Anti-cancer Drug Anlotinib Promotes Autophagy and Apoptosis in Breast Cancer

Background : Anlotinib, a multi-target tyrosine kinase inhibitor, has significant anti-cancer effects on breast cancer (BC), lung cancer, colon cancer and ovarian cancer, but its mechanism has not been investigated in BC. Methods : The cell viability and growth of human non-triple negative BC cell line MCF-7 and triple negative BC cell line MDA-MB-231 with the treatment of anlotinib were tested by Cell Counting Kit-8 (CCK-8) assay and Ki67 staining. The alteration of genes related to apoptosis and autophagy were investigated by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), western blots and immunocytochemistry (ICC). Cell apoptosis was valued by TUNEL staining and flow cytometry. Further, mouse breast tumour cell lines AT-3 cells were subcutaneously injected into C57BL/6 mice, and the effect of anlotinib intragastrically on tumour growth in vivo was examined. Results : We found that anlotinib suppressed the cell viability and depressed Ki67 staining in MCF-7 and MDA-MB-231 cell lines. Besides, the drug also enhanced cell autophagy and apoptosis of MCF-7 and MDA-MB-231 cell lines, which could be rescued by autophagy inhibitors wortmannin (wort) and 3-methyladenine (3-MA), and BECN1 knockdown. Furthermore, Akt/GSK-3 α pathway was inactivated by anlotinib treatment, while rescued by wort, 3-MA and silencing of BECN1 in the MCF-7 or MDA-MB-231 cells. We also found that anlotinib inhibited implanted tumour growth of BC in syngeneic mice. Conclusions : Our study demonstrated that anlotinib inhibited breast cancer cell growth in vitro and in vivo . Anlotinib promoted cell apoptosis and inactivated Akt/GSK-3 α pathway of BC cells by inducing cell autophagy. It indicated that anlotinib may be an effective new drug for BC treatment.


Introduction
Breast cancer (BC) is one of the most common and high-risk female malignant tumors worldwide. Its incidence rises each year, and the age of onset tends to be younger. At present, BC had surpassed lung cancer and became the most common cancer in the world, accounting for 11.7% of all new cancer cases in 2020 [1]. BC was also the most frequent cancer in female in 2020 in China and was estimated to be the fourth most common cancer diagnosed, replacing liver cancer [2]. Although the treatment of BC has improved in recent years, postoperative tumour cell recurrence, metastasis and drug resistance have resulted in short tumour-free survival and low 5-year survival rates in highrisk populations, which seriously threaten the lives and health of patients [2][3][4]. Targeted therapy, as a new therapeutic strategy, has the advantages of strong specificity, remarkable curative effects and few side effects. Hence, targeted therapy has been recognized as an effective and selective method to kill tumour cells, and it is gradually becoming a hot spot and trend in the field of cancer therapy.
Anlotinib hydrochloride, a novel oral multi-target tyrosine kinase receptor inhibitor that targets vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR), shows broad-spectrum inhibitory effects on tumour angiogenesis and growth [5,6]. In vitro studies have shown that anlotinib selectively inhibits VEGFR2/kinase insert domain receptor (KDR) and VEGFR3, with an inhibition rate 20 times that of sunitinib and 500 times that of sorafenib, respectively. In addition, the dysregulated FGF/FGFR axis promotes cancer progression and enhances the angiogenic potential of the tumour microenvironment, leading to an invasive phenotype of cancer cells [7][8][9], and FGF/FGFR signalling changes are associated with chemoresistance and adverse clinical prognosis of cancers. Part of reports shows that anlotinib inactivates FGFRs obviously, especially FGFR2, compared with the effects of sorafenib in preclinical trials. Recently, it is found that anlotinib also inhibits c-Kit receptor, glial cell-derived neurotrophic factor receptor tyrosine kinase, Aurora-B kinase, c-Fms kinase and discoid domain receptor 1, which are involved in the cell proliferation of colon cancer, lymphoma, and acute T cell leukaemia or in the progression of lung, breast, and ovarian cancers [10][11][12][13]. In vivo experiments also demonstrates that anlotinib has broad inhibitory effects against xenograft tumors from transplanted human colon, ovarian, kidney and lung cancer cells and glioma cells [14].
It has been shown that anlotinib has anti-cancer effects on many malignant tumors remarkably, such as non-small cell lung cancer, renal cell carcinoma and hepatocellular carcinoma in clinical trials. Recently, the clinical trials of triple-negative BC and metastatic HER2-negative BC have been carried out, however, there is still a lack of in-depth research on the role of anlotinib in BC and its underlying mechanism. Especially, BC can be divided into three main sub-types at least, including triple negative BC, HER2 Enriched BC and Luminal (A and B) BC. Hence, in this study, we used triple negative BC cell line MDA-MB-231 and non-triple negative, luminal, BC cell line MCF-7 to explore the effects of anlotinib on the growth of different types of BC and its underlying mechanism, which provided a new choice for targeted therapy of BC.

