HJC0152 suppresses human non–small‐cell lung cancer by inhibiting STAT3 and modulating metabolism

Abstract Objectives Signal transducer and activator of transcription 3 (STAT3) is constitutively activated and overexpressed in many cancers, including non–small‐cell lung cancer (NSCLC). We recently developed HJC0152 as an orally active STAT3 inhibitor. This study focused on investigating HJC0152's effect and mechanism of action in NSCLC. Materials and methods We analysed cell proliferation by MTT assays, cell migration by wound healing and transwell assays, protein levels by Western blot, and apoptosis and reactive oxygen species (ROS) level by flow cytometry. A nude mouse tumorigenesis model was established for in vivo experiment. UHPLC‐QTOF/MS was used for untargeted metabolomic relative quantitation analysis. Results We found that HJC0152 exhibited activity against human NSCLC cells in vitro and NSCLC xenograft tumours in vivo via regulating STAT3 signalling and metabolism. HJC0152 efficiently reduced NSCLC cell proliferation, promoted ROS generation, induced apoptosis, triggered DNA damage and reduced motility in A549 and H460 NSCLC cells. Moreover, HJC0152 significantly inhibited the growth of A549 xenograft tumours in vivo. HJC0152 also affected metabolism, significantly decreasing and perturbating levels of several metabolites in the purine, glutathione and pyrimidine metabolism pathways. Conclusions HJC0152 reduces cellular capacity to scavenge free radicals, leading to ROS generation and accumulation and apoptosis. This study provides a rationale for further developing HJC0152 as a potential therapy for NSCLC and provides insights into the mechanisms by which HJC0152 exerts its anti‐cancer effects.


| INTRODUC TI ON
Malignant tumours pose a great threat to human health and life and are one of the leading causes of death in the world. 1 Lung cancer is the second most frequently diagnosed malignant tumour, and the 5-year survival rate of advanced lung cancer is about 15%. 1 Worldwide in 2018, lung cancer occurred in 2.1 million people and resulted in 1.76 million deaths. 2 This makes it the most common cause of cancer-related death in men and the second most common in women after breast cancer. 2 Lung cancer can be divided into 2 main pathological types: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. NSCLC accounts for 80%-85% of all lung cancer cases. 3 In recent years, although some progress has been made in the treatment of NSCLC with surgery, radiotherapy, chemotherapy and targeted therapy, the 5-year survival rate remains low. 1 Therefore, there is an urgent need for more effective treatments for NSCLC.
While the aetiology of NSCLC is considered multifactorial, aberrant activation of multiple oncogenes and inactivation of tumour suppressor genes are the primary contributors to abnormal proliferation and reduced apoptosis of pulmonary epithelial cells.
Accumulating evidence has shown that signal transducer and activator of transcription 3 (STAT3) is constitutively activated and overexpressed in a variety of malignant tumours, including NSCLC. STAT3 is implicated in cancer cell proliferation and invasion, apoptosis and the cell cycle 4-6 and making it a potential therapeutic target.
However, targeting STAT3 with its inhibitors has been a challenge in the past decade due to off-target effects or poor bioavailability associated with in-developing STAT3 inhibitors.
Accumulating evidence shows that cellular metabolism plays an important role in the development of cancer; thus, metabolism is a new focus for research on the pathogenesis of cancer and for the search for cancer biomarkers. 7 Metabolomics is a branch of systems biology similar to genomics, transcriptomics and proteomics.
Metabolomic research has contributed important insights to cancer biology and clinical practice in oncology. 8 Metabolomic studies can use highly sensitive, high-throughput instrumentation to qualitatively and quantitatively determine changes in the abundance of metabolites during carcinogenesis and tumour development and to screen and identify novel tumour biomarkers. 9 Recently, metabolomics has been used in research on prostate cancer, 10 hepatocellular carcinoma, 11 colorectal cancer, 12 papillary thyroid carcinoma 13 and other malignant tumours, 14,15 leading to an ever-growing list of metabolites that may be cancer biomarkers or therapeutic targets.
Importantly, STAT3 involves in regulating key metabolism pathways in normal and cancer cells.
We and others recently determined that niclosamide, a Food and Drug Administration-approved anthelmintic drug, 16 effectively suppresses the activation, nuclear translocation and transactivation of STAT3. 17,18 However, the further clinical development of niclosamide for cancer therapy is hindered by its poor water solubility and low oral bioavailability. We previously developed a series of O-alkylamino-tethered niclosamide derivatives, including HJC0152 ( Figure 1A). 19 HJC0152 has significantly better (up to 680fold) aqueous solubility and oral bioavailability. It also has stronger STAT3-inhibiting activity than niclosamide. 19,20 However, little is known about whether HJC0152 has anti-lung cancer activity, and particularly whether it has effects against NSCLC. In this study, we investigated the anti-cancer effect of HJC0152 on human NSCLC cell lines in vitro and NSCLC xenograft tumours in vivo. We observed significant inhibition of proliferation and motility in cultured HJC0152-treated NSCLC cells and inhibition of xenograft tumour growth in mice. Further, STAT3 was found to be involved in NSCLC carcinogenesis and in mediating HJC0152's anti-NSCLC effects. We then delineated the metabolic alterations that occur in NSCLC cells treated with HJC0152 in this report.
F I G U R E 1 HJC0152 inhibits proliferation of human lung cancer cells. A, Chemical structure of HJC0152. B, Equal amounts of protein from lung cancer cell lines A549, H460, and H1299 were analysed by Western blotting for expression of p-STAT3 (Tyr705) and STAT3. GAPDH was used as a loading control. C-E, Results of MTT assays of cell survival. A549 (C), H460 (D), and H1299 (E) cells were treated with the indicated doses of HJC0152 for 24, 48 or 72 h and subjected to MTT assays. Results are presented as the mean ± standard error of 3 independent experiments. F-H, 24-h IC 50 values for HJC0152 in A549 (F), H460 (G), and H1299 (H) cells. I-J, Representative photographs of cell viability assays (100× magnification). A549 (I) and H460 (J) human NSCLC cells were treated with the indicated concentrations of HJC0152 for 24 h. Cell viability was determined by crystal violet staining. K-L, Representative photographs of colony formation assays of A549 and H460 cells treated with the indicated concentrations of HJC0152. Results are presented as the mean ± standard error from 3 independent experiments. * P < .05, ** P < .01, *** P < .001 vs control

