S3I-201

lncARSR promotes liver cancer stem cells expansion via STAT3 pathway

Abstract
Liver cancer stem cells (CSCs) have important functions in tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). lncARSR has been reported to play an important role in the maintenance and self-renewal of renal cancer stem cells, but its role in liver cancer stem cells (CSCs) remains obscure. Herein, we observed high expression of lncARSR in chemoresistant hepatocellular carcinomas (HCCs). A remarkable increase of lncARSR expression in EpCAM or CD133–positive liver CSCs as well as in CSC-enriched hepatoma spheres. Interference lncARSR suppressed liver CSC expansion by inhibiting the dedifferentiation of hepatoma cells and decreasing the self-renewal ability of liver CSCs. Mechanistically, we found STAT3 as the downstream of lncARSR in HCC cells. The special STAT3 inhibitor S3I-201 abolished the discrepancy in liver CSC proportion and the self-renewal capacity between lncARSR knockdown hepatoma cells and control cells, which further confirmed that STAT3 was required in lncARSR promoted liver CSCs expansion. More importantly, interference lncARSR HCC cells were more sensitive to sorafenib or cisplatin treatment. This maybe means that patients with low lncARSR levels benefited from cisplatin or sorafenib treatment, but patients with high lncARSR expression did not. Conclusion: lncARSR was upregulated in liver CSCs and could promote HCC cells dedifferentiation and liver CSCs expansion by targeting STAT3 signaling.

Introduction
Hepatocellular carcinoma (HCC) is the most common malignancies in liver cancer (1). It remains the sixth most common malignant tumor and the second highest cause of cancer-related death in the world (2). There are about 700000 new cases of liver cancer occurring in the world and more than half of the new patients are in China (2, 3). Most HCC patients diagnosis at advanced stage due to the unobvious symptoms and the metastasis and recurrence rate remains very high (4). Increasing appreciation of heterogeneity and hierarchical organization in liver cancer supported the theory of liver cancer stem cells (CSCs) (5). HCC is a highly aggressive liver tumor containing cancer stem cells (CSCs) that participate in tumor propagation, resistance to conventional therapy, and promotion of tumor recurrence, causing poor patient outcomes (6). Liver CSCs can be enriched with several defined surface markers, including epithelial cell adhesion molecule (EpCAM), CD24, CD90 and CD133 (7-10). CD24 can drive self-renewal and tumor initiation of liver CSCs through STAT3-mediated Nanog regulation (8). Tumors that harbor an abundant CSC population or have high expression of stemness-related genes may signal a poor clinical outcome in HCC patients (11). Therefore, the underlying mechanisms of liver CSCs expansion are urgent to be explored.

Long non-coding RNA (lncRNA) is a heterogeneous class of transcripts with a minimum length of 200 bases and limited protein-coding potential (12). lncRNAs have important roles in multilevel regulation of gene expression, including transcriptional regulation by recruiting chromatin-modifying complexes and post-transcriptional regulation by interacting with miRNAs, mRNAs, or proteins (13, 14). Emerging evidence supports the notion that lncRNAs could act as oncogenes or tumor suppressors in tumors dependent on special conditions (15, 16). lncRNAs are also involved in regulating the progression of cancers and CSCs. Recent studies have shown that lncARSR play an important role in the maintenance and self-renewal of renal cancer stem cells (17). They observed highly abundant lncARSR in sunitinib-resistant RCC cells and high levels of lncARSR in RCC patients correlated with poor response to sunitinib therapy (18). However, the regulatory role of lncARSR in liver CSCs remains unknown.In the present study, we for first find that lncARSR was upregulated in liver CSCs. Next, by using loss-of-function analysis in HCC cells, we demonstrate that lncARSR promoted the self-renewal capacity of liver CSCs. Further mechanism study reveals that lncARSR regulated STAT3 signaling in HCC cells. STAT3 special inhibitor S3I-201 could abolish the self-renewal discrepancy between lncARSR knockdown cells and control cells. We also found that lncARSR could affect the drug resistance of HCC cells to sorafenib and cisplatin. Collectively, our study showed that lncARSR was a novel cancer stem cell marker that plays a key role in liver CSCs expansion and drug resistance of HCC.

