Anlotinib inhibits the development of esophageal squamous cell carcinoma by regulating the PI3K/AKT signaling pathway

doi:10.21203/rs.3.rs-2600904/v1

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Anlotinib inhibits the development of esophageal squamous cell carcinoma by regulating the PI3K/AKT signaling pathway

https://doi.org/10.21203/rs.3.rs-2600904/v1

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Background: Anlotinib, a small molecule multi-targeted tyrosine kinase inhibitor, is widely used in the treatment of various diseases. This study aimed to investigate the potential therapeutic effects and possible molecular mechanisms of anlotinib on esophageal squamous cell carcinoma (ESCC) .

Methods: Cell viability was detected by CCK-8 assay. Wound-healing and transwell assay were used to evaluate the migratory-invasive ability of ESCC cells. Apoptosis was detected by Hoechst33342 fluorescence staining. Transcript data were obtained by RNA sequencing. Transcript data were analyzed for differentially expressed genes (DEGs) using the DESeq2 program. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using R package clusterProfiler. Western blotting was used to detect the protein expression level.

Results: The results of functional experiments showed that anlotinib can inhibit the viability of KYSE30 cells with time- and dose-dependent toxicity. The IC50 at 24h, 48h and 72h were 124.85μM, 21.226μM and 13.03μM, respectively. Anlotinib also inhibited cell migration and invasion, and induced apoptosis. Transcriptome sequencing results showed that anlotinib resulted in 359 genes were up-regulated and 156 down-regulated in KYSE30 cells. GO analysis of these DEGs showed that GO mainly included biological processes and molecular functions. KEGG enrichment analysis showed that PI3K/AKT pathway was the most critical signaling pathway. Western blot demonstrated that anlotinib inhibited the phosphorylation of AKT, and the total protein expression of PI3K and AKT.

Conclusion: Anlotinib inhibits the progression of ESCC cells by modulating the PI3K/AKT signaling pathway.

anlotinib

esophageal squamous cell carcinoma

single-cell RNA sequencing

PI3K/AKT pathway

PDGFRB

Esophageal cancer is a common malignant tumor of the digestive tract, and esophageal squamous cell carcinoma (ESCC) is the most important histological type [1-3]. The effective treatments for patients with advanced esophageal cancer are radiotherapy and chemotherapy. Current treatment options remain limited for those patients who cannot tolerate or respond poorly [4, 5].

Anlotinib is a small molecule multi-targeted tyrosine kinase inhibitor (TKI) that targets vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) [6]. Preclinical studies have shown that anlotinib blocks the extracellular regulated protein kinases (ERK) / protein kinase B (AKT) signaling pathways by inhibiting the phosphorylation of VEGFR2, PDGFRB, c-kitproto-oncogeneprotein (c-KIT) and other kinases, thereby exerting anti-angiogenesis and inhibition of cell proliferation [7, 8]. Clinically, anlotinib is included in the "CSCO Guidelines for the diagnosis and treatment of esophageal cancer" as a category 2A recommendation for the second-line treatment of unresectable locally advanced esophageal cancer. However, there are few reports on its effects on the biological phenotype of ESCC cells and mechanisms.

In this study, we carried out relevant functional experiments and RNA transcriptome sequencing to explore how anlotinib plays an anti-tumor effect in ESCC, hoping to provide a more reliable experimental basis to guide clinical practice.

2.1 Drugs and Antibodies

Anlotinib was donated by China Tai Tianqing Company (Nanjing, Jiangsu, China). In western blot ,the primary antibody use : anti-PI3K(1:1000 dilution; cat.no.ab191606; Abcam), anti-phosphorylate(p)-AKT(1:1000 dilution; cat.no.ab192623; Abcam), anti-AKT(1:20000 dilution; cat.no.ab179463; Abcam) and anti-β-actin (1:2,000 dilution; cat. no. 60008-1-lg; Proteintech Group, Inc.), the secondary antibody use : Goat anti-mouse IgG (1:5,000 dilution; cat. no. S0002; Affinity Biosciences Co., Ltd), goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.).

2.2 Cell Selection, Culture and Drug Treatment

Human esophageal squamous cell carcinoma (KYSE-30) was obtained from Wuhan Procell Life Technology Co., LTD (Wuhan, Hubei, China). It was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) which containing 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Solarbio, Beijing, China), it was placed in an incubator at 37℃ and 5% CO₂ saturated humidity to observe the cells. Digestion and passage are carried out when the confluence of cell growth reaches about 80. All experiments used logarithmic growth phase cells. Cancer cells were treated with a series of different concentration gradients of anlotinib.

