Carrying G allele in rs786204926 involved in alternative splicing of PTEN is associated with chemosensitivity in breast cancer


 Background

Chemoresistance is still the main reason for the failure of breast cancer treatment and is the main cause of death of breast cancer patients. Although many studies have shown the association between genetic polymorphisms of PTEN and chemoresistance, the molecular mechanism of breast cancer chemotherapy has not been further studied. This study aims to investigate the potential association between novel PTEN gene polymorphism and breast chemoresistance in Chinese population, and explore whether alternative splicing of the PTEN gene is affected by the gene polymorphism.
Methods

The study included 234 patients with breast cancer chemotherapy, 157 chemosensitive cases and 77 chemoresistant case. rs786204926, rs701848, rs12402181, rs35770269 were analysed using Sanger sequence and Sequenom MassArray typing technology. Silicon analysis was used to predict whether and how the polymorphism affects alternative splicing. Semi-quantitative RT-PCR and Western blot were further used to validate the silicon analysis.
Results

It is showed that there was a significant association between rs786204926 polymorphism and breast cancer chemoresistance. Carrying G allele or AG genotype will increase the risk of chemosensitivity in breast cancer. Additionally, Logistic multivariate regression analysis showed that age, lymph node metastasis and rs786204926 genotype are risk factors for breast cancer chemoresistance. Furthermore, Silico analysis showed that carrying G allele or AG genotype in chemosensitivity samples produced a new receptor site of alternative splicing, which increases the possibility of a new mutant PTEN isoform production. Interestingly, further experiments also verify this possibility.
Conclusion

We speculate that the mechanism of breast cancer chemosensitivity might be caused by a change in alternative splicing caused by the rs786204926 of PTEN gene. Thus, our study might provide theoretical guidance for the individualized treatment of clinical breast cancer patients.


Abstract Background
Chemoresistance is still the main reason for the failure of breast cancer treatment and is the main cause of death of breast cancer patients. Although many studies have shown the association between genetic polymorphisms of PTEN and chemoresistance, the molecular mechanism of breast cancer chemotherapy has not been further studied. This study aims to investigate the potential association between novel PTEN gene polymorphism and breast chemoresistance in Chinese population, and explore whether alternative splicing of the PTEN gene is affected by the gene polymorphism.

Methods
The study included 234 patients with breast cancer chemotherapy, 157 chemosensitive cases and 77 chemoresistant case. rs786204926, rs701848, rs12402181, rs35770269 were analysed using Sanger sequence and Sequenom MassArray typing technology. Silicon analysis was used to predict whether and how the polymorphism affects alternative splicing. Semi-quantitative RT-PCR and Western blot were further used to validate the silicon analysis.

Results
It is showed that there was a signi cant association between rs786204926 polymorphism and breast cancer chemoresistance. Carrying G allele or AG genotype will increase the risk of chemosensitivity in breast cancer. Additionally, Logistic multivariate regression analysis showed that age, lymph node metastasis and rs786204926 genotype are risk factors for breast cancer chemoresistance. Furthermore, Silico analysis showed that carrying G allele or AG genotype in chemosensitivity samples produced a new receptor site of alternative splicing, which increases the possibility of a new mutant PTEN isoform production. Interestingly, further experiments also verify this possibility.

Conclusion
We speculate that the mechanism of breast cancer chemosensitivity might be caused by a change in alternative splicing caused by the rs786204926 of PTEN gene. Thus, our study might provide theoretical guidance for the individualized treatment of clinical breast cancer patients.