HCC cell lines SMMC7721 and HCCLM3 were purchased form Chinese Academy of Sciences, Shanghai, China. The HCC cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, and 25 µg/ml of gentamicin and maintained at 37°C in 5% CO2 incubator. The culture cells were digested with 0.5% trypsin and moved to a new six-well plate twice a week.SMMC7721 or HCCLM3 cells were seeded into a six-well plate until they reached 60-70% confluence. Transfection of si-lncARSR or its negative control was performed in each well in the absence of serum with siRNA transfection reagent according to the manufacturer’s instructions (Polyplus, Illkirch, France). The sequence of si-lncARSR is: sense sequence 5′- CAACCCUGGAUCCAAAGUATT-3′ and antisense sequence 5′- UACUUUGGAUCCAGGGUUGTT-3′. These siRNAs were purchased from Shanghai GenePharma (Shanghai, China).SMMC7721 si-lncARSR or HCCLM3 si-lncARSR cells and their control cells were treated with 100 nM S3I-201 (Cat. no. S1155; Selleck.cn) or not, and then subjected to Spheroid formation or flow-cytometric analysis.One thousand SMMC7721 or HCCLM3 si-lncARSR and their control cells were cultured in a 6-well or 96-well ultra-low attachment culture plate for 7 days, and the total number of spheres was counted under the microscope.

Various numbers of SMMC7721 or HCCLM3 si-lncARSR and their control cells (64, 32, 16, 8, 4 cells per well) were seeded into 96-well ultra-low attachment culture plates for 7 days. CSC proportions were analyzed using Poisson distribution statistics and the L-Calc Version 1.1 software program (Stem Cell Technologies, Inc., Vancouver, Canada) as described (19).Hepatoma cells were diluted serially to the desired doses, and were mixed with matrigel gel (1:1). Then the mixed cells were injected subcutaneously into eight NOD-SCID mice each group. After two months, the number of tumors was counted, and the frequency of CSCs was assessed using ELDA software.The total cells RNA were extracted by using Trizol reagent (Invitrogen, 15596-018). Total cDNAs were synthesized by ThermoScript TM RT-PCR system (Invitrogen, 11146-057). The total mRNA amount presented in the cells was measured by RT-PCR using the ABI PRISM 7300.The hepatoma cells were lysed with cell lysis buffer (Beyotime) followed by supersonic splitting. The total protein was quantified using the BCA Protein Quantification kit. A total of 25 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. The membranes were blocked with 10% non-fat milk and incubated with the primary antibodies overnight. The protein band, specifically bound to the primary antibody, was detected using an IRDye 800CW-conjugated secondary antibody and LI-COR imaging system (LI-COR Biosciences, Lincoln, NE, USA). The primary antibodies were p-STAT3 (1:1000; #9145, Cell Signaling Technology), STAT3 (1:1000; #9139, Cell Signaling Technology) and GAPDH (1:5000; #5174, Cell Signaling Technology).

