| Abstract|| |
Background and Aims: Epithelial stromal interaction protein-1 (EPSTI-1) is originally identified as stromal-fibroblast – induced gene in breast cancer. It was found to be involved in promotion of EMT, breast cancer invasion, metastasis and anchorage-independent growth in vitro. Strong expression was observed in various tissues as well as higher expression was observed in invasive breast cancer compared to normal breast. EPSTI-1 expression was evaluated from 106 pre-therapeutic breast cancer patients. EPSTI-1 expression was correlated with known clinico-pathological parameters of breast cancer to explore its role in breast carcinogenesis. Subjects and Methods: EPSTI-1 expression was analyzed from the collected synchronous tissues [tumors, Malignant Lymph nodes (LN) and adjacent normal tissues (ANT)] of breast carcinoma patients (N = 106). The statistical correlation was performed using SPSS 16.0. Results: In this study EPSTI-1 was significantly higher in LN compared to tumors (P < 0.001), and in tumors compared to ANT (P < 0.01) which is also reflected in ROC curve analysis (P < 0.0001). Further the small tumor size, stage I, grade I and tumors without stromal involvement exhibited significant lower expression compared to their counter parts. Conclusion: EPSTI-1 may have significant role in epithelial stromal interaction and disease extension. Moreover, it may be responsible for aggressive tumor behavior and involved in metastatic process which needs to be validated in larger cohort.
Keywords: Breast cancer, epithelial mesenchymal transition, epithelial stromal interaction, epithelial stromal interaction protein-1
|How to cite this article:|
Raval AP, Desai UN, Joshi JS, Shah FD. Role of epithelial - Stromal interaction protein-1 expression in breast cancer. Indian J Pathol Microbiol 2020;63:382-7
|How to cite this URL:|
Raval AP, Desai UN, Joshi JS, Shah FD. Role of epithelial - Stromal interaction protein-1 expression in breast cancer. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Jun 14];63:382-7. Available from: https://www.ijpmonline.org/text.asp?2020/63/3/382/291684
| Introduction|| |
Breast cancer is the most frequent type of cancer in women worldwide. The breast tumor is heterogeneous in nature at both epidemiological and at molecular levels. The higher mortality rate of the disease is due to metastasis to distant organ. Metastatic cascade includes epithelial stromal interaction (ESI) and epithelial mesenchymal transition (EMT). Epithelial stromal interaction is considered as the pre event of the epithelial mesenchymal transition leading to metastasis, governed by certain molecules and EPSTI-1 is one of these genes. The epithelial stromal interaction occurs during the advancement of the tumor when tumor cells interact extensively with the surrounding stroma.,
The EPSTI-1 is an interferon (IFN) response gene located on chromosome 13q13.3. It contains 11 exons and spans 104.2 Kb. The higher expression of this gene was noted in spleen, germinal center in lymphatic tissues, small intestine, salivary gland, testes and placenta that involved high stromal activities suggest that EPSTI-1 is a communicator between epithelial cells and stromal compartment and hence, the name is epithelial stromal interaction protein-1., However, the role of EPSTI-1 gene is less explored. Here in this study, we try to explore the role of this molecule in breast cancer where we have checked the transcript expression of EPSTI-1 in breast cancer patients and correlated the expression with the known clinico-pathologic prognosticator of breast cancer.
| Subjects and Methods|| |
A total number of 106 breast cancer tissue samples were analyzed in the present study. The clinico-pathological parameters classifying patients are described in [Table 1]. The study was performed according to the ethical standards of the 1975 Declarations of Helsinki, as revised in 2008. Informed consent was obtained from individual cases prior to sampling as the study was approved by the Institutional Review board and Ethics committee at the Gujarat Cancer and Research Institute. Fresh tissue samples including Tumors (N = 106), Lymphnodes (wherever possible; N = 67(N+ = 47 and N- = 20) and the Adjacent normal tissues (ANT; N = 106) were collected at the time of surgeries (simple and radical mastectomies).
