Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice
2018; Elsevier BV; Volume: 59; Issue: 5 Linguagem: Inglês
10.1194/jlr.m080879
ISSN1539-7262
AutoresLiansheng Zhong, Ji Na Kong, Michael B. Dinkins, Silvia Leanhart, Zhihui Zhu, Stefka D. Spassieva, Haiyan Qin, Hsuan-Pei Lin, Ahmed Elsherbini, Rebecca Wang, Xue Jiang, Mariana Nikolova‐Karakashian, Guanghu Wang, Erhard Bieberich,
Tópico(s)Phagocytosis and Immune Regulation
ResumoSphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis. Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis. Sphingolipids, especially ceramide, sphingosine, and sphingosine-1-phosphate, play essential roles in cell signaling (1.Ogretmen B. Hannun Y.A. Biologically active sphingolipids in cancer pathogenesis and treatment.Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1007) Google Scholar, 5.Nagahashi M. Matsuda Y. Moro K. Tsuchida J. Soma D. Hirose Y. Kobayashi T. Kosugi S. Takabe K. Komatsu M. et al.DNA damage response and sphingolipid signaling in liver diseases.Surg. Today. 2016; 46: 995-1005Crossref PubMed Scopus (26) Google Scholar). They are modulators of various aspects of biology and pathology, including aging and cancer. In cancer, sphingolipids regulate tumor initiation, progression, metastasis, and drug resistance (1.Ogretmen B. Hannun Y.A. Biologically active sphingolipids in cancer pathogenesis and treatment.Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1007) Google Scholar, 3.Pralhada Rao R. Vaidyanathan N. Rengasamy M. Mammen Oommen A. Somaiya N. Jagannath M.R. Sphingolipid metabolic pathway: an overview of major roles played in human diseases.J. Lipids. 2013; 2013: 178910Crossref PubMed Scopus (3) Google Scholar, 5.Nagahashi M. Matsuda Y. Moro K. Tsuchida J. Soma D. Hirose Y. Kobayashi T. Kosugi S. Takabe K. Komatsu M. et al.DNA damage response and sphingolipid signaling in liver diseases.Surg. Today. 2016; 46: 995-1005Crossref PubMed Scopus (26) Google Scholar, 6.Dubois N. Rio E. Ripoche N. Ferchaud-Roucher V. Gaugler M.H. Campion L. Krempf M. Carrie C. Mahe M. Mirabel X. et al.Plasma ceramide, a real-time predictive marker of pulmonary and hepatic metastases response to stereotactic body radiation therapy combined with irinotecan.Radiother. Oncol. 2016; 119: 229-235Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7.Krishnamurthy K. Wang G. Rokhfeld D. Bieberich E. Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide.Breast Cancer Res. 2008; 10: R106Crossref PubMed Scopus (43) Google Scholar, 8.Bradley E. Dasgupta S. Jiang X. Zhao X. Zhu G. He Q. Dinkins M. Bieberich E. Wang G. Critical role of spns2, a sphingosine-1-phosphate transporter, in lung cancer cell survival and migration.PLoS One. 2014; 9: e110119Crossref PubMed Scopus (53) Google Scholar, 9.Kong J.N. He Q. Wang G. Dasgupta S. Dinkins M.B. Zhu G. Kim A. Spassieva S. Bieberich E. Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells.Int. J. Cancer. 2015; 137: 1610-1620Crossref PubMed Scopus (64) Google Scholar). Excess ceramide leads to cancer cell death, reduced growth, and senescence, and may serve as a real-time predictive marker for radiation treatment response (6.Dubois N. Rio E. Ripoche N. Ferchaud-Roucher V. Gaugler M.H. Campion L. Krempf M. Carrie C. Mahe M. Mirabel X. et al.Plasma ceramide, a real-time predictive marker of pulmonary and hepatic metastases response to stereotactic body radiation therapy combined with irinotecan.Radiother. Oncol. 2016; 119: 229-235Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7.Krishnamurthy K. Wang G. Rokhfeld D. Bieberich E. Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide.Breast Cancer Res. 2008; 10: R106Crossref PubMed Scopus (43) Google Scholar, 9.Kong J.N. He Q. Wang G. Dasgupta S. Dinkins M.B. Zhu G. Kim A. Spassieva S. Bieberich E. Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells.Int. J. Cancer. 