Remarkable Effect of Increasing Glutamine Metabolism on Caveolin-1 in Breast Cancer: A Brief Report

doi:10.21203/rs.3.rs-2086201/v1

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Remarkable Effect of Increasing Glutamine Metabolism on Caveolin-1 in Breast Cancer: A Brief Report

https://doi.org/10.21203/rs.3.rs-2086201/v1

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Background

Caveolin-1(CAV-1) is a key molecule in cancer metabolism and progression and decrease in highly metastatic breast cancer cells. Moreover, glutamine is a crucial amino acid in the tumor microenvironment, and cancer cells are addicted to it. The effect of glutamine metabolism on caveoline-1 was not completely clarified. However, metformin has been demonstrated in separate studies to impair glutamine metabolism, and also affect caveoline-1 expression. Hence, the focus of this study is to evaluate the effect of glutamine metabolism on caveoline-1 expression, and to examine if metformin could affect caveoline-1 in high glutamine media.

Methods

In this study, 4T1 breast cancer cell line was cultured in different concentrations of glutamine. Caveolin-1 expression was assessed, using immunocytochemistry test. We also conducted MTT assay to evaluate optimum dose for metformin. To understand if metformin could regulate caveoline-1 expression in high glutamine media, 5mM of metformin was administrated and compared with control.

Results

our results indicated that glutamine metabolism significantly decreased CAV-1. On the other hand, administration of 5mM metformin for 24 hours efficiently re-expressed CAV-1.

Conclusion

High level of glutamine significantly decreased CAV-1, which may up-regulate the possibility of breast tumor speared, metastasis and metabolic reprogramming. Metformin, however, could be a promising drug, targeting glutamine metabolism to re-express caveoline-1, inhibiting tumor progression and metastasis.

Caveolin 1

Glutamine

Metformin

Neoplasm

Tumor Microenvironment

Catabolism of glucose and glutamine is necessary for production of carbon intermediates as building blocks, and ATP generation. In this regard, glutamine plays a crucial role in providing carbon and nitrogen for cancer cell rapid growth. Nitrogen provided by glutamine is also essential for producing purine and pyrimidine nucleotides, glucosamine-6-phosphate, and nonessential amino acids(1). Increased in glutamine consumption in cancer cells was first shown by Eagle who demonstrated that Hela cells need glutamine 10 to 100 fold more than normal cells(2). As shown by Yang et al. increase in glutamine metabolism disrupted cancer therapy, also de novo glutamine synthesis in cancer cells or stromal cells in the TME limits glutamine depletion therapies (3). On the other hand, anti neoplastic mechanism of metformin has been widely studied in recent years (4, 5). Moreover, a sturdy by Saladini et al. (6) clarified that metformin impaired glutamine metabolism via down regulation of glutaminase (GLS), affecting the proliferation of MCF7, MDA-MB-231, and cervical cancer cell line.

Furthermore, CAV-1 is located in the lipid raft structure of cell membrane involved in cell proliferation, regulation of cellular metabolism, and signaling. Regardless of the role of this molecule in cellular mechanisms, loss of CAV-1 is correlated by up-regulation of nitric oxide syntase (eNOS), and ROS generation. Increased in ROS, in turn derives mitochondrial dysfunction, and metabolic reprogramming toward glycolysis. Loss of CAV-1 in cancer cell results in increased antioxidant machinery and consequently chemoresistance, resistance to apoptosis, and finally metastasis(7).

Several reasons have been discussed about the loss of CAV-1, including autophagy, oxidative stress, and cytokines of TME (8). However, the effect of glutamine metabolism on loss of CAV-1 has not been completely clarified. Moreover, several articles confirmed the loss of caveoline-1 in cancer stroma and its poor clinical outcome (9-11), but the status of CAV-1 in breast cancer cells is still controversial. To obtain more insight into this issue, we studied the relationship between increased glutamine and loss of CAV-1, as well as evaluating therapeutic effect of metformin.

