Characterization of Potential Target Genes of Borneol in Increasing Trastuzumab Sensitivity in HER2+ Trastuzumab-Resistant Breast Cancer: Bioinformatics and In Vitro Studies

Objective: The long-term use of trastuzumab (TRZ), a therapeutic agent for human epidermal growth factor receptor 2 (HER2)+ breast cancer subtype (HER2+ BC), induces resistance. Borneol (BOR) exerts anticancer effects on various types of cancer. However, its anticancer effect on HER2+ BC remains unknown. This study aimed to determine the potential target genes of BOR and its effect on overcoming the resistance of HER2+ BC to TRZ. Methods: The hub gene of BOR’s potential target on HER2+ BC cells was determined via a bioinformatics approach. Resistant HCC1954 cells (HCC1954-TR) were obtained through repeated inducement of HCC1954 cancer cells with TRZ. The cells were then subjected to cytotoxic tests involving single compounds and their combinations. Then, the hub gene expression was determined using quantitative reverse-transcription polymerase chain reaction. The interaction between BOR and selected proteins was measured through molecular docking. Results: Hub genes IL6, TNF, ESR1, IL1B, CYP19A1, AR, NR3C1, RELA, CYP17A1, and GPT were obtained via a bioinformatics approach. HCC1954-TR cells were successfully established. The TRZ–BOR combination treatment of parental HCC1954 (400 µg/mL and 25 µM) and HCC1954-TR (800 µg/mL and 100 µM) yielded considerably better results compared with BOR or TRZ alone. The expressions of AR, GPT, and ESR1 under the TRZ–BOR combination were notably different compared with those under single exposure. The molecular docking study of CYP19A1, CYP17A1, NR3C1, and IL-1β highlighted the potential interaction between BOR and such proteins. Conclusion: BOR improved the cytotoxic effects of TRZ on HCC1954 and HCC1954-TR cell lines, where it specifically targets AR, ESR1, and GPT genes. In addition, the BOR effect, which counteracted the resistance of HCC1954-TR cells to TRZ, was mediated by genes CYP19A1, CYP17A1, NR3C1, IL-1, and RELA. However, additional research is required to validate their role in BOR activity to circumvent the resistance of HER2+ BC to TRZ.


Introduction
Breast cancer (BC) refers to the most diagnosed cancer in women worldwide [1]; it has surpassed lung cancer as the most frequently diagnosed cancer [2].The human epidermal growth factor receptor 2 (HER2)+ BC subtype (HER2+ BC) shows a poor prognosis, such as high aggressiveness, recurrence, and low survival [3].Trastuzumab (TRZ), which is used in HER2+ BC therapy, binds to the extracellular domain of HER2 and suppresses the intracellular HER2 signaling pathway [4].
the problem of TRZ resistance.BOR is a bicyclic monoterpene compound [7].This substance can be developed to overcome the problem of TRZ resistance because of its anticancer activity.The combination treatment of BOR with cisplatin and paclitaxel induced apoptosis in glioma cells [8] and esophageal squamous carcinoma cells [9].Meanwhile, combination BOR with curcumin and selenocysteine can induce apoptosis in melanoma cells [10] and hepatocellular carcinoma cells [11].BOR increases the efficacy of doxorubicin in glioma cells [12].BOR increased the anticancer efficiency of temozolomide in glioma cells [13], the efficacy of cisplatin in glioma cells [8], and the efficacy of paclitaxel in esophageal squamous carcinoma cells [9].BOR can also reverse the resistance to mitoxantrone [14] and increase the sensitivity of cisplatin with a resistance experience [15].A recent study revealed the importance of the regulation of receptor tyrosine kinase-RAS-PI3K/ AKT by BOR in overcoming BC resistance to tamoxifen through bioinformatics analysis [16].We speculate that BOR can overcome the resistance of human BC cells to TRZ.To date, no studies have been conducted on TRZ therapy with the addition of BOR in BC.This study aimed to investigate the effect of BOR on the improving the responsiveness of TRZ in HER2+ BC cells.Moreover, it aimed to identify the primary target of BOR in HER2+ BC to overcome TRZ resistance in HER2+ BC.