Compounds and Antibodys
Anlotinib was kindly given as a gift by Chia Tai Tianqing Co., Ltd. (Nanjing, JS, China). Wortmannin (Wort) and 3-methyladenine (3-MA) were purchased from APExBIO (Houston, TX, USA). The antibodies for western blotting, immunocytochemistry and immunohistochemistry were listed in the Supplementary Table 1.

Cell Culture and Transfection
The human BC cell lines MCF-7 and MDA-MB-231 were purchased from the cell bank of the Chinese Academy of Science. MCF-7 was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) and antibiotics (penicillin 100 U/mL and streptomycin 100 mg/mL) in a 37 • C humidified atmosphere with 5% CO 2 . MDA-MB-231 was cultured in Leibovitz's L-15 medium with 10% FBS and antibiotics at 37 • C in a free gas exchange environment with atmospheric air. The mouse breast carcinoma cell line AT3 were obtained from Roswell Park Cancer Institute (Buffalo, NY, USA) [15], and were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS and antibiotics in a 37 • C humidified atmosphere with 5% CO 2 .
For silencing of BECN1, siRNAs were transiently transfected into cells using jetPRIME® transfection reagent according to the manufacturer's instructions (Polyplus transfection®SA, France).

Cell Viability Assay
A Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Briefly, 10000 cells for MCF-7 cell and 5000 cells for MDA-MB-231 cell per well were seeded in 96-well plates and incubated with anlotinib at various concentrations and for different time points. The CCK-8 reagent was then added at a 1:10 dilution and incubated for 1.5 h, and the absorbance at 450 nm was measured on a microplate reader to calculate the cell viability and IC50.

Quantitative Real-time PCR (qRT-PCR)
RNA was extracted from the cells or tissues using the TRIzol-trichloromethane-isopropanol method and reverse transcribed into cDNA using ReverTra Ace qPCR RT Kit (TOYOBO CO., LTD., Osaka, Japan). The mixtures contained samples of 10 ng (1 µL), SYBR Green of 5 µL, and primers of 2 µL. The sequences of the primers are listed in Supplementary Table 2.

Western Blot Analysis
Protein samples from cancer cells and tissues were resolved by SDS-PAGE (10% or 12%), electrotransferred onto Immobilon-P membranes, blocked, and incubated with primary and secondary antibodies. Densitometric quantification of the protein bands was analysed using Im-ageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA).

Immunocytochemistry (ICC) and TUNEL Staining Assays
Cells were fixed in 4% paraformaldehyde (PFA) for 15 min and then permeabilized with 0.1% Triton X-100 for 20 min. After blocking with 5% goat serum for 2 h, the cells were incubated with primary antibodies against Ki67, BECN1 and LC3B overnight at 4 • C. Then, the cells were washed and incubated with secondary antibodies and finally incubated with DAPI for nuclei staining. Images were taken with an Olympus microscope (OLYMPUS, Tokyo, Japan). For the TUNEL staining assay, cells were fixed with 4% PFA first and then stained using TUNEL BrightRed Apoptosis Detection Kit (Vazyme, Nanjing, JS, China).

