Investigation of the effects of small VCP-interacting protein (SVIP) on the migration and invasion of breast cancer cells.

doi:10.21203/rs.3.rs-2614791/v1

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Investigation of the effects of small VCP-interacting protein (SVIP) on the migration and invasion of breast cancer cells.

https://doi.org/10.21203/rs.3.rs-2614791/v1

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Background: Endoplasmic reticulum-associated protein degradation (ERAD) eliminates misfolded proteins. Numerous illnesses, including cancer, cystic fibrosis, diabetes, and neurological diseases, are influenced by ERAD. Although there are many proteins involved in ERAD, we focused on small VCP-interacting protein (SVIP).The presence and the function of SVIP in breast cancer types has not been studied yet. The aim of the study was to investigated the expression of SVIP, in breast cancer types and to determine whether SVIP contribute to the migration and invasion of breast cancer cells.

Material and methods: Normal (MCF 10A), estrogen positive (MCF-7) and triple negative (MDA-MB 231) breast cancer cell lines were used in this study. Presence and cellular localization of SVIP in breast cancer cells were examined by immunohistochemistry. Western blot analysis was used to evaluate protein expression after SVIP siRNA transfection of the cells. The consequences of this suppression on cell invasion and migration were identified by using the xCELLigence real-time analysis system (RTAC).

Results: The ımmunoexpression of SVIP was observed in the cytoplasm of MCF 10A, MCF-7, and MDA-MB 231 breast cancer cell lines. The transfection of the cells with SVIP siRNA led to a reduction in the protein expression of SVIP. SVIPsi RNA suppression results in decreased invasion and migration of MCF 10A cells on the other hand increased invasion and migratory potential of MDA-MB 231.

The MCF 7 cell index increased in the invasion setting, although migration was decreased.

Conclusion: We showed that the protein expression and cellular localization of SVIP in normal, estrogen positive and triple negative breast cancer cell lines. The capacity of breast cancer cells to migrate and invade changed when the three cell types were transfected with SVIPsi.

Breast cancer cell lines (MCF 10A

MCF-7 and MDA-MB 231)

Endoplasmic reticulum-associated protein degradation (ERAD)

SVIP

invasion

migration

The most frequent type of cancer among women is breast cancer. The expression of estrogen and progesteron receptors (ER and PR) and human epidermal growth factor receptor 2 (HER2) are the most important markers in patient focused therapy [1]. Patients with invasive or noninvasive breast cancer have a wide range of difficult therapy choices. The Clinical Practice Guidelines in Oncology for Breast Cancer guide recommends determining ER and PR status in all primary invasive breast cancers patients, regardless of the patient's age, axillary lymph node status, or whether they have received adjuvant chemotherapy [2]. Triple-negative breast cancer (TNBC) is a diverse group of cancers and patients with TNBC, lacking ER and PR expression as well as HER2 amplification [3,4].

The biology of breast cancer has become more dependent on androgen receptor (AR) signaling. According to reports, the androgen receptor (AR), a hormone receptor, is utilized to predict the prognosis of breast cancer.AR is nuclear steroid hormone receptor and also expressed in normal breast tissue [5]. ER and AR regulate cell proliferation and differentiation in breast cancer. Although AR was also detected in ER-negative breast cancer, AR and ER are typically expressed together. In ER positive breast cancer cells, androgens suppress cell proliferation while they stimulate it in ER negative breast cancer cells. According to reports, a crucial molecule in prognosis of breast cancer is AR [6,7].

An established technique for eliminating misfolded or unfolded proteins in order to regulate the quality of proteins is endoplasmic reticulum-associated degradation (ERAD).For both lipid metabolism and protein homeostasis, ERAD must be tightly regulated. A key protein in the retrotranslocation stage of ERAD is p97/VCP. The various binding partners of p97/VCP, including the endogenous ERAD inhibitor small VCP-interacting protein, are essential for the multifunctionality of this protein (SVIP) [8].The 76 amino acids that make up the 9-kDa protein SVIP are involved in the ERAD pathway [9].

