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Inhibition of GPX4 or mTOR overcomes resistance to Lapatinib via promoting ferroptosis in NSCLC cells
Jiangwei Ni, Kun Chen, Jiandong Zhang, Xiang Zhang*
Department of Thoracic Surgery, The First Afﬁliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

A R T I C L E I N F O

Article history:
Received 12 June 2021
Accepted 14 June 2021
Available online 20 June 2021

Keywords:
Non-small cell lung cancer GPX4
mTOR
Lapatinib Ferroptosis

A B S T R A C T

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase and mutations in EGFR is a major driver force of lung cancer. EGFR tyrosine kinase inhibitors (TKIs) are group of promising agents to treat cancer patients with EGFR mutations. However, the application of TKIs is often hampered by the development of drug-resistance. In the present study, we studied the role of Glutathione peroxidase 4 (GPX4) and mammalian target of rapamycin (mTOR) in regulation of lung cancer cells response to Lapatinib (Lap). Lap resistant NSCLC cells A549/Lap and H1944/Lap were created and GPX4 was knockdown by lentivirus shGPX4. Change of cell viabilities and cell death were measured by MTT and ﬂow cytometry, respectively. ROS, MDA, GSH and Fe2þ were detected by commercial kits. Xenograft mice was used to assay the in vivo effects of GPX4 on the sensitivity of Lap. We found that GPX4 and mTORC1 signalling was upregulated in Lap resistant NSCLC cells when compared to Lap sensitive NSCLC cells. Mechanistically, upregulation of GPX4 was due to enhanced activation of mTORC1 in Lap resistant NSCLC cells. Inhibition of mTORC1 led to the downregulation of GPX4 which promoted Lap induced ferroptosis as evidenced by increase of ROS, MDA, Fe 2þ and decrease of GSH. Rescue experiments conﬁrmed the role of GPX4 in regulation of Lap induced ferroptosis. In vivo experiments also indicated that silencing of GPX4 enhanced the anticancer effect of Lap via promoting ferroptosis. Overall, targeting GPX4 might be a potential strategy to enhance antitumor effects of Lap.

1. Introduction

Lung cancer is the second most common death and the leading cause of cancer-related death worldwide for many years [1]. Non- small cell lung cancer (NSCLC) is the major subtype of lung can- cer. Mutations in EGFR is one of the most frequency of oncogenic mutations of NSCLC [2]. EGFR tyrosine kinase inhibitors (TKIs) could block the phosphorylation of EGFR and thereby inhibit the downstream events that contributed to the development of cancers [3]. However, the majority of NSCLC patients will ultimately develop resistance to TKIs [4]. Hence, its necessary to develop strategies to overcome TKI resistance.
Ferroptosis is a novel cell death that is distinct from well-known types of cell death. Ferroptosis is characterized by iron-dependent initiated cell death and progressed with accumulation of lipid peroxide and generation of reactive oxygen species (ROS) [5]. GPX4

(glutathione peroxidase 4) is the only glutathione peroxidase that used in liposome peroxides [6]. During lipid peroxidation, GPX4 converts peroxy bonds to hydroxyl groups and thereby abrogates the activity of peroxides [6]. When cells undergo ferroptosis, both the expression and activity of GPX4 will be decreased in the anti- oxidant system [7]. In a recent study, it was reported that synthesis of GPX4 protein was subjected to the regulation of mTORC1 sig- nalling pathway [8].
Lapatinib (Lap) is a dual tyrosine kinase inhibitor which was initially approved for the treatment of advanced breast cancer [9]. Recently, clinical trials showed that Lap is well-tolerated and showed promising anti-tumour effects in combination with another agent against NSCLC [10]. However, whether mTOR-GPX4- mediated ferroptosis is involved in the NSCLC cells response to Lap has not been reported yet. The aim of this study was to investigate the role of GPX4 and mTOR in regulation of response to Lap in NSCLC cells.

