NT157 Inhibits Hepatocellular Carcinoma Growth and Sensitizes Cells to Sorafenib

Background: Sorafenib has been recognized as the standard therapy for advanced hepatocellular carcinoma (HCC). Besides, ecacy of sorafenib was unsatisfactory and vast patients are resistant to sorafenib. Thus, molecular mechanisms underlying regulation of sorafenib resistance and seeking potential strategy to improve its ecacy have attracted much attention. As a small-molecule inhibitor of IGF-1R, NT157 has potent antitumor activity against some human cancers. However, whether NT157 has potential anti-tumor effects and its molecular mechanisms in HCC remain poorly understood. Methods: We assessed the effects and explored the mechanism of NT157 and sorafenib as single agents or in combination with sorafenib in HCC cells and mouse model. Further, we further demonstrated that NT157 reversed resistance to sorafenib in HCC. Results: Here, we found NT157 inhibited HCC growth and induced apoptosis in vitro and in vivo. In terms of mechanism, NT157 phosphorylated IRS-1 through ERK-MAPK signaling to be degraded by the ubiquitin-proteasome pathway, lowered p-AKT to deactivate IGF-1R signaling to inhibit proliferation and induce apoptosis. Surprisingly, we further demonstrated that NT157 acted synergistically with sorafenib to inhibit proliferation and contributed to sensitize HCC cells to sorafenib by down-regulation of p-AKT. Conclusions: Overall, our ndings provide a translational rationale for inhibition of IGF-1R and downstream signaling pathways by NT157 as a novel targeted therapy alone or combined with sorafenib in HCC. our that combining and offered increased benets against likely through the mechanism of inhibiting NT157-induced ERK activation after a sequential treatment. However, the long-term effectiveness of sorafenib is still controversial because the acquired resistance has been observed. Activation of alternative pathways to counteract therapies leads sorafenib resistance. Similarly to our data, evidence implies important role of IGF-1R and AKT signaling in conferring resistance in human malignancies, consisting of a study which highlighted the role of AKT in sorafenib resistance 13,15 . Our results showed that NT157 could negatively regulate p-AKT. Therefore, we further focused on exploring the effect of NT157 on sorafenib-resistant HCC cells. We found that sorafenib activated AKT in sorafenib-resistant HCC cells. Surprisingly, NT157 successfully decreased sorafenib dosage and sensitized cells to sorafenib with the reduction of p-AKT in sorafenib-resistant HCC cells. These data have suggested that IGF-1R/AKT pathway activation maybe a sorafenib-resistant mechanism and NT157 could serve as a novel targeted therapy combined with sorafenib in HCC.


NT157 inhibits HCC growth in vivo and in vitro.
In search of acquiring the potential value of IGF-1R as a therapeutic target in HCC, we analyzed in silico the correlation between IGF-1R RNA expression and overall survival (OS). Using of TCGA database, IGF-1R overexpression was found to be associated with poor overall survival in HCC (Supplementary Fig. 1, Supplementary Table1).
Next, we directly detected potential therapeutic value of NT157 in vivo and in vitro experiments.
Different HCC cells were incubated with various concentrations of NT157 for various time. The data showed that NT157 signi cantly inhibited the proliferation of HCC cells in a dose-dependent and timedependent manner (Fig. 1a). Colony formation assays corroborated the inhibition of proliferation by NT157 treatment (Fig. 1b). To test whether NT157 could induce apoptosis, we also measured the apoptotic ratio of HCC cells in response to NT157 treatment. Results showed that NT157 treatment resulted in marked increases in apoptosis as measured by Annexin V and caspases-3 and PARP cleavage, suggesting the activation of the apoptotic cascade ( Fig. 1c-d). Furthermore, to verify the antitumor effects of NT157 against HCC in vivo, we conducted subcutaneous mouse xenograft model by intraperitoneal and intratumor administration, respectively. In both administration methods, NT157-treated mice exhibited signi cant tumor growth inhibition in sharp contrast to control groups (Fig. 1e).
