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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 1  |  Issue : 2  |  Page : 105-110

Elevated mRNA expression levels of inflammation-related genes in triple-negative breast cancer: A pilot study from North East India


1 Multi-Disciplinary Research Unit, Fakhruddin Ali Ahmed Medical College and Hospital, Barpeta, Assam, India
2 Department of Biochemistry, Fakhruddin Ali Ahmed Medical College and Hospital, Barpeta; Department of Biochemistry, Diphu Medical College and Hospital, Karbi Anglong, Assam, India

Date of Submission25-Oct-2021
Date of Decision10-Nov-2021
Date of Acceptance11-Nov-2021
Date of Web Publication22-Dec-2021

Correspondence Address:
Dr. Rizwana Sultana
Multi-Disciplinary Research Unit, Fakhruddin Ali Ahmed Medical College and Hospital, Barpeta - 781 301, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/aort.aort_25_21

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  Abstract 


BACKGROUND: The prevalence of Triple-Negative breast cancer (TNBC) accounts for a large percentage of breast cancer cases in India. TNBC is associated with poor prognosis, higher mortality rate, ill-defined molecular etiology, and hence limited therapeutic interventions.
AIM: The aim of this study is to evaluate the association of certain inflammatory markers with TNBC pathogenesis.
MATERIALS AND METHODS: Prospectively collected resected breast cancer tissue samples along with adjacent normal control (n = 100) were prospectively collected in RNA Later. Differential mRNA expression analysis of inflammatory-related genes namely; inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX2), and Nuclear Factor Kappa B (NFκB) and were evaluated in non-TNBC and TNBC tissues samples along with adjacent normal control tissue samples with the help of mRNA specific primers using reverse transcription-polymerase chain reaction. Statistical analysis was performed using SPSSv13.0 software.
RESULTS: A total of 60 non-TNBC and 40 TNBC tissue samples along with adjacent normal control were included for the study with informed consent and clinical details. The mean age of the TNBC patients was 39 ± 9 years, All the breast cancer cases were clinically staged as Infiltrating Duct Carcinoma (non-TNBC-invasive ductal carcinoma [IDC] II [n = 29 cases], non-TNBC-IDC III [n = 31 cases], TNBC-IDCII [n = 22 cases], TNBC-IDC III [n = 18 cases]). The results showed an upregulation of all the markers in TNBC cases compared to non-TNBC vis-avis non-neoplastic adjacent control area. Second, significant changes in iNOS mRNA expression were found to be associated with severity of TNBC cases (P = 0.020), while the expression of constitutively expressed eNOS was comparative between IDC-II and IDC-III stages of TNBC.
CONCLUSIONS: The present study indicates that the mRNA-based differential expression results showed an upregulation of all the markers (iNOS, eNOS, COX2, and NFκB) in TNBC cases compared to non-TNBC cases vis-a-vis non-neoplastic adjacent control area. Significant changes in iNOS mRNA expression were found to be associated with severity of TNBC cases (P = 0.020), depicting the role of iNOS-induced inflammation in the pathogenesis of TNBC.

Keywords: Breast, cancer, mRNA, triple negative


How to cite this article:
Sultana R, Salman Chisty SJ. Elevated mRNA expression levels of inflammation-related genes in triple-negative breast cancer: A pilot study from North East India. Ann Oncol Res Ther 2021;1:105-10

How to cite this URL:
Sultana R, Salman Chisty SJ. Elevated mRNA expression levels of inflammation-related genes in triple-negative breast cancer: A pilot study from North East India. Ann Oncol Res Ther [serial online] 2021 [cited 2022 Jan 18];1:105-10. Available from: http://www.aort.com/text.asp?2021/1/2/105/333308




  Introduction Top


Triple-negative breast cancer (TNBC) refers to tumors lacking the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptorer-2 (HER-2), and is typically associated with poor prognosis and aggressive tumor phenotype.[1],[2] TNBC accounts for 15% of all breast carcinomas in Asian and Western populations.[3] TNBC is prevalent in a significant proportion of patients from India;[4],[5],[6] and North East India alone accounts for 31.9% of all breast cancer patients, and is reported to be present at an early age and are associated with high-grade large tumors with high rate of node positivity.[7]

Inflammation is a pathophysiological phenomenon which has been recognized to be involved in the onset and development of various cancers including breast cancer. Numerous studies revealed that pro-inflammatory markers are significantly upregulated in the stroma of breast tumors compared to normal tissue.[8] Unchecked adiposity commonly modulates a variety of physiological responses which include altered steroid hormone production and chronic subclinical inflammation.[9],[10],[11] These pathophysiological effects have been associated with increased oxidative stress which in turn is involved in breast cancer carcinogenesis. The emergence of neoplastic tumors in selected sites of breast indicates the presence of heterogeneous microenvironments in the mammary glands. Thus it can be assumed that there are susceptible sites in the mammary gland of high-risk women where persistent inflammatory conditions could play a crucial role in inducing cellular proliferation, cytotoxicity, and tissue injury that initiate the process of carcinogenesis.

