Fabio Marra

Inghilesi, A. L.

Annals of Hepatology

English

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305SALLuting a new biomarker in hepatocellular carcinoma. , 2014; 13 (2): 305-307SALLuting a new biomarker in hepatocellular carcinomaAndrea L. Inghilesi, Fabio MarraDipartimento di Medicina Sperimentale e Clinica, University of Florence, Florence, Italy.Correspondence and reprint request: Fabio Marra, MD, PhDDipartimento di Medicina Sperimentale e ClinicaUniversity of FlorenceLargo Brambilla, 3. I50134 Florence, ItalyPhone: +390557945425. Fax: +39055417123E-mail: fabio.marra@unifi.itManuscript recived: December 14, 2013.Manuscript accepted: December 14, 2013.March-April, Vol. 13 No. 2, 2014: 305-307LIVER NEWS ELSEWHEREArticle commented:Yong KJ, Gao C, Lim JS, Yan B, Yang H, DimitrovT, Kawasaki A, et al. Oncofetal gene SALL4 inaggressive hepatocellular carcinoma. N Engl J Med2013; 368: 2266-76.Comment:Hepatocellular carcinoma (HCC) is the sixthmost prevalent cancer worldwide and the third mostfrequent cause of cancer-related death, with increas-ing incidence in traditionally low-frequency regionslike the United States and Northern Europe.1 Arelevant proportion of patients with HCC presentwith advanced disease, and the only approved therapyin this subgroup is the multi-kinase inhibitor sorafe-nib, approved in 2008.2 The lack of novel and/or se-cond-line therapies, and the limited survival benefitprovided by sorafenib underscore the urgent needfor novel treatment options in advanced HCC. Thisgoal may be more easily achieved with a better un-derstanding of the molecular pathogenesis of thiscancer, which is characterized by a marked hetero-geneity. Unfortunately, the molecular mechanismsleading to HCC diversity and to its variable progno-sis are still far to be defined.The paper by Yong, et al., recently published inThe New England Journal of Medicine,3 describesthe possible role of SALL4 as a biomarker of aggres-sive hepatocellular carcinoma and a possible noveltarget for therapy in HCC. SALL4 (Sal-like 4) is oneof the human homologues of the homeotic gene spalt(Sal) in Drosophila, and encodes for a zinc-fingertranscription factor. SALL4 has a key role in themaintenance of pluripotency and self-renewal of em-bryonic stem cells,4-6 and mutations in this gene areassociated with the Okihiro syndrome, characterizedby multiple organ defects and a phenotype similarto thalidomide embryopathy.7 SALL4 has beenpreviously associated with regulation of liver deve-lopment. Its expression has been described in fetalhepatoblasts but not in adult hepatocytes, andexpression levels of SALL4 gradually fall duringliver maturation.8 In adulthood, SALL4 expressionhas been reported in various types of cancer9-11 andmay play a significant role in their development.Yong, et al. constructed a panel of tissue mi-croarrays consisting of 179 surgically resected HCC,and matched, archived, non-neoplastic liver speci-mens obtained from the National University Hospi-tal of Singapore, and compared the differentialexpression of SALL4. Immunohistochemical analysisshowed a significantly increase in the number of cellswith SALL4 expression in HCC specimens comparedto non-neoplastic tissue. 55.6% of HCC showedSALL4 positivity, although the levels of expressionwere variable. These findings were validated in anindependent group of specimens obtained from 228patients in Hong Kong, and analyzing data from pu-blic gene expression databases. The Authors next es-tablished whether expression of SALL4 had aprognostic role for patients with HCC. In both theSingapore and Hong Kong cohorts, a multivariateanalysis identified SALL4 expression as an indepen-dent prognostic factor for overall survival (in bothseries) and early recurrence (only in the Hong Kongcohort).In the past few years, transcriptional profiling ofhuman HCC has identified several subgroups, includ-ing tumors with phenotypic features of stem cells orprogenitor cells, which are particularly aggressiveand associated with a poor prognosis.12 In a tumor,cells with stem-cell properties are indicated astumor-initiating cells or cancer stem cells, and arebelieved to play a pivotal role in initiation, mainte-nance and recurrence of different types of cancer,© 2019, Fundación Clínica Médica Sur, A.C. Published by Elsevier España S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Inghilesi AL, et al. , 2014; 13 (2): 305-307306including HCC.13 Yong, et al. performed a hierarchi-cal cluster analysis of gene expression from humanprimary hepatocytes, fetal liver tissue and HCC,showing that tumors with high SALL4 cluster-ed with fetal livers, whereas low-SALL4 HCC clusteredwith normal hepatocytes. In addition, high-SALL4tumors were enriched in genes upregulated in HCCwith poor survival or with embryonic stem cell sig-natures. To provide more mechanistic