SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). In this study, we show that hepatocyte-specific SND1 transgenic mice (Alb/SND1 mice) develop spontaneous HCC with partial penetrance and exhibit more highly aggressive HCC induced by chemical carcinogenesis. Livers from Alb/SND1 mice exhibited a relative increase in inflammatory markers and spheroid-generating tumor-initiating cells (TIC). Mechanistic investigations defined roles for Akt and NF-κB signaling pathways in promoting TIC formation in Alb/SND1 mice. In human xenograft models of subcutaneous or orthotopic HCC, administration of the selective SND1 inhibitor 3′, 5′-deoxythymidine bisphosphate (pdTp), inhibited tumor formation without effects on body weight or liver function. Our work establishes an oncogenic role for SND1 in promoting TIC formation and highlights pdTp as a highly selective SND1 inhibitor as a candidate therapeutic lead to treat advanced HCC. Cancer Res; 77(12); 3306–16. ©2017 AACR.

Staphylococcal nuclease and tudor domain containing 1 (SND1) is a multifunctional protein that regulates transcription, mRNA splicing, RNA editing, and miRNA-mediated mRNA degradation as a nuclease in RNA-induced silencing complex (RISC; refs. 1–8). SND1 is overexpressed in multiple cancers, where it functions as an oncogene (9–14). In hepatocellular carcinoma (HCC), SND1 overexpression was identified in approximately 74% cases (14). Overexpression of SND1 promotes and knockdown of SND1 inhibits proliferation, invasion, angiogenesis, and in vivo tumorigenesis by human HCC cells (14–17). Our studies document that SND1 exerts its function in HCC cells by a variety of mechanisms. SND1 overexpression contributed to increased RISC activity in HCC cells, resulting in augmented degradation of tumor suppressor mRNAs that are targets of oncogenic miRNAs (14). SND1 promotes angiogenesis by activating NF-κB, resulting in the induction of miR-221 and subsequently angiogenic factors angiogenin and CXCL16 (16). SND1 binds to 3′ untranslated region (3′-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 to activation of ERK, Smad2, and subsequently TGFβ signaling pathway promoting epithelial–mesenchymal transition (EMT), migration, and invasion by human HCC cells (15). SND1 interacts with monoglyceride lipase (MGLL), resulting in MGLL degradation, which leads to activation of Akt and stimulation of cell proliferation and cell-cycle progression in HCC cells (17). Overall, SND1 plays a dynamic role in regulating expression of genes crucial to hepatocarcinogenesis by employing diverse transcriptional 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 tudor domain and a nuclease domain (8). The nuclease domains function as RNase, while the tudor domain is involved in protein–nucleic acid interaction (8). Enzymatic activity of SND1 is required for its function in RISC (7). However, whether the other functions of SND1 require enzymatic activity remains to be determined. Structurally, SND1 is unique in the human proteome with no close homolog as revealed by BLAST search (18). The closest homolog of SND1 is EBNA2, which is not transcribed. SND1 has highly electropositive SN domains, to which binds the negatively charged 3′, 5′-deoxythymidine bisphosphate (pdTp) molecule inhibiting SND1 nuclease activity (7, 8, 19, 20). The absence of a close homolog of SND1 and availability of a specific SND1 inhibitor pdTp suggests that pdTp might be developed as a potential HCC therapeutic with little side effects. Indeed, we showed that pdTp inhibited growth of human HCC cells in vitro (14). However, in vivo antitumor efficacy of pdTp remains to be determined. In addition, pdTp might also serve as a valuable tool to distinguish enzymatic and nonenzymatic functions of SND1.

In this article, we employ a hepatocyte-specific transgenic mouse overexpressing SND1 (Alb/SND1) to obtain insights into the oncogenic function of SND1 in an in vivo context. We document that Alb/SND1 mice develop spontaneous HCC with expansion of tumor-initiating cells (TIC). We also demonstrate in vivo safety and antitumor efficacy of pdTp, thereby paving the way for its further characterization as an anti-HCC agent. We thus identify SND1 as a valid molecular target in HCC and present SND1 inhibition as a promising approach to curb hepatocarcinogenesis.