There is a great chance that production of low nitric oxide (NO) for a prolonged period generates excessive reactive nitrogen species/reactive oxygen species (RNS/ROS), which damage the DNA or post-translationally modify proteins of nearby epithelial and stromal cells at these susceptible sites in high-risk mammary glands.[10] This could induce infiltration of inflammatory cells, which in turn release RNS, ROS and cytokines to aggravate the inflammatory process. Endothelial NO Synthase (eNOS), has also been found to modulate the expression of the pro-inflammatory molecules such as cyclooxygenase-2 (COX-2) and vice-versa.[12] The inflammatory molecules also stimulate inducible NO Synthase (iNOS) in this complex site of inflammation in breast cancer tumors, which further damage DNA and alter protein structure and function. Inflammation and its association with TNBC tumorigenesis had also been implicated in few sporadic reports[13],[14],[15] and is often associated with poor overall survival and relapse of the disease.[13] Among numerous pro-inflammatory and inflammatory molecular markers, iNOS (iNOS/NO synthase [NOS2]), eNOS (eNOS/NOS3), COX-2, and Nuclear Factor Kappa B (NFκB) had gained immense attention in breast cancer research.

Inducible nitric oxide synthase (inducible nitric oxide synthase/nitric oxide synthase-2)

iNOS is one of three crucial enzymes involved in the production of NO from the amino acid L-arginine.[16] The effect of iNOS in the process of inflammation is a complex process and often increased iNOS expression seems to play an important in the promotion of cancer including breast cancer. Previous studies have reported high iNOS expression correlation with increased DCIS grade, aggressiveness, and poor prognosis of breast cancer.[17],[18],[19],[20] A recent study found that iNOS was usually increased in invasive TNBC and correlated with poor overall survival of TNBC patients.[21]

Endothelial nitric oxide synthase (endothelial nitric oxide synthase/nitric oxide synthase 3/constitutive nitric oxide synthase)

eNOS is a critical regulator which is involved in the initiation and promotion of breast cancer progression.[22],[23] Very sporadic reports are found regarding the expression levels of eNOS in breast cancer. However, few epidemiological studies have indicated aberrantly high expression of eNOS being predominantly present in breast cancer and positively correlated with aggressiveness and poor survival of the patients.[22],[23],[24],[25] Further, eNOS had also been found to influence the expression of the pro-inflammatory molecules like COX-2, which can also modulate the expression of eNOS.[12]

Cyclooxygenase 2

COX-2 is one of the three key members of myeloperoxidases family which are located at the luminal side of the endoplasmic reticulum and nuclear membrane. COX-2 is an inducible isoform, which is overexpressed during inflammation and neoplastic processes and is regulated by growth factors and various cytokines.[26] Several studies had reported COX-2 overexpression in invasive breast cancer and ductal carcinoma in situ, and the aberrantly high expression of COX-2 had been identified to be associated with aggressive histological and clinical features.[26],[27],[28],[29],[30],[31],[32],[33],[34] Previous studies had indicated an overexpression of COX-2 in TNBC and HER2-positive breast cancer and associated with a worse prognosis.[35],[36],[37],[38],[39],[40]

Nuclear factor-Kappa B

NFκB is a transcription factor that is known to play an essential role in inflammatory responses. It is one of the most important molecules which link chronic inflammation to cancer and its activity is tightly regulated by several mechanisms. The activation of NFκB is transiently and rapidly and stimulated by oxidative stress and pro-inflammatory cytokines. Previous studies have shown upregulation of NFκβ in breast tumors.[41]

Inflammation plays a crucial role in pathogenesis of cancer of all etiologies and has been proven to be either an associative or independent risk factor, with literatures supporting its role on breast cancer pathogenesis including sporadic reports on TNBC. The aim of this study is to evaluate the association of certain inflammatory markers with TNBC pathogenesis.


