HILPDA, a New Player in NASH-driven HCC, Links Hypoxia Signaling with Ceramide Synthesis.

HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxiaJournal of HepatologyVol. 79Issue 2PreviewThe prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. Full-Text PDF See Article, pages 378–393 See Article, pages 378–393 Non-alcoholic fatty liver disease is a spectrum of liver disorders that begin with the accumulation of lipids in hepatocytes and can progress to non-alcoholic steatohepatitis (NASH). Besides steatosis, NASH is characterized by inflammation, hepatocellular damage and fibrosis, and can progress to hepatocellular carcinoma (HCC), the most common type of liver cancer. The incidence of NASH-driven HCC is expected to continue rising throughout the world due to its association with the obesity and type 2 diabetes epidemic. Overweight and obesity are known risk factors for the development of several types of cancers, including HCC.1Bianchini F. Kaaks R. Vainio H. Overweight, obesity, and cancer risk.Lancet Oncol. 2002; 3: 565-574Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar, 2Calle E.E. Rodriguez C. Walker-Thurmond K. Thun M.J. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults.N Engl J Med. 2003; 348: 1625-1638Crossref PubMed Scopus (6212) Google Scholar, 3Forner A. Reig M. Bruix J. Hepatocellular carcinoma.Lancet. 2018; 391: 1301-1638Abstract Full Text Full Text PDF PubMed Scopus (3306) Google Scholar, 4Forner A. Reig M. Bruix J. Hepatocellular carcinoma.Lancet. 2018; 391: 1301-1314Abstract Full Text Full Text PDF PubMed Scopus (3447) Google Scholar Despite important improvements in HCC management, effective treatment options, such as local ablative therapies and resection, are limited to early disease stages.[5]Bruix J. Reig M. Sherman M. Evidence-based diagnosis, staging, and treatment of patients with hepatocellular carcinoma.Gastroenterology. 2016; 150: 835-853Abstract Full Text Full Text PDF PubMed Scopus (1251) Google Scholar,[6]Villanueva A. Hernandez-Gea V. Llovet J.M. Medical therapies for hepatocellular carcinoma: a critical view of the evidence.Nat Rev Gastroenterol Hepatol. 2013; 10 (34-4)Crossref Scopus (270) Google Scholar This therapeutic limitation is mainly due to our incomplete understanding of the molecular events involved in HCC pathogenesis. Hence, it is urgent to understand the biochemical basis for HCC progression in order to identify new therapeutic targets to prevent HCC initiation and progression. Hypoxia is an inherent and key feature of solid tumors that reflects the disorganized structure and architecture of tumor vasculature, resulting in irregular and inefficient oxygen delivery; it is considered a negative prognostic factor for response to treatment and survival of patients with cancer.7Beyoglu D. Imbeaud S. Maurhofer O. Bioulac-Sage P. Zucman-Rossi J. Dufour J.F. Idle J.R. Tissue metolomics of hepatocellular carcinoma: tumor energy metabolism and the role of transcriptome classification.Hepatology. 2013; 58 (PMID: 23463346. PMCID): 229-238Crossref PubMed Scopus (154) Google Scholar, 8Lasante V. Choy P.M. Fung S.W. Liu Y. Chai J.G. Dyson J. Del Rio A. D´Santos C. Williams R. Chokshi S. Anders R.A. Bubici C. Papa S. PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation.Nat Commun. 2015; 6: 7882Crossref PubMed Scopus (161) Google Scholar, 9Lluis J.M. Burichi F. Chiarugi P. Morales A. Fernandez-Checa J.C. Dual role of mitochondrial reactive oxygen species in hypoxia signaling: activation of nuclear factor-kappaB via s-SRC and oxidant-dependent cell death.Cancer Res. 2007; 67: 7368-7377Crossref PubMed Scopus (189) Google Scholar HCC is a heterogenous cancer, whose development is determined largely by hypoxia.[10]Ma I. Hernandez M.O. Zhao Y. Mehta M. Tran B. Kelly M. Rae Z. Hernandez J.M. Davis J.L. Martin S.P. Kleiner D.E. Hewitt S.M. Ylaya K. Wood B.J. Greten T.F. Wang X.W. Tumor cell biodiversity drives microenvironmental reprogramming in liver cancer.Cancer Cell. 2019; 36: 418-430Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar,[11]Ma L. Craig A.M. Heinrich S. Hypoxia is a key regulator in liver cancer progression.J Hepatol. 