Zinc Complex of 3,5-di-tert-butyl Salicylate Inhibits the Proliferation, Migration, and Invasion of Triple-Negative Breast Cancer Cells

: Zinc complex of 3,5-di-tert-butyl salicylate (Zn{[CH 3 ) 3 C] 2 Sal} 22- ) is a zinc ion chelate of salicylate. In this study, we found that it inhibits the proliferation, invasion, migration and induce apoptosis of triple-negative breast cancer 4T1 cells. The results of RNA-seq showed that expression of 17 genes was up-regulated and 26 genes was down-regulated significantly by Zn{[CH 3 ) 3 C] 2 Sal} 22- treatment. Further GO analysis and KEGG analysis showed that the activity of Zn{[CH 3 ) 3 C] 2 Sal} 22- against triple-negative breast cancer cells may be involved JAK-STAT3, HIF-1, and TNF signaling pathways. The expression of key genes was verified by RT-PCR. The phosphorylation of STAT3 and its upstream SRC decreased drastically upon Zn{[CH 3 ) 3 C] 2 Sal} 22-treatment as analyzed by western blot. Our results indicate that Zn{[CH 3 ) 3 C] 2 Sal} 22- inhibits the activity of TNBC cells by down-regulating STAT3 signaling pathway.


Introduction
Breast cancer is the most common cancer among women and accounts for the second largest number of cancer deaths in women worldwide. About 10 to 20 percent of new cases of breast cancer are triple-negative breast cancer (TNBC) 1,2 . TNBC lacks targeted therapies because it does not express estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) 3 . Compared with other types of breast cancer, TNBC patients have a poor prognosis due to higher risk of metastasis and recurrence 4 . In addition to surgical treatment, chemotherapy is the only FDA approved method for the treatment of TNBC 5 . TNBC is sensitive to chemotherapy in the initial treatment 6 , subsequently it become chemotherapy resistant. Therefore, it remains crucial to find the new chemotherapeutic agents in the treatment of TNBC. Salicylic acids and derivatives are mostly used in antipyretic, analgesic and anti-inflammatory in clinical practice and belong to non-steroidal anti-inflammatory drugs. It also has a certain therapeutic effect on tumors such as breast cancer 7 , pancreatic cancer 8 , lung cancer 9 , ovarian cancer and prostate cancer 10 . It has been found that small doses of aspirin can reduce the risk of multiple cancers, and in vitro aspirin inhibits the growth of various cancer cells including breast cancer through COX-dependent and COX-independent pathway 11 . This inhibition may associate with the salicylic acid group. During the process of breast cancer metastasis, aspirin can resist platelet activation and thus inhibit platelet-induced epithelial transformation, migration and invasion ability of breast cancer cells 12 . Asprin can also inhibit the activation of the transcription factor-1 (AP-1) and the nuclear factor κB (NF-κB), thus affecting cell proliferation, differentiation and apoptosis, transformation, invasion and metastasis of tumor cells 13 . However, there are few studies on the role of heavy metal chelate of salicylate against cancer. Sorenson et al. found that copper salicylate and its derivatives can inhibit the growth and lung metastasis of ascites cancer in animal experiments, induce the differentiation of tumor cells, and thus prolong the survival period of the body 14 . In addition, combined use of copper salicylate can significantly improve the anti-cancer efficacy of cisplatin and reduce its cytotoxicity. O'Connor et al. reported that copper complex of salicylate inhibits the proliferation of MCF-7, DU145, HT29 and SKOV3 cancer cells 15 . Our previous study showed that copper complex of phenanthroline salicylate can induce apoptosis in TNBC cells by down-regulating the expression of anti-apoptosis proteins such as Bcl-2 、 Bcl-xL and Survivin in vitro and in vivo 16 . Until now, the function of Zn{[CH3)3C]2Sal}2 2-is under investigation. The activation of signal transduction and transcription (STAT) protein family is closely related to the occurrence and development of many tumors. Among the various STAT members, STAT3 is often over-expressed in tumor cells and tissue samples, and regulates the expression of many oncogenes that control growth and metastasis 17 . As a gathering point of several tumorigenic signaling pathways, STAT3 is considered to have an important role in tumor cells and in the tumor microenvironment. The activation of STAT3 pathways is related to the aggressiveness of breast cancer, and is involved in cell proliferation, apoptosis, metastasis and chemotherapy resistance. Both preclinical and clinical studies have shown that STAT3 plays a crucial role in the progression and metastasis of TNBC 18

Enrichment analysis of differentially expressed genes.
The Significant Differentially expressed genes (SDEGs) between the control and Zn{[CH3)3C]2Sal}2 2-treated 4T1 cells were subjected to functional GO and KEGG pathway analyses. For GO classification, three different categories, including Molecular Function (MF), Cellular Component (CC), and Biological Process (BP), were used to enrich the SDEGs. As shown in Fig.3C, in the MF category, the SDEGs were primarily enriched in Zinc ion binding, Calcium ion binding, metalloendopeptidase activity, fibronectin binding and MRF binding. In the CC category, they were predominantly enriched in the anchored component of extracellular space and proteinaceous extracellular matrix. In the BP category, they were mainly enriched in terms of response to hypoxia and estradiol, cellular response to interleukin-1, collagen catabolic process and angiogenesis. The top 10 of the 23 enriched KEGG pathways are presented in Fig.3D . Pathways in cancer ranked the highest. Numerous well-established signaling pathways were also enriched, including JAK-STAT signaling pathway, HIF-1 signaling pathway and TNF signaling pathway.

