Oncogenic Role of SND1 in Development and Progression of … · document that Alb/SND1 mice develop spontaneous HCC with expansion of tumor-initiating cells (TIC). We also demonstrate

Therapeutics, Targets, and Chemical Biology

Oncogenic Role of SND1 in Development andProgression of Hepatocellular CarcinomaNidhi Jariwala1, Devaraja Rajasekaran1, Rachel G. Mendoza1, Xue-Ning Shen1,Ayesha Siddiq1, Maaged A. Akiel1, Chadia L. Robertson1, Mark A. Subler1,Jolene J.Windle1, Paul B. Fisher1,2,3, Arun J. Sanyal4, and Devanand Sarkar1,2,3

Abstract

SND1, a subunit of the miRNA regulatory complex RISC, hasbeen implicated as an oncogene in hepatocellular carcinoma(HCC). In this study, we show that hepatocyte-specific SND1transgenic mice (Alb/SND1 mice) develop spontaneous HCCwith partial penetrance and exhibit more highly aggressiveHCC induced by chemical carcinogenesis. Livers from Alb/SND1 mice exhibited a relative increase in inflammatory mar-kers and spheroid-generating tumor-initiating cells (TIC).Mechanistic investigations defined roles for Akt and NF-kB

signaling pathways in promoting TIC formation in Alb/SND1mice. In human xenograft models of subcutaneous or ortho-topic HCC, administration of the selective SND1 inhibitor 30,50-deoxythymidine bisphosphate (pdTp), inhibited tumor for-mation without effects on body weight or liver function. Ourwork establishes an oncogenic role for SND1 in promotingTIC formation and highlights pdTp as a highly selective SND1inhibitor as a candidate therapeutic lead to treat advancedHCC. Cancer Res; 77(12); 3306–16. �2017 AACR.

IntroductionStaphylococcal nuclease and tudor domain containing 1

(SND1) is a multifunctional protein that regulates transcription,mRNA splicing, RNA editing, and miRNA-mediated mRNA deg-radation as a nuclease in RNA-induced silencing complex (RISC;refs. 1–8). SND1 is overexpressed in multiple cancers, where itfunctions as an oncogene (9–14). In hepatocellular carcinoma(HCC), SND1 overexpression was identified in approximately74% cases (14). Overexpression of SND1 promotes and knock-down of SND1 inhibits proliferation, invasion, angiogenesis, andin vivo tumorigenesis by human HCC cells (14–17). Our studiesdocument that SND1 exerts its function in HCC cells by a varietyof mechanisms. SND1 overexpression contributed to increasedRISC activity in HCC cells, resulting in augmented degradation oftumor suppressor mRNAs that are targets of oncogenic miRNAs(14). SND1 promotes angiogenesis by activating NF-kB, resultingin the induction of miR-221 and subsequently angiogenic factorsangiogenin and CXCL16 (16). SND1 binds to 30 untranslatedregion (30-UTR) of angiotensin II type I receptor (AT1R) mRNA,

increases AT1R mRNA stability, and increases AT1R protein level(1). We documented that this increase in AT1R by SND1 leads toactivation of ERK, Smad2, and subsequently TGFb signalingpathway promoting epithelial–mesenchymal transition (EMT),migration, and invasion by human HCC cells (15). SND1 inter-acts with monoglyceride lipase (MGLL), resulting in MGLL deg-radation, which leads to activation of Akt and stimulation of cellproliferation and cell-cycle progression inHCCcells (17).Overall,SND1 plays a dynamic role in regulating expression of genescrucial to hepatocarcinogenesis by employing diverse transcrip-tional as well as posttranscriptional molecular mechanisms(15, 18).

SND1 is composed of four staphylococcal nuclease (SN)domains and a single fusion domain, consisting of a tudordomain and a nuclease domain (8). The nuclease domainsfunction as RNase, while the tudor domain is involved in pro-tein–nucleic acid interaction (8). Enzymatic activity of SND1 isrequired for its function in RISC (7). However, whether the otherfunctions of SND1 require enzymatic activity remains to bedetermined. Structurally, SND1 is unique in the humanproteomewith no close homolog as revealed by BLAST search (18). Theclosest homolog of SND1 is EBNA2, which is not transcribed.SND1 has highly electropositive SN domains, to which binds thenegatively charged 30, 50-deoxythymidine bisphosphate (pdTp)molecule inhibiting SND1 nuclease activity (7, 8, 19, 20). Theabsence of a close homolog of SND1 and availability of a specificSND1 inhibitor pdTp suggests that pdTpmight be developed as apotential HCC therapeutic with little side effects. Indeed, weshowed that pdTp inhibited growth of human HCC cells in vitro(14). However, in vivo antitumor efficacy of pdTp remains to bedetermined. In addition, pdTp might also serve as a valuable toolto distinguish enzymatic and nonenzymatic functions of SND1.

In this article, we employ a hepatocyte-specific transgenicmouse overexpressing SND1 (Alb/SND1) to obtain insightsinto the oncogenic function of SND1 in an in vivo context. We

1Department of Human and Molecular Genetics, Virginia Commonwealth Uni-versity, Richmond, Virginia. 2VCU Massey Cancer Center, Virginia Common-wealth University, Richmond, Virginia. 3VCU Institute of Molecular Medicine(VIMM), Virginia CommonwealthUniversity, Richmond, Virginia. 4Department ofInternal Medicine, Virginia Commonwealth University, Richmond, Virginia.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Devanand Sarkar, Virginia Commonwealth University,School of Medicine, 1220 East Broad St, Box 980035, Richmond, VA 23298.Phone: 804-827-2339; Fax: 804-628-1176; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-0298

�2017 American Association for Cancer Research.

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document that Alb/SND1 mice develop spontaneous HCC withexpansion of tumor-initiating cells (TIC). We also demonstratein vivo safety and antitumor efficacy of pdTp, thereby pavingthe way for its further characterization as an anti-HCC agent.We thus identify SND1 as a valid molecular target in HCC andpresent SND1 inhibition as a promising approach to curbhepatocarcinogenesis.