According to reports, androgens control the ERAD pathway in prostate cancer cells and in response to androgen that only SVIP is decreased, but other ERAD proteins are up-regulated. Furthermore, SVIP has also been defined as an ERAD inhibitor [10]. According to research by Bao et al, nervous system cell lines have significant levels of AR protein expression. In addition, tumor tissue from glioma patients shows elevated AR expression whereas SVIP expression is lowered [11].

Many diseases, including cancer, cystic fibrosis, diabetes, and neurodegenerative disorders, are influenced by ERAD [12].Several molecules have been investigated in breast cancer prognosis [7,13].

However, SVIP expression in breast cancer subtypes has not been studied yet. The purpose of this study was to examine the expression of SVIP, one of the ERAD pathway proteins, as well as its effects on breast cancer invasion and migration.

Cell culture and transfections

Normal human mammary epithelial cells (MCF 10A) estrogen-positive (MCF-7), and triple-negative (MDA-MB 23) breast cancer cell lines obtained from ATCC. The cells were grown in DMEM-F12 with 8% fetal bovine serum (Gibco), 1% antibiotics (penicillin/streptomycin), and 5% horse serum in a humidified incubator at 37 °C. Following the manufacturer's instructions, transfections were carried out in 12-well plates using Lipofectamine 2000 (Invitrogen).MCF 10A, MCF-7, and MDA-MB 231 cells were plated at 50,000 cells per well the day before the transfection. According to the manufacturer's recommendations, serum-free medium, transfection reagent, and either SVIP siRNA (AM16708, 508716, Invitrogen) or a negative control siRNA (AM4611, Ambion, Thermo Fischer) were combined. After 6 hours of transfection, fresh medium (500 mL/well) were added. The cells were transfected, and 48 hours later they were lysed for western blotting.At least three duplicates of each transfection were performed.

Immnohistochemistry

For immunocytochemical studies, cells were cultured in 12-well dishes. After washing the cells 3 times with PBS, they were fixed in 4% formalin for 15 minutes. The cells were washed with PBS 3 times for 5 minutes and treated with blocking solution for 1 hour. After blocking, without washing the cells were incubated with the primary antibody (SVIP, 1:50, HPA039807; Sigma) at 4 °C overnight. The next day, they were washed 3 times with PBS for 5 minutes, incubated for one hour at room temperature with secondary antibodies, and washed again with PBS. DAB chromogen was used to incubate the cells and image taken through a light microscope (Olympus BX43).

Immunofluorescence

40,000 cells per well in 12-well plates with glass coverslips were seeded. Cells were then permeabilized with 0.1% saponin in PBS after being fixed in 4% formaldehyde for 15 min. The coverslips were washed with PBS before being incubated with SVIP primary antibody (1:50, HPA039807; Sigma) for overnight at 4 °C. FITC-conjugated anti-rabbit secondary antibody (1:500 dilution; Abcam) and Texas red conjugated anti-mouse secondary antibodies (1:500 dilution, ab6787; Abcam) were used to incubate on the coverslips after PBS washes. At room temperature, the coverslips were mounted using DAPI-containing mounting medium in 1X PBS.Under a fluorescence microscope (Olympus BX53), at magnifications of 40× .the SVIP protein's intracellular location was investigated.

Western blot

Normal human mammary epithelial cells (MCF 10A), estrogen-positive (MCF-7), and triple-negative cells (MDA-MB 231)were used for Western Blot. Trypsin (Pan Biotech, Aidenbach, Germany) was used to separate the cells after they had been washed in DPBS (ThermoFischer Scientific, Waltham, MA, USA).The cell pellet was resuspended in phosphatase and protease inhibitor (Roche Applied Science, Penzberg, Germany) containing RIPA buffer (Sigma-Aldrich, USA). The cell lysate solution was centrifuged at 14,000 rpm for 40 min at 4 °C after the cells were mechanically killed.