2021; 75: 736-737Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Hypoxia-inducible factor (HIF)-1 plays a central role in oxygen sensing and is the master regulator that mediates the genetic reprogramming involved in the adaptation of cancer cells to hypoxia.[12]Giaccia A. Siim B.G. Johnson R.S. HIF-1 as a target for drug development.Nat Rev Drug Discov. 2003; 2: 803-811Crossref PubMed Scopus (544) Google Scholar,[13]Semenza G.L. Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5509) Google Scholar HIF-1 comprises a stable β subunit (HIF-1β/Arnt) and a labile α subunit (HIF-1α), which encompasses several family members, such as HIF-1α and HIF-2α. HIF-1α/2α are degraded in normoxia by the proteasome, which is preceded by the hydroxylation of HIF-1α at proline residues by prolyl hydroxylase (PHDs). Although PHDs are dependent on iron and 2-oxoglutarate, PHDs primarily function as oxygen sensors and become inactivated in low oxygen tension. Hypoxia signaling orchestrates metabolic reprogramming, which is an essential event in cancer (and more specifically HCC) development.[10]Ma I. Hernandez M.O. Zhao Y. Mehta M. Tran B. Kelly M. Rae Z. Hernandez J.M. Davis J.L. Martin S.P. Kleiner D.E. Hewitt S.M. Ylaya K. Wood B.J. Greten T.F. Wang X.W. Tumor cell biodiversity drives microenvironmental reprogramming in liver cancer.Cancer Cell. 2019; 36: 418-430Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar,[11]Ma L. Craig A.M. Heinrich S. Hypoxia is a key regulator in liver cancer progression.J Hepatol. 2021; 75: 736-737Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Much of the metabolic reprogramming induced by HIF1α/2α in cancer cells impacts on mitochondrial pathways and bioenergetics, the stimulation of glycolysis and reliance on glutamine metabolism, and recent findings also revealed a hypoxic regulation of lipid metabolism, consistent with the known formation of lipid droplets (LDs) in hypoxic tumors.[14]Koizume S. Miyagi Y. Lipid droplets: a key celular organelle associated with cancer cell survival under normoxia and hypoxia.Int J Mol Sci. 2016; 17Crossref PubMed Scopus (129) Google Scholar,[15]VandeKopple M.J. Wu J. Auer E.N. Giaccia A.J. Denko N.C. Papandreou I. HILPDA regulates lipid metabolism, lipid droplet abundance, and response to microenvironmental stress in solid tumor.Mol Cancer Res. 2019; 17: 2089-2101Crossref PubMed Scopus (38) Google Scholar In this regard, emerging evidence has shown that hypoxia-inducible lipid droplet associated protein (HILPDA) regulates LD homeostasis and free fatty acid (FFA) flux by inhibiting adipose triglyceride lipase, the rate-limiting enzyme for intracellular triglyceride (TG) hydrolysis. In the hypoxic environment, HILPDA mRNA expression is induced by the binding of HIF-1 to the hypoxia response element in its promoter.[16]Gimm T. Wiese M. Teschemacher B. Deggerich A. Schödel J. Knaup K.X. Hackenbeck T. Hellerbrand C. Amann K. Wiesener M.S. Höning S. Eckardt K.U. Warnecke C. Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia inducible factor-1.FASEB J. 2010; 24: 4443-4458Crossref PubMed Scopus (121) Google Scholar In solid tumors, such as colorectal and renal cancer, the activation of HILPDA results in LD accumulation, preventing the release and oxidation of FFAs, and promoting cancer cell survival.[16]Gimm T. Wiese M. Teschemacher B. Deggerich A. Schödel J. Knaup K.X. Hackenbeck T. Hellerbrand C. Amann K. Wiesener M.S. Höning S. Eckardt K.U. Warnecke C. Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia inducible factor-1.FASEB J. 2010; 24: 4443-4458Crossref PubMed Scopus (121) Google Scholar However, the impact of HILPDA in NASH-driven HCC development remained unknown. In this issue of the Journal of Hepatology, using transcriptomic, lipidomic and single-cell RNA sequencing analysis, Povero et al. identified an increased expression of HILPDA in patients and mice with NASH-driven HCC.17Povero D. Chen Y. Johnson S.M. McMahon C.E. Pan M. Bao H. Petterson X.T. Blake E. Lauer K.P. O'Brien D.R. Yu Y. Graham R.P. Taner T. Han X. Razidlo G.L. Liu J. HILPDA promotes NASH-driven HCC development by restraining intracelular fatty acid flux in hypoxia.J Hepatology. 