Patients with pre-existing hormonal disorders or an HIV/HBsAg positive status was not included. The presence of primary breast tumor was verified histo-pathologically and only those samples that were conceded the criteria were included in the study. Moreover, following sampling, the tissues were sectioned into two mirror image specimens. One of the sections was always evaluated by the pathology unit, in order to confirm the presence or absence of malignancy in the tumor specimen. The samples thus acquired were scraped and cleaned and then snap frozen in liquid nitrogen for storage. The other tissue specimens were immediately frozen in liquid nitrogen after the biopsy and stored at -80°C until further use. These tissues were processed for the RNA extraction and cDNA synthesis. The copy numbers obtained by qRT-PCR analysis were expressed as copy numbers/μgRNA.
RNA extraction and cDNA synthesis
The snap frozen tissues were powdered with a Mikrodismembrator (B. Braun, Germany). Total RNA extraction was performed from 50 mg of powdered tumor tissue using RNeasy Mini kit (Qiagen). Due to low cellularity of the adjacent normal tissue, 100 mg of powdered tissue was processed with RNeasy Lipid Tissue Mini Kit (Qiagen). Company provided protocols were used in both instances. On column DNA digestion was carried out for all samples with RNase–Free DNase set (Qiagen). The Extracted RNA was quantified and qualified with spectrophotometry using the Multiskan Spectrum (Thermo Labsystems). The RNAs (5 μg) were reverse transcribed using High-Capacity cDNA Archive Kit (Applied Biosystems) using the temperature cycles of 25°C for 10 minutes and 37°C for 120 minutes. The obtained cDNA was stored at -20°C until analysis.
Real time PCR
ABI Prism 7000 Sequence Detection System was used for absolute quantification of synthesized cDNA. 20 μg/20 μl reaction volume was used for each assay. Taqman primers and probes for EPSTI-1 (Hs015667890234244_m1) and for GAPDH (HKG) (Hs99999905_m1) with purified plasmid standards ranging from 100 to 1,000,000 copy numbers were designed (Applied Biosystems, Hilden, Germany), and assayed simultaneously in each experiment. The real time PCR was programmed for 40 cycles: 2 minutes at 50°C for uracil-N-glycosylase (UNG) activation; 10 minutes hold at 95°C for DNA polymerase activation and; 15 sec at 95°C and 1 minute at 60°C for 40 cycles for DNA melting and annealing/extension, respectively. All samples were duplicated and the levels of cDNA (copies/μl) were reported using plasmid standards. Negative Template Controls were used in every experiment. The copy numbers obtained from every sample were converted to per μg RNA for further analysis.
Statistical analysis was done using SPSS ver. 16.0. Student 't' test was used to calculate the mean levels of EPSTI-1. ROC curve analysis was performed for EPSTI-1 using values from the adjacent normal tissue for comparison. P value ≤0.05 was considered statistically significant level.
| Results|| |
The clinico-pathological details are showed in [Table 1]. In the study cohort, patient's age ranged from 20 to 80 years (mean = 47; median = 46). A prevalence of breast cancer patients was higher within age group of 40 to 60 years (68.9%). Metastatic node positive disease was observed in 59.4% patients. A predominance of patients with tumor size between 2 – 5 cm and stage II disease was observed. Invasive/Infiltrative Ductal Carcinomas (IDC) were observed in 86.8% patients and Invasive/Infiltrative Lobular Carcinomas (ILC) were seen in 2.8% patients while in 10.4% patients other breast diseases (Mucinous Carcinoma, Paget's disease, Papillary Carcinoma etc.) were detected. Maximum numbers of patients were diagnosed at stage II and majority of the tumors were moderately differentiated, of ductal origin, and were positive for the stromal reaction with different intensity suggests that the tumors are aggressive in nature.