2015; 137: 1610-1620Crossref PubMed Scopus (64) Google Scholar, 10.Ordoñez R. Fernández A. Prieto-Domínguez N. Martínez L. García-Ruiz C. Fernández-Checa J.C. Mauriz J.L. González-Gallego J. Ceramide metabolism regulates autophagy and apoptotic cell death induced by melatonin in liver cancer cells.J. Pineal Res. 2015; 59: 178-189Crossref PubMed Scopus (78) Google Scholar). On the other hand, sphingosine-1-phosphate promotes inflammation, metastasis, proliferation, vasculogenesis, and drug resistance (4.Proia R.L. Hla T. Emerging biology of sphingosine-1-phosphate: its role in pathogenesis and therapy.J. Clin. Invest. 2015; 125: 1379-1387Crossref PubMed Scopus (365) Google Scholar, 5.Nagahashi M. Matsuda Y. Moro K. Tsuchida J. Soma D. Hirose Y. Kobayashi T. Kosugi S. Takabe K. Komatsu M. et al.DNA damage response and sphingolipid signaling in liver diseases.Surg. Today. 2016; 46: 995-1005Crossref PubMed Scopus (26) Google Scholar, 8.Bradley E. Dasgupta S. Jiang X. Zhao X. Zhu G. He Q. Dinkins M. Bieberich E. Wang G. Critical role of spns2, a sphingosine-1-phosphate transporter, in lung cancer cell survival and migration.PLoS One. 2014; 9: e110119Crossref PubMed Scopus (53) Google Scholar, 11.Nagahashi M. Yuza K. Hirose Y. Nakajima M. Ramanathan R. Hait N.C. Hylemon P.B. Zhou H. Takabe K. Wakai T. The roles of bile acids and sphingosine-1-phosphate signaling in the hepatobiliary diseases.J. Lipid Res. 2016; 57: 1636-1643Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Mainly three pathways generate ceramide, namely the de novo pathway, the salvage pathway, and the SM cycle (12.Airola M.V. Hannun Y.A. Sphingolipid metabolism and neutral sphingomyelinases.Handb. Exp. Pharmacol. 2013; 215: 57-76Crossref PubMed Scopus (115) Google Scholar, 13.Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2451) Google Scholar, 14.Hannun Y.A. The sphingomyelin cycle and the second messenger function of ceramide.J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar). Several enzymes are involved in the SM cycle, including the SM synthases (SMSs) (SMS1 and SMS2) that catalyze the conversion of ceramide to SM, and several SMases that hydrolyze SM to generate ceramide (13.Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2451) Google Scholar, 15.Shamseddine A.A. Airola M.V. Hannun Y.A. Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological processes.Adv. Biol. Regul. 2015; 57: 24-41Crossref PubMed Scopus (132) Google Scholar, 16.Stoffel W. Jenke B. Block B. Zumbansen M. Koebke J. Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4554-4559Crossref PubMed Scopus (134) Google Scholar, 17.Stoffel W. Jenke B. Holz B. Binczek E. Gunter R.H. Knifka J. Koebke J. Niehoff A. Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1-driven smpd3 transgene expression.Am. J. Pathol. 2007; 171: 153-161Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18.Stoffel W. Hammels I. Jenke B. Binczek E. Schmidt-Soltau I. Brodesser S. Schauss A. Etich J. Heilig J. Zaucke F. Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.Cell Death Dis. 2016; 7: e2488Crossref PubMed Scopus (28) Google Scholar). Acid SMase, encoded by SM phosphodiesterase (Smpd1), is a lysosomal enzyme the deficiency of which is associated with Niemann-Pick disease (19.Schuchman E.H. Wasserstein M.P. Types A and B Niemann-Pick disease.Best Pract. Res. Clin. Endocrinol. Metab. 2015; 29: 237-247Crossref PubMed Scopus (57) Google Scholar, 20.Schuchman E.H. Wasserstein M.P. Types A and B Niemann-Pick disease.Pediatr. Endocrinol. Rev. 2016; 13: 674-681PubMed Google Scholar). Four mammalian neutral SMases (nSMases) have been identified so far, among which nSMase2 (encoded by Smpd3) appears to be the predominant nSMase in various tissues and associated pathophysiologies (12.Airola M.V. Hannun Y.A. Sphingolipid metabolism and neutral sphingomyelinases.Handb. Exp. Pharmacol. 2013; 215: 57-76Crossref PubMed Scopus (115) Google Scholar, 15.Shamseddine A.A. Airola M.V. Hannun Y.A. Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological processes.Adv. Biol. Regul. 2015; 57: 24-41Crossref PubMed Scopus (132) Google Scholar, 21.Clarke C.J. Wu B.X. Hannun Y.A. The neutral sphingomyelinase family: identifying biochemical connections.Adv. Enzyme Regul. 