The 4T1 mouse breast cancer cell line was purchased from the Pasture Institute, Cell Bank of Iran (NCBI, Tehran, Iran). Glutamine free RPMI 1640 was purchased from Thermo Fisher Scientific (Catalog # 21870076). The cells were maintained in this media with 10% Fetal Bovine Serum (FBS), and penicillin 100 units/mL-streptomycin 100 µg/mL (Invitrogen). For low glutamine experiments the media was supplemented by 300 mg/L glutamine (Thermo Fisher Scientific), and high glutamine media was supplemented with 600 mg/L glutamine (2 fold increase in glutamine concentration), Sigma-Aldrich (Catalog # G8540). All the cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. Caveolin 1 polyclonal antibody was purchased from Thermo Fisher Scientific (Catalog # PA1-064). Secondary monoclonal antibody FITC conjugated was also purchased from Thermo Fisher Scientific (Catalog # 65-6111).

In this study, cytotoxicity of metformin for tumor and PBMC was assessed using MTT assay kit, which purchased from the Roche Company. Briefly, the cells were seeded in 98 wells plate and incubated for 24 h in humidified CO2 incubator. Then treated with serial concentrations of metformin and incubated 24 hours in humidified incubator. Following on, 100 µl of MTT reagent (0.5 mg/ml in PBS) was added to each well and then the plates were returned in the incubator for 4 hrs. The water insoluble formazan crystals were formed during incubation period that solublized by adding 100 µl of the solubilization buffer (10% SDS in 0.01 M HCl) to each well. After overnight incubation in above mentioned conditions, the absorbances of the solublized formazan dyes were measured using an ELISA plate reader

Venous blood (10 mL) was collected aseptically from a healthy donor in preservative free heparin tubes. The blood was diluted with phosphate buffered saline (PBS) pH 7.4, and layered onto Ficoll-(Paque™ PREMIUM) at the ratio of diluted blood with Ficoll 2:1. After centrifugation at 400 x g for 50 minutes, the lymphocytes were collected at the interface and washed three times with the PBS. The cells were re-suspended in RPMI with 10% (v/v) fetal bovine serum (FBS) and antibiotics. Before MTT test was conducted

12× 10³ 4T1 cells were seeded in 4 wells plate and incubated for 24 hours in humidified CO2 incubator before ICC test. Briefly, cells were treated with 5 mM metformin (Sigma-Aldrich), then fixed 30 minutes at room temperature in 2% paraformaldehyde diluted in PBS, then permeablized with triton X100 2% for 20 minutes. After that, cells were blocked with BSA 1% for 45 minutes. Primary antibody was incubated over night, and cells washed 3 times with PBS 1X before incubation with fluorescent conjugated (secondary) antibody for 3 hours. Finally, cells were washed 3 times at room temperature with PBS1X, therefore were ready for fluorescence microscopy (Olympus IX70 inverted microscope).

Average values were analyzed by GraphPad Prism 8 software, and the data expressed as mean ± standard error of the mean (StEM). Mean fluorescence intensity (MFI) of each photo was calculated by Image J software version 1.53s. Then, One way Annova test was used to compare the difference between the three groups. Differences were considered statistically significant when p < 0.05.

Decrease in cell viability, while increasing metformin concentration was seen in MTT test (Fig. 2).

We found that supplementation of media with 300mg/L glutamine (low glutamine) didn’t decrease CAV-1, and 4T1 cells expressed high level of CAV-1 in their cell membrane (P value: 0/0003). In the comparison with low glutamine group, the group of 4T1 cells cultured in high glutamine media (600mg/L) significantly decreased in CAV-1 (P value: 0/0003). Due to the crucial effect of CAV-1 in mitochondrial function, it is probable that loose of CAV-1 may contribute in mitochondrial mediated metabolic reprogramming of 4T1 cells.

4T1 cells seeded in 4 wells plate and after 24hours treated with 5mM metformin, after 24hours of treatment plate were ready for ICC test and fluorescent microscopy. We have found that treatment with metformin improved the expression of CAV-1, compare with control (a group of untreated cells cultured in high glutamine media) (P value: 0/0392) (Fig. 1). It is probable that metformin by inhibiting glutamine metabolism affect CAV-1 expression, which may also affect cancer metabolism and behavior. Also re-expression of CAV-1 by metformin could rearrange the shape of cancer cell membrane as shown in Fig. 2.

