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An official publication of the Middle-Eastern Association for Cancer Research
Clinical Cancer Investigation Journal
Year: 2021   |   Volume: 10   |   Issue: 6   |   Page: 318-330     View issue
Exposure to water-pipe smoking dysregulates a set of genes associated with breast cancer development and an unfavorable outcome
Vanessa Lopez-Ozuna, Ishita Gupta, Ryan Chen Kiow, Emad Matanes, Amber Yasmeen, Semir Vranic, Ala-Eddin Al Moustafa

Background: Water-pipe smoking (WPS), a predominant method of tobacco consumption, is common amongst young females in the Middle East. WPS smoke consists of toxins analogous to the ones that exist in cigarette smoke and frequently correlates with the onset of several types of human cancers including breast. However, the potential target genes and their underlying mechanisms in the initiation and/ or progression of human cancers, especially breast, due to WPS exposure are still unknown. Materials and Methods: In this investigation, we explored the effect of WPS chronic exposure on human normal mammary epithelial cells and analyzed alterations in the differentially ex-pressed gene (DEG) targets using the NanoString nCounter PanCancer Pathways Panel consisting of 770 gene transcripts and a quantitative real-time polymerase chain reaction (PCR) analysis. Results: Our NanoString analysis identified 13 genes dysregulated under the effect of WPS exposure involved in regulating signal transduction, cell cycle, cell motility, proliferation and migration/invasion as well as the inflammatory response. We further performed an in silico analysis to investigate the effect of the identified genes in the prognosis of breast cancer patients and reported those DEGs that directly correlated with smoking and were upregulated in breast cancer in comparison with normal tissue. Moreover, the Kaplan–Meier curve analysis showed a significant correlation be-tween WPS-dysregulated genes (MX1, CCL8, GNGT1 and MMP9) and relapse-free survival in breast cancer patients. Conclusions: Our data clearly suggest that exposure to WPS can alter the expression of key regulator genes involved in the pathogenesis of breast cancer, thereby affecting the breast cancer prognosis.

Breast cancer, gene deregulation, mammary epithelial cells, smoking, water-pipe


Tobacco smoking is an avoidable risk factor for various noncommunicable diseases including pulmonary, diabetes, cardiovascular and different types of cancer; it is also responsible for the rise in mortality rates.[1],[2] Various forms of tobacco intake include water-pipe smoking (WPS), cigarettes, cigars and electronic cigarettes (E-cigarettes). Currently, WPS along with E-cigarettes are becoming global trends[3],[4] as they are generally more preferred publicly than cigarette smoking especially among youths and women[5] mainly due to entertainment recreation.[3],[6] On average, around 100 million smokers use WPS on a regular basis,[7] resulting in nearly five million deaths annually.[8] Remarkably, Middle Eastern people as well as those with a Middle Eastern origin residing in the West consume WPS as an integral part of their culture and ethnicity, thus escalating this tendency in Western countries.[5]

In WPS, charcoal-heated air is passed across a perforated aluminum foil and through flavored tobacco to turn into smoke,[9] which consists of toxins similar to those present in cigarettes including carbon monoxide, tar, nicotine, hydrocarbons and other toxicants.[10],[11] Compared with cigarette smokers, the plasma concentration of nicotine in individuals smoking a WP once a day is analogous to consuming 10 cigarettes in a day.[12],[13] Therefore, in spite of the general belief that WPS is less toxic than cigarette smoking, investigations point out that the consumption of both WPS and cigarette smoking leads to severe health problems including nicotine/tobacco addiction and an increased risk for a variety of systemic serious human diseases.[14],[15],[16],[17],[18] WPS also induces significant embryotoxicity on the early stage of embryogenesis, thereby causing serious complications in early pregnancy.[19]

Previous investigations have revealed that WPS exposure can have an important impact on the development of various human cancers including head and neck, oral and breast cancers.[20],[21],[22],[23] Prolonged WPS exposure induces gene alterations regulating DNA stability and repair and detoxification as well as xenobiotic metabolism, thereby enhancing cancer susceptibility.[24],[25] Exposure to WPS can stimulate the transition from an epithelial to a mesenchymal phenotype and increase the cell invasive ability of breast cancer cells through the Erk1/Erk2 pathways.[22] Nevertheless, it is important to highlight that altered genes in normal mammary tissues exposed to WPS that can potentially participate in the onset and/or development of human breast cancer have not been explored yet. Therefore, in this investigation, we examined the outcome of chronic exposure to WPS on a set of known carcinogenesis-related gene targets and molecular pathway profiles in human normal mammary epithelial (HNME) cells.

Materials and Methods

Smoking machine protocol and preparation of the water-pipe smoking solution

The Aleppo method, a standard smoking procedure, was used as previously reported.[19],[22],[23] Briefly, the water-pipe was set by packing the head with 10 g of a brand of tobacco mixture (Two Apples, Paterson, NJ, USA) and concealing it with perforated aluminum foil to permit air passage. The quick-light block charcoal (Tree Kings, Paterson, NJ, USA) was inflamed and positioned on top of the head at the start of the smoking session. Post 1 h of smoking, the smoking condensate was collected using a regular laboratory filter paper attached to the mouthpiece. Filters were then parched and weighed before and after collecting the smoke. Later, smoked filters were dissolved in a phosphate-buffer saline (1×) (PBS) or keratinocyte serum-free medium (KSFM) (1×) (Gibco®, Life Technologies, Burlington, ON, Canada) to a final concentration of 20 mg/mL of smoking particles. Several previous investigations outlined the detailed yield of a WPS session;[26],[27] however, in this study, the overall effect of WPS was under investigation not the individual components. Based on previous studies,[19],[22],[23] the collectable WPS particulates were dissolved in the mentioned solvents. The PBS and KSFM solutions were then filtered using size 0.45 μm filters (Costar, Washington, DC, USA) to obtain the final extractable WPS solution.

Cell Lines

HNME cells[28] were grown and maintained in KSFM (1×) (Gibco®, Life Technologies, Burlington, ON, Canada) with heregulin (5 ng/mL), bovine pituitary extract (BPE) (5 mg/100 mL) (Life Technologies, Burlington, ON, Canada) and penicillin–streptomycin (100 μg/mL) (Invitrogen, Life Technologies, Burlington, ON, Canada). Cells were exposed to 150 μg/mL of WPS dissolved in either the PBS or KSFM solution for 48 h and maintained at a temperature of 37°C in a 5% CO2 humidified atmosphere.