| Cell proliferation assays with crystal violet staining
Following 24 hours of HJC0152 treatment at different concentrations, cells were fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS) for 10 minutes. After being washed with PBS, cells were incubated with 0.1% crystal violet solution for 10 minutes. Cells were then gently washed with distilled water and air-dried.
Each experiment was conducted independently and repeated at least 3 times.

| Colony formation assays
A549 or H460 cells were plated in 6-well plates (800 cells/well) and allowed to attach overnight. The cells were then incubated in the presence or absence of HJC0152 (0, 1.25, 2.5, or 5 μmol/L) at 37°C in 5% CO 2 for 24 hours. The cell culture medium was replaced every 3 days. After 14 days, cells were washed twice in cold PBS, fixed with methanol and stained with 0.1% crystal violet. Digital images of the plates were obtained as a permanent record of colony counting. Colonies with >50 cells per field were analysed by ImageJ software.

| Flow cytometry
To determine the apoptosis rate, cells were treated with HJC0152 were acquired under a fluorescence microscope, and the mean fluorescence intensity of DCFH-DA was measured using a flow cytometer (Accuri C6, BD Biosciences) as previously described. 21

| Scratch assays
For the scratch assay, A549 or H460 cells were seeded into 6-well
After 16 hours, cells that had not penetrated the membrane and those on the bottom of the upper chamber were removed with cotton swabs.
The remaining cells, those that had migrated through the pores, were fixed with 4% paraformaldehyde in PBS for 20 minutes and stained with 0.1% crystal violet solution for 15 minutes. Cellular migration and invasion were evaluated by counting the number of cells penetrating through the membrane onto the lower surface, which was performed using a light microscope (Olympus, CX31) at 100× magnification. Five fields were randomly selected for analysis.

| Protein extraction and Western blotting
After treatment, cells were prepared in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Roche) at 4°C for 30 minutes. The lysates were then centrifuged (13 000 × g, 4°C) for 15 minutes to obtain total protein lysates.
Protein expression was analysed using Western blotting as described previously. 17,20,21 The expression level of GAPDH was used as the loading control.