data, SALL4expression was knocked down in SNU-398 HCCcells with specific short hairpin RNA (shRNA). Whilegene expression of SALL4-knocked down cells clus-tered with human hepatocytes, SNU-398 cells trea-ted with a control, scrambled shRNA clustered withhuman fetal liver tissue, further confirming the roleof the SALL4 transcription factor in conferringstemness features. Moreover, SALL4 knockdown de-creased cell viability and limited tumorigenesis inan experimental animal model.Transcription factor activity is usually more diffi-cult to be therapeutically targeted compared to pro-tein kinases, which are effectively modulated bysmall molecule inhibitors. To demonstrate a poten-tial translatability of their experimental observations,the authors used as a competitive inhibitor a re-cently described SALL4 12-AA peptide, that blocksthe interaction between SALL4 and NuRD (nucleo-some remodeling and histone deacetylase [HDAC]complex).14 Interaction between SALL4 and NuRDresults in downregulation of the tumor suppressorPTEN, which acts on the PI3-kinase/Akt pathway.When SNU-398 cells were treated with the SALL4antagonistic peptide, a reduction of cell viabilitywas observed, demonstrating the efficacy of thisapproach. Finally, the Authors tested the in vivoeffects of the SALL4 12-AA peptide in a xenograftmodel of HCC in which SNU-398 cells had beentransplanted subcutaneously. A significant reductionof tumor load was observed in mice treated with theantagonistic peptide compared to the group treatedwith a control peptide.The paper by Yong, et al. is of great interest inthe field of HCC for a number of reasons. SALL4has received considerable attention in the past fewyears for its role as a determinant and marker ofembryonic stem cells and of cancer stem cells indifferent tissues. Remarkably, the results of the studypublished in The New England Journal of Medi-cine3 are in agreement with those of two otherpapers which have appeared in 2013. Oikawa, et al.15found expression of SALL4 in HCC, cholangiocarci-noma, and mixed forms of liver tumor, and showedthat expression of this molecule is associated withpoor prognosis and with increased expression ofstem cell markers. Moreover, a Japanese group hasrecently reported that SALL4 is activated in a sub-type of HCC with stem cell features, and again con-firmed poor prognosis compared to SALL4-negativepatients.16 An intriguing finding from this study isthe observation that SALL4-positive HCC is signifi-cantly associated not only with markers of hepaticstem cells but also with HBV infection.One of the most interesting aspects emergingfrom the work of Yong, et al.3 and from the other re-cent studies on this molecule15,16 is the dual role ofSALL4 as a biomarker and a potential new targetfor therapy. This latter aspect was analyzed in allthree studies by combined in vitro and in vivoapproaches, including an injectable 12-AA peptide.While a strategy directly targeting SALL4 deservesfurther analysis, elucidation of its downstream effec-tors could provide alternative ways to interfere withthe aggressiveness of this subtype of cancers.SALL4 signals through NuRD, a complex associatedwith histone deacetylase activity, and histone deace-tylase inhibitors are currently investigated in HCC.Similarly, the fact that SALL4 decreases PTEN andresults in boosted activity of the PI3-kinase/Akt/mTOR pathway could pave the way to the use ofspecific related inhibitors17 in patients with highSALL4 expression. In addition, in the Hong Kongcohort of the Yong study,3 expression of SALL4 wasassociated with cancer recurrence. Along these li-nes, the observation that some SALL4 expression isdetectable even in non-neoplastic tissue may contri-bute to explain the higher risk of risk of recurrence,as demonstrated for other gene signatures in the ci-rrhotic liver.18An equally important aspect is the significance ofSALL4 as a biomarker to detect cancers with a moreaggressive phenotype, in line with other stem-cellgene expression profiles. Serum alpha-fetoproteinhas been indicated as a marker of HCC with proge-nitor-like phenotype and poor prognosis,12 and couldpotentially substitute for measurement of SALL4 ina liver biopsy. In the correspondence followingpublication of the SALL4 paper, Yong, et al. reportthat serum alpha-fetoprotein levels greater than 100ng/mL can identify patients with high SALL4 expres-sion in tumor tissue with sensitivity and specificityof 66.7 and 68.4%, respectively.19 The diagnosticaccuracy of the measurement of circulating SALL4levels in HCC, as reported for breast cancer,20 needsfurther evaluation in the near future.A final remark that is inspired by this excellentstudy is the re-evaluation of the role of liver biopsy307SALLuting a new biomarker in hepatocellular carcinoma. , 2014; 13 (2): 305-307for a more accurate stratification of the differentsubtypes