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T) and Expression in Breast cancer Patients
Gene Reports 17 (2019) 100512
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PIK3CA Polymorphism (rs17849079 C/T) and Expression in Breast cancer Patients
T
Shaden Muawia Hanafya, Reham Abdel Haleem Abo Elwafab, Ahmed Abdellatif Abdelkaderc, ⁎ Nabil Hadi Alowiria, a
Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt c Surgical Oncology Unit, Department of General Surgery, Faculty of Medicine, Alexandria University, Alexandria, Egypt b
ARTICLE INFO
ABSTRACT
Keywords: PIK3CA SNP rs17849079 Expressions Breast cancer
The phosphatidylinositol-3-kinase (PI3K) pathway is an important signaling pathway in cells and is involved in essential cellular functions and plays a main role in development and progression of breast cancer. The present study aimed to assess the frequency of PIK3CA rs17849079 polymorphism in breast cancer patients and its relationship with the PIK3CA gene expression. Methods: A total of 50 healthy controls and 50 breast cancer patients were included in this study. Genomic DNA was extracted from peripheral blood leukocytes and genotyping was performed using a TaqMan® SNP readymade genotyping assay. RNA was extracted from breast carcinoma tissues and adjacent normal tissues. The Quantitative Real Time PCR was used to quantify expressions of PIK3CA. Chi-square test, odds ratios (ORs) and 95% CIs were used to determine associations. Results: The frequency of SNP rs17849079 was significantly higher in the BC cases (16%) than controls group (2%), (P = 0.019, OR = 12.25, 95%, CI = 1.50 – 99.8). The study results showed no significant association between SNP rs17849079 and PIK3CA expression. Conclusion: Significant association was observed between the PIK3CA rs17849079 polymorphism and breast cancer risk. PIK3CA gene expression is found to be significantly higher in carcinoma tissues than in normal tissues.
1. Introduction Breast cancer (BC) is the most frequent cancer and the second leading cause of cancer death among women worldwide, causing approximately 626,679 deaths in the world and 40,920 deaths in the United States (US) in 2018 (Siegel et al., 2018; Bray et al., 2018). Breast cancer occurs because of an interaction between various environmental and genetic factors (Kamińska et al., 2015). Approximately 2.09 million breast cancer cases diagnosed in 2018 in the world, and an estimated 266,120 new cases of breast cancer diagnosed among US women (Siegel et al., 2018; Bray et al., 2018). The molecular biomarkers and their correlation with clinicopathological characteristics are important for investigations, prognosis, and prevention of cancer (Patani et al., 2013). A single nucleotide polymorphism (SNP), is a variation at a single
position in a DNA sequence between individuals, and the most common type of variation in the human genome (Wang et al., 1998). Most SNPs in protooncogenes and tumor suppressor genes (TSG) are associated with genetic susceptibility to cancer. These SNPs exist in genes promoters, exons, introns, or 5- and 3- UTRs, and may also located in the non-coding regions of genes, leading effects on gene expression. Therefore, some SNPs have an important role in diagnostic and therapeutic biomarkers in many cancer types (Deng et al., 2017). Previous studies reported many of SNPs in the coding region of PIK3CA and showed an association of these SNPs with protein expression level and apoptosis in the tissue (Xing et al., 2012). PIK3CA encodes P110α catalytic subunit of phosphatidylinositol-3kinase (PI3K). PI3K consists of catalytic subunit P110α and regulatory subunit P85α (Engelman et al., 2006) and is an important signaling
Abbreviations: BC, Breast cancer; SNP, A single nucleotide polymorphism; TSG, Tumor suppressor genes; UTRs, Untranslated regions; PI3K, Phosphatidylinositol-3kinase; PIK3CA, Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; qPCR, Quantitative Real Time PCR; χ2, Chi-square test; Z, Wilcoxon signed ranks test; H, Kruskal Wallis test; OR, Odds ratio ⁎ Corresponding author. E-mail address: [email protected] (N.H. Alowiri). https://doi.org/10.1016/j.genrep.2019.100512 Received 21 July 2019; Received in revised form 4 September 2019; Accepted 14 September 2019 Available online 24 October 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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S.M. Hanafy, et al.
pathway for the regulation of many essential cellular functions and processes, such as proliferation, survival, motility, activity, differentiation, growth, angiogenesis, apoptosis, and cell death, and plays a central role in carcinogenesis (Bellacosa et al., 2005). PI3K/AKT signaling pathway is activated in many cancer types, such as breast cancer, colorectal cancer, lung cancer, ovarian cancer, and others (Coutte et al., 2012). Many of PIK3CA mutations affect downstream of PIK3CA/AKT/ mTOR signaling pathway, several previous studies reported an association between the genetic polymorphisms of PIK3CA and some types of cancer such as breast cancer, gastric cancer, non-small cell lung cancer, endometrial cancer, rectal cancer, and head neck squamous cell carcinoma (Pang et al., 2014; Lin et al., 2014; Pu et al., 2011; Wang et al., 2012; Rivera et al., 2010; Slattery et al., 2010). The PIK3CA rs17849079 is a silent polymorphism in the codon 1025 in exon 20 of PIK3CA gene which changes from ACC to ACT, but both codons encode threonine (Naguib et al., 2011), and this SNP was reported at first time in retinoblastomas (Cohen et al., 2009). This study aims to evaluate the association of PIK3CA rs17849079 polymorphism with susceptibility to breast cancer and to assess the PIK3CA gene expression and relation with the SNP rs17849079 in breast cancer.
the manufacture instructions.
