https://orcid.org/0000-0002-3082-1892
Figures
Abstract
Background
Breast cancer is a multifactorial disease which involves number of molecular factors that are critically involved in proliferation of breast cancer cells. MEN1 gene that is traditionally known for its germline mutations in neuroendocrine tumors is associated with high risk of developing breast cancer in females with MEN1 syndrome. However, the paradoxical role of MEN1 is reported in sporadic breast cancer cases. The previous studies indicate the functional significance of MEN1 in regulating breast cells proliferation but its relevance in development and progression of breast cancer is still not known. Our study targets to find the role of MEN1 gene aberration and its clinical significance in breast cancer.
Methods
Breast tumor and adjacent normal tissue of 142 sporadic breast cancer patients were collected at the time of surgery. The expression analysis of MEN1 mRNA and protein was done through RT-PCR, immunohistochemistry and western blotting. Further to find the genetic and epigenetic alterations, automated sequencing and MS-PCR was performed respectively. Correlation between our findings and clinical parameters was determined using appropriate statistical tests.
Results
MEN1 expression was found to be significantly increased in the breast tumor tissue with its predominant nuclear localization. The elevated expression of MEN1 mRNA (63.38% cases) and protein (60.56% cases) exhibited a significant association with ER status of the patients. Most of the cases had unmethylated (53.52%) MEN1 promoter region, which can be a key factor responsible for dysregulated expression of MEN1 in breast cancer cases. Our findings also revealed the significant association of MEN1 mRNA overexpression with Age and lymph node status of the patients.
Citation: Massey S, Khan MA, Rab SO, Mustafa S, Khan A, Malik Z, et al. (2023) Evaluating the role of MEN1 gene expression and its clinical significance in breast cancer patients. PLoS ONE 18(7): e0288482. https://doi.org/10.1371/journal.pone.0288482
Editor: Abdul Rauf Shakoori, University of the Punjab, PAKISTAN
Received: April 6, 2023; Accepted: June 27, 2023; Published: July 12, 2023
Copyright: © 2023 Massey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Safia Obaidur Rab will be providing APC under Large Research Group Project under grant number RGP.02/339/44. the author has played role editing and proof reading of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The recent decade has witnessed a tremendous rise in incidence of breast cancer and according to globocan data; it is the 2nd leading cause of death among women globally [1]. Predominantly the breast cancer can originate by hyper-proliferation of milk producing glands or ductal epithelial cells of the breast [2, 3]. The multifarious nature of origin and progression of the disease makes it more challenging for the treatment. Hence, meticulous understanding of breast cancer associated genes as well as their pathogenic mechanism is crucial to develop preventive and therapeutic methods [4, 5].
Multiple Endocrine Neoplasia 1 (MEN1) gene that encodes menin protein having three isoforms and is located on chromosome 11 (11q13.1) is classically known for germline inactivating mutations in several endocrine tumors [6–8]. The functional importance of menin has been thoroughly defined in previous studies, highlighting its decisive role in number of epigenetic regulations, DNA repair and metabolic pathways [9, 10]. MEN1 has an enigmatic character and can function as an oncogene in cancers including leukaemia, prostate cancer, and hepatocellular carcinoma or as a tumor suppressor in endocrine tumors [11, 12]. Apart from mutational anomalies, expressional variations of MEN1 are widely recognised in MEN1 related disorders. Patients with overexpression of MEN1 in prostate and hepatocellular carcinoma reportedly show poor survival when compared to patients with low MEN1 expression [13, 14]. Also, the oncogenic behaviour of MEN1, through its direct interaction with MLL to promote leukemogenesis is well documented in prior studies [15]. MEN1 deficiency is widely associated with an increased risk of developing breast cancer in female patients with MEN1 syndrome [16]. However, a paradoxical role of menin is reported in sporadic breast cancer cases, where it shows proliferative function and is also linked with resistance to drug and endocrine therapy [16–18].
Although research in this field is ongoing, the role of MEN1 in the onset and progression of breast cancer is not fully known. The expression of MEN1 in the mammary glands may be controlled by the level of released hormones, such as prolactin, and have a negative response in the process of milk protein synthesis via PI3K/Akt/mTOR [19]. Disruption in hormonal balance due to MEN1 mutations or its altered expression can potentially contribute to breast cancer development. In recent studies, direct interaction of menin with AF2 domain of ERα in estrogen positive cell line has been reported and it might promote ERα dependent transcription and proliferation. Moreover, for antiestrogen therapy drugs that bind to the AF2 domain of the ERα, menin’s interaction with this region could be the reason in patients with MEN1 overexpression to develop resistance to adjuvant therapy [17, 20]. Apart from being co-activator for ERα, it also regulates expression of ESR in ER+ breast cancer cell that further supports its proliferative function in sporadic breast cancer cases [21].
