IMPLICATION OF P 53 MUTATION IN THE DYSREGULATION OF METABOLIC , APOPTOTIC AND OXIDANT / ANTIOXIDANT EQUILIBRIUM IN BREAST CANCER BY

Breast cancer is the most worldwide frequent invasive tumor diagnosed in women. No single genomic or metabolic condition can be regarded as decisive for its progression. Whoever, few key players can be pointed out among them the tumor suppressor p53, one of the most frequent mutated gene in human malignances. The current study aimed to explore the influence of p53 mutation in the regulation of metabolic, apoptotic and oxidant/antioxidant pathways in breast cancer. In the present study, tumor specimens were obtained from 40 women in different grades of primary breast carcinoma. Another 10 non-malignant (marginal) breast tissue samples were used as controls. Both mutant p53 (mutp53) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins were assessed by Western blotting, while the mRNA levels of both the proapoptotic caspase-3 and the antiapoptotic Bcl-2 were assessed by RT-PCR. The breast tissue levels of MDA, NO and GSH besides SOD activity were assayed calorimetrically. Results: Our results revealed that the tumor associated mutp53 overexpression is accompanied on the one hand by Bcl-2 up-regulation and caspase-3 downregulation reflecting a dysfunctional apoptosis. On the other hand, excessive mutp53 was associated with GAPDH redundant-expression indicating an increased glycolysis. Significant disruptions in the oxidant/antioxidant balance were also coincided with tumor-inherent p53 mutation. Conclusion: Our findings concluded that p53 mutation in breast cancer could not only perturb the tumor suppressive potential of the wild type p53 (wtp53) but also could induce dominant-negative effects over its apoptotic and metabolic functions besides its endeavor in oxidant/antioxidant equilibrium. This overview could have valuable clinical applications in establishing novel strategies for cancer therapy.