Notably, HCC cell lines were sensitive to NT157, whereas normal hepatocyte LO2 was tolerant to NT157 treatment ( Supplementary Fig. 2a Fig. 3a), suggesting that tumor burden may be decreased after NT157 treatment. Further, to seek the e cacy biomarker of NT157, RNA-seq, qRT-PCR and ow uorimetry were performed. And CCL20 and CXCL8 had the most noteworthy change in the NT157-treatment group (Supplementary Fig. 3b-c). In addition, the relative expression in CXCL8, CCL20, and AFP had remarkably negative correlations ( Supplementary Fig. 3d), which intensively implied that CXCL8 and CCL20 may be an e cacy biomarker of NT157. Taken together, these data demonstrated that NT157 may inhibit HCC growth in vivo and in vitro effectively and safely.
NT157 targets IGF-1R/AKT pathway by activation of the ERK/MAPK pathway To determine the mechanisms of NT157, HCC cells were subjected to RNA-seq analysis to quantitatively detect expression pro le changes in mRNA. A total of 3738 differentially expressed genes (DEGs) were identi ed (Fig. 2a). DEGs were mostly involved in cell cycle progression, proliferation, and apoptosis (Fig.  2b). And Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis implied the signi cant pathway, MAPK signaling pathway (Fig. 2c).
We found that NT157 induced extensive phosphorylation of IRS1 proteins in HepG2 and SMMC-7721 cells (Fig. 3a). And, with time increasing, p-IRS induced by NT157 decreased (Fig. 3b). Furthermore, to explore the mechanism of p-IRS degradation, we combined treatment with NT157 and MG132, a proteasome inhibitor, led to p-IRS1 accumulation (Fig. 3c), suggesting that NT157 induced IRS1 phosphorylation and subsequent IRS1 degradation. Surprisedly, the downstream activation of AKT was also suppressed in HepG2 cells treated with NT157 (Fig. 3d). It has been reported that shutting off IGF-1R signaling depended on the targeting of IRS1 phosphorylation and ubiquitin proteasome-dependent degradation [8][9] . Hence, it showed that targeting IGF-1R by NT157 depended on degrading p-IRS1and subsequently inhibiting downstream AKT. Besides, we found that NT157 could induced ERK pathway activation (Fig. 3a). To detect the relationship between IRS1 and ERK pathway, treatment of HepG2 cells with the RAF/ERK pathway inhibitor PLX4720 showed that PLX4720 abolished NT157-induced increase in phosphorylation of IRS1 and ERK (Fig. 3e). It proved NT157-induced phosphorylation of IRS1 was dependent on the ERK pathway. Besides, combined treatment of PLX4720 abrogated the anti-proliferative effects of NT157 (Fig. 3f). Thus, these results indicated that NT157 induced the activation of the ERK-MAPK pathway, leading to phosphorylation and degradation of IRS1, and in turn blocked IGF-1R/IRS1/AKT signaling to induce cell death.
Combining NT157 and sorafenib exhibits synergy against HCC via ERK signaling.
A recent report indicates that ERK signaling is involved in enhancing sensitivity to the sorafenib 10 . As NT157 treatment results in ERK activation, we then evaluated the effect of combined treatment of NT157 and sorafenib, trying to gure out whether it is additive, synergistic or antergic. First, we veri ed that sorafenib, which has been proved to block ERK signaling pathway 11 , inhibited HCC cell proliferation and lowered ERK expression ( Supplementary Fig. 4a-b). Then, we found that monotherapy with either sorafenib or NT157 reduced HCC cell growth, and combination of sorafenib and NT157 intriguingly exerted greater growth inhibition than either agent alone, suggesting that NT157 treatment signi cantly enhanced sorafenib cytotoxicity (Fig. 4a). Meanwhile, we observed a synergistic effect by NT157 and sorafenib, as demonstrated in Loewe plots and calculated Bliss combination indices (CIs) 11 (Fig. 4a). Importantly, although a synergistic effect was observed in HCC cell lines, no signi cantly cooperative lethality appeared in normal hepatocytes ( Supplementary Fig. 4c). Moreover, apoptotic rate was also found to be increased as measured by ow cytometer and apoptotic related proteins in HCC cell lines. We found that compared with untreated control, NT157 or sorafenib alone induced apoptosis to some extent, while the combination induced more apoptosis (Fig. 4b). Similarly, the cleavage of caspase-3 and PARP were increased in HCC cells exposed to NT157 or sorafenib alone and were further enhanced in cells treated with the combination (Fig. 4c).