The analysis of gene expression was performed using the NanoString PanCancer Pathways Panel (NanoString 125 Technologies, Seattle, WA, USA) comprising of 770 gene probes associated with tumorigenic pathways derived from The Cancer Genome Atlas (TCGA) data. Raw data (RCC files) from NanoString runs were processed and normalized using the standard protocols (nSolver User Manual) of the nSolver Analysis Software (NanoString Technologies, Seattle, WA, USA) as previously described by our group.[23] The obtained data were normalized once again to the geometric mean of the housekeeping genes. Following normalization, data were log2-transformed and then transported to Microsoft Excel for an analysis.

Based on previous studies, a fold-change analysis of 1.5-or 2-fold in addition to a P < 0.05 is frequently used as the cutoff value for identifying differentially expressed genes (DEGs).[29],[30] Hence, genes were chosen based on a 1.5-fold change or higher with P < 0.05.

RNA extraction and quantitative reverse transcriptase real-time polymerase chain reaction

The extraction of total RNA was done from WPS exposed and unexposed HNME cells using RNeasy Mini Kit spin columns (Qiagen, Valencia, CA, USA) as previously described by our group.[23] In brief, a synthesis of the first strand of cDNA was performed using the 5X All-In-One MasterMix (MasterMix-LR, Diamed, Mississagua, Ontario, Canada according to the manufacturer's instructions. Quantitative reverse transcriptase real-time PCR (qRT-PCR) was carried out using iTaq Universal SYBR Green Supermix (BioRad, Hercules, CA, USA). The primer sequences used in this study were designed using Primer ExpressTM Software v3.0.1 (ThermoFisher Scientific, Franklin, MA, USA) [Table 1].{Table 1}

Gene profile and in silico analysis

The DEGs identified by the NanoString study were then subjected to an in silico analysis used to further support and validate our findings. The Oncomine TM database (, November 14, 2020) is a large, public and widely available database that consists of around 65 gene expression datasets;[31] we investigated the differential gene expression in breast cancer in comparison with normal tissues and clinicopathological parameters. From the Oncomine TM database, we used the TCGA Finak and Zhao datasets to analyze the mRNA expression of the identified DEGs in normal versus malignant patient samples. Furthermore, the Bittner breast dataset was used to evaluate the difference in the log2 median-concentrated intensity between smoker breast cancer patients compared with nonsmokers with breast cancer. Parameters were set and the program produced levels of gene expression per dataset. Based on the analysis, statistically significant deregulated genes were selected. Moreover, we used a cohort of breast carcinoma samples from the PanCancer RNA-seq dataset (Kaplan–Meier plotter database) to evaluate the clinical outcome of patients in relation to individual genes.[32]

The GOBO database[33] was then used to evaluate the association between WPS-deregulated genes and breast cancer molecular subtypes in 1881 breast cancer patient samples according to PAM50 or Hu subclassifications.

The association between gene expression and molecular subtypes was presented as a boxplot where the band inside the box exemplified the median and the top (high expression) and bottom (low expression) of the box implied the distance between the different quantiles. Outliers were presented as circles. The level of significance provided by the database was calculated using an ANOVA test.[33]

Network and pathway interaction

We used the Search Tool for the Retrieval of Interacting Genes (STRING v9.1) (, November 10, 2020) to analyze the network and interaction between the altered WPS deregulated genes and their biological function as previously performed by our group.[23] This tool was used to underline the vitality of plausible networks linking obtained genes to understand the underlying mechanisms of breast cancer progression under the effect of smoking.

Statistical analysis

All in vitro experiments were carried out in triplicates of at least three independent experiments and the results were expressed as means ± standard error mean. The Student's t-test was performed to calculate the statistical significance. GraphPad Prism (Version 8.4.3) and nSolver analysis software were used to carry out the statistical analysis. A Kaplan–Meier survival analysis was done to determine the association between WPS-dysregulated genes and survival (relapse-free survival [RFS] and overall survival (OS)); significance was achieved at a P < 0.05 (log-rank test).


Identification of a set of breast cancer-associated differentially expressed genes deregulated by water-pipe smoking in human normal mammary epithelial cells

To analyze the detrimental outcome of WPS exposure on human breast carcinogenesis, we investigated the effect of WPS on HNME cells.[28] Our data showed that exposure to WPS slightly induces Epithelial Mesenchymal Transition (EMT) where HNME cells display a mesenchymal phenotype compared with the matched unexposed controls. As shown in [Figure 1], compared with the unexposed cells, WPS exposed ones were found to become elongated in appearance with a diminished cell-to-cell contact [Figure 1]. We found that exposure of HNME cells to 100 μg/mL of a WPS solution for 48 h disrupted the regulation of cell proliferation and the progression of the cell cycle in HNME cells in comparison with untreated ones (Data not shown).{Figure 1}

We further identified gene deregulated by WPS exposure in the development of human breast cancer. We performed a differential gene expression analysis on HNME cells (exposed and unexposed to WPS) using the NanoString nCounter PanCancer Pathways Panel comprising of probes for 770 genes involved in tumorigenic pathways. A NanoString analysis revealed 13 DEGs in WPS exposed versus unexposed HNME cells: CCL5, MX1, CCL21, IFNγ, ALOX5, CCL8, GNGT1, MMP9, TNFSF14, PTGR1, CCL4, IL3 and TLR9 (1.5-fold change or higher, P < 0.05).

Post the identification of plausible candidate DEGs, we performed qRT-PCR to validate our obtained gene panel from NanoString data. The panel of DEGs correlated with the NanoString analysis with 13 genes (CCL5, MX1, CCL21, IFNγ, ALOX5, CCL8, GNGT1, MMP9, TNFSF14, PTGR1, CCL4, IL3 and TLR9) upregulated by a fold-change varying from 1.6-to 24-fold [Figure 2].{Figure 2}

Moreover, based on functional annotations and molecular pathways underpinning carcinogenesis, we found the 13 DEGs to directly regulate cell cycle, cell proliferation, cell survival, cell migration/invasion, cell death (apoptosis), signal transduction and the inflammatory response [Table 2].{Table 2}

Differentially expressed genes by water-pipe smoking are upregulated in invasive breast cancer samples in comparison with normal tissue

For further evaluation of the role of our top DEGs deregulated by WPS in our in vitro study, we try to validate those DEGs in patients' samples using in silico approach. To achieve this, we primarily investigated the expression patterns of those DEGs in samples obtained from normal tissues and compare its expression from samples obtained from invasive breast tumor patients using many databases included in the publicly available Oncomine database.