INTRODUCTION
Breast cancer represents one of the most frequently diagnosed invasive women's malignancies with approximately 400.000 worldwide annual deaths (Walerych et al., 2012).In Egypt, breast cancer is one of the most common types of cancer, representing 15.4% of the total cancer cases among both sexes and 38.8% of all types of cancer in females (Ibrahim et al., 2014).In spite of the lower incidence than in the developed countries, a continuous increase in the incidence of breast cancer has been observed in Egypt with a substantial elevation in the associated mortality projection (Shulman et al., 2010).Furthermore, the current demographic trends favor the prospect that breast cancer will represent an eminent health issue among Egyptian women in the future (Dey, 2009).
It is suggested that tumor cells acquire their malignant lineaments through the accumulation of events linked to gene activation and/or inactivation over long periods of time.Of these genes is p53 tumor suppressor gene, a negative regulator of cell cycle with a pivotal role in the protection of the genome from the consequences of DNA damages.p53 inactivation was reported as a critical step in cellular transformation and its mutation was frequently reported in several types of cancers (Agrawal et al., 2011;Olivier et al., 2010).The principle p53 protein exhibits a very short half-life and is present only in minute amounts in normal tissues and cells.However, malignant cells originated mutant p53 (mutp53) protein is usually a product of a missense point mutation in the p53 gene in which a single amino acid substitution leads to significantly prolonged half-life and increased accumulation of the mutp53 in the infected cells (Radha et al., 2014).
Besides its repertoire of activities, p53 has recently been reported to affect cellular metabolic pathways such as energy production, most likely via affecting the levels of series of gene products that regulate metabolic fates.For instance, p53 was reported to play a role in the slowdown of glycolysis and promotion of oxidative phosphorylation (Puzio-Kuter, 2011).Despite, glycolysis generates adenosine triphosphate (ATP) less efficiently than oxidative phosphorylation, malignant cells depend on glycolysis for ATP synthesis, even in the presence of abundant oxygen (Warburg effect) (Lu et al., 2015;Srinivasan et al., 2015).Accordingly, an upset energy metabolism has been considered as one of the "hallmarks of cancer" (Ganapathy-Kanniappan and Geschwind, 2013).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most important enzymes involved in cell energy metabolism.It is a glycolytic enzyme which specifically catalyzes the reversible conversion of glyceraldehyde-3-phosphate (G-3-P) to 1, 3-diphosphoglycerate.In addition to its glycolytic effects, GAPDH participates in numerous cellular functions as nuclear tRNA export, DNA replication and repair, endocytosis, exocytosis, cytoskeletal organization, iron metabolism, carcinogenesis and cell death (Colell et al., 2009;Sheokand et al., 2014).
Another important function of p53 is its ability to activate apoptosis which if disrupted, can promote tumor progression and chemoresistance (Fridman and Lowe, 2003).The most important effectors of apoptosis are cysteine aspartic acid-specific proteases (caspases), pro-apoptotic proteins, which participate in a tightly regulated proteolytic cascade (Adams and Cory, 2002).Among the most extensively studied caspases is caspase-3 which was found to play important roles in both the death receptors pathway and the mitochondrial pathway (Bellarosa et al., 2001;Keane et al., 1999).Apoptosis is also governed by other effectors including the B cell lymphoma 2 (Bcl-2) family proteins that include both anti-and pro-apoptotic proteins.A slight change in the dynamic balance of these proteins could either inhibit or promote cell death (Ola et al., 2011).Bcl-2 is a founder member of the Bcl-2 family of apoptosis regulator proteins which has been elucidated in tumor development by dysfunction in apoptotic pathways (Hanahan and Weinberg, 2000).It regulates the mitochondrial membrane potential and blocks the cytochrome-c and apoptosis inducing factor (AIF) release into the cytoplasm (Brunelle and Letai, 2009).
In spite of the above mentioned p53-linked cancer susceptibility and development, the risk factors associated with breast carcinogenesis may exert their effects via generation of reactive oxygen species (ROS) that are recognized to induce oxidative DNA damage and neoplastic transformation.Of late, ROS are being increasingly documented to be implicated in breast cancer development (Behrend et al., 2003;Kang, 2002).One of the most important functions of p53 is its involvement in the regulation of intracellular ROS levels (Ladelfa et al., 2011).The antioxidant function of p53 is represented in its ability to induce the expression of proteins acting to lower ROS levels, which prevents DNA damage and tumorgenesis under stressful conditions (Sablina et al., 2005).
The current study aimed to investigate the impact of mutp53 in breast cancer mainly, its role in metabolic regulation by investigating the glycolytic enzyme GAPDH, its apoptotic function by investigating the pro-apoptotic caspase-3 and anti-apoptotic Bcl-2 and its response to oxidative stress by investigating the tissue levels of some oxidative stress markers including malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH) and superoxide dismutase (SOD) activity.Another goal of the present study was to clarify the importance of GAPDH as a predictive biomarker of prognosis in breast cancer patients and to elucidate its role in tumor pathogenesis.

The study population
The present study included 40 women with primary breast carcinoma in the age range of 33 to 63 years that were randomly selected from those admitted to the inpatient Surgical Oncology Department, South Egypt Cancer Institute, Assiut University, in the period between March 2013 and January 2015.
Prior to initiation, the current study was approved by the Research Committee at the Faculty of Medicine Assiut University (R. Nr. 02/02/2014).All participants were informed about the goal of the study and gave written consents for participation.Clinical data were obtained from patient's data sheets including age, tumor location, tumor size, tumor grade, evidence of axillary lymphadenopathy, presence of distant metastasis, local changes of aggressiveness and history of recurrence.
Tumor tissue specimens were obtained from all subjects through either the use of a biopsy or following a surgical mastectomy.In addition, 10 normal (marginal) breast tissue samples were obtained to be used as controls.Immediately after surgical removal, each fresh tissue specimen was dissected into 4 portions.The first part was kept in 10% neutral buffered formalin for subsequent histological examinations and classification in accordance to the criteria of Elston and Ellis grading system (Elston and Ellis, 1993).The second part was immediately homogenized in ice-cold phosphate buffer saline pH 7.0, centrifuge at 4000 rpm for 15 minutes at 4 °C and subsequently stored at -70 °C until colorimetric assays of the oxidant/antioxidant indices.The third and the fourth portions were promptly frozen in liquid nitrogen and stored at -70 °C until use in Western blot and PCR assays.