Materials and MethodsGeneration of Alb/SND1 mouse and induction of chemicalcarcinogenesis

Alb/SND1 transgenic mouse in B6CBAF1 background wasgenerated by directing the expression of C-terminal Myc-taggedhuman SND1 under an upstream enhancer region (�10400 to�8500) fused to the 335-bp core region of mouse albuminpromoter (Supplementary Fig. S1; ref. 21). Microinjection andmanipulation procedures were performed according to standardprotocols in the VCUMassey Cancer Center Transgenic/KnockoutMouse Core. For induction of chemical carcinogenesis, a singleintraperitoneal injection of 10 mg/g body weight of N-nitroso-diethylamine (DEN)was given at 14 days of age tomalewild-type(WT) and Alb/SND1 littermates (22). The animals were sacrificedat 32 weeks of age and liver, internal organs, and blood werecollected. Serum liver enzymes were analyzed in the MolecularDiagnostic Laboratory, Department of Pathology, VCU (Rich-mond, VA) using standard procedures. All experiments wereperformed using sibling littermates, fed regular chow diet duringlight cycle. All animal studies were approved by the InstitutionalAnimal Care and Use Committee at Virginia CommonwealthUniversity and were conducted in accordance with the AnimalWelfare Act, the PHS Policy on Humane Care and Use of Labo-ratory Animals, and the U.S. Government Principles for theUtilization and Care of Vertebrate Animals Used in Testing,Research, and Training.

Cells, culture condition, sphere formation, and Matrigelinvasion assays and chemicals

Primary mouse hepatocytes were isolated from adult male WTand Alb/SND1 littermates, cultured without passage as described,and were mycoplasma free (22). The human HCC cell line QGY-7703 was developed at Fudan University (Shanghai, China),obtained from Dr. Zhao-Zhong Su in 2008, and cultured asdescribed previously (23). Generation and characterization ofQGY-7703 cells expressing luciferase (QGY-luc) have beendescribed previously (24, 25). Early passage (>5) cultures ofQGY-7703 and QGY-luc cells were stored in liquid nitrogen, andin vivo studies described in this article were performedwith freshlythawed culture of the cells after confirmingmycoplasma free usingMycoplasma Detection Kit (Thermo Fisher Scientific). Hepato-cytes were cultured in Essential 8 Medium (Thermo Fisher Sci-entific; catalog # A1517001) for enrichment of TICs using ultra-low attachment plates. Sphere formation was monitored, andspheres containing more than 50 cells were quantified micro-scopically. Nonproliferative spheres were excluded as abortivespheres. Matrigel invasion assay using primary hepatocytes wasperformed as described previously (23, 26). pdTp was purchasedfrom Axxora (catalog # BLG-T012-05). BMS-3445541 (catalog #B9935) and LY294006 (catalog # L9908) were purchased fromSigma-Aldrich, and U0126 (catalog # 9903) was purchased fromCell Signaling Technology.

IHC and immunofluorescenceIHC using formalin-fixed paraffin-embedded (FFPE) sections

and immunofluorescence in primary hepatocytes were perform-ed as described previously (23). For IHC, the sections wereblocked in PBST using 10% normal goat serum for rabbit andmouse polyclonal antibodies. Primary antibodies were diluted inPBST containing 5%blocking serum. Theprimary antibodies usedwere SND1 (rabbit polyclonal; 1:200; Sigma), PCNA (mousemonoclonal; 1:200; Cell Signaling Technology), AFP (rabbitpolyclonal; 1:50; Santa Cruz Biotechnology), F4/80 (rat poly-clonal; 1:200; Bio-Rad), CD31 (rabbit polyclonal; 1:50; AbCam),cleaved caspase-3 (rabbit polyclonal; 1:300; Cell Signaling Tech-nology), p-p65 (rabbit polyclonal; 1:400; Cell Signaling Technol-ogy), EpCAM (rabbit polyclonal; 1:400; Sigma), CD133 (rabbitpolyclonal; 1:500; Proteintech), and CD44 (mouse monoclonal;1:250; AbCam). Biotin-conjugated secondary antibodies werediluted in PBST containing corresponding 2.5% blocking serum.Sections were stained using avidin–biotin–peroxidase complexestreated with a DAB substrate solution (Vector Laboratories). IHCimageswere quantifiedbyH-scorewithWT score normalized to 1.For immunofluorescence, the primary antibody was p65 (mousemonoclonal; 1:400; Cell Signaling Technology), and the second-ary antibody was Alexa546-conjugated anti-mouse IgG (goat;1:400; Invitrogen). The slides were mounted in VectaShieldfluorescence mounting medium containing 4,6 -diamidino-2-phenylindole (Vector Laboratories). Images were analyzed usinga Zeiss confocal laser scanning microscope.

Western blottingLysates were prepared by lysing cells in 1.5% n-dodecyl -D-

maltoside buffer supplemented with protease and phosphataseinhibitor cocktail (Pierce). Lysates from liver tissue were preparedby mechanical homogenization using the same buffer. Westernblot analysis was performed as described previously (23). Primaryantibodies used were SND1 (rabbit polyclonal; 1:1,000; Sigma),GAPDH (mouse monoclonal; 1:1,000; Santa Cruz Biotechno-logy), CD133 (rabbit polyclonal; 1:1,000; Proteintech), CD44(mouse monoclonal; 1:1000; Abcam), Myc-Tag, p-Akt, Akt, ERK,p-ERK, p-GSK3b, GSK3b, p-p65, p65 (rabbit polyclonal; 1:1,000;Cell Signaling Technology), AT1R (rabbit polyclonal; 1:1000;Abnova), MGLL (rabbit polyclonal; 1:1,000; Thermo Fisher Sci-entific). Densitometric analysis was performed by ImageJsoftware.

Total RNA extraction, cDNA preparation, and quantitativeRT-PCR

Total RNA was extracted using the Qiagen miRNeasy Mini Kit(Qiagen). cDNA preparation was done using ABI cDNA SynthesisKit (Applied Biosystems). qRT-PCR was performed using an ABIViiA7 Fast Real-Time PCR System and TaqMan gene expressionassays according to the manufacturer's protocol (AppliedBiosystems).

Flow cytometryWT and Alb/SND1 hepatocytes were isolated and washed in

PBS. Hepatocytes (1 � 106 per sample) were stained withfluorochrome-conjugated primary antibodies in 2% BSA inPBS at room temperature for 1 hour. CD133 (mouse-APC;1:50; Miltenyi Biotec), CD44 (rat-FITC; 1:50; Abcam), andEpCAM (rat-PE; 1:20; BD Pharmingen) were used as per themanufacturer's protocol. Cells were washed twice in PBS and

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resuspended in 2% BSA in PBS for flow cytometric analysisusing BD FACSCanto II.

