Synergistic effects of Nrf2 inhibitor, brusatol treatment in combination with lapatinib, a selective HER2 tyrosine kinase inhibitor in HER2-positive cancers

doi:10.21203/rs.3.rs-1564524/v1

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Synergistic effects of Nrf2 inhibitor, brusatol treatment in combination with lapatinib, a selective HER2 tyrosine kinase inhibitor in HER2-positive cancers

https://doi.org/10.21203/rs.3.rs-1564524/v1

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Overexpression of human epidermal growth factor receptor 2 (HER2), a member of the HER family of receptor tyrosine kinases (RTKs), is found in many solid tumors. The dual tyrosine kinase (EGFR/HER2) inhibitor lapatinib is currently used to clinically treat HER2-positive breast cancer. However, a majority of patients do not respond to lapatinib therapy within 6 months when using lapatinib alone. Therefore, potentiating the anti-tumor effect of lapatinib by combination treatment has a great potential to overcome the obstacle. Our findings revealed that brusatol potently enhanced the anti-tumor effect of lapatinib against SK-BR-3, SK-OV-3 and AU565 cancer cells in a synergistic manner. Furthermore, we found that lapatinib plus brusatol more effectively decreased Nrf2 level and induced ROS generation in both SK-BR-3 and SK-OV-3 cells. Moreover, we also observed a significant reduction on the phosphorylation of HER2, EGFR, AKT and ERK1/2 in SK-BR-3 and SK-OV-3 cells when treated with lapatinib plus brusatol compared to either agent alone. More importantly, brusatol significantly augmented the anti-tumor effects of lapatinib in the SK-OV-3 xenograft model. In summary, these data provide a potential rationale for the combination of brusatol and lapatinib on the treatment of HER2-positive cancers.

Nrf2

Brusatol

Lapatinib

HER2

ROS

Overexpression of HER2 was observed in approximately 20% of breast and gastric cancers [1]. Interference with the phosphorylation of HER2 will lead to regression of several downstream signaling cascade(s), such as PI3K/AKT and mitogen-activated protein (MAPK) signaling[2]. Lapatinib was approved by FDA in 2007 as a dual-tyrosine kinase inhibitor against HER2 and EGFR that functions by blocking the intracellular ATP binding site of the tyrosine kinase domain and inhibiting the phosphorylation of EGFR and HER2 [3]. In clinical trials, lapatinib has shown relatively weaker cardiotoxicity compared to trastuzumab, the other FDA-approved HER2-targeted therapeutic [4]. However, primary and acquired resistance also limits the clinical application of lapatinib [5–8]. A majority of breast cancer patients develop acquired resistance within 6 months when using lapatinib alone [9]. Collectively, it is necessary to test the new therapeutic strategies for enhancing the efficacy of lapatinib and improving the survival of breast cancer patients.

Nuclear factor erythroid 2-related factor 2 (Nrf2) was an important transcription factor regulating anti-oxidant response element (ARE), thereby limiting the production of reactive oxygen species (ROS) to maintain cellular redox status [10]. In recent years, more and more studies disclosed that Nrf2 signaling pathway is activated in a variety of cancers such as lung, pancreatic and breast cancers [11]. Moreover, Nrf2 overexpression is tightly associated with tumor invasiveness, metastasis, chemotherapy resistance and poor clinical outcomes in many cancer patients [12]. Notably, our team also found that silencing Nrf2 sensitizes HER2-positive cancer cells to trastuzumab treatment, suggesting that Nrf2 may be a new potential target in treating breast cancers [13].

Brusatol was extracted from the seeds of Brucea javanica, which exhibited the potent inhibitory activity against Nrf2 transcriptional signature [14–16]. Xiang et al reported that brusatol potentiated gemcitabine-induced apoptosis and growth inhibition in pancreatic cancer cells in vitro and in vivo [17]. Recently, we also disclosed that trastuzumab markedly enhanced brusatol-induced ROS accumulation and apoptosis level, which resulted in markedly greater anti-tumor effects [13]. Collectively, brusatol has a great potential to be an adjuvant drug in combination with other molecular targeted drugs in treating cancers.

