Determination Of Selective Cytotoxicity From Ncb-0846 In The Treatment Of Triple Negative Breast Cancer

doi:10.21203/rs.3.rs-4689289/v1

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Determination Of Selective Cytotoxicity From Ncb-0846 In The Treatment Of Triple Negative Breast Cancer

https://doi.org/10.21203/rs.3.rs-4689289/v1

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BACKGROUND

It was aimed to investigate the therapeutic effect of NCB-0846 inhibition of TNIK, which is the activator kinase of Wnt/β-catenin signaling pathway, on triple negative breast cancer at the molecular stage.

METHODS

MDA-MB-231 cells were used as triple negative breast cancer cell line and MCF-10A cells were used as control cell line. WST-1 analysis showed the cytotoxic effect of NCB-0846, Annexin V analysis showed its apoptotic effect, cell cycle analysis showed its effect on cell cycle, and Acridine Orange staining showed its effect on cell morphology. In addition, the effect of NCB-0846 on CTNNB1 (β-catenin) gene expression was demonstrated by RT-PCR analysis.

RESULTS

The viability rates in NCB-0846, MDA-MB-231 cells decreased significantly in a time and dose dependent manner (p < 0.01). The lowest viability rates for MDA-MB-231 cells were determined as 42,20% at the 3 µM dose after 72 hours of incubation. After 72 hours of incubation in MCF-10A cells, the viability rate was determined as 53,92% at 3 µM dose (p < 0.01). Apoptotic cell rates were determined as 60,5% and 39,33% for 3 µM NCB-0846 incubation in MDA-MB-231 and MCF-10A cells, respectively. In the RT-PCR analysis, while the expression level of CTNNB1 was decreased in MDA-MB-231 cells, it was determined that it increased significantly in MCF-10A cells.

CONCLUSION

NCB-0846 was shown to induce apoptosis while inhibiting viability in MDA-MB-231 cells. Our results suggest that NCB-0846 may be a suitable candidate for cancer therapy, but further in vitro and in vivo studies are required to better understand its mechanisms of action.

Triple negative breast cancer

Wnt/β-catenin

TNIK

NCB-0846

Breast cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in women. According to 2020 World Health Organization data (GLOBOCAN), breast cancer is the most commonly diagnosed cancer with 2.3 million new cases per year. The lifetime risk of developing breast cancer is one in 8 women in the United States and one in 10 women in Europe [1].

Breast cancers are divided into 4 subtypes immunohistochemically based on estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor (HER2) status: Luminal A, Luminal B, HER2-positive and triple negative. Triple-negative breast cancer (TNBC) has the highest mortality rate among breast cancer subtypes. While there are targeted therapies such as trastuzumab (Herceptin) against HER2-positive breast cancers and tamoxifen against ER-positive breast cancers, such therapeutic agents cannot be used because these receptors are not present in TNBC.

The Wnt gene was first obtained from integrase-1 and the wingless gene of Drosophila in mouse breast cancer [2]. The WNT/β-Catenin signaling pathway is a key signaling pathway involved in the regulation of basic cellular functions such as cell proliferation, differentiation, cell migration, genetic stability, apoptosis and stem cell renewal. In addition, the pathway has many pathological processes such as migration, invasion, epithelial-mesenchymal transition (EMT) and cancer cell stem [3]. The therapeutic effect of inhibition of different steps of the Wnt/β-catenin signaling pathway in colorectal cancer, lung cancer and breast cancer has been demonstrated by different studies and its importance as a potential target in cancer treatment has been emphasized [4–6]. The WNT pathways consist of non-canonical and canonical pathways [7]. Non-canonical WNT pathway is independent of β-catenin-T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) [2]. The canonical Wnt pathway, also known as the Wnt/β-catenin pathway, involves β-catenin-mediated nuclear translocation and TCF/LEF transcription factors [2].

Traf2 (TNF receptor associated factor 2) and TNIK (Nck interacting kinase) are important regulatory components of the WNT/β-catenin signaling pathway. TNIK phosphorylates TCF4 by interacting with the β-Catenin/TCF4 complex at the nucleus. The anti-cancer therapeutic effect of TNIK inhibition on colorectal cancer and lung cancer was investigated [8]. NCB-0846 is a newly identified small molecule TNIK inhibitor [9, 10]. NCB-0846 has anti-WNT/β-Catenin activity. Chemical name of NCB-0846 is cis-4-(2-(3H-Benzo[d]imidazol-5-ylamino)quinazolin-8-yloxy) cyclohexanol [10]. It was found that the stereochemistry at the chiral terminal hydroxyl group of the cyclohexane moiety is important for the inhibition of the TNIK enzyme [10]. It inhibits the phosphorylation of TCF4 and the auto-phosphorylation of TNIK.

