|Ahead of print publication
Transcriptional elucidation of tumor necrosis factor-α-mediated nuclear factor-κB1 activation in breast cancer cohort of Pakistan
Javeria Qadir, Syeda Kiran Riaz, Namood-e Sahar, Durkhshan Aman, Muhammad Jawad Khan, Muhammad Faraz Arshad Malik
Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
Muhammad Faraz Arshad Malik,
Department of Biosciences, COMSATS Institute of Information Technology, Islamabad
Source of Support: None, Conflict of Interest: None
Background: Initiation, promotion, progression, and metastasis of mammary tumors are mediated by dysregulation of multiple genes involved in various signaling pathways. Expressional variation of these molecules significantly influences cancer cell proliferation in breast cancer. Aims and Objectives: In the current study, tumor necrosis factor-alpha (TNF-α) and its downstream effector nuclear factor kappa-B1 (NF-κB1) mean transcript levels were explored and associated with molecular subtypes in breast cancer cohort of Pakistan. Freshly excised tumors (n = 150) along with background tissues were collected for RNA isolation and cDNA synthesis. Materials and Methods: Quantitative polymerase chain reaction was carried out for quantification of TNF-α, NF-κB1, and β-actin gene transcripts along with estrogen receptor, progesterone receptor, HER2, and Ki-67, followed by statistical analysis. Results: For TNF-α and NF-κB1, 95% and 77% of the cohort was found to be positive, respectively. Both of these molecules were found to be significantly upregulated in tumors when compared against their respective controls (P < 0.0001). Association of TNF-α and NF-κB1 with late clinical stages, poorly differentiated tumors, increased tumor size, nodal involvement, and metastasis was observed to be statistically significant (P < 0.05). Strong positive correlation was established between TNF-α and NF-κB1(r = 0.465, P< 0.05). Moreover, mean transcript levels of TNF-α and NF-κB1 were significantly elevated in Luminal A and Luminal B subtypes of breast cancer patients, respectively. Conclusion: Strong positive correlation between TNF-α and NF-κB1 proposed the putative role of these molecules as prognostic biomarkers in breast cancer.
Keywords: Breast cancer, nuclear factor kappa B1, prognosis, quantitative polymerase chain reaction, tumor necrosis factor-α
|How to cite this URL:|
Qadir J, Riaz SK, Sahar Ne, Aman D, Khan MJ, Malik MF. Transcriptional elucidation of tumor necrosis factor-α-mediated nuclear factor-κB1 activation in breast cancer cohort of Pakistan. J Can Res Ther [Epub ahead of print] [cited 2020 Jan 22]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269911
| > Introduction|| |
Tumor growth, progression, and metastasis are influenced by interplay of tumor cells with their inflammatory microenvironment. Tumor microenvironment variations may persist between different classes and stages of tumors consisting of numerous cell types and factors. Of all these, a pivotal part has been played by cytokines in breast adenocarcinoma. Moreover, an extensive intercommunication occurs between inflammatory cells and tumor cells leading to breast carcinogenesis. Numerous cytokines involving components of the transforming growth factor-beta and tumor necrosis factor (TNF) lineage have been involved in underlying process of breast cancer metastasis.
TNF-α is one of the major pro-inflammatory cytokines which may either act as tumor promoter associating inflammation with carcinogenesis or tumor suppressor, as it instigates cancer cell death due to perpetual c-Jun N-terminal kinase activation. Involvement of TNF-α either as promoter or suppressor is influenced by molecular cross talks, type of organ involved, and tumor host immune response.
Cellular responses to TNF-α are mediated through transcription factor called nuclear factor kappa-B (NF-κB). NF-κB is a group of transcription factors constituted of five members, i.e., the precursor proteins NF-κB1 (p105), NF-κB2 (p100), RelA (p65), RelB, and c-Rel. Stimulation of NF-κB1 signaling in response to TNF-κ assists tumor cells in addressing cytotoxicity and cell survival. Constitutively, impairment of NF-κB1 function is related to inflammatory disorders as well as cancer.