Background
Breast cancer is a malignant tumour that occurs in breast epithelial tissue and is the most common malignant tumour in women [1]. Breast cancer has become a serious health problem threatening women all over the world [2,3]. Although chemotherapy is one of the main treatments for breast cancer, chemoresistance is still the main reason for the failure of breast cancer treatments [4][5][6]. Therefore, the identi cation of genetic mechanisms involved in chemoresistance is crucial for predicting the drug response of tumour with genetic mutations [7,8].
gene related to chemoresistance, and study how the polymorphism affects chemoresistance. In recent years there are some reports that polymorphism can affect the process of alternative splicing (AS), which lead to the occurrence and development of human diseases [22][23][24][25]. A small number of studies have found that the PTEN gene can produce new isoforms, such as retained intron 3 regions (3a, 3b, 3c) and intron 5 regions (5a, 5b, 5c); excluded part of exon 5 (DelE5) or all of exon 6 (DelE6); PTENα and PTENβ [26,27], but the abnormal AS of PTEN gene about chemoresistance is not reported.
Although many studies have shown the association between genetic polymorphisms of PTEN and drug resistance [28,29], the underlying molecular mechanism of PTEN gene polymorphism leading to breast cancer chemoresistance is still unclear. As far as we know, mutations in speci c sites of genes can cause certain diseases and affect the AS process of genes, but the association between SNP and AS of PTEN in Chinese population with the risk of breast cancer chemoresistance has not been reported. Therefore, this study aims to explore the molecular mechanism of the potential relationship between novel PTEN gene polymorphism and breast chemoresistance. Our work might provide theoretical guidance for individualized treatment of breast cancer chemotherapy in Chinese population.

Study population
This study enrolled 234 blood samples and randomly selected 9 tissue samples, all from the former Lanzhou General Hospital of Lanzhou Military Region between September 2013 and April 2020. All samples were clinically diagnosed and received chemotherapy. All chemotherapy regimens were based on anthracyclines, samples who had not received treatment, did not use anthracyclines, or treated less than 2 courses were excluded. Usually anthracycline-based chemotherapy regimens consist of 5-uorouracil (5-FU), anthracycline compounds and cyclophosphamide (FAC-FEC-EC or AC regimen). After treatment, the chemotherapy e cacy after anthracycline treatment was evaluated and scored according to the Response Evaluation Criteria in Solid Tumour (RECIST) criteria [30]. In this study, chemosensitivity were considered as patients with complete or partial remission, and patients with stable or progressing disease were classi ed as chemoresistance. The ethics committee of Lanzhou University School of Basic Medicine approved the study.

Extraction of genomic DNA and genotyping
Collected peripheral blood of all samples in sterile tubes with EDTA and stored at -80°C. Used the phenol/chloroform method to isolate genomic DNA from white blood cells. The extracted DNA was stored at -80°C until use. The concentration and purity of DNA were measured using NanoDrop 2000c spectrophotometer (Thermo Fisher Scienti c, Lenexa, KS, USA). Four SNPs (rs786204926, rs701848, rs12402181, rs35770269) were genotyped using Sequenom MassArray typing technology for samples with a DNA concentration of more than 20ng/μl (Beijing Bomiao Biotechnology Co., Ltd., China). Used Assay Design 3.1 software to design the primers required for Sequenom MassArray (Supplementary Table 1). After PCR ampli cation reaction, SAP-PCR reaction, puri cation reaction, and spotting the puri ed extension product, Spectro CHIP was prepared. After spotting, used MALDI-TOF mass spectrometer to detect Spectro CHIP, and used TYPER 4.0 software to analyse the results.

Semi-quantitative RT-PCR and sequencing
We randomly selected 6 breast cancer tissues samples after chemotherapy. Total RNA was extracted from the tissue using TRIzol (Invitrogen, Carlsbad, CA, USA), and cDNA was reverse transcribed using RT MasterMix (Cwbiotech, Beijing, China). PCR ampli cation of the cDNA using primers: PTEN-Wild-F: AATTGCAGAGTTGCACAATATCC; PTEN-Mutation-F: GTTATCTTTTTACCACGGTTGC; PTEN R: GTCTCTGGTCCTTACTTCCCC; PCR was initially activated at 94° C for 3min, followed by 32 cycles of 30s at 94°C ; 30s at 60° C; 25s at 72° C; nally at 72° C Extend for 5min. Analysed the product on a 1% agarose gel and used Quanity One software for quantitative analysis. PCR ampli ed products were veri ed by sequencing (General Biosystems (Anhui) Co., Ltd.).

Western blot analysis
We randomly selected 9 breast cancer tissues samples after chemotherapy. The total protein was extracted using radio immunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentration was measured using a protein concentration determination kit (Solarbio, Beijing, China). Then the proteins were quanti ed according to different concentrations, separated by polyacrylamide gel electrophoresis, In silico analysis In order to study the correlation between PTEN mutant genome and epirubicin drug sensitivity in breast cancer, we used Genomics of Drug Sensitivity in Cancer version 8.3 (GDSC, https://www.cancerrxgene.org/) [31].