CAV-1 is a pivotal molecule in cellular functions, including metabolism, cell growth, and cell death(7). Previous studies confirmed that decrease in stromal CAV-1 abrogate the outcome of breast cancer (12). However, no study has verified whether the expression of CAV-1 in cancer cells is related to the metabolism of glutamine. In this study, we found for the first time that increase in glutamine metabolism down-regulates CAV-1, which is approximately compensated by administrating 5 mM metformin. Based on previous studies, decrease in CAV-1 promoted breast cancer cell progression via increase in aerobic glycolysis pathway, MCT4 expression, and matrix metalloproteinase MMP9 and MMP2 (13). Moreover, CAV-1 is considered as a prognosis marker in breast cancer, and knockdown of endogenous CAV-1 resulted in stimulating the proliferation of breast cancer cells. However, decrease in CAV1 is a strong predictor of tumor recurrence(14). Not only activating pro-survival signals such as PI3K/Akt and MAPK, but also inactivation of tumor suppressors BRCA1, P53 and PTEN were occurred as a result of loss of CAV-1(15).

Glutamine, however, compensate for the lack of enough nutritious and serve rapid tumor growth via glutaminolysis (GLS) pathway (conversion of glutamine to α-ketoglutarate). Therefore, the glutamine metabolism is urgent for tumor progression, metastasis, and drug resistance. Hence, tumor cells are known as glutamine addicted cells(16). Metformin by reducing the glucose metabolism improved the efficacy of BPTES-nanoparticles (GLS inhibitor) in hindering tumor growth. This combination therapy also decreased lactate level, glucose-6-phosphate, glucose-1-phosphate, and UDP-glucose, regarding metabolic stress and lack of enough ATP for tumor proliferation (17). In metabolic compartments, myofibroblasts lacking CAV-1 secrete more glutamine for cross-feeding of cancer cells. In addition, culturing cancer associated fibroblasts (CAFs) in high glutamine media suppressed CAV-1 via lysosomal degradation (18). However, study on 4T1 cell line revealed that highly metastatic 4T1.2 and 4T1.13 tumors express very low CAV1, and after inoculation of these cells into the mammary gland, moderate level of CAV-1 could suppress tumor growth and metastasis, and subsequently high level of CAV-1 could inhibit metastasis into bone, and lung(19). Thereby, metformin via re-expressing CAV-1 may hinder tumor metastasis.

Moreover, metformin as a promising drug in cancer therapy effectively improved the efficacy of anti cancer drugs via up-regulation of CAV-1 (20). In addition, re-expression of CAV-1 promotes better overall survival, and delayed onset age of breast cancer patients by metabolic reprogramming in cancer stem cells(21). Notably, metformin impaired glutamine metabolism (by down regulating glutaminase activity) in breast cancer cell lines, resulted in inhibiting tumor growth (6). Therefore, as we shown it is probable that glutamine metabolism affect CAV-1 expression and function, and on the other side, metformin by modulating glutamine metabolism re-expressed CAV-1. Hence, metformin may impact on tumor growth, and metastasis via metabolic reprogramming and modulation of CAV-1. However, further studies need to evaluate this idea in detail.

Funding

This work was supported by the National Institute for Medical Research Development (NIMAD; Grant No. 995813).

Conflicts of interest/Competing interests

All authors declare no conflict of interest.

Ethics approval

Not applicable

Informed consent

Not applicable.

Acknowledgements

This work was supported by the National Institute for Medical Research Development (NIMAD; Grant No. 995813).

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No competing interests reported.

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Version 1

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Remarkable Effect of Increasing Glutamine Metabolism on Caveolin-1 in Breast Cancer: A Brief Report

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Cell Culture And Antibodies

Mtt Assay

Pbmc Isolation

Immunocytochemistry And Fluorescent Microscopy

Statistics

Cell Viability Test Revealed Significant Anti Tumor Effect Of Metformin

Increased Glutamine Metabolism Significantly Restricts Cav-1 In 4t1

The potential healing effect of metformin on re-expression of CAV-1 in high glutamine media

Declarations

References

Additional Declarations

Status:

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