TNF-α enhances tumor cell proliferation through NF-κB1 activation. It also mediates tumor cell survival by inducing genes that encode for NF-κB1 dependent anti-apoptotic factors. Within macrophages, TNF-α cell to cell signaling via NF-κB1 constitutes an inflammatory microenvironment that promotes cells to metastasize in cancer. Hence, it is interesting to assess both these molecules in breast cancer patients of Pakistan to establish their probable involvement in carcinogenesis.
The aim of the current study was to explore the expression profiles of TNF-α and NF-κB1 in breast cancer cohort of Pakistan. Any possible association of these molecules with clinicopathological features of the cohort was assessed. Based on the findings, the potential of TNF-α and/or NF-κB1 as early breast cancer prognostic biomarkers may also be suggested. This study will be helpful to understand potential involvement of TNF-α/NF-κB1 signaling in breast cancer progression and metastasis with respect to molecular subtypes.
| > Methodology|| |
Tissue specimen and data collection
The current study was conducted in agreement with prior approval from the Ethical Review Committee following guidelines set forth by Institutional Biosafety and Bio-regulations Committees of both COMSATS University and collaborating hospitals. The cohort comprised 150 freshly excised tumors along with their background nontumor tissues (2 cm away from the tumor area) collected with informed patient consent. Clinicopathological characteristics (grade, stage, tumor size, nodal status, and metastasis) and demographic data (age at disease onset and menopausal status) of these patients were also retrieved. These biological samples were immediately immersed in Falcon tubes containing RNA Later® solution to minimize RNA degradation. Subsequently, these tubes were stored at −85°C until further usage.
RNA extraction from both tumor as well as control samples was performed using Trizol® (Invitrogen, California, USA), following an already established protocol.
Reverse transcription for cDNA synthesis
RevertAid first strand cDNA synthesis kit (Thermo Scientific™, California, USA) was used for generating cDNA from extracted RNA in accordance with manufacturer's instructions. Synthesis of cDNA was confirmed by amplification with β-actin primers through conventional polymerase chain reaction (PCR).
Primer designing and quantitative real-time polymerase chain reaction
Quantitative elucidation of TNF-α and NF-κB1 expression was conducted employing real-time PCR technology with gene-specific primers. Primers for TNF-α, NF-κB1, and β-actin were designed using designed using IDT(Integrated DNA Technology, IA, USA) software. The primer set designed for TNF-α amplicon (162 bps) contains forward 5'-AAGAATTCAAACTGGGGCCT-3' and reverse 3'-GAGGAAGGCCTAAGGTCCAC-5'sequences, whereas for NF-κB1 amplicon (106 bps), the primer set contains forward 5'-CTCCACAAGGCAGCAAATAGA-3' and reverse 3'-ACTGGTCAGAGACTCGGTAAA-5' sequences. Reaction conditions for amplification of molecules TNF-α, NF-κB1, and β-actin were optimized following range of annealing temperatures. β-actin was used as an internal control. Each reaction was performed in triplicates in an attempt to ensure the accuracy and precision of the results obtained.
Comparative mRNA expression of TNF-α, NF-κB1, and β-actin was evaluated by means of the Livak method.
The results were statistically analyzed through nonparametric tests using the Wilcoxon signed-rank test (paired), Mann–Whitney test, and Spearman rank correlation coefficient.
| > Results|| |
Clinicopathological and demographic characteristics of breast cancer patients
Clinicopathological characteristics and demographic details of the cohort have been indicated in [Table 1]. The mean age for these breast cancer affected women was calculated to be 45 with their ages ranging between 23 and 75 years.
|Table 1: Demographic and clinicopathological categorization of the cohort (n=150)|
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Relative gene expression of tumor necrosis factor-α and nuclear factor kappa-B1 in breast tumor tissue in comparison to controls
Transcriptional profiling showed approximately 4 and 3 folds upregulation in tumor tissues of breast cancer patients in comparison to their respective controls for TNF-α and NF-κB1, respectively. Within the cohort, 95% tumor samples were positive for TNF-α, while 77% of tumor samples were observed to be positive for NF-κB1 [Table 1] and [Figure 1], [Figure 2].