2. Materials and methods
2.6. Statistical analysis
2.1. Patients and samples
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp) Qualitative data were described using the number and percent. Quantitative data were described using range (minimum and maximum), mean, standard deviation and median. The chi-square test (χ2), Wilcoxon signed ranks test (Z), Kruskal Wallis test (H), Odds ratio (OR), and Hardy-Weinberg were performed for statistical examination.
2.5. Quantitative expression examination Quantitative Real Time PCR (qPCR) was used to quantify PIK3CA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA with the Applied Biosystem 7500 Fast Dx Real- Time PCR System from Applied Biosystem. GAPDH, as the appropriate housekeeping gene, was used to control for variations in RNA concentration and integrity. The qPCR was performed using the Maxima SYBR Green qPCR Master Mix (2×) kit (Fermentas, Thermo Fisher Scientific Inc.). Catalog Number (K0251). Specific primers were used to quantify PIK3CA and GADPH genes, the primers were synthesized in the Ferments, Thermo Fisher Scientific Inc. The sequences of primers were: PIK3CA Forward: GGCCACTGTGGTT GAATTGGGA, Reverse: AGTGCAC-CTTTCAAGCCGCC, GAPDH Forward: AAGGTCGGAGTCAACGG-ATTTG, and Reverse: GCCATGGG TGGAATCATATTGG. The amplicons sizes of the amplified products were: PIK3CA 250 pb, and GAPDH 150 pb. The Real-Time PCR System was programmed as follows: one cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s.
Fifty BC patients were included in this study, selected from the surgical oncology unit of Main University Hospital, Alexandria University, Alexandria, Egypt. Peripheral blood, fresh breast tumor tissue, and the adjacent normal tissue were collected from each patient. The tissue samples were immediately stored at −80 °C. Also collected 50 peripheral blood samples from healthy individuals to be as controls for SNP analysis.
3. Results
2.2. Genomic DNA extraction
This study included 50 patients with breast cancer, and they had age ranged between 19 and 80 years, and 50 healthy individuals as control, their ages ranged from 19 to 40 years. We performed Hardy-Weinberg analysis for PIK3CA rs17849079 polymorphism and found it consistent with Hardy-Weinberg equilibrium in both controls and cases (p > 0.05). Data of allele and genotype frequencies of PIK3CA rs17849079 polymorphism were listed in the Table 1, that showed the frequency of CT genotype was significantly higher in the BC cases (8 out 50, 16%) than controls group (1 out 50, 2%), (P = 0.034, OR = 9.80, 95% CI = 1.17–81.6), whereas, the TT genotype was found in only 2 BC patients only and absence in the controls group. T allele showed significant association with breast cancer, the T allele frequency was significantly higher in BC patients (12%) than in control group (1%) (P = 0.13, OR = 13.50, 95% CI = 1.72–105.9). PIK3CA rs17849079 polymorphism did not show any significant association with Clinicopathological characteristics of breast cancer (P > 0.05), Table 2. PIK3CA gene expression was a significantly higher expression in carcinoma tissues than in normal tissues (P < 0.001). The median value for PIK3CA/GADPH ratio in breast carcinoma tissues was 42.35 compared to 1.12 PIK3CA/GADPH in normal breast tissues, Table 3. PIK3CA mRNA expression was increased in 47 out of 50 carcinoma tissue of breast cancer patients (94%). Clinicopathological characteristics did not show any significant association with PIK3CA gene expression. The median value of BC PIK3CA gene expression was 46.54 in patients with CC genotype, 21.58 in patients with CT genotype, and 247.06 in patients with TT genotype. There was no significant statistical association between SNP rs17849079 and PIK3CA gene expression (P = 0.08), Table 4.
Two ml of peripheral blood was collected by vein-puncture in EDTA sterile tubes from each patient and healthy control for DNA extraction. Genomic DNA was extracted from whole blood according to the manufacturer's instructions of the kit (Invitrogen™, Carlsbad, CA 92008, USA). 2.3. Genotyping Genotyping and allelic discrimination of rs17849079 were performed using a TaqMan® SNP ready-made genotyping assay, TaqMan® Genotyping Master Mix, and the 7500 Fast Dx Real-Time PCR System from Applied Biosystem. A reaction prepared a reaction mixture by adding 12.5 μl of TaqMan genotyping master mix, 1.25 μl of TaqMan SNP genotyping assay, and 11.25 μl of nuclease-free H2O, then 3 μl genomic DNA (20–100 ng) was added to the PCR tube containing the reaction mixture. 2.4. RNA extraction and cDNA synthesis The RNA was extracted using PureLink® RNA Mini Kits for purification of total RNA (Invitrogen ™, Carlsbad, CA 92008, USA). Catalog Number (12183018A) according to the manufacture instructions. Fifty mg of frozen tissue was used to extract the total RNA, the tissues were homogenized and grinded by a Tissue Lyser LT (Qiagen, Germany). It was operated for 5 min at 50 Hz. The quantity and purity of extracted RNA was determined by NanoDrop ND-2000/2000c spectrophotometer (ThermoScientific, USA). High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis (Applied Biosystems, Carlsbad, CA 92008, USA) according to 2
Gene Reports 17 (2019) 100512
S.M. Hanafy, et al.