2.3 Cell Viability Assay

Cell Counting Kit-8 (CCK-8; Dojindo, Molecular Technologies, Kumamoto, Japan) was used to detect the effect of anlotinib on cell viability of KYSE30 cells. KYSE30 cells were evenly plated in 96-well plates at 5×10³ cells per well and cultured overnight at 37°C and 5% CO₂. After the cells adhered, the cells were treated with different concentrations of anlotinib (0μM, 5μM, 10μM, 20μM and 40μM) for 24h, 48h or 72h, then 10μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2h in a dark incubator. The absorbance at 450nm was measured with a microplate reader (Tecan Group), and the cell viability was estimated according to the formula for calculating the drug survival rate. The median inhibitory concentration (IC50) was calculated using SPSS 26.0 (IBM, Armonk, NY, USA).

2.4 Cell Migration and Invasion Assays

Cell migration and invasion abilities were determined using transwell chambers (Corning, NY, USA) coated or uncoated with Matrigel. KYSE30 cells in 6 well plates were treated with 2ml DMSO and 5μM anlotinib for 24h. Adjust the cell concentration of each group to 5×10⁴ cells/well with serum-free DMEM medium and inoculate them in a small chamber. Incubate for 24h in a 37°C, 5% CO₂ humidified incubator. Cells that migrated or invaded the lower chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The passing cells were observed with an IX71 (Olympus, Tokyo, Japan) inverted microscope and images were counted by ImageJ (NIH, MD, USA) software.

Wound-healing assays were used to determine the collective migration rate of cells. KYSE30 cells were seeded in 6-well plates at 6×10⁵ cells/well. After the cells are approximately 90% confluent, use a yellow pipette tip to draw a line along the center of the plate with a ruler. After removal of slip cells, cells were treated with 5μM anlotinib and medium containing 1% FBS for 0h, 24h, 48h. Use an IX71 inverted microscope to take pictures at the corresponding times. Finally, ImageJ was used to measure the distance of the wound edge.

2.5 Analysis of Apoptosis

To investigate the apoptosis of ESCC cells after anlotinib treatment, we used Hoechst33342 staining to observe the morphology of apoptotic cells. KYSE30 cells were seeded at 4×10⁵ cells/well in 6-well plates and they were treated with 5μM anlotinib for 24 hours. Fluorescent detection of apoptosis was performed by staining with Hoechst33342 (Solarbio, Beijing, China) fluorescent dye. Cells were observed photographed under a fluorescence microscope IX71.

2.6 RNA Sequencing and Data Preprocessing

RNA-Seq were completed by Shanghai Zhongke New Life Co., Ltd. Briefly, to meet the requirements of RNA sequencing, NanoPhotometer^® Spectrophotometer (IMPLEN, CA, USA), Qubit^® RNA Assay (Life Technologies, CA, USA) and RNA Nano 6000 Assay (Agilent Technologies, CA, USA) were used to assess and measure RNA purity, content and integrity. RNA-seq libraries are constructed after samples are qualified and magnetic beads with oligonucleotides (dT) are used to enrich eukaryotic mRNA for cDNA synthesis. The synthesized cDNA was then ligated into the cBot cluster generation system. Finally, sequencing was performed on the Illumina Hiseq 2500 platform (Illumina, Inc.) to generate 150 bp matched end reads. The raw image data files obtained by high-throughput sequencing were converted into raw sequencing sequences by CASAVA base calling analysis. Reads containing poly-N, adapters, and low-quality reads were removed from the raw data to obtain high-quality clean reads. Principal component analysis (PCA) was subsequently performed to assess between-group differences and within-group sample replication.

2.7 Differentially Expression Analysis

Use Bioconductor's DESeq2 software (v1.34.0) to identify differentially expressed gene (DEGs) in the R language (http://www.bioconductor.org/). The significant difference standard was set as p-value < 0.05 and |log2 fold change| >1.

2.8 Gene Ontology and Pathway Enrichment Analysis DEGs

Gene Ontology (GO) annotation for DEGs and function and pathway enrichment for Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed by clusterProfiler R package [9]. GO (http://geneontology.org/) is an ontology widely used in the field of bioinformatics, which covers three aspects of biology, namely cellular components, molecular functions, and biological processes. KEGG (http://www.kegg.jp/) is a database resource used to understand the advanced functions and uses of biological systems, such as cells, organisms, and ecosystems. The clusterProfiler R package was used to enrich DEGs into various KEGG pathways. P-value < 0.05 was the cut-off point for selecting significantly enriched GO terms and KEGG pathways.