* Corresponding author. Department of thoracic surgery, The ﬁrst afﬁliated Hospital of Wenzhou Medical University, 325035, Wenzhou, Zhejiang, China.
E-mail address: [email protected] (X. Zhang).

https://doi.org/10.1016/j.bbrc.2021.06.051

2. Materials and methods

2.1. Cell culture and chemicals

Human NSCLC cell lines A549 and H1944 cells obtained from ATCC (USA) were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum, Gibco, USA) at 37 ◦C, 5% CO2 and 95% humidity. In order to create Lap-resistant cells, A549 and H1944 cells were cultured in the medium with increasing doses (from 1 mM to 5 mM) of Lap for 10 months. Finally, the Lap-resistant A549 (A549/Lap) and H1944 (H1944/Lap) were cultured in medium with 5 mM Lap. Lap and Torin1 were purchased from Selleck Chemicals (Shanghai, China). The lentivirus against GPX4 (shGPX4) and negative control (shNC) were purchased from GenePharma (Shanghai, China). After transfection, cells were treated with 2 mg/ ml puromycin for 2 weeks and stable transfected cells were veriﬁed by western blotting and RT-PCR assay. For the overexpression of GPX4, vector and vector containing full-length cDNA of GPX4 were obtained from GenePharma. In order to create synonymous mu- tation of GPX4 that resistant to shGPX4, site-directed mutagenesis was created using the QuickChange II site-directed mutagenesis kit (Thermo Scientiﬁc, USA) according to the manufacturer’s guide. Cells were transfected with vectors using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer’s guide. All other routine chemicals were obtained from Sigma-Aldrich (USA).

2.2. RT-PCR assay

Total RNA was extracted using the Trizol (Life Technologies, USA) and reverse transcribed into cDNA using the PrimeScript RT Master Mix (Takara, China). RT-PCR was performed on the CFX
Connect™ Real-Time System (Bio-Rad, USA). The mRNA expression was normalized to GAPDH and data was analyzed using the 2—DDCt
method.

2.3. MTT assay

Cell viability was measured using the MTT assay as described previously [11].

2.4. Measurement of cellular death

Cellular death was measured using the Annexin V and propi- dium iodide (PI) staining kit (Sigma). Positively stained cells were counted by the FACSCalibur (BD Bioscience, USA) and the results were analyzed by the FlowJo7.6 software (TreeStar, USA).

2.5. ROS assay

Cells were incubated with 2 ml 20,70-dichloroﬂuorescin diacetate for 1 h at 37 ◦C in the dark. Then cells were centrifuged at 1000 g for 5 min and the pellets were resuspended in 500 ml sterile PBS. The results were measured by the FACSCalibur.

2.6. Measurement of MDA, GSH and Fe2þ

The MDA, GSH and Fe2þ levels were measured using the Lipid Peroxidation (MDA) Assay Kit, GSH Assay Kit and Iron asay Kit, respectively (Abcam, USA).

2.7. Western blotting

Total proteins were separated by the 12% SDA-PAGE and trans- ferred to the PVDF membrane and blocked with 5% skimmed milk for 1 h at room temperature. Primary antibodies were

administrated overnight at 4 ◦C, and the secondary antibody was applied for 1 h at room temperature. The results were visualized using the enhanced chemiluminescence kit (Life Technologies, USA).

2.8. Xenograft model

Six-week-old male BALB nude mice were purchased from Ani- mal Model research centre of Nanjing University (Nanjing, China). Mice were randomly divided into four groups: shNC, shNC Lap, shGPX4, and shGPX4 Lap, with 6 mice in each group. Stable transfected cells were created as described above. 2 10 6 cells were inoculated into the right side of mice back. When the size of the inoculated tumours reached around 200 mm3, tumour volumes were measured every three days as described before [12]. Lap (10 mg/kg, once two days) were intraperitoneally injected into shNC Lap and shGPX Lap groups for 20 days. On the 30th days of inoculation, mice were sacriﬁced and tumours were collected and subjected to different analysis using the Total Tissues Lysis Kit (Solarbio, China). All animal experiments were approved by the Animal Care and Use committee of Wenzhou Medical University and were conducted according to the U.K. Animals (Scientiﬁc Pro- cedures) Act, 1986 and associated guidelines.