The HCC cells were incubated with the primary anti-CD24 (Cat. no. ab64064; Abcam, USA) or anti-CD133 (Cat. no. 18470-1-AP; Proteintech) for 30 min at room temperature. The cells were then subjected to flow cytometry using a MoFlo XDP cell sorter from Beckman Coulter (Indianapolis, IN, USA) according to the manufacturer’s instructions.The SMMC7721 or HCCLM3 si-lncARSR and their control cells were incubated with the primary anti-CD133 (Cat. no. 18470-1-AP; Proteintech) or anti-EpCAM (Cat. no. ab8666; Abcam, USA) for 30 min at room temperature. Flow cytometric analysis was performed using a MoFlo XDP cell sorter from Beckman Coulter according to the manufacturer’s instructions.SMMC7721 or HCCLM3 si-lncARSR and their control cells were treated with sorafenib (10 mM) or cisplatin (4 μg/ml) for 48 hours, followed by staining with Annexin V and PI for 15 min at 48C in the dark. Apoptotic cells were determined by an Annexin VFITC Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA) and flow cytometer according to the manufacturer’s instructions.HCC cells were transfected with STAT3 response element-luciferase reporter (STAT3-luc) plasmids. Luciferase activity was measured using a Synergy 2 Multidetection Microplate Reader (BioTek Instruments, Inc.). Data were normalized for transfection efficiency by dividing firefly luciferase activity by Renillaluciferase activity.GraphPad Prism (GraphPad Software, Inc. La Jolla, USA) was used for all statistical analyses. Statistical analysis was carried out using t test or Bonferroni Multiple Comparisons Test: *p<0.05. A p value of less than 0.05 was considered significant. Results Epithelial cell adhesion molecule (EpCAM) and Cluster of differentiation 133 (CD133) are well-accepted liver CSCs marker (8, 9). To determine the expression of lncARSR in liver CSCs, we enriched CSCs by flow cytometry sorting or sphere formation. As expected, lncARSR levels were upregulated in sorted EpCAM+ or CD133+ primary HCC cells (Fig. 1A&B). Compared with adherent cells, lncARSR expression was increased in HCC spheres derived from human primary HCC cells (Fig. 1C). Considering the close association of liver CSCs with HCC chemoresistance, we investigated the expression of lncARSR in liver CSCs. Cisplatin-resistant HCC xenografts were established as described. In comparison with control tumors, lncARSR expression was markedly increased in the cisplatin-resistant HCC residual, indicating that lncARSR expression was associated with chemoresistance (Fig. 1D). As expected, CD24+ and CD133+ liver CSCs sorted from trypsinized spheres of hepatoma cells displayed upregulated lncARSR expression (Fig. 1E&F). Moreover, the expression of lncARSR was dramatically increased in the self-renewing spheroids compared with the attached cells (Fig. 1G). Intriguingly, lncARSR levels could be partially restored during reattachment in parallel with the differentiation (Fig. 1H), which further suggested that lncARSR expression was upregulated in liver CSCs. To explore the function of lncARSR in liver CSCs regulation, SMMC7721 and HCCLM3 cells were transfected with small interference RNA of lncARSR (si-lncARSR) or siNC (Fig. 2A). Flow-cytometric analysis revealed a decreased proportion of liver CSCs in lncARSR siRNA transfected hepatoma cells (Fig. 2 B&C). Consistently, SMMC7721 si-lncARSR and HCCLM3 si-lncARSR cells formed small and fewer spheroids than control cells (Fig. 2D). An in vitro limiting dilution assay illustrated that lncARSR kncokdown dramatically decreased the CSC population in hepatoma cells (Fig. 2E). Furthermore, an in vivo limiting dilution assay illustrated that lncARSR interference dramatically decreased the CSC population in hepatoma cells (Fig. 2F). It was reported that the stem cell associated genes, including OCT4, SOX-2 and Nanog and so on, have important roles in liver CSCs regulation (20-22). Consistently, interfenrence lncARSR expression in HCC cells downregulated the expression of stemness-associated transcription factor (Fig. 3A&B) and liver CSC markers in hepatoma cells (Fig. 3C&D), which further supported that STAT3 was the downstream of lncARSR in HCC cells.Numerous studies have reported that JAK/STAT3 pathway was involved in cancer stem cells regulation (23, 24). In this study, our data demonstrated that the phosphorylation of the STAT3 molecule was evidently inactivated in both the SMMC7721 si-lncARSR and HCCLM3 si-lncARSR cells (Fig. 4A). STAT3 reporter assay further confirmed the effects of lncARSR on STAT3 activation (Fig. 4B). To further confirm the role of STAT3 in lncARSR-mediated expansion of liver CSCs, the special STAT3 inhibitor SI3-201 was used (25). As expected, SI3-201 diminished the difference in liver CSC proportion between lncARSR knockdown hepatoma cells and control cells (Fig. 4C). Consistently, SI3-201 