Although in vitro studies demonstrated the success of NCB-0846 in different types of cancer, data on non-cancerous cell lines were missing in almost all available studies. Therefore, the literature created the misconception that NCB-0846 might have selective cytotoxicity on cancerous cells. Therefore, in our study, we compared the cytotoxicity and apoptotic effects of NCB-084 on TNBC (MDA-MB-231) and non-cancerous breast epithelial (MCF-10A) cells.

Cell Culture Conditions

The MDA-MB-231 (ATCC® HTB-26TM) human TNBC cell line was purchased commercially from the American Type Culture Collection (ATCC). MDA-MB-231 cell line was cultured in Dulbecco Modified Eagle Media (DMEM) medium with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin in a T₇₅ flask in a 5% CO₂ incubator at 37°C. After the adherent cell culture was confluent in growth medium, it was washed with phosphate buffered saline (PBS) solution for removal and incubated with 0.25% Trypsin-EDTA (Gibco, USA). The related cells were treated wıth ncb-0846. Ncb-0846 (commercıally obtaıned from Sellechem Cayman Cat No:33268)

Cell Viability Analysis

Cells were seeded into 96-well culture plates at 2x104 cells per well using 100 µL of medium and incubated for 24 hours to ensure adequate confluency. Cells were treated with different concentrations (0–3 µM) of NCB-0846 and incubated for 24, 48, and 72 h. Then, the cell medium was removed and 10 µL of WST-1 Reagent (BioVision, CA) was added to the fresh medium with a final volume of 100 µL and incubated. After incubation, cell viability analysis was performed by reading the absorbance values of the cells at 450 nm with a microplate reader (Allsheng, China). The viability of the control group without NCB-0846 was accepted as 100%, and the viability of the experimental cells was expressed as a percentage compared to the control group.

Annexin-V Analysis

Annexin-V Analysis was performed to determine the apoptotic effect of NCB-0846 on MDA-MB-231 cell line. Cells were seeded in 6-well plates with 1x10⁵ cells/well. Cells were treated with NCB-0846 at the most effective dose and time according to the data obtained from WST-1 analysis were removed by trypsinization. The removed cells were centrifuged and washed 2 times with PBS. The obtained cell pellet was incubated with Muse Annexin V; Dead Cell Assay Kit (Merck, Germany) stain for 20 minutes at room temperature in the dark. After incubation, the stained cells were analyzed in Muse® Cell Analyzer (Merck Millipore, Germany) and apoptotic death rates were determined.

Cell Cycle Analysis

Cell cycle analysis was performed to determine in which phase of the cell cycle NCB-0846 is active in the MDA-MB-231 cell line. The MDA-MB-231 cell line treated with the effective dose and time of NCB-0846 according to WST-1 assay. The cells were fixed with 1 mL of 70% cold ethanol at -20°C for at least 3 hours. After fixation, cell pellets were stained with 200 µL Muse® Cell Cycle Reagent according to the protocol of Muse® Cell Cycle Kit (Merck Millipore, Germany) and incubated at room temperature in the dark for 30 minutes. Then they were analyzed on Muse® Cell Analyzer (Merck Millipore, Germany).

Acridine-Orange/Ethidium Bromide (AO/EB) Staining

AO/EB staining was performed to observe the morphological changes caused by NCB-0846 in the MDA-MB-231 cell line. For this purpose, equal numbers of cells (5x10⁶) were seeded in 6-well plates and incubated for 24 hours. At the end of the incubation period, cells were treated with NCB-0846 at the effective dose and time determined by the data obtained from the WST-1 assay. After treatment, the medium was removed and cells were fixed with 4% Praformaldehyde (PFA). After fixation, cells were stained with Acridine Orange (Sigma-Aldrich, USA)/Ethidium Bromide (BioVision, USA) solution for 30 minutes at room temperature and in the dark. The stained cells were washed three times with PBS buffer and visualized with EVOS™ FLoid (Life Technologies, USA) cell imaging system.