|Figure 1: Association of tumor necrosis factor-α mRNA expression with clinicopathological parameters and intrinsic molecular subtypes in breast cancer cohort|
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|Figure 2: Association of nuclear factor kappa-B1 mRNA expression with clinicopathological parameters and intrinsic molecular subtypes in breast cancer cohort|
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Association of tumor necrosis factor-α and nuclear factor kappa-B1 gene expression with tumor grade
For both TNF-α and NF-κB1, statistically significant differences were noticed between well-differentiated/moderately-differentiated and poorly-differentiated tumors (P< 0.05) [Table 2] and [Figure 1], [Figure 2].
|Table 2: Correlation of Tumor necrosis factor-alpha and nuclear factor kappa-B1+ expression with clinicopathological characteristics of the cohort|
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Association of tumor necrosis factor-α and nuclear factor kappa-B1 gene expression with clinical stage
Expressional differences among tumor stages demonstrated statistically significant overexpression of TNF-α and NF-κB1 in late clinical stages, i.e., Stage III/IV when compared against Stage I/II (P< 0.05) [Table 2] and [Figure 1], [Figure 2].
Association of tumor necrosis factor-α and nuclear factor kappa-B1 gene expression with age at disease onset
Transcript levels for TNF-α and NF-κB1 were quantified to be significantly elevated in patients with age group >45 years, when compared against patients <45 years of age (P< 0.05), as depicted in [Table 2].
Association of tumor necrosis factor-α and nuclear factor kappa-B1 gene expression with menopausal state
TNF-α and NF-κB1 mRNA quantification displayed significantly increased levels in patients who appeared with postmenopause as compared to those in the premenopausal state (P< 0.05) [Table 2].
Association of tumor necrosis factor-α and nuclear factor kappa-B1 gene expression with metastasis
Statistically significant results (P< 0.05) were obtained for higher levels of TNF-α and NF-κB1 transcripts in patients exhibiting distant metastasis, indicating their prominent role in breast cancer progression and lymphovascular invasion [Table 2] and [Figure 1], [Figure 2].
Association of tumor necrosis factor-α and nuclear factor kappa-B1 gene expression with intrinsic molecular subtypes of breast cancer
Relative expression analysis of TNF-α and NF-κB1 revealed statistically significant upregulation in mean transcript levels among intrinsic molecular subtypes of breast cancer cohort. Highest TNF-α expression was seen in Luminal A subtype, whereas NF-κB1 showed most elevated mRNA levels among Luminal B breast cancer patients [Figure 1] and [Figure 2].
Correlation between tumor necrosis factor-α and nuclear factor kappa-B1 at transcript level
In the present study, the correlation between the two entities, i.e., TNF-α and NF-κB1 were assessed at mRNA level. The relationship came out to be strongly positive and statistically significant [Figure 3].
|Figure 3: Correlation between tumor necrosis factor-α and nuclear factor kappa-B1 mRNA expression in breast cancer cohort|
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| > Discussion|| |
Breast cancer remains the most life-threatening malignancy affecting women across the world. Despite improving survival, it still contributes a major proportion toward cancer-related death in women. In Pakistan, breast cancer accounts for 34.6% of all female cancers.
Inflammation has been reported as “seventh hallmark of cancer.” TNF-α is an essential stimulator of inflammation and cancer. For TNF-α-mediated tumor progression, NF-κB1 is crucial, and its activity is regulated through multiple signaling pathways., Almost all cells undergo NF-κB1 activation (transient or sustained) when exposed to TNF-α. This stimulation triggers various characteristics of oncogenesis including uncontrolled proliferation, loss of apoptosis, angiogenesis, and metastasis.
The objective of the current study was to explore the expressional profile of TNF-α in breast cancer cohort of Pakistani origin and its association with clinicopathological parameters (encompassing tumor grade, stage, metastasis, etc.) as well as intrinsic molecular subtypes of breast cancer. To elucidate the signaling cascade activated by TNF-α in breast cancer progression, expression analysis of NF-κB1 (a transcription factor activated by TNF-α) was also conducted.