The previously published literature signifies the distinctive functions of MEN1 in breast cancer patients and also provides scope to evaluate its anomalous expression and connection with clinical parameters. Thereby our study targets to untangle MEN1 expression at mRNA and protein level and also evaluate its epigenetic and polymorphic alterations in sporadic breast cancer patients. The findings of our study provide better insight in understanding the clinical significance of MEN1 gene in the breast cancer cases.
Material and methods
Study samples
This study includes 142 clinically confirmed sporadic breast cancer cases that are genetically unrelated Indian females of age 20 to 79 years and with breast tumor as the primary site. The patients who have received chemotherapy or radiotherapy or had multiple tumors were excluded from the study. At the time of surgery aliquot of tumor along with adjacent normal tissues were taken in PBS, RNA later and formalin for further processing. The written informed consent was taken from the patients volunteering to participate in the study and the clinicopathological parameters considered at time of diagnosis were procured from patient’s record maintaining the patient’s confidentiality. The ethical approval to conduct this research was provided by Institute Ethics Committee, AIIMS, New Delhi (IEC-849/03.12.2021) and Institutional Ethics Committee, JMI, New Delhi (25/7/236/JMI/IEC/2019). All the experiments were conducted in the year 2022 to 2023 after obtaining the ethical approval from both the institutions and strictly following the ethical guidelines. The enrolment details and clinical profile of the patients are provided in the S1 Table.
mRNA expression analysis
RNA was isolated from the tissue samples stored in RNA later at -80°C. Tissue were homogenized in TRIZOL using the homogenizer followed by the phase separation, precipitation and washing step as instructed in the manufacturer’s protocol. The obtained RNA pellet was dissolved in the RNAase free water and was treated with DNAse to eliminate any DNA contamination. The purity and the concentration of isolated RNA was assessed through nanodrop spectrophotometer at 260/280 ratio. RNA samples having purity ~2.0 were used for cDNA synthesis. cDNA were constructed using Verso cDNA synthesis kit, 1000 ng of RNA was used per 20 ul reaction.
The constructed cDNA was subjected to Real Time Polymerase Chain Reaction (RT-PCR) using specific primers designed for MEN1 and ACTB was taken as internal control (Table 1). The RT-PCR reaction was carried out using KAPA SYBR® FAST (cat no: KK4610) master mix, following the steps provided in manufacturer’s protocol. The RT-PCR data was analyzed by the comparative Ct method using formula: Fold Change = 2-ΔΔCt; ΔΔCt = (Cttargeted gene−CtACTB) targeted sample—(Cttargeted gene−CtACTB) calibration sample.
DNA isolation
Genomic DNA was isolated by homogenizing 50–100 ug of tissue in the tissue lysis buffer and overnight incubation at 45°C [22]. Further, Phenol-chloroform method was used for DNA extraction from the tissue lysate. The qualitative and quantitative analysis of the obtained genomic DNA was done through gel electrophoresis using 0.7% agarose gel. The purity of the DNA was confirmed by nanodrop spectrophotometer by taking absorbance ratio at 260/280 A. The isolated genomic DNA having purity ~1.8 were used for further experiments.
MS-PCR
To determine the promoter methylation status of MEN1 gene through Methylation specific PCR (MS-PCR), firstly genomic DNA was bisulfite modified using EZ DNA Methylation-Lightning Kit (cat: D5030). Two sets of primers specific to methylated and unmethylated promoter region of MEN1 gene were designed using methprimers (Table 1) (Fig 1). The PCR reaction of 25 ul was prepared containing 100 ng of bisulfite-converted DNA, 1.5 mM MgCl2, 200 μM of each deoxynucleotide triphosphates (dNTPs: dATP, dCTP, dGTP, and dTTP), 0.5 μM of each forward and reverse oligonucleotide primers, 1 x PCR buffer, and 1 unit of Hot Start Taq DNA Polymerase (Qiagen, Hilden, Germany). The PCR amplification of bisulfite modified DNA was carried out by using following the standardized protocol of our laboratory [23, 24]. The qualitative analysis of PCR product was done by gel electrophoresis using 2% agarose gel followed by visualization under UV-transilluminator.