Functional, disease-gene, and drug-gene association annotations
WebGestalt was used in the analysis of functional, disease-gene, and drug-gene association annotations (http://www.webgestalt.org/)[26].For functional annotation, symbols of intersecting genes of BOR and HER2+ BC-related gene clusters were submitted to WebGestalt and analyzed using the "over-representation analysis (ORA)" default settings.Disease-gene and druggene associations were analyzed using WebGestalt using several selected parameters, such as the following: set "method of interest" to "over-representation," "functional database" to "disease," and "Select a function database name" with "DisGeNet" for the analysis of disease-gene associations.The "functional database" was changed to "drug" and "Select a function database name" to "DrugBank" for drug-gene association analysis.A p-value of less than 0.05 was selected as the selection criterion [26].

Protein-protein interaction (PPI) and hub gene selection
PPI analysis involved the use of STRING-DB v11.5 (https://string-db.org/)[27].The symbols of intersecting genes of BOR and HER2+ BC-related gene clusters were submitted to STRING database, and several parameters, such as "multiple proteins," and set "organisms" to "Homo sapiens" were selected.PPIs obtained from STRING-DB were exported to Cytoscape v3.9.1 (https://cytoscape.org/) to determine the hub gene (top gene) and ranked by degree score using the Cytohubba plugin [28] in Cytoscape.

Genetic Alteration and Prognostic Analysis
cBioportal (https://www.cbioportal.org/)[29] was used to determine genetic alteration and survival of hub genes.Hub genes from previous experiments were submitted to cBioportal via studies related to "BC." OncoPrint was used in viewing genetic alterations [30].The survival of each hub gene was compared with the data on patients with unaltered genes, and the results were combined in the form of Kaplan-Meier curves using Graphpad Prism [31].

Cell Culture
HCC1954, which comprised HER2+ BC cell lines, was collected from the APS Laboratory, Faculty of Pharmacy, Universitas Gadjah Mada.HCC1954 cells were cultured in Roswell Park Memorial Institute 1640 media (Sigma, Germany) supplemented with 10% fetal bovine serum (Sigma, Germany) with 1% penicillin-streptomycin (USA) and subjected a 5% CO 2 humidified atmosphere at 37 °C until the 70%-80% confluency [32].HCC1954 TRZ-resistant (HCC1954-TR) cells were collected from the APS Laboratory, Faculty of Pharmacy, Universitas Gadjah Mada and induced with a low concentration of TRZ (Roche, Germany) for 72 h.The media were replaced with a new one and then induced again [33,34].This process was repeated to obtain TRZ-resistant HCC1954 cells (HCC1954-TR).

Cytotoxicity assay
Bioinformatics findings were validated through in vitro methods, starting with the cytotoxicity assay.The cytotoxicity of TRZ in HCC1954 parental and HCC1954-TR cells were initially checked, and statistically significant variations (p < 0.05) were observed across all concentrations of TRZ except at 40 µg/mL (Figure 2A).Thus, HCC1954 cells with TRZ resistance were successfully generated.A single treatment of BOR was applied to parental HCC1954 and HCC1954-TR cells.The subjects were exposed to various doses of BOR from 10 µM to 320 µM (Figure 2B).The half maximal inhibitory concentration (IC 50 ) of BOR against the parental HCC1954 cell line was 130 µM, and that against HCC1954-TR was above 320 µM.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
HCC1954-TR cells were grown, incubated, and treated with the combination of 50 µM BOR and 800 µg/mL TRZ for 72 h.RNA was isolated in accordance with the instructions of RNeasy Kits (Qiagen).Total RNA was obtained and used for cDNA synthesis.cDNA synthesis was accomplished following the instructions of SensiFAST cDNA Synthesis Kit (Meridian Bioscience).qRT-PCR was performed in accordance with the instructions of SensiFAST™ SYBR® No-ROX Kit with the synthesized cDNA as the sample [39] and using the selected primers (Supplementary Table 1).Gene expression measurement accordance with the comparative threshold cycle (ΔΔCT) method.Statistical analysis of the combination treatments included one-way analysis of variance (ANOVA) with Tukey's multiple comparison test.The significance levels are as follows: *p < 0.05, ***p < 0.001.

Molecular Docking
The three-dimensional (3D) structures of cytochrome P450 family 19 subfamily (CYP19A1) (PDB ID: 3S7S), cytochrome the P450 family 17 subfamily A member 1 (CYP17A1) (PDB ID: 6CHI), RelA (PDB ID: 1NFI), NR3C1 (PDB ID: 7PRX), and interleukin (IL)-1β (PDB ID: 5R8O) were downloaded from the RCSB protein database (https://www.rcsb.org/).Docking protocol was performed in accordance with a previous study [40,41] and using the Molecular Operating Environment v2020.Briefly, proteins were prepared with "Protonate 3D" at 310 K and pH 7.4, and the forcefield was assigned under default settings.BOR, the test ligand, was also prepared using the MOPAC system with default settings.Each protein was redocked with its corresponding native ligand, and the root mean square deviation (RMSD) was calculated.The RMSD <2.0 Å corresponds to valid docking protocol and was thus applied to dock all proteins with BOR.The docking results were analyzed based on the best pose and score.The 2D and 3D interactions were visualized in accordance with previous studies [42,43].