RNA Isolation and Quantitative PCR Analysis

The effect of NCB-0846 on the Wnt/β-Catenin pathway in the MDA-MB-231 cell line was determined by quantitative PCR analysis. mRNA extraction from groups treated and untreated with NCB-0846 was carried out in accordance with the TRIzol™ Reagent (Invitrogen, USA) kit protocol. The resulting mRNAs were converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA).

CTNB1 gene expression levels were evaluated from specifically obtained cDNA samples using the appropriate commercially available primer. The following concentrations were used for each sample: 100 ng of cDNA, TaqMan™ Fast Advanced Master Mix (Applied Biosystems, USA), 1 µL of primer CTNNB-1 (β-catenin) (Hs00355045_m1), and 20 µL final volume with RNase-. β-actin (ACTB)(Hs99999903_m1) was used as the reference gene and comparisons were made between the experimental groups and the control group. Relative expression was measured using the 2 − ΔΔCt method. GraphPad Prism 9.0.0 was used to visualize the relative expression of genes of interest in experimental groups

Statistical Analysis

The data obtained were statistically analyzed using the SPSS 21.0 program. p < 0.05 values were considered statistically significant. Differences in expression change were determined by Paired Sample t-Test.

Evaluation of the Cytotoxic Effect of NCB-0846

Our results showed that cancerous cells (MDAMB231) and non-cancerous (MCF10A) cells were equally affected by NCB-0846 treatment (Fig. 1A-B). NCB0846 did not affect cellular metabolism in either cell line at 24 and 48 hours. However, this effect reduced the viability at 1 µM dose for MCF10A and 3 µM dose for MDA–MB231 to 39.74% and 42.20%, respectively, compared to the control group, over 72 hours of treatment (p < 0,05).As a result, higher doses of NCB0846 resulted in more cell death in cancer cells than in normal cells (p < 0.01). Interestingly, NCB-0846 increased cell viability in MCF10A at increasing concentrations. The IC50 value of NCB0846 determined for MDA-MB-231 increased viability in MCF10A by 35.68% compared to the 1 µM dose (Fig. 1C). In conclusion, we believe that increasing doses of NCB-0846 will exhibit selective cytotoxicity in cancer cells.

Evaluation of the Apoptotic Effect of NCB-0846

In the Annexin V analysis performed on MDA-MB-231 cells, total apoptotic cell death rates were determined as 49.95%, 51.85% and 60.5% in the experimental groups treated with 1 µM, 2 µM and 3 µM NCB-0846 for 72 hours (Fig. 2). Additionally, it was analyzed that the total amount of apoptotic death in each experimental group increased statistically significantly compared to the control group (p < 0.01), (Fig. 3).

In MCF-10A cells, NCB-0846 treatment at 1, 2 and 3 µM concentrations for 72 hours increased total apoptotic cell death by 57.30%, 46.10% and 39.44%, respectively, compared to the control group (p < 0.01). The results were correlated with decreasing cytotoxicity depending on the increasing dose, and the highest apoptotic activity was observed in MDAMB231 cells at a concentration of 3 µM, while apoptotic activity was observed to decrease in MCF10A cells at this concentration.

Evaluation of the Changes Caused by NCB-0846 in the Cell Cycle

The amount of cells arrested in G2/M phase in NCB0846-treated MDAMB231 was determined to be 43.5%, 42.0%, and 44.7% for 1, 2 and 3 µM concentration, respectively (Fig. 4). The increase in cell retention in the G2/M phase in NCB-0846 triple negative breast cancer cells compared to the control group (22.6%), respectively, is statistically significant (p < 0,01), (Fig. 5).

While the cell population in the G0/G1 phase in MCF-10A cells was 82.3% in the control group, it was determined as 803%, 762% and 832% in cells treated with 1 µM, 2 µM and 3 µM NCB-0846, respectively (p < 0.01). However, despite its toxic effect on MCF-10A cells, NCB-0846 did not cause a significant increase in the G2/M phase of the cell cycle.

Determination of Morphological Changes Caused by NCB-0846 on Cells

Morphological changes induced by NCB-0846 in MDA-MB-231 and MCF-10A cells were visualized by AO/PI staining (Fig. 6).

DNA fragmentation, chromatin condensation and cell shrinkage were observed in MDA-MB-231 cells depending on the increasing dose of NCB-0846. In addition, degenerative changes in the cytoplasm as well as cell membrane ruptures (apoptotic body) were observed due to the increase in the concentration of NCB-0846, which is active on pathways targeting adherent junctions.