Generation of Alb/SND1 mouse and induction of chemical carcinogenesis

Alb/SND1 transgenic mouse in B6CBAF1 background was generated by directing the expression of C-terminal Myc-tagged human SND1 under an upstream enhancer region (−10400 to −8500) fused to the 335-bp core region of mouse albumin promoter (Supplementary Fig. S1; ref. 21). Microinjection and manipulation procedures were performed according to standard protocols in the VCU Massey Cancer Center Transgenic/Knockout Mouse Core. For induction of chemical carcinogenesis, a single intraperitoneal injection of 10 μg/g body weight of N-nitrosodiethylamine (DEN) was given at 14 days of age to male wild-type (WT) and Alb/SND1 littermates (22). The animals were sacrificed at 32 weeks of age and liver, internal organs, and blood were collected. Serum liver enzymes were analyzed in the Molecular Diagnostic Laboratory, Department of Pathology, VCU (Richmond, VA) using standard procedures. All experiments were performed using sibling littermates, fed regular chow diet during light cycle. All animal studies were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University and were conducted in accordance with the Animal Welfare Act, the PHS Policy on Humane Care and Use of Laboratory Animals, and the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.

Cells, culture condition, sphere formation, and Matrigel invasion assays and chemicals

Primary mouse hepatocytes were isolated from adult male WT and 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 as described previously (23). Generation and characterization of QGY-7703 cells expressing luciferase (QGY-luc) have been described previously (24, 25). Early passage (>5) cultures of QGY-7703 and QGY-luc cells were stored in liquid nitrogen, and in vivo studies described in this article were performed with freshly thawed culture of the cells after confirming mycoplasma free using Mycoplasma Detection Kit (Thermo Fisher Scientific). Hepatocytes were cultured in Essential 8 Medium (Thermo Fisher Scientific; catalog # A1517001) for enrichment of TICs using ultralow attachment plates. Sphere formation was monitored, and spheres containing more than 50 cells were quantified microscopically. Nonproliferative spheres were excluded as abortive spheres. Matrigel invasion assay using primary hepatocytes was performed as described previously (23, 26). pdTp was purchased from Axxora (catalog # BLG-T012-05). BMS-3445541 (catalog # B9935) and LY294006 (catalog # L9908) were purchased from Sigma-Aldrich, and U0126 (catalog # 9903) was purchased from Cell Signaling Technology.

IHC and immunofluorescence

IHC using formalin-fixed paraffin-embedded (FFPE) sections and immunofluorescence in primary hepatocytes were performed as described previously (23). For IHC, the sections were blocked in PBST using 10% normal goat serum for rabbit and mouse polyclonal antibodies. Primary antibodies were diluted in PBST containing 5% blocking serum. The primary antibodies used were SND1 (rabbit polyclonal; 1:200; Sigma), PCNA (mouse monoclonal; 1:200; Cell Signaling Technology), AFP (rabbit polyclonal; 1:50; Santa Cruz Biotechnology), F4/80 (rat polyclonal; 1:200; Bio-Rad), CD31 (rabbit polyclonal; 1:50; AbCam), cleaved caspase-3 (rabbit polyclonal; 1:300; Cell Signaling Technology), p-p65 (rabbit polyclonal; 1:400; Cell Signaling Technology), EpCAM (rabbit polyclonal; 1:400; Sigma), CD133 (rabbit polyclonal; 1:500; Proteintech), and CD44 (mouse monoclonal; 1:250; AbCam). Biotin-conjugated secondary antibodies were diluted in PBST containing corresponding 2.5% blocking serum. Sections were stained using avidin–biotin–peroxidase complexes treated with a DAB substrate solution (Vector Laboratories). IHC images were quantified by H-score with WT score normalized to 1. For immunofluorescence, the primary antibody was p65 (mouse monoclonal; 1:400; Cell Signaling Technology), and the secondary antibody was Alexa546-conjugated anti-mouse IgG (goat; 1:400; Invitrogen). The slides were mounted in VectaShield fluorescence mounting medium containing 4,6 -diamidino-2- phenylindole (Vector Laboratories). Images were analyzed using a Zeiss confocal laser scanning microscope.

