Research article

The Effect of Engineered-TP53 on Breast Cancer Cells Growth by The Advantage of Adeno-associated Viral Particles Delivery System

Negar Moghare Dehkordi^1,2, Sadegh Paydari Rostami^1,2, Mohammad Reza Bolouri^3,4, Azam Samei⁵, Yazdan Asgari¹, Reza Falak^3,4 and Gholam Ali Kardar^1,2*

¹Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Iran

²Immunology Asthma & Allergy Research Institute, Tehran University of Medical Sciences, Iran

³Immunology Research Center, Iran University of Medical Sciences, Iran

⁴Department of Immunology, School of Medicine, Iran University of Medical Sciences, Iran

⁵Department of Clinical Laboratory Sciences, School of Allied Medical Sciences, Kashan University of medical Sciences, Iran

Submission: January 17, 2021; Published:February 11, 2022

*Corresponding author:Gholam Ali Kardar, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

How to cite this article:Negar Moghare D, Sadegh Paydari R, Mohammad R B, Azam S, Yazdan A, et al. The Effect of Engineered-TP53 on Breast Cancer Cells Growth by The Advantage of Adeno-associated Viral Particles Delivery System. Int J Cell Sci & Mol Biol. 2022; 7(1): 555705. DOI: 10.19080/IJCSMB.2022.07.555705

Abstract

Accumulation of mutations in the genome, especially in the tumor suppressor p53 (TP53) sequence can gradually promote normal cells toward cancer development. As a treatment strategy, regeneration of TP53 could induce apoptosis and cell cycle arrest in tumor cells. In this study, by developing, an adeno associated virus-based vector (AAV) with an engineered TP53 sequence (AAV-TP53), we tried to remove the inhibitory effects of cellular factors on TP53 as well as enhancing TP53 homo-tetramerization, and consequently, increase its tumor suppressor effects. After optimization of the target residues in the TP53 sequence (vector designation) and production of AAV-TP53 in HEK293T cells, the virus was transduced in SKBR3 breast cancer cells. The expression/activation of engineered TP53 downstream genes, including p21 (as the most critical downstream gene), and BAX (as an apoptosis-related gene) were examined by relative Real-time PCR. Apoptosis rate was also measured by AnnexinV-PE/ 7AAD staining. The engineered TP53 was highly expressed in SKBR3 cells and consequently, the expression level of p21 and BAX genes were significantly increased (p<0.0001). Furthermore, transduced cells demonstrated a higher apoptotic rate than control and mock samples (p<0.0001). We showed that using engineered TP53 could be an effective method among many TP53-related gene therapy methods for cancer treatment.

Keywords: Tumor suppressor protein p53; Gene therapy; Adeno-associated virus; Apoptosis; Breast cancer

Abbreviation: TP53: Tumor Suppressor Protein 53; p21 (CDKN1a): Cyclin-Dependent Kinase Inhibitor; MDM2: Mouse Double Minute2; HPV: Human Papillomavirus; AAV: Adeno Associated Virus; CPP: Cell-Penetrating Protein; HCQ: Hydroxychloroquine

Introduction

Cancer is characterized by uncontrollable proliferation of cells and is the second cause of death after cardiovascular disease [1]. Gene therapy is an attractive cancer treatment method and currently several FDA-approved gene therapy products are available [2]. Human tumor suppressor p53 (TP53) as the guardian of the genome plays crucial roles in cellular and genomic integration and exerts its anti-tumor effects by controlling the expression level of cell proliferation-related genes, cell cycle arrest and apoptosis [3]. Accordingly, cyclin-dependent kinase inhibitor 1 (p21) is the major downstream factor of TP53, which controls cell cycle arrest [4,5]. In more than 50% of cancer cases, various genetic alterations including missense somatic mutations, especially in DNA binding domain hotspots’ of TP53 were observed [6]. Moreover, uncontrolled proteasomal degradation of TP53 have been observed in some cases.