In MCF-10A cells, especially in cells treated with 2 µM NCB-0846, cell morphology was disrupted and cell junction breaks and nuclear fragmentation were observed compared to the control group. In cells treated with 3 µM NCB-0846, less damage was observed compared to other doses. However, it was determined that damage occurred in MCF-10A cells in general due to the toxic effect of NCB-0846.

Evaluation of Changes in CTNNB1 Gene Expression Level

The effect of NCB-0846, which plays an active role in cell adhesion, on CTNNB1 (β-catenin) gene expression in MDA-MB-231 and MCF-10A cells was determined by RT-PCR analysis and the findings are presented in Fig. 7. In addition, the effects of different concentrations of NCB-0846 on gene expression in MDA-MB-231 and MCF-10A cells were normalized by 2-ΔΔCT method with ACTB gene for each group.

Although the expression level of CTNNB1 gene, which encodes β-catenin protein as an important target of the Wnt pathway, increased 2.83-fold (p < 0.01) after 1µM NCB-0846 treatment for 72 hours in MDA-MB-231 cells, it was determined as 0.29 and 0.70-fold after 2 µM and 3 µM NCB-0846 treatment, respectively.

The mRNA levels of CTNNB1 gene in MCF-10A cells after treatment with 1 µM, 2µM and 3 µM NCB-0846 for 72 hours were 1.83, 3.12 and 4.08-fold, respectively (p < 0.01).

As a result, although CTNNB1 expression level decreased in MDA-MB-231 cells due to increasing dose of NCB-0846, CTNNB1 mRNA level increased significantly in MCF-10A cells.

TNBC has the worst prognosis among other breast cancer subtypes [11]. Indications for surgery, radiotherapy, adjuvant and neoadjuvant therapy are similar to other breast cancer subtypes [11]. However, while targeted therapy is available in medical treatment in other breast cancer subtypes, receptor-targeted therapy is not available in TNBC due to ER(-), PR(-), HER-2(-). Systemic cytotoxic agents are used in the treatment of TNBC [12]. Our study aimed to reveal the cytotoxicity of NCB-0846, which has anti-cancer activity on TNBC cells, on non-cancerous breast epithelial cells.

TNBC responds well to standard chemotherapies in the early stages [13]. However, in advanced stages and relapses, targeted treatments are needed in addition to standard chemotherapy. Therefore, studies have targeted the inhibition of TNBC through Wnt/β-catenin signaling pathways, which are involved in tumor migration, invasion, and metastasis [14]. Therefore, NCB-0846 is of interest as a chemotherapeutic agent. However, literature showing that NCB-0846 demonstrates selective cytotoxicity in TNBC is lacking.

In our study, WST1 viability assay results evaluating the cytotoxic effects of NCB0846 showed that NCB0846 was dose and time dependent on MDAMB231 cells, whereas it was only time dependent on MCF10A cells. NCB0846 was found to be non-cytotoxic in MDAMB231 and MCF10A at doses administered at 24 and 48 hours. TNIK, identified as a regulatory component of the transcriptional complex of β-catenin and TCF4, regulates Wnt signaling at the nuclear stage. Masude et al revealed that NCB-0846 affected the migration of TCF4 phosphorylated by TNIK in the concentration range of 01–03 uM [10]. Masuda et al reported that NCB-0846 affected the migration of TCF4 phosphorylated by TNIK in the concentration range of 01–03 µM and completely inhibited the phosphorylation of TCF4 at a concentration of 3 µM [10]. Our results showing that NCB0846 halved cell viability in MDAMB231 cells at a concentration of 3uM are consistent with the literature.

When the apoptotic effect of NCB-0846's 72-hour application, which is the most effective period, on MDA-MB-231 was evaluated, it was found that apoptotic cell rates were higher at all doses compared to the control group. When morphological changes were evaluated, degenerative changes in the cytoplasm as well as cell membrane ruptures (apoptotic body) were observed due to the increase in concentration. Morphologic changes and WST-1 viability analysis were consistent with apoptosis. This indicated that TNBC may be sensitive to Wnt/β-Catenin-targeted therapy. However, the results also showed for the first time that NCB-0846 also triggered cell death in MCF-10A cells at the active time. Flow cytometry was performed to reveal the mechanism of NCB0846-induced cell growth inhibition, and the results showed that both apoptosis and non-apoptotic cell death occurred after low-dose NCB0846 treatment at the effective hour. Necrotic cells were observed at the corresponding indicated doses. This suggests that this necrotic induction of NCB0846 in mammary epithelial cells may have occurred through the WntβcateninNFκB axis. Indeed, many studies have proven that Wnt/βcatenin signaling mediates necroptosis [15, 16].