Verification of gene expression change by RT-PCR.
To confirm the RNA-seq results, expression level of some interesting gene was verified by RT-PCR. As we expected, results of RT-PCR were consistent with RNA-seq analysis. As shown in Fig.3E, the mRNA expression level of MT1 and MT2, which was related Metal ion transportation, were up-regulated nearly 10 times. The expression of Angpt16 and Angp14, which was closely related to angiogenesis, was also significant up-regulated. Meanwhile, Mmp3, Mmp9, Mmp16 and Mmp10, which belong to MMP family and are closely related to tumor metastasis, were down-regulated. Besides that, JAK2, which was an important component of STAT signaling pathway, was down-regulated significantly.

Effect of Zn{[CH3)3C]2Sal}2 2-on lung metastasis in vivo.
Lung metastasis was measured at day 16. As shown in Fig.5, the pulmonary nodules of metastasis in zinc salicylate treatment group and MK2206 treatment group were similar to the control group. In addition, no inhibitory effect on lung metastasis was observed from group treated with Zn{[CH3)3C]2Sal}2 2-and MK2206. The weight of Zn{[CH3)3C]2Sal}2 2-treated mice decreased slightly compared with the control group, however, Akt inhibitor MK2206 alone or combination treatment caused weight loss significantly.

Discussion
The prognosis of TNBC is poor because of high rate of relapse and metastasis. Drugs that target metastasis are of great significance. In this study, we demonstrated that low dose Zn{[CH3)3C]2Sal}2 2-inhibits migration and invasion of mouse TNBC cells 4T1, while high dose Zn{[CH3)3C]2Sal}2 2-inhibits cell proliferation and induces apoptosis. In addition, we explored anti-TNBC mechanism of Zn{[CH3)3C]2Sal}2 2 -using RNA-seq and bioinformatic analyses. Furthermore, we demonstrated that Zn{[CH3)3C]2Sal}2 2-can inhibit the activation of the STAT3 signaling pathway by western blot analysis. By utilizing GO and KEGG pathway analyses, We found expression changes of genes associated with Zinc ion binding, metalloendopeptidase activity, fibronectin binding, proteinaceous extracellular matrix, collagen catabolic process and angiogenesis in 4T1 cells. The RT-PCR results verified that the transcription levels of genes related to metal ion transport, angiogenesis, and tumor metastasis have been significantly altered. It indicates that the anti-tumor effect of Zn{[CH3)3C]2Sal}2 2-may be related to those genes. In the pathway analyses, many enriched pathways were associated with proliferation, migration, and Invasion, which were consistent with our functional analyses. For instance, JAK-STAT signaling pathway, is important in tumor metastasis. The establishment of a database of RNA expression after Zn{[CH3)3C]2Sal}2 2treatment in 4T1 cell lines could provide a broad foundation to guide focused studies of anti-TNBC research. The TNBC metastasis mechanism is regulated by multiple signaling pathways, such as: NF-κB signaling pathway 19 , Wnt/beta-Catenin signaling pathway 20 , PI3K/AKT signaling pathway, FAK/c-Src pathway 21 and JAK/STAT3 signaling pathway. STAT signaling pathway is the focal point of multiple carcinogenic signaling channels in the body, STAT3 activation is short and strictly controlled, but STAT3 protein appears in contitutively activating in multiple tumor cells 22 . Specifically, Studies have shown that STAT3 affects important processes of tumorigenesis and development of TNBC such as occurrence and metastasis and is highly activated in TNBC 23 . By screening the transcriptional differences between TNBC cells and normal breast cells, Zhan L. et al. and other researchers observed the abnormal expression of multiple signaling proteins of the IL6/JAK2/STAT3 pathway in TNBC, and constitutive activation of JAK2/STAT3 may be an important factor in the distal metastasis of TNBC 24 . Tyrosine kinase SRC also plays an important role in cell proliferation 25 , cell cycle, adhesion 26 , migration, and invasive signaling pathways, SRC was overexpressed or highly active in various solid tumor cell lines and pathological tissues, and SRC inhibitors inhibited breast cancer cell invasion and metastasis 27 . It has been found that STAT3 are activated by both SRC and JAK2 in breast cancer 22 . Intriguingly, we found Zn{[CH3)3C]2Sal}2 2-can inhibit the activation of both SRC and STAT3 and consequently affect migration and invasion ability of 4T1 TNBC cell. This paves the way to develop this chemical as therapeutic drug for TNBC. We have not detected the anti-metastasis effect of Zn{[CH3)3C]2Sal}2 2-in animal work and we condider that the drug was not delivered poperly. Further study with nano-particle delivery is warranted.