The TCGA dataset (137 patient samples) revealed that the expression of CCL5 (P < 0.001), MX1 (P < 0.001), MMP9 (P < 0.001), IFNγ (P < 0.001), ALOX5 (P < 0.001), GNGT1 (P < 0.001), TNFSF14 (P = 0.031), IL3 (P < 0.001) and TLR9 (P = 0.004) were significantly higher in invasive breast carcinoma compared with the normal tissue [Supplementary Figure 1]a. Moreover, the Finak dataset (59 patient samples) showed CCL4 (P < 0.001), CCL8 (P < 0.001) and CCL21 (P < 0.001) to be upregulated in invasive breast carcinoma [Supplementary Figure 1]b. The Zhao dataset (39 patient samples) reveled PTGR1 (P = 0.018) to be overexpressed in invasive breast carcinomas [Supplementary Figure 1]c.[INLINE:1]

Differentially expressed genes are highly expressed in smoking breast cancer patients in comparison with nonsmoker patients

To further analyze the correlation amongst our identified WPS-deregulated genes and smoking as a risk factor in breast cancer development, we explored the fold-change of the 13 deregulated genes in breast cancer samples in smoker against nonsmoker breast cancer patients using the Bittner breast dataset of the Oncomine database. Remarkably, our data confirmed that of the 13 identified WPS-deregulated genes, the expression of 9 genes were upregulated in smoking patients with breast cancer in comparison with those who had never smoked. These genes included CCL5, MXI, CCL21, ALOX5, PTGR1, TNFSF14, CCL4, IL3 and TLR9 (P ≤ 0.05). Unfortunately, the smoking status was unavailable for MMP9, IFNγ, GNGT1 and CCL8 in the database [Supplementary Figure 2].[INLINE:2]

Water-pipe smoking-deregulated genes and their relation to breast cancer molecular subtypes

Breast cancer, a heterogenous disease, is categorized into five intrinsic molecular subtypes (Luminal A, Luminal B, HER2-positive, normal-like and basal-type).[34],[35] Hence, we further analyzed WPS-deregulated gene expressions in relation to different breast cancer molecular subtypes using clinical cases available from the GOBO database (1881 patients). Remarkably, using the PAM50 subclassification, we found most of those genes to be more expressed in the highly aggressive basal subtype including CCL5, MX1, MMP9, CCL8 and CCL4 [Figure 3]a. Similarly, CCL21, TNFSF14 and IL3 expressions showed higher expression in the basal subtypes according to the Hu subclassification [Figure 3]b.{Figure 3}

Water-pipe smoking-deregulated genes have a direct impact on the prognosis of breast cancer patients

We then assessed the plausible impact of WPS-deregulated DEGs on the prognosis of breast cancer patients. We evaluated the correlation between the expression of the DEGs' mRNA levels and the outcome of patients, described as RFS, using a large breast cancer cohort (n = 1764 patients) from the Kaplan–Meier plotter database.

Our results showed conflicting data regarding the association between individual genes and the survival of patients. While MXI (P = 0.0049), CCL8 (P < 0.001), GNGT1 (P = 0.012) and MMP9 (P = 0.0039) showed a significant association with a poor outcome of patients presented as a shortened RFS, other genes showed a significant association with a favorable outcome presented as prolonged patient survival [Figure 4]. These findings clearly indicated the central role of WPS in modulating breast cancer cells that might affect their behavior leading to a more aggressive phenotype and a worse outcome.{Figure 4}

On the other hand, WPS-induced genes were not associated with OS [Supplementary Figure 3].[INLINE:3]

Water-pipe smoking-deregulated genes are commonly involved in immune response pathways

Subsequently, we further investigated major gene interactions between WPS-deregulated DEGs and their plausible pathway enrichment [Figure 5].{Figure 5}

We found that these genes cooperated in major pathways including signal transduction, ligand bindings and the synthesis of lipoxins, leukotrienes, interleukins and interferon [Table 3]. Moreover, these DEGs were also part of molecular functions that included chemokine, cytokine and protein binding receptors, phospholipases, phosphotransferases and kinases with catalytic activity [Table 3].{Table 3}


To our knowledge, this study was the first cancer gene expression profiling study on the effect of WPS treatment in HNME cells. Similar to the present data, WPS enhanced the progression of EMT and invasion of breast cancer through the Erk1/2 pathway accompanied by E-cadherin and FAK gene deregulation in human breast cancer cells.[22] Moreover, cigarette smoking enhanced EMT in several human carcinoma cells[36],[37],[38,[39],[40] and, hence, smoking was a significant etiological factor in the onset and progression of various human cancers including breast.[22],[41],[42],[43],[44] Our present study implied that WPS exposure could play a vital role in the onset and possible progression of human breast cancer.

Indeed, in this investigation, the NanoString nCounter PanCancer Pathways Panel of 770 gene transcripts scattered in 13 biological pathways was used to identify the gene targets of WPS exposed HNME cells. Our data revealed significant alterations in the expression of 13 genes as targets for WPS exposure in HNME cells, which were further confirmed by qRT-PCR in addition to the Oncomine TM database. Subsequently, we determined the prognostic outcome of WPS-deregulated genes on breast cancer prognosis using the PanCancer RNA-seq dataset of the Kaplan–Meier plotter database. More significantly, this study indicated that these DEGs were found for the first time as targets of WPS exposure in human normal mammary cells. The discovered genes were involved in cell cycle, cell proliferation, cell migration/invasion, cell apoptosis, signal transduction and the inflammatory response and were thus likely involved in the neoplastic transformation of normal mammary epithelial cells leading to the onset of breast cancer.