2011; 51: 51-58Crossref PubMed Scopus (57) Google Scholar). Mice deficient in nSMase2 have multiple developmental defects, including dwarfism and delayed puberty, attributed to hypothalamic pituitary deficiency (16.Stoffel W. Jenke B. Block B. Zumbansen M. Koebke J. Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4554-4559Crossref PubMed Scopus (134) Google Scholar, 17.Stoffel W. Jenke B. Holz B. Binczek E. Gunter R.H. Knifka J. Koebke J. Niehoff A. Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1-driven smpd3 transgene expression.Am. J. Pathol. 2007; 171: 153-161Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18.Stoffel W. Hammels I. Jenke B. Binczek E. Schmidt-Soltau I. Brodesser S. Schauss A. Etich J. Heilig J. Zaucke F. Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.Cell Death Dis. 2016; 7: e2488Crossref PubMed Scopus (28) Google Scholar). nSMase2 is implicated in diverse cellular functions, including inflammation and pathophysiology of pulmonary, circulatory, cardiac, and neurological systems (15.Shamseddine A.A. Airola M.V. Hannun Y.A. Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological processes.Adv. Biol. Regul. 2015; 57: 24-41Crossref PubMed Scopus (132) Google Scholar). It is also implicated in cancers, such as leukemia, breast cancer, and liver cancer (22.Kim W.J. Okimoto R.A. Purton L.E. Goodwin M. Haserlat S.M. Dayyani F. Sweetser D.A. McClatchey A.I. Bernard O.A. Look A.T. et al.Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias.Blood. 2008; 111: 4716-4722Crossref PubMed Scopus (68) Google Scholar, 23.Bhati R. Patterson C. Livasy C.A. Fan C. Ketelsen D. Hu Z. Reynolds E. Tanner C. Moore D.T. Gabrielli F. et al.Molecular characterization of human breast tumor vascular cells.Am. J. Pathol. 2008; 172: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). A recent genome-wide study indicates that Smpd3 is a potential repressor of hepatocellular carcinoma (HCC) (24.Revill K. Wang T. Lachenmayer A. Kojima K. Harrington A. Li J. Hoshida Y. Llovet J.M. Powers S. Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma.Gastroenterology. 2013; 145: 1424-1435.e1-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Sphingolipids play essential roles in stem cell and cancer stem-like cell (CSC) biology (2.Oskouian B. Saba J.D. Cancer treatment strategies targeting sphingolipid metabolism.Adv. Exp. Med. Biol. 2010; 688: 185-205Crossref PubMed Scopus (110) Google Scholar, 25.Spassieva S. Bieberich E. The gut-to-breast connection - interdependence of sterols and sphingolipids in multidrug resistance and breast cancer therapy.Anticancer. Agents Med. Chem. 2011; 11: 882-890Crossref PubMed Scopus (8) Google Scholar, 26.Bieberich E. Silva J. Wang G. Krishnamurthy K. Condie B.G. Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants.J. Cell Biol. 2004; 167: 723-734Crossref PubMed Scopus (138) Google Scholar, 27.Bieberich E. Ceramide in stem cell differentiation and embryo development: novel functions of a topological cell-signaling lipid and the concept of ceramide compartments.J. Lipids. 2011; 2011: 610306Crossref PubMed Google Scholar, 28.Bieberich E. Ceramide and sphingosine-1-phosphate signaling in embryonic stem cell differentiation.Methods Mol. Biol. 2012; 874: 177-192Crossref PubMed Scopus (8) Google Scholar, 29.Gupta V. Bhinge K.N. Hosain S.B. Xiong K. Gu X. Shi R. Ho M.Y. Khoo K.H. Li S.C. Li Y.T. et al.Ceramide glycosylation by glucosylceramide synthase selectively maintains the properties of breast cancer stem cells.J. Biol. Chem. 2012; 287: 37195-37205Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). CSCs, also termed cancer-initiating cells or tumor-initiating cells, are a subpopulation of cells in the tumor that possess stem cell characteristics (30.Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J. Clin. Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 31.Yamashita T. Kaneko S. Orchestration of hepatocellular carcinoma development by diverse liver cancer stem cells.J. Gastroenterol. 2014; 49: 1105-1110Crossref PubMed Scopus (34) Google Scholar). They are derived from either reactivation of dormant progenitor cells or dedifferentiation of somatic cells (30.Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J. Clin. Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 31.Yamashita T. Kaneko S. Orchestration of hepatocellular carcinoma development by diverse liver cancer stem cells.J. Gastroenterol. 2014; 49: 1105-1110Crossref PubMed Scopus (34) Google Scholar, 32.Shackleton M. Quintana E. Fearon E.R. Morrison S.J. Heterogeneity in cancer: cancer stem cells versus clonal evolution.Cell. 2009; 138: 822-829Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar). Mounting evidence has demonstrated that CSCs contribute significantly to tumor initiation, metastasis, treatment resistance, and relapse in many cancer types, including liver cancer (30.Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J. Clin. Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 31.Yamashita T. Kaneko S. Orchestration of hepatocellular carcinoma development by diverse liver cancer stem cells.J. Gastroenterol. 2014; 49: 1105-1110Crossref PubMed Scopus (34) Google Scholar). Thus, identifying and targeting CSCs is a promising new strategy for cancer therapy (33.Wolf B. Krieg K. Falk C. Breuhahn K. Keppeler H. Biedermann T. Schmid E. Warmann S. Fuchs J. Vetter S. et al.Inducing differentiation of premalignant hepatic cells as a novel therapeutic strategy in hepatocarcinoma.Cancer Res. 2016; 76: 5550-5561Crossref PubMed Scopus (11) Google Scholar). Signal transducer and activator of transcription 3 (Stat3) is a key transcription factor in maintaining mouse embryonic stem cell self-renewal and CSC stemness (34.Niwa H. Burdon T. Chambers I. Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1251) Google Scholar, 35.Ghoshal S. Fuchs B.C. Tanabe K.K. STAT3 is a key transcriptional regulator of cancer stem cell marker CD133 in HCC.Hepatobiliary Surg. Nutr. 2016; 5: 201-203Crossref PubMed Google Scholar, 36.Won C. Kim B.H. Yi E.H. Choi K.J. Kim E.K. Jeong J.M. Lee J.H. Jang J.J. Yoon J.H. Jeong W.I. et al.Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma.Hepatology. 2015; 62: 1160-1173Crossref PubMed Scopus (124) Google Scholar, 37.Bradley E. Bieberich E. Mivechi N.F. Tangpisuthipongsa D. Wang G. Regulation of embryonic stem cell pluripotency by heat shock protein 90.Stem Cells. 2012; 30: 1624-1633Crossref PubMed Scopus (45) Google Scholar). Stat3 is phosphorylated and activated by Janus kinases (30.Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J. Clin. Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar). The activated Stat3 translocates into the nucleus and binds to promoters of stemness genes, including the liver CSC marker protein, CD133, leading to their expression (30.Yamashita T. Wang X.W. Cancer stem cells in the development of liver cancer.J. Clin. Invest. 2013; 123: 1911-1918Crossref PubMed Scopus (399) Google Scholar, 35.Ghoshal S. Fuchs B.C. Tanabe K.K. STAT3 is a key transcriptional regulator of cancer stem cell marker CD133 in HCC.Hepatobiliary Surg. Nutr. 2016; 5: 201-203Crossref PubMed Google Scholar). We report here the potential function of sphingolipids in liver tumor development using the nSMase2-deficient (fragilitas ossium, fro/fro) mouse line (38.Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.L. et al.A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (140) Google Scholar, 39.Wang G. Dinkins M. He Q. Zhu G. Poirier C. Campbell A. Mayer-Proschel M. Bieberich E. Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4): potential mechanism of apoptosis induction in Alzheimer disease (AD).J. Biol. Chem. 2012; 287: 21384-21395Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). This mouse line was discovered in a random-bred stock of mice after treatment with the chemical mutagen, tris(1-aziridinyl)phosphine-sulphine (40.Guenet J.L. Stanescu R. Maroteaux P. Stanescu V. Fragilitas ossium: a new autosomal recessive mutation in the mouse.J. Hered. 1981; 72: 440-441Crossref PubMed Scopus (34) Google Scholar), which caused a deletion in the Smpd3 gene leading to loss of enzyme activity (38.Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.L. et al.A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (140) Google Scholar). These mice show similar phenotypes as nSMase2 knockout mice generated through gene-targeting approaches (16.Stoffel W. Jenke B. Block B. Zumbansen M. Koebke J. Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4554-4559Crossref PubMed Scopus (134) Google Scholar, 17.Stoffel W. Jenke B. Holz B. Binczek E. Gunter R.H. Knifka J. Koebke J. Niehoff A. Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1-driven smpd3 transgene expression.Am. J. Pathol. 2007; 171: 153-161Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18.Stoffel W. Hammels I. Jenke B. Binczek E. Schmidt-Soltau I. Brodesser S. Schauss A. Etich J. Heilig J. Zaucke F. Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.Cell Death Dis. 2016; 7: e2488Crossref PubMed Scopus (28) Google Scholar). In our study, liver tumors were observed in 27.3% of aged male fro/fro mice (9 of 33), accompanied by increased proliferation and apoptosis. Lipidomics studies demonstrate a marked increase of C16-SM, C18-SM, C24:1-SM, C16-ceramide, and dihydro (dh)C16-ceramide in the tumor tissue. The number of CSCs was increased in fro/fro liver tumors, as determined by quantitative (q)PCR and immunocytochemistry of marker genes CD133 and/or EpCAM. Interestingly, CD133(+) cells were labeled for high levels of SM and ceramide. Furthermore, fro/fro liver tumors exhibited a significant increase of ceramide synthase (CerS)5 expression and lipid droplet (LD) content, suggesting that the lack of nSMase2 was associated with a compensatory upregulation of sphingolipid and neutral lipid synthesis, which may play a role in CSC proliferation/survival and tumor formation in the fro/fro mouse. Oil Red O (ORO), propylene glycol, and fluoroshield supplemented with DAPI were from Sigma-Aldrich (St. Louis, MO). Anti-Ki67 was from Novocastra (Buffalo Grove, IL). Lysenin and anti-lysenin antibody were from Peptide Institute, Inc. (Osaka, Japan). Anti-CD133 was from EMD Millipore (Billerica, MA). Anti-phosphorylated (p)Stat3 and anti-total (t)Stat3 were from Cell Signaling Technology (Danvers, MA). Anti-ceramide rabbit IgG was generated in our laboratory as described previously (41.Krishnamurthy K. Dasgupta S. Bieberich E. Development and characterization of a novel anti-ceramide antibody.J. Lipid Res. 2007; 48: 968-975Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). The in situ TUNEL fluorescence staining kit was from Roche (Indianapolis, IN). The nSMase2-deficient (fro/fro) mouse line (C3H and C57Bl6 mixed background), carrying a deletion in the Smpd3 gene, was a gift from Dr. Christophe Poirier, Indiana University, Indianapolis, IN (38.Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.L. et al.A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (140) Google Scholar, 39.Wang G. Dinkins M. He Q. Zhu G. Poirier C. Campbell A. Mayer-Proschel M. Bieberich E. Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4): potential mechanism of apoptosis induction in Alzheimer disease (AD).J. Biol. Chem. 2012; 287: 21384-21395Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). These mice were maintained in the Laboratory Animal Service facility of the Medical College of Georgia at Augusta University according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Augusta University Institutional Animal Care and Use Committee. Tumor and adjacent normal tissues were collected immediately after surgery by either snap-freeze in −80°C or fixation with 4% PFA. Frozen tissues were never allowed to thaw after initial freezing until used for further experiments. Fixed tissues were either embedded in optimal cutting temperature medium or paraffin for sectioning. H&E staining and RNA analysis were used for quality control of the integrity of the samples. For histology studies, paraffin-embedded tissues were sectioned into 10 μm slices and stained by H&E in the Electron Microscopy and Histology Core Laboratory at Augusta University (directed by Dr. Sylvia B. Smith). For immunohistochemistry, frozen or paraffin sections were immunolabeled with