Table 1 Genotype and allele frequencies of SNP rs17849079 in BC patients and healthy controls. SNP rs17849079
Cases (n = 50)
CC CT TT CC CT + TT Allele frequency C T
Controls (n = 50)
OR
95% C. I
No.
%
No.
%
40 8 2 40 10
80.0 16.0 4.0 80.0 20.0
49 1 0 49 1
98.0 2.0 0.0 98.0 2.0
1.000 9.80⁎ – 0.081⁎ 12.25⁎
– 1.17–81.6 – 0.010–0.665 1.50–99.8
88 12
88.0 12.0
99 1
99.0 1.0
0.074⁎ 13.50⁎
0.009–0.5813 1.72–105.9
p
L.L – U. L – 0.034⁎ 0.290 0.019⁎ 0.013⁎
OR: Odd's ratio, L.L: Lower limit, U.L: Upper limit, C·I: Confidence interval. ⁎ Statistically significant at p ≤ 0.05
as breast, ovary, colon, liver, stomach, brain, and lung (Samuels et al., 2004). The phosphatidylinositol-3-kinase (PI3K) pathway is an important signaling pathway in cells and is involved in essential cellular functions and plays an important role in the development and progression of breast cancer (Boyault et al., 2012). PI3K converts the phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol3,4,5-trisphosphate (PIP3) that leads to downstream signaling through a PI3K/AKT/mTOR pathway and activates it (Wang et al., 2011). PIK3CA somatic mutations have been commonly found in several types of cancers, mutations in exon 20 are the most common of these mutations, and there were some silent mutations and missense mutations identified in breast cancer (Samuels et al., 2004; Palimaru et al., 2013). SNPs are the most variations in the genome, and situated in various regions of genes (exons, introns, promoters, silencers, enhancers, 5UTRs and 3- UTRs). SNPs in exon regions can be classified to synonymous and non-synonymous as per their impact on the encoded amino acid. Non-synonymous coding SNPs alter the protein structures and functions due to amino acid substitution, the most of these SNPs as result to changes in the first two bases of a codon, whereas the synonymous SNPs do not change amino acid sequence, for example, some substitution in the third base in a codon do not alter the amino acid because the wild and variant codons encode the same amino acid (for example the silent mutation), however, they may affect gene function and expression by changing the neighboring genes expressions or affect the binding of microRNA (miRNA) which regulate of gene expression with the mRNA sequences (Deng et al., 2017). The SNPs which are not located in the exons have an indirect role impact on proteins functions by may effecting in the genes expressions (Wang et al., 2005). According to previous studies, there were some PIK3CA polymorphisms showed susceptibility to breast cancer, non-small cell lung cancer, endometrial cancer, oral squamous cell carcinoma, esophageal squamous cell carcinoma, rectal cancer, and gastric cancer (Pang et al., 2014; Pu et al., 2011; Wang et al., 2012; Wan et al., 2015; Lin et al., 2014; Slattery et al., 2010). In the current study, the CT genotype of rs17849079 polymorphism was significantly associated with BC (P = 0.034, OR = 9.80, 95% CI = 1.17–81.6), the frequency of this genotype was 16% in BC patients, the variants genotypes (CT + TT) of rs17849079 were 20% in BC patients, while in healthy controls were 2%, and the T allele frequency was 12% in BC patients and 1% in healthy controls, all these results indicate to significant association of PIK3CA rs17849079 polymorphism with susceptibility to BC. The SNP rs17849079 is located in the exon 20 of PIK3CA gene and was initially reported in retinoblastomas with no obvious functional effects (Cohen et al., 2009), and although it is a silent polymorphism in the exon 20 of PIK3CA gene (did not alter the amino acid) because its wild type codon ACC and variant type codon ACT encode the same amino acid (threonine), but it showed highly significant association with BC, the impact of SNP rs17849079 on PIK3CA expression may be due to the effect on the binding of hsamiR-4324 to the PK3CA mRNA, the hsa-miR-4324 regulate the PIK3CA
Table 2 Relationship between the genotype frequency of rs17849079 and the clinicopathological features of breast cancer (BC) patients. Parameters
SNP rs17849079
Number, N (%) Age, median (range) Tumor size, mm, N (%) ≤ 20 > 20 Histology, N (%) Ductal Lobular Tumor grade, N (%) I II III ER status, N (%) Positive Negative PR status, N (%) Positive Negative HER2 status, N (%) 1+ 2+ 3+ Lymph node status,N (%) Metastasis No metastasis
Significance X2
P value
0.587
0.386 1.000
0.719
1.000
0 (0.0) 12 (100) 0 (0.0)
6.189
0.110
1 (12.5) 7 (87.5)
0 (0.0) 2 (100)
1.886
0.377
19 (47.5) 21 (52.5