Functional, disease-gene, and drug-gene associations
Gene Ontology (GO) revealed the role of BGBC in biological process (BP), cellular component (CC), and    After determining the effect of BOR treatment alone, a combination test was performed to determine the capability of the compound to kill parental HCC1954 cells in combination with TRZ, and the results revealed a significant difference (p < 0.05) when using 25 µM BOR and 400 µg/mL TRZ compared with TRZ (*) or BOR (#) alone but not when using BOR 50 µM and TRZ 400 µg/mL (Figure 2C).The combination test was also performed on HCC1954-TR cells (Figure 2D), with 25 or 50 µM BOR and 800 µg/mL TRZ exhibiting significant differences compared with TRZ alone (#).Moreover, the combination with the highest concentration, namely, 100 µM BOR and 800 µg/mL TRZ, effectively eradicated HCC1954-TR cells.

qRT-PCR
To verify the findings of bioinformatics analysis, we conducted a comprehensive test and assessed the expression of the central gene via quantitative qRT-PCR.GPT, AR, and ESR1 under combined BOR and TRZ treatment exhibited significant mRNA expression differences (p<0.05)(Figure 3A-C) compared with those treated with TRZ alone.These results indicate the importance of ESR, AR, and GPT in the effect of BOR on overcoming the resistance of BC cell toward TRZ.IL6 and TNFA gene expressions under combined TRZ-BOR treatment showed no significant difference (p > 0.05) compared with those under TRZ treatment alone (Figures 3D-3E).