2023; 79: 378-393Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar HILPDA expression was not only induced in human HCC, as revealed by immunohistochemical and quantitative reverse-transcription PCR analyses, but it was associated with poor prognosis. Detailed spatial transcriptomic analyses of the expression of HILPDA in histopathologically proven human NASH-driven HCC and adjacent non-cancerous tissue revealed that the expression of HILPDA colocalized with glutamine synthase and HIF-1α in tumors, and paralleled the size and number of fat vacuoles resembling LDs. In vitro studies using HCC cells and 3D spheroids revealed that hypoxia increased the expression of HILPDA mRNA and protein and immunofluorescence staining demonstrated the colocalization of HILPDA on the surface of LDs in hypoxic Huh7 cells treated with oleic acid. Exposure of Huh7 cells to hypoxia and media enriched in FFAs indicated a synergistic effect in the induction of HILPDA. The deletion of HILPDA by CRISPR-Cas9 in Huh7 and Hep3B cells led to a dramatic decrease in the size and number of LDs and TG levels, which was accompanied by reduced cell survival and enhanced caspase 3/7 activity, suggesting that HILPDA sustains HCC survival by protecting against metabolic stress-induced apoptosis under hypoxia. Lipidomic analyses revealed that the deletion of HILPDA disrupted lipid homeostasis and phospholipid composition with a significant decrease in phosphatidylserine (PS) species, as well as phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylglycerol linked to a significant increase in cell death and decreased in vivo HCC development. The decrease seen in the PS levels is of interest vis a vis recent findings revealing that impaired transfer of PS from the endoplasmic reticulum (ER) to mitochondria contributes to HCC development.[18]Hernández-Alvarez M.I. Sebastián D. Vives S. Ivanova S. Bartoccioni P. Kakimoto P. Plana N. Veiga S.R. Hernández V. Vasconcelos N. Peddinti G. Adrover A. Jové M. Pamplona R. Gordaliza-Alaguero I. Calvo E. Cabré N. Castro R. Kuzmanic A. Boutant M. Sala D. Hyotylainen T. Orešič M. Fort J. Errasti-Murugarren E. Rodrígues C.M.P. Orozco M. Joven J. Cantó C. Palacin M. Fernández-Veledo S. Vendrell J. Zorzano A. Deficient endoplasmic reticulum-mitochondrial phosphatidylserine transfer causes liver disease.Cell. 2019; 177: 881-895Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar Further studies will be required to address whether HILPDA regulates the ER-mitochondrial PS transfer, which may underlie the relevance of HILPDA in HCC progression. In addition to the alterations in phospholipid homeostasis, HILPDA deletion also caused increased ceramide levels as revealed by the lipidomic data. In hypoxic Huh7 cells, HILPDA deletion enhanced the levels of ceramides C16, C18 and C24 that paralleled the reduction in serine content. Although ceramide can be generated by several mechanisms, including sphingomyelin hydrolysis,[20]Morales A. Lee H. Goñi F.M. Kolesnick R. Fernandez-Checa J.C. Sphingolipids and cell death.Apoptosis. 2007; 12: 923-939Crossref PubMed Scopus (195) Google Scholar the increase in ceramides was due to their de novo synthesis in the ER from serine and palmitoyl-CoA (Fig. 1), as hypoxic Huh7 exhibited increased expression of serine palmitoyltransferase (SPT), the rate-limiting enzyme, as well as ceramide synthases (CERS) 2 and 6. Consistent with the known function of ceramide as a proapoptotic lipid,[19]García-Ruiz C. Colell A. Marí M. Morales A. Fernández-Checa J.C. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial gutathione.J Biol Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar,[20]Morales A. Lee H. Goñi F.M. Kolesnick R. Fernandez-Checa J.C. Sphingolipids and cell death.Apoptosis. 