Expression of EPSTI-1 in breast cancer
[Table 2] shows the expression of EPSTI-1 in tumor tissues, lymphnode tissues and adjacent normal tissues. EPSTI-1 transcripts levels were significantly higher in lymphnodes (P < 0.001) as compared to tumors (P < 0.001). Further the EPSTI-1 transcript levels were significantly higher in tumors compared to ANTs (P < 0.001). Thus, transcript levels in lymph nodes were the highest followed by tumors and then adjacent normal tissues. A severe upregulation of EPSTI-1 was observed in tumor tissues as compared to ANTs, while the lymphnodes showed the highest EPSTI-1 expression amongst all and was even significantly higher than the tumor tissues.
Receiver Operative characteristic Curve analysis (ROC) for EPSTI-1 expression in breast cancer
In ROC curve analysis, a strong demarcation was observed between tumors and ANTs (P = 0.0001, [Figure 1]) with the cutoff level of 561207 CN/μg RNA and area under curve (AUC) is 0.915. The specificity and the sensitivity of the curve were 88.68% and 85.85%, respectively. The positive predictive value and the negative predictive value were 88.3 and 86.2, respectively.
Associations of EPSTI-1 expression with clinico-pathological parameters
[Table 3] shows the association o f the EPSTI-1 expression and clinico-pathological parameters. The lymphnodes showed the highest expression in all 3 age groups. Significantly higher levels were observed in tumor tissues of all age groups as compared to the corresponding ANTs (young and middle age: P < 0.001; older age: P < 0.01). The expression was significantly higher in lymphnodes as compared to tumors of middle age group patients (P < 0.02). Correlating the expression with the menopausal status, the lymphnodes of the pre- menopausal patients showed the higher expression as compared to the tumors (P < 0.01). The tumors of all the menopausal groups showed the higher expression as compared to the adjacent normal tissues (Pre M and Post M:p < 0.001;Peri M:p < 0.02).
The EPSTI-1 expression was correlated with pathological variables like tumor size, nodal status, stage, histological type and histological grade. T1 tumors showed significantly lower expression as compared to T2 (P < 0.001), T3 (P < 0.05) and T4 (P < 0.01) tumors. The tumoral expression was higher for all tumor sizes as compared to the adjacent normal tissues [T1 (P < 0.01), T2 (P < 0.001), T3 (P < 0.01) and T4 (P < 0.001) small (P < 0.01) and large-sized (P < 0.001)]. The T4 (P < 0.05) and large-sized tumors (P < 0.01) depicts significantly lower EPSTI-1 expression as compared to their respective lymphnodes. The node negative and node positive tumors demonstrated higher EPSTI-1 expression as compared to ANT (P < 0.001). Further, the lymphnodes of both the groups showed highest expression as compared to their respective tumors (P < 0.05). Stage I tumors expressed the lowest levels of EPSTI-1 as compared to all other stages (P < 0.001). The tumors of all stages demonstrated the higher EPSTI-1 expression as compared to their relevant ANTs (stage I-p < 0.01, stage II, III-p < 0.001, stage IV-p < 0.02). Stage III tumors showed lower expression as compared to their lymphnodes (P < 0.05). The IDC (P < 0.001) and others (P < 0.01) type tumors showed significant higher expression compared to their respective ANTs. Further the lymphnodes corresponds to the IDC tumors showed the higher expression compared to the tumors indicates the invasive potential of these tumors (P < 0.01). The tumors of all the grades expressed the higher EPSTI-1 expression compared to their corresponding ANTs (P < 0.001). The well differentiated tumors showed the lowest expression as compared to the moderate (P < 0.02), poor (P < 0.05) and non- well differentiated tumors (P < 0.01). The lymphnodes showed the higher expression compared to their respective tumors in poorly (P < 0.001) and in non-well differentiated (P < 0.001) tumors.