Immunohistochemistry
Dissected tumour tissues were preserved in 4% PFA at 4 • C for 24 h, dehydrated with xylenes and alcohols, and embedded in paraffin. Sections were cut at a thickness of 5 µm, dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. Antigens were unmasked, blocked, and incubated with primary antibodies overnight at 4 • C. Then sections were incubated with secondary antibodies and DAPI. Images were taken with an Olympus microscope.

Syngeneic Mouse Model
Sixteen male wild-type C57BL/6 mice were purchased from the Laboratory Animal Resources of Chinese Academy of Sciences (Shanghai, China). Mice were maintained in specific pathogen free (SPF) conditions at the Fudan University Animal Experimental Centre, compliant with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiment was approved by the Institutional Animal Care and Utilization Committee of Fudan University (Approval number: 20190703). AT-3 cells were harvested and resuspended at 1 × 10 7 cell/mL, and the 0.1 mL of suspension was subcutaneously injected into the 16 male mice. On day 7 after injection, 10 mice randomly were administered 2.5 mg/kg anlotinib intragastrically once daily for 3 weeks (anlotinib group, n = 10), while other 6 mice were treated with the same volume of 0.25% DMSO (DMSO group, n = 6). The mice were sacrificed after 4 weeks of cell injection, and the tumors were removed and measured.

Statistical Analysis
Statistical analysis was performed by Student's t-test for comparisons between the DMSO and anlotinib groups and two-factor Analysis of Variance (ANOVA) for comparisons among the four groups, followed by a subsequent post-hoc test. Growth curves descripted in the CCK-8 assay were analysed with two-factor ANOVA (Treatment × time). The experiments were repeated at least 3 times independently. The data were presented as mean ± SD and analysed using GraphPad Prism 8.0.1 (GraphPad, San Diego, CA, USA), and p < 0.05 was considered statistically significant.

Anlotinib Suppressed the Cell Viability and Proliferation of MCF-7 and MDA-MB-231 Cells
The CCK-8 assay was used to assess cell viability and the half maximal inhibitory concentration (IC50) in MCF-7 and MDA-MB-231 cells. The results showed that anlotinib inhibited cell viability in a dose-and time-dependent manner.  (Fig. 1B). The IC50 value of anlotinib in MCF-7 is smaller than that in MDA-MB-231 in a timedependent manner, indicating that MCF-7 is more sensitive to anlotinib. We also performed Ki67 staining, and the results showed that number of positive staining of MCF-7 and MDA-MB-231 cells was decreased with anlotinib administration ( Fig. 1C-D, n = 6, * * p < 0.01 vs DMSO). Therefore, anlotinib suppressed the proliferation of MCF-7 and MDA-MB-231 cells.