The typical Bradford test was used to calculate the protein concentration. 30 g of total protein were loaded onto a 4-12% Bis-Tris gel (Invitrogen) and transferred onto a nitrocellulose membrane after being boiled for 10 minutes in a denaturing sample solution. 3% milk in PBS was used for membrane blocking. The primary antibody rabbit polyclonal anti-SVIP was used to detect SVIP (1:500; HPA039807; Sigma). The membrane was rinsed with PBS-Tween for 30 minutes after being incubated with the primary antibody for an entire night. The membrane was rinsed with PBS-Tween for 30 minutes after being incubated with the primary antibody for an entire night. A gel documentation system (UVP, Cambridge, UK) was used to view the Western blot bands, and IMAGEJ Version 1.46 was used to assess the band densities.

Analysis of Invasion and Migration with xCELLigence Real-Time Cell Analyzer system (RTCA)

The matrigel which mimics the extracellular matrix is used in the invasion tests. The purpose of these tests is; to determine whether the cells pass from one side of the matrigel to the other by degrading the matrigel in the environment by secreting various proteases. Cells are put into FBS-free media. The medium was then switched to serum-free medium for 6 hours, and a 1x10⁴/90 µL cell suspension was made. 165 μL of 20% FBS medium were used to fill the lower chamber for the migration assay. After that, the two compartments were secured, and the upper chamber was filled with 90 μL of the serum-free medium. The RTCA instrument was filled with the CIM-plates 16 (ACEA Biosciences) and allowed to equilibrate for 1 hour. 90 µL of cell suspension were placed in each well of the CIM-plate 16 before being observed for 96 hours in the RTCA instrument. The same numbers of cells were seeded into the top transwell chambers for the invasion assay after coating them with matrigel [14].

Statistical Analysis

Immunocytochemistry, Western blot, and density determination studies were performed with the Image J (NIH) program, the expression degrees were shown as mean ± standard error. Invasion-migration value was evaluated using the GraphPad Prism software (Graphpad, San Diego,CA). Data between groups were analyzed for significance using an ANOVA followed by Turkey’s multiple comparison tests. Statistical significance level was accepted as p<0.05

Immunohistochemistry and Immunofluorescence Results

There is presently no information on the potential location of SVIP in breast cancer subtypes. Therefore, we determined the cellular localization and immunoexpression of SVIP in breast cancer cells. SVIP was detected in the cytoplasm of normal (MCF 10A), estrogen positive (MCF-7) and triple negative (MDA-MB 231) breast cancer cell lines shown in Figure 1. MCF-7 cells had intense SVIP staining in the cytoplasm compare to MCF 10A and MDA-MB 231.

Western Blot Results

The protein expression of SVIP was determined in MDA-MB 231, MCF-7 and MCFA10A shown in Figure 2A. Control si (Csi) and SVIP siRNA (SVIP si) transfection was performed intoMDA-MB 231 MCF-7 and MCF 10A breast cancer cells to determine whether the protein expression of SVIP was affected as a result of this suppression. After transfection with Csi and SVIP si RNA, western blot results revealed that SVIP si protein expression was decreased in three breast cancer cell types. Furthermore the decreasing in the protein expression of SVIP si was significant in MDA-MB 231 and MCFA10A cells compare to control (Figure 2B).

Invasion and Migration Findings with xCELLigence (RTCA)

xCELLigence Real-Time Cell Analyzer system was used to determine the invasion and the migration characteristics of in three breast cell (MDA-MB 231, MCF-7 and MCF 10A) lines. Analyzes were performed in CIM 16-well plates. Invasion analyzes were performed with matrigel however migration analyzes performed in wells without matrigel. The cell indices were shown in graphs.

The RTCA assay showed that the invasion and migration capacity of MDA-MB 231 was increased (Figure 3). In the invasion setting the cell index of MCF 7 increased on the other hand the migration was decreased (Figure 4) The decrease of cell migration and invasion of MCF10Awas shown in Figure 4.