Investigation of mutp53 and GAPDH proteins expression using Western blotting
Breast tissue samples were homogenized in ice-cold Tris-HCl buffer (pH 7.4) incorporating protease inhibitor cocktail (cell signaling technology, Inc, MA, USA) with the aid of Potter-Elvehjem rotor-stator homogenizer, fitted with a Teflon Pestle (Omni International, Kennesaw, GA, USA).
The total proteins in each breast tissue homogenate were thermally denatured at 95°C for 5 minutes in 2× Laemmli buffer containing 5% (v/v) 2-mercaptoethanol.SDS-PAGE electrophoresis was carried out by loading 50 μg protein in each lane at 50 volts through the (5%) stacking gel followed by 125 volts through the (12.5%) resolving gel during around 2 hours and subsequently transferred to a PVDF membrane using T-77 ECL semidry transfer unit (Amersham Biosciences UK Ltd) for 2 hours.Immunoblotting was carried out via incubating the PVDF membrane in TBST buffer containing 0.1% Tween and 5% non-fat milk for one hour at 4 °C, followed by overnight incubation at 4 °C with mouse monoclonal anti-mutp53 anti body (Novus Biologicals, LLC, Littleton, CO, USA) at 1: 1500 dilution and mouse monoclonal anti-GAPDH antibody (Novus Biologicals, LLC, Littleton, CO, USA) at 1: 1000 dilution, for p53 and GAPDH respectively.Subsequent to three times wash with TBST buffer, each membrane was incubated for an hour with an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Novus Biologicals, LLC, Littleton, CO, USA) at room temperature in a dilution of 1:5000.After four times wash with TBST buffer, the membrane bound antibody was visualized with a commercially available BCIP/NBT substrate detection kit (Genemed Biotechnologies, INC, CA, USA).Equivalent protein loading for each lane was confirmed by stripping and re-blotting each membrane against mouse monoclonal anti β-actin antibody (Santa Cruz Biotechnology, INC, CA, USA) at 4 °C in a dilution of 1:5000.All analyses were repeated to assure reproducibility of the results.Corresponding quantification of each analysis was performed using image J software and expressed as a β-actin %.

Detection of caspase-3 and Bcl-2 mRNA by reverse transcriptase PCR (RT-PCR)
Breast tissue samples were harvested in a specific lysis buffer supplied in total RNA purification kit (Jena Bioscience GmbH Jena, Germany) using Potter-Elvehjem rotor-stator homogenizer, fitted with a Teflon Pestle (Omni International, Kennesaw, GA, USA) according to the manufacturer's recommendations.To inhibit any active RNAses, the used stationary glass tube and inner tuning Teflon shaft of the homogenizer were washed with 0.1% diethylpyrocarbonate (DEPC) treated water, incubated overnight at 37 °C, and then autoclaved for 15 minutes to eliminate residual DEPC.
The isolated RNA purity (A260/A280 ratio) and concentration were determined using a GeneQuant 1300 Spectrophotometer (Uppsala, Sweden).RNA quality was confirmed by gel electrophoresis.The first-strand cDNA was then synthesized from 4 µg of total RNA using an Oligo (dT) 18 primer and RevertAid M-MuL V Reverse Transcriptase kit (Thermo Fisher Scientific Inc, Waltham, MA, USA).The mixture was incubated at 42 °C for an hour, followed by incubation for 5 minutes at 70 °C to terminate the reaction.The obtained cDNA was amplified by PCR.RT-PCR of β-actin was achieved in parallel as an internal control.The RT-PCR products were analyzed by electrophoresis using 2% molecular screening agarose gel (Roche Diagnostics, GmbH, Mannheim, Germany), stained with ethidium bromide and visualized by UV light.Corresponding quantification of each gel analysis was further performed using Image J software and expressed as a β-actin %.NCBI reference sequence, were used to design primers for human caspase-3, Bcl-2 and β-actin as shown in table 1.