Treatment of human HCC xenografts in NSG miceQGY-7703 cells (6.5 � 105 cells suspended in 50 mL of Matri-

gel) were injected subcutaneously in the flanks of adult male NSGmice. Tumor volume was measured twice a week with a caliperand calculated using the formula p/6� larger diameter� (smallerdiameter)2. After the tumors reached approximately 100 mm3

(requiring a week), different doses of pdTp (0.16, 0.32, and 0.8mg/kg) were administered to the mice intraperitoneally twice aweek for 4 weeks. Sixmice per groupwere used for each treatmentgroup and 8mice were used for control vehicle-treated group. Fororthotopic xenografts, QGY-luc cells (1� 106) were implanted byintrahepatic injection in adult male NSG mice (24, 25). Tumorgrowth was monitored by bioluminescence imaging (BLI) with aXenogen IVIS imager once a week. After one week, when well-established tumors were detected by BLI, different doses of pdTp(0.8 and 1.6 mg/kg) were administered to the mice intravenouslytwice a week for 4 weeks. Six mice per group were used.

Statistical analysisData were represented as the mean � SEM and analyzed for

statistical significance using Student paired t test. A P value of<0.05 was considered as statistically significant.

ResultsAlb/SND1 mice manifest aggressive hepatocarcinogenesis

We have created a hepatocyte-specific C-terminal Myc-taggedhuman SND1-expressing transgenic mouse (Alb/SND1) by usingthe mouse albumin promoter/enhancer element to drive SND1expression in a B6CBAF1 background. This particular strain ofmouse is very sensitive to DEN-induced hepatocarcinogenesis(22). The expression of SND1 in the livers of Alb/SND1mice wasconfirmed by Western blot analysis, TaqMan qRT-PCR, and IHC(Fig. 1A–C; Supplementary Fig. S2A). Alb/SND1 mice developand reproduce normally, and no physiologic abnormalities andsignificant differences in body weight compared with WT litter-mates were observed. Histopathologic analysis at 2months of agedid not reveal any difference in liver architecture betweenWT andAlb/SND1 mice (Fig. 1C). However, at one year of age, 6 of 14(�42%) Alb/SND1 mice developed hepatic nodules, which wereconfirmed as HCC upon histologic examination with loss ofhepatic architecture, and AFP expression (Fig. 1D–F; Supplemen-tary Fig. S2A; Table 1). However, no such nodules were observedin WT littermates. Compared with WT, a significant increase inmRNAs for c-Myc, TNFa, and IL6, known drivers of HCC, wasdetected in Alb/SND1 livers at 2 and 12months of age, except forIL6, which showed an increase only at 12 months (Fig. 1G).

We next checked the response of Alb/SND1 mice to DEN-induced HCC. At 32 weeks after DEN injection, Alb/SND1 miceshowed a profound tumorigenic response, with tumorigenesisaffecting the entire liver, compared with WT littermates, in whichthere were either no nodules or nodules that were <5 mm in size(Fig. 2A; Table 1). Liver weight, reflecting higher tumor load, andserum AST, ALT, and total protein were significantly elevated inAlb/SND1 mice versus WT (Fig. 2B and C). A marked increase inmRNA of HCC markers AFP and CD36 was detected in DEN-treated Alb/SND1 livers compared with DEN-treated WT(Fig. 2D). Histologically, DEN-treated Alb/SND1 livers showed

loss of architecture and increased expression of CD31 (angiogen-esismarker) andPCNA(proliferationmarker) versusDEN-treatedWT (Fig. 2E; Supplementary Fig. S2A). Increased activation ofERK, Akt, and its downstream GSK3b was observed in DEN-treated and spontaneous Alb/SND1 tumors compared withDEN-treated WT (Fig. 2F; Supplementary Fig. S2B). ERK and Aktare activated by SND1 (15, 17) and also play a critical role inHCC(27) thus might be crucial in mediating SND1-induced HCC.

Alb/SND1 hepatocytes show increased activation of NF-kBChronic inflammation is a central event in hepatocarcinogen-

esis, and NF-kB plays a pivotal role in promoting inflammation(28). Increased expression of IL6 and TNFa (Fig. 1G) indicatesthat SND1 overexpression results in a chronic inflammatory stateleading to HCC. We previously documented activation of NF-kBin SND1-overexpressing humanHCC cells (16). NF-kB activationis marked by phosphorylation of serine residue 536, allowingnuclear translocation of p65 subunit, which then functions as atranscription factor to modulate expression of inflammatorygenes. We checked nuclear localization of p65 NF-kB in WT andAlb/SND1 hepatocytes, treated or untreated with lipopolysaccha-ride (LPS), as serumLPS levels are elevated inHCCpatients,whichpromotes inflammation. Alb/SND1 hepatocytes, but not WT,showed nuclear p65 under basal condition, indicating constitu-tive activation of NF-kB (Fig. 3A). LPS treatment resulted innuclear translocation of p65 in both WT and Alb/SND1 hepato-cytes. Inhibition of enzymatic activity of SND1 by pdTp resultedin marked downregulation of p65 levels with simultaneousinhibition of p65 nuclear translocation in both WT and Alb/SND1hepatocytes, suggesting a central role of SND1 in regulatingp65 expression (Fig. 3A).Western blot analysis revealed increasedbasal level of phosphorylated p65 (p-p65) in Alb/SND1 hepato-cytes compared with WT (Fig. 3B). In WT hepatocytes, upon LPStreatment, increased p-p65 was observed at 10 minutes, whichgradually waned down over a period of 2 hours (Fig. 3B).However, in Alb/SND1 hepatocytes, increased p-p65 levelremained sustained during the assay period (Fig. 3B). Increasedinflammation was also indicated by increased infiltration ofmacrophages, detected by staining for F4/80 marker, in DEN-treated and 1-year-old tumor-bearing Alb/SND1 livers versuscorrespondingWT(Fig. 3C; Supplementary Fig. S2C). In addition,a marked increase in immune checkpoint molecule PD-L1 and asignificant increase in its receptor PD-1 were observed in DEN-treated and 1-year-old tumor-bearing Alb/SND1 livers versuscorresponding WT, indicating a strong carcinogenic responseinduced by SND1 overexpression (Supplementary Fig. S2D).