Given the strong synergy of these 2 drugs in HCC, we next investigated the possible mechanisms of combination therapy. We found that when sorafenib was administrated, NT157-induced ERK activation was attenuated (Fig. 4d). Combined treatment resulted in inhibition of both ERK and AKT pathways. Importantly, a sequential treatment of NT157 and sorafenib was crucial to gain a synergic effect.
Potentiated cytotoxicity by NT157 plus sorafenib might be partially due to ERK suppression by sorafenib in HCC cells. The synergic effect in vitro prompted an evaluation in vivo. In our xenograft model, animals were randomly treated with vehicle, NT157, sorafenib, or NT157 and sorafenib in combination. We found that both NT157 and sorafenib signi cantly suppressed HCC xenograft growth, while suppressed more signi cantly in the combination cohort compared with single agent (Fig. 4e). Meanwhile, no signi cant weight loss and signi cantly cooperative lethality of vital organs were observed compared with sorafenib alone during this treatment period (Fig. 4f, Supplementary Fig.5), indicating that the combination was well tolerated. Immunohistochemical analysis showed increased TUNEL-positive tumor cells and decreased Ki67 in the combination cohort (Fig. 4g). Overall, the combination of NT157 and sorafenib exhibited signi cant synergy against HCC, likely through p-ERK suppression.
NT157 can sensitize HCC cells to sorafenib by down-regulation of p-AKT.
After long term exposure to sorafenib, almost all patients would emerge acquired resistance, and eventually progress [3][4] . To this end, we developed HCC cell line resistant to sorafenib in vitro, HepG2-SR. This cell line was cloned from the parental HCC cell line HepG2, and the relative resistance index was 2.10 times higher in sorafenib-resistant HepG2 cells than parental HepG2 cells (Fig. 5a). Apoptotic rate of HepG2 cells was more than 2-fold higher than HepG2-SR cells with 5 and 10 μmol/L of sorafenib, respectively (Fig. 5b).
Surprisingly, we found a signi cant increase of p-AKT and its downstream p-mTOR in HepG2-SR cells, compared with the corresponding parental HepG2 cells, which are sensitive to sorafenib (Fig. 5c). The results indicated that sustained exposure to sorafenib would lead to AKT activation, conforming the previous studies 6, [13][14][15] . As we found the suppression of AKT signaling in HepG2 cells treated with NT157 (Fig. 3d), which prompted us to investigate whether NT157 can reverse sorafenib-resistance of HCC.
Furthermore, we assessed the e cacy of NT157 and sorafenib, in combination and as single agents, on established sorafenib-resistant HCC cells in vitro. NT157 strikingly inhibited cell viability, and the inhibitive effect of combination of these 2 drugs on cell viability was more remarkable in HepG2-SR cells (Fig. 5d). Besides, we also demonstrated a combination of sorafenib and NT157 signi cantly increased apoptosis in HepG2-SR cells (Fig. 5e). The increased apoptosis shown by the expression of caspase-3 and PARP indicated that sorafenib-resistant cells were refractory to sorafenib-induced apoptosis through caspase-dependent and -independent ways, while NT157 sensitized resistant cells to sorafenib-induced cell death (Fig. 5f). Moreover, in HepG2-SR cells NT157 induced striking p-AKT inhibition, and combined treatment with NT157 and sorafenib attenuated p-AKT increase compared with HepG2-SR cells treated with sorafenib, a nding that suggests NT157 could sensitize HCC cells to sorafenib by inhibiting AKT signaling pathway (Fig. 5f). In summary, we found p-AKT level was increased in sorafenib-resistant HCC cells and NT157 may be a potential drug for reversing sorafenib resistance by inhibiting AKT signaling in HCC.