Cancer cells must have balanced protein synthesis, folding, quality control, and degradation in order to grow and endure a variety of cell-intrinsic and extrinsic stresses, such as hypoxia or food restriction. These altered cells dependent on proper protein homeostasis due to their faster rate of protein synthesis and cell cycle [15].

The ER is able to maintain protein homeostasis. High protein synthesis rates and a rise in misfolded proteins in cancer cells might cause an ER stress response that will eventually cause apoptosis [16]. The UPR and ERAD pathways are primary ER stress adaptation mechanisms [17]. To reduce the proteotoxic stress, they both result in global protein attenuation and the ER clearance of misfolded proteins. Numerous studies have been conducted on the UPR network in relation to human malignancies [18].

The physiological importance of SVIP in the adrenal gland was investigated by Ilhan et al using human adrenal corticocarcinoma H295R cell line. Also, they used rat adrenal gland tissues to assess the postnatal developmental levels and zonal expression pattern of SVIP. Their results suggest that SVIP has an unique role in the production of cortisol and DHEA as well as the homeostasis of cells in the adrenal cortex [8].

Llinàs-Arias et al investigated the endogenous ERAD inhibitor SVIP experiences DNA hypermethylation-associated silence in cancer. They have concluded that the recovery of SVIP is linked to increased ER stress and growth inhibition, and demonstrated its tumor suppressor characteristics.. Cancer cells with epigenetic loss of SVIP are found to have low levels of mitochondrial enzymes and oxidative respiration activity, according to proteomic and metabolomic investigations. SVIP restoration results in the reversal of this phenotype [19].

It has been demonstrated that testosterone regulates the ERAD pathway in androgen-responsive prostate cancer cells. Prostate tumor development has been found to be positively correlated with higher ERAD proteolytic activity, which is increased when component levels are controlled [10].

The cell cycle is regulated by the tumor suppressor p53, which is frequently inactivated in many cancers. As a result, p53 mutations impair apoptosis and cause genomic instability. Approximately 10% of initial glioblastoma multiform have p53 mutations [20].

A well-known tumor suppressor called p53 can inhibit cell proliferation and encourage apoptosis. When SVIP was overexpressed, it increased apoptosis, and siRNA removal decreased the protein level in the p53^wt cell line without affecting the p53 mRNA. This demonstrated that SVIP at least partially suppressed cell proliferation in a p53-dependent way. They discovered that androgens stimulate AR signaling, which promotes glioma cell proliferation. SVIP, an AR response gene, was then transcriptionally negatively regulated. Despite the mutual repression of AR-p53, decreased SVIP expression may cause decreased p53wt expression and facilitate the course of the malignancy that AR antagonists may be able to stop [19].

It is known that some breast tumors show a different aggressive course. Since clinical data are insufficient to predict prognosis in these cases, new prognostic factors were determined by ımmunohistochemical and molecular techniques. Some factors, such as steroid receptors, are widely used in routine practice, but the status of these receptor-associated SVIP in breast cancer subtypes is unknown. In our study, we first determined the immunoexpression of SVIP in the cytoplasm of normal MCF10A and breast cancer subtypes MCF7 and MD 231.

AR is expressed as about 60–90% of all breast cancer cases [21]. It has known that androgens are used as tumor suppressors by their anti-proliferative effects in ER positive breast cancers. Several cancer types and diseases such as prostate cancer, breast cancer, diabetes, metabolic syndrome, cardiovascular diseases and Alzheimer's are associated with androgen signaling for this reason, it is important to reveal the biological mechanisms that are regulated by androgens [22].

It has been reported that SVIP has an inhibitory effect on ERAD in cancer cells. In most of human tumors, silencing of SVIP results in up-regulation of VCP, to prevents programmed cell death in ERAD [23].