Western blotting

Lysates were prepared by lysing cells in 1.5% n-dodecyl -D-maltoside buffer supplemented with protease and phosphatase inhibitor cocktail (Pierce). Lysates from liver tissue were prepared by mechanical homogenization using the same buffer. Western blot analysis was performed as described previously (23). Primary antibodies used were SND1 (rabbit polyclonal; 1:1,000; Sigma), GAPDH (mouse monoclonal; 1:1,000; Santa Cruz Biotechnology), CD133 (rabbit polyclonal; 1:1,000; Proteintech), CD44 (mouse monoclonal; 1:1000; Abcam), Myc-Tag, p-Akt, Akt, ERK, p-ERK, p-GSK3β, GSK3β, 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 Scientific). Densitometric analysis was performed by ImageJ software.

Total RNA extraction, cDNA preparation, and quantitative RT-PCR

Total RNA was extracted using the Qiagen miRNeasy Mini Kit (Qiagen). cDNA preparation was done using ABI cDNA Synthesis Kit (Applied Biosystems). qRT-PCR was performed using an ABI ViiA7 Fast Real-Time PCR System and TaqMan gene expression assays according to the manufacturer's protocol (Applied Biosystems).

Flow cytometry

WT and Alb/SND1 hepatocytes were isolated and washed in PBS. Hepatocytes (1 × 106 per sample) were stained with fluorochrome-conjugated primary antibodies in 2% BSA in PBS at room temperature for 1 hour. CD133 (mouse-APC; 1:50; Miltenyi Biotec), CD44 (rat-FITC; 1:50; Abcam), and EpCAM (rat-PE; 1:20; BD Pharmingen) were used as per the manufacturer's protocol. Cells were washed twice in PBS and resuspended in 2% BSA in PBS for flow cytometric analysis using BD FACSCanto II.

Treatment of human HCC xenografts in NSG mice

QGY-7703 cells (6.5 × 105 cells suspended in 50 μL of Matrigel) were injected subcutaneously in the flanks of adult male NSG mice. Tumor volume was measured twice a week with a caliper and calculated using the formula π/6 × larger diameter × (smaller diameter)2. After the tumors reached approximately 100 mm3 (requiring a week), different doses of pdTp (0.16, 0.32, and 0.8 mg/kg) were administered to the mice intraperitoneally twice a week for 4 weeks. Six mice per group were used for each treatment group and 8 mice were used for control vehicle-treated group. For orthotopic xenografts, QGY-luc cells (1 × 106) were implanted by intrahepatic injection in adult male NSG mice (24, 25). Tumor growth was monitored by bioluminescence imaging (BLI) with a Xenogen 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 intravenously twice a week for 4 weeks. Six mice per group were used.

Statistical analysis

Data 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.

Alb/SND1 mice manifest aggressive hepatocarcinogenesis

We have created a hepatocyte-specific C-terminal Myc-tagged human SND1-expressing transgenic mouse (Alb/SND1) by using the mouse albumin promoter/enhancer element to drive SND1 expression in a B6CBAF1 background. This particular strain of mouse is very sensitive to DEN-induced hepatocarcinogenesis (22). The expression of SND1 in the livers of Alb/SND1 mice was confirmed by Western blot analysis, TaqMan qRT-PCR, and IHC (Fig. 1A–C; Supplementary Fig. S2A). Alb/SND1 mice develop and reproduce normally, and no physiologic abnormalities and significant differences in body weight compared with WT littermates were observed. Histopathologic analysis at 2 months of age did not reveal any difference in liver architecture between WT and Alb/SND1 mice (Fig. 1C). However, at one year of age, 6 of 14 (∼42%) Alb/SND1 mice developed hepatic nodules, which were confirmed as HCC upon histologic examination with loss of hepatic architecture, and AFP expression (Fig. 1D–F; Supplementary Fig. S2A; Table 1). However, no such nodules were observed in WT littermates. Compared with WT, a significant increase in mRNAs for c-Myc, TNFα, and IL6, known drivers of HCC, was detected in Alb/SND1 livers at 2 and 12 months of age, except for IL6, 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 mice showed a profound tumorigenic response, with tumorigenesis affecting the entire liver, compared with WT littermates, in which there were either no nodules or nodules that were <5 mm in size (Fig. 2A; Table 1). Liver weight, reflecting higher tumor load, and serum AST, ALT, and total protein were significantly elevated in Alb/SND1 mice versus WT (Fig. 2B and C). A marked increase in mRNA 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 (angiogenesis marker) and PCNA (proliferation marker) versus DEN-treated WT (Fig. 2E; Supplementary Fig. S2A). Increased activation of ERK, Akt, and its downstream GSK3β was observed in DEN-treated and spontaneous Alb/SND1 tumors compared with DEN-treated WT (Fig. 2F; Supplementary Fig. S2B). ERK and Akt are activated by SND1 (15, 17) and also play a critical role in HCC (27) thus might be crucial in mediating SND1-induced HCC.