Of the thirteen differentially expressed genes, five (CCL5, CCL4, CCL8, CCL21 and TNFSF14) of these genes were a part of the chemokine family. Upregulated levels of CCL5 significantly correlated with breast cancer progression, metastasis and/or relapse[45],[46] in addition to drug resistance,[47] signifying a fundamental role in cancer progression.[48] Recent investigations have shown an upregulation of CCL5 in breast cancer tissues compared with normal ones.[49] Increased CCL5 levels can recruit monocytes in the tumor microenvironment, thus promoting breast cancer progression.[50] CCL5 also enhances breast cancer progression in a p53-dependent manner through CCR5.[51] Similar to our data obtained from the PAM50 classification analysis, other studies have also found elevated CCL5 levels in triple-negative breast cancer (TNBC).[48],[49] Concordantly, our results showed an enhanced CCL5 expression, thus indicating a plausible association of CCL5 with breast cancer progression upon WPS use. Indeed, this association between CCL5 expression and aggressive and non-remissive breast cancer might be due to its ability to trigger the release of matrix-metalloproteinase,[49] MMP9;[52] our data analysis identified MMP9 as a target gene. An earlier study showed that the overexpression of MMP9 linked with the progression of dysplasia to breast cancer;[53] its elevated expression is found in breast cancer[49] and correlates with poor disease prognosis.[54] On the other hand, the overexpression of CCL5 is involved in enhancing tumor tolerance leading to poor prognosis in breast cancer.[55] CCL5 upregulation is also associated with non-remissive and later stage breast cancer.[46],[56] This could be due to its ability to augment MMP9 and monocyte migration, thus promoting angiogenesis and tumor progression.[57] Previous reports have shown a positive MMP9 to be associated with a shorter RFS and breast cancer-related survival.[58],[59] Our results also demonstrated that CCL5 as well as MMP9 were targets of WPS in human normal mammary cells. Intriguingly, ALOX5 facilitated an invasion via MMP9 stimulation; enhanced ALOX5 expression plays a role in tumor pathogenesis.[60] Furthermore, tumor-initiating genes associated with ALOX5 expression enhance mitogenesis, mutagenesis, angiogenesis, cell survival, immunosuppression and metastasis in breast cancer.[61] An earlier study by Wculek et al. reported that neutrophils enhanced ALOX5-dependent breast cancer lung metastasis.[62] Moreover, the ALOX5 inhibitor, Zileuton, significantly decreased breast cancer metastasis,[62] further supporting our finding of a suggestive role of ALOX5 in breast cancer initiation and progression. Moreover, a recent investigation showed the activation of ALOX5 was linked with HER2 expression, which regulates ALOX5 expression and enhances breast cancer growth and migration.[63] This was similar to our data where we found upregulated ALOX5 significantly correlated with the HER2-positive breast cancer subtype.

Analogous to CCL5, CCL4 has a comparable role in cancer progression; CCL4 induces breast cancer metastasis.[64] Recently, a study revealed that smoking in addition to CCL4 polymorphism could pose an elevated risk of breast cancer.[65] Similarly, we reported that WPS enhanced the CCL4 expression resulting in an augmented inflammatory response, thus promoting tumor development and progression. Remarkably, CCL8, a monocyte chemo-attractant protein-2, deregulates several cellular processes including proliferation, apoptosis and differentiation as well as enhances the progression of EMT.[66],[67] CCL8 can also trigger fibroblasts, thus creating a pro-tumor environment specifically in the TNBC stroma and promotes breast cancer metastasis.[49],[68] Our data were confirmed by previous investigations that found elevated CCL8 expression in breast cancer tissues to be significantly associated with negative hormone receptors, TNBC subtypes, basal-like subtypes, high grade breast cancers and a worse prognosis.[49],[69] The other identified chemokine, CCL21, also plays a vital role in regulating cellular proliferation, invasion, apoptosis and metastasis.[70],[71] Smoking enhances blood and bronchioalveolar lavage fluid levels of the CCR7 ligands CCL19 and CCL21[55] as well as contributing to the migration of lung cancer cells[72] via the EMT and ERK1/2 signaling pathways.[51] Numerous reports have shown that CCL21 plays a role in the migratory properties of breast cancer cells.[73] Concordant to the data reported by Chen and colleagues, in our study high levels of CCL21 significantly correlated with the basal-like subtype.[69] Interestingly, previous studies reported cross-talk of various CC chemokines in breast cancer including CCL8/21; cross-talk between CCL8 and CCL21 is involved in the development and progression of breast cancer and correlates with patient prognosis.[69] In concordance with our data, we showed the presence of CCL8/21 in normal mammary epithelial cells when exposed to WPS indicating its role in the transition to cancerous ones. Tumor-necrosis factor superfamily member 14 (TNFSF14), also known as LIGHT, is an inflammatory cytokine and plays a role in the anti-tumor immune response.[74] Ganstev and colleagues reported an upregulation of TNFSF14 in newly formed lymph nodes in breast cancer;[75] this was in concordance with our data and thus suggested a role of TNFSF14 in the onset and progression of breast cancer. Moreover, studies have shown that smoking enhances the expression of TNFSF14,[76],[77] which is upregulated in female smokers while the expression of TNFSF14 is absent in male smokers.[76] This finding supported our data as TNGSF14 expression was enhanced in WPS-induced breast cancer.[78],[79]

Subsequently, in our study we identified TLR9, a gene involved in the innate immune system. Studies have shown an elevated expression of TLR9 in breast cancer, which was associated with tumor grade.[80],[81],[82] An in vitro study by Merrell et al. reported upregulated TLR9 expression in the TNBC cell line (MDA-MB-231) and indicated a plausible role of TLR9 in tumor growth, progression and metastasis.[81] Intriguingly, several investigations have reported cigarette/e-cigarette smoke to elevate TLR9 expression;[83],[84],[85] these studies correlated with our data where we demonstrated exposure to WPS smoke-induced TLR9 expression in breast cancer. Furthermore, we identified prostaglandin reductase 1 (PTGR1), a metabolic enzyme involved in blocking a chemotactic factor, leukotriene B4. A previous study showed elevated PTGR1 expression in several breast cancer cell lines including HER2-positive and TNBC cell lines with the highest expression present in the TNBC cell line, HCC1937,[86] further supporting our data. Another investigation also reported the expression of PTGR1 to correlate with TNBC pathogenicity; the silencing of PTGR1 with licochalcone A decreased the TNBC pathogenicity.[87]

On the contrary, cytokine IL3, a selective growth factor, is released by a subset of tumor-infiltrating T cells in breast cancer tissues stimulating tumor angiogenesis.[88] In our study we found an increase of IL3 expression in human mammary epithelial cells upon WPS exposure. Additionally, the upregulated expression of IL3 has been shown to play a role in breast cancer bone metastasis;[89] this further confirmed the strong association between WPS upregulated genes and breast cancer tumor progression. In this study, we also identified another type of cytokine, interferon gamma (IFNG). Our data showed elevated IFNG expression in mammary epithelial cells exposed to WPS. An earlier investigation showed that breast cancer cells exhibited enhanced IFNG expression[90] thus promoting cancer invasion and angiogenesis.[91] In breast cancer, the key pathway associated with a prolonged RFS focuses on the immune response with IFNG signaling being one crucial pathway.[92] On the other hand, we also identified an interferon-related gene, MX1, which is upregulated in breast cancer;[93] concordant with our finding, MX1 levels were elevated in mammary epithelial cells exposed to smoke. Furthermore, similar to our data, mRNA and protein levels of MX1 were found to be increased in both in vivo and in vitro tamoxifen and fulvestrant resistance experimental models[94],[95],[96] indicating MX1 involvement in RFS and a poor prognosis. Recently, a study showed the involvement of the PIK3/AKT pathway in enhancing MX1 expression, which can be linked with the stimulation of growth signaling pathways in relapsing patients.[97]

