, R. J. and colleagues illustrated a model to explain the apparent paradox that loss of liver kinase B1 (LKB1); an apical-basal cell polarity protein also known as Par 4; in tumors can result in increased cell growth under basal conditions, yet LKB1 deficient cells readily undergo apoptosis under the condition that elevate AMP [8]. The model suggests that LSR deficient tumor cells are close to LKB1 deficient tumor cells in biological features. Interestingly, Sebbagh and colleagues have reported that E-cadherin regulated LKB1 localization at the cell membrane and the expression of phospho-AMPKα (Thr172) in apical-basal polarized epithelial cells [9]. Therefore, we investigated whether LSR also promotes LKB1-mediated AMPK phosphorylation and changes the response under energy stress in EOC cells.
In this paper, we evaluated AMPK activity in EOC cells expressing LSR via Western blotting and examined LKB1 localization using immunocytochemistry. In summary, LSR increased EOC cell survival through the LKB1-AMPK pathway under energy stress.
Section snippets
Materials
5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and antibodies recognizing for LSR, AMPK alpha subunits (AMPKα) (Thr172), phosphor-AMPKα (Thr172), phospho-ACC (Ser79), ACC, phospho-stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185), SAPK/JNK, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Tokyo, Japan). Ionomycin, 2-Deoxy-d-glucose (2-DG) and antibodies recognizing for FLAG M2 were purchased from
LSR is required for the growth of EOC tumor xenografts in vivo
To examine the effect of LSR expression on EOC tumor growth in vivo, we first established clones of OVISE cells stably expressing control scramble shRNA (OVISE-scramble) or stably expressing LSR shRNA (OVISE-LSR #1 and OVISE-LSR #2) via shRNA-expressing plasmid vector transfection. LSR levels were confirmed using Western blotting (Fig. 1a). After establishing tumor xenografts of these clones in athymic nude mice, we found mice inoculated with OVISE-LSR #1 (114.2 ± 15.9 mm3, 23 ± 4 mg) or
Discussion
High expression of LSR is a poor prognosis factor in EOC patients. Interestingly, previous reports suggested that LSR had an indispensable role in the growth of tumor xenografts of several cell lines in vivo [[3], [4], [5]]. In the present study, we demonstrated that LSR promoted EOC cell survival under glucose deprivation through the LKB1-AMPK pathway.
In previous reports, AMPK activation by LKB1 was reported to suppress ATP consumption by inhibiting anabolic pathways and stimulating catabolic
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by Japan Agency for Medical Research and Development (AMED) under Grant No. JP17im0210606, JP20im0210111 and the Japanese Ministry of Education, Science, Sports, and Culture Grant-in-Aid for challenging Exploratory Research (16K14637).
We would like to thank M. Inoue for useful discussions. We also thank Y. Kanazawa and M. Yamamoto for their secretarial assistance and R. Nishi and for technical assistance. The authors would like to thank Enago (www.enago.jp) for the
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