Immunohistochemistry
Differential expression of menin was analyzed through immunohistochemistry using specific antibody for menin (A500-003A). Formalin fixed tissue blocks of breast cancer tissue were prepared and sectioned on poly-L-lysine coated slides. Different grades of xylene were used for deparaffinizing the sections and then rehydrated through series of ethanol. 0.3% hydrogen peroxide was used to block endogenous peroxidase activity, further Ag retrieval was done through boiling in citrate buffer. The slides were subjected to overnight incubation at 4°C with primary antibody at dilution 1:200 followed by incubating with secondary antibody at 37°C for 1 hour. 3,3’- Diaminobenzidine (DAB) treatment was given for visualizing the antibody binding site and Hematoxylin was used for counter staining.Normal breast tissue were considered as the positive control and the sections processed following identical steps excluding primary antibody incubation served as negative control. The immunohistological staining was then interpreted by expert histopathologists and were graded as follows: (a) no expression- 0%, (b) mild expression- 1% - 10%, (c) moderate expression- 10% - 50%, (d) high expression- >50% [25].
Western blotting
Total protein was isolated from tissue samples using RIPA buffer. The quantification of the protein was done through Bradford method using BSA concentration as the standard curve. Equal amount protein (30μg) was resolved on 10% SDS gel and was transferred to nitrocellulose membrane. One hour blocking in fat free milk was done followed by overnight primary antibody incubation for menin (D45B1) and control gene beta actin (sc-4778). After 3 times washing in TBST, membrane was incubated for 1 hour in suitable HRP conjugated secondary antibodies. Clarity western ECL substrate (cat: 170–5060) was used for protein detection by chemi-luminescence (Bio Rad).
Automated DNA sequencing
MEN1 mutations are prominently known in human cancers. MEN1 gene comprises of total 10 exons of which exon 8 (136bp), exon 9 (165bp) and exon 10 (1,301bp) of MEN1 gene are identified for their critical relevance in menin interaction with various proteins and also coding nucleus localizing signals. The mentioned exons were amplified using specific primers and PCR product were purified using Qiagen MinElute PCR Purification kit (Cat. No.28004). The amplified products were subjected to direct sequencing at Macrogen Inc., South Korea Lab using both forward and reverse primers. The sequencing results were analyzed using the software clustal omega. The sequencing was repeated to rule out any contamination or false results.
METABRIC data analysis
METABRIC data set comprises of the clinical profiles, survival data, copy number variants, expression and SNP genotypes of the patients participating in the METABRIC trial. We analyzed the MEN1 expression in the breast cancer patients included in the METABRIC trial data set available at cBioPortal and compared the survival data of the participants with low MEN1 and high MEN1 expression. Survival curve was plotted using graph pad prism.
Statistical analysis
The statistical correlation of our molecular findings and clinical parameters was done using SPSS-IBM (version 22.0) and graph pad prism. The mRNA expression data in the study have been expressed as mean ± standard error of mean. Non-parametric test i.e. Wilcoxon signed-ranked test and Kruskal wallis test were performed to evaluate the significant difference between mRNA expression of MEN1/ACTB and compare the mRNA expression with clinical parameters respectively. The correlation of protein expression and methylation status with clinical parameters was performed using chi square test and the p value <0.05 is considered to be significant.
Results
Clinical profiling of the enrolled patients
In current study, breast tissue samples from female breast cancer patients (N = 142) were taken at the time of surgery. Majority of the patients enrolled in the study are postmenopausal (87/142), indicating a higher frequency of breast cancer among postmenopausal females. The status of the hormone receptors ER, PR, and Her2 neu of the recruited patients was screened, and were categorized into molecular subtypes accordingly. Most of the patients (101/142) were at the advanced stage (III and IV), signifying poor diagnosis of the disease (Table 2).
Upregulated expression of MEN1 mRNA in breast tumors
Our analysis of mRNA expression using real-time PCR shows that MEN1 mRNA is upregulated in breast tumor samples as compared to the nearby normal tissue samples. The housekeeping gene ACTB was used to normalize the expression of MEN1, and a mean fold change of 5.17 was observed in the upregulated cases. Overall, 63.38% (90/142) cases exhibited the overexpression expression of MEN1 at mRNA level and on correlating the elevated expression with clinical parameters; the significant association was observed with estrogen receptor status (p = 0.015), Age of the patient (p = 0.028) and lymph node status (p = 0.024). However, in our results we found that correlation of MEN1 upregulation with molecular subtype (p = 0.118) and clinical stage (p = 0.341) of breast cancer is not significant (Table 3) (Fig 2).