| Immunofluorescence microscopy
A total of 2 × 10 4 cells was grown with cell culture medium and treated with designated concentrations of HJC0152 on chambered slides. At designated times after treatment, cells were fixed with 4% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X-100 (Solarbio) in PBS for 20 minutes at room temperature. Cells were subsequently washed and blocked with 5% BSA (Amresco) in PBS for 60 minutes at room temperature.
Primary antibodies against γ-H2AX were applied to the cells and incubated at 4°C overnight. After being washed with PBS 3 times, the cells were incubated with fluorescence-conjugated secondary antibodies for 60 minutes at room temperature and then washed 3 times with PBS. Cell nuclei were counterstained with DAPI. Cells were visualized using a fluorescent microscope (Thermo Fisher) or confocal microscope.

| Immunohistochemical staining
Details of the immunohistochemical analyses were described previously. 20 Briefly, tumour tissues were harvested, fixed in 4% paraformaldehyde/PBS, dehydrated, embedded in paraffin and cut into 4-μm-thick sections. Deparaffinized sections were rehydrated and stained using specific antibodies against p-STAT3 (Tyr705), Ki-67, or cleaved caspase-3, then stained with biotinylated secondary antibodies. Detection was performed with an avidin-biotin horseradish peroxidase complex and 3,3′-diaminobenzidine (BD Biosciences) as the chromogen. All slides were analysed, and representative photographs were taken using an Olympus CX31 microscope (Olympus America) at 400× magnification. Tumours were allowed to grow to an average volume of 100 mm 3 before initiation of therapy, as described previously. 17,22 Tumourbearing mice were randomly divided into 2 treatment groups (5 mice per group): vehicle control (dimethyl sulfoxide) or HJC0152 (7.5 mg/kg). The schedules for the administration of HJC0152 are provided in the figure legends. Tumour size was measured every three days using a vernier calliper, and the tumour volume was calculated from two-dimensional measurements (mm) using the following formula: Tumour volume = length × width 2 /2. The tumour volume doubling time (TVDT) was calculated using the following

| Untargeted metabolomic relativequantitative analyses
The cell samples were collected in 15-mL Vacutainer tubes and then centrifuged for 15 minutes (1500 × g, 4°C). Aliquots of the samples were stored at −80°C for use in ultra-high-performance liquid chro- The raw mass spectrometry data (wiff.scan files) were converted to MzXML files using ProteoWizard MSConvert before being imported into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z = 25 ppm, peakwidth = c (10, 60), prefilter = c (10, 100). For peak grouping, the parameters bw = 5, mzwid = 0.025, minfrac = 0.5 were used. In the extracted ion features, only variables having more than 50% of the nonzero measurement values in at least 1 group were kept. Metabolites were identified by comparing their mass spectra with an in-house database established using available authentic standards.
After normalization to the total peak intensity, the processed data were uploaded and imported into SIMCA-P (version 14 The methods used for the bioinformatic analysis of metabolism are described in the Appendix S1.

| Statistical analysis
All experiments were performed with at least 3 independent replicates. All data are presented as mean ± standard error of the mean.
Statistical comparisons between 2 groups were made using the Student t test. Differences between experimental groups were assessed for statistical significance using a 1-way analysis of variance with repeated measures followed by post hoc comparisons using Tukey's multiple paired comparison test. Differences were considered statistically significant at P < .05. All analyses were performed using GraphPad Prism software.

| HJC0152 inhibits the proliferation of human lung cancer cells
We initially determined p-STAT3 (Tyr705) level in non-squamous NSCLC cells, A549, H460, and H1299. All tested lines had constitutively activated p-STAT3 (Tyr705) ( Figure 1B). Particularly, strong activation of STAT3 was evident in the A549 and H460 cells. Next, we characterize the effect of HJC0152 on NSCLC cell proliferation, and A549, H460 and H1299 cells were treated with HJC0152 in vitro for 24, 48 or 72 hours, and cell viability was determined by MTT assays.
As shown in Figure 1C To further evaluated the anti-proliferation activity of HJC0152 in 2 human NSCLC cell lines that with high level of p-STAT3 (Tyr705), A549 and H460 cells were treated with different concentrations of HJC0152 for 24 hours, and significant morphological changes and cell death were observed ( Figure 1I and J). In addition, HJC0152-treated NSCLC cells formed significantly fewer colonies and significantly smaller colonies than did untreated control cells ( Figure 1K and L). Collectively, these results demonstrate that HJC0152 has a strong inhibitory effect on the growth of NSCLC cells.

