Anlotinib Inhibits Cisplatin Resistance in Non-Small-Cell Lung Cancer Cells by Inhibiting MCL-1 Expression via MET/STAT3/Akt Pathway

Background Anlotinib is an effective targeted therapy for advanced non-small-cell lung cancer (NSCLC) and has been found to mediate chemoresistance in many cancers. However, the underlying molecular mechanism of anlotinib mediates cisplatin (DDP) resistance in NSCLC remains unclear. Methods Cell viability was assessed by the cell counting kit 8 assay. Cell proliferation, migration, and invasion were determined using the colony formation assay and transwell assay. The mRNA expression levels of mesenchymal-epithelial transition factor (MET) and myeloid cell leukemia-1 (MCL-1) were measured by quantitative real-time PCR. Protein expression levels of MET, MCL-1, and STAT3/Akt pathway-related markers were examined using western blot analysis. Results Our data showed that anlotinib inhibited the DDP resistance of NSCLC cells by regulating cell proliferation and metastasis. Moreover, MET and MCL-1 expression could be decreased by anlotinib treatment. Silencing of MET suppressed the activity of the STAT3/Akt pathway and MCL-1 expression. Furthermore, MET overexpression reversed the inhibitory effect of anlotinib on the DDP resistance of NSCLC cells, and this effect could be eliminated by MCL-1 knockdown or ACT001 (an inhibitor for STAT3/Akt pathway). Conclusion Our results confirmed that anlotinib inhibited DDP resistance in NSCLC cells, which might decrease MCL-1 expression via mediating the MET/STAT3/Akt pathway.


Introduction
Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80% of the total lung cancer [1,2].NSCLC is characterized by easy recurrence, micrometastasis, and poor prognosis, so its treatment is very difcult [3,4].At present, chemotherapy and molecular targeted therapy are the main treatment methods for NSCLC, but the occurrence of drug resistance is a major problem to be solved in clinical practice [5][6][7].Terefore, it is of great signifcance to explore the molecular mechanism of drug resistance for efective treatment of NSCLC.
Anlotinib, a novel multitarget tyrosine kinase inhibitor (TKI), has inhibition efects on tumor angiogenesis and growth [8,9].In addition to being used as a third-line treatment for patients with advanced NSCLC [10], the antitumor efects of anlotinib have been found in many other types of cancer, such as oral squamous cell carcinoma [11] and thyroid cancer [12].Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that regulates cell proliferation, apoptosis, metastasis, and drug resistance [13].Some research studies have shown that MET is overexpressed in osteosarcoma tumor tissues, and anlotinib suppresses osteosarcoma progression and cisplatin (DDP) resistance by reducing MET expression and phosphorylation [14,15].Moreover, anlotinib has been confrmed to decrease MET expression and phosphorylation to hinder colorectal carcinoma cell growth and oxaliplatin resistance [16].A recent study suggests that anlotinib combined with osimertinib therapy represses the osimertinib resistance in NSCLC [17].However, whether anlotinib mediates NSCLC progression and DDP resistance through MET-related pathways has not been studied.
It has been reported that silencing of CSE1L inhibits lung cancer proliferation and metastasis through the MET/ STAT3/PD-L1 pathway [18].Besides, MET knockdown suppresses pancreatic ductal adenocarcinoma proliferation and metastasis by inhibiting the Akt-related signaling activity [19].In osteosarcoma, MET has been shown to regulate osteosarcoma progression by mediating the activity of the STAT3/Akt pathway [14].Terefore, MET, as a key molecule for tumor progression, may regulate cell biological functions through the STAT3/Akt signaling pathway.Myeloid cell leukemia-1 (MCL-1), a member of the Bcl-2 gene family, is highly expressed in many tumor tissues and participates in regulating multidrug resistance [20,21].A previous study showed that targeted inhibition of MCL-1 might promote DDP-induced mitochondrial apoptosis, thereby enhancing DDP sensitivity in NSCLC [22].In addition, genipine had been suggested to promote mitochondrial dysfunction in gastric cancer by inactivating of STAT3/MCL-1 axis [23].Terefore, we speculated that MET mediated the STAT3/Akt signaling pathway to regulate MCL-1 expression, thereby participating in the DDP resistance of NSCLC.
Tis study aimed to reveal the underlying molecular mechanism by which anlotinib inhibited chemoresistance in NSCLC and to provide new ideas for NSCLC treatment.Combined with the above, we proposed the hypothesis that anlotinib might repress DDP resistance in NSCLC through the MET/STAT3/Akt/MCL-1 pathway.

