Nicotine Synergizes with High-Fat Diet to Induce an Anti-Inflammatory Microenvironment to Promote Breast Tumor Growth

Breast cancer results from a complex interplay of genetics and environment that alters immune and inflammatory systems to promote tumorigenesis. Obesity and cigarette smoking are well-known risk factors associated breast cancer development. Nicotine known to decrease inflammatory signals also modulates immune responses that favor breast cancer development. However, the mechanisms by which nicotine and obesity contribute to breast cancer remain poorly understood. In this study, we examined potential mechanisms by which nicotine (NIC) and high-fat diet (HFD) promote growth of HCC70 and HCC1806 xenografts from African American (AA) triple negative (TN) breast cancer cells. Immunodeficient mice fed on HFD and treated with NIC generated larger HCC70 and HCC1806 tumors when compared to NIC or HFD alone. Increased xenograft growth in the presence of NIC and HFD was accompanied by higher levels of tissue-resident macrophage markers and anti-inflammatory cytokines including IL4, IL13, and IL10. We further validated the involvement of these players by in vitro and ex vivo experiments. We found a proinflammatory milieu with increased expression of IL6 and IL12 in xenografts with HFD. In addition, nicotine or nicotine plus HFD increased a subset of mammary cancer stem cells (MCSCs) and key adipose browning markers CD137 and TMEM26. Interestingly, there was upregulation of stress-induced pp38 MAPK and pERK1/2 in xenografts exposed to HFD alone or nicotine plus HFD. Scratch-wound assay showed marked reduction in proliferation/migration of nicotine and palmitate-treated breast cancer cells with mecamylamine (MEC), a nicotine acetylcholine receptor (nAchR) antagonist. Furthermore, xenograft development in immune-deficient mice, fed HFD plus nicotine, was reduced upon cotreatment with MEC and SB 203580, a pp38MAPK inhibitor. Our study demonstrates the presence of nicotine and HFD in facilitating an anti-inflammatory tumor microenvironment that influences breast tumor growth. This study also shows potential efficacy of combination therapy in obese breast cancer patients who smoke.