| STAT3 activation is positively correlated with NSCLC, and HJC0152 inhibits constitutive STAT3 activation in NSCLC cells
We assessed the prognostic value of STAT3 expression in clinical samples using the Kaplan-Meier plotter platform (www.kmplot. com). 23 The samples were grouped according to the median (or upper or lower quartile) expression of STAT3 gene, and then, the two groups were compared by a Kaplan-Meier plot. The patients in the cohorts were filtered using the term "adenocarcinoma" before running the analysis. The results showed that high levels of STAT3 expression were associated with shorter progression-free survival durations in patients with lung adenocarcinoma (Figure 2A). Data from the Oncomine database 24 confirmed that STAT3 was more highly expressed in lung adenocarcinoma tissues than in corresponding normal lung tissue and was also expressed significantly more highly in NSCLC than in squamous cell lung carcinoma ( Figure 2B and C).
Next, we determined the expression level of STAT3 and 2 other key members of the STAT family of transcription factors, STAT1 and STAT5. A STAT3 siRNA was used to block STAT3 expression, and the levels of the 3 STAT proteins were determined. As shown in Figure 2D, STAT3 siRNA inhibited the expression of STAT3 and p-STAT3 (Tyr705). Lower p-STAT3 (Tyr705) protein levels were found in HJC0152-treated A549 and H460 cells than in control cells, whereas no difference in the levels of STAT1 and STAT5 was found ( Figure 2D). Furthermore, HJC0152 treatment inhibited p-STAT3 (Tyr705) expression in NSCLC cells in a dose-dependent manner ( Figure 2E).

| HJC0152 induces ROS accumulation and apoptosis in human NSCLC cells
Reactive oxygen species are normal products of cell metabolism, 25 but they also play an important role in tumorigenesis and cancer progression. 26 Therefore, to gain further insight into the mechanism of HJC0152-induced cytotoxicity in NSCLC cells, we evaluated intracellular ROS levels using the fluorescent probe DCFH-DA. As the concentration of HJC0152 increased, the fluorescence intensity observed by fluorescence microscopy in A549 and H460 cells gradually increased over that of control cells ( Figure 3A and B). Flow cytometry also indicated a significant dose-dependent increase of intracellular ROS generation in treated A549 and H460 cells ( Figure 3C and D). These results indicate that HJC0152 induces ROS accumulation in NSCLC cell

lines.
Previously, we found that oxidative stress is an early upstream event of apoptosis in breast cancer cells. 17 To assess whether HJC0152 induces apoptosis in lung cancer cells, we performed annexin V-FITC/PI double staining and analysis using flow cytometry. 17 The results showed that treatment with increasing concentrations of HJC0152 induced a corresponding increase in the percentage of apoptotic cells ( Figure 3E and F). This finding, that HJC0152-induced apoptosis in A549 and H460 cells, was supported by Western blotting. Protein levels of Bcl-2, a marker of apoptotic cells, decreased in a dose-dependent manner after HJC0152 treatment ( Figure 4D). Taken together, these findings showed that HJC0152 induces ROS accumulation and apoptosis in NSCLC cells.
In addition, pre-treatment with antioxidants N-acetyl-L-cysteine (NAC), a ROS scavenger, 27 completed blocked ROS induction by HJC0152 ( Figure 3G and H), and partially protected cells from HJC0152-induced apoptosis ( Figure 3G and H). These results suggest that quenching ROS by NAC partly abrogated HJC0152-induced apoptosis. Therefore, HJC0152-induced ROS partially contributes to apoptosis of A549 cells.

| HJC0152 triggers DNA damage in human lung cancer cells
Many anti-cancer drugs work by inducing DNA damage in rapidly dividing cancer cells. 28 Figure 4D). Together, these data showed that HJC0152 induces DNA damage in NSCLC cells.