Alb/SND1 hepatocytes show increased activation of NF-κB

Chronic inflammation is a central event in hepatocarcinogenesis, and NF-κB plays a pivotal role in promoting inflammation (28). Increased expression of IL6 and TNFα (Fig. 1G) indicates that SND1 overexpression results in a chronic inflammatory state leading to HCC. We previously documented activation of NF-κB in SND1-overexpressing human HCC cells (16). NF-κB activation is marked by phosphorylation of serine residue 536, allowing nuclear translocation of p65 subunit, which then functions as a transcription factor to modulate expression of inflammatory genes. We checked nuclear localization of p65 NF-κB in WT and Alb/SND1 hepatocytes, treated or untreated with lipopolysaccharide (LPS), as serum LPS levels are elevated in HCC patients, which promotes inflammation. Alb/SND1 hepatocytes, but not WT, showed nuclear p65 under basal condition, indicating constitutive activation of NF-κB (Fig. 3A). LPS treatment resulted in nuclear translocation of p65 in both WT and Alb/SND1 hepatocytes. Inhibition of enzymatic activity of SND1 by pdTp resulted in marked downregulation of p65 levels with simultaneous inhibition of p65 nuclear translocation in both WT and Alb/SND1 hepatocytes, suggesting a central role of SND1 in regulating p65 expression (Fig. 3A). Western blot analysis revealed increased basal level of phosphorylated p65 (p-p65) in Alb/SND1 hepatocytes compared with WT (Fig. 3B). In WT hepatocytes, upon LPS treatment, increased p-p65 was observed at 10 minutes, which gradually waned down over a period of 2 hours (Fig. 3B). However, in Alb/SND1 hepatocytes, increased p-p65 level remained sustained during the assay period (Fig. 3B). Increased inflammation was also indicated by increased infiltration of macrophages, detected by staining for F4/80 marker, in DEN-treated and 1-year-old tumor-bearing Alb/SND1 livers versus corresponding WT (Fig. 3C; Supplementary Fig. S2C). In addition, a marked increase in immune checkpoint molecule PD-L1 and a significant increase in its receptor PD-1 were observed in DEN-treated and 1-year-old tumor-bearing Alb/SND1 livers versus corresponding WT, indicating a strong carcinogenic response induced by SND1 overexpression (Supplementary Fig. S2D).