2.9 Western Blot Analysis

KYSE30 cells were treated with 5μM anlotinib or DMSO for 48 hours, and then the total protein of each group of cells was extracted with RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Take the supernatant containing total protein, centrifuge at 14,000×g, 4 degrees Celsius for 17 minutes, and detect the total protein concentration by Bradford protein (BCA) (Solarbio, Beijing, China) assay. Then use 10% or 12% SDS-PAGE to separate the different proteins and transfer them to the nitrocellulose membrane. After blocking with 5% skim milk in 0.05% Tween-20 TBS at room temperature for 50 minutes, incubate the membrane with the primary antibody overnight at 4°C, and then incubate the membrane with the secondary antibody (HRP) for a further 30 minutes. Finally, use the ECL kit (GE Healthcare) and E-Gel imager (Universal Hood II; Bio-Rad Laboratories, Shanghai, China) to visualize the target protein, and use the β-actin as a control.

2.10 Statistical Analysis

Data were analyzed using SPSS 26.0 (IBM, Armonk, NY, USA)software and GraphPad Prism 8.0 program (GraphPad Software, La Jolla, CA,USA). One-way analysis of variance (ANOVA) and Student's t-test were used to analyze differences between groups. The p-value < 0.05 (two-sided) was considered as statistically significant.

3.1 Anlotinib Inhibits Cell Proliferation in KYSE30

The effect of anlotinib on the viability of esophageal squamous cell carcinoma cells was determined by CCK-8 cell proliferation assay. CCK-8 experiments showed that KYSE30 cells were dose- and time-dependent toxicity to anlotinib. The IC50 at 24h, 48h and 72h were 124.85μM, 21.226μM and 13.03μM, respectively (Figure 1).

3.2 Anlotinib Modulates KYSE30 Migration and Invasion In Vitro

To verify whether anlotinib affects tumor migration and invasion, esophageal squamous carcinoma cells were treated with or without anlotinib. The closing speed of the wound edge of the cells with anlotinib treatment was significantly reduced (Figure 2A, B), indicating that anlotinib could significantly inhibit the migration of KYSE30 cells. The transwell experiment showed similar results. After anlotinib treatment, the number of cells that penetrated the ventricular membrane was significantly reduced, which means that the invasion and migration ability of KYSE30 cells was significantly inhibited (Figure 2C, D).

3.3 Anlotinib Induces the Apoptosis of KYSE30

KYSE30 cells were stained with Hoechst33342 after anlotinib treatment for 48 hours. Under the microscope, strong blue fluorescence and increased nuclear pyknosis and mitosis could be observed, suggesting that anlotinib could promote apoptosis of KYSE30 cells (Figure 3A, B).

3.4 Identification and Functional Annotation of DEGs

In order to identify the differentially expressed genes related to anlotinib, the transcriptome profile of KYSE-30 cells treated with or without anlotinib was analyzed after RNA sequencing. PCA showed that the gene expression profile of esophageal squamous cells treated with anlotinib was significantly different from that without anlotinib (Figure 4A). Compared with the control, 359 genes were up-regulated and 156 genes were down-regulated in anlotinib-treated KYSE30 cells (Figure 4B). Cluster analysis showed that there were obvious differences between the two groups (Figure 4C).

To explore which biological functions and pathways DEGs are involved in, we performed GO and KEGG enrichment analysis. GO enrichment analysis for DEGs found that GO items mainly include biological process (BP) and molecular function (MF) (Figure 5A, 5B). BP mainly includes positive regulation of cytokine production, external encapsulating structure organization and positive regulation of fibroblast proliferation. MF mainly includes extracellular matrix structural components, transmembrane receptor protein kinase activity and transmembrane receptor protein tyrosine kinase activity.

To further verify the enriched pathways of DEGs, we performed KEGG pathway analysis. According to the number of enriched DEGs, the KEGG pathway mainly includes PhosphoInositide-3 Kinase/Protein kinase B (PI3K/AKT) signaling pathway, calcium signaling pathway, and thiamine metabolism (Figure 5C). The PI3K/AKT signaling pathway is the most enriched pathway, and a total of 13 DEGs are enriched in this pathway, containing PDGFD, SPP1, FN1, PPP2R2B, FGF2, CREB5, PDGFRB, CSF3R, MYB, COL6A3, FLT4, ITGB7 and COL9A2 (Table 1).

3.5 Anlotinib Inhibits protein expression in PI3K/AKT Signaling Pathway

Studies have shown that the PI3K/AKT signaling pathway plays an important role in the occurrence, development and invasion of malignant tumors such as esophageal cancer, colon cancer, and gastric cancer [10-12]. In order to determine whether anlotinib is involved in the activation of PI3K and AKT, western blot was used to analyze the protein expression level involved in the PI3K/AKT signaling pathway. The results showed that anlotinib inhibited not only the phosphorylation of AKT, but also the total protein expression of PI3K and AKT (Figure 5D, 5E). These findings indicate that anlotinib may have a positive therapeutic effect on esophageal squamous cell carcinoma by antagonizing PI3K/AKT signaling.