2.9. Statistical analysis

Statistical analyses were performed with SPSS 12.0 (IBM, Chi- cago, IL, USA). Data are expressed as the mean ± SD. Student’s t-test was used for comparisons between groups, and multiple groups were analyzed with one-way analysis of variance (ANOVA), fol- lowed by Tukey’s post-hoc test. P value < 0.05 (two-tailed) was considered statistically signiﬁcant.

3. Results

3.1. GPX4 and mTORC1 signalling were upregulated in lap-resistant NSCLC cells

NSCLC cell lines A549 and H1944 were selected to create Lap- resistant strains (A549/Lap and H1944/Lap). Cell viabilities assays showed that A549/Lap and H1944/Lap cells are less sensitive to Lap when compared to A549 and H1944 cells (Fig. 1A). Then the expression of GPX4 were measured and it was found that both protein (Fig. 1B) and mRNA (Fig. 1C) levels of GPX4 were upregu- lated in A549/Lap and H1944/Lap cells when compared to the sensitive strains. Because it was reported that activation of mTORC1 promoted synthesis of GPX4 protein [8]. Thus, the status of mTORC1 was also examined and it was found that mTORC1 sig- nalling was enhanced in A549/Lap and H1944/Lap cells when compared to the sensitive strains (Fig. 1B). To evaluate the corre- lation between the expression of GPX4 and Lap sensitivity, we knockdown the GPX4 in A549/Lap and H1944/Lap cells (Fig. 1D). RT-PCR also conﬁrmed that the mRNA levels of GPX4 were down- regulated in shGPX4 groups when compare to the shNC groups (Fig. 1E). However, downregulation of GPX4 had no effects on mTORC1 activation (Fig. 1D). The effects of mTORC1 activation on Lap sensitivity were also examined by administration of Torin1, a selective ATP-competitive inhibitor of mTOR. It was found that phosphorylation of P70 and 4EBP1 were markedly suppressed after exposure to Torin1 in A549/Lap and H1944/Lap cells (Fig. 1F). At the same time, treatment of Torin 1 led to the downregulation of GPX4 (Fig. 1F). Cell viability assays were then conducted and it was found that after treated with various doses of Lap for 24 h, viabilities of shGPX4 groups were signiﬁcantly decreased when compared to the shNC groups (Fig. 1G). Meanwhile, it was also observed that

Fig. 1. Silencing of GPX4 or inhibition of mTOR overcome resistance to Lap in NSCLC cells.
A, A549, A549/Lap, H1944 and H1944/Lap cells were treated with various doses of Lap for 24 h, cell viabilities were measured. B, A549, A549/Lap, H1944 and H1944/Lap cells lysates were subjected to western blots with indicated antibodies. C, GPX4 mRNA levels were measured by RT-PCR in indicated cells. D, indicated protein levels were measured. E, mRNA levels of GPX4 were measured. F, A549/Lap and H1944/Lap cells were treated with DMSO or Torin1 for 24 h, total cellular lysates were subjected to Western blot with indicated antibodies. G, shNC and shGPX4 groups of A549/Lap and H1944/Lap cells were treated with various doses of Lap for 24 h, cell viabilities were measured. H, A549/Lap and H1944/Lap cells were treated with DMSO or Torin1 (5 mM) for 4 h, then cells were further treated with various doses of Lap for another 24 h, then cell viabilities were measured. I, cells were treated as indicated, cell death was measured by ﬂow cytometry. J, shNC and shGPX4 groups of Lap resistant NSCLC cells were treated with 20 mM of Lap for 24 h, in the presence of Fer-1 (5 mM) or Lip-1 (5 mM). Cell death was measured. K, A549/Lap and H1944/Lap cells were treated with DMSO or Torin1 (5 mM) for 4 h, then cells were further treated with 20 mM of Lap for 24 h, in the presence of Fer-1 (5 mM) or Lip-1 (5 mM). Cell death was measured. The data was presented as mean ± SD. *P < 0.05; **P < 0.01.