Briefly, reactivation of TP53 could be a reliable approach for treatment of cancer. Restoration of wild type TP53 (wt-TP53) functionality increases the rate of apoptosis in tumor cells but would be harmless for normal cells [7]. Mutated TP53 consists of complex proteins with different characteristics and targeting this mutant protein and simultaneously restoring a wild type of form is rather a complicated process [8]. Meanwhile, it was shown that substitution of some amino acids might reduce TP53 interaction with its inhibitor, mouse double minute-2 (MDM2), and restore TP53 activity and consequently increase the expression of downstream genes such as p21 and Fas ligand [9]. Oligomerization of mutated TP53 with a wt-TP53 will form a nonfunctional structure, which is a challenging issue in gene therapy using wt-TP53. A coiled-coil (CC) structure was previously found in the breakpoint cluster region protein (BCR) that can form a tetrameric shape similar to the tetramerization domain (TD) in wt-TP53. This structure could be a good substitution and might provide a tetramerization domain for oligomerization of TP53 molecules. Okal et al. demonstrated that TP53-CC could be expressed without any interference with mutant TP53 [10-12]. Furthermore, the accelerated anti-tumor effect of TP53-CC in comparison with wt-TP53 was proven by viral delivery of TP53- CC to xenograft tumor model of breast cancer [13].

The HPV-E6 protein has inhibitory effects on TP53 through direct binding and promoting its ubiquitination. Thus, tumors with E6 expression have resistance to TP53 therapy. Heideman et al. [14] showed that substitution of asparagine with aspartate (N268D) in murine models will result in no interaction of TP53 with E6 protein and simultaneously, would not affect TP53 functionality [11,14]. Cell-penetrating peptides (CPPs) such as those with 11 arginine residues (11R) are suitable candidates to transfer proteins to cytoplasm. Several studies showed the 11RTP53 fusion improves the efficiency of protein entrance to cancer cells in-vitro [15,16].

Gene therapy, through providing a standard form of the gene, could be used for molecular correction of genetic mutations. For an efficient gene therapy, it is critical to adjust target gene expression levels to reduce any possible side effects. Additionally, providing a mechanism for gene silencing is essential to prevent cellular damages [17]. Viral vectors are one of most effective and pioneering delivery methods for in-vivo gene therapy, among them; adeno-associated virus (AAV) vectors are the safest vectors and have been utilized in many gene therapy clinical trials [18]. The first AAV-based drug was “Glybera,” which was approved in 2012 by European medicines agency for treatment of lipoprotein lipase deficiency. Later on, in 2017, “Luxturna” was approved by the FDA for treatment of inherited retinal dystrophy caused by bi-allelic RPE65 gene mutation [19,20]. Despite those excellent improvements, there is still a lack of knowledge on how to deliver AAV vectors to the host cell nucleus without any interference. The virus will encounter with inhibitory macromolecules such as proteases and also should resist against the host’s immune system as another limiting factor for successful transduction [18,21]. It is believed that Hydroxychloroquine (HCQ) as an inhibitor of antiviral pattern recognition receptor may enhance the AAV transduction rate [21] and metallic ions such as copper and zinc could augment AAV entrance to several cell lines [18].

In this study, we designed an engineered TP53 expressing construct and through an AAV-based system, we delivered it to cancer cells containing mutant TP53. Consequently, the efficacy of the construct in promoting cell apoptosis and expression of downstream genes, especially p21 and BAX were studied.

Material and Methods

Cell culture and reagents

HEK293T and SKBR3 cell lines were purchased from the Pasteur Institute of Iran (Tehran, Iran) and cultured in high glucose DMEM (Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and penicillin-streptomycin (Gibco, USA) (complete medium) and maintained at 37°C in a humidified incubator containing 5% CO2 (optimum condition).