Cell Counting Kit 8 (CCK8)
Assay.A549/DDP and H1299/DDP cells were cultured in 96-well plates and then treated with anlotinib or DDP for 24 h at the indicated concentrations.After that, cells were incubated with CCK8 solution (Dojindo, Kumamoto, Japan), and then cell viability was assessed at 450 nm by using a microplate reader.In functional experiments, transfected and treated A549/DDP and H1299/DDP cells were seeded into 96-well plates and then hatched with CCK8 solution to measure cell viability.

Colony Formation
Assay.A549/DDP and H1299/DDP cells were plated in 6-well plates and cultured for 14 days.Afterwards, cells were fxed with 4% paraformaldehyde (Beyotime) and stained with crystal violet (Beyotime, Shanghai, China).Te number of colonies was counted under a microscope to assess cell proliferation.
2.5.Transwell Assay.A549/DDP and H1299/DDP cells suspended with a serum-free medium were seeded into the top of transwell chambers coated with or without Matrigel (for detecting cell invasion or migration, respectively).Ten, a complete medium was added to the lower chamber.After 24 h, the migrated and invaded cells were fxed with 4% paraformaldehyde and stained with crystal violet, followed by counted under a microscope.

Statistical Analysis.
Data are expressed as the mean ± SD.GraphPad Prism 7.0 was utilized for data analysis.Diferences were calculated using Student's t-test or ANOVA.P < 0.05 was considered signifcant diference.Canadian Respiratory Journal increasing DDP concentration, the viability of A549 and H1299 cells was signifcantly lower than that of A549/DDP and H1299/DDP cells, respectively.By detecting the medium inhibitory concentration (IC50) of DDP on cells, we confrmed that A549/DDP and H1299/DDP cells had higher DDP resistance than A549 and H1299 cells.In this, 2 μM DDP had no signifcant efect on cell viability, so it was used for subsequent functional experiments.In addition, we explored the optimal concentration of anlotinib-treated A549/ DDP cells and confrmed that 1 μM anlotinib had no signifcant efect on cell viability and could be used in subsequent functional experiments.After cells were treated with 1 μM anlotinib and 2 μM DDP, we determined that the viability of A549/DDP and H1299/DDP cells was signifcantly reduced (Figure 1(c)), indicating that anlotinib might inhibit DDP resistance of A549/DDP and H1299/DDP cells.Furthermore, we measured cell proliferation using the colony formation assay and confrmed that anlotinib and DDP alone had no signifcant efect on cell proliferation, while the combination of the two treatments markedly reduced cell proliferation (Figure 1(d)).Above all, anlotinib reduced DDP resistance in A549/DDP and H1299/DDP cells.Tese data also confrmed that MCL-1 was the downstream target of MET, and its expression was regulated by MET.Moreover, we found that the promotion efects of oe-MET on cell viability, proliferation, migration, and invasion in anlotinib and DDP-induced A549/DDP and H1299/DDP cells also were reversed by MCL-1 knockdown (Figures 4(e)-4(g)).

Anlotinib Suppresses MET Expression to
All data showed that anlotinib suppressed DDP resistance in NSCLC cells through regulating MET/MCL-1 axis.

Discussion
Chemotherapy is one of the main treatment methods for NSCLC, but the emergence of chemoresistance greatly reduces the efcacy of chemical drugs [5].Terefore, it is urgent to clarify the potential mechanisms afecting chemoresistance in NSCLC and provide new ideas for overcoming NSCLC chemoresistance.Targeted therapy is characterized by strong pertinence and small side efects and has shown great advantages in cancer treatment in recent years [24,25].
Canadian Respiratory Journal