In this study, we are the first to investigate the effects of Nrf2 inhibition in combination with dual HER2/EGFR inhibitor, lapatinib in HER2-positive cancers. Our results for the first time showing that brusatol synergistically enhanced the growth-inhibitory effect of lapatinib against HER2-positive cancer cells including SK-BR-3, AU565 and SK-OV-3 cell lines in vitro. Brusatol act as a potent inhibitor of Nrf2 signaling [18, 19]. Interestingly, we found that the combination of lapatinib and brusatol more effectively inhibited Nrf2 signaling and induced ROS accumulation in both SK-BR-3 and SK-OV-3 cells. Notably, we also observed a more significant decrease on the phosphorylation of HER2, EGFR, AKT and ERK1/2 in SK-BR-3 and SK-OV-3 cells when treated with lapatinib plus brusatol compared to either agent alone. More importantly, brusatol potently augmented the anti-tumor effects of lapatinib in SK-OV-3 tumor xenografts. In summary, above-mentioned results provide a potential regimen by combining brusatol and lapatinib to improve the therapeutic efficacy of lapatinib in HER2-positive cancers.

2.1. Cell lines

The human breast cancer cell line AU565, SK-BR-3 and ovarian cancer cell line SK-OV-3 were purchased from the American Type Culture Collection (ATCC).

2.2. Agents

Lapatinib was purchased from Rhawn. Inc (Shanghai, China). Brusatol was purchased from Yuanye Biotech Corporation (Shanghai, China). It is over 95% pure determined by HPLC. The stock solution of brusatol was prepared by dissolving in DMEM with 0.25% dimethyl sulfoxide (DMSO). N-acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich. Inc (Mo, USA). The antibodies were utilized as following: Nrf2 (1:1000; cat.no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat.no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)‑HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)‑EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176‑2‑AP; ProteinTech Group, Inc.), p‑AKT (Ser473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β‑actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase‑conjugated goat anti‑mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001‑1 or SA00001‑2; ProteinTech Group, Inc.).

2.3. Animals

Six-week female BALB/c nude mice were obtained from the Beijing Vital River Laboratory Animal Technology (Beijing, China). The animal research was conducted according to the Principle of Laboratory Animal Care (NIH Publication No. 85 − 23, revised in 1985). All experimental protocols were approved by the Animal Experimentation Ethics Committee of Xinxiang Medical University and all efforts were made to minimize animal suffering and reduce the number of animals used. Animals were treated in accordance with the guideline of the Animal Care and Use Committee of Xinxiang Medical University.

2.4. In vitro cytotoxicity assay

Breast cancer SK-BR-3 and AU-565 cells and ovarian cancer SK-OV-3 cells were plated in 96-well plates (5×10³ cells per well) and incubated with lapatinib, brusatol or lapatinib in combination with brusatol for 48h. Cell viability was then determined by CCK-8 kit (Dojindo). The percentage of surviving cells was calculated using the following formula: [(A450 of experiment – A450 of background)/(A450 of control – A450 of background)] × 100. Combination index (CI) values were calculated using the Chou-Talalay method by Compusyn Software. Drug synergy, addition, and antagonism are defined by C.I. values less than 1.0, equal to 1.0, or greater than 1.0, respectively.

2.5. Immunoblotting

Firstly, cells were lysed in RIPA lysis buffer containing 50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% SDS, 1% NP-40, 0.5% deoxycholic acid sodium salt (w/v)] supplemented with 2 µL/mL protease inhibitor cocktail (Sigma) and 10 µL/mL phosphatase inhibitor cocktail (Sigma). The protein concentration of cell lysates was measured by a QuantiPro BCA protein assay kit (Sigma Aldrich). After denaturation, total cell lysates were subjected to SDS–polyacrylamide and immunoblotted with primary antibodies and HRP-conjugated secondary antibody as mentioned above. After wash of the membrane, the bands were detected using the sensitive ECL reagent (Millipore), and visualized using an ChemiDoc imaging system (Bio-Rad Laboratories, Inc.).