4 (50) 4 (50)
2 (100) 0 (0.0)
1.790
0.612
15 (37.5) 12 (30) 13 (32.5)
4 (50) 2 (25) 2 (25
2 (100) 0 (0.0) 0 (0.0)
2.408
0.758
12 (30) 28 (70)
2 (25) 6 (75)
0 (0.0) 2 (100)
0.560
1.000
CC
CT
TT
40 (80) 54 (19–80)
8 (16) 48 (45–71)
2 (4) 64 (61–68)
26 (65) 14 (35)
5(62.5) 3 (37.5)
1 (50) 1 (50)
36 (90) 4 (10)
7 (87.5) 1 (12.5)
2 (100) 0 (0.0)
3 (7.5) 20 (50) 17 (42.5)
3 (37.5) 3 (37.5) 2 (25)
14 (35) 26 (65)
χ2: Chi square test p: p value for comparing between the different categories. Table 3 Descriptive analysis of the studied cases according to PIK3CA expression (n = 50). Carcinoma tissues PIK3CA expression Min. – Max. Mean ± SD. Median
5.98–2288.2 131.4 ± 328.6 42.35
Normal tissues 0.03–335.5 11.45 ± 48.12 1.12
Z 5.700
p ⁎
< 0.001⁎
Z, p: p value for Wilcoxon signed ranks test for comparing between pathogenic and normal. ⁎ Statistically significant at p ≤ 0.05.
4. Discussion In the present study, we assessed the PIK3CA gene expression and polymorphism (rs17849079) in the BC patients. PIK3CA gene is located on chromosome 3q26.32 and encodes the catalytic subunit of PI3K. PIK3CA has been found amplified or mutated in many cancer types such 3
Gene Reports 17 (2019) 100512
S.M. Hanafy, et al.
Table 4 Relationship between the genotypes of the SNP rs17849079 and PIK3CA expression for of BC patients (n = 50). Carcinoma tissue
PIK3CA expression Min. – Max. Mean ± SD. Median
SNP rs17849079 CC (n = 40)
CT (n = 8)
TT (n = 2)
5.98–2288.20 146.06 ± 362.09 46.54
13.36–68.59 29.45 ± 18.43 21.58
78.25–415.87 247.06 ± 238.73 247.06
H
p
5.054
0.080
H, p: H and p values for Kruskal Wallis test for comparing between the different categories.
expression and bind to the mRNA sequences at exon 20 of PIK3CA gene, which contains this SNP (Goff et al., 2009; miRBase, n.d.). This miRNA may be bind only to the sequences containing the wild-type allele (ACC) and not bind to the sequences containing the variant codon (ACT), and this might affect the regulation of PIK3CA gene expression (Karakas et al., 2013). In a previous study, karakas et al. studied the prevalence of PIK3CA rs17849079 polymorphism in 81 Arab BC patients and 189 cancer-free individuals as controls. They found a high prevalence of this SNP in the BC patients compared to the healthy controls group, the frequency of rs17849079 was 8.6% in BC patients and 1.05% in healthy individuals (Karakas et al., 2013). In another study, Hong-Guang et al. at 2015 studied the PIK3CA polymorphisms associated with the susceptibility to hepatocellular carcinoma (HCC). They showed the rare T allele of SNP rs17849079 was more frequent in HCC patients (29.7%) than in healthy controls (21.7%) (Li et al., 2015). Besides, we have measured the mRNA expression of PIK3CA gene in breast carcinoma tissue and adjacent normal tissue by using the quantitative method by real-time PCR. the PIK3CA mRNA expression was significantly higher in breast cancer tissue than in normal breast tissue, the median value of PIK3CA expression in normal tissue was 1.12, whereas its expression in breast carcinoma was 42.35 (P < .001). This may support and indicate for the PI3K pathway role in development and progression of cancer. Palimaru I. et al. studied the Expression of PIK3CA, PTEN mRNA and PIK3CA mutations in primary breast cancer: association with lymph node metastases, they showed the PIK3CA expression was significantly higher in breast cancer tissue than in normal breast tissue, the median value of PIK3CA/HMBS ratio expression in breast carcinoma tissue was 56 compared to 23 in breast normal tissue (Palimaru et al., 2013). Also, there was a previous study showed high the PIK3CA expression in breast cancer tissue with immunohistochemistry (IHC) method (Aleskandarany et al., 2010). In conclusion, the high prevalence of PIK3CA rs17849079 polymorphism in the present study indicates this SNP was significantly associated with BC risk, which may be used as a molecular marker for early diagnosis of breast cancer susceptibility. The PIK3CA expression showed highly significant increase in breast carcinoma tissue than normal breast tissue, this result supports the role of PI3K pathway in development and progression of BC. We didn't note any significance between rs17849079 polymorphism and clinical parameters of BC, also didn't show a significant association between this SNP and PIK3CA expression, these may be due to the small sample size which studied in this study, so these results need to further studies with larger sample sizes.