SND1 overexpression results in expansion of TICsAs Alb/SND1mice develop spontaneous HCC, we checked the

effect of SND1 overexpression on TICs. In a sphere formationassay in ultralow attachment plates,WThepatocytes formed smallabortive spheres, while Alb/SND1 hepatocytes formed robustspheres that gradually increased in size and number, indicatingan expansion of TICs (Fig. 4A). Indeed, TICs positive for threemarkers, EpCAM, CD44, and CD133, were significantly more inAlb/SND1 livers versus WT (Fig. 4B). Alb/SND1 hepatocytesisolated from 2-month-old mice showed significant increase inmRNA levels of EpCAM (�2-fold), CD44 (�4-fold), and CD133(�4-fold) compared with WT (Supplementary Fig. S3A). Theseincreases in mRNA levels were further augmented, for example,EpCAM (�2.5-fold), CD44 (�6-fold), and CD133 (�4-fold), in

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Alb/SND1 hepatocytes isolated from 12-month-old mice versusWT (Supplementary Fig. S3A). IHC analysis showed increasedexpression of EpCAM,CD44, andCD133 in 1-year-old Alb/SND1livers, with or without tumor, compared with WT littermates(Supplementary Fig. S3B and S3C). Increased CD133 expressionwas observed in DEN-treated and spontaneous tumors in Alb/SND1 mice when compared with DEN-treated WT livers (Sup-plementary Fig. S3D). Treatment with pdTp significantly inhib-ited sphere formation by both WT and Alb/SND1 hepatocytes,indicating that enzymatic function of SND1 is necessary topromote expansion of TICs (Fig. 4C). As expected from studiesin human cell lines, naïve Alb/SND1 livers showed increased

activation of ERK and Akt (Fig. 3D). To check the effects of thesignaling pathways activated by SND1 in regulating TICs, weperformed sphere formation assay with Alb/SND1 hepatocytesupon treatment with BMS-3445541(IkB kinase inhibitor to blockNF-kB activation), LY294006 (PI3K inhibitor to block Akt acti-vation), and U0126 (MEK1/2 inhibitor to block ERKactivation; Fig. 4E and F). Inhibition of NF-kB and Akt activation,but not ERK activation, significantly abrogated sphere formationby Alb/SND1 hepatocytes with corresponding decrease in CD133expression (Fig. 4E and F; Supplementary Fig. S4A). We interro-gated the potential role of ERK activation in SND1-inducedphenotypes other than sphere formation. We previously

Figure 1.

SND1 overexpression in vivo results in neoplastic transformation. SND1 overexpression in Alb/SND1 (TG) liver was confirmed by Western blot analysis(A), TaqMan qRT-PCR (B), and IHC (C). SND1 mRNA levels were normalized by GAPDH mRNA levels. A–C, Liver samples from 2-month-old mice were used.D, Photographs of representative livers of 1-year-old mice. Arrow, liver nodule. E, Histologic analysis and AFP immunostaining of FFPE liver sections from 1-year-oldWT and Alb/SND1 mice with or without tumors. F, Relative expression of AFP mRNA by TaqMan qRT-PCR in 1-year-old WT and Alb/SND1 mice with tumor(3mice/group).G, Relative expression of the indicatedmRNAs by TaqMan qRT-PCR in livers of 2-month and 1-year-old mice (3mice/group). A.U., arbitrary unit. Forgraphs, data represent mean � SEM; � , P < 0.01 versus WT.

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demonstrated a potential role of ERK activation in mediatingSND1-induced invasion of human HCC cells (15). Although WThepatocytes did not invade through Matrigel, Alb/SND1 hepato-

cytes acquiredMatrigel invasion property, whichwas significantlyabrogated upon treatment with U0126 (Fig. 4G).

SND1 inhibitor pdTp significantly abrogates human HCCxenografts in vivo

pdTp specifically inhibits the nuclease activity of SND1, butdoes not affect the oligonucleotide binding function of the tudordomain. We previously documented that pdTp inhibits prolifer-ation of human HCC cells in vitro (14). As yet, in vivo efficacy andtoxicity has not been tested for pdTp. We injected multiple dosesof pdTp (calculated from our in vitro studies) to WT B6CBA miceintraperitoneally twice a week for 4 weeks (a total of 8 doses). Atthe highest dose of 0.8 mg/kg, no difference in body and liverweights, serum liver enzymes, total protein, albumin, and glob-ulin was observed versus vehicle at the end of the treatment cycle(Fig. 5A and B). Bilirubin levels (conjugated and unconjugated)were normal and did not show any increase upon pdTp treatment(data not shown). Histologic analysis of internal organs also didnot show any abnormality (Fig. 5C). We established subcutane-ous xenografts of QGY-7703 cells in NSGmice and evaluated theeffect of intraperitoneal administration of different doses pdTpontumor development. A significant decrease in tumor volume andtumor weight was observed with 0.32 and 0.8 mg/kg pdTp at theend of the treatment (Fig. 5D). We next established orthotopicxenografts of QGY-luc cells (QGY-7703 cells expressing lucifer-ase) in the livers of NSG mice and evaluated the effect of intra-venous administration of pdTp on tumor development by BLI. Asignificant inhibitory effect on tumor progression was observedwith pdTp treatment compared with vehicle (Fig. 5E and F). IHCanalysis of subcutaneous tumor sections revealed that pdTptreatment resulted in a dose-dependent decrease in PCNA,CD133, CD44, and p-p65 staining, and an increase in apoptosis,determined by staining for cleaved caspase-3 (Fig. 6A; Supple-mentary Fig. S4B). Western blot analysis of tumor samples iden-tified that pdTp treatment resulted in downregulation of CD133

Table 1. Number of nodules in WT and Alb/SND1 (TG) mice

Nodules (mm)ID 1–2 >3 >10

1 year old TG1 7 0 1TG2 No noduleTG3 No noduleTG4 No noduleTG5 3 0 0TG6 No noduleTG7 No noduleTG8 No noduleTG9 0 0 1TG10 0 1 0TG11 No noduleTG12 No noduleTG13 0 1 0TG14 0 1 0

DEN treated WT1 0 0 1WT2 3 5 0WT3 0 5 0WT4 0 0 10WT5 No noduleWT6 No noduleWT7 No noduleWT8 No noduleTG1 4 27 44TG2 0 0 41TG3 Entire liverTG4 Entire liverTG5 Entire liverTG6 Entire liverTG7 Entire liverTG8 Entire liverTG9 Entire liverTG10 Entire liver

Figure 2.