SND1 overexpression results in expansion of TICs

As Alb/SND1 mice develop spontaneous HCC, we checked the effect of SND1 overexpression on TICs. In a sphere formation assay in ultralow attachment plates, WT hepatocytes formed small abortive spheres, while Alb/SND1 hepatocytes formed robust spheres that gradually increased in size and number, indicating an expansion of TICs (Fig. 4A). Indeed, TICs positive for three markers, EpCAM, CD44, and CD133, were significantly more in Alb/SND1 livers versus WT (Fig. 4B). Alb/SND1 hepatocytes isolated from 2-month-old mice showed significant increase in mRNA levels of EpCAM (∼2-fold), CD44 (∼4-fold), and CD133 (∼4-fold) compared with WT (Supplementary Fig. S3A). These increases in mRNA levels were further augmented, for example, EpCAM (∼2.5-fold), CD44 (∼6-fold), and CD133 (∼4-fold), in Alb/SND1 hepatocytes isolated from 12-month-old mice versus WT (Supplementary Fig. S3A). IHC analysis showed increased expression of EpCAM, CD44, and CD133 in 1-year-old Alb/SND1 livers, with or without tumor, compared with WT littermates (Supplementary Fig. S3B and S3C). Increased CD133 expression was observed in DEN-treated and spontaneous tumors in Alb/SND1 mice when compared with DEN-treated WT livers (Supplementary Fig. S3D). Treatment with pdTp significantly inhibited sphere formation by both WT and Alb/SND1 hepatocytes, indicating that enzymatic function of SND1 is necessary to promote expansion of TICs (Fig. 4C). As expected from studies in human cell lines, naïve Alb/SND1 livers showed increased activation of ERK and Akt (Fig. 3D). To check the effects of the signaling pathways activated by SND1 in regulating TICs, we performed sphere formation assay with Alb/SND1 hepatocytes upon treatment with BMS-3445541(IκB kinase inhibitor to block NF-κB activation), LY294006 (PI3K inhibitor to block Akt activation), and U0126 (MEK1/2 inhibitor to block ERK activation; Fig. 4E and F). Inhibition of NF-κB and Akt activation, but not ERK activation, significantly abrogated sphere formation by Alb/SND1 hepatocytes with corresponding decrease in CD133 expression (Fig. 4E and F; Supplementary Fig. S4A). We interrogated the potential role of ERK activation in SND1-induced phenotypes other than sphere formation. We previously demonstrated a potential role of ERK activation in mediating SND1-induced invasion of human HCC cells (15). Although WT hepatocytes did not invade through Matrigel, Alb/SND1 hepatocytes acquired Matrigel invasion property, which was significantly abrogated upon treatment with U0126 (Fig. 4G).

SND1 inhibitor pdTp significantly abrogates human HCC xenografts in vivo

pdTp specifically inhibits the nuclease activity of SND1, but does not affect the oligonucleotide binding function of the tudor domain. We previously documented that pdTp inhibits proliferation of human HCC cells in vitro (14). As yet, in vivo efficacy and toxicity has not been tested for pdTp. We injected multiple doses of pdTp (calculated from our in vitro studies) to WT B6CBA mice intraperitoneally twice a week for 4 weeks (a total of 8 doses). At the highest dose of 0.8 mg/kg, no difference in body and liver weights, serum liver enzymes, total protein, albumin, and globulin 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 did not show any abnormality (Fig. 5C). We established subcutaneous xenografts of QGY-7703 cells in NSG mice and evaluated the effect of intraperitoneal administration of different doses pdTp on tumor development. A significant decrease in tumor volume and tumor weight was observed with 0.32 and 0.8 mg/kg pdTp at the end of the treatment (Fig. 5D). We next established orthotopic xenografts of QGY-luc cells (QGY-7703 cells expressing luciferase) in the livers of NSG mice and evaluated the effect of intravenous administration of pdTp on tumor development by BLI. A significant inhibitory effect on tumor progression was observed with pdTp treatment compared with vehicle (Fig. 5E and F). IHC analysis of subcutaneous tumor sections revealed that pdTp treatment 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; Supplementary Fig. S4B). Western blot analysis of tumor samples identified that pdTp treatment resulted in downregulation of CD133 and CD44 levels and decreased phosphorylation of Akt and p65 (Fig. 6B; Supplementary Fig. S4C). Total p65 level was also decreased upon pdTp treatment (Fig. 6B). Interestingly, no change in ERK activation was observed upon treatment with pdTp. We previously documented that increased RISC activity resulting from SND1 overexpression augments oncomiR-mediated degradation 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 treatment resulted in significant increases in PTEN, TGFBR2, and CDKN1C mRNA levels in tumors compared with vehicle (Fig. 6C). Collectively, these findings reveal that pdTp inhibits proliferation and inflammation, induces apoptosis, and downregulates TICs.

In this research article, we report the oncogenic role of SND1 in HCC development and progression by pursuing studies in a novel hepatocyte-specific SND1-overexpressing transgenic mouse model. Our previous in vitro studies established that SND1 overexpression positively regulates multiple hallmarks of cancer, including proliferation, migration, invasion, angiogenesis, EMT, and inhibition of tumor suppressor gene expression (14–17). In the current in vivo studies, we document that SND1 causes spontaneous hepatocarcinogenesis by increasing TICs within the liver and creating a proinflammatory microenvironment, and sensitizes hepatocytes toward DEN-induced HCC. As a corollary, chemical inhibition of SND1 enzymatic activity by pdTp reduced tumor-initiating potential of hepatocytes and rescued proinflammatory signaling caused by SND1 overexpression, resulting in significant abrogation of tumor growth in a xenograft model.