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PIK3CA Polymorphism (rs17849079 C/T) and Expression in Breast cancer Patients
T
Shaden Muawia Hanafya, Reham Abdel Haleem Abo Elwafab, Ahmed Abdellatif Abdelkaderc, ⁎ Nabil Hadi Alowiria, a
Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt c Surgical Oncology Unit, Department of General Surgery, Faculty of Medicine, Alexandria University, Alexandria, Egypt b
ARTICLE INFO
ABSTRACT
Keywords: PIK3CA SNP rs17849079 Expressions Breast cancer
The phosphatidylinositol-3-kinase (PI3K) pathway is an important signaling pathway in cells and is involved in essential cellular functions and plays a main role in development and progression of breast cancer. The present study aimed to assess the frequency of PIK3CA rs17849079 polymorphism in breast cancer patients and its relationship with the PIK3CA gene expression. Methods: A total of 50 healthy controls and 50 breast cancer patients were included in this study. Genomic DNA was extracted from peripheral blood leukocytes and genotyping was performed using a TaqMan® SNP readymade genotyping assay. RNA was extracted from breast carcinoma tissues and adjacent normal tissues. The Quantitative Real Time PCR was used to quantify expressions of PIK3CA. Chi-square test, odds ratios (ORs) and 95% CIs were used to determine associations. Results: The frequency of SNP rs17849079 was significantly higher in the BC cases (16%) than controls group (2%), (P = 0.019, OR = 12.25, 95%, CI = 1.50 – 99.8). The study results showed no significant association between SNP rs17849079 and PIK3CA expression. Conclusion: Significant association was observed between the PIK3CA rs17849079 polymorphism and breast cancer risk. PIK3CA gene expression is found to be significantly higher in carcinoma tissues than in normal tissues.
1. Introduction Breast cancer (BC) is the most frequent cancer and the second leading cause of cancer death among women worldwide, causing approximately 626,679 deaths in the world and 40,920 deaths in the United States (US) in 2018 (Siegel et al., 2018; Bray et al., 2018). Breast cancer occurs because of an interaction between various environmental and genetic factors (Kamińska et al., 2015). Approximately 2.09 million breast cancer cases diagnosed in 2018 in the world, and an estimated 266,120 new cases of breast cancer diagnosed among US women (Siegel et al., 2018; Bray et al., 2018). The molecular biomarkers and their correlation with clinicopathological characteristics are important for investigations, prognosis, and prevention of cancer (Patani et al., 2013). A single nucleotide polymorphism (SNP), is a variation at a single
position in a DNA sequence between individuals, and the most common type of variation in the human genome (Wang et al., 1998). Most SNPs in protooncogenes and tumor suppressor genes (TSG) are associated with genetic susceptibility to cancer. These SNPs exist in genes promoters, exons, introns, or 5- and 3- UTRs, and may also located in the non-coding regions of genes, leading effects on gene expression. Therefore, some SNPs have an important role in diagnostic and therapeutic biomarkers in many cancer types (Deng et al., 2017). Previous studies reported many of SNPs in the coding region of PIK3CA and showed an association of these SNPs with protein expression level and apoptosis in the tissue (Xing et al., 2012). PIK3CA encodes P110α catalytic subunit of phosphatidylinositol-3kinase (PI3K). PI3K consists of catalytic subunit P110α and regulatory subunit P85α (Engelman et al., 2006) and is an important signaling
Abbreviations: BC, Breast cancer; SNP, A single nucleotide polymorphism; TSG, Tumor suppressor genes; UTRs, Untranslated regions; PI3K, Phosphatidylinositol-3kinase; PIK3CA, Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; qPCR, Quantitative Real Time PCR; χ2, Chi-square test; Z, Wilcoxon signed ranks test; H, Kruskal Wallis test; OR, Odds ratio ⁎ Corresponding author. E-mail address: [email protected] (N.H. Alowiri). https://doi.org/10.1016/j.genrep.2019.100512 Received 21 July 2019; Received in revised form 4 September 2019; Accepted 14 September 2019 Available online 24 October 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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pathway for the regulation of many essential cellular functions and processes, such as proliferation, survival, motility, activity, differentiation, growth, angiogenesis, apoptosis, and cell death, and plays a central role in carcinogenesis (Bellacosa et al., 2005). PI3K/AKT signaling pathway is activated in many cancer types, such as breast cancer, colorectal cancer, lung cancer, ovarian cancer, and others (Coutte et al., 2012). Many of PIK3CA mutations affect downstream of PIK3CA/AKT/ mTOR signaling pathway, several previous studies reported an association between the genetic polymorphisms of PIK3CA and some types of cancer such as breast cancer, gastric cancer, non-small cell lung cancer, endometrial cancer, rectal cancer, and head neck squamous cell carcinoma (Pang et al., 2014; Lin et al., 2014; Pu et al., 2011; Wang et al., 2012; Rivera et al., 2010; Slattery et al., 2010). The PIK3CA rs17849079 is a silent polymorphism in the codon 1025 in exon 20 of PIK3CA gene which changes from ACC to ACT, but both codons encode threonine (Naguib et al., 2011), and this SNP was reported at first time in retinoblastomas (Cohen et al., 2009). This study aims to evaluate the association of PIK3CA rs17849079 polymorphism with susceptibility to breast cancer and to assess the PIK3CA gene expression and relation with the SNP rs17849079 in breast cancer.
the manufacture instructions.