Alb/SND1 mice develop aggressive DEN-induced HCC. WT and Alb/SND1 littermates were injected intraperitoneally with DEN (10 mg/g) at 2 weeks of age,and livers were harvested at 32 weeks postinjection when subsequent analyses were performed. A, Photographs of representative livers. Measurement ofliver wt (B), and serum AST, ALT, and total protein (C; WT, n ¼ 8; Alb/SND1, n ¼ 10). D, Analysis of AFP and CD36 mRNA levels by TaqMan qRT-PCR innaïve and DEN-treated livers. Normalized by GAPDH. E, Histologic analysis and IHC for CD31 and PCNA in FFPE liver sections. F, Western blot analysis for theindicated proteins in the livers of DEN-treated WT and Alb/SND1 littermates at 32 weeks and spontaneous tumors in Alb/SND1 livers at 1 year. For graphs,data represent mean � SEM; � , P < 0.01 versus WT.

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and CD44 levels and decreased phosphorylation of Akt and p65(Fig. 6B; Supplementary Fig. S4C). Total p65 level was alsodecreased uponpdTp treatment (Fig. 6B). Interestingly, no changein ERK activation was observed upon treatment with pdTp. Wepreviously documented that increased RISC activity resultingfrom SND1 overexpression augments oncomiR-mediated degra-dation of tumor suppressor mRNAs, such as PTEN, target of miR-221 and miR-21, CDKN1C (p57), target of miR-221, CDKN1A(p21), target of miR-106b, SPRY2, target of miR-21, and TGFBR2,target of miR-93 (14). As a corollary, in vivo pdTp treatmentresulted in significant increases in PTEN, TGFBR2, and CDKN1C

mRNA levels in tumors compared with vehicle (Fig. 6C). Collec-tively, these findings reveal that pdTp inhibits proliferation andinflammation, induces apoptosis, and downregulates TICs.

DiscussionIn this research article, we report the oncogenic role of SND1 in

HCCdevelopment and progression by pursuing studies in a novelhepatocyte-specific SND1-overexpressing transgenicmousemod-el. Our previous in vitro studies established that SND1 overexpres-sion positively regulates multiple hallmarks of cancer, including

Figure 3.

NF-kB activation is exaggerated in Alb/SND1 hepatocytes. A, Hepatocytes were harvested from 1-year-old WT and Alb/SND1 littermates and treated withLPS (200 ng/mL) for 30 minutes. In a second experiment, hepatocytes were treated with pdTp (200 mmol/L) for 18 hours before LPS treatment. Thehepatocytes were immunostained for p65 NF-kB, and immunofluorescence analysis was performed by confocal microscopy. B, Hepatocytes were harvestedfrom 2-month-old WT and Alb/SND1 littermates and treated with LPS (200 ng/mL) for the indicated time points. Western blot analysis of the indicated proteinswas performed. C, FFPE sections of DEN-treated and 1-year-old WT and Alb/SND1 livers were stained for macrophage marker F4/80.

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proliferation, migration, invasion, angiogenesis, EMT, and inhi-bition of tumor suppressor gene expression (14–17). In thecurrent in vivo studies, we document that SND1 causes sponta-neous hepatocarcinogenesis by increasing TICs within the liverand creating a proinflammatory microenvironment, and sensi-tizes hepatocytes toward DEN-induced HCC. As a corollary,chemical inhibition of SND1 enzymatic activity by pdTp reducedtumor-initiating potential of hepatocytes and rescued proinflam-matory signaling caused by SND1 overexpression, resulting insignificant abrogation of tumor growth in a xenograft model.

SND1 is a multifunction protein that regulates gene expressionat transcriptionalaswellasposttranscriptional level. Inflammatorycytokine TNFa is constitutively upregulated in Alb/SND1 liver,whereas IL6 is upregulated in an age-dependent manner. Boththese cytokines are known to activateNF-kB signaling and are alsoinduced by NF-kB. As expected, Alb/SND1 hepatocytes manifestan exaggerated NF-kB activation, both constitutive and upon LPStreatment. LPS is detected at notably high levels in HCC patients,thusmaking SND1-mediated augmentation inNF-kB activation aclinically relevant scenario.Activationof residentKupffer cells andinvasion of liver with macrophages is a crucial event in HCC.Secretion of inflammatory cytokines by SND1-overexpressing

hepatocytes might contribute to NF-kB signaling and activationinmacrophages. Thus, SND1overexpressioncreatesanunderlyingproinflammatory condition within liver, which aggravates withage, creating conducive preexisting pathology for tumorigenesis.

We observe that constitutive overexpression of SND1 predis-poses Alb/SND1 animals to risk of HCC development in lateadulthood, in the absence of any carcinogen exposure, withexpansion of CD133þ, CD44þ, and EpCAMþ TICs. Our inhibitorstudies unravel that active Akt and NF-kB signaling is required tomaintain tumor initiation potential of SND1-overexpressinghepatocytes. A recent report documented that in a hepatitis Bmodel, AFP-induced upregulation of CD133þ, CD44þ, andEpCAMþ TICs is dependent on PI3K/Akt signaling (29). Clinicalstudy analyzing protein marker expression in liver from morethan 100 HCC patients showed a significant negative correlationwith PTEN and positive correlation with Akt levels with TICmarker proteins CD133, EpCAM, and CD90 (30). Univariate andmultivariate analysis showed significant correlation between lossof PTEN and high AFP, Akt, CD133, and EpCAM levels withoverall survival of HCC patients (30). CD133 expression iscorrelated with sorafenib resistance in human HCC patients(31, 32). CD133 confers chemoresistance and radioresistance via

Figure 4.

SND1 overexpression results in the expansion of TICs. A, Hepatocytes were harvested from 1-year-old WT and Alb/SND1 littermates and cultured underultralow attachment conditions to form spheroids and observed over a period of 3 days. Left, photomicrograph of representative spheres; right, graphicalquantification of the spheres. B, Hepatocytes were harvested from 1-year-old WT and Alb/SND1 littermates, immunostained with fluorochrome-conjugatedEpCAM, CD44, and CD133 antibodies, and subjected to flow cytometry. C, Hepatocytes were harvested from 2-month-old WT and Alb/SND1 littermates,treatedwith pdTp (200mmol/L), and sphere formationwasmeasured over a period of 7 days.D,Western blot analysis of the indicated proteins in the livers of 1-year-old WT and Alb/SND1 littermates. Two independent mice per group were used. E and F, Hepatocytes from 2-month-old Alb/SND1 mice were harvested andtreated with BMS-3445541 (5 mmol/L), LY294006 (25 mmol/L), and U0126 (10 mmol/L) in enriching conditions. Western blot analysis using the indicatedantibodies was performed (E). Sphere formation was compared between untreated and treated Alb/SND1 hepatocytes over a period of 3 days (F). G,Matrigel invasion using WT and Alb/SND1 (TG) hepatocytes treated or untreated with U0126 (10 mmol/L) was performed. HPF, high-power field. For graphs, datarepresent mean � SEM; �, P < 0.01 versus WT in A and G, vehicle (Veh) in C, and no Rx in F.