Introduction
Molecular and cellular alterations influenced by genetic as well as environmental factors contribute to breast cancer development [1]. Poor understanding of the complexity of breast cancer has resulted in ineffective treatment strategies and high mortality rate. In addition, factors that increase the risk for breast cancer development also remain understudied. A diverse array of engineered models including single cell sequencing has revealed highly plastic nature of breast cancer cells, which generate heterogeneous dynamic clones that change over time [2,3]. In addition, these clones cross-talk with complex breast tumor microenvironment, which actively not only participate in tumor development but also manipulate therapeutic responses [4,5]. Despite these challenges, prognosis for breast cancer types that present estrogen receptor (ER), progesterone receptor (PR), and/or HER2/neu has improved due to targeted drug development. However, specific pharmaceutical countermeasures have not been successful in treating breast cancers that are negative for ER, PR, and Her2 neu, called triple-negative breast cancers (TNBC). However, 330,000 American women develop breast cancer and 41,760 die of the disease per year; thus, research needs to focus on prevention that will require a deeper understanding of the environmental factors that influence breast tumor development.
Both active and passive smoking has been shown to be a major risk factor for the initiation and progression of breast cancer [6][7][8]. Burning of the 600 ingredients in cigarettes produces more than 4,000 chemicals of which 60 are known carcinogens [9]. Nicotine, the main addictive ingredient in cigarettes, is also present in nicotine-replacement therapy (NRT), which is available to many smokers who want to stop smoking. Furthermore, since nicotine cessation leads to weight gain, especially in women, who have stopped smoking and continue on NRT to prevent weight gain [10], the consequences of nicotine from cigarettes versus from NRT on breast cancer development are difficult to delineate in humans, thus emphasizing the importance of animal models. Nicotine, a tumor-promoting compound, binds to the nicotinic acetylcholine receptors (nAchR) to promote tumor growth by increasing angiogenesis and metastasis [11]. Breast tumors with higher levels of nAchR are usually more aggressive and have poor prognosis [12]. Nicotine influences the development of various subtypes of breast tumors through PI3K/Akt/Ras/Raf and STAT1/STAT3 pathways [13,14]. However, more alarming is the fact that nicotine could contribute to breast tumor metastasis by increasing the expression of a GTPase, cdc42, as well as vimentin, which promotes epithelial to mesenchymal transition (EMT), a key step in metastasis [15]. Another mechanism by which nicotine promotes breast tumor development is by inducing galectin-3, an antiapoptotic β-galactoside-binding lectin that binds α9-nAchR to activate the STAT signaling pathway [16].
Interestingly, there are reports of nicotine increasing aldehyde dehydrogenase 1-(ALDH1-) expressing mammary cancer stem cell population (MCSC) in MCF7 breast cancer cell line [15]. Mechanistically, nicotine increased Hes-1 expression, a target gene involved in the Notch signaling pathway to facilitate stem cell renewal [17]. MCSCs are a small stem cell population in breast tumors that have the potential to promote initiation and progression of breast tumors [18]. Heterogeneous populations of MCSCs express markers like ALDH1, CD44, and/or CD133, which has enabled their identification [19]. Alarmingly, there are high levels of ALDH1 expression in aggressive Ghanaian and African American (AA) TNBC [20]. Although a large population of AA women who develop breast tumors also smoke, the influence of nicotine-induced various cytokines on MCSCs and tumor growth remains understudied.
Obesity is a known risk factor for breast cancer [21,22]. Breast tumors develop in an adipose-rich microenvironment, which has proven to be an active participant in the tumor development [23]. Cytokines, hormones, and growth factors secreted from adipocytes can induce phenotypic changes and aggressive behavior in breast cancer cells [24]. A cross-talk between cancer cells and adipocytes in tumor microenvironment generate Cancer-Associated Adipocytes (CAA) that undergo morphological changes and acquire additional secretory capability to influence breast tumor development [25]. Oncostatin M (OSM), a paracrine secretion from CAA, increased STAT3 phosphorylation subsequently inducing angiogenesis in breast cancer xenografts [26]. Conditioned media from CAA were sufficient to induce migration of MCF-7, demonstrating potency of secreted cytokines [27]. Obese people have high circulating cytokines as well as pockets of cytokine secreting cells that attract immune cells, such as macrophages, resulting in sustained inflammatory sites that could be the fertile ground for cancer development [28]. However, breast tumors are heterogeneous where pockets of both M1 macrophages and proinflammatory cytokines (TNFα, IL-12, and IL-6) as well as M2 macrophages and anti-inflammatory cytokines exist [28]. Despite implications of both M1 and M2 macrophages in breast cancer progression, M2 macrophages with its wound healing properties promote angiogenesis and metastasis in aggressive breast tumors [29]. M2 macrophages have also been reported to cross-talk with beige/brown adipocytes, which are upregulated by nicotine. We have recently demonstrated an increase in beige adipocytes in xenografts from breast cancer cells and patient tumors, where it contributes to tumor growth [30].
Recent studies suggest that obese AA women are more susceptible to develop an aggressive form of TNBC for which there is no treatment [31]. In addition, a large number of AA women who are obese and develop aggressive form of breast cancer also smoke [32]. Mechanisms by which smoking contributes to the development of this aggressive disease in obese AA women remains poorly understood. In this study, we used immune-deficient nude mice to examine mechanisms by which nicotine (NIC) and high-fat diet (HFD) promote growth of xenografts from AA TN breast cancer cells. We examined whether nicotine promotes conversion of HFDinduced increased adipose tissue to beige adipocytes, as well as in-filtered M1 macrophages to tumor promoting M2 phenotype.

Aldefluor Assay and Flow Cytometry.
Aldefluor assay was carried out as described previously [35] according to the manufacturer's (cat# 01700, Stem cell Technologies, Vancouver, Canada) guidelines. Briefly, cells suspended in Aldefluor assay buffer were incubated with 1.5 μM bodipyaminoacetaldehyde (BAAA) (ALDH substrate) for 40 min at 37°C. A fraction of the cells with BAAA was incubated with 10-fold molar excess of diethyl amino benzaldehyde DEAB, which is an ALDH inhibitor, under identical conditions and was used as a control. ALDH + and ALDH − cells were analyzed in a BD-LSR II analyzer (UCLA core lab). OCT4 and SOX2 expressing cells were quantitated by suspension in PBS followed by incubation with PE conjugated anti-OCT4 (cat# 3A2A20, Bio Legend, San Diego) or PEconjugated anti-SOX2 antibodies (cat# 60447, Cell Signaling Technology, Inc., Boston) for 2 hours and subjected to flow cytometry analysis (FACSCalibur analyzer, UCLA core lab).
2.6. Mammosphere Formation. HCC70 cells after various treatments (NIC and ±PAL, 48 h) were suspended in mammosphere media (DMEM supplemented with 10μg/ml insulin and 25 ng/ml fibroblast growth factor) and plated on ultralow attachment 6-well plates (Costar™ 3471), at a density of 50,000 cells/well as described previously [36]. We harvested the spheres at 48 h and number of mammospheres counted using an Olympus BX43 motorized microscope.