We herein identified the G Protein Subunit Gamma Transducin 1 (GNGT1) gene, which encodes for the guanine nucleotide binding protein (G protein) as a target of WPS. Although previous studies have demonstrated an enhanced expression of GNGT1 in head and neck squamous cell carcinomas,[98] lung cancer[99],[100] as well as liver cancer,[101] this was the first study that reported the overexpression of GNGT1 in breast cancer. As smoking is considered to be a key risk factor for lung cancer,[102] we suggested a plausible role for GNGT1 in WPS-induced breast cancer. Furthermore, GNGT1 correlated with poor overall and progression-free survival in serous ovarian cancer,[103],[104] thus supporting our data.

While smoking is a vital etiological factor in the onset and progression of various human cancers including lung and oral as well as breast,[22],[39],[40],[41],[44],[105],[106] a previous study demonstrated that WPS exposure can stimulate the cell invasion of breast cancer cells.[22] However, an increase in WPS consumption leads to rising levels of toxicant intake; it is postulated that WPS can be a carcinogenic and therefore it can play a plausible role in the development and progression of various types of human cancers as well as cancer-related mortality in comparison with cigarette smoking. Moreover, in this study we identified DEGs that could be plausible therapeutic targets; nevertheless, future studies are essential to validate and determine the mechanisms underpinning WPS-induced breast carcinogenesis.


In our study, we revealed for the first time that WPS could plausibly play a role in inducing EMT in HNME cells along with the deregulation of a set of genes responsible of the development and progression of human breast carcinogenesis and RFS. Thus, WPS could enhance breast cancer development and/or its progression predominantly due to its effect on key regulatory carcinogenic genes that have a direct impact on the outcome of breast cancer patients. However, further research is warranted to further elucidate the underlying mechanism underpinning WPS-induced human breast carcinogenesis.

Data availability statement

Data supporting the reported results are contained within the article or Supplementary Materials.


We would like to thank A. Kassab for her critical reading of the manuscript.

Financial support and sponsorship

This work is supported by Qatar University, grant numbers: QUCG-CMED-20/21-2 and QUHI-CMED-19/20-1. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Conflicts of interest

There are no conflicts of interest.


World Health Organization. (↱2018)↱. WHO global report on trends in prevalence of tobacco smoking 2000-2025, 2nded. World Health Organization. License: CC BY-NC-SA 3.0 IGO.

Onor IO, Stirling DL, Williams SR, Bediako D, Borghol A, Harris MB, et al. Clinical effects of cigarette smoking: Epidemiologic impact and review of pharmacotherapy options. Int J Environ Res Public Health 2017;14:E1147.

Maziak W, Taleb ZB, Bahelah R, Islam F, Jaber R, Auf R, et al. The global epidemiology of waterpipe smoking. Tob Control 2015;24 Suppl 1:i3-12.

Jawad M, McEwen A, McNeill A, Shahab L. To what extent should waterpipe tobacco smoking become a public health priority? Addiction 2013;108:1873-84.

Akl EA, Gunukula SK, Aleem S, Obeid R, Jaoude PA, Honeine R, et al. The prevalence of waterpipe tobacco smoking among the general and specific populations: A systematic review. BMC Public Health 2011;11:244.

Maziak W, Nakkash R, Bahelah R, Husseini A, Fanous N, Eissenberg T. Tobacco in the Arab world: Old and new epidemics amidst policy paralysis. Health Policy Plan 2014;29:784-94.

Wolfram RM, Chehne F, Oguogho A, Sinzinger H. Narghile (water pipe) smoking influences platelet function and (iso-) eicosanoids. Life Sci 2003;74:47-53.

Neergaard J, Singh P, Job J, Montgomery S. Waterpipe smoking and nicotine exposure: A review of the current evidence. Nicotine Tob Res 2007;9:987-94.

Javed F, ALHarthi SS, BinShabaib MS, Gajendra S, Romanos GE, Rahman I. Toxicological impact of waterpipe smoking and flavorings in the oral cavity and respiratory system. Inhal Toxicol 2017;29:389-96.

Eissenberg T, Shihadeh A. Waterpipe tobacco and cigarette smoking: Direct comparison of toxicant exposure. Am J Prev Med 2009;37:518-23.

Cobb CO, Shihadeh A, Weaver MF, Eissenberg T. Waterpipe tobacco smoking and cigarette smoking: A direct comparison of toxicant exposure and subjective effects. Nicotine Tob Res 2011;13:78-87.

Maziak W, Ward KD, Eissenberg T. Factors related to frequency of narghile (waterpipe) use: The first insights on tobacco dependence in narghile users. Drug Alcohol Depend 2004;76:101-6.

Rastam S, Eissenberg T, Ibrahim I, Ward KD, Khalil R, Maziak W. Comparative analysis of waterpipe and cigarette suppression of abstinence and craving symptoms. Addict Behav 2011;36:555-9.

Joseph S, Pascale S, Georges K, Mirna W. Cigarette and waterpipe smoking decrease respiratory quality of life in adults: Results from a national cross-sectional study. Pulm Med 2012;2012:868294.

Radwan G, Hecht SS, Carmella SG, Loffredo CA. Tobacco-specific nitrosamine exposures in smokers and nonsmokers exposed to cigarette or waterpipe tobacco smoke. Nicotine Tob Res 2013;15:130-8.

Ali M, Jawad M. Health effects of waterpipe tobacco use: Getting the public health message just right. Tob Use Insights 2017;10:1179173X17696055.

Layoun N, Saleh N, Barbour B, Awada S, Rachidi S, Al-Hajje A, et al. Waterpipe effects on pulmonary function and cardiovascular indices: A comparison to cigarette smoking in real life situation. Inhal Toxicol 2014;26:620-7.

Javed F, Al-Kheraif AA, Rahman I, Millan-Luongo LT, Feng C, Yunker M, et al. Comparison of clinical and radiographic periodontal status between habitual water-pipe smokers and cigarette smokers. J Periodontol 2016;87:142-7.