SND1 is a multifunction protein that regulates gene expression at transcriptional as well as posttranscriptional level. Inflammatory cytokine TNFα is constitutively upregulated in Alb/SND1 liver, whereas IL6 is upregulated in an age-dependent manner. Both these cytokines are known to activate NF-κB signaling and are also induced by NF-κB. As expected, Alb/SND1 hepatocytes manifest an exaggerated NF-κB activation, both constitutive and upon LPS treatment. LPS is detected at notably high levels in HCC patients, thus making SND1-mediated augmentation in NF-κB activation a clinically relevant scenario. Activation of resident Kupffer cells and invasion of liver with macrophages is a crucial event in HCC. Secretion of inflammatory cytokines by SND1-overexpressing hepatocytes might contribute to NF-κB signaling and activation in macrophages. Thus, SND1 overexpression creates an underlying proinflammatory condition within liver, which aggravates with age, creating conducive preexisting pathology for tumorigenesis.

We observe that constitutive overexpression of SND1 predisposes Alb/SND1 animals to risk of HCC development in late adulthood, in the absence of any carcinogen exposure, with expansion of CD133+, CD44+, and EpCAM+ TICs. Our inhibitor studies unravel that active Akt and NF-κB signaling is required to maintain tumor initiation potential of SND1-overexpressing hepatocytes. A recent report documented that in a hepatitis B model, AFP-induced upregulation of CD133+, CD44+, and EpCAM+ TICs is dependent on PI3K/Akt signaling (29). Clinical study analyzing protein marker expression in liver from more than 100 HCC patients showed a significant negative correlation with PTEN and positive correlation with Akt levels with TIC marker proteins CD133, EpCAM, and CD90 (30). Univariate and multivariate analysis showed significant correlation between loss of PTEN and high AFP, Akt, CD133, and EpCAM levels with overall survival of HCC patients (30). CD133 expression is correlated with sorafenib resistance in human HCC patients (31, 32). CD133 confers chemoresistance and radioresistance via upregulation of PI3K/AKT signaling (32, 33). Interaction between hyaluronan and CD44 in extracellular matrix promotes carcinogenic signaling and results in chemoresistance (34). Thus, a close interplay between AKT signaling and TIC expansion is predicted in Alb/SND1 livers, rendering them therapeutically resistant. It would be interesting to investigate the potential of SND1 targeting in overcoming chemoresistance in HCC for future studies.

Studies have shown that IL8, a NF-κB downstream gene, increases CXCL1 expression, upregulates MAPK signaling, and sustains cancer stemness that eventually expands CD133+ population in liver (35). Autocrine IL6 signaling is also implicated in protumorigenic properties of HCC progenitor cells (36). IL6 signaling promoted by tumor-associated macrophages is found to promote expansion of CD44+ cells, potentiating growth of xenograft tumors, and inhibition of IL6/STAT3 signaling in this model, reduced tumorigenic potential of CD44+ cells (37). In SND1-overexpressing HCC, we report an increased invasion of macrophages, which can be predicted to upregulate IL6 secretion and thus promote expansion of TICs. As yet, the molecular mechanism by which SND1 activates NF-κB remains to be determined. pdTp treatment studies clearly demonstrate that SND1 enzymatic activity is required to activate NF-κB (Fig. 3A). However, pdTp treatment also resulted in a decrease in total p65 level, indicating that SND1 regulates not only NF-κB activation but also p65 expression. Inflammatory cytokines induce SND1 expression and SND1 promoter contains consensus NF-κB binding sites (38, 39). Thus, chronic inflammation preceding HCC might result in induction of SND1, which facilitates expansion of TICs and further aggravates inflammation, thereby establishing a scenario for the development of HCC.