Cell apoptosis detection.
Apoptosis of 4T1 cells was determined by Flow cytometry with Annexin V-FITC/PI staining. Cells were plated into six-well plates, after treatment with Zn{[CH3)3C]2Sal}2 2-for 24 h, cells were washed with PBS then harvested and further stained according to the manufacturer's instructions. Analysis was performed using flow cytometry (BD Biosciences, San Jose, CA, USA).

Wound Healing Assay.
Logarithmic growth phase cells were plated into six-well plates and allow the cells reach 90% confluence. A "scratch" was made by 10 µl pipette tip, and then added different concentration of Zn{[CH3)3C]2Sal}2 2-. Photograph of the "scratch" fusion was taken in Zn{[CH3)3C]2Sal}2 2-treated group and the control group each 12 h. Image J software was used for Image data analysis.

Cell Invasion Assay.
Transwell chambers with matrigel-coated membranes were placed in 24-well plates and incubated with 500 ul serum free medium at 37°C for 2 hr. Then, 4T1 cells (1×10 5 ) in 500 µl of serum-free medium were seeded into the top chamber and 750 µl of RPMI-1640 medium with 10% FBS were added into the down compartment of the transwell chambers as the chemical attractant. After incubation for 22 hr at 37°C, the cells were fixed and stained with crystal violet. The invaded cells in five randomly chosen fields of each well were counted.

RNA-sequencing.
The cDNA library construction, library purification and transcriptome sequencing were carried out according to the Suzhou GENEWIZ Company's instructions. Three samples were used in each group for RNA-sequencing assay.

GO and KEGG Pathway Enrichment Analysis.
Differential expression analysis used the DESeq2 Bioconductor package, a model based on the negative binomial distribution. the estimates of dispersion and logarithmic fold changes incorporate data-driven prior distributions, Padj of genes were setted <0.05 to detect differentially expressed ones. GOSeq (v1.34.1) was used identifying Gene Ontology (GO) terms that annotate a list of enriched genes with a significant padj less than 0.05. And top GO was used to plot DAG. KEGG (Kyoto Encyclopedia of Genes and Genomes) is a collection of databases dealing with genomes, biological pathways, diseases, drugs, and chemical substances (http://en.wikipedia.org/wiki/KEGG). We used scripts in house to enrich significant differential expression gene in KEGG pathways.

RNA extraction and Quantitative Real-time RT-PCR.
Total RNA was extracted from 4T1 cells using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) to analyze differentially expressed gene. cDNA was prepared from 1 μg of total RNA using SuperScript® III First-Strand Synthesis System kit (Invitrogen) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in CFX Connect TM Real-time System (Bio-rad,Singapore) using iTaq Universal SYBR Green Supermix (Hercules, CA, USA). Primers used in this study are listed in Supplemental Table S1. The 2 (−ΔΔCt) method was used to analyze relative expression changes of mRNA and β-actin was used as internal reference.

Western blotting assay.
After treated with different concentrations of Zn{[CH3)3C]2Sal}2 2-or MK2206(AKT inhibitor)for a specific period of time, the cell protein was extracted according to the experimental instructions, separated by SDS-PAGE, transferred to the nitrocellulose filter membrane (Pall, Pensacola, USA).The membranes were incubated with the specific primary antibody, and then incubated with the with IRDye secondary antibodies (Licor, NB, USA). The membranes were scanned and the images were captured with Odyssey SA (Licor, NB, USA).

Animal model for lung metastasis.
Animal experiments were approved by the ethics committee of Jianghan University and in accordance with relevant guidelines and regulations including ARRIVE guidelines. The experimental mice were purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd. 4T1 cells (5x10 4 cells in 100 μl volume) were injected through tail vein of 6-week-old female BALB/c mice. Zn{[CH3)3C]2Sal}2 2-(15mg/kg) was administered by intraperitoneal injection three times a week after transplantation. For AKT inhibitor, mice were gavaged with 0.1mL MK2206 (120mg/kg) three times a week. Combination treatment was done accordingly. Animals were humanely sacrificed on day 16 after 4T1 transplantation, and the number of tumor nodules in lungs was recorded for analysis of metastasis.

Statistical Analysis.
All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA). Comparison between two groups was performed using Student's t-test. Statistical significance was defined as * p < 0.05 and ** p < 0.01.Data were expressed as the mean ± standard deviation, n ≥ 3,unless otherwise stated.