Ashour AA, Haik MY, Sadek KW, Yalcin HC, Bitharas J, Aboulkassim T, et al. Substantial toxic effect of water-pipe smoking on the early stage of embryonic development. Nicotine Tob Res 2018;20:502-7.

Fouad H, Awa FE, Naga RA, Emam AH, Labib S, Palipudi KM, et al. Prevalence of tobacco use among adults in Egypt, 2009. Glob Health Promot 2016;23:38-47.

Rastam S, Li FM, Fouad FM, Al Kamal HM, Akil N, Al Moustafa AE. Water pipe smoking and human oral cancers. Med Hypotheses 2010;74:457-9.

Sadek KW, Haik MY, Ashour AA, Baloch T, Aboulkassim T, Yasmeen A, et al. Water-pipe smoking promotes epithelial-mesenchymal transition and invasion of human breast cancer cells via ERK1/ERK2 pathways. Cancer Cell Int 2018;18:180.

López-Ozuna VM, Gupta I, Kiow RL, Matanes E, Kheraldine H, Yasmeen A, et al. Water-pipe smoking exposure deregulates a set of genes associated with human head and neck cancer development and prognosis. Toxics 2020;8:E73.

Waziry R, Jawad M, Ballout RA, Al Akel M, Akl EA. The effects of waterpipe tobacco smoking on health outcomes: An updated systematic review and meta-analysis. Int J Epidemiol 2017;46:32-43.

Montazeri Z, Nyiraneza C, El-Katerji H, Little J. Waterpipe smoking and cancer: Systematic review and meta-analysis. Tob Control 2017;26:92-7.

Bou Fakhreddine HM, Kanj AN, Kanj NA. The growing epidemic of water pipe smoking: Health effects and future needs. Respir Med 2014;108:1241-53.

Kim KH, Kabir E, Jahan SA. Waterpipe tobacco smoking and its human health impacts. J Hazard Mater 2016;317:229-36.

Yasmeen A, Alachkar A, Dekhil H, Gambacorti-Passerini C, Al Moustafa AE. Locking Src/Abl tyrosine kinase activities regulate cell differentiation and invasion of human cervical cancer cells expressing E6/E7 oncoproteins of high-risk HPV. J Oncol 2010;2010:530130.

Zhao B, Erwin A, Xue B. How many differentially expressed genes: A perspective from the comparison of genotypic and phenotypic distances. Genomics 2018;110:67-73.

Thomas JG, Olson JM, Tapscott SJ, Zhao LP. An efficient and robust statistical modeling approach to discover differentially expressed genes using genomic expression profiles. Genome Res 2001;11:1227-36.

Rhodes DR, Kalyana-Sundaram S, Mahavisno V, Varambally R, Yu J, Briggs BB, et al. Oncomine 3.0: Genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia 2007;9:166-80.

Györffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat 2010;123:725-31.

Ringnér M, Fredlund E, Häkkinen J, Borg Å, Staaf J. GOBO: Gene expression-based outcome for breast cancer online. PLoS One 2011;6:e17911.

Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature 2000;406:747-52.

Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001;98:10869-74.

Sun X, Deng Q, Liang Z, Liu Z, Geng H, Zhao L, et al. Cigarette smoke extract induces epithelial-mesenchymal transition of human bladder cancer T24 cells through activation of ERK1/2 pathway. Biomed Pharmacother 2017;86:457-65.

Pillai S, Trevino J, Rawal B, Singh S, Kovacs M, Li X, et al. β-arrestin-1 mediates nicotine-induced metastasis through E2F1 target genes that modulate epithelial-mesenchymal transition. Cancer Res 2015;75:1009-20.

Liu Y, Luo F, Xu Y, Wang B, Zhao Y, Xu W, et al. Epithelial-mesenchymal transition and cancer stem cells, mediated by a long non-coding RNA, HOTAIR, are involved in cell malignant transformation induced by cigarette smoke extract. Toxicol Appl Pharmacol 2015;282:9-19.

Dinicola S, Masiello MG, Proietti S, Coluccia P, Fabrizi G, Catizone A, et al. Nicotine increases colon cancer cell migration and invasion through epithelial to mesenchymal transition (EMT): COX-2 involvement. J Cell Physiol 2018;233:4935-48.

Chen PC, Lee WY, Ling HH, Cheng CH, Chen KC, Lin CW. Activation of fibroblasts by nicotine promotes the epithelial-mesenchymal transition and motility of breast cancer cells. J Cell Physiol 2018;233:4972-80.

Andersen ZJ, Jørgensen JT, Grøn R, Brauner EV, Lynge E. Active smoking and risk of breast cancer in a Danish nurse cohort study. BMC Cancer 2017;17:556.

Inoue-Choi M, Hartge P, Liao LM, Caporaso N, Freedman ND. Association between long-term low-intensity cigarette smoking and incidence of smoking-related cancer in the national institutes of health-AARP cohort. Int J Cancer 2018;142:271-80.

Lee PN, Thornton AJ, Hamling JS. Epidemiological evidence on environmental tobacco smoke and cancers other than lung or breast. Regul Toxicol Pharmacol 2016;80:134-63.

Liu M, Zhou C, Zheng J. Cigarette smoking impairs the response of EGFR-TKIs therapy in lung adenocarcinoma patients by promoting EGFR signaling and epithelial-mesenchymal transition. Am J Transl Res 2015;7:2026-35.

Bièche I, Lerebours F, Tozlu S, Espie M, Marty M, Lidereau R. Molecular profiling of inflammatory breast cancer: Identification of a poor-prognosis gene expression signature. Clin Cancer Res 2004;10:6789-95.

Niwa Y, Akamatsu H, Niwa H, Sumi H, Ozaki Y, Abe A. Correlation of tissue and plasma RANTES levels with disease course in patients with breast or cervical cancer. Clin Cancer Res 2001;7:285-9.

Yi EH, Lee CS, Lee JK, Lee YJ, Shin MK, Cho CH, et al. STAT3-RANTES autocrine signaling is essential for tamoxifen resistance in human breast cancer cells. Mol Cancer Res 2013;11:31-42.

Lv D, Zhang Y, Kim HJ, Zhang L, Ma X. CCL5 as a potential immunotherapeutic target in triple-negative breast cancer. Cell Mol Immunol 2013;10:303-10.

Thomas JK, Mir H, Kapur N, Bae S, Singh S. CC chemokines are differentially expressed in Breast Cancer and are associated with disparity in overall survival. Sci Rep 2019;9:4014.

Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 2007;449:557-63.