Table 1 KEGG enrichment analysis of differentially expressed genes
ID	Description	GeneRatio	P-value	GeneID
hsa00730	Thiamine metabolism	4/108	3.62287E-05	ALPG/ALPP/AK5/AK8
hsa04020	Calcium signaling pathway	11/108	0.000331861	PDGFD/FGF2/P2RX2/CACNA1A/SPHK1/PDGFRB/GRIN2A/RET/NTRK3/ADORA2A/FLT4
hsa04151	PI3K/AKT signaling pathway	13/108	0.000802648	PDGFD/SPP1/FN1/PPP2R2B/FGF2/CREB5/PDGFRB/CSF3R/MYB/COL6A3/FLT4/ITGB7/COL9A2

Despite improvements in treatment in recent years, esophageal cancer remains a lethal disease with an increasing incidence worldwide [13, 14]. It is well known that drug resistance, recurrence and metastasis of tumor cells are the main reasons for chemotherapy failure, which makes chemotherapy drugs lose their efficacy, increases the difficulty of tumor treatment, and reduces the survival rate of tumor patients [15]. With the emergence of molecular targeted drugs such as trastuzumab [16], apatinib [17, 18], and anlotinib [4], the treatment of ESCC patients has brought about dawn [19]. We reviewed the literature and found that there is little basic research on the use of these drugs, especially anlotinib, in esophageal squamous cell carcinoma, so we carried out related research.

Through biological function experiments, we found that anlotinib has obvious inhibitory effects on the proliferation, migration and invasion of esophageal squamous cell carcinoma. Previous studies have also shown that anlotinib inhibits the viability of human lung cancer cells by blocking the JAK2/STAT3/VEGFA signaling pathway, inducing their apoptosis, and enhancing their anti-angiogenic effect [20]. It also exerts antitumor effects by inhibiting osteosarcoma cell growth, metastasis and angiogenesis by dual blocking VEGFR2 and MET signaling [21]. These are all similar to our experimental results.

In this study, we focused more on exploring the DEGs and signaling pathways that anlotinib affects the occurrence and development of esophageal squamous cell carcinoma, so we performed subsequent transcriptome sequencing and bioinformatics analysis. GO and KEGG analysis showed that the PI3K/AKT signaling pathway was the most enriched pathway during anlotinib treatment of KYSE30, and 13 DEGs such as PDGFRB were significantly enriched in the PI3K/AKT pathway. PDGFRB is an important target of anlotinib and an important receptor in cell signaling pathways [6, 22, 23]. The PI3K/AKT signaling pathway is involved in various oncogenic processes, including cell proliferation, survival, epithelial mesenchymal transition, enhanced motility and angiogenesis [24-27]. Pharmacological inhibition of the PI3K/AKT pathway can counteract the increased invasiveness and motility of ESCC cells caused by Id1 overexpression [28]. Most importantly, previous studies have shown that PDGFRB can activate the PI3K/AKT signaling pathway through the phosphorylation of PI3KR1, and induce cell proliferation and migration [29, 30]. Therefore, we speculate that the PDGFRB/PI3K/AKT pathway is closely related to the occurrence and development of esophageal cancer. Anlotinib may block the downstream signal transduction by affecting the expression of PDGFRB/PI3K/AKT, thereby exerting anti-tumor effects. This is confirmed by our experiments. The results of Western blot showed that anlotinib significantly inhibited the phosphorylation of AKT, and the total protein expression of PI3K and AKT. This implies that anlotinib may have a corresponding therapeutic effect by antagonizing the PI3K/AKT pathway. However, it remains to be further verified how PDGFRB acts on the PI3K/AKT pathway and thus affects the development of esophageal cancer. We will conduct relevant experiments in the future to fully elucidate the molecular mechanism of anlotinib in esophageal squamous cell carcinoma.

Taken together, the results of this study indicate that anlotinib inhibits the proliferation, migration and invasion of esophageal squamous cell carcinoma cells by antagonizing the PI3K/AKT signaling pathway. Our study provides new evidence that anlotinib may be a promising treatment for ESCC.

Specific author contributions

SJ, XZ, and ZZ designed the experiments. SJ, HW, AL, WH, QS, and JL performed the experiments. SJ and YX analyzed and interpreted the data. SJ drafted the manuscript. XZ and ZZ revised the manuscript. SJ prepared the figures. Tib provided financial support and supervised the project. All the authors reviewed and approved the final version of the manuscript.

Financial support

Key Project of Natural Science Foundation of Hebei province of China (H2017209233) and the Graduate Student Innovation Fund of North China University of Science and Technology (2019S12).

Potential competing interests

The authors declare that they have no conflict of interests.

Ethical Approval

This study does not involve ethical relevance and therefore is not applicable.

Availability of data and materials

All study data will subsequently be available in a public database.

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No competing interests reported.

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Anlotinib inhibits the development of esophageal squamous cell carcinoma by regulating the PI3K/AKT signaling pathway

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2. Material And Methods

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