treatment of Torin1 signiﬁcantly decreased cells viabilities when compare to DMSO treated A549/Lap and H1944/Lap cells after exposure to Lap (Fig. 1H). Next, we examined the cell death by annexin V-PI staining. It was found that Lap induced signiﬁcantly more cellular death of shGPX4 groups than shNC groups (Fig. 1I). Similarly, Lap also triggered more cellular death of Torin1 incubated A549/Lap and H1944/Lap cells when compared to the control groups (Fig. 1I). To examined whether Lap induced cell death was correlated with the ferroptosis, ferroptosis speciﬁc inhibitors Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lip-1) were applied. As shown in Fig. 1J, cell death induced by Lap in shNC and shGPX4 in A549/Lap and H1944/Lap cells were both markedly inhibited by Fer-1 and Lip-1. In addition, increased cell death caused by Torin1 was also signiﬁcantly abrogated by Fer-1 and Lip-1 (Fig. 1K). These ﬁndings suggested that knockdown of GPX4 or inhibition of mTORC1 increased sensitivity to Lap via promotion of ferroptosis in NSCLC cells.

3.2. Silencing of GPX4 or inhibition of mTORC1 promoted lap- induced ferroptosis in NSCLC cells

In order to further examine the role of ferroptosis in GPX4 and mTORC1's regulation of sensitivity to Lap in NSCLC cells. Several ferroptosis markers were examined. After treated with Lap, it was found that knockdown of GPX4 and inhibition of mTOR increased the accumulation of ROS than control groups (Fig. 2A). In addition, it was also observed that MDA levels and intracellular Fe2þ con- centrations were signiﬁcantly increased and GSH levels were inhibited shGPX4 and Torin1-treated groups of A549/Lap and H1944/Lap cells (Fig. 2B, C, D). Noteworthy, knockdown of GPX4 alone also increased the levels of MDA, Fe2þ and decrease the level of GSH in both A549/Lap and H1944/Lap cells (Fig. 2B, C, D). However, treatment of Torin1 alone without Lap had little effects on the levels of MDA, Fe2þ and GSH in A549/Lap and H1944/Lap cells (Fig. 2B, C, D). The above results conﬁrmed that silencing of GPX4 and inhibition of mTORC1 promoted ferroptosis induced by

Fig. 2. Silencing of GPX4 and inhibition of mTOR promoted Lap induced ferroptosis in NSCLC cells.
A, A549/Lap and H1944/Lap cells were treated as indicated for 6 h, ROS levels were measured by ﬂow cytometry. B, MDA levels were measured. C, intracellular Fe 2þ levels were measured. D, GSH levels were measured. The data was presented as mean ± SD. *P < 0.05.

Lap in NSCLC cells.

3.3. Overexpression of GPX4 rescued resistance to lap in NSCLC cells

To further verify the role GPX4 in the sensitivity of NSCLC cells to Lap, we created a vector containing the full length of GPX4 cDNA harbouring synonymous mutations which cannot be recognized by the shGPX4 (Suppl. Table 1). Overexpression of vector containing mutant GPX4 (mut GPX4) signiﬁcantly upregulated both the mRNA (Fig. 3A) and protein (Fig. 3B) levels of GPX4 in shGPX4 groups of A549/Lap and H1944/Lap cells. As expected, overexpression of GPX4 signiﬁcantly increased the cell viabilities of A549/Lap and H1944/Lap cells under the treatment of Lap (Fig. 3C). The effects of Torin1 on cell viabilities were also reversed by forced expression of GPX4 in Lap treated A549/Lap and H1944/Lap cells (Fig. 3C). Meanwhile, cell death induced by Lap was greatly reduced after overexpression of GPX4 in shGPX4 and Torin1 treated groups of cells (Fig. 3D). Furthermore, upregulation of GPX4 also reversed the effects of Lap on levels of ROS, MDA and GSH in shGPX4 and Torin1 treated groups cells (Fig. 3E, F, G, H). Taken together, those data suggested that GPX4 determines the sensitivity of NSCLC cells to ferroptosis induced by Lap.