  Materials and Methods Top


Patient enrolment, sample collection, and stratification

All the enrolled histopathologically breast cancer patients had attended as new cases in the Surgical Oncology Department of Dr. B. Borooah Cancer Institute (BBCI), Guwahati, India, and had undergone surgical resection between 2012 and 2013. This study was approved by an institutional medical ethics committee of BBCI, Guwahati, India vide Memo No. BBCI/IEC-21/02 dated August 19, 2014. Tumor tissue samples along with adjacent controls were collected after surgical resection in RNA Later from the Department of Surgical Oncology, Dr BBCI. The patient's tissue samples were histopathologically and immunohistochemically confirmed and categorized as non-TNBC (n = 60) and TNBC (n = 40) along with non-neoplastic control. Each enrolled patient was interviewed and informed consent was obtained from all individual participants included in the study.

Evaluation of the role of certain inflammatory genes in the pathogenesis of triple-negative breast cancer

Differential mRNA expression analysis of inflammatory genes namely iNOS, eNOS, COX2, and NFκB in all the breast cancer tissues samples along with adjacent normal control tissue samples was studied with the help of mRNA specific primers and using β-actin as normalization internal control, by reverse transcription-polymerase chain reaction (PCR). Total RNA was extracted by standard Trizol method using TNBC tissue samples along with adjacent non-neoplastic adjacent controls. The quality of the total RNA was checked by Nanodrop spectrophotometry and was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The relative mRNA expression of inflammatory genes namely; iNOS, eNOS, COX2 and NFκB was analyzed by real-time PCR (Applied Biosystems 7500 Fast Real-Time PCR System) using specific primers and SYBR Green PCR Master mix, and β-actin as internal normalization control. Fold-change was evaluated by the 2−ΔΔCt method.

Statistical analysis

The statistical analysis for differential mRNA expression was studied using SPSS13.0 software (SPSS version 13.0 software (SPSS Inc., Chicago, IL).


  Results Top


Demographical and clinical profile

A total of 100 breast cancer tissue samples along with adjacent normal control were included for the study with informed consent and clinical details. The samples were divided into two subcategories namely; non-TNBC (ER/PR/HER2 positive) and TNBC cases. The mean age of the non-TNBC and TNBC patients was 45 ± 11 and 39 ± 9 years, respectively. All the breast cancer cases were clinically staged as Infiltrating Duct Carcinoma (non-TNBC- invasive ductal carcinoma [IDC] II [n = 29 cases], non-TNBC-IDC III [n = 31 cases], TNBC-IDC II [n = 22], and TNBC-IDC III [n = 18 cases]).

Evaluation of the role of inflammatory-related genes in the pathogenesis of triple-negative breast cancer

A panel of inflammatory-related genes viz., iNOS, eNOS, COX-2, and NFκB was evaluated for differential expression at mRNA level by real-time PCR using SyBr green chemistry [Figure 1] and [Figure 2]. The result showed an upregulation of all the four inflammatory-related genes namely; iNOS, eNOS, COX2 and NFκβ in TNBC cases compared to non-TNBC vis-a-vis controls. The iNOS (P = 0.002) was significantly higher in TNBC cases compared to non-TNBC cases [[Figure 1] (L)]. The results also showed upregulation of all the four inflammatory-related genes namely iNOS, eNOS, COX2, and NFκB in IDC-III cases compared to IDC-II cases vis-a-vis controls in all breast cancer samples. Further, upregulation of all the markers in TNBC cases compared to non-neoplastic adjacent control area. Significant changes in iNOS mRNA expression were found to be associated with severity of TNBC cases (P = 0.020), while the expression of constitutively expressed eNOS was comparative between IDC-II and IDC-III stages of TNBC [Figure 2]L]; depicting the role of iNOS-induced inflammation in the pathogenesis of TNBC.
Figure 1: (Left [L] to Right [R]): (L): Box-plot analysis showing that the upregulated mRNA expression of the four inflammatory-related genes namely inducible nitric oxide synthase, endothelial nitric oxide synthase, cyclooxygenase-2, and Nuclear Factor Kappa B in triple-negative breast cancer cases compared to non- triple-negative breast cancer. The inducible nitric oxide synthase (P = 0.002) was significantly higher in triple-negative breast cancer cases compared to nontriple-negative breast cancer cases. (R): Box-plot analysis showing that the upregulated mRNA expression of the four inflammatory-related genes namely inducible nitric oxide synthase, endothelial nitric oxide synthase, cyclooxygenase-2, and Nuclear Factor Kappa B in invasive ductal carcinoma-III cases compared to invasive ductal carcinoma-II cases. The inducible nitric oxide synthase (P = 0.002) was significantly higher in triple-negative breast cancer cases compared to non- triple-negative breast cancer cases