2. Materials and methods
2.6. Statistical analysis
2.1. Patients and samples
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp) Qualitative data were described using the number and percent. Quantitative data were described using range (minimum and maximum), mean, standard deviation and median. The chi-square test (χ2), Wilcoxon signed ranks test (Z), Kruskal Wallis test (H), Odds ratio (OR), and Hardy-Weinberg were performed for statistical examination.
2.5. Quantitative expression examination Quantitative Real Time PCR (qPCR) was used to quantify PIK3CA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA with the Applied Biosystem 7500 Fast Dx Real- Time PCR System from Applied Biosystem. GAPDH, as the appropriate housekeeping gene, was used to control for variations in RNA concentration and integrity. The qPCR was performed using the Maxima SYBR Green qPCR Master Mix (2×) kit (Fermentas, Thermo Fisher Scientific Inc.). Catalog Number (K0251). Specific primers were used to quantify PIK3CA and GADPH genes, the primers were synthesized in the Ferments, Thermo Fisher Scientific Inc. The sequences of primers were: PIK3CA Forward: GGCCACTGTGGTT GAATTGGGA, Reverse: AGTGCAC-CTTTCAAGCCGCC, GAPDH Forward: AAGGTCGGAGTCAACGG-ATTTG, and Reverse: GCCATGGG TGGAATCATATTGG. The amplicons sizes of the amplified products were: PIK3CA 250 pb, and GAPDH 150 pb. The Real-Time PCR System was programmed as follows: one cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s.
Fifty BC patients were included in this study, selected from the surgical oncology unit of Main University Hospital, Alexandria University, Alexandria, Egypt. Peripheral blood, fresh breast tumor tissue, and the adjacent normal tissue were collected from each patient. The tissue samples were immediately stored at −80 °C. Also collected 50 peripheral blood samples from healthy individuals to be as controls for SNP analysis.
3. Results
2.2. Genomic DNA extraction
This study included 50 patients with breast cancer, and they had age ranged between 19 and 80 years, and 50 healthy individuals as control, their ages ranged from 19 to 40 years. We performed Hardy-Weinberg analysis for PIK3CA rs17849079 polymorphism and found it consistent with Hardy-Weinberg equilibrium in both controls and cases (p > 0.05). Data of allele and genotype frequencies of PIK3CA rs17849079 polymorphism were listed in the Table 1, that showed the frequency of CT genotype was significantly higher in the BC cases (8 out 50, 16%) than controls group (1 out 50, 2%), (P = 0.034, OR = 9.80, 95% CI = 1.17–81.6), whereas, the TT genotype was found in only 2 BC patients only and absence in the controls group. T allele showed significant association with breast cancer, the T allele frequency was significantly higher in BC patients (12%) than in control group (1%) (P = 0.13, OR = 13.50, 95% CI = 1.72–105.9). PIK3CA rs17849079 polymorphism did not show any significant association with Clinicopathological characteristics of breast cancer (P > 0.05), Table 2. PIK3CA gene expression was a significantly higher expression in carcinoma tissues than in normal tissues (P < 0.001). The median value for PIK3CA/GADPH ratio in breast carcinoma tissues was 42.35 compared to 1.12 PIK3CA/GADPH in normal breast tissues, Table 3. PIK3CA mRNA expression was increased in 47 out of 50 carcinoma tissue of breast cancer patients (94%). Clinicopathological characteristics did not show any significant association with PIK3CA gene expression. The median value of BC PIK3CA gene expression was 46.54 in patients with CC genotype, 21.58 in patients with CT genotype, and 247.06 in patients with TT genotype. There was no significant statistical association between SNP rs17849079 and PIK3CA gene expression (P = 0.08), Table 4.
Two ml of peripheral blood was collected by vein-puncture in EDTA sterile tubes from each patient and healthy control for DNA extraction. Genomic DNA was extracted from whole blood according to the manufacturer's instructions of the kit (Invitrogen™, Carlsbad, CA 92008, USA). 2.3. Genotyping Genotyping and allelic discrimination of rs17849079 were performed using a TaqMan® SNP ready-made genotyping assay, TaqMan® Genotyping Master Mix, and the 7500 Fast Dx Real-Time PCR System from Applied Biosystem. A reaction prepared a reaction mixture by adding 12.5 μl of TaqMan genotyping master mix, 1.25 μl of TaqMan SNP genotyping assay, and 11.25 μl of nuclease-free H2O, then 3 μl genomic DNA (20–100 ng) was added to the PCR tube containing the reaction mixture. 2.4. RNA extraction and cDNA synthesis The RNA was extracted using PureLink® RNA Mini Kits for purification of total RNA (Invitrogen ™, Carlsbad, CA 92008, USA). Catalog Number (12183018A) according to the manufacture instructions. Fifty mg of frozen tissue was used to extract the total RNA, the tissues were homogenized and grinded by a Tissue Lyser LT (Qiagen, Germany). It was operated for 5 min at 50 Hz. The quantity and purity of extracted RNA was determined by NanoDrop ND-2000/2000c spectrophotometer (ThermoScientific, USA). High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis (Applied Biosystems, Carlsbad, CA 92008, USA) according to 2
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Table 1 Genotype and allele frequencies of SNP rs17849079 in BC patients and healthy controls. SNP rs17849079
Cases (n = 50)
CC CT TT CC CT + TT Allele frequency C T
Controls (n = 50)
OR
95% C. I
No.