2.6. Reactive Oxygen Species (ROS) Detection

The production of ROS in solution was routinely detected with 2’, 7’ - dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma Aldrich) according to the instruction of supplier. Briefly, the cells (1×10⁵/well) were treated with PBS, brusatol, lapatinib or brusatol plus lapatinib at 37°C for 6 hours. Then, 1 mM DCFH-DA was added to the cells (final concentration of DCFH-DA will be 10 µM). After incubation for 30 min, the cells were washed with phosphate buffer saline (PBS) and collected. Fluorescence signal intensities indicating ROS levels were recorded by flow cytometer (BD Biosciences) using excitation and emission spectra of 488/525nm.

2.7. Apoptosis analysis

Cells were seeded in 6-well plates at a density of 2×10⁶/well and exposed to different drugs. After incubation for 48 hours, the cells were washed with ice-cold PBS and incubated with Annexin V and propidium iodide (PI) (Sigma Aldrich) for 15 min at room temperature in the dark. The rate of apoptotic cells was determined by flow cytometer (BD Biosciences) and analyzed by FlowJo software. The percentage of early apoptosis was calculated by annexin V (+) / PI (-), while the percentage of late apoptosis was calculated by annexin V (+) / PI (+).

2.8. In vivo therapy study

SK-OV-3 cells (1 × 10⁷ per mouse) were inoculated subcutaneously into the right flank of female BALB/c nude mice. When tumor volumes reached an average of about 150 mm³ approximately on day 12 after inoculation, the mice were randomly divided into 4 groups of 6 mice each. Mice were intraperitoneally injected with PBS or brusatol (2 mg/kg) once per day for 18 days. We treated mice with lapatinib (100 mg/kg) daily via oral gavage. Tumors were measured with digital calipers, and tumor volumes were calculated by the formula: Volume = [Length × (Width)²]/2. Mice were euthanized with CO₂ asphyxiation.

2.9. Statistical analysis

Statistical analysis was performed with the Graphpad Prism version 5.0 (Graphpad software). Data are shown as mean ± SD. Statistical analysis was performed by Student’s unpaired t test to identify significant differences unless otherwise indicated. Nonlinear regression analyses were used to fit curves.

3.1. Brusatol synergistically enhanced growth inhibition caused by lapatinib in SK-BR-3, SK-OV-3 and AU565 cells

In the previous study, we found that brusatol exerted anti-tumor effects against HER2-overexpressed cancer cells by repressing HER2-AKT/ERK1/2 signaling pathway [13]. Based on these results, we hypothesize that addition of brusatol to lapatinib may result in a therapeutic benefit in treating HER2-positive cancers. Results revealed that lapatinib in combination with brusatol exhibited an significantly enhanced inhibitory activity than that of either agent alone in all three HER2-positive cancer cells including SK-BR-3, SK-OV-3 and AU565 cancer cells (Fig 1A). Moreover, the superior effects of lapatinib plus brusatol was synergistic on SK-BR-3, SK-OV-3 and AU-565 cells by utilizing the method of Chou and Talalay to establish drug C.I. values (Fig 1B).

3.2. Brusatol sensitizes HER2-positive cells to lapatinib by inducing the up-regulation of the ROS level in SK-BR-3 and SK-OV-3 cancer cells

Recent studies illustrated that brusatol might provide potential clinical benefit especially when combined with anti-tumor drugs that stimulate ROS production. [17, 20]. Therefore, we also examined the effects of combinatorial treatment on ROS accumulation utilizing FACS assay. Results revealed that co-treatment of lapatinib with brusatol resulted in a significantly greater increase on ROS production than brusatol or lapatinib treatment alone in both SK-OV-3 and SK-BR-3 cancer cells (Fig 2). To further provide additional evidence for these results, N-acetyl-L-cysteine (NAC), a type of anti-oxidant agent that can effectively reduce the ROS level was added to SK-OV-3 and SK-BR-3 cells treated with a combination of lapatinib and brusatol. As expected, the co-administration of the anti-oxidant NAC antagonized the elevation in ROS production from both SK-OV-3 and SK-BR-3 cell lines treated with brusatol plus lapatinib (Fig 2). To conclude, brusatol plus lapatinib was more effective in inducing ROS production than either agent alone, which may explain the superior anti-tumor effects of the combinatorial treatment.