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upregulation of PI3K/AKT signaling (32, 33). Interaction betweenhyaluronan and CD44 in extracellular matrix promotes carcino-genic signaling and results in chemoresistance (34). Thus, a closeinterplay betweenAKT signaling andTIC expansion is predicted inAlb/SND1 livers, rendering them therapeutically resistant. Itwouldbe interesting to investigate thepotential of SND1 targetingin overcoming chemoresistance in HCC for future studies.

Studies have shown that IL8, a NF-kB downstream gene,increases CXCL1 expression, upregulates MAPK signaling, andsustains cancer stemness that eventually expands CD133þ pop-ulation in liver (35). Autocrine IL6 signaling is also implicated inprotumorigenic properties of HCC progenitor cells (36). IL6signaling promoted by tumor-associated macrophages is foundto promote expansion of CD44þ cells, potentiating growth ofxenograft tumors, and inhibition of IL6/STAT3 signaling in thismodel, reduced tumorigenic potential of CD44þ cells (37). In

SND1-overexpressing HCC, we report an increased invasion ofmacrophages, which can be predicted to upregulate IL6 secretionand thus promote expansion of TICs. As yet, the molecularmechanism by which SND1 activates NF-kB remains to be deter-mined. pdTp treatment studies clearly demonstrate that SND1enzymatic activity is required to activate NF-kB (Fig. 3A). How-ever, pdTp treatment also resulted in a decrease in total p65 level,indicating that SND1 regulates not only NF-kB activation but alsop65 expression. Inflammatory cytokines induce SND1 expressionand SND1 promoter contains consensus NF-kB binding sites (38,39). Thus, chronic inflammation preceding HCC might result ininduction of SND1, which facilitates expansion of TICs andfurther aggravates inflammation, thereby establishing a scenariofor the development of HCC.

SND1 overexpression activates Akt, ERK, and NF-kB signaling.We document that pdTp treatment inhibited Akt and NF-kB

Figure 5.

pdTp is nontoxic and inhibits humanHCC xenografts.WTB6/CBAmice (2months old)were injected intraperitoneallywith 0.8mg/kg pdTp twice aweek for 4weeks.Body and liver weights (A), and serum liver enzymes (AST, ALT, and AP), total protein (TP), albumin (Alb), and globulin (Glo; B) were measured at the endof the treatment. C, Histology of internal organs at the end of pdTp treatment. D, QGY-7703 cells were injected subcutaneously in NSG mice to establishxenografts and treated with the indicated doses of pdTp intraperitoneally twice a week for 4 weeks (n ¼ 6/dose). Tumor volume and weight were measured atthe end of the treatment. E and F, QGY-luc cells were injected orthotopically in the livers of NSG mice to establish xenografts and treated with the indicateddoses of pdTp intravenously twice a week for 4 weeks (n ¼ 6/dose). BLI of the orthotopic tumors before and after pdTp treatment (E). Quantification ofphoton counts from the mice (F). For graphs, data represent mean � SEM; � : P < 0.01 versus pdTp 0 mg/kg (A, B, and D); � , P < 0.05 versus pdTp 0 mg/kgafter treatment (F).

SND1 Promotes HCC





activation but not ERK activation in human HCC cells (Fig. 6B)and inhibited sphere formation by WT and Alb/SND1 hepato-cytes (Fig. 4C). In addition, inhibition of Akt and NF-kB, but notthat of ERK, abrogated sphere formation by Alb/SND1 hepato-cytes (Fig. 4E). These findings suggest that enzymatic activity ofSND1 is necessary for the expansion of TICs and is not necessaryfor ERK activation. Binding of SND1 to 30-UTR of AT1R mRNAincreases AT1R mRNA stability and protein translation, resultingin increased ERK activation (1, 15). This function of SND1 doesnot require its enzymatic activity and is not inhibited by pdTptreatment. SND1 activates Akt by multiple mechanisms. By aug-menting RISC activity, it downregulates PTEN, a negative regu-

lator of Akt signaling (14). Protein–protein interaction betweenSND1 and MGLL results in MGLL degradation (17). MGLLselectively interacts with phosphatidic acid and phosphoinositidederivatives, leading to inhibition of PI3K/Akt signaling (40). Assuch, MGLL degradation results in Akt activation. SND1 interactswith MGLL via its SN domains, to which pdTp binds (17). pdTpmight interfere with SND1/MGLL interaction, thereby blockingAkt activation. Thus, SND1-induced Akt activation requires enzy-matic and nonenzymatic activities of SND1, both of which mightbe interfered by pdTp. It should be noted that downregulation ofMGLL and upregulation of AT1R are also preserved in Alb/SND1livers compared with WT (Supplementary Fig. S5).

Figure 6.

pdTp reduces expression of proliferation, inflammation and TIC markers, and upregulates apoptosis and expression of selective tumor suppressor genes.A, FFPE sections of vehicle- and pdTp-treated subcutaneous xenograft tumors were analyzed histologically and immunostained for the indicated proteins.B,Western blot analysis of the indicated proteins in vehicle- and pdTp-treated subcutaneous xenograft tumor samples. Tumor samples from two independent miceper group were used. C, Relative expression of the indicated mRNAs was measured by TaqMan qRT-PCR in vehicle- and pdTp-treated subcutaneousxenograft tumors (3 mice/group). Normalized by GAPDH. Data represent mean � SEM; � , P < 0.01.