entirely depleted the discrepancy of self-renewal capacity between lncARSR knockdown hepatoma cells and control cells (Fig. 4D). Collectively, these data suggest distinct regulation of STAT3 by lncARSR in liver CSCs. Liver CSCs were also involved in HCC chemoresistance and recurrence (26). So we next explored the role of lncARSR in chemoresistance of HCC to sorafenib and cisplatin. As expected, we found that lncARSR expression was upregulated in cisplatin-resistant or sorafenib-resistant hepatoma cells (Fig. 5A&B); suggesting lncARSR was involved in drug resistance. Furthermore, lncARSR knockdown dramatically increased the sensitivity of HCC cells to the same dosages of sorafenib or cisplatin (Fig. 5C&D). In addition, the population of apoptotic cells was also significantly increased in hepatoma cells with si-lncARSR when exposed to sorafenib or cisplatin (Fig. 5E). Moreover, western blot analysis showed that the protein expression of PARP in HCC cells with lncARSR knockdown was significantly increased when they were exposed to different doses of sorafenib or cisplatin when compared with control HCC cells (Fig. 5F). Taken together, these results demonstrated that drug sensitivity of HCC to sorafenib and cisplatin was significantly increased when lncARSR was interference, suggesting a possible role of lncARSR in the treatment of HCC drug resistance. Discussion Hepatocellular carcinoma (HCC) is a worldwide and lethal cancer with high incidence and recurrence (27). The traditional therapies of HCC patients include surgery, TACE and sorafenib (28-30). While most HCC patients lost their best optimal surgery time and only a few HCC patients are benefited from TACE or sorafenib treatment (26). Most liver cancer therapies fail to eradicate tumors due to the existence of CSCs. However, the understanding of regulatory mechanisms for CSCs is limited. In this study, we demonstrated that lncARSR is highly expressed in a subpopulation of HCC cells with stem-like characteristics and is essential for maintaining self-renewal and oncogenic potential of this type of cancer cell.Accumulating evidence shows that lncRNAs have important functions in the initiation and progression of human cancers (31), and may prove to be a novel marker for the diagnosis and treatment of cancers. It was reported that lncARSR worked as an oncogene in renal tumors (18). They found a feed-forward loop between lncARSR and YAP activity promotes expansion of renal tumour-initiating cells (17). However, the potential role of lncARSR in liver CSCs has not been reported. In our above work, we found that lncARSR knockdown by siNRA inhibited liver CSCs self-renew and dedifferentiation. The existence of CSCs has been confirmed by numerous studies, and these cells have the ability to self-renew and the potential for generating heterogeneous malignant progenies (30). It was accepted that liver CSCs are contributed to the chemoresistance. In this study, liver CSCs were enriched by establishing chemoresistant HCC xenograft tumors, and expression of lncARSR in these chemoresistant xenografts was notably up-regulated. Considering the importance of CSCs in tumor chemoresistance, we investigated the function of lncARSR on liver CSCs. We found that lncARSR expression was upregulated in hepatoma spheroids. Our data showed that lncARSR levels increased in CD24+ or CD133+ liver CSCs. Moreover, lncARSR interference in hepatoma cells inhibited the self-renewal capacity of liver CSCs, and downregulated stemness-associated genes and liver CSC markers. We also observed that lncARSR knockdown HCC cells are more sensitivity to sorafenib and cisplatin treatment. It reminders that lncARSR-low expression HCC patients may benefited from sorafenib or cisplatin treatment. The STAT3 signaling pathway is an important cascade in the regulation of organ size control and homeostasis (32, 33). The overactive of STAT3 pathway can lead to tumorigenesis (34). Moreover, STAT3 is also required to sustain self-renewal and tumor initiation of CSCs in various cancers (35). Herein, our study demonstrates that inhibition of lncARSR impairs STAT3 activity and attenuates renal CSCs propagation. In additional, STAT3 special inhibitor S3I-201 could abolish the discrepancy of the self-renewal ability between lncARSR knockdown hepatoma cells and their control cells, which further confirmSTAT3 was the downstream of lncARSR in regulating liver CSCs expansion.Here, we showed that lncARSR was upregulated in liver CSCs, which in turn promoted the expansion of liver CSCs. Moreover, lncARSR promoted liver CSCs expansion via STAT3 signaling in vitro. The findings of the S3I-201 present study not only shed a new light on the mechanism of liver CSCs but suggest a novel prognostic marker and a potential therapeutic target against HCC.