Canadian Respiratory Journal
Anlotinib is an antiangiogenic targeted therapy approved for treating locally advanced, metastatic NSCLC in patients who have progressed or relapsed following at least two prior regimens of systemic chemotherapy [26,27].Previous study had suggested that anlotinib could overcome multidrug resistance in many cancers [16,28].More importantly, a recent study shows that anlotinib combined with platinum-chemotherapy has efective antitumor activity as the frstline treatment for extensive-stage small-cell lung cancer [29].
In our study, we demonstrated that anlotinib suppressed DDP resistance in NSCLC cells by regulating cell proliferation and metastasis.In terms of mechanism, we confrmed that anlotinib decreased MET expression to inactivate the STAT3/ Akt pathway, thereby reducing expression.
MET plays a proto-oncogene role in many cancers, and activation of the MET-related pathway plays a vital role in cancer development [30,31].Multiple previous research studies had indicated that MET amplifcation might lead to EGFR-TKI resistance, and its inhibition could overcome resistance to EGFR-TKI in NSCLC [32].Anlotinib had been confrmed to restrain cancer cell chemoresistance via blocking MET expression and phosphorylation [14,16].Consistent with these results [14,16], we pointed out that anlotinib repressed DDP resistance in NSCLC via decreasing MET expression.STAT3/Akt is a key pathway regulating tumor progression and is closely related to the occurrence of many tumors, such as neuroblastoma [33] and cervical cancer [34].It has been reported that MET is involved in regulating the cancer malignant phenotype by STAT3 and Akt [14,18,19].Here, we found that MET knockdown inhibited the activity of STAT3/Akt pathway, and targeted inhibition of the STAT3/Akt pathway using ACT001 could abolish the promotion efect of MET on anlotinib-mediated DDP resistance in NSCLC cells.Te above data further revealed that MET regulated the STAT3/Akt pathway to participate in the regulation of anlotinib on DDP resistance in NSCLC.
MCL-1 may be a key molecule leading to drug resistance in patients, and it is involved in regulating apoptosis and chemoresistance in cancers [20,35].Studies had shown that MCL-1 was highly expressed in NSCLC, and its inhibition reduced NSCLC cell proliferation and DDP resistance [36,37].Novel SHP2 degrader SHP2-D26 in combination with the EGFR inhibitor osimertinib (AZD9291) could overcome osimertinib resistance in NSCLC by reducing MCL-1 expression [38].Tese evidences suggested that a high expression of MCL-1 was associated with drug resistance in NSCLC.In this, MCL-1 expression could be reduced by anlotinib and MET knockdown.Functional experiments showed that MCL-1 knockdown reversed the enhancing efect of MET on anlotinib-mediated DDP resistance, confrming that anlotinib might suppress DDP resistance by regulating MCL-1 through MET.Besides, we also found that STAT3/Akt pathway inhibitor ACT001 could decrease MCL-1 mRNA and protein expression, which further indicated that MET regulated the STAT3/Akt pathway to mediate MCL-1 expression.

Conclusions
Our study suggests a new mechanism by which anlotinib inhibits chemoresistance in NSCLC.Our research pointed out that anlotinib restrained DDP resistance in NSCLC through the MET/STAT3/Akt/MCL-1 pathway.Te discovery of MET/STAT3/Akt/MCL-1 axis provides a new idea for overcoming DDP resistance in NSCLC and has important clinical signifcance.
Cells.To confrm the DDP resistance of A549/DDP and H1299/DDP cells, we assessed cell viability under diferent concentrations of DDP treatment.As shown in Figure1(a)

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Regulate the DDP Resistance of NSCLC Cells.Anlotinib treatment signifcantly inhibited MET mRNA expression (Figure2(a)).At the protein level, the expression of p-MET/MET also was reduced after anlotinib treatment (Figure2(b)).To explore whether anlotinib restrained DDP resistance in NSCLC cells by regulating MET expression, we performed the rescue experiments.Firstly, oe-MET was transfected in A549/DDP and H1299/DDP cells to overexpress the MET level and phosphorylation.Ten, A549/DDP and H1299/DDP cells were transfected with oe-MET and then treated with anlotinib and DDP.Our data showed that anlotinib and DDP treatment markedly repressed MET mRNA expression and p-MET/MET protein expression, while these efects could be abolished by oe-MET (Figures2(c) and 2(d)).In functional experiments, anlotinib and DDP treatment signifcantly repressed cell viability, the number of colonies, as well as the numbers of migrated and invaded cells.However, these efects were partially eliminated by MET overexpression (Figures2(e)-2(g)).All data showed that anlotinib inhibited MET expression and phosphorylation to suppress the DDP resistance of NSCLC cells.
3.4.MET Participates in the Regulation of Anlotinib on DDP Resistance in NSCLC Cells through Regulating MCL-1 Level.MET and MCL-1 mRNA expression, as well as p-MET/MET and MCL-1 protein expression, were signifcantly inhibited in both anlotinib group and anlotinib + DDP group, while DDP alone treatment had no efect on this (Figures4(a) and 4(b)).