Discussion
This study aimed to determine the effect of BOR on the increased sensitivity of TRZ and its potential target genes in overcoming the resistance of HER2+ BC toward TRZ.The results of this study demonstrate the top 10 hub genes, including IL6, TNF, ESR1, IL1B, CYP19A1, AR, NR3C1, RELA, CYP17A1, and GPT.IL6, TNF, ESR1, and IL1B, which encode IL-6, tumor necrosis factor (TNF), ER-α, and IL-β1, respectively.IL-1β induces the expression of hypoxia-inducible factor 1 (HIF-1α) or nuclear factor (NF)-κB, which are major hypoxia-sensitive pathways that participate in tumor progression by promoting angiogenesis, cell migration, and invasiveness of cancer cells [47].Glutamate-pyruvate transaminase 1 (GPT) (also called ALT1 or ALAT1) contributes to glutamine catabolism in tumor cells and the generation of reactive oxygen species necessary for Kras-induced anchorage-independent growth through regulation of the signal-regulated kinase/mitogen-activated protein kinase signaling pathway [48].NR3C1 encodes the glucocorticoid receptor (GR), which can act as a tumor suppressor in breast tissue [49].
The gene CYP19A1 encodes CYP19A1, an enzyme that belongs to the cytochrome P450 monooxygenase family and contributes to the conversion of C19 androgens, specifically androst-4-ene-3,17-dione (androstenedione) and testosterone, into C18 estrogens, namely, estrone and estradiol, respectively [50].AR encodes the androgen receptor (AR), which is a ligand-activated transcription component that regulates gene expression in eukaryotic cells and takes part in the control of cellular proliferation and differentiation in specific tissues [51].RELA encodes the RELA proto-oncogene, an NF-kB subunit and a family of transcription factors that is activated and enters the nucleus during cell stimulation involving extracellular signals, such as TNFα and IL1 [52].CYP17A1 encodes CYP17A1, an enzyme that performs a crucial function in the steroidogenic pathway via 17-alpha-hydroxylase and 17,20-lyase activities [53].CYP17A1 accounts for the production of progestins, mineralocorticoids, glucocorticoids, androgens, and estrogens [53].
The cytotoxicity of TRZ against parental HCC1954 cells compared with HCC1954-TR cells showed significant differences (p < 0.05) at all concentrations, except 40 µg/mL.Exposure to various concentrations of TRZ yielded significant differences.In addition, an extremely high IC 50 indicates that TRZ induction produced TRZ-resistant HCC1954 cells (HCC1954-TR).HCC1954 exhibited resistance to TRZ as a result of PIK3CA mutations [54].
Glutaminolysis provide cancer cells with energy [57,58].GPT/ALT1 converts glutamate (from glutamine) to α-ketoglutarate, which then enters the tricarboxylic acid/ Krebs and produces energy to facilitate tumor growth [59].The high expression of GPT is directly proportional to cancer cell survival and growth [60].The results of this study revealed significantly higher GPT expression in cells treated with TRZ alone under compared with the TRZ-BOR combination treatment.This finding indicates the more effective treatment effect of TRZ-BOR exposure than TRZ alone.Other studies also mentioned the high expression of other glutaminolysis agents in BC [61].However, to date, no research related to GPT expression in BC and relationship with BOR compounds has been conducted, which will be an interesting topic for future studies.
ERα, which is encoded by ESR1, is an alternative pathway to the HER2 pathway for BC cell growth and survival [62].HER2 blockade in HER2+ BC upregulates or reactivates a ER-regulated gene transcription, called a compensatory mechanism [63].The results of this study revealed the significantly higher ESR1 expression under TRZ-BOR exposure than that treated with TRZ or BOR alone.This finding indicates the capability of TRZ-BOR to suppress HER2 better than TRZ alone.Currently, studies on the effect of BOR on ER signaling are lacking, which necessitate further studies in the future.
AR signaling pathways are in crosstalk with PI3K/ AKT [64].The PI3K/AKT signaling pathway serves as a contributor to cancer growth, cell proliferation, and survival [65].Another study showed activated AR signaling due to PI3K inhibition in prostate cancer [64].The results of this work indicate a significantly increased AR expression (*p < 0.05) during exposure to TRZ-BOR compared with single exposure.We speculated that BOR exerts a synergistic effect on TRZ by inhibiting the PI3K/ AKT pathway.However, in this study, the expressions of IL6 and TNF genes in the TRZ-BOR treatment showed no significant differences compared with those exposed to TRZ or BOR alone.Further studies are needed to determine the protein levels of IL6, AR, and TNFα and PI3K/AKT regulatory proteins in cells treated with the combination of BOR and TRZ.
The crystal structure of CYP19A1 used in this study bound to the ligand EXM (exemestane; aromasin) as an inhibitor.EXM is an aromatase inhibitor and applied in BC therapy [66].The results of docking study demonstrate the hydrogen bonds formed by the carbonyl group of EXM and hydroxyl group of BOR with the same amino acid residue (Met374) on CYP19A1.However, BOR exhibited various interactions with abiraterone, a CYP17A1 inhibitor [67].The interaction of the hydroxyl group of BOR with Val482 transpired through hydrogen bond formation.Meanwhile, the carbonyl and hydroxyl groups of abiraterone showed interaction with Asn202 and Arg 239.The NF-κB transcription factor RelA/ p65 shows activity in the canonical NF-κB pathway, which is responsible for survival, proliferation, tumor development, and inflammation.Therefore, cancer drug development can target the selective inhibition of the canonical NF-κB pathway [68].Another study revealed the capability of BOR to reduce p65 levels.This finding correlates with the inhibition of the NF-κB pathway, which can suppress neuroinflammation in mice [69].
The results of docking study indicate that the hydroxyl group of BOR bound to Gln220 through hydrogen binding.NR3C1/GR performs two roles during activation, namely, transactivation and transrepression.A transrepression mechanism enables GR to inhibit transcription factors [70].The capability of GR to inhibit transcription factors (proinflammatory) is applied in the therapy of several cancers, such as lymphoid cancer, leukemia, and prostate cancer [71].Velsecorat, as the native ligand of the GR crystal structure, provides selective support to the role of GR in the transrepression pathway [72].Although more amino acid residues bound to velsecorat, the hydroxyl group of BOR showed interaction with same amino acid residue, Gln570.IL-1β alleviates the aggressive nature of BC through the increased levels of NF-kβ.In addition, IL-1β plays contributes to the initiation of metastasis and promotion of BC metastasis in bones [73].BOR can also inhibit IL-1β signaling [74].S8P, the native ligand of IL-1β, interacts with three different amino acid residues.Among these residues, Tyr24 bound to the hydroxyl group of BOR via hydrogen binding.Overall, the docking results demonstrate that BOR occupied all protein binding sites with similar orientations.In addition, the hydroxyl group of BOR presented a predominant interaction with amino acid residues.Therefore, expectedly BOR exhibits a similar bioactivity.BOR achieved lower docking scores.However, native ligands are not endogenous ligands.
Thus, we assumed that BOR can still bind to the proteins regardless of the negative scores.
We proposed the mechanism of genes involved in the action of BOR when overcoming the TRZ resistance of HER2+ BC (Figure 6).The results of this study unveiled the involvement of eight genes, namely, AR, ESR1, GPT, CYP19A1, CYP17A1, NR3C1, IL1B, and RELA.A reciprocal mechanism regulates the AR and PI3K pathways, given that the inhibition of one inactivates the other, which allows cancer cell survival and progression.The inactivation of AR leads to the overregulation of PI3K/ AKT.AR inhibition reduces FKBP5 levels and impairs the function of PHLPP [75], which dephosphorylates and inactivates AKT [76].CYP19A1 contributes to estrogen synthesis.The ligand-dependent transcription factor ERα regulates the expressions of various genes, such as Bcl-2, which can act as a barrier to apoptosis and facilitate tumor progression and resistance to cancer therapy [62].BOR can also downregulate Bcl-2 expression [15].IL-1β activates various proinflammatory pathways, including that of NF-κB.IL-1β induces the expression of HIF-1α and NF-κB, which are major hypoxia-sensitive pathways and participate in tumor progression by promoting angiogenesis, cell migration, and invasiveness [47].In addition to inhibiting IL-1β signaling [15], BOR may support transrepression work on GR/NR3C1, which can suppress proinflammatory transcription factors, such as NF-κB.BOR can suppress GPT given the greatly decreased gene expression during exposure to combined TRZ-BOR treatment compared with that during exposure to TRZ alone.However, this mechanism needs to be further investigated.
In conclusion, the combination of 100 µM BOR and 800 µg/mL TRZ provided significant results on HCC1954-TR and showed that BOR exerted a synergistic effect that increased the TRZ sensitivity of HCC1954-TR cells.In addition, this study revealed the ten genes that are most likely to be the target hub genes of BOR: GPT, IL6, IL1B, ESR1, AR, NR3C1, TNF, RELA, CYP19A1, and CYP17A1.Among the hub genes, AR, ESR1, and GPT are most likely to play significant roles in the BOR's effect on overcoming TRZ resistance.CYP19A1, CYP17A1, NR3C1, IL-1β, and RELA genes may be possibly involved, but further in vitro and in vivo research are needed to confirm their role in BOR's activity in overcoming HER2+ BC resistance toward TRZ.