%
No.
%
40 8 2 40 10
80.0 16.0 4.0 80.0 20.0
49 1 0 49 1
98.0 2.0 0.0 98.0 2.0
1.000 9.80⁎ – 0.081⁎ 12.25⁎
– 1.17–81.6 – 0.010–0.665 1.50–99.8
88 12
88.0 12.0
99 1
99.0 1.0
0.074⁎ 13.50⁎
0.009–0.5813 1.72–105.9
p
L.L – U. L – 0.034⁎ 0.290 0.019⁎ 0.013⁎
OR: Odd's ratio, L.L: Lower limit, U.L: Upper limit, C·I: Confidence interval. ⁎ Statistically significant at p ≤ 0.05
as breast, ovary, colon, liver, stomach, brain, and lung (Samuels et al., 2004). The phosphatidylinositol-3-kinase (PI3K) pathway is an important signaling pathway in cells and is involved in essential cellular functions and plays an important role in the development and progression of breast cancer (Boyault et al., 2012). PI3K converts the phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol3,4,5-trisphosphate (PIP3) that leads to downstream signaling through a PI3K/AKT/mTOR pathway and activates it (Wang et al., 2011). PIK3CA somatic mutations have been commonly found in several types of cancers, mutations in exon 20 are the most common of these mutations, and there were some silent mutations and missense mutations identified in breast cancer (Samuels et al., 2004; Palimaru et al., 2013). SNPs are the most variations in the genome, and situated in various regions of genes (exons, introns, promoters, silencers, enhancers, 5UTRs and 3- UTRs). SNPs in exon regions can be classified to synonymous and non-synonymous as per their impact on the encoded amino acid. Non-synonymous coding SNPs alter the protein structures and functions due to amino acid substitution, the most of these SNPs as result to changes in the first two bases of a codon, whereas the synonymous SNPs do not change amino acid sequence, for example, some substitution in the third base in a codon do not alter the amino acid because the wild and variant codons encode the same amino acid (for example the silent mutation), however, they may affect gene function and expression by changing the neighboring genes expressions or affect the binding of microRNA (miRNA) which regulate of gene expression with the mRNA sequences (Deng et al., 2017). The SNPs which are not located in the exons have an indirect role impact on proteins functions by may effecting in the genes expressions (Wang et al., 2005). According to previous studies, there were some PIK3CA polymorphisms showed susceptibility to breast cancer, non-small cell lung cancer, endometrial cancer, oral squamous cell carcinoma, esophageal squamous cell carcinoma, rectal cancer, and gastric cancer (Pang et al., 2014; Pu et al., 2011; Wang et al., 2012; Wan et al., 2015; Lin et al., 2014; Slattery et al., 2010). In the current study, the CT genotype of rs17849079 polymorphism was significantly associated with BC (P = 0.034, OR = 9.80, 95% CI = 1.17–81.6), the frequency of this genotype was 16% in BC patients, the variants genotypes (CT + TT) of rs17849079 were 20% in BC patients, while in healthy controls were 2%, and the T allele frequency was 12% in BC patients and 1% in healthy controls, all these results indicate to significant association of PIK3CA rs17849079 polymorphism with susceptibility to BC. The SNP rs17849079 is located in the exon 20 of PIK3CA gene and was initially reported in retinoblastomas with no obvious functional effects (Cohen et al., 2009), and although it is a silent polymorphism in the exon 20 of PIK3CA gene (did not alter the amino acid) because its wild type codon ACC and variant type codon ACT encode the same amino acid (threonine), but it showed highly significant association with BC, the impact of SNP rs17849079 on PIK3CA expression may be due to the effect on the binding of hsamiR-4324 to the PK3CA mRNA, the hsa-miR-4324 regulate the PIK3CA
Table 2 Relationship between the genotype frequency of rs17849079 and the clinicopathological features of breast cancer (BC) patients. Parameters
SNP rs17849079
Number, N (%) Age, median (range) Tumor size, mm, N (%) ≤ 20 > 20 Histology, N (%) Ductal Lobular Tumor grade, N (%) I II III ER status, N (%) Positive Negative PR status, N (%) Positive Negative HER2 status, N (%) 1+ 2+ 3+ Lymph node status,N (%) Metastasis No metastasis
Significance X2
P value
0.587
0.386 1.000
0.719
1.000
0 (0.0) 12 (100) 0 (0.0)
6.189
0.110
1 (12.5) 7 (87.5)
0 (0.0) 2 (100)
1.886
0.377
19 (47.5) 21 (52.5
4 (50) 4 (50)
2 (100) 0 (0.0)
1.790
0.612
15 (37.5) 12 (30) 13 (32.5)
4 (50) 2 (25) 2 (25
2 (100) 0 (0.0) 0 (0.0)
2.408
0.758