Anlotinib Suppressed BC Growth in vivo
To examine the therapeutic significance in vivo, mice were subcutaneously injected with AT-3 cells to generate syngeneic tumors followed by continuous 3-week intragastric treatment with anlotinib. The anlotinib group mice showed significantly decreased body weight, tumour weight and tumour volume compared to those of the DMSO group. The body weight of mice fed with anlotinib was not significantly different from that of mice without anlotinib in the first two weeks. However, the weight of mice treated with anlotinib was increased significantly during the last two weeks. The result comprehensively demonstrated that anlotinib inhibited tumour growth (Fig. 6A-D, * p < 0.05 vs DMSO, DMSO group n = 6, anlotinib group n = 10). Consistent with the cell-based assay, the mRNA levels of LC3B, ATG4B and ATG5 in tumour from the anlotinib group were upregulated (Fig. 6E, * * p < 0.01 vs DMSO), and the ratio of LC3BII to LC3BI protein also increased, suggesting that anlotinib induced autophagy of BC in vivo. The proapoptotic protein cleaved caspase-3 was expressed at higher levels in the tumour from the anlotinib group than in the DMSO group, and the anti-apoptotic protein Bcl-2 showed the opposite result. These results indicated that induction of apoptosis occurred in anlotinib-treated syngeneics ( Fig. 6F-G, * p < 0.05, * * p < 0.01 vs DMSO, DMSO = 6, anlotinib = 8). Anlotinib group mice had fewer Ki67positive cells compared to those of DMSO group mice, indicating that the number of proliferating cells was reduced (Fig. 6H, * p < 0.05, * * p < 0.01, DMSO = 6, anlotinib = 8). These data demonstrated that anlotinib inhibited BC growth in vivo.

Discussion
Anlotinib, an inhibitor of multiple tyrosine kinase receptors, inhibits tumour progression by inhibiting angiogenesis [17], but there is no published literature on the inhibitory mechanism of anlotinib on BC.
Many studies have reported that anlotinib exerts antitumour effects to inhibit cell viability and proliferation in hepatocellular carcinoma (HCC), lung cancer, thyroid cancer, and osteosarcoma [16,[18][19][20]. Our study found that anlotinib also inhibited the cell viability and proliferation of BC cells, which was consistent with the effects in other tumors.
Autophagy is an important cellular mechanism that plays a "housekeeping" role in normal physiological processes, including the removal of longevity, aggregation and misfolded proteins, removal of damaged organelles, and the regulation of growth and ageing. In tumour cells, autophagy is usually activated during anticancer treatments such as radiation therapy, chemotherapy, and targeted therapy. This may be a cytoprotective mechanism that also causes excessive autophagy in the cell, namely, excessive self-digestion, and induces phagocytic cell death, which is also known as type II programmed cell death [21]. A study found that anlotinib induced autophagy in human lung cancer cells in a time-and concentration-dependent manner and increased the ratio of LC3BII/I protein and the protein expression level of BECN1. The autophagy inhibitors 3-MA and small interfering RNA of BECN1 reversed the autophagy effect induced by anlotinib, unexpectedly, they both enhanced the inhibitory effect of anlotinib on cell proliferation, making the anticancer effect of anlotinib more sensitive and strengthening its inhibition of angiogenesis [19]. These findings suggested that the induction of autophagy in human lung cancer cells by anlotinib is a cytoprotective effect. In our study, anlotinib also induced autophagy in MCF-7 and MDA-MB-231 human BC cells. These results showed significantly increased mRNA and protein expression levels of LC3B and BECN1 and the ratio of protein LC3BII/I and BECN1 levels and decreased P62 levels, suggesting that anlotinib promotes autophagic cell death in BC cells.