SND1 overexpression activates Akt, ERK, and NF-κB signaling. We document that pdTp treatment inhibited Akt and NF-κB activation but not ERK activation in human HCC cells (Fig. 6B) and inhibited sphere formation by WT and Alb/SND1 hepatocytes (Fig. 4C). In addition, inhibition of Akt and NF-κB, but not that of ERK, abrogated sphere formation by Alb/SND1 hepatocytes (Fig. 4E). These findings suggest that enzymatic activity of SND1 is necessary for the expansion of TICs and is not necessary for ERK activation. Binding of SND1 to 3′-UTR of AT1R mRNA increases AT1R mRNA stability and protein translation, resulting in increased ERK activation (1, 15). This function of SND1 does not require its enzymatic activity and is not inhibited by pdTp treatment. SND1 activates Akt by multiple mechanisms. By augmenting RISC activity, it downregulates PTEN, a negative regulator of Akt signaling (14). Protein–protein interaction between SND1 and MGLL results in MGLL degradation (17). MGLL selectively interacts with phosphatidic acid and phosphoinositide derivatives, leading to inhibition of PI3K/Akt signaling (40). As such, MGLL degradation results in Akt activation. SND1 interacts with MGLL via its SN domains, to which pdTp binds (17). pdTp might interfere with SND1/MGLL interaction, thereby blocking Akt activation. Thus, SND1-induced Akt activation requires enzymatic and nonenzymatic activities of SND1, both of which might be interfered by pdTp. It should be noted that downregulation of MGLL and upregulation of AT1R are also preserved in Alb/SND1 livers compared with WT (Supplementary Fig. S5).

We present SND1 inhibition as a new avenue for targeted therapeutic research in HCC management. The nontoxicity, specificity for SND1 inhibition, and strong therapeutic efficacy make pdTp an attractive reagent for clinical use. More stringent pharmacokinetic and biodistribution studies and medicinal chemistry analysis to generate more potent pdTp analogues should be pursued further. However, pdTp may not block all aspects of SND1 function, especially its nonenzymatic function, thereby requiring an alternative approach to inhibit SND1. We have recently demonstrated therapeutic utility of a hepatocyte-specific nanoparticle delivering siRNA against an oncogene in orthotopic xenograft models of HCC (24). Similar approaches might be employed to knockdown SND1 and in combination with pdTp might bring forth complete and sustained inhibition of SND1 function. These approaches might also be combined with current standard-of-care chemotherapies for HCC. Our current study opens up a wide avenue of preclinical and clinical research on testing efficacy of a novel targeted treatment approach.

A.J. Sanyal is the President at Sanyal Biotechnology. No potential conflicts of interest were disclosed by the other authors.

Conception and design: N. Jariwala, D. Rajasekaran, M.A. Subler, J.J. Windle, D. Sarkar

Development of methodology: N. Jariwala, D. Sarkar

Acquisition 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. Sarkar

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Jariwala, D. Rajasekaran, M.A. Akiel, C.L. Robertson, P.B. Fisher, A.J. Sanyal, D. Sarkar

Writing, review, and/or revision of the manuscript: N. Jariwala, D. Rajasekaran, M.A. Subler, P.B. Fisher, A.J. Sanyal, D. Sarkar

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. Jariwala, R.G. Mendoza, X.-N. Shen, D. Sarkar