| HJC0152 suppresses the migration and invasion of NSCLC cells
We next measured the effect of HJC0152 on the motility of NSCLC cell lines. In scratch assays, HJC0152 treatment delayed wound healing in A549 and H460 cell lines ( Figure 5A-D). Consistent with the results of the scratch assays, HJC0152 inhibited NSCLC cell migration and invasion in a dose-dependent manner in Transwell assays ( Figure 5E-F, and Figure S1). These results suggest that HJC0152 suppresses the migration and invasion ability of NSCLC cells.
The matrix metalloproteinase (MMP) family plays a key role in facilitating tumour metastasis. In particular, reduction of the secretion and activity of MMP-2 and MMP-9 has been shown to inhibit cancer cell motility and metastasis. 30 In both A549 and H460 cell lines, HJC0152 inhibited the protein expression of MMP2 and MMP9, suggesting that HJC0152 attenuates the ability of these MMPs to degrade the extracellular matrix ( Figure 5G and H). Epithelial-mesenchymal transition (EMT), a biological process in which epithelial cells transform into mesenchymal cells, is regarded as one of the most important mechanisms of cell migration and tumour metastasis, which is closely related to clinical outcomes. 31,32 EMT is characterized by the loss of expression of proteins that promote cell-to-cell contact, such as E-cadherin, and gain of mesenchymal markers, such as N-cadherin and vimentin. Therefore, we determined whether the expression of these EMT- Taken together, these results demonstrate that HJC0152 inhibits migration and invasion of NSCLC cells by regulating the EMT process.

| HJC0152 significantly suppresses the growth of NSCLC xenograft tumours in vivo
We previously reported that HJC0152 has anti-cancer effects in animal models of human breast cancer, 19 head and neck squamous cell carcinoma, 20 glioma, 33 and gastric cancer. 34 To establish the in vivo anti-cancer efficacy of HJC0152 against human lung cancer, we generated a mouse xenograft lung cancer model by subcutaneously injecting A549 NSCLC cells into mice ( Figure 6A). HJC0152 given to A549-bearing mice significantly retarded the tumour growth rate compared to that of the control group. Tumour growth appeared slow, as indicated by the mean tumour volume doubling time (TVDT), which was 2.03 and 4.62 days in the control and HJC0152 groups, respectively ( Figure 6B and C). The tumour weight in mice treated with HJC0152 was significantly lower than that of control mice ( Figure 6D and E). These results demonstrate that HJC0152 has significant antitumour activity in a NSCLC xenograft model raised from A549 cells. Immunohistochemical assays showed decreased intensity of Ki67, p-STAT3 (Tyr705), and cleaved caspase-3 staining in tumours from mice treated with HJC0152 ( Figure 6F and G).
Collectively, these results suggest that HJC0152 significantly suppressed the in vivo growth of human lung cancer xenografts in mice.

| HJC0152 reprograms metabolism of human NSCLC cells
Our previous studies and a report from other groups have shown that STAT3 is a primary target of HJC0152 in breast cancer, head and neck squamous cell carcinoma, glioma, and gastric cancer. 19,20,33,34 In this study, we showed that STAT3 expression is a prognostic factor for NSCLC patients (Figure 2A-C), and that STAT3 is activated in NSCLC cells ( Figure 2D and E). We hypothesized that HJC0152 alters cellular metabolism by targeting STAT3 and its signalling during lung carcinogenesis. We therefore characterized the metabolic alterations in A549 NSCLC cells treated with HJC0152 using UHPLC-QTOF/MS technology. In total, 5160 positive-ion-mode and 5227 negative-ion-mode features were identified in 10 samples from A549 cells treated with or without HJC0152. As shown in Figure 7A

| Altered metabolic pathways in HJC0152treated NSCLC cell line A549
Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was performed for the metabolites whose accumulation was increased in HJC0152-treated A549 cells.
The KEGG enrichment analysis showed that the metabolites that were significantly changed after HJC0152 treatment were highly associated with purine metabolism, ATP-binding cassette (ABC) transport, pyrimidine metabolism and 17 other signalling pathways, as shown in Figure 8C. The major perturbed pathways are described in detail below.

| Purine metabolism
Purines and their derivatives are widely involved in biological processes, including host-tumour interactions. 35,36 In the NSCLC A549 cell line, the metabolic pathway most affected by HJC0152 was purine metabolism, with significantly decreased levels of all identified intermediates, including adenosine monophosphate, adenosine, adenine, inosine, hypoxanthine, adenosine 5'-diphosphate and L-glutamine (Table 1), suggesting that HJC0152 downregulates purine metabolism.