Figure 1 .
Figure 1.In silico Test via a bioinformatics approach.(A) Gene slice showing the BOR gene targets and gene associated with HER2+ BC (BGBC).(B) GO, (C) gene-disease association analysis, and (D) gene-drug association analysis of BGBC, as analyzed using the ORA of WebGestalt.(E) Protein-protein interaction of BGBC, as analyzed by STRING.(F) Top ten hub genes, as analyzed by the CytoHubba plugin of Cytoscape based on degree score.(G) Breast cancer studies found in cBioportal server.(H) OncoPrint analysis of genetic alterations of hub genes using the Metastatic Breast Cancer (INSERM, Plos Med 2016) study in cBioportal server.(I) Survival curve related to genetic alterations of hub genes determined using the TCGA study in cBioportal server.The results are shown as a Kaplan-Meier curves generated using GraphPad Prism 9, with * indicating statistical significance with a p-value of less than 0.05.

Figure 2 .
Figure 2. (A) Cytotoxicity of TRZ toward HCC1954 and HCC1954-TR cells in the MTT Assay.(B) BOR cytotoxicity toward parental HCC1954 and HCC1954-TR cells.Cytotoxicity of combined TRZ-BOR treatment toward (C) parental HCC1954 or (D) HCC1954-TR cells.Cells were treated with TRZ or BOR for 72 h.Results are displayed as the mean percentage and standard deviation (SD) of cell viability for each concentration group.(n = 3, statistical analysis using one-way ANOVA with Tukey's multiple comparison; significant: p < 0.05).

Figure 3 .
Figure 3. Effects of TRZ, BOR, and their combination on the mRNA Levels of (A) GPT, (B) AR, (C) ESR1, (D) IL-6, and (E) TNFA.Gene expression was quantified via q-RT PCR.Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control in this study.The data were evaluated via a ΔΔCT method and presented as a fold change compared with the control.The results are presented as the arithmetic mean ± SD of three separate and distinct experiments.(n = 3; statistical analysis was performed via one-way ANOVA with Tukey's multiple comparison test; *p < 0.05; ***p < 0.001; ns = not significant).