Stromal reaction is an important parameter for tumor development and advancement. Tumors without stromal involvement (Absent) showed significantly lower expression compared to the tumors with stromal reaction (present) (P < 0.001) and its different intensities (+p < 0.001;++p < 0.01;+++p < 0.05). In all the comparisons, the tumors showed the higher expression as compared to their ANTs. The tumors with stromal involvement and tumors with ++ intensity showed lower expression compared to their respective lymphnodes (P < 0.01).
| Discussion|| |
Despite involvement of EPSTI-1 in EMT, there is dearth of data in expression of EPSTI-1 in breast cancer. Breast cancer metastasis is the resultant effect of the epithelial mesenchymal transition, initiated by the epithelial stromal interaction. The epithelial stromal interaction includes the interaction of the epithelial cells and cells of the collagenous stromal tissue of the breast. This interaction is critical for the malignancy and is governed by certain molecules.
However, the accurate mechanism for the process is not known. It was speculated that at the upstream of TGFβ,,,, and KLF-8 may be involved in the process that triggers EPSTI-1 to initiate the epithelial stromal interaction and EMT.,, The activation of EPSTI-1 triggers NFκB  and downstream events that may lead the cell towards epithelial mesenchymal transition. In the current study, the cohort possess aggressive tumor characteristics. Lymphnodes showed maximum expression compared to tumors and ANTs in overall cohort as well as in parametric analysis for stage, lymphocytic infiltration, and for stromal reaction positivity. The tumoral expression was significantly higher compared to ANT. Lymphnode positive tumors and lymphnodes showed the higher expression compared to node negative tumors and lymphnodes. EPSTI-1 expression is lower in small sized tumors as well as in well differentiated tumors as compared to their counter groups. The lymphnodes with the highest stromal intensity showed the highest expression, which is a noteworthy observation.
The results are suggestive of the aggressive phenomena that correlate with the higher EPSTI-1 expression. Particularly, it is well correlated with the lymphnode metastasis. Hence, it is speculated that EPSTI-1 may induce tumorigenesis and might be important in disease advancement leading to metastasis by introducing epithelial stromal interaction. However, the current study did not reveal the role of any other speculated molecule involved in the process. As per the best knowledge to us this is the first study estimated the gene expression of EPSTI-1 in 106 breast cancer patients, wherein the role of EPSTI-1 in epithelial stromal interaction and in disease extension is clearly evident.
EPSTI-1 is one of these genes that upregulated while interaction between tumor cells and stromal cells in tumor environment assay. It was demonstrated that stromal cells expressed higher levels of EPSTI-1 expression in tumors and ANT. Correlating this study with the current study the EPSTI-1 expression was higher in the tumors with higher stromal reaction suggest that EPSTI-1 activity increases with the higher stromal component. This view explains that the aggressive tumors may showed the higher stromal activity. Hence, a higher epithelial stromal interaction add strength to the possibility that EPSTI-1 reflects an important molecular event associated with organ development, tissue remodeling and neoplasia, but it needs to be elucidated that the higher expression of EPSTI-1 or interaction of epithelial cells to stromal cells is an early event or a late event in the malignant transformation. The lymphnodes showed the highest expression compared to tumors in the current study suggest the distant metastasis. The distant metastasis occurs due to the invasion/breakage of tumor boundaries/may be due to the Involvement of cancer associated fibroblast (CAFS) as a part of stromal epithelial interaction considered as a pre event of epithelial mesenchymal transition. This result and associated speculation is supported by the study of Li et al., where the authors have found EPSTI-1 serves as one of the driving forces and a support for lung metastasis and tumor invasion from breast.
Further, the transcriptional activation of KLF-8 to EPSTI-1 is connected with tumor microenvironment which is critical for breast cancer progression. Both of these molecules can induce EMT that release the stromal factors, stimulates the expression of KLF-8, and plays a role in the large tumor size in breast cancer. In accordance with the study of Wang et al. (2011), the result of our study demonstrate lower expression in stage I compared to other stages as well as the highest expression was found in lymphnodes of the patients having large tumors.