3.3. Brusatol in combination with lapatinib potently potentiated apoptosis in SK-BR-3 and SK-OV-3 cells

As we know, Nrf2 inhibition by Nrf2-targeted agents renders cancer cells susceptible to apoptosis [19, 21]. Thus, we evaluated the apoptotic percentage in SK-BR-3 and SK-OV-3 cells when were exposed to brusatol plus lapatinib. Compared to brusatol or lapatinib alone, a combination of brusatol with lapatinib significantly potentiated apoptosis in SK-BR-3 cells (Fig 3A and B). Notably, as shown in Figure 3C and D, combinatorial therapy has not exhibited the superiority in SK-OV-3 cells compared to brusatol group, which suggested that apoptosis activation may be not the main mechanism explaining the superior growth-inhibitory effects in combinatorial group.

3.4. Lapatinib plus brusatol exhibited a significant effect on regressing Nrf2/HO-1 anti-oxidant and EGFR/HER2-AKT/ERK1/2 signaling pathways in SK-BR-3 and SK-OV-3 cancer cells

To further explore the mechanism involved in synergistic anti-tumor effect, we examined the core protein level involved in Nrf2/HO-1 signaling and EGFR/HER2-AKT/ERK1/2 signaling pathway in both SK-BR-3 and SK-OV-3 cancer cells. Western blot analysis showed that brusatol in combination with lapatinib was more effective in inhibiting phosphorylation of EGFR and HER2 than either single-drug treatment. Moreover, weaker phosphorylation level of AKT and MAPK was observed in the two cell lines treated with contaminant treatment, respectively, compared with single agent treatments (Fig 4). Notably, nearly complete AKT deactivation was observed in the condition of contaminant treatment compared with single-agent treatments, while ERK1/2 activation was only partly inhibited (Fig 4). It suggested that the combinatorial treatment may mainly preclude HER2/EGFR-AKT signaling activation, thereby resulted in cell growth repression.

3.5. Combination therapy of lapatinib and brusatol is superior to single-agent treatment in SK-OV-3 xenografts

To further evaluate the therapeutic effects in vivo, brusatol plus lapatinib was injected in nude mice bearing established SK-OV-3 xenograft tumors. Nude mice bearing SK-OV-3 xenografts were randomized and treated either with lapatinib (100 mg/kg), brusatol (2 mg/kg), or both. Results showing that contaminant treatment of brusatol and lapatinib resulted in a significant benefit over either brusatol or lapatinib alone in SK-OV-3 xenografts (Fig 5A). Meanwhile, we found that the mice in combinatorial group did not significantly lose body weight and behaved normally in the treatment period (Fig 5B). As is shown in Figure 5C and 5D, the treatment with lapatinib and brusatol combination resulted in a 50 % reduction in tumor weight in comparison with control. Besides these, hematoxylin and eosin (H&E) staining also showed that no marked liver toxicity was observed in SK-OV-3 tumor-bearing mice upon treatment with lapatinib plus brusatol (Fig S1). Overall, these results above revealed that the combinatorial therapy shows stronger inhibitory effects and appear to be well tolerated in HER2-positive tumor xenografts.

Lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase activity, has already been approved by the US Food and Drug Administration as a treatment for HER2-positive metastatic breast cancer [22]. Despite the effectiveness, primary and acquired resistance dramatically limits the usage of lapatinib. Therefore, developing a strategy to enhance its therapeutic efficacy is urgent in clinics. Brusatol has been reported to sensitize cancer cells to carboplatin, 5-fluorouracil, gemcitabine, etoposide, and paclitaxel by regressing Nrf2-dependent anti-oxidant response pathway [15, 17, 23]. In the study, we for the first time found that brusatol was capable of enhancing the anti-tumor effects of lapatinib in HER2-positive SK-BR-3, AU565 and SK-OV-3 cancer cells in a synergistic manner. Moreover, the synergistic effects were verified in mice bearing SK-OV-3 tumors, which was characterized as a classical model to examine HER2-targeted drugs [24, 25]. Further study will be aimed to explore the effects of lapatinib plus brusatol in lapatinib-resistant cancer cell lines. Overall, our results provide evidence for rational strategies that can promote the efficacy of lapatinib.