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Figure 2: ((Left [L] to Right [R]): (L): Box-plot analysis showing that the upregulated mRNA expression of the inducible nitric oxide synthase gene in invasive ductal carcinoma-III cases compared to invasive ductal carcinoma-II cases in triple-negative breast cancer samples. (R): Box-plot analysis showing that the mRNA expression of the inducible nitric oxide synthase, endothelial nitric oxide synthase, cyclooxygenase-2, and Nuclear Factor Kappa B gene in invasive ductal carcinoma-III cases compared to invasive ductal carcinoma-II cases in nontriple-negative breast cancer samples

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  Discussion Top


TNBC is a high risk-breast cancer as it manly affects women belonging to reproductive age group, lacks effective targeted therapy, and has a poor survival rate compared to other subtypes of breast cancer.[42],[43] The prevalence of TNBC cases in North-East India is alarmingly high depicting that a significant portion of breast cancers exclusively belong to this part of the country.[4],[5],[6],[7] It is commonly found in younger age group and associated with high-grade large tumors and high rate of node positivity. IDC NOS is found to be the most common histological subtype in TNBC.[7] Our study also depicted a similar picture. We found that the majority of the women suffering from TNBC belong to the reproductive age group and were IDC positive histopathologically. Our study is the first study of its type from Northeast India with an ethnically distinct population from the rest of India focusing on the role of certain inflammatory-related genes namely, iNOS, eNOS, COX2 and NFκB; and its association with TNBC pathogenesis.

Inflammation plays a crucial role in pathogenesis of cancer of all etiologies and has been proven to be either an associative or independent risk factor, with literatures supporting its role on breast cancer pathogenesis.[44] Accumulating scientific evidence are suggestive of the role of inflammatory markers, adipocytes and in turn, the expressed adipokines and adipocytokines in chronic inflammation which is critically linked to carcinogenesis, including sporadic reports on TNBC.[45],[46],[47] Hence, the mRNA expression profiling of certain inflammatory-related genes was done and correlated with the severity of the disease.

A panel of inflammatory-related genes namely; iNOS, eNOS, COX-2, and NFκB was evaluated for differential expression at mRNA level by real-time PCR using SyBr green chemistry. Significant changes in iNOS mRNA expression were found to be associated with the severity of TNBC cases (P = 0.020), while the expression of constitutively expressed eNOS was comparative between IDC-II and IDC-III stages of TNBC depicting the role of iNOS-induced inflammation in the pathogenesis of TNBC. This is consistent with the reports submitted by Granados-Principal et al., 2015[21] which states that iNOS was usually increased in invasive TNBC and correlated with pathogenesis of TNBC patients.

Our findings have shown mRNA-based differential expression profile of all the inflammatory markers (iNOS, eNOS, COX2, and NFκB) in TNBC cases compared to non-TNBC cases vis-a vis non-neoplastic adjacent control area. Significant changes in iNOS mRNA expression were found to be associated with the severity of TNBC cases (P = 0.020), depicting the role of iNOS-induced inflammation in the pathogenesis of TNBC.

Since it was a pilot study we wish to restrict our findings on the establishment of the significance of mRNA expression profiling of the TNBC cases compared to non-TNBC cases and thereby not reveal the survival status of patients for this particular study.


  Conclusions Top


The present study, is first of its kind on TNBC from northeast India, indicates that the mRNA-based differential expression results showed an upregulation of all the markers (iNOS, eNOS, COX2, and NFκB) in TNBC cases compared to non-TNBC cases vis-à-vis non-neoplastic adjacent control area. Significant changes in iNOS mRNA expression were found to be associated with the severity of TNBC cases (P = 0.020), depicting the role of iNOS-induced inflammation in the pathogenesis of TNBC.

Acknowledgments

The authors would like to acknowledge the support of the staff of the Pathology and Surgical oncology departments of Dr. B. Borooah Cancer Institute, Guwahati for their active help in executing the study. The authors acknowledge the Department of Health Research, Government of India (DHR/ADC/73/Assam/2013) for the infrastructural support required for the work. Dr. Rizwana Sultana acknowledges Dr. Uddip Talukdar, Nodal Officer, Multi-Disciplinary Research Unit; for the support during the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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