Box plots representing significant change in expression of MEN1 mRNA (A) Breast tumor and adjacent normal tissue. (B) ER negative and ER positive cases (C) Lymph node negative and lumph node positive cases (D) Age upto 50 and age more than 50.
Loss of promoter methylation in cases with MEN1 overexpression
MS-PCR of bisulfite modified DNA revealed unmethylated CpGs in the MEN1 promoter region of 53.52% (76/142) breast tumor samples, with the majority (68.42%) having elevated menin expression. The lack of promoter methylation was found to have a significant correlation (p<0.001) with MEN1 expression, with 72.37% (55/76) cases exhibiting upregulated MEN1 expression. Furthermore, our promoter methylation study shows aberrant promoter methylation in 29 cases, out of which only 09 cases had MEN1 mRNA overexpression. In addition, 37 cases had no change in promoter methylation status and were considered as unaltered. In advanced stages III and IV of breast cancer, a strong correlation (p = 0.034) with unmethylated promoter region was observed, with 80.26% (61/76) cases having unmethylated MEN1 promoter region. Also, the age of the patients at menopause exhibited significant association (p = 0.009) and 34.48% (30/45) early menopause cases had unmethylated CpG’s in MEN1 promoter region (Table 4) (Fig 3).
(A) Representative Agarose gel picture for MS-PCR showing promoter methylation (product size: M = 161) and unmethylation status (product size: UM = 160) of MEN1 in tumor tissue and adjacent normal breast tissue. (B) MS-PCR results exhibiting the MEN1 promoter methylation and unmethylation status of breast tumor tissue.
MEN1 protein expression and its association with clinicopathological parameters in breast cancer
Immunohistochemistry and western blotting results were compared and analysed to determine the expression of the MEN1 gene at the protein level. Menin was found to be overexpressed in 86 of 142 patients, with its predominant nuclear localization. The Menin protein was elevated in 60.56% of cases, with its significant upregulation in breast tumor as compared to normal tissue. Our findings indicate a strong association between increased Menin expression and ER positive cases (p = 0.002), with 46 of 61 ER positive cases showing elevated Menin expression (Table 5) (Figs 4 and 5).
Representative panel of immunohistochemical images taken at 20X magnification for MEN1 protein detection in breast tissue (A) Normal Breast tissue (B) Breast cancer tissue showing low expression of menin protein (C) Breast cancer tissue with high expression of menin protein.
MEN1 protein expression analysis through western blot (A) elevated expression of menin in tumor tissue as compared to its paired normal breast tissue (B) comparative analysis of menin/Beta actin expression in normal and breast tumor tissues.
MEN1 is not mutated in Indian breast cancer patients
MEN1 mutations are frequent and aggressive C-terminus of MEN1 protein forms the finger domain and contains three nucleus localizing sequences. The disruption in C-terminus is known to have a vital role in tumorigenesis [9, 26, 27]. However, our automated sequencing analysis did not show any mutation in the exons coding this domain of MEN1 gene (Fig 6).
High MEN1 expression is associated with poor survival
The METABRIC data analysis to compare the survival among the patients with low MEN1 and high MEN1 expression was done [28]. Poor survival was observed amongst the participants having higher expression of MEN1 gene when compared to participants with low MEN1 expression (Fig 7).
Discussion
Even after rigorous research and advancement made in diagnosis and treatment, the steady rise in breast cancer incidence has compelled clinicians and researchers to revisit our traditional approaches in managing this disease [29]. Even though most of the patients share common histological features at the time of diagnosis, the underlying molecular aspects of the disease leads to varied clinical outcomes in response to traditional therapies [30, 31]. Moreover, these traditional therapies are much focused on ER, PR and Her2 expression status to treat the disease rather than evaluating complex expression patterns of related genes. To ensure more assertive clinical outcomes and survival of the patients, personalized approach of treatment with better understanding of molecular drivers of chief pathways involved in the progression of breast cancer is needed [32–34].
Earlier studies on women with sporadic breast cancer and those with the MEN1 syndrome have underlined the contradicting role of MEN1 in the disease. The anti-proliferative function of MEN1 is reported in normal mammary epithelium cells and females with MEN1 syndrome are at high risk of developing cancer. However, the contradictory role of MEN1 in sporadic breast cancer cases is observed where it is linked to tumorigenesis through coactivation of ERα [35–37]. While the molecular functions of MEN1 are being widely explored in previous researches, we aimed to evaluate the MEN1 gene expression pattern and determine its relevance to clinical parameters in 142 sporadic breast cancer patients.