the antibodies listed and images taken using a Zeiss LSM 510 upright or a Zeiss LSM 780 inverted confocal laser scanning microscope equipped with a two photon argon laser at 488 nm (Cy2), 543 nm (Cy3), and 633 nm (Cy5, Alexa Fluor 647), respectively. LSM 510 Meta 3.2 software was used for image acquisition and processing. Samples labeled with antibodies against lipids were only PFA-fixed and frozen for cryosectioning. Images obtained with secondary antibodies were only used as negative controls to correct for background intensity of a particular laser channel. Antigen-specific immunolabeling was quantified by counting cells that showed signals 2-fold or more above background fluorescence. The percentage of Ki67- and TUNEL-positive cells was counted at ×400 magnification in at least five random fields where tumors existed. Fixed frozen sections were washed with distilled water. Excess water was drained and depleted by rinsing twice using propylene glycol. ORO was applied and incubated for 7 min at 60°C. Specimens were destained with 85% propylene glycol, rinsed in distilled water, and counterstained using hematoxylin. After sufficient washing with water, specimens were mounted with aqueous mounting media and microscopic images taken using an epifluorescence microscope. RNA was extracted using the TriZol reagent (Invitrogen). First strand cDNA was generated by the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time qPCR was performed using SYBR green/Rox qPCR master mix on the CFX96 Touch real-time system (Bio-Rad). The primers for qPCR were: mCerS5-forward, TAGAGAGCAGCTGAGAGGAAGAAGA; mCerS5-reverse, GAACCCAGAGTCTCAAGAGCCATGGC; mCD133-forward, CAGCAATCACTGAAATTTGTG; mCD133-reverse, ACATCCTCTGAATCCATCCTG; mEpCAM-forward, TATTTTGAAAAAGATGTGAAG; mEpCAM-reverse, ATTAAGCTCTCTGTGGATCTC. Liver tissues were analyzed at the Lipidomics Core of the Medical University of South Carolina according to published protocols on a Thermo Fisher TSQ Quantum triple quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode (42.Bielawski J. Szulc Z.M. Hannun Y.A. Bielawska A. Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.Methods. 2006; 39: 82-91Crossref PubMed Scopus (420) Google Scholar). Briefly, similar amounts of tissue were homogenized, protein concentration determined, aliquots of the homogenates equivalent to 1.5 mg protein fortified with internal standards (ISs), and lipids from the fortified homogenates extracted twice with 2 ml ethyl acetate, isopropanol, and water (60:30:10 v/v/v) solvent. Lipid extracts were evaporated and reconstituted in 150 μl of 1 mM ammonium formate in 0.2% formic acid in methanol before being subjected to LC-MS/MS on the HP1100/TSQ Quantum LC-MS/MS system. The samples were gradient-eluted from the BDS Hypersil C8, 150 × 3.2 mm, 3 μm particle size column, with a 1.0 mM methanolic ammonium formate per 2 mM aqueous ammonium formate mobile phase system. Peaks corresponding to the target analytes and ISs were collected and processed using the Xcalibur software system. Quantitative analysis was based on the calibration curves generated by spiking an artificial matrix with the known amounts of the target analyte synthetic standards and an equal amount of the ISs. The target analyte/IS peak area ratios were plotted against analyte concentration. The target analyte/IS peak area ratios from the samples were similarly normalized to their respective IS and compared with the calibration curves using a linear regression model. Results were normalized to lipid phosphate. The statistical significance was calculated using Student's t-test with Excel or one-way ANOVA and Tukey's post hoc test with GraphPad Prism. P < 0.05 was considered significant. To determine a potential involvement of nSMase2 and sphingolipids in hepatic cancer, we analyzed liver tissue in aged fro/fro mice (38.Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.L. et al.A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (140) Google Scholar, 39.Wang G. Dinkins M. He Q. Zhu G. Poirier C. Campbell A. Mayer-Proschel M. Bieberich E. As
Referência(s)