Statistically significant overexpression of TNF-α and NF-κB1 in tumor versus controls was observed. It may be due to increased stability of TNF-α transcript through its 3'untranslated region. Besides, NF-κB1 has been reported to increase mammary epithelial cells multiplication by mediating transcription of genes encoding for cyclins.
TNF-α-mediated NF-κB1 activation has been reported to render the cells immortal. It is through NF-κB1 that TNF-α builds up interaction between tumor cells and macrophages, thus, increasing tumor invasion and metastasis. The current findings also signify TNF-α as a mitogen triggering proliferation of mammary cells in NF-κB1 dependent manner.
Earlier studies indicated significant association of TNF-α and NF-κB1 in elderly patients. Likewise, statistically significant overexpression of TNF-α and NF-κB1 transcripts were observed in patients with age group >45 years in the cohort. Comparison between premenopausal and postmenopausal patients demonstrated significantly elevated mRNA levels of TNF-α and NF-κB1 in women with postmenopause. These findings were consistent with data reported earlier for expression of these molecules at protein level., It may be attributed to aggravated expression of estrogen receptor and progesterone receptor associated with elevated TNF-α and NF-κB1 transcript levels.
Appertaining to tumor grades, TNF-α and NF-κB1 mRNA concentration was significantly higher in patients with poorly differentiated tumors compared to those with well-differentiated and moderately differentiated tumors, therefore, supporting the fact that TNF-α greatly influences tumor cell differentiation in addition to its primary function as a mutagen inducing tumor cell proliferation and survival., Inhibition of NF-κB1 has been reported to induce terminal differentiation of tumor cells.,, Furthermore, these findings confirm a crucial role of NF-κB1 in tumor dedifferentiation.
Earlier studies showed statistically significant overexpression of TNF-α and NF-κB1 with progressive stages in breast cancer patients.,, In the present study, statistically significant increase in TNF-α mRNA levels in early stages of breast tumorigenesis was observed. Additionally, NF-κB1 has been communicated to induce cell cycle progression resulting in accelerated proliferation, i.e., a typical feature of breast cancer. Activation of NF-κB1 at mRNA level has significantly been coassociated with growing tumor stages in breast cancer., Coextending previous research findings, the present study also affirmed high NF-κB1 transcript levels in later stages of breast cancer validating its role in cancer progression and metastasis through activation of various cytokines and growth factors.
Tumor size measurement is an established variable employed widely in cancer research. In the past studies on breast cancer, higher NF-κB1 activity has been significantly associated with increasing tumor size. In line with the previous findings, the present study also revealed statistically significant association of high TNF-α and NF-κB1 levels with increasing tumor size indicating their critical part in breast tumor growth.
Published data on TNF-α and NF-κB1 indicated their significant association with lymph node metastasis. Similarly, mRNA expression of both these molecules was found to be significantly higher in patients with greater number of lymph nodes involved (i.e., >04) predicting its imperative role in late stages of lymph node metastasis.
In this study, significantly elevated levels of TNF-α and NF-κB1 transcripts were observed in patients exhibiting breast cancer metastasis, validating their role in secondary tumor development. Higher levels of NF-κB1 have also been reported to be positively correlated with metastasis. Possibly, these molecules are responsible for localization and transition of breast tumor into more invasive form.
For elucidating the correlation between TNF-α and NF-κB1 at transcript level, Spearman rank correlation coefficient was employed. The association between the two biological entities showed up to be strongly positive at par with the findings published already. This suggested that NF-κB1 activation in breast cancer cohort was probably mediated through TNF-α.
| > Conclusion|| |
Strong positive correlation between TNF-α and NF-κB1 proposed the putative role of these molecules as prognostic biomarkers. However, arrays of in vitro and in vivo assays are required to establish the clinical significance of these findings and validate involvement of TNF-α and NF-κB1 in breast cancer therapy.
The authors want to acknowledge Dr. Jahangir Sarwar Khan and Dr. Faryal from the Department of General Surgery, Holy Family Hospital, Rawalpindi, Pakistan. We are also grateful to all the participants involved in this study.
Financial support and sponsorship
This study was financially supported by COMSATS Institute of Information Technology, Islamabad and Higher Education Commission (HEC) of Pakistan under the project ID 2989.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]