Apoptosis is a common programmed cell death and plays a key role in the development of diseases, including cancer. Cancer cells evade apoptosis, thereby achieving excessive proliferation and surviving under hypoxic conditions and with drug resistance [22]; thus, promoting tumour cell apoptosis has become an important strategy for the treatment of cancer. Studies have shown that anlotinib exerts its antitumour effects on HCC, thyroid cancer, osteosarcoma, and lung cancer by promoting apoptosis [16,[18][19][20]23]. Anlotinib significantly inhibited colony formation and promoted apoptosis in HCC and thyroid cancer in vitro [16,18]. It upregulated the pro-apoptotic molecule Bax and inhibited the anti-apoptotic proteins Bcl-2 and Survivin to kill tumour cells. In addition, animal experiments demonstrated that anlotinib reduced the volumes and weights of transplanted tumors [18]. In thyroid cancer, anlotinib caused abnormal spindle assembly and G2/M arrest, promoted the activation of cleaved-Caspase 3 and cleaved PARP, and activated TP53 [16]. Like the above experimental results, we found that anlotinib increased the mRNA and protein levels of proapoptotic proteins and inhibited the mRNA and protein levels of the anti-apoptotic protein Bcl-2 in MCF-7 and MDA-MB-231 BC cells, thereby exerting an antitumour effect.
Autophagy and apoptosis often occur in the same cells with the same upstream cellular signals activated by the endoplasmic reticulum, such as extracellular regulated protein kinases (ERK)/activating transcription factor 4 (ATF4), Inositol-requiring enzyme-1α, ATF6, and Ca 2+ . Autophagy blocks the induction of apoptosis by inhibiting the activation of apoptosis-associated caspases or by reducing cell damage. Meanwhile, activation of apoptosisrelated proteins also suppresses autophagy by degrading autophagy-related proteins such as BECN1, autophagyrelated protein 4D (ATG4D), ATG3 and ATG5, but the specific mechanisms between the interaction in BC cells need further study [24]. However, in our study, we utilized the autophagy inhibitors wort and 3-MA before anlotinib treatment and found that inhibition of autophagy reversed anlotinib-induced apoptosis in BC cells. The similarity results also have been found in the MDA-MB-231 cells transfected with BECN1 siRNA, indicating that loss of BECN1 recued anlotinib-induced apoptosis. We obtained the opposite results compared with that reported in lung cancer cells [19]. This is probably due to the different concentrations of autophagy inhibitors and different cells, but it remains unclear and needs further study to clarify the crosstalk between apoptosis and autophagy.
Our study indicates that anlotinib has both effects on triple negative breast cancer cells and non-triple negative breast cancer cells. The IC50 value of anlotinib in MCF-7  is smaller than that in MDA-MB-231 in a time-dependent manner, demonstrating that anlotinib has a stronger effect to kill neoplasm cells in non-triple negative breast cancer. The apoptosis induction analysis revealed that a larger proportion of early and late apoptosis was counted in the MCF-7 cells compared with MDA-MB-231 cells. These results may reveal that anlotinib is more effective in the treatment of non-triple negative breast cancer.
Evidence indicates that Akt is a key molecule in both autophagy and apoptosis because it is the upstream signal of mammalian target of rapamycin complex and JNK [25][26][27]. Previous studies reported that anlotinib inhibits Erk and Akt signal transduction pathways to regulate cell growth in HCC cells [18]. Akt/GSK-3 signals can regulate cell autophagy, apoptosis, and cell cycle. Furthermore, level of Akt (S473) and GSK-3α/β(S21/S9) phosphorylation was positive correlated with the level of apoptosis and autophagy in a lung adenocarcinoma cell line [28]. Hence, we investigated Akt signalling and found that inactivated Akt/GSK-3α signalling in anlotinib-induced cells was reversed by autophagy inhibitors and BECN1 deficiencies, suggesting that anlotinib-induced autophagy regulated Akt/GSK-3α signalling (Fig. 7). We conjecture that autophagy and Akt might regulate each other, forming a closed circuit.

Conclusions
In summary, our study demonstrated that anlotinib inhibited the growth of BC in vitro and in vivo, especially in non-triple negative, luminal, BC cell line MCF-7 cells. Anlotinib promoted cell apoptosis and inactivated Akt/GSK-3α pathway of BC cells, by autophagy induction. Therefore, anlotinib may be an effective new drug for BC treatment.

Author Contributions
PZ (Ping Zhou) and HY conceived the idea and designed the research study. SC designed and performed most experiments, analyzed the data, and wrote the manuscript. YG repeated some experiments. PZ (Ping Zhu) and YJ provided help and advice on animal experiment. XW and LZ analyzed the data. XZ provided advice on the study and manuscript.

Ethics Approval and Consent to Participate
All animal care and experimental procedures complied with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiment was approved by the Institutional Animal Care and Utilization Committee of Fudan University (Approval number: 20190703).