| ATP-binding cassette transporters
ATP-binding cassette transporters are a large class of transmembrane proteins involved in substance transport 37 and use ATP F I G U R E 6 HJC0152 exhibits antitumour activity in an in vivo model of lung cancer. A, Mice bearing NSCLC xenograft tumours developed from A549 cells were treated with vehicle (control) or HJC0152 (7.5 mg/kg/d), as illustrated in the diagram. B, Tumour growth curves of groups as indicated. C, Tumour volume doubling time (time for a tumour to double in volume) of groups. Data are presented as mean ± standard error. *** P < .001 vs control. D, Images of dissected tumours from nude mice (n = 5). E, Weight of resected xenograft tumours from nude mice. Data are presented as mean ± standard error. ** P < .01 vs control. F, Representative images (400× magnification) of A549 xenografts stained for Ki-67, p-STAT3 (Tyr705) and cleaved caspase-3. G, Quantification of Ki-67, p-STAT3 (Tyr705) and cleaved caspase-3. *** P < .001 vs control hydrolysis to transport diverse substrates (eg, amino acids, lipids, lipopolysaccharides, inorganic ions, polypeptides, carbohydrates and various drugs) across cell and organelle membranes, thus in association with many diseases and cancers. 38 In HJC0152-treated A549 cells, 8 altered metabolites were involved in ABC transporter pathways (Table 1). Among them, l-glutamate and glutathione, which are involved in the glutathione metabolism pathway, were significantly reduced. The lower levels of glutathione found in HJC0152-treated A549 cells suggest that HJC0152 decreases the cellular capacity to scavenge free radicals, resulting in enhanced ROS generation. These results suggest that the metabolic changes in response to HJC0152 are highly associated with ROS levels in A549 cells.

| Pyrimidine metabolism
Pyrimidine synthesis is important for DNA replication in tumour cells. 39 The abundance of several metabolites involved in pyrimidine metabolism was decreased in A549 cells after  (Table 1). These results demonstrate that HJC0152 causes perturbation of pyrimidine metabolism in A549 cells.

| D ISCUSS I ON
Based on the results obtained from this study, we concluded that HJC0152 exerts its anti-cancer effect on human NSCLC at least partially via regulating STAT3 signalling and metabolic processes regulated by STAT3. We speculate that inhibition of STAT3 activa-  More specifically, we uncovered marked changes in the levels of several important metabolites, including glutamine, and in metabolic pathways including purine metabolism, ABC transporters and pyrimidine metabolism.
Recent reports have shown that glutamine, which is important for the synthesis of glutathione, 47,48 is an abundant and versatile nutrient that participates in the proliferation of cancer cells and their metabolism. Glutathione is riched in antioxidants in cancer cells and is important for cellular redox homeostasis and cancer cell survival in response to oxidative stress. 49 Our results showed that HJC0152 treatment reduces the abundance of glutamine and glutathione among the metabolites in A549 cells. HJC0152 also decreases the antioxidant capacity of A549 cells and increases ROS levels. When ROS is accumulated to a level higher than the antioxidant capacity of cells, oxidative stress occurs, leading to apoptosis. 17 Our results also showed that HJC0152 induces apoptosis in A549 NSCLC cells. Collectively, these observations demonstrate that the metabolic changes induced by HJC0152 cause apoptosis by promoting ROS generation in A549 cells. These findings are consistent with those of our recent report on metastatic breast cancer cells. 17,20 Additionally, we found a markedly high level of sphingomyelin (d18:1/18:0) in HJC0152-treated A549 cells, strongly suggesting that HJC0152 also acts by targeting the key role of sphingomyelin in maintaining the integrity of the cell membrane for the protection of cells from apoptosis.
The findings of the current study support the notion that the anti-cancer effect of HJC0152 against NSCLC may be primarily mediated by STAT3 signalling, a key regulator of cellular metabolism and ROS production. It has recently been shown that glutamine per se activates STAT3, which promotes cancer cell proliferation, and that STAT3 activation is independent of glutamine metabolism. 50 This is not inconsistent with our results, which show that HJC0152 regulates not only STAT3, but also metabolites including glutamine in NSCLC cells.
With its efficacy against multiple cancer types, HJC0152 has the promise to be developed as a targeted therapy for single use or in combination with standard-of-care therapies. This study provides a solid rationale for the further investigation of HJC0152 as a treatment for lung cancer, particularly NSCLC. In addition, further study of how HJC0152 regulates explicit metabolite pathways is currently ongoing.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare that they have no potential conflicts of interest.

AUTH O R CO NTR I B UTI O N S
LL and QS conceived the study and generated hypotheses. LL designed and performed the experiments, and analysed the data with the help from HL, XW and JR provided assistance for metabonomic analysis. JZ provided HJC0152. LL, QS and SJF wrote the manuscript. All authors critically reviewed the content and approved the final version for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.