Plasmid’s construct

Engineered TP53, composed of 1242 bp, encoding 413 amino acid residues were designed and cloned in AAV-ITR vector (Vector Builder, Chicago, USA). The amino acid sequences of Thr18, Ser20 and Asn268 were modified to aspartate and the tetramerization domain of the TP53 was substituted with BCR coiled-coil domain expressing 72 amino acids. G4S sequence was considered as the linker and 11R peptide (composed of 11 arginine residues) was added to the C terminal region of TP53 for better cellular penetration of the protein. To facilitate a high expression rate, the EF1α promoter was included, and as demonstrated in the vector schematic map (Figure 1), the IRES sequence was followed by EGFP protein sequence to monitor the rate and efficiency of transfection and transduction steps. Other necessary vectors including pAAV-DJ (Rep/cap) and pHelper (pDeltaAdF6) were purchased from Cell Biolab (Santiago, CA USA). All vectors were propagated in competent E. coli strains./p>

AAV production and titration

One day before transfection, 4-5×106 HEK293T cells were plated in 10Cm cell culture dishes (Biofil, Korea) to achieve 70-80% confluency at the transfection time. Cells were transfected with a mixture of three vectors necessary for AAV production. Three plasmids including pHelper, pAAV-DJ, and the plasmid harboring the gene of interest/mock (pAAV-TP53 or pAAV-IRES-GPF as mock sample) (Cell Biolab, Santiago, USA) were transfected at the ratio of 1:1:1 (8μg each) using 40-50μg polyethyleneimine (PEI; Sigma, UK) with 2μg/μl concentration as transfection reagent. Twentyfour hours post-transfection, the whole media was replaced with DMEM containing 2-3% FBS. Approximately, 72h after successful transfection, appropriate volume of 0.5M EDTA solution was added to medium to obtain a final concentration of 10mM and the plate was incubated for three min in RT. Then, the cells were scraped by a cell scraper (SPL, Korea), and centrifuged for 5min at 400g and the pellet was suspended in a desired amount of DMEM. Then the cell lysate was prepared by five times freezing and thawing in liquid nitrogen followed by short incubation on 37°C thermomixer (Eppendorf, Hamburg, Germany). Following centrifugation at 13000g for 10min, the supernatant containing AAV particles (AAV-TP53, AAV-IRES-GFP) were collected and used in virus purification and concentrating steps. For long-term storage, the supernatant was aliquoted and kept at -80°C. AAV titration was done by Quantitative PCR (qPCR) analysis, using the Qiagen rotor gene Q5 plex HRM real-time PCR system (Dusseldorf, Germany). To determine a linear dynamic range for AAV-TP53 in QPCR, serial dilutions of AAV-TP53 were prepared within 6 logs (based on the standard plasmid titration in the range of 2×103 to 2×109 plasmid copies), with each dilution being assayed in 10 replicates according to Aurnhammer et al. recommendation [22]. ITR primers used in the QPCR are shown in Table 1.

Toxicity assessment

For better transduction rate with the least cellular toxicity, we applied a suitable amount of HCQ as AAV transduction enhancer. Cell proliferation assay with 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma, USA) was performed to determine the optimum concentration of HCQ [21]. Briefly, 3.5×103 SKBR3 cells were seeded in 200μl complete medium in 96-well culture plate (Nunc, Denmark). Next day, cells were treated with diluted HCQ ranging from 1.5 to 48μM in DMEM with 5% FBS in triplicate manner. After 48h, 20μl MTT stock solution (5mg/ml; Sigma, USA) was added to each well. After 3h, the media contained MTT solution was substituted with 100μl dimethyl sulfoxide (DMSO; Sigma, USA) and the plate was maintained on an orbital shaker for 15min. Then the absorbance was read at 570nm versus 630nm as a reference wavelength.