Assessment of tissue levels of oxidant/antioxidant markers
Lipid peroxidation was determined spectrophotometrically in breast tissue homogenates at 535 and 520 nm in the form of thiobarbituric acid reacting substance (TBARS) and is expressed as equivalents of MDA, using 1, 1, 3, 3 tetramethoxypropane as standard (Mihara and Uchiyama, 1978).Results were expressed as µmol/g protein after estimation of the total protein content in the breast tissue homogenates (Lowry et al., 1951).
GSH was assayed spectrophotometrically in the breast tissue homogenates at 412 nm using Ellman assay method (Ellman, 1959).Results were expressed as µmol/mg protein.SOD activity in the breast tissue homogenates was assayed using kinetic procedures that based on the ability of SOD to inhibit the autooxidation of pyrogallol at alkaline medium (pH 8.2) (Mathupala et al., 1997).The enzymatic activity is expressed as U/mg protein.One unit is equivalent to the amount of SOD required to inhibit 50% of pyrogallol autooxidation.Based on the Griess reaction, NO was assayed spectrophotometrically in the breast tissue homogenates in the form of its stable metabolites, particularly, nitrite (NO 2 ) and nitrate (NO 3 ) (Sessa et al., 1994).Results were expressed as µmol/mg protein.

Statistical analysis
Statistical analyses of the obtained data were carried out using GraphPad prism version 5.0 (Graph pad software San Diego, USA).Data comparisons were performed using analysis of variance (ANOVA) followed by Tukey ' s t-test.The levels of significance were accepted with p<0.05 and all relevant results were graphically displayed as mean ± SEM.

Clinical findings
The clinicopathological features of the patients are summarized in Table 2. Tumor samples were histopathologically typified according to the World Health Organization (WHO) "Classifications of Breast Tumors" into invasive ductal carcinoma (IDC), lobular carcinoma, and medullary carcinoma (Ellis et al., 1992).All cases of breast cancer were histopathologically classified into grade-I (well differentiated carcinoma), grade-II (intermediately differentiated carcinoma), and grade-III (poorly differentiated carcinoma) in accordance to the published criteria of the WHO (Elston and Ellis, 1993).

Immunoblotting detection of both mutp53 and GAPDH proteins
As illustrated in Figure 1A, mutp53 protein showed over-expression in all grades of breast cancer in comparison to the normal marginal tissues.This tumor associated mutp53 over-expression was gradually increased from grade-I (low grade) to grade-III (high grade) (Figure 1A).
In comparison to the normal marginal tissues, GAPDH protein showed overexpression in all grades of breast cancer with gradual increase from grade-I (low grade) to grade-III (high grade) (Figure 1B).

RT-PCR assessment of the mRNA levels of both caspase-3 and Bcl-2
RT-PCR gel analysis showed an evident decrease in caspase-3 mRNA levels in all grades of breast cancer when compared to the normal marginal tissues (Figure 2A).
On the other hand, Bcl-2 mRNA levels were higher in tissues from all grades of breast carcinoma in comparison to the nonmalignant marginal tissues.Of note that, the observed decreases in caspase-3 mRNA levels as well as the increase in Bcl-2 mRNA levels were relatively proportional to the tumor grade (Figure 2B).

Assessment of oxidant/antioxidant markers
In comparison to the nonmalignant control tissues, MDA and NO were significantly increased in grade-I (p<0.01 and p<0.01 respectively), grade-II (p<0.001 and p<0.001, respectively) and grade-III (p<0.001 and p<0.001, respectively) breast carcinoma.These increase in breast tissue contents of MDA and NO were significant in grade-II (p<0.01 and p<0.01, respectively) and grade-III (p<0.001 and p<0.001, respectively) when compared to grade-I.In grade-III, breast tissue contents of MDA and NO showed significant increase (p<0.01 and p<0.01, respectively) as compared to grade-II (Table 3).
Conversely, the GSH content and SOD activity were significantly reduced in the breast tissue homogenates of grade-I (p<0.05 and p<0.01, respectively), grade-II (p<0.001 and p<0.001, respectively), and grade-III (p<0.001 and p<0.001, respectively) in comparison to the control group (Table 3).When compared to grade-I, the decrease in GSH content and SOD activity were significant only in grade-III (p<0.001 and p<0.001, respectively) but not in grade-II.In comparison to grade-II, the decrease in GSH content and SOD activity in the breast tissue homogenates was significant in grade-III (p<0.05 and p<0.01, respectively).

