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Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome. Hepatocellular carcinoma HCC is the fifth most prevalent primary cancer and the third leading cause of cancer-related mortality worldwide. NAFLD is characterized by hepatic fat accumulation and is closely associated with central obesity, diabetes, and other features of the metabolic syndrome 2. The endoplasmic reticulum ER stress response has recently been reported to play a crucial role in the development of NAFLD and to be linked to dysfunction of lipolysis, insulin resistance, inflammation, and cell apoptosis 4.
Clinical histological assessments describe NAFLD as a macrovesicular steatosis in which the nucleus is displaced by large lipid droplets LD to the edge of the cell 5. The ER regulates lipid metabolic processes. Lipolysis has been classically recognized to occur through the actions of the cytosolic neutral lipase that hydrolyzes LD-stored triglycerides TGs into free fatty acids FFAs and glycerol. Recognition of similarities in regulation and function of lipolysis and macroautophagy led to the identification of a specific form of autophagy termed lipophagy that degrades LD-sequestered lipids 7.
Considering the important role of lipid metabolism in liver homeostasis, dysfunctional lipophagy may be a contributing factor in hepatic lipid metabolism during the development of NASH and HCC 8. On the other hand, the breakdown of stored lipids from LDs into FFA not only provides energy to the cell, but also modulates other cellular processes, such as activating carcinogenic signaling pathways that contribute to cancer development 8.
Growing evidence supports the notion that macroautophagy can be used by hepatic cancer cells for tumor progression. In fact, increased levels of macroautophagy markers, such as light chain 3 LC3 in hepatocarcinoma, have been associated with poor prognoses and higher rates of recurrence after surgery 9 , The molecular mechanisms linking the disordered metabolic programming to liver tumorigenesis remain elusive.
We discovers the hitherto unrecognized function of Nogo-B as a liver metabolic regulator that reprograms the lipophagy-mediated LD degradation to enhance oncogenic Yes-associated protein YAP activity. These findings demonstrate the functional significance of Nogo-B and provide a strong impetus for therapeutic interventions.
Ten genes were differentially expressed 2 fold-difference in tumors and paired adjacent normal tissues Fig. However, little is known about its ER-related function in tumorigenesis and related metabolic processing. Nogo-B is highly expressed and promotes tumorigenesis in HCC.
The tumors were collected after 4 weeks. In all, 1. The mice were sacrificed after 5 weeks. Source data are provided as a Source Data file.
Additionally, we checked the Nogo-B gene amplification level in HCC patients using the cBioportal and Oncomine websites and found that the DNA copy number of Nogo-B in HCC tumors was higher compared with adjacent non-tumor liver tissues or normal livers from healthy donors in three cohorts.
To assess the functional role of Nogo-B in oncogenesis, colony formation and cell proliferation assays were performed. After subcutaneous injection, LO2 cells with Nogo-B overexpression displayed faster and more sustainable tumor growth in the xenograft model compared with empty vector control group Fig.
We further verified these in vivo findings using another orthotopic model in which 1. Both the tumor volume and weight of the Nogo-B orthografts were increased while Nogo-B knockdown orthografts were decreased when compared with vector or control orthografts, respectively Fig. Taken together, these in vitro and in vivo results suggest that Nogo-B exhibits strong oncogenic properties in HCC. To explore the underlying molecular mechanisms of Nogo-B upregulation, we compared the Nogo-B expression level in tumors from HCC patients with different etiological factors.
The cluster of differentiation 36 Cd36 was identified as the most stimulated gene Fig. A well-known scavenger receptor, CD36 has been reported as a potential diagnostic and treatment target in NAFLD progression for hepatic inflammation via mediating uptake of FFA and some modified low-density lipoprotein, especially for oxidized low-density lipoprotein oxLDL 13 , Notably, knockdown of Nogo-B in the livers of obese mice Fig.
The reduced tumorigenicity was associated with a significant reduction in hepatic steatosis, hepatocyte ballooning, hepatocellular lipid accumulation, and hepatic inflammation Fig. Notably, knockdown of Nogo-B in the livers of obese mice significantly reduced the levels of these lipids except oxLDL Fig. These findings indicate that Nogo-B enhances carcinogenesis, possibly through metabolic modulations.