AAV transduction

Twenty-four hours before transduction, SKBR3 cells were seeded at 1.5×105 cells/well in 6 well plate with complete media and incubated overnight at optimum condition. The next day, one hour before transduction, the media was substituted with 1ml DMEM containing 5% FBS and 20μM HCQ as the optimized concentration. After one hour incubation, the HCQ concentration was reduced to 10μM by adding desired amount of AAV-TP53 or AAV-IRES-GFP to specified wells. Forty-eight hours posttransduction, the media was refreshed, and the incubation was continued for 24h. To analyze the transduction rate, the GFP expression was examined under the invert fluorescent microscope after 72h. Moreover, the cells were detached by trypsinization and studied by flow cytometry.

Real-time PCR (RT-qPCR)

Total RNA was extracted using TRIzol^® reagent (GeneAll, Seoul, South Korea) 72 h post-transduction and the RNA quality was evaluated with NanoDrop 2000c spectrophotometer (Thermofisher, USA). Then, cDNA was synthesized from 1μg/μl of RNA according to the manufacturer’s instructions (SMOBIO technology, Taiwan). The real-time PCR was performed in 20μl reaction volumes, containing 10μl of 2X SYBR-Green master mix (Biofact, Daejeon, South Korea), 1μl cDNA, and 1μl of both reverse and forward primers. Two-step thermal cycling consisted of 1 cycle of 95°C for 15min, followed by 40 cycles of 95°C for 20s, and variable annealing temperatures according to each primer, for 40s. The details of the designed primers are shown in Table 1. HPRT1 was considered as the housekeeping gene and the 2-(ΔΔCT) value was calculated to get the relative expression rate.

Apoptosis assay

Approximately 6×104 SKBR3 cells were cultured in 12-well plate and incubated at optimum condition for 24h. Then, cells were transduced by AAV-TP53 or AAV-IRES-GFP, according to the transduction protocol mentioned above. After 72-96h posttransduction, cells were trypsinized and Annexin V-PE/7AAD staining was performed by a commercial kit (IQ products, the Netherlands). Briefly, 100μl Annexin V binding buffer, 6μl of Annexin V conjugated with PE and 6μl of 7-AAD were added to cell suspension. After 15-20min incubation at RT, in a dark place, 300μl of phosphate-buffered saline (PBS) was added to each tube and the percentage of the apoptotic and necrotic cells were measured by flow cytometry.

Statistical analysis

Statistical parameters and tests are reported in the description of each figure. All gene expression level data were presented as mean (±SD). One-way ANOVA and Bonferroni analysis was performed for all datasets that required comparison among more than two independent groups. GraphPad Prism 8 (San Diego, CA, USA) and Flowjo Software were used to present the statistical and flowcytometry data, respectively.

Results

Transfection efficiency and AAV titration

The transfection rate of HEK293T with triple vector system including pAAV-TP53, pHelper, and pAAV-DJ was more than 90% as studied 72h post-transfection (Figure 2A & 2B). Moreover, 72h post-transfection the morphological evaluation of HEK293T cells revealed them as round cells, pointing out to production of AAV particles (Figure 2C & 2D). AAV-TP53 titration by qPCR, showed that a six-log serial dilution of standard plasmid, ranging from 2×103 to 2×109 particles exhibited a linear correlation coefficient (RSq) of 0.997, a slope of -3.221, and an efficiency of 104% for ITR sequence.

Toxicity assessment demonstrated appropriate concentration of HCQ for SKBR3 cells

The MTT assay demonstrated that HCQ at concentrations above 20μM could be toxic for SKBR3 cells compared to the control group. Treatment of SKBR3 cells with 20μM HCQ is a necessary step in transduction. However, we found that after onehour treatment, reducing the final concentration of HCQ to 10μM could be beneficial and could prevent cell toxicity (Figure 3).

The AAV-engineered TP53 transduction efficiency on SKBR3 cells

Based on AAV titration quality in each transfection test of Hek293T cells, the result of transduction before following tests including Real-time PCR and apoptosis assay were differed. According to flowcytometry analysis, AAV transduction rate before Real-time PCR and apoptosis assay in AAV-engineered TP53 treated samples was 88.7% and 28.2% respectively (Figure 4A & 4B).