Distributions of the clinical characteristics in breast cancer patients
The study included 234 breast cancer patients with 157 (67.1%) chemosensitive and 77 (32.9%) chemoresistant. Figure 1A is the imagological examination data of chemosensitivity and chemoresistance.

Statistical Analysis Of Four Snps Genotyping Results
MassArray genotyping was performed on the rs786204926, rs701848, rs12402181, rs35770269 polymorphism of 234 patients. We analysed the genotype frequency of SNP, rs786204926, rs701848, rs35770269 accords with the HW equilibrium state (Table 2). We further statistically analysed the genotype results and compared the distribution of genotypes under different genetic models. The results were listed in  Table 3). There is no correlation between rs701848, rs12402181, rs35770269 and breast cancer chemosensitivity. We randomly selected some samples to verify the genotyping results using Sanger sequencing method. Figure 1B showed the image of PCR products of some samples after agarose gel electrophoresis. The wild homozygous, heterozygous, and mutant homozygous sequence of the rs786204926 polymorphism were shown in Figure 1C.

Logistic Regression Analysis
The chemoresistance was used as the dependent variable, and related factors (sex, age, lymph node metastasis, rs786204926 genotype, etc.) were used as independent variables. Logistic regression analysis showed that age (P=0.000) was risk factors for breast cancer chemoresistance. lymph node staging (P=0.023) and rs786204926 AA/GG/AG genotype (P=0.000) were protective factors for breast cancer chemoresistance ( Figure 1D).

Linkage Disequilibrium (Ld) And Haplotype Analysis
SHEsis software was used to analyse the LD of the two polymorphisms of PTEN. Complete LD was detected in the polymorphisms of rs701848 and rs786204926 (r 2 =0.222, D'=1.000) (Figure 2A, 2B and 2C). At the same time, haplotype analysis was performed, the chemosensitive and chemoresistant groups with haplotype estimated frequency less than 3% were excluded from further analysis. We found that TA, TG haplotypes (rs701848, rs786204926) were related to chemosensitivity. Carrying TA might increase the risk of chemoresistance. Carrying TG might decrease the risk of chemoresistance. Other haplotypes were not signi cantly different between the chemosensitive and chemoresistant groups ( Figure 2D).

rs786204926 affects a new PTEN isoform expression association with drug sensitivity
According to The cBioPortal for Cancer Genomics (http://cbioportal.org) database analysis of samples with mutation data in invasive breast cancer (TCGA, Firehose Legacy) [33,34], PTEN had mutations in 9% of patients ( Figure 3A), and mutations in PTEN are associated with overall survival ( Figure 3C). In order to study the correlation between the PTEN mutant genome and epirubicin drug sensitivity in breast cancer, we used GDSC online software for analysis. The results showed that PTEN mutant genome would increase the sensitivity of epirubicin drug in breast cancer ( Figure 3C). The database had reported that there are two splice sites (X70_splice, X212_splice) on the PTEN gene. We predicted that the rs786204926 is also a splice site ( Figure 3D). Mutations at the splicing site might affect AS of genes, and these effects had been detected by Alamut Visual v.2.15 and HSF. HSF predicted whether the rs786204926A>G mutation affected AS. The prediction results showed that rs786204926 is a splice acceptor site, when carrying G allele, it will destroy the wild-type acceptor site and affect splicing (Supplementary Table 4). Further using Alamut Visual v.2.14 showed that the rs786204926A>G mutation destroyed the acceptor site and created a new acceptor site. It also showed the changes between the exon splicing enhancers (ESE) after mutation, the results showed that the ESE binding ability is weakened after mutation ( Figure 3E).