The findings of our study reveal the upregulated expression of MEN1 mRNA in nearly 63% of breast cancer cases. Interestingly, 46 of 61 ER+ patients included in the study had elevated levels of MEN1 mRNA, showing a strong correlation between MEN1 mRNA overexpression and ER+ status. Further, when the protein expression was evaluated with IHC and western blotting, similar outcome was observed. Almost 60% cases exhibited the elevated expression of menin protein with its prominent nuclear localization and showed significant association with ER status of the patients. MEN1 is majorly a nuclear protein and is known to be crucial regulator of gene transcription and cellular pathways [38]. Its direct interaction with various histone modifiers like PRMT5, MLL, and SUV39H1 is well established in the previous studies. Also, MEN1 interacts with NFKβ, JunD, cMyc transcription factors and control the expression of associated genes [10, 39]. These diverse interactions of MEN1 are responsible for its dual behavior in tumorigenesis and also its tissue specific functioning. In the recently published literature, probable role of MEN1 in ESR expression is reported in ER+ breast cancer cell [18, 20, 21]. These connections indeed support our findings and MEN1 could be a key player of cellular proliferation in ER+ cells.
The upregulated expression of MEN1 mRNA was also found to be significantly associated with lymph node status and age of the patients. The positive lymph node score of axillary lymph nodes is considered as important prognostic marker in breast cancer and is also known to have critical role in tumor free survival of the patients [40]. The role of menin in epithelial to mesenchymal transition is reported previously and can lead to metastatic response via TGF-β/Menin/C/EBPβ regulatory axis [41]. In most of the patients included in our study, higher MEN1 mRNA expression was observed in lymph node positive cases, indicating its possible role in poor survival of the patients. To further investigate epigenetic or polymorphic alteration that could possibly be involved in the anomalous expression of MEN1 in breast tumors, MS-PCR and Sanger sequencing was performed. Mutations in the MEN1 gene can cause menin function to be lost or altered, compromising the normal regulation of cell growth and division [9, 42]. The C terminal of MEN1 protein has nucleus localizing sequences that are critical for its nuclear import; however there are very limited instances where MEN1 mutations in cases of sporadic breast cancer are reported [9, 43]. We did not find any alteration in these sequences in breast cancer cases included in our study. To ensure continuous growth, cancer cells undergo multiple changes to alter gene expression programming of which DNA methylation alterations across the genome have major role. DNA methylation status is known to have strong connection with mRNA levels [44, 45]. Our promoter methylation study for MEN1 gene depicts the hypomethylation in majority of the cases and when compared with the clinical parameters of the patients, majority of the unmethylated cases were of advanced clinical stages. In persistence, when the promoter hypomethylation was compared with MEN1 mRNA and protein expression, a significant correlation was seen.
The critical association of MEN1 with Androgen receptor signalling pathway and its oncogenic role in prostate cancer is well documented. Its higher expression is reported to be linked with poor survival and developing resistance to the treatment in various malignancies including hepatocellular carcinoma and prostate cancer [11–13, 46]. We further evaluated the significance of MEN1 expression in survival of the breast cancer patients. METABRIC data was analyzed and the patients with higher expression of MEN1 were found to have poor survival in comparison to cases with low MEN1 expression. In future, case studies could be designed on larger population to evaluate the differential expression of MEN1 gene in different phenomenon like disease free survival, metastasis and resistance to hormone and drug therapy. This will give better insight in understanding clinical significance of MEN1in breast cancer patients.
Conclusion
In conclusion, our study demonstrates the higher expression of MEN1 in sporadic breast cancer patients. Previous researches indicated the enigmatic role of MEN1 in breast cancer, where its positive association is evident with ERα and ESR [21]. The overexpression of the MEN1 gene in Indian breast cancer patients is reported for the first time in our study and it exhibits substantial correlation with the ER+ status of the patients. In most of the cases MEN1 promoter region was unmethylated and had significant association with high expression of MEN1 gene. We also report that MEN1 mutations, especially at NLS are not prominent in Indian breast cancer patients included in our study. Our findings can be pioneer for further research to determine if MEN1 plays a tumor suppressive or oncogenic role in breast cancer.
Supporting information
S1 Table. Clinical profile of patients included in the study (2015–2022).
https://doi.org/10.1371/journal.pone.0288482.s001
(PDF)
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