In a study by Neegraard et al. (2010), the role of EPSTI-1 was checked in regulation of tumor cell properties and epithelial mesenchymal transition. The authors have found no EPSTI-1 staining in normal breast epithelial cells while in breast cancer tissues it was present in ~50% (22/40 biopsies) cases. Further, the EPSTI-1 is directly associated with the apoptosis rate and increase in the proliferation rate of the cells. They have also demonstrated that this molecule can replace peritumoral activated fibroblasts in tumor environment assay. Hence, the EPSTI-1 is un-appreciated regulator of tumor cell properties and can increase the spread and invasion. Additionally, they have also observed that EPSTI-1 increases tumor sphere formation suggesting its 'stem cell characteristics'.
In conclusion, the EPSTI-1 is strongly involved in the epithelial stromal interaction that gives the aggressive nature to the tumors in breast cancer patients. The molecule is significantly associated with the disease extension (node positivity) and responsible for the invasive nature of the tumor suggesting its significance in the process of metastasis. However, more extensive study is warranted that includes protein expression to elucidate the role of EPSTI-1 in breast cancer.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
The authors are thankful to The Gujarat Cancer and research Institute and The Gujarat cancer society for all the support and funding.
Financial support and sponsorship
The Gujarat Cancer and research Institute and The Gujarat cancer society.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lukong KE. Understanding breast cancer–The long and winding road. BBA Clin 2017;7:64-77.
De Neergaard M, Kim J, Villadsen R, Fridriksdottir AJ, Rank F, Timmermans-Wielenga V, et al.
Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment. Am J Pathol 2010;176:1229-40.
Wu Y, Guo X, Brandt Y, Hathaway HJ, Hartley RS. Three-dimensional collagen represses cyclin E1 via β1 integrin in invasive breast cancer cells. Breast Cancer Res Treat 2011;127:397-406.
Gray J, Zhao J. Implications of epithelial–stromal interaction 1 in diseases associated with inflammatory signaling. Cell Communication Insights 2016;8:1-6.
Wang X, Zheng M, Liu G, Xia W, McKeown-Longo PJ, Hung MC, et al.
Krüppel-like factor 8 induces epithelial to mesenchymal transition and epithelial cell invasion. Cancer Res 2007;67:7184-93.
Zhao J, Bian ZC, Yee K, Chen BP, Chien S, Guan JL. Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Molecular cell 2003;11:1503-15.
Wang X, Zhao J. KLF8 transcription factor participates in oncogenic transformation. Oncogene. 2007;26:456.
Zhang H, Liu L, Wang Y, Zhao G, Xie R, Liu C, et al.
KLF8 involves in TGF-beta-induced EMT and promotes invasion and migration in gastric cancer cells. J Cancer Res Clin Oncol 2013;139:1033-42.
Yang T, Cai SY, Zhang J, Lu JH, Lin C, Zhai J, et al.
Krüppel-like factor 8 is a new Wnt/beta-catenin signaling target gene and regulator in hepatocellular carcinoma. PLoS One 2012;7:e39668.
Gudjonsson T, Rønnov-Jessen L, Villadsen R, Bissell MJ, Petersen OW. To create the correct microenvironment: Three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia. Methods 2003;30:247-55.
Nielsen HL, Rønnov-Jessen L, Villadsen R, Petersen OW. Identification of EPSTI1, a novel gene induced by epithelial–stromal interaction in human breast cancer. Genomics 2002;79:703-10.
Li T, Lu H, Shen C, Lahiri SK, Wason MS, Mukherjee D, et al.
Identification of epithelial stromal interaction 1 as a novel effector downstream of Krüppel-like factor 8 in breast cancer invasion and metastasis. Oncogene 2014;33:4746.
Wang X, Lu H, Urvalek AM, Li T, Yu L, Lamar J, et al.
KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9. Oncogene 2011;30:1901.
Franky D Shah
The Stem Cell Biology Lab, The Gujarat Cancer and Research Institute, NCH Campus, Asarawa, Ahmedabad - 380 016, Gujarat
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]