3.4. Silencing of GPX4 enhanced the anti-tumour effect of lap in vivo

Finally, we evaluated the effects of downregulation of GPX4 in combination with Lap on tumorigenesis in vivo. After establish the xenografts in nude mice, silencing of GPX4 reduced the growth of tumours and further enhanced the inhibitory effect of Lap on tumour growth (Fig. 4A). 30 days later, mice were sacriﬁced and tumours were collected and subjected to various assays. As indi- cated in Fig. 4B, C and D, Lap treatment led to the upregulation of MDA, Fe 2þ and downregulation of GSH in tumour tissues and silencing of GPX4 enhanced these effects of Lap. Next, the tumours tissues were subjected to western blotting analysis. As shown in Fig. 4E, treatment of Lap slightly increased the expression of GPX4. Moreover, Lap decreased the expression of Ki-67 and silencing of GPX4 could further enhance the inhibitory effects of Lap on Ki-67 (Fig. 4E). Taken together, those data suggest that silencing of GPX4 enhanced the anti-tumour effect of Lap in vivo via promoting ferroptosis.

Fig. 3. Overexpression of GPX4 inhibited ferroptosis in NSCLC cells
A, Lap resistant NSCLC cells were transfected with empty vector or synonymous mutant GPX4 (mut GPX4) for 24h, mRNA levels of GPX4 were measured by RT-PCR. B, Lap resistant NSCLC cells were transfected with empty vector or synonymous mutant GPX4 (mut GPX4) for 24h, protein levels of GPX4 were measured by western blots. C, Lap resistant NSCLC cells were stably transfected with shGPX4 or treated with Torin1 (5 mM), cells were transfected with empty vector or synonymous mutant GPX4 (mut GPX4) for 24h, then cells were treated with Lap (20 mM) for another 24 h, cell viabilities were measured. D, cells were treated as described above, cellular death was measured. E, Lap resistant NSCLC cells were transfected with empty vector or synonymous mutant GPX4 (mut GPX4) for 24h, then cells were treated with Lap (20 mM) for another 6 h, ROS levels were measured. F, cells were treated as above, MDA levels were measured. G, GSH levels were measured. H, intracellular Fe 2þ levels were measured. The data was presented as mean ± SD. *P < 0.05; **P < 0.01.

4. Discussion

NSCLC is a worldwide health issue that is still lack of effective treatments. Although TKIs provide a promising treatment option in EGFR mutation positive patients, most NSCLC patients will acquire drug-resistance which hampers the application of TKIs [13]. Lap is a dual inhibitor of EGFR and ERBB2 and ﬁrstly approved for the treatment of breast cancer. Recently, both clinical trials and in vitro studies showed that Lap in combination with other agents showed encouraging anti-tumour effects against NSCLC [10,14]. However, the mechanisms that affect Lap sensitivity in NSCLC cells are still largely elusive.
One of the main strategies to treat cancer is to induce apoptosis in cancer cells. However, cancer cells often develop resistance to apoptosis and the induction of other forms of regulated cell death such as ferroptosis becoming another strategy to ﬁght against cancers under this scenario [15]. In the current study, it was found that GPX4 was upregulated in Lap resistant NSCLC cells and later investigation showed that knockdown of GPX4 will increase NSCLC cells sensitivity to Lap via promotion of ferroptosis. Our ﬁndings are in accordance with a recent study which suggested that GPX4 is positively correlated with resistance to Lap in various cancer cells [16]. In order to determine the form of cell death, Fer-1 and Lip-1 were administrated and it was found that cell death triggered by inhibition of GPX4 and Lap was fully inhibited. In addition, upre- gulation of ROS, MDA, Fe2þ and downregulation of GSH were also observed after silencing of GPX4 and treatment of Lap. In order to further conﬁrm the role of GPX4 in regulation of Lap sensitivity in