Higher levels of new isoforms of PTEN in chemosensitive samples of breast cancer
We randomly selected 6 breast cancer tissues after chemotherapy (2 GG types, 2 AG types, and 2 AA types). Perform RT-PCR ampli cation on the sample, and agarose gel electrophoresis on the products and sequence veri cation, the results showed that PTEN wild type had no difference among alleles (Figure 4). In PTEN mutant isoforms, AG and AA genotypes are statistically signi cant compared with GG alleles (P<0.05). The results show that rs786204926 affects the production of PTEN isoforms, and the levels of new PTEN subtypes are higher in breast cancer chemosensitive samples.
We randomly selected 9 breast cancer tissues after chemotherapy (3 GG types, 3 AG types, and 3 AA types). WB results showed that in PTEN wild type and mutant type, AG and AA genotypes were statistically signi cant compared with GG alleles (P<0.05). The AG and AA genotype in SRP40 and ASF/SF2 were statistically signi cant compared with the GG allele (P<0.05) ( Figure 5). The results showed that SNP and splicing factor related to breast cancer chemosensitivity, and the levels of PTEN-W and PTEN-M are higher in breast cancer chemosensitive samples.

Discussion
PTEN is the rst reported tumour suppressor gene with phosphatase activity, which plays a vital role in cell apoptosis, cell cycle arrest and cell migration, and is closely related to the occurrence and development of a variety of human malignant tumours [35][36][37]. Mutations or deletions of PTEN in a variety of malignant tumours, resulting in weakened or lost tumour suppressor function [15]. PTEN has a high deletion or mutation rate in breast cancer, and its loss of function is closely related to the malignant transformation of breast cancer. Liang H et al. found that the PTEN gene can produce new isoforms PTENβ, PTENβ protein speci cally locates in the nucleolus, binds to nucleolin and regulates its phosphorylation level, thereby regulating rDNA transcription and ribosome production, and inhibiting cell proliferation [27]. Agrawal S et al. found that there are new isoforms in sporadic breast cancer tissues. They are produced a short-lived structure PTEN by different splicing methods of PTEN introns 3 and 5, and its phosphatase activity is also limited. The short-lived structure PTEN plays an important role in the occurrence and development of sporadic breast cancer [26]. In addition, many studies have reported that PTEN gene plays an important role in multi-drug resistance in cancers, PTEN can speci cally dephosphorylate PIP3 in the cell membrane, thereby antagonizing the PI3K/AKT signaling pathway, leading to the development of drug resistance [38]. Previously, people thought that the high expression of PTEN was related to chemosensitivity, but people did not notice the expression of PTEN isoforms. In our research, we discovered a new PTEN isoform, which is closely related to chemosensitivity in breast cancer.
Previous studies have shown that SNP of PTEN might be a candidate pharmacogenomic factor to assess the susceptibility of breast cancer and response and prognosis prediction for chemotherapy in breast cancer [39]. Therefore, PTEN gene polymorphism was related to chemoresistance of breast cancer. In recent years, it has been found that PTEN gene polymorphism and chemoresistance are different in breast cancer. However, the potential association between rs786204926, rs701848, rs12402181, rs35770269 and chemoresistance in breast cancer has not been reported. In our research, we selected rs786204926, rs701848, rs12402181, rs35770269 to study the potential association between gene polymorphism and chemoresistance in breast cancer. Our research showed that rs786204926 of PTEN was closely related to breast cancer chemoresistance, and carrying the G allele or AG genotype will increase the risk of chemosensitive in breast cancer. Previous studies have con rmed that rs701848, rs12402181, and rs35770269 are related to drug resistance [39][40][41], but we have not found any correlation with breast cancer chemoresistance. It might be the association between genetic polymorphisms and breast cancer varies greatly among different races, indicating that different genetic backgrounds have different mutation frequencies [42]. In addition, studies have shown that genetic SNP might change the response to drugs, and there are signi cant differences between different races [43]. This indicates that mutations have different therapeutic effects on people with different genetic backgrounds. China has a vast territory and a large population. Different regions have different eating habits, cultural backgrounds, living environments, and genetic backgrounds. Our samples mainly collected patients from Northwest China. All samples came from patients in the hospital, so we analysed patients who did not meet the HW equilibrium state. This study is the rst time to detect and analyse the PTEN gene polymorphism of rs786204926 in Northwest China. It provides important theoretical data for establishing a database of PTEN gene polymorphism in Northwest China and promoting pharmacogenomics research to achieve personalized medicine.