Mice were sacrificed at 28 weeks of age. As hepatic lipid overload and accumulation are major characteristics of NAFLD 16 , we investigated the role of Nogo-B on lipid accumulation. Unexpectedly, we found there was much less lipid accumulation in the tumorous areas compared with that in the para-tumorous areas from obese mice, which showed a negative correlation with the Nogo-B level Fig.
Considering the high energy and nutrient demands of cancer cells, we therefore speculated that Nogo-B might promote hepatocarcinogenesis by consuming excessive lipids as nutrient supplies and inducing metabolic reprogramming. To verify that Nogo-B resides on LDs in the hepatocyte, we first isolated LDs from mice that fasted overnight using subcellular fractionation and found Nogo-B was expressed in LDs in primary hepatocytes Supplementary Fig.
Consistent with the negative correlation between Nogo-B and lipid accumulation in vivo, Nogo-B knockdown induced greater expression of the lipid marker PLIN2 in vitro Supplementary Fig.
Interestingly, Nogo-B overexpression or depletion did not affect LD formation as demonstrated by the fact that no difference in the amount of LD was observed between Nogo-B and vector cells Fig. LD breakdown leads to degradation of TGs into FFAs, which is a critical step during cellular metabolic reprograming and energy support It has been reported that a transmembrane protein, Nogo-B receptor NgBR , recognizes the other terminal RHD domain of Nogo-B and could mediate Nogo-B-mediated chemotaxis and development of angiogenesis, and is also involved in hepatic lipogenesis 19 , Additionally, both ectopic expression and knockdown experiments showed that Nogo-B did not affect the expressions of triglyceride lipase and hormone-sensitive lipase involved in TG hydrolysis 21 Supplementary Fig.
Nogo-B promotes lipid droplet degradation in HCC cells. Since lipophagy represents a major mechanism for LD degradation and most cancer cells exhibit higher levels of basal autophagy than normal cells 7 , 22 , we postulated that Nogo-B-promoted LD breakdown is lipophagy-dependent. To better indicate the lipophagy progress, we performed electron microscopy to clarify that autophagosomes enveloping LDs, and found double-membrane vesicles analogous to autophagosomes arrow heads around LDs arrows and degradative structures enriched in LDs asterisks, Supplementary Fig.
RAB7 has been implicated in the initiation of autophagy-centric LD catabolism by mediating docking of autophagosomes AP and lysosomes to LDs during lipophagy As shown in Fig.
Moreover as expected, Nogo-B overexpressing stable cell line showed obvious higher Nogo-B expression than vector control cell line Supplementary Fig. Additionally, after treatment of these stable cells with autophagy inhibitors wortmannin wort and chloroquine CQ , We found that wortmannin inhibited Nogo-B induced p62 degradation and LC3 conversion while there were a slightly higher LC3-II level and the same p62 level in Nogo-B overexpressing stable cells compared with control vector cells after CQ treatment Fig.
Nogo-B promotes lipophagy in HCC cells. We next determined the mechanism by which Nogo-B regulates the autophagic catabolism of LDs. We speculated that as an ER-residential protein, Nogo-B might recruit the phagophore components for autophagy flux to the LD surface.
To further clarify the autophagy level in NAFLD-associated HCC, we performed western blot using the tumorous and adjacent non-tumorous tissues of the mouse model. These results suggest that Nogo-B promotes hepatocellular proliferation through lipophagy-mediated LD breakdown. To elucidate the oncogenic mechanism, we employed a luciferase reporter-based cancer pathways array in LO2 cells to interrogate the signaling pathways that could be regulated by Nogo-B overexpression.
Among the 11 pathways, Hippo signaling was most highly activated upon ectopic Nogo-B expression Fig. Concordant with the luciferase activity data, Nogo-B decreased the protein level of inactive phosphorylated YAP and increased the transcript levels of its downstream targets connective tissue growth factor CTGF and cysteine-rich protein 61 CYR61 Fig.