The BAX and p21 as downstream genes of engineered- TP53 expressed significantly after transduction in SKBR3 cell line

As expected, the expression rate of HPRT1 as the normalizer gene was equal in the transduced and control samples. Gene expression of p21 was analyzed 72 h post-transduction, which was significantly higher than control and mock samples (Figure 5A, p < 0.0001 and p < 0. 001, respectively). Similarly, the expression rate of BAX as one of main pro-apoptotic genes was significantly higher than control and mock samples (Figure 5B, p < 0.0001 for both of them). Obviously, the expression of the engineered TP53 was considerably higher than controls (Figure 5C, both p < 0.0001). However, for all mentioned genes, the expression rate in control and mock samples were similar, and we did not observe any difference between them.

Engineered TP53 stimulated apoptosis in SKBR3 cells

Total apoptosis by Annexin V-PE/7AAD in negative control, mock and AAV-engineered-TP53 treated samples were 3.29, 18.65, and 35.4% respectively, and the cells became granulated in comparison with the mock sample. Moreover, Expression of engineered TP53 significantly induced apoptosis of SKBR3 cells compared with negative control and mock samples (p<0.0001) (Figure 6).

Discussion

TP53, as a tumor suppressor gene, prevents cancer development by controlling the cell cycle, apoptosis, senescence, and DNA repair-related genes. Therefore, in cancers with TP53 mutation, gene therapy could be an interesting idea for treating the patients. Currently, some gene therapy studies have focused on transferring wt-TP53 into cancer cells using various delivery methods. However, in cancer cells, mutant TP53 effectively inactivates wt-TP53 by developing a dominant-negative property due to oligomerization with the wild-type form. To solve this problem, some previous studies focused on the replacement of TP53 tetramerization domain with the CC domain derived from BCR protein. This resulted in inhibition of the dominant-negative effect, which caused apoptosis in many cancer cells regardless of their TP53 status [13].

A comparative study showed that although wt-TP53 and TP53- CC have no difference in gene expression profile and apoptosis rate, they differed in transcriptional activation of downstream genes. It means that unlike wt-TP53, TP53-CC could better identify promoters of related genes and successfully bypassed the transdominant inhibition by endogenous mutant TP53 [23].

Another research with Ad-TP53-CC was done in MDA-MB-468 (TP53-dominant-negative breast cancer) mouse tumor model. The results showed a significant reduction (nearly 100%) in breast tumor size in the right mammary fat pad after 12 days postinfection. Moreover, Western-blot analysis demonstrated higher levels of caspase-3 in Ad-TP53-CC treated mice compared with the Ad-wt-TP53 treated group [23]. Due to the mentioned data, we added CC domain instead of tetramerization domain and got the same effective and significant results in our analysis, especially in apoptosis test.

MDM2 suppression, as an important inhibitor of TP53, is a great challenge in gene therapy with wt-TP53. Substitution of amino acids Thr18 and Ser20, with aspartate at MDM2/TP53 interaction site results in a 25- and 11-fold increase in p21 protein and p21 mRNA compared to wt-TP53 group [9]. According to our results, the TP53 function after mentioned substitution was remarkable in activating the downstream genes.

The E6 protein in the HPV oncolytic virus is another inhibitor of TP53. Based on a study, asparagine substitution with aspartate at 268th residue, which resides in E6-Tp53 interaction site, can alter the conformation of the E6 binding site in TP53 protein. Consequently, the TP53-E6 complex was not formed, resulting in a 30-fold increase in engineered TP53 activity compared to the wild type [14]. Based on this study, we included this substitution in our construct, which resulted in TP53 normal activity. Moreover, we used the 11-arginine (11R) sequence, as CPPs, infusion with TP53 as a way to increase the efficiency of TP53 in cancer cells. This modification was done based on Wang J et al. study who showed that viral transduction of TP53-11R in the mouse tumor model results in higher survival up to 84 days compared to 28 days in control samples. They also demonstrated that cancer cells apoptosis could increase up to 66.75% in case of TP53- 11R treatment [16]. However, our results demonstrated that although engineered-TP53 including 11R is functional, the study’s limitation is the lack of performance assay of engineered TP53 without 11R. However, in future studies, we are going to use this system in the mouse tumor model and 11R will be expected to be effective in tumor growth inhibition.