NSCLC cells. We successfully rescued the expression of GPX4 by overexpressed a synonymous mutated GPX4. Not surprisingly, overexpression of GPX4 decreased the sensitivity of shGPX4 NSCLC cells to Lap. In addition, ROS, MDA and Fe2þ levels were decreased and GSH level was upregulated after overexpression of GPX4. Those ﬁndings suggested that GPX4 affected NSCLC cells sensitivity to Lap via regulation of ferroptosis. Similarly, GPX4 has also been reported to affect antitumor effect of sorafenib via regulation of ferroptosis in liver cancer cells [17]. Our ﬁndings suggested that GPX4 could also be applied as a biomarker for the NSCLC cells response to Lap. It would be interesting to test the impact of GPX4 on the sensitivity of NSCLC cells to other TKIs.
In the current study, it also found that pharmacologic inhibition of mTOR could enhance sensitivity to Lapatinib in NSCLC cells. This ﬁnding is in line with a previous study which showed that inhibi- tion of mTOR improved Lap sensitivity in HER2-overexpressing breast cancer cells [18]. Meanwhile, we also observed that enhanced mTOR signalling was positively correlated with upregu- lation of GPX4. This is in accordance with a recent study which reported that synthesis of GPX4 was relied on the activation of mTOR [8]. Noteworthy, it was also reported that inhibition of mTOR resulted in elevated GPX4 protein levels [19]. This discrepancy revealed the complex relationship between mTOR and GPX4. More investigation into the correlation between mTOR and GPX4 is necessary.
The role of Lap in regulation of oxidative stress and ferroptosis is still not fully understood. In our scenario, it was found that Lap induced accumulation of ROS and oxidative stress in NSCLC cells.

Fig. 4. Knockdown of GPX4 promoted the anti-tumour effect of Lap in vivo.
A, Mice were injected subcutaneously with shNC or shGPX4 groups of NSCLC cells. Each point represents the mean ± SD of the tumour volumes of ﬁve mice in each group. Mice were treated with DMSO or Lap (10 mg/kg) 5 days a week for four weeks. B, Mice were sacriﬁced, MDA levels in tumour tissues were measured. C, GSH levels in tumour tissues were measured. D, Fe 2þ levels in tumour tissues were assayed. E, tumour tissues were subjected to Western blot analysis with indicated antibodies. The data was presented as mean ± SD.
*P < 0.05; **P < 0.01.

Our observations are in line with a previous study which also showed that Lap treatment caused accumulation of ROS and fer- roptosis in glioma and lung cancer cells [20]. Interestingly, another study showed that Lap could eradicate ROS and protect neurons against ferroptosis [21]. This discrepancy might due to different cell types since cancerous cells had higher levels of ROS than normal cells [22]. Therefore, it is necessary to further examine the role of Lap in regulation of oxidative stress and ferroptosis in more cell types. Finally, we also revealed that knockdown of GPX4 reduced the growth rate of cancer in xenograft models, and further enhanced the inhibitory effect of Lap. In line with the in vitro

studies, silencing of GPX4 also promoted ferroptosis induced by Lap in vivo. Our ﬁndings are in accordance with a previous study which also found that inhibition of GPX4 repressed the growth of breast cancer in vivo [23].
In summary, our study revealed that silencing of GPX4 and in- hibition of mTOR increase the sensitivity of NSCLC cells to Lap via enhancement of ferroptosis. It was also found that GPX4 expression was positively correlated with activation of mTOR. Although some investigations suggested that Lap hold potential ability to treat NSCLC, but there is lack of biomarkers to predict its clinical beneﬁt [10,14]. Based on our ﬁndings in the current study, it will be

meaningful to examine whether the GPX4 and mTOR status is correlated with the prognosis of individual NSCLC patients that received Lap treatment.

Declaration of competing interest

None.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2021.06.051.

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