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We present SND1 inhibition as a new avenue for targetedtherapeutic research in HCCmanagement. The nontoxicity, spec-ificity for SND1 inhibition, and strong therapeutic efficacy makepdTp an attractive reagent for clinical use. More stringent phar-macokinetic and biodistribution studies andmedicinal chemistryanalysis to generate more potent pdTp analogues should bepursued further. However, pdTp may not block all aspects ofSND1 function, especially its nonenzymatic function, therebyrequiring an alternative approach to inhibit SND1. We haverecently demonstrated therapeutic utility of a hepatocyte-specificnanoparticle delivering siRNA against an oncogene in orthotopicxenograft models of HCC (24). Similar approaches might beemployed to knockdown SND1 and in combination with pdTpmight bring forth complete and sustained inhibition of SND1function. These approaches might also be combined with currentstandard-of-care chemotherapies for HCC. Our current studyopens up a wide avenue of preclinical and clinical research ontesting efficacy of a novel targeted treatment approach.

Disclosure of Potential Conflicts of InterestA.J. Sanyal is the President at Sanyal Biotechnology. No potential conflicts of

interest were disclosed by the other authors.

Authors' ContributionsConception and design: N. Jariwala, D. Rajasekaran, M.A. Subler, J.J. Windle,D. Sarkar

Development of methodology: N. Jariwala, D. SarkarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):N. Jariwala, D. Rajasekaran, R.G. Mendoza, A. Siddiq,M.A. Akiel, C.L. Robertson, M.A. Subler, J.J. Windle, D. SarkarAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Jariwala, D. Rajasekaran, M.A. Akiel, C.L. Robert-son, P.B. Fisher, A.J. Sanyal, D. SarkarWriting, review, and/or revision of the manuscript: N. Jariwala, D. Rajase-karan, M.A. Subler, P.B. Fisher, A.J. Sanyal, D. SarkarAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):N. Jariwala, R.G.Mendoza, X.-N. Shen,D. SarkarStudy supervision: D. Sarkar

Grant SupportThis study was supported in part by NCI grant R21 CA183954 and The

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)grant 1R01DK107451-01A1 to D. Sarkar. C.L. Robertson was supported by aNational Institute of Diabetes and Digestive and Kidney Diseases grantT32DK007150. Services in support of this project were provided by the VCUMassey Cancer Center Transgenic/Knock-outMouse Facility and flow cytometrycore facility, supported in part with funding from NIH-NCI Cancer CenterSupport grant P30 CA016059.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 30, 2017; revised March 29, 2017; accepted April 14, 2017;published OnlineFirst April 20, 2017.

References1. Paukku K, Kalkkinen N, Silvennoinen O, Kontula KK, Lehtonen JY. p100

increases AT1R expression through interaction with AT1R 30-UTR. NucleicAcids Res 2008;36:4474–87.

2. Paukku K, Yang J, Silvennoinen O. Tudor and nuclease-like domainscontaining protein p100 function as coactivators for signal transducer andactivator of transcription 5. Mol Endocrinol 2003;17:1805–14.

3. Yang J, Aittomaki S, Pesu M, Carter K, Saarinen J, Kalkkinen N, et al.Identification of p100 as a coactivator for STAT6 that bridges STAT6 withRNA polymerase II. EMBO J 2002;21:4950–8.

4. Yang J, Valineva T, Hong J, Bu T, Yao Z, Jensen ON, et al. Transcriptionalco-activator protein p100 interacts with snRNP proteins and faci-litates the assembly of the spliceosome. Nucleic Acids Res 2007;35:4485–94.

5. Garcia-Lopez J, Hourcade Jde D, Del Mazo J. Reprogramming of micro-RNAs by adenosine-to-inosine editing and the selective elimination ofedited microRNA precursors in mouse oocytes and preimplantationembryos. Nucleic Acids Res 2013;41:5483–93.

6. Scadden AD. The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage. Nat Struct Mol Biol 2005;12:489–96.

7. Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AM, Tops BB,et al. A micrococcal nuclease homologue in RNAi effector complexes.Nature 2003;425:411–4.

8. Li CL, YangWZ, Chen YP, Yuan HS. Structural and functional insights intohuman Tudor-SN, a key component linking RNA interference and editing.Nucleic Acids Res 2008;36:3579–89.

9. Blanco MA, Aleckovic M, Hua Y, Li T, Wei Y, Xu Z, et al. Identification ofstaphylococcal nuclease domain-containing 1 (SND1) as a Metadherin-interacting protein with metastasis-promoting functions. J Biol Chem2011;286:19982–92.

10. Emdad L, Janjic A, Alzubi MA, Hu B, Santhekadur PK, Menezes ME, et al.Suppression of miR-184 in malignant gliomas upregulates SND1 andpromotes tumor aggressiveness. Neuro Oncol 2015;17:419–29.

11. Tsuchiya N, Ochiai M, Nakashima K, Ubagai T, Sugimura T, Nakagama H.SND1, a component of RNA-induced silencing complex, is up-regulated inhuman colon cancers and implicated in early stage colon carcinogenesis.Cancer Res 2007;67:9568–76.

12. Wan L, Lu X, Yuan S, Wei Y, Guo F, ShenM, et al. MTDH-SND1 interactionis crucial for expansion and activity of tumor-initiating cells in diverseoncogene- and carcinogen-induced mammary tumors. Cancer Cell 2014;26:92–105.

13. Kuruma H, Kamata Y, Takahashi H, Igarashi K, Kimura T, Miki K, et al.Staphylococcal nuclease domain-containing protein 1 as a potential tissuemarker for prostate cancer. Am J Pathol 2009;174:2044–50.

14. Yoo BK, Santhekadur PK, Gredler R, Chen D, Emdad L, Bhutia S, et al.Increased RNA-induced silencing complex (RISC) activity contributes tohepatocellular carcinoma. Hepatology 2011;53:1538–48.

15. Santhekadur PK, Akiel M, Emdad L, Gredler R, Srivastava J, Rajasekaran D,et al. Staphylococcal nuclease domain containing-1 (SND1) promotesmigration and invasion via angiotensin II type 1 receptor (AT1R) andTGFbeta signaling. FEBS Open Bio 2014;4:353–61.

16. Santhekadur PK,Das SK,Gredler R, ChenD, Srivastava J, RobertsonC, et al.Multifunction protein staphylococcal nuclease domain containing 1(SND1) promotes tumor angiogenesis inhumanhepatocellular carcinomathrough novel pathway that involves nuclear factor kappaB andmiR-221. JBiol Chem 2012;287:13952–8.

