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 significantly inhibited the proliferation of HCC cells in a dose-dependent and time-dependent 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 significant 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). In both subcutaneous mouse xenograft model, NT157 had no significant effect on the body weight of tumor-bearing mice (Supplementary Fig. 2b). Furthermore, NT157 had no obvious effect on routine blood test and liver and renal function test (Supplementary Table2). Serum concentrations of Alpha-fetoprotein protein (AFP) are commonly and classically used for the evaluation of tumor burden of HCC patients. To evaluate the effect of NT157, AFP level was assessed by ELISA. Results showed NT157 reduced the AFP levels in HCC cells (Supplementary Fig. 3a), suggesting that tumor burden may be decreased after NT157 treatment. Further, to seek the efficacy biomarker of NT157, RNA-seq, qRT-PCR and flow fluorimetry 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 efficacy 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 profile changes in mRNA. A total of 3738 differentially expressed genes (DEGs) were identified (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 significant 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 degradation8-9. 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 sorafenib10. As NT157 treatment results in ERK activation, we then evaluated the effect of combined treatment of NT157 and sorafenib, trying to figure out whether it is additive, synergistic or antergic. First, we verified that sorafenib, which has been proved to block ERK signaling pathway11, 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 significantly 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 significantly cooperative lethality appeared in normal hepatocytes (Supplementary Fig. 4c). Moreover, apoptotic rate was also found to be increased as measured by flow 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 significantly suppressed HCC xenograft growth, while suppressed more significantly in the combination cohort compared with single agent (Fig. 4e). Meanwhile, no significant weight loss and significantly 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 significant 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 progress3-4. 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 significant 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 studies6,13-15. 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 efficacy 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 significantly 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 finding 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.