2007; 12: 923-939Crossref PubMed Scopus (195) Google Scholar HILPDA deletion in Huh7 cells stimulated reactive oxygen species generation, phospholipid peroxidation, decreased mitochondrial membrane potential as well as caspase 3/7 activity, causing the demise of HCC cells. This sequence of events was further established by myriocin and fumonisin B1, which inhibit SPT and CERS, respectively, preventing the synthesis of ceramide, and thus protecting against reactive oxygen species generation, phospholipid peroxidation and cell death. Similar findings were observed in 3D Huh7 spheroids, indicating that HILPDA deletion impaired anchorage-independent growth and survival. To address the relevance of the in vitro observations with HCC cells, Povero et al. generated mice with hepatocyte-specific HILPDA deletion (HilpdaΔHep) to induce a dietary mediated model of HCC development. While Hilpdaf/f mice fed a western diet plus fructose and glucose in the drinking water for 24 weeks followed by weekly doses of carbon tetrachloride exhibited increased NAFLD scores, this outcome was markedly decreased in HilpdaΔHep mice. Hilpa deletion in hepatocytes attenuated diet-induced steatosis and ballooning but increased iron oxidation products and oxidative stress, though it did not affect plasma liver injury markers and plasma TG levels. Quite intriguingly, the deletion of Hilpda in hepatocytes did not have a significant impact on inflammation and fibrosis, suggesting that Hilpda regulates the early stage of NAFLD, including steatosis and liver injury, and that the onset of inflammation and fibrosis are independent of Hilpda in mice. In line with the reduced NAFLD activity score, Hilpda deletion ameliorated NASH-driven HCC with a decrease in the number of nodules observed, which exhibited lower expression of the bona fide HCC marker glypican-3 as well as markers of cell survival (Oct4) and stemness (Sox2), and increased levels of the tumor suppressor and intracelular iron storage promoter ferritin heavy chain 1, compared to Hilpdaf/f livers. In contrast to the findings in HCC cells, targeted lipidomics analysis of mice with Hilpda deletion in hepatocytes indicated a lack of significant increase in the levels of several sphingolipids, including C14-, C16-, C18:1-ceramides as well as sphingosine-1-phosphate. Single-cell RNA-sequencing analysis demonstrated that Hilpda regulates the immune microenvironment of NASH-driven HCC, as HilpdaΔHep livers exhibited reduced total cell count and lower relative numbers of Kupffer cells, NASH-associated macrophages and cancer cells. Although no difference in T cell number was observed between Hilpdaf/f livers and HilpdaΔHep livers, the expression of genes involved in T-cell receptor signaling and the cytokine response were significantly downregulated in T cells isolated from HilpdaΔHep mice. Thus, overall, these findings indicate that HILPDA promotes NASH-HCC by preventing ceramide synthesis in the ER and by stimulating a tumor-prone immune microenvironment. Further investigation will be required to determine whether these two cues affected by HILPDA, namely, ceramide homeostasis and tumor immune microenvironment are independent of each other, and if the use of nanoliposomes encapsulating ceramide, which have been shown to be effective in other cancer types, including HCC,[21]Qi X. Wu F. Kim S.H. Kaifi J.T. Kimchi E.T. Snyder H. Illendula A. Fox T. Kester M. Staveley-O'Carroll K.F. Li G. Nanoliposome C6-Ceramide in combination with anti-CTLA4 antibody improves anti-tumor immunity in hepatocellular cancer.FASEB J. 2022; 36e22250Crossref Scopus (8) Google Scholar mimic the effects seen upon Hilpda deletion. Overall, despite these limitations, the study of Povero et al. identifies a novel target for the treatment of NASH-driven HCC, which deserves further investigation. We acknowledge the support from grants PID2019-111669RB-100 and PID2020-115055RB-I00 from Plan Nacional de I+D funded by the Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER) and from the CIBEREHD, the European Horizon´s research and innovation program HORIZON-HLTH-2022-STAYHLTH-02 under agreement No 101095679, and the Project 201916/31 Contribution of mitochondrial oxysterol and bile acid metabolism to liver carcinogenesis 2019 by Fundació Marató TV3. The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details. CGR, ST and JCFC wrote the manuscript and designed the figure. The following are the supplementary data to this article: Download .pdf (1.04 MB) Help with pdf files Multimedia component 1