Compared with previous studies related to chemoresistance, our research not only nd new SNP related to chemoresistance, but also used software to predict the correlation between rs786204926 and AS. Up to 50% of all mutations leading to genetic diseases result in abnormal splicing [44]. It has been found that PTEN splicing aberrations might be caused by genetic mutations at the junction of the splicing sites and deep in the introns [45]. The function of splice sites is to "signal" the assembling snRNAs and auxiliary splicing factors to recognize the "staging area" in order to initiate the assembly of the spliceosome eventually leading to the excision of intron and joining of exons to yield a mature mRNA [46]. This is one of the important mechanisms of AS involved in the occurrence and development of various diseases. When an A>G mutation occurs at this site, it will destroy the wild-type acceptor site to create a new acceptor site and affect AS. After mutation, there are 18 bases from pre-mRNA intron 4 will be retained on the mature mRNA, and the exon splicing enhancer (ESE) binding ability will also change, which might eventually cause the mutant protein to have 6 amino acids more than the wild type. In the structure of PTEN protein, the N-terminal phosphatase domain composed of N-terminal amino acids 1-185 is closely related to its phosphatase function [47]. The 6 amino acids produced by the mutant are exactly in this domain, which will cause the protein structure to change. After mutation, we found that there are varying degrees of protein structure changes in the amino acids. PTEN is a tumour suppressor gene with phosphatase activity [36]. On the cell membrane, PTEN mediates the conversion of 3,4,5-phosphatidylinositol triphosphate (PIP3) to PIP2, and inhibits the PI3K/Akt pathway to arrest the cell cycle In the G1 phase,lead to the development of drug resistance [48][49][50]. When rs786204926A>G is mutated, AS may cause changes in mRNA and protein, resulting in a new isoform of PTEN-M, which will eventually lead to the reduction or loss of the function of PTEN phosphatase. Loss of PTEN function will lose its negative regulatory effect on PI3K/Akt, Akt will be continuously activated, the cell cycle will be accelerated, and cell growth and proliferation will be accelerated, reducing the risk of breast cancer chemoresistance. Previously, people thought that PTEN was related to chemosensitivity, but people did not notice the expression of PTEN isoforms. In our research, we have discovered a new PTEN-W isoform in breast cancer. In the past, when studying PTEN, whether it was RT-PCR or WB, the sum of the wild and isoforms of PTEN was regarded as the expression level of PTEN. In our research, we have done a more detailed study, and the results suggest that we should pay attention to the respective expression levels of PTEN-W and PTEN-M isoforms. We analysed the collected clinical samples and found the correlation between rs786204926 polymorphism and chemosensitivity in breast cancer, and the G allele was associated with chemosensitivity in breast cancer. Later, we performed transcript and protein level veri cation. Carrying G allele is easier to produce PTEN-W in chemosensitivity patients. There is no difference in the transcription level of PTEN-W, but there is a signi cant difference in the protein level. PTEN-M has no difference in transcription and protein levels. Therefore, we found that PTEN-W is not related to chemosensitivity, PTEN-M is related to chemosensitivity, and the higher expression of PTEN-M, the more sensitive it is. At the same time, through software analysis, we found that PTEN-M has a stronger binding ability with PIP3 and is easier to exert its dephosphorylation effect, thereby inhibiting the PI3K-AKT pathway and leading to chemosensitivity. Therefore, we suspect that chemosensitivity of breast cancer patients is mainly caused by the AS of PTEN gene rs786204926 polymorphism (Figure 7).
Previous studies mainly collected clinical information and blood samples, test results, and statistical analysis to obtain the association between gene polymorphism and breast cancer chemoresistance. However, the mechanism of chemoresistance has not been further studied. Here, we have not only conducted association studies, but also predicted the function of SNPs in chemotherapy resistance-related genes by silico analysis, and further experiments also verify this possibility. We speculate that the mechanism of breast cancer chemosensitivity might be caused by a change in AS caused by rs786204926 of PTEN gene.

Conclusions
In conclusion, we hypothesize that the mechanism of breast cancer chemosensitivity might be caused by a change in AS caused by the rs786204926 of the PTEN gene. Our work not only provides theoretical guidance for the individualized treatment of clinical breast cancer patients in the Chinese population, but also provides new ideas for the mechanism of breast cancer chemoresistance.

Consent for publication
All authors have agreed on the contents of the manuscript.

Availability of data and material
All the data and materials are available.       Schematic representation of mechanism of chemosensitivity in breast cancer

Supplementary Files
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