Discussion
In the study, we found that NT157 inhibits HCC growth both in vitro and in vivo. NT157 phosphorylates IRS-1 through ERK-MAPK signaling to degrade by the ubiquitin-proteasome pathway, lowers p-AKT to turn off IGF-1R signaling to inhibit proliferation. Furthermore, NT157 and sorafenib provide synergistic effects to inhibit proliferation, and NT157 can sensitize cells to sorafenib by lowering p-AKT levels. Our ndings not only illustrate the mechanism of NT157-mediated growth inhibition by targeting IGF-1R/IRS1/AKT signaling but also provide a novel strategy of combining NT157 and sorafenib, which may be a promising therapeutic approach in HCC (Fig. 6).
Given the observed negative correlation between IGF-1R expression and OS in patients with HCC, we focused on the possibility of targeting IGF-1R by NT157 in HCC. Our results showed that NT157 signi cantly inhibited HCC growth and induced apoptosis in vitro and in vivo. In terms of mechanism, we demonstrated that NT157 induced ERK-MAPK activation to phosphorylate IRS1. Then phosphorylated IRS1 underwent degradation to shut off long-lasting IGF-1R/IRS1/AKT signaling to exert effect. IGF-1R is highly homologous to insulin receptor (IR) in structure and function. It has been demonstrated that monoclonal antibodies of IGF-1R can induce compensatory activation of IR, leading to drug resistance 16 . As IRS1/2 mediates signaling pathway from both IGF-1R and IR, NT157 leads to the disruption of signaling downstream of both receptors induced by IGF1, IGF2 or insulin and reduce the probability of drug resistance. Taken together, these data demonstrated that NT157 was well effective by inhibiting IGF-1R/IRS/AKT signaling to prevent HCC growth.
In our study, we found ERK activation after NT157 treatment. As it has been previously shown that the higher the p-ERK level is in HCC, the more sensitive the tumor is to sorafenib, suggesting that p-ERK is a potential predictor of sensitivity to sorafenib when treating HCC 10 , we further explored the possibility and rationale of combination treatment with sorafenib and NT157. We demonstrated a synergistic effect between NT157 and sorafenib in HCC cell lines, and combination therapy yielded reductions in tumor growth in HCC. In addition, our ndings showed that combining sorafenib and NT157 offered increased bene ts against HCC likely through the mechanism of inhibiting NT157-induced ERK activation after a sequential treatment. However, the long-term effectiveness of sorafenib is still controversial because the acquired resistance has been observed. Activation of alternative pathways to counteract targeted therapies leads to sorafenib resistance. Similarly to our data, growing evidence implies an important role of IGF-1R and AKT signaling in conferring resistance in human malignancies, consisting of a study which highlighted the role of AKT in sorafenib resistance 13,15 . Our results showed that NT157 could negatively regulate p-AKT. Therefore, we further focused on exploring the effect of NT157 on sorafenib-resistant HCC cells. We found that sorafenib activated AKT in sorafenib-resistant HCC cells. Surprisingly, NT157 successfully decreased sorafenib dosage and sensitized cells to sorafenib with the reduction of p-AKT in sorafenib-resistant HCC cells. These data have suggested that IGF-1R/AKT pathway activation maybe a sorafenib-resistant mechanism and NT157 could serve as a novel targeted therapy combined with sorafenib in HCC.
Although targeted IGF-1R inhibition alone or in combination with other therapies has achieved antitumor responses in a wealth of different tumors, IGF-1R inhibitors are associated with fatal events and a narrow therapeutic index [16][17][18] . Given these constraints and therapeutic potential, exploring novel therapeutic strategies is warranted.

Conclusions
Our ndings provide rationale for testing NT157 and developing the combination of sorafenib in HCC. In our study, we found NT157 had good safety no matter monotherapy or combination. Therefore, it provides the possibility for clinical application in the future as its promising clinical e cacy and safety. Taken together, our ndings provide a translational rationale for inhibition of IGF-1R and downstream signaling pathways by NT157 as a novel targeted therapy alone or combined with sorafenib in HCC. And NT157 may potentially act as an enhancer of sorafenib to inhibit HCC growth.