Study supervision: D. Sarkar

This 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 a National Institute of Diabetes and Digestive and Kidney Diseases grant T32DK007150. Services in support of this project were provided by the VCU Massey Cancer Center Transgenic/Knock-out Mouse Facility and flow cytometry core facility, supported in part with funding from NIH-NCI Cancer Center Support grant P30 CA016059.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Paukku
K
,
Kalkkinen
N
,
Silvennoinen
O
,
Kontula
KK
,
Lehtonen
JY
. 
p100 increases AT1R expression through interaction with AT1R 3′-UTR
.
Nucleic Acids Res
2008
;
36
:
4474
87
.
2.
Paukku
K
,
Yang
J
,
Silvennoinen
O
. 
Tudor and nuclease-like domains containing protein p100 function as coactivators for signal transducer and activator 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 with RNA polymerase II
.
EMBO J
2002
;
21
:
4950
8
.
4.
Yang
J
,
Valineva
T
,
Hong
J
,
Bu
T
,
Yao
Z
,
Jensen
ON
, et al
Transcriptional co-activator protein p100 interacts with snRNP proteins and facilitates the assembly of the spliceosome
.
Nucleic Acids Res
2007
;
35
:
4485
94
.
5.
Garcia-Lopez
J
,
Hourcade Jde
D
,
Del Mazo
J
. 
Reprogramming of microRNAs by adenosine-to-inosine editing and the selective elimination of edited microRNA precursors in mouse oocytes and preimplantation embryos
.
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
,
Yang
WZ
,
Chen
YP
,
Yuan
HS
. 
Structural and functional insights into human 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 of staphylococcal nuclease domain-containing 1 (SND1) as a Metadherin-interacting protein with metastasis-promoting functions
.
J Biol Chem
2011
;
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 and promotes 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 in human 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
,
Shen
M
, et al
MTDH-SND1 interaction is crucial for expansion and activity of tumor-initiating cells in diverse oncogene- 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 tissue marker 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 to hepatocellular 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) promotes migration and invasion via angiotensin II type 1 receptor (AT1R) and TGFbeta signaling
.
FEBS Open Bio
2014
;
4
:
353
61
.
16.
Santhekadur
PK
,
Das
SK
,
Gredler
R
,
Chen
D
,
Srivastava
J
,
Robertson
C
, et al
Multifunction protein staphylococcal nuclease domain containing 1 (SND1) promotes tumor angiogenesis in human hepatocellular carcinoma through novel pathway that involves nuclear factor kappaB and miR-221
.
J Biol Chem
2012
;
287
:
13952
8
.
17.
Rajasekaran
D
,
Jariwala
N
,
Mendoza
RG
,
Robertson
CL
,
Akiel
MA
,
Dozmorov
M
, et al
Staphylococcal nuclease and tudor domain containing 1 (SND1) promotes hepatocarcinogenesis by inhibiting monoglyceride lipase (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 in oncogenesis (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 native enzyme and an enzyme-inhibitor complex at 4 A resolution
.
Proc Natl Acad Sci U S A
1969
;
64
:
420
7
.
20.
Weber
DJ
,
Mullen
GP
,
Mildvan
AS
. 
Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR
.
Biochemistry
1991
;
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 expression at the transcriptional level in culture and in transgenic mice
.
Mol Cell Biol
1994
;
14
:
2809
21
.
22.
Srivastava
J
,
Siddiq
A
,
Emdad
L
,
Santhekadur
PK
,
Chen
D
,
Gredler
R
, et al
Astrocyte elevated gene-1 promotes hepatocarcinogenesis: novel insights from 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 development 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 Elevated Gene-1 (AEG-1) and All-trans Retinoic Acid (ATRA): an effective therapeutic strategy for Hepatocellular Carcinoma (HCC)
.
Bioconjug Chem
2015
;
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 gene therapeutic 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 hepatocarcinogenesis 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-associated cancer
.
Nature
2004
;
431
:
461
6
.
29.
Zhu
M
,
Li
W
,
Lu
Y
,
Dong
X
,
Lin
B
,
Chen
Y
, et al
HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through 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 of components of PTEN/AKT/mTOR pathway with cancer stem cell markers and 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
CD133+ liver cancer stem cells modulate radioresistance in human hepatocellular carcinoma
.
Cancer Lett
2012
;
315
:
129
37
.
33.
Ma
S
,
Lee
TK
,
Zheng
BJ
,
Chan
KW
,
Guan
XY
. 
CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway
.
Oncogene
2008
;
27
:
1749
58
.
34.
Bourguignon
LY
,
Shiina
M
,
Li
JJ
. 
Hyaluronan-CD44 interaction promotes oncogenic signaling, microRNA functions, chemoresistance, and radiation resistance in cancer stem cells leading to tumor progression
.
Adv Cancer Res
2014
;
123
:
255
75
.
35.
Tang
KH
,
Ma
S
,
Lee
TK
,
Chan
YP
,
Kwan
PS
,
Tong
CM
, et al
CD133(+) liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling
.
Hepatology
2012
;
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 progression depends 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 to promote expansion of human hepatocellular carcinoma stem cells
.
Gastroenterology
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 of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFalpha response in 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-suppressive role of monoglyceride lipase in human colorectal cancer
.
Oncogene
2013
;
32
:
234
41
.