17. Rajasekaran D, Jariwala N, Mendoza RG, Robertson CL, Akiel MA, Doz-morov M, et al. Staphylococcal nuclease and tudor domain containing 1(SND1) promotes hepatocarcinogenesis by inhibiting monoglyceridelipase (MGLL). J Biol Chem 2016;291:10736–46.

18. Jariwala N, Rajasekaran D, Srivastava J, Gredler R, Akiel MA, Robertson CL,et al. Role of the staphylococcal nuclease and tudor domain containing 1 inoncogenesis (review). Int J Oncol 2015;46:465–73.

19. Arnone A, Bier CJ, Cotton FA, Hazen EE Jr, Richardson DC, Richardson JS.The extracellular nuclease of Staphylococcus aureus: structures of the nativeenzyme andan enzyme-inhibitor complex at 4A resolution. ProcNatl AcadSci U S A 1969;64:420–7.

20. Weber DJ, Mullen GP, Mildvan AS. Conformation of an enzyme-boundsubstrate of staphylococcal nuclease as determined by NMR. Biochemistry1991;30:7425–37.

21. Ramirez MI, Karaoglu D, Haro D, Barillas C, Bashirzadeh R, Gil G.Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expres-sion at the transcriptional level in culture and in transgenic mice. Mol CellBiol 1994;14:2809–21.

SND1 Promotes HCC





22. Srivastava J, Siddiq A, Emdad L, Santhekadur PK, Chen D, Gredler R, et al.Astrocyte elevated gene-1 promotes hepatocarcinogenesis: novel insightsfrom a mouse model. Hepatology 2012;56:1782–91.

23. Yoo BK, Emdad L, Su ZZ, Villanueva A, Chiang DY, Mukhopadhyay ND,et al. Astrocyte elevated gene-1 regulates hepatocellular carcinoma devel-opment and progression. J Clin Invest 2009;119:465–77.

24. Rajasekaran D, Srivastava J, Ebeid K, Gredler R, Akiel M, Jariwala N, et al.Combination of nanoparticle-delivered siRNA for Astrocyte ElevatedGene-1 (AEG-1) and All-trans Retinoic Acid (ATRA): an effective thera-peutic strategy for Hepatocellular Carcinoma (HCC). Bioconjug Chem2015;26:1651–61.

25. Chen D, Siddiq A, Emdad L, Rajasekaran D, Gredler R, Shen XN, et al.Insulin-like growth factor-binding protein-7 (IGFBP7): a promising genetherapeutic for hepatocellular carcinoma (HCC). Mol Ther 2013;21:758–66.

26. Srivastava J, Siddiq A, Gredler R, Shen XN, Rajasekaran D, Robertson CL,et al. Astrocyte elevated gene-1 and c-Myc cooperate to promote hepato-carcinogenesis in mice. Hepatology 2015;61:915–29.

27. Villanueva A, Llovet JM. Targeted therapies for hepatocellular carcinoma.Gastroenterology 2011;140:1410–26.

28. Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, et al. NF-kappaB functions as a tumour promoter in inflammation-associatedcancer. Nature 2004;431:461–6.

29. Zhu M, Li W, Lu Y, Dong X, Lin B, Chen Y, et al. HBx drives alphafetoprotein expression to promote initiation of liver cancer stem cellsthrough activating PI3K/AKT signal pathway. Int J Cancer 2017;140:1346–55.

30. Su R, Nan H, Guo H, Ruan Z, Jiang L, Song Y, et al. Associations ofcomponents of PTEN/AKT/mTOR pathway with cancer stem cell markersand prognostic value of these biomarkers in hepatocellular carcinoma.Hepatol Res 2016;46:1380–91.

31. Hagiwara S, Kudo M, Nagai T, Inoue T, Ueshima K, Nishida N, et al.Activation of JNK and high expression level of CD133 predict a poor

response to sorafenib in hepatocellular carcinoma. Br J Cancer 2012;106:1997–2003.

32. Piao LS, Hur W, Kim TK, Hong SW, Kim SW, Choi JE, et al. CD133þ livercancer stem cells modulate radioresistance in human hepatocellular car-cinoma. Cancer Lett 2012;315:129–37.

33. Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY. CD133þ HCC cancer stemcells confer chemoresistance by preferential expression of the Akt/PKBsurvival pathway. Oncogene 2008;27:1749–58.

34. Bourguignon LY, Shiina M, Li JJ. Hyaluronan-CD44 interaction promotesoncogenic signaling, microRNA functions, chemoresistance, and radiationresistance in cancer stemcells leading to tumor progression.AdvCancer Res2014;123:255–75.

35. Tang KH,Ma S, Lee TK, Chan YP, Kwan PS, Tong CM, et al. CD133(þ) livertumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling. Hepatology2012;55:807–20.

36. He G, Dhar D, Nakagawa H, Font-Burgada J, Ogata H, Jiang Y, et al.Identification of liver cancer progenitors whose malignant progressiondepends on autocrine IL-6 signaling. Cell 2013;155:384–96.

37. Wan S, Zhao E, Kryczek I, Vatan L, Sadovskaya A, Ludema G, et al. Tumor-associated macrophages produce interleukin 6 and signal via STAT3 topromote expansion of human hepatocellular carcinoma stem cells. Gas-troenterology 2014;147:1393–404.

38. Armengol S, Arretxe E, Rodriguez L, Ochoa B, Chico Y, Martinez MJ. NF-kappaB, Sp1 and NF-Y as transcriptional regulators of human SND1 gene.Biochimie 2013;95:735–42.

39. Arretxe E, Armengol S, Mula S, Chico Y, Ochoa B, Martinez MJ. Profiling ofpromoter occupancy by the SND1 transcriptional coactivator identifiesdownstream glycerolipid metabolic genes involved in TNFalpha responsein human hepatoma cells. Nucleic Acids Res 2015;43:10673–88.

40. Sun H, Jiang L, Luo X, Jin W, He Q, An J, et al. Potential tumor-suppressiverole of monoglyceride lipase in human colorectal cancer. Oncogene2013;32:234–41.


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2017;77:3306-3316. Published OnlineFirst April 20, 2017.Cancer Res Nidhi Jariwala, Devaraja Rajasekaran, Rachel G. Mendoza, et al. Hepatocellular CarcinomaOncogenic Role of SND1 in Development and Progression of

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Oncogenic Role of SND1 in Development and Progression of … · document that Alb/SND1 mice develop spontaneous HCC with expansion of tumor-initiating cells (TIC). We also demonstrate