DISCUSSION
Like the vast majority of other malignancies, breast cancer has a ramified complex heterogeneous genetic and biochemical background with no solitary definite metabolic or genomic implication.Up-to-date, some key players can be pointed out to exhibit a role in this regard; among them is p53 tumor suppressor gene In order to assess its role in the development and progression of breast cancer, the current study aimed to investigate the expression pattern of mutp53 in different grades of breast cancer in comparison to histologically normal tumor-surrounding tissues.Results of the current study have established that there was over-expression of mutp53 protein in all grades of breast cancer in comparison to the normal marginal tissues with gradual increase from grade-I to reach its maximum in grade-III.Similar findings were also reported in other investigations (Redondo et al., 2003 ;Skarlos et al., 2005).The currently underlined steadily increasing mutp53 expression in tumor tissues inspires that p53 mutation might be a decisive event in the development of breast cancer that is maintained during tumor progression after its initiation at the early phases of the disease.This can be explained by the fact that the majority of p53 missense mutations take place in the DNA binding domain (DBD) of p53 protein leading to marked alterations in the p53 tertiary structure and accordingly loss of its specific DNA-binding activity that in turn deactivate p53 as a transcription factor that directly regulates gene expression (Xu et al., 2011).On the basis of these findings, mutp53 is proposed to gain oncogenic functions, i.e., promote tumor growth and cancer cell survival.
Another aspect of the current study was to investigate the impact of p53 mutation in the regulation of breast cancer cell metabolism mainly glycolysis, via assessment of the expression pattern of GAPDH, an important component of glycolysis.Our results revealed that there was an apparent over-expression of GAPDH in breast cancer tissues, especially grade-II and grade-III, in comparison to the marginal normal tissues suggesting tumor associated up-regulation of glycolysis.It is well known today that the increased glucose metabolism is an important feature of the progression of many types of cancers.This could be explained by the fact that cancer cells up-regulate a number of different pathways including glycolysis to keep up with the demands of providing biomass for rapidly dividing transformed cells to sustain growth and at the same time live under stressful hypoxic conditions (Vander Heiden et al., 2009).
According to the Warburg effect, cancer cell switch its metabolic pathways towards an increased dependence on glycolysis for ATP synthesis, even in the presence of abundant oxygen, rather than using the more effective oxidative phosphorylation (Lu et al., 2015;Srinivasan et al., 2015).This switch has even been termed a hallmark of cancer that explains how cancerous processes prepare substrates for active cell growth and division (Puzio-Kuter, 2011).Additionally, the apparent harmony between the increased rate of glycolysis, as evidenced by GAPDH over-expression and the p53 malfunction, as evidenced by mutp53 over-expression, could be explained by the fact that p53 retard glycolysis and reinforce oxidative phosphorylation opposing the Warburg effect.It plays this role via regulating series of genes that affect metabolic fates and metabolic products providing a mechanism of blocking tumorgenesis that is mechanistically different from either apoptosis or senescence (Bensaad and Vousden, 2007).However, loss of p53 functions in breast cancer due to mutation, as evidenced here by mutp53 over-expression, strongly confirm a switch from aerobic respiration to glycolysis.These metabolic alterations, particularly the metabolic reprogramming to aerobic glycolysis (i.e., the Warburg effect) and the reprogramming of mitochondrial metabolism, represent a hallmark of cancer that contributes to malignant transformation as well as the growth and maintenance of tumors (Hanahan and Weinberg, 2011; Ward and Thompson, 2012).
Another important goal of the current study was to explore the influence of p53 mutation in the apoptotic pathway in breast cancer.RT-PCR gel analyses showed marked decrease in the mRNA level of the pro-apoptotic caspae-3 concomitantly with apparent increase in in the mRNA level of the anti-apoptotic Bcl-2 in all grades of breast cancer in comparison to the non-neoplastic marginal tissues.The observed alterations in the mRNA levels of these apoptosis effectors commensurate with the tumor grade.These results are in agreement with previous reports that mutp53 enhance the expression of Bcl-2 protein levels in estrogen receptor positive breast cancer cells (Pratt et al., 2007).Tumor-derived mutp53 has also been reported to impair the activation of the procaspase-3 resulting eventually in down-regulation of active caspase-3 (Frank et al., 2011).