Supernatant and cell lysate were collected for detection. Although the survival period data did not show statistically significant differences between low and high expression of the individual genes, we found that concomitant upregulation of the three genes was strongly associated with poor patient survival Fig. Accumulating reports have shown that obesity-related steatosis exerts a huge public health burden worldwide. As a central hub for lipid metabolism, liver diseases are closely related to metabolic disorders Although there are several models for cytosolic LD biogenesis in cells, one shared feature is that LDs are formed in association with the ER.
A number of membrane-bound proteins, mostly identified in animals and yeast, have been shown to be involved in the biogenesis of LDs and localized at the ER, mainly at ER-LD junctions 29 , Nogo-B is primarily known to play important roles in pathological vascular conditions in response to vascular injuries such as ischemia and atherosclerosis 31 , However, the molecular actions of Nogo-B in liver metabolism that induces tumorigenesis remain elusive.
Our functional study using knockdown and ectopic expression approaches has confirmed the oncogenic role of Nogo-B in vitro and in vivo, and that Nogo-B promotes cancer cell growth through dysregulated metabolic programming.
Considering the vital role of ER in LD dynamics, we further determined whether these functions are dependent on the location of Nogo-B.
Both LD degradation analysis and co-immunoprecipitation assay indicated that ER-retention characteristic of Nogo-B is required for its promoted effect on lipophagy-mediated lipid metabolism. The role of autophagy in HCC is paradoxical. Basal autophagy acts as a tumor suppressor by removing damaged mitochondria and mutated cells, thus maintaining genomic stability. However, once a tumor is established, unbalanced autophagy will contribute to HCC cell survival under various stress conditions and in turn promotes tumor growth In support of this notion, the liver-specific Atg5-deficient mice developed only hepatic adenoma but not cancer In NAFLD-associated HCC, the role of autophagy is more complex because of the complicated linkages of lipid metabolism, inflammation and tumorigenesis.
Although mTORC1-regulated autophagy is reported to be necessary and sufficient for starvation-induced LD biogenesis, another study showed that the blockade of autophagy by pharmacological inhibitor or silencing ATG5 caused the reduction of LD and TG breakdown 7 , In our study, we revealed the lipolytic role of Nogo-B in the liver is dependent on autophagy-mediated LD degradation instead of LD formation.
Our data supports the role of autophagy in promoting lipid utilization and tumorigenesis. As a fatty acid translocase, CD36 is widely expressed in various tissues and mainly involved in lipid metabolism 39 , As hypoxia has been reported to increase lipid uptake and upregulate the expression of oxLDL receptor CD36, the increased Nogo-B levels could be a reflection of hypoxia in tumors Cancer cells reprogram their metabolic pathways to meet their abnormal demands for proliferation and survival.
Cancer cells are exposed to intermittent hypoxic episodes in the acidic microenvironment, which interferes with the metabolism of oxygenated cancer cells. Two recent studies demonstrated that cancer cells subjected to an acidic environment pH 6.
The consequences of this rewiring of metabolism are the preferred consumption of fatty acids to provide energy, but also the reduction of ROS production, which together support cancer cell proliferation and tumor growth In the absence of available nutrients, cancer cells can withstand long periods of nutrient deprivation via the self-catabolic process of macroautophagy to liberate free amino and fatty acids In fatty liver-related HCC, tumor cells tend to breakdown LD to provide fatty acids to the starved cells On the other hand, lipid metabolism in cancer cells also stimulates the common oncogenic signaling pathways, and is believed to be important for the initiation and progression of tumors 46 , Although autophagy has been demonstrated to be favorable for tumor growth and progression in advanced tumor stages 17 , 48 , the role of lipophagy in cancer cells has not been identified.
Recent reports have identified links between canonical Wnt signaling and the Hippo pathway, and several genes have been shown to take part in the crosstalk between the two pathways 50 , In our luciferase assay shown in Fig. Increasing evidence supports the notion that oncoproteins directly reprogram the metabolism of tumor cells, which makes them excellent chemotherapeutic targets for cancer treatment.
Sustainable Production: Novel Trends in Energy, Environment and Material Systems
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