Our previous study revealed that brusatol was effective in inhibiting tumor growth and potently enhanced the anti-tumor activity of trastuzumab in HER2-positive cancers [13]. It suggested that dual inhibition of Nrf2 anti-oxidant signaling and HER2-AKT/ERK1/2 signaling is crucial for the anti-tumor effect of HER2-targeted therapeutics in HER2-overexpressed tumors. Therefore, we examined the effects of brusatol plus lapatinib in HER2-positive cancer cells in vitro and in vivo. As a result, we also observed the synergistic effects of brusatol plus lapatinib in the three cancer cell lines. Until now, resistance to HER2-targeted therapeutics such as lapatinib or trastuzumab during therapy period is still a major obstacle to the successful treatment of breast cancer [26, 27]. Thus, these results provided a potential therapeutic strategy by combining brusatol with other HER2-targeted therapeutics including antibody or small-molecule antagonists to overcome tolerance.

In our study, we also attempted to elucidate the mechanism by which lapatinib in combination with brusatol blocks the activity of Nrf2. It is known that Nrf2 protein is suppressed by an association with Keap1 under homeostatic conditions, but it is activated when cells are exposed to oxidative or electrophilic stress. Indeed, the level

of Keap1 was not affected (data not shown), and thus we assumed that inhibition of Nrf2 is independent of Keap1. Consistent with the previous study, our study also demonstrate that modulating redox homeostasis is a new mechanism underlying the observed synergism between brusatol and lapatinib in HER2-positive cancers [28].

Collectively, at least 2 possible mechanisms will contribute to the synergistic anti-tumor effect of brusatol and lapatinib. The first involves attenuated phosphorylation of HER2 and EGFR and regressed activation of downstream signaling relevant to tumor growth. Additionally, inhibition of Nrf2/HO-1 anti-oxidant signaling and resultant ROS-dependent apoptosis may be involved. To conclude, brusatol potently enhance the sensitivity of HER2-positive cancer cells to lapatinib both in vitro and in vivo, providing a potential rationale for the combination of brusatol with lapatinib on the treatment of HER2-positive cancers.

No potential conflicts of interest were disclosed.

Conflict of interest statement

All authors declare no conflict of interest or previous publication. The authors fulfill the conditions required for authorship.

Author contributions

Y.Y. and Z.Y.T. designed the experiments and wrote the manuscript. Z.Y.T, Y.Y., H.W., Y.Y.C, H.J., L.Z., R.J.H., Y.B.J., B.Z., C.P.G., Y.X.S. and Y.Y. carried out the experiments. Y.Y. and Y.X.S. supervised and corrected the manuscript.

Ethics Statement

This study was carried out in accordance with the Principle of Laboratory Animal Care (NIH Publication No. 85-23, revised 1985). The protocol was approved by the Animal Ethics Committee of Xinxiang Medical University.

Data Availability Statements

Data arising from this study are contained within the manuscript.

Funding Statement

This work was supported by grants from the Program for Science＆Technology Innovation Talents in Universities of Henan Province (22HASTIT049), Natural Science

Foundation of Henan (202300410322 and 222300420266), Training Plan for Young Backbone Teachers in Universities of Henan Province (2020GGJS143), Key Scientific Research Project of Higher Education of Henan Province (22A310007 and 22B310011), Science & Technology Project for Young Talents of Henan Province (2020HYTP048), National College Students' Innovation and Entrepreneurship Training Program (202110472008) and Innovation project of Graduate in Xinxiang Medical University (YJSCX202130Y).

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Synergistic effects of Nrf2 inhibitor, brusatol treatment in combination with lapatinib, a selective HER2 tyrosine kinase inhibitor in HER2-positive cancers

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cell lines

2.2. Agents

2.3. Animals

2.4. In vitro cytotoxicity assay

2.5. Immunoblotting

2.6. Reactive Oxygen Species (ROS) Detection

2.7. Apoptosis analysis

2.8. In vivo therapy study

2.9. Statistical analysis

3. Results

4. Discussion

Declarations

References

Supplementary Files

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