These findings strongly suggests that the caspase-3 down-regulation and/or Bcl-2 up-regulation could be implicated in breast cancer development and progression via rendering breast cancer cells resistant to apoptosis and thus may affect the outcome and progression of the disease.Based on these results, we claimed that there was an entertaining association between p53, glycolysis, and apoptosis, where p53 mutation in breast cancer is accompanied by enhanced rate of glycolysis and dysfunctional apoptosis.This can be attributed to the fact that p53-mediated PUMA expression is restrained by high glucose level (Zhao et al., 2008).p53 inactivation and PUMA induction is initiated by glucose metabolism that then promote an antiapoptotic balance supporting cancer cell survival (Coloff et al., 2011).Additionally, p53 hinders glycolysis and promotes oxidative phosphorylation by regulating the levels of a series of genes that are under its transcriptional control.Thus, p53 mutation can influence aspects of apoptosis, glycolysis and oxidative phosphorylation, and therefore is significantly important in contributing to the Warburg effect.
Finally, the present study examined the tissue levels of some oxidant/antioxidant markers to clarify their link with p53 mutation in breast cancer development and progression.Outcomes of our study revealed a significant increase in the tissue level of MDA, the lipid peroxidation product and NO, a free radical in all grades of breast cancer when compared to the non-malignant marginal tissues.This could be attributed to the tumor associated over-production of ROS.These results agree with previous studies which have shown increased lipid peroxidation and No levels in solid tumors and malignant cell lines including breast tumors (Kumaraguruparan et al., 2005;Tupurani et al., 2013).In addition, our results exhibited that the changes in the tissue levels of both MDA and NO were more pronounced in grade-III compared to grade-I and grade-II, indicating that there was a positive relation between their levels and tumor grade suggesting the involvement of ROS in tumor progression.On the other hand, the tumor associated increases in lipid peroxidation and NO levels were accompanied by obvious decrease in the antioxidant pool as manifested by diminished GSH levels and SOD activity in all grades of breast cancer tissues when compared to controls.This diminished GSH l levels and SOD activity are in accordance with previous records in breast cancer patients (Kumaraguruparan et al., 2002).We also observed that there was a negative relation between the tissue levels of these parameters and tumor grade suggesting that the defect in the antioxidant mechanism may contribute to tumor development and progression.Therefore, our results support the proposition that the generation of reactive oxygen and nitrogen species increase oxidative damage and decrease the antioxidant detoxifying capacity that could induce a high frequency of p53 mutations contributing to increased risk of cancer.This agree well with the results of some previous reports which showed that the expression of mutp53 was correlated to increased lipid peroxidation and lowered antioxidant pool in the breast cancer patients (Milicevic et al., 2014).
In conclusion, these findings collectively propose that the harmonious interaction between p53 mutation and each of up-regulated glycolysis and dysfunctional apoptosis as well as the oxidant/antioxidant imbalance might be potentially implicated in the pathogenesis and invasiveness of breast cancer.Future investigations of breast cancer biology from this overview could have valuable clinical applications not only in reconnoitering new tumor markers but also in the establishing novel strategies for cancer therapy.

Figure 1 :
Figure 1: Representative Western blotting analysis of mutp53 (A) and GAPDH (B) in breast tissue homogenates of different groups.β-actin was used in parallel as an internal control.The right panels represent corresponding quantification of each analysis measured by Image J software and expressed as a β-actin ratio.

Figure 2 :
Figure 2: Representative RT-PCR detection of mRNA fragments of caspase-3 (A) and Bcl-2 (B) in breast tissue homogenates of different groups.β-actin was used in parallel as an internal control.The right panels represent corresponding quantification of each analysis measured by Image J software and expressed as a β-actin ratio.

(
Amelio et al., 2016; Walerych et al., 2012).p53 is the most frequently mutated gene in human malignancies (Agrawal et al., 2011; Basu and Murphy, 2016).Mutant p53 (mutp53) has been reported to exhibit a longer half-life than the wild type p53 (wtp53) protein, leading to the accumulation of nonfunctioning p53 protein in the cell nucleus (Radha et al., 2014).