Mañes S, Mira E, Colomer R, Montero S, Real LM, Gómez-Moutón C, et al. CCR5 expression influences the progression of human breast cancer in a p53-dependent manner. J Exp Med 2003;198:1381-9.

Swamydas M, Ricci K, Rego SL, Dréau D. Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases. Cell Adh Migr 2013;7:315-24.

Ha HY, Moon HB, Nam MS, Lee JW, Ryoo ZY, Lee TH, et al. Overexpression of membrane-type matrix metalloproteinase-1 gene induces mammary gland abnormalities and adenocarcinoma in transgenic mice. Cancer Res 2001;61:984-90.

Vilen ST, Salo T, Sorsa T, Nyberg P. Fluctuating roles of matrix metalloproteinase-9 in oral squamous cell carcinoma. ScientificWorldJournal 2013;2013:920595.

Zhang JF, Li Y, Zhang AZ, He QQ, Du YC, Cao W. Expression and pathological significance of CC chemokine receptor 7 and its ligands in the airway of asthmatic rats exposed to cigarette smoke. J Thorac Dis 2018;10:5459-67.

Luboshits G, Shina S, Kaplan O, Engelberg S, Nass D, Lifshitz-Mercer B, et al. Elevated expression of the CC chemokine regulated on activation, normal T cell expressed and secreted (RANTES) in advanced breast carcinoma. Cancer Res 1999;59:4681-7.

Azenshtein E, Luboshits G, Shina S, Neumark E, Shahbazian D, Weil M, et al. The CC chemokine RANTES in breast carcinoma progression: Regulation of expression and potential mechanisms of promalignant activity. Cancer Res 2002;62:1093-102.

Li HC, Cao DC, Liu Y, Hou YF, Wu J, Lu JS, et al. Prognostic value of matrix metalloproteinases (MMP-2 and MMP-9) in patients with lymph node-negative breast carcinoma. Breast Cancer Res Treat 2004;88:75-85.

Pellikainen JM, Ropponen KM, Kataja VV, Kellokoski JK, Eskelinen MJ, Kosma VM. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in breast cancer with a special reference to activator protein-2, HER2, and prognosis. Clin Cancer Res 2004;10:7621-8.

Kummer NT, Nowicki TS, Azzi JP, Reyes I, Iacob C, Xie S, et al. Arachidonate 5 lipoxygenase expression in papillary thyroid carcinoma promotes invasion via MMP-9 induction. J Cell Biochem 2012;113:1998-2008.

Kennedy BM, Harris RE. Cyclooxygenase and lipoxygenase gene expression in the inflammogenesis of breast cancer. Inflammopharmacology 2018;26:909-23.

Wculek SK, Malanchi I. Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Nature 2015;528:413-7.

Zhou X, Jiang Y, Li Q, Huang Z, Yang H, Wei C. Aberrant ALOX5 activation correlates with HER2 status and mediates breast cancer biological activities through multiple mechanisms. Biomed Res Int 2020;2020:1703531.

Sasaki S, Baba T, Nishimura T, Hayakawa Y, Hashimoto S, Gotoh N, et al. Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis. Cancer Lett 2016;378:23-32.

Hu GN, Tzeng HE, Chen PC, Wang CQ, Zhao YM, Wang Y, et al. Correlation between CCL4 gene polymorphisms and clinical aspects of breast cancer. Int J Med Sci 2018;15:1179-86.

Zhou J, Zheng S, Liu T, Liu Q, Chen Y, Tan D, et al. MCP2 activates NF-κB signaling pathway promoting the migration and invasion of ESCC cells. Cell Biol Int 2018;42:365-72.

Bryja A, Dyszkiewicz-Konwińska M, Huang Y, Celichowski P, Nawrocki MJ, Jankowski M, et al. Genes involved in regulation of cellular metabolic processes, signaling and adhesion are the markers of porcine buccal pouch mucosal cells long-term primary cultured in vitro. J Biol Regul Homeost Agents 2018;32:1129-41.

Farmaki E, Chatzistamou I, Kaza V, Kiaris H. A CCL8 gradient drives breast cancer cell dissemination. Oncogene 2016;35:6309-18.

Chen B, Zhang S, Li Q, Wu S, He H, Huang J. Bioinformatics identification of CCL8/21 as potential prognostic biomarkers in breast cancer microenvironment. Biosci Rep 2020;40:BSR20202042.

Hwang TL, Lee LY, Wang CC, Liang Y, Huang SF, Wu CM. CCL7 and CCL21 overexpression in gastric cancer is associated with lymph node metastasis and poor prognosis. World J Gastroenterol 2012;18:1249-56.

Xiong Y, Huang F, Li X, Chen Z, Feng D, Jiang H, et al. CCL21/CCR7 interaction promotes cellular migration and invasion via modulation of the MEK/ERK1/2 signaling pathway and correlates with lymphatic metastatic spread and poor prognosis in urinary bladder cancer. Int J Oncol 2017;51:75-90.

Kuźnar-Kamińska B, Mikuła-Pietrasik J, Sosińska P, Książek K, Batura-Gabryel H. COPD promotes migration of A549 lung cancer cells: The role of chemokine CCL21. Int J Chron Obstruct Pulmon Dis 2016;11:1061-6.

Tutunea-Fatan E, Majumder M, Xin X, Lala PK. The role of CCL21/CCR7 chemokine axis in breast cancer-induced lymphangiogenesis. Mol Cancer 2015;14:35.

Mauri DN, Ebner R, Montgomery RI, Kochel KD, Cheung TC, Yu GL, et al. LIGHT, a new member of the TNF superfamily, and lymphotoxin alpha are ligands for herpesvirus entry mediator. Immunity 1998;8:21-30.

Gantsev SK, Umezawa K, Islamgulov DV, Khusnutdinova EK, Ishmuratova RS, Frolova VY, et al. The role of inflammatory chemokines in lymphoid neoorganogenesis in breast cancer. Biomed Pharmacother 2013;67:363-6.

Faner R, Gonzalez N, Cruz T, Kalko SG, Agustí A. Systemic inflammatory response to smoking in chronic obstructive pulmonary disease: Evidence of a gender effect. PLoS One 2014;9:e97491.

Yun JH, Morrow J, Owen CA, Qiu W, Glass K, Lao T, et al. Transcriptomic analysis of lung tissue from cigarette smoke-induced emphysema murine models and human chronic obstructive Pulmonary disease show shared and distinct pathways. Am J Respir Cell Mol Biol 2017;57:47-58.