It is necessary to know the expression levels of the proteins that belongs to the ERAD family in different breast cancer subtypes in order to reduce the toxicity and the spread of breast cancer in the most effective way and to ensure its use in prognosis and treatment. There are many proteins (p97/VCP, HRD1, Derl1, gp78, grp78, ubiquitin) play important roles in the ERAD [24].

In the current study we demonstrated the protein expression of SVIP in MDA-MB 231, MCF-7 and MCFA10A by using Western blot. Three breast cancer cell types were transfected with control si and SVIP si RNA to determine whether the protein expression of SVIP was affected as a result of this suppression. Western blot analyses were carried out 48 hours after transfection, it was discovered that the levels of SVIP si protein were much lower than those of control si in MDA-MB 231, MCF-7, and MCF 10A.

Cancer cell viability, migration, and invasion can be tracked using conventional assays such the MTT. The invasive potential of bladder cancer cells can now be objectively measured and monitored in xCELLigence Real-Time Cell Analysis (RTCA) [25,26]. The greater sensitivity of RTCA techniques enables a better identification of novel molecules involved in the development of metastatic lesions and urothelial carcinoma of the bladder progression [27,28].

Head and neck squamous cell carcinoma, which is aggressive and has a high recurrence rate, is the sixth most common cancer in the world. In a variety of human cancers, the anti-proliferative, pro-apoptotic, and anti-metastatic properties of S adenosyl L methionine (AdoMet) have been thoroughly investigated. In one study, the researchers examined the underlying mechanisms and the impacts of AdoMet on the invasion and migration of head and neck squamous carcinoma cells. They discovered that AdoMet triggers cell cycle arrest and reduces the capacity of invasion and migration of HNSCC, oral Cal-33 and laryngeal JHU-SCC011 cells [29].

Triclosan has been taken by humans as a result of its pervasive use as an antibacterial component in household and personal care products. It may be linked to breast cancer due to its prevalence in human tissues, including milk, and its oestrogen-disrupting characteristics. One study used human breast epithelial cell lines MCF-10F, MCF-7, and DA-MB-231 to analyze how triclosan affected cell invasion and migration. All three cell lines increased their migration and invasion after prolonged exposure to 10^− 7 M of triclosan, as determined by xCELLigence technology [30].

Erzurumlu and Ballar discovered that inhibiting the expression of the ERAD genes Hrd1, SVIP, and gp78 decreased the migration and malignant transformation of prostate cancer cell lines (LNCaP) cells in vitro. Their findings imply that androgens influence the expression of ERAD components, promoting its proteolytic activity, which is favorably correlated with development of prostate tumors [10].

Increased cell invasion and migration are crucial to the metastatic tumor's progression [31]. The suppressive effects of SVIPsi on cell invasion and migration were examined in the current investigation using the xCELLigence real-time analysis system. The results shown that the invasion and migration capacity of MDA-MB 231 was increased, while the same effect results in the decreased of cell index of MCF10 A. On the other hand the invasion of the cell index of MCF 7 increased and migration was decreased. According to the literature investigations in the current study we discovered that the suppression of SVIP results in the decreasing migration capacity of MCF 10 A and MCF 7 however increased in the invasion and migration of MDA-MB 231.

In conclusion in this study we demonstrated that cellular localization and protein expression of SVIP in MDA-MB 231, MCF10 A and MCF7 breast cancer cell lines. Furthermore when three cell types were transfected with SVIPsi affects the protein expression and migration and invasion capacity of MDA-MB 231, MCF10 A and MCF7 cells.

Funding: This study was founded by Ankara Yıldırım Beyazıt University Scientific Research Projects (grant number TSA-2020-2030).

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

Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest: Author Bahar Kartal declares that she has no conflict of interest. Author Ebru Alimoğulları declares that she has no conflict of interest. Author Sevil Çaylı declares that she has no conflict of interest.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Bahar Kartal, Ebru Alimoğulları and Sevil Çaylı. The first draft of the manuscript was written by Bahar Kartal and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Investigation of the effects of small VCP-interacting protein (SVIP) on the migration and invasion of breast cancer cells.

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