Due to the importance of TP53 gene in cell apoptosis, AnnexinVPE/ 7AAD staining test was used to evaluate the percentage of apoptotic and necrotic cells. One of the results that showed the significant and non-random effect of engineered TP53 on the growth of tumor cells is the correlation between the transduction rates, and consequently the expression of engineered TP53, and the cancer cell growth level. In real-time PCR experiment, which was done after 88.7% transduction, the effect on downstream genes including p21 and BAX was significant (both p = 0.0001). However, in the experiment of apoptosis on the same cells, due to the 28.9% of transduction, the rate of cells, which have experienced apoptosis, was just 35.4% that is compatible with the transduction result. Therefore, it is obvious that the impact of engineered TP53 on apoptosis would be more if the transduction rate was higher.

The results of a study in which three major types of breast cancer cell lines were treated with AAV2, showed that Rep78 protein, as the main protein in the virus, induced apoptotic pathways and activated the expression of tumor inhibitors such as TP53 in cells due to DNA damage. In addition, cell viability analysis showed that after 7 days post-transduction, their survival was significantly reduced compared to the mock sample [24]. According to this study and previous studies on the high tropism of AAV-Dj to enter cells compared to other strains and having several capsid proteins from different strains including AAV2 [25], We used this strain as a carrier of engineered TP53 construct resulting in promoting the amount of transduction, increasing viral entrance into the target cell and apoptosis enhancement.

According to a study performed on HCQ as a viral transduction enhancer, probably accelerated endosomal escape resulted in the prevention of cytoplasmic proteases on the virus before it enters the nucleus, the HCQ was used in MEF cells and the transduction rate in these cells increased from 38% to 65% and 74% in the sample treated with 3 and 18μM HCQ, respectively [21]. Since the effect of HCQ varies according to the cell type, in our study, a concentration of 1.5 to 48μM was tested to obtain the exact amount of toxicity and IC50 of this substance on SKBR3 cells. According to the results, the rate of transduction has increased from 23% to 88.7% in the optimum concentration of 20μM.

In this study, we tried to investigate the effect of engineered TP53 on breast cancer cells’ growth and apoptosis by designing an AAV-based construct including all the mentioned modifications for increasing TP53 efficiencies. Fortunately, according to the results, the percentage of apoptosis and the expression of main downstream factors of TP53 like p21 and BAX were significantly higher than mock and control groups. With further research on the modifications and the use of other types of cancer cell lines (with mutated TP53) in comparison with normal cells to confirm the engineered TP53 functionality, it is possible to use this structure in clinical trials.

Conclusion

The findings of this study demonstrated that the use of engineered TP53 by the advantage of AAV delivery system could be an effective method among many other TP53-based gene therapy methods. With further optimizations and studies, it could be implemented in clinical studies.

Ethical approval

The Research Ethics Committees of Tehran University of Medical Science approved the study proposal protocols with registration number I.R.TUMS.VCR.REC.1397.971.

Funding Information

This study was supported by grant Number 39410 from Tehran University of Medical Sciences, Tehran, Iran.

Acknowledgment

We thank Dr. Ravanshad (Associated professor at the medical virology Department, Tarbiat Modarres University, Tehran, Iran) for his kind gift and consulting. Special thanks to Mrs. Maryam Sadri and Mrs. Nesa Rashidi (PhD students at immunology Department, Iran University of medical sciences, Tehran, Iran) for their kind assistance in laboratory experiments of this study.

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