of HCC and of the related prognosis, asshown by the comparison of SALL4-high and lowHCC. HCC is one of the few solid tumors for whichtreatment is not guided by molecular characteriza-tion. The possibility to diagnose HCC in a cirrhoticliver based only on imaging features has certainlyhelped the clinical management of the patients, buthas indirectly represented a hurdle to advance ourknowledge in the molecular characterization of thistumor, and in the design of personalized treatments.This is clearly reflected in the recently publishedphase II study evaluating tivantinib in patients withHCC failing sorafenib treatment,21 where drug-induced prolongation of the time-to-progressionwas markedly more evident in patients with highexpression of c-met in tumor tissue. Studies like thosedescribing the significance of SALL4 shouldencourage to obtain biopsies at least in the settingof controlled clinical trials.REFERENCES1. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma.Lancet 2012; 379: 1245-55.2. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, BlancJF, et al. Sorafenib in advanced hepatocellular carcinoma.N Engl J Med 2008; 359: 378-90.3. Yong KJ, Gao C, Lim JS, Yan B, Yang H, Dimitrov T, et al.Oncofetal gene SALL4 in aggressive hepatocellular carci-noma. N Engl J Med 2013; 368: 2266-76.4. Elling U, Klasen C, Eisenberger T, Anlag K, Treier M. Murineinner cell mass-derived lineages depend on Sall4 function.Proceedings of the National Academy of Sciences of theUnited States of America 2006; 103: 16319-24.5. Yang J, Gao C, Chai L, Ma Y. A novel SALL4/OCT4 trans-criptional feedback network for pluripotency of embryo-nic stem cells. PloS one 2010; 5: e10766.6. Rao S, Zhen S, Roumiantsev S, McDonald LT, Yuan GC, Or-kin SH. Differential roles of Sall4 isoforms in embryonicstem cell pluripotency. Molecular and Cellular Biology2010; 30: 5364-80.7. Kohlhase J, Chitayat D, Kotzot D, Ceylaner S, Froster UG,Fuchs S, et al. SALL4 mutations in Okihiro syndrome (Dua-ne-radial ray syndrome), acro-renal-ocular syndrome, andrelated disorders. Hum Mutat 2005; 26: 176-83.8. Oikawa T, Kamiya A, Kakinuma S, Zeniya M, NishinakamuraR, Tajiri H, et al. Sall4 regulates cell fate decision in fetalhepatic stem/progenitor cells. Gastroenterology 2009;136: 1000-11.9. Ma Y, Cui W, Yang J, Qu J, Di C, Amin HM, et al. SALL4,a novel oncogene, is constitutively expressed in humanacute myeloid leukemia (AML) and induces AML in transgenicmice. Blood 2006; 108: 2726-35.10. Cui W, Kong NR, Ma Y, Amin HM, Lai R, Chai L. Differen-tial expression of the novel oncogene, SALL4, in lympho-ma, plasma cell myeloma, and acute lymphoblasticleukemia. Modern pathology: an official journal of theUnited States and Canadian Academy of Pathology, Inc2006;19: 1585-92.11. Kobayashi D, Kuribayshi K, Tanaka M, Watanabe N. SALL4is essential for cancer cell proliferation and is overexpres-sed at early clinical stages in breast cancer. Internatio-nal Journal of Oncology 2011; 38: 933-9.12. Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, CalvisiDF, et al. A novel prognostic subtype of human hepatocel-lular carcinoma derived from hepatic progenitor cells. NatMed 2006; 12: 410-6.13. Rountree CB, Mishra L, Willenbring H. Stem cells in liverdiseases and cancer: recent advances on the path to newtherapies. Hepatology 2012; 55: 298-306.14. Gao C, Dimitrov T, Yong KJ, Tatetsu H, Jeong HW, Luo HR,et al. Targeting transcription factor SALL4 in acute myeloidleukemia by interrupting its interaction with an epige-netic complex. Blood 2013; 121: 1413-21.15. Oikawa T, Kamiya A, Zeniya M, Chikada H, Hyuck AD, Ya-mazaki Y, et al. Sal-like protein 4 (SALL4), a stem cell bio-marker in liver cancers. Hepatology 2013; 57: 1469-83.16. Zeng SS, Yamashita T, Kondo M, Nio K, Hayashi T, Hara Y,et al. The transcription factor SALL4 regulates stemnessof EpCAM-positive hepatocellular carcinoma. J Hepatol2014; 60: 127-34.17. Moeini A, Cornella H, Villanueva A. Emerging SignalingPathways in Hepatocellular Carcinoma. Liver Cancer 2012;1: 83-93.18. Hoshida Y, Villanueva A, Kobayashi M, Peix J, Chiang DY,Camargo A, et al. Gene expression in fixed tissues and out-come in hepatocellular carcinoma. N Engl J Med 2008;359: 1995-2004.19. Yong KJ, Chai L, Tenen DG. Oncofetal gene SALL4 inaggressive hepatocellular carcinoma. N Engl J Med 2013;369: 1171-2.20. Panis C, Pizzatti L, Herrera AC, Cecchini R, Abdelhay E.Putative circulating markers of the early and advancedstages of breast cancer identified by high-resolution la-bel-free proteomics. Cancer Lett 2013; 330: 57-66.21. Santoro A, Rimassa L, Borbath I, Daniele B, Salvagni S,Van Laethem JL, et al. Tivantinib for second-line treat-ment of advanced hepatocellular carcinoma: a randomi-sed, placebo-controlled phase 2 study. Lancet Oncol2013; 14: 55-63.

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