12 (30) 28 (70)
2 (25) 6 (75)
0 (0.0) 2 (100)
0.560
1.000
CC
CT
TT
40 (80) 54 (19–80)
8 (16) 48 (45–71)
2 (4) 64 (61–68)
26 (65) 14 (35)
5(62.5) 3 (37.5)
1 (50) 1 (50)
36 (90) 4 (10)
7 (87.5) 1 (12.5)
2 (100) 0 (0.0)
3 (7.5) 20 (50) 17 (42.5)
3 (37.5) 3 (37.5) 2 (25)
14 (35) 26 (65)
χ2: Chi square test p: p value for comparing between the different categories. Table 3 Descriptive analysis of the studied cases according to PIK3CA expression (n = 50). Carcinoma tissues PIK3CA expression Min. – Max. Mean ± SD. Median
5.98–2288.2 131.4 ± 328.6 42.35
Normal tissues 0.03–335.5 11.45 ± 48.12 1.12
Z 5.700
p ⁎
< 0.001⁎
Z, p: p value for Wilcoxon signed ranks test for comparing between pathogenic and normal. ⁎ Statistically significant at p ≤ 0.05.
4. Discussion In the present study, we assessed the PIK3CA gene expression and polymorphism (rs17849079) in the BC patients. PIK3CA gene is located on chromosome 3q26.32 and encodes the catalytic subunit of PI3K. PIK3CA has been found amplified or mutated in many cancer types such 3
Gene Reports 17 (2019) 100512
S.M. Hanafy, et al.
Table 4 Relationship between the genotypes of the SNP rs17849079 and PIK3CA expression for of BC patients (n = 50). Carcinoma tissue
PIK3CA expression Min. – Max. Mean ± SD. Median
SNP rs17849079 CC (n = 40)
CT (n = 8)
TT (n = 2)
5.98–2288.20 146.06 ± 362.09 46.54
13.36–68.59 29.45 ± 18.43 21.58
78.25–415.87 247.06 ± 238.73 247.06
H
p
5.054
0.080
H, p: H and p values for Kruskal Wallis test for comparing between the different categories.
expression and bind to the mRNA sequences at exon 20 of PIK3CA gene, which contains this SNP (Goff et al., 2009; miRBase, n.d.). This miRNA may be bind only to the sequences containing the wild-type allele (ACC) and not bind to the sequences containing the variant codon (ACT), and this might affect the regulation of PIK3CA gene expression (Karakas et al., 2013). In a previous study, karakas et al. studied the prevalence of PIK3CA rs17849079 polymorphism in 81 Arab BC patients and 189 cancer-free individuals as controls. They found a high prevalence of this SNP in the BC patients compared to the healthy controls group, the frequency of rs17849079 was 8.6% in BC patients and 1.05% in healthy individuals (Karakas et al., 2013). In another study, Hong-Guang et al. at 2015 studied the PIK3CA polymorphisms associated with the susceptibility to hepatocellular carcinoma (HCC). They showed the rare T allele of SNP rs17849079 was more frequent in HCC patients (29.7%) than in healthy controls (21.7%) (Li et al., 2015). Besides, we have measured the mRNA expression of PIK3CA gene in breast carcinoma tissue and adjacent normal tissue by using the quantitative method by real-time PCR. the PIK3CA mRNA expression was significantly higher in breast cancer tissue than in normal breast tissue, the median value of PIK3CA expression in normal tissue was 1.12, whereas its expression in breast carcinoma was 42.35 (P < .001). This may support and indicate for the PI3K pathway role in development and progression of cancer. Palimaru I. et al. studied the Expression of PIK3CA, PTEN mRNA and PIK3CA mutations in primary breast cancer: association with lymph node metastases, they showed the PIK3CA expression was significantly higher in breast cancer tissue than in normal breast tissue, the median value of PIK3CA/HMBS ratio expression in breast carcinoma tissue was 56 compared to 23 in breast normal tissue (Palimaru et al., 2013). Also, there was a previous study showed high the PIK3CA expression in breast cancer tissue with immunohistochemistry (IHC) method (Aleskandarany et al., 2010). In conclusion, the high prevalence of PIK3CA rs17849079 polymorphism in the present study indicates this SNP was significantly associated with BC risk, which may be used as a molecular marker for early diagnosis of breast cancer susceptibility. The PIK3CA expression showed highly significant increase in breast carcinoma tissue than normal breast tissue, this result supports the role of PI3K pathway in development and progression of BC. We didn't note any significance between rs17849079 polymorphism and clinical parameters of BC, also didn't show a significant association between this SNP and PIK3CA expression, these may be due to the small sample size which studied in this study, so these results need to further studies with larger sample sizes.
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