The molecular mechanism of NCB0846 in non-cancerous breast cells is quite interesting. NCB0846 as a TNIK inhibitor suppressed βcatenin expression in MDAMB231 cells, but the corresponding gene expression increased in MCF10A cells at 1 and 2 µM concentrations after treatment (p < 0.01). As a matter of fact, it was observed that the apoptotic and non-apoptotic effects decreased in normal epithelial cells as the dose increased. Our qPCR analyzes suggest that this is due to the development of a possible resistance mechanism. Therefore, elucidating this mechanism will contribute to the therapeutic potential of wnt/βcatenin-targeted studies.

NCB-0846 demonstrated potent chemotherapeutic activity in different types of cancer. In a study showing the apoptotic effects of NCB-0846, it was found that apoptosis was induced via PARP-1 in the HCT-116 cell line [9]. Toru H. et al. showed that NCB-0846 administration increased apoptosis in osteosarcoma cells by increasing PARP-1 degradation [17]. We found that NCB-0846 caused cell arrest in G2/M phase in MDA-MB-231 cells due to its increased concentration. A similar study reported that NCB-0846 caused sub-G1 phase arrest in osteosarcoma cell [17]. In the study of Mari M. et al., NCB-0846 was shown to induce sub-G1 phase arrest in the HCT116 cell line [10]. However, our results showed that the dose of NCB-0846 that is cytotoxic in TNBC may also be toxic in noncancerous breast epithelium. This would limit the clinical benefit of NCB-0846, especially in TNBC. However, our data revealed that normal mammary epithelium had decreased CTNB1 expression and necrosis and increased cell viability at the IC50 for MDA-MB-231.

A more comprehensive understanding of the critical differences between breast cancer cells and normal cells in NCB-0846 treatment is needed. Understanding target function and the impact of target blockade, especially in the general molecular framework of normal and cancer cells, is crucial in selecting the most clinically relevant molecular targets for the individual patient [18].

In conclusion, in the treatment of triple negative breast cancer, NCB-0846 reduced proliferation and increased apoptosis due to Beta-catenin expression. However, our results revealed that NCB-0846 did not show selective cytotoxicity. Therefore, it is hoped that our study will contribute to the literature in the development of targeted therapeutics using NCB-0846 in combination with agents that have low bioavailability alone but are highly effective and tolerable in the treatment of breast cancer.

Acknowledgments

I would like to extend my sincere thanks to Gamze Guney Eskiler

Conflicts of Interest

The authors declare that they have no conflicts of interest

Funding

This study was funded by a grant from the Scientific Research Projects Foundation (BAP) of the Sakarya University of Turkey [Project No: 2021-7-25-9].

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Data Availability Statement

No Data associated in the manuscript.

Author Contribution

ATH, NK: study concept and design, acquisition of data, analysis, and interpretation of data, drafting and revision of the article, wrote the main manuscript text, final approval for the submission. NK: cell line analysis and preparation of all figuresED: data acquisition, drafting, and revision of the article, final approval for the submission. BK: acquisition of data, revision of the article,MY: data acquisition, article revision

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No competing interests reported.

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Determination Of Selective Cytotoxicity From Ncb-0846 In The Treatment Of Triple Negative Breast Cancer

Status:

Version 1

Abstract

BACKGROUND

METHODS

RESULTS

CONCLUSION

Figures

INTRODUCTION

MATERIAL METHOD

Cell Culture Conditions

Cell Viability Analysis

Annexin-V Analysis

Cell Cycle Analysis

Acridine-Orange/Ethidium Bromide (AO/EB) Staining

RNA Isolation and Quantitative PCR Analysis

Statistical Analysis

RESULTS

Evaluation of the Cytotoxic Effect of NCB-0846

Evaluation of the Apoptotic Effect of NCB-0846

Evaluation of the Changes Caused by NCB-0846 in the Cell Cycle

Determination of Morphological Changes Caused by NCB-0846 on Cells

DISCUSSION

Declarations

Author Contribution

References

Additional Declarations

Status:

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