Maker AV, Ito H, Mo Q, Weisenberg E, Qin LX, Turcotte S, et al. Genetic evidence that intratumoral T-cell proliferation and activation are associated with recurrence and survival in patients with resected colorectal liver metastases. Cancer Immunol Res 2015;3:380-8.

Maker AV. Precise identification of immunotherapeutic targets for solid malignancies using clues within the tumor microenvironment-evidence to turn on the LIGHT. Oncoimmunology 2016;5:e1069937.

González-Reyes S, Marín L, González L, González LO, del Casar JM, Lamelas ML, et al. Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis. BMC Cancer 2010;10:665.

Merrell MA, Ilvesaro JM, Lehtonen N, Sorsa T, Gehrs B, Rosenthal E, et al. Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity. Mol Cancer Res 2006;4:437-47.

Berger R, Fiegl H, Goebel G, Obexer P, Ausserlechner M, Doppler W, et al. Toll-like receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors. Cancer Sci 2010;101:1059-66.

Foronjy RF, Salathe MA, Dabo AJ, Baumlin N, Cummins N, Eden E, et al. TLR9 expression is required for the development of cigarette smoke-induced emphysema in mice. Am J Physiol Lung Cell Mol Physiol 2016;311:L154-66.

Nadigel J, Préfontaine D, Baglole CJ, Maltais F, Bourbeau J, Eidelman DH, et al. Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8(+) T cells in chronic obstructive pulmonary disease. Respir Res 2011;12:149.

Li J, Huynh DL, Tang MS, Simborio H, Huang J, Kosmider B, et al. Electronic cigarettes induce mitochondrial DNA damage and trigger toll-like receptor 9-mediated atherosclerosis. bioRxiv Arterioscler Thromb Vasc Biol; 2021;41:839-853.

Liu M, Liu Y, Deng L, Wang D, He X, Zhou L, et al. Transcriptional profiles of different states of cancer stem cells in triple-negative breast cancer. Mol Cancer 2018;17:65.

Roberts LS, Yan P, Bateman LA, Nomura DK. Mapping novel metabolic nodes targeted by anti-cancer drugs that impair triple-negative breast cancer pathogenicity. ACS Chem Biol 2017;12:1133-40.

Dentelli P, Rosso A, Calvi C, Ghiringhello B, Garbarino G, Camussi G, et al. IL-3 affects endothelial cell-mediated smooth muscle cell recruitment by increasing TGF beta activity: Potential role in tumor vessel stabilization. Oncogene 2004;23:1681-92.

Mora EM, Torres D, Tari AM. Inhibition of the interleukin-3 receptor decreases the growth of bone-metastatic breast carcinoma cells. Cancer Res 2006;66:386.

Yaghoobi H, Azizi H, Oskooei VK, Taheri M, Ghafouri-Fard S. Assessment of expression of interferon γ (IFN-G) gene and its antisense (IFNG-AS1) in breast cancer. World J Surg Oncol 2018;16:211.

Ni L, Lu J. Interferon gamma in cancer immunotherapy. Cancer Med 2018;7:4509-16.

Bedognetti D, Hendrickx W, Marincola FM, Miller LD. Prognostic and predictive immune gene signatures in breast cancer. Curr Opin Oncol 2015;27:433-44.

Liu YP, Suksanpaisan L, Steele MB, Russell SJ, Peng KW. Induction of antiviral genes by the tumor microenvironment confers resistance to virotherapy. Sci Rep 2013;3:2375.

Johansson HJ, Sanchez BC, Forshed J, Stål O, Fohlin H, Lewensohn R, et al. Proteomics profiling identify CAPS as a potential predictive marker of tamoxifen resistance in estrogen receptor positive breast cancer. Clin Proteomics 2015;12:8.

Becker M, Sommer A, Krätzschmar JR, Seidel H, Pohlenz HD, Fichtner I. Distinct gene expression patterns in a tamoxifen-sensitive human mammary carcinoma xenograft and its tamoxifen-resistant subline MaCa 3366/TAM. Mol Cancer Ther 2005;4:151-68.

Huber M, Bahr I, Krätzschmar JR, Becker A, Müller EC, Donner P, et al. Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r. Mol Cell Proteomics 2004;3:43-55.

Chai Y, Huang HL, Hu DJ, Luo X, Tao QS, Zhang XL, et al. IL-29 and IFN-α regulate the expression of MxA, 2',5'-OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells. Mol Biol Rep 2011;38:139-43.

Ghias K, Rehmani SS, Razzak SA, Madhani S, Azim MK, Ahmed R, et al. Mutational landscape of head and neck squamous cell carcinomas in a South Asian population. Genet Mol Biol 2019;4242:526-42.

Zhang JJ, Hong J, Ma YS, Shi Y, Zhang DD, Yang XL, et al. Identified GNGT1 and NMU as combined diagnosis biomarker of non-small-cell lung cancer utilizing bioinformatics and logistic regression. Dis Markers 2021;2021:6696198.

Juarez-Flores A JM. Squamous cell carcinoma of the lung: Gene expression and network analysis during carcinogenesis. Int J Clin Exp Med 2019;12:6671-83.

Qian Z, Zhang G, Song G, Shi J, Gong L, Mou Y, et al. Integrated analysis of genes associated with poor prognosis of patients with colorectal cancer liver metastasis. Oncotarget 2017;8:25500-12.

O'Keeffe LM, Taylor G, Huxley RR, Mitchell P, Woodward M, Peters SAE. Smoking as a risk factor for lung cancer in women and men: A systematic review and meta-analysis. BMJ Open 2018;8:e021611.

Zhang YB, Jiang Y, Wang J, Ma J, Han S. Evaluation of core serous epithelial ovarian cancer genes as potential prognostic markers and indicators of the underlying molecular mechanisms using an integrated bioinformatics analysis. Oncol Lett 2019;18:5508-22.

Mucaki EJ, Zhao JZ, Lizotte DJ, Rogan PK. Predicting responses to platin chemotherapy agents with biochemically-inspired machine learning. Signal Transduct Target Ther 2019;4:1.

Strumylaite L, Kregzdyte R, Poskiene L, Bogusevicius A, Pranys D, Norkute R. Association between lifetime exposure to passive smoking and risk of breast cancer subtypes defined by hormone receptor status among non-smoking Caucasian women. PLoS One 2017;12:e0171198.

White AJ, D'Aloisio AA, Nichols HB, DeRoo LA, Sandler DP. Breast cancer and exposure to tobacco smoke during potential windows of susceptibility. Cancer Causes Control 2017;28:667-75.

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