Immunohistochemical Analyses.
Xenografts excised from nude mice were fixed in 5% formalin overnight, after which they were dehydrated in ethanol and embedded in paraffin.
These data indicate that both independent and interdependent actions of NIC and HFD in an additive and synergistic manner may have contributed to increased xenograft growth.

Nicotine and Palmitate Increase Mammary Cancer Stem
Cells, Beige Markers, and Anti-Inflammatory Cytokines in Human Breast Cancer Cells In Vitro. Since protein expressions of MCSCs and macrophages were examined using HCC70 xenografts, we set up in vitro models using HCC70 cells to further validate our in vivo data. The effect of NIC and HFD on MCSC, macrophage populations, and antiinflammatory cytokines was examined using in vitro models.

Mediators of Inflammation
Due to solubility issues of high fat in aqueous solvents, we used palmitate (Pal) conjugated with bovine serum albumin (BSA) for our in vitro experiments. We examined HCC70 cells treated with NIC or Pal alone or in combination for MCSCs, as well as key pro-or anti-inflammatory cytokines and macrophage markers. We enriched MCSC from HCC70 cell line by propagating under low-attachment plates as mammospheres, which are high in mammary stem cell population. There was a significant increase in the number and size of mammospheres with 300 nM NIC and 200 μM Pal treatments either alone or in combination when compared to ethanol or BSA groups (Figure 3(a)). The analysis of total number of mammospheres per 50,000 cells/well from various treatment groups showed a significant increase in NIC (4.45-fold; p ≤ 0:05), Pal (2.5-fold; p ≤ 0:05), and NIC + Pal (7.5-fold; p ≤ 0:01) groups compared to control groups (Figure 3(b)). Quantitation of ALDH1-positive population in these groups by Aldefluor assay further shows a significant increase in NIC (2.3-fold, p ≤ 0:01), Pal (4.4-fold, p ≤ 0:001) and NIC + Pal (6.1-fold, p ≤ 0:001) groups compared to the control group (Figure 3 (Figure 3(d)). We further examined total macrophage population as well as tissue-resident macrophages, which require anti-inflammatory cytokines for their maintenance in breast cancer cells treated with NIC and Pal either alone or in combination. Increased the expression of tissue-resident macrophages (CD163) expression (NIC: 3.2-fold; Pal: 1.4-fold; NIC + Pal: 3.4-fold) compared to the control group (Figure 3(d)). Since NIC has been reported to increase browning or beige adipocytes [39], which are also upregulated by anti-inflammatory cytokines, expression of beige marker TMEM26 was examined in these cells after various treatment. There was increased expression of TMEM26 in cells treated with NIC (2.4-fold) as well as NIC+ Pal (3.2-fold) treatment groups (Figure 3(d)). Since stress kinase pp38MAPK levels were upregulated in xenografts grown in the presence of HFD as well as NIC + HFD groups, we further examined pp38 MAPK in breast cancer cells following various in vitro treatments. There was an increase in pp38 MAPK protein expression (Pal: 2.2-fold; NIC + Pal: 3.4-fold) when compared to ethanol or BSA control groups (Figure 3(d)). Our data, therefore, show that both NIC and Pal either alone or in combination significantly increase MCSC population, anti-inflammatory cytokines, and key browning marker TMEM26 in HCC70 breast cancer cells.

Treatment with nAchR Antagonist
Reduces NIC plus Pal-Mediated Aggressive Breast Tumor Properties. We next examined the expression of various isoforms of nicotinic acetylcholine receptors (nAchR) in HCC70 and HCC1806 breast cancer cells to determine their contribution in tumor growth in the presence of NIC and HFD. Quantitative gene expression analysis showed a significant increase in α-7 nAchR gene expression with 200 μM Pal (3:7 ± 0:3-fold; p ≤ 0:05), which was further increased in the NIC + Pal (5:2 ± 0:4-fold; p ≤ 0:01) treatment group compared to the control group ( Figure 5(a), left panel). In addition, α-9 nAchR and α-10 nAchRα gene expression was found to be significantly induced only in the NIC + Pal (5:8 ± 0:2-fold; p ≤ 0:01) and Pal (7:2 ± 0:2-fold; p ≤ 0:01) groups, respectively ( Figure 5(a), left panel). Quantitative gene expression analysis of HCC1806 cells following various treatments also 8 Mediators of Inflammation  . Also, α-9 nAchR and α-10 nAchRα gene expression was found to be significantly induced only in NIC + Pal (4:2 ± 0:2-fold; p ≤ 0:05) and Pal (2:8 ± 0:3-fold; p ≤ 0:05) groups, respectively ( Figure 5(a), right panel). Since HCC70 breast cancer cells lack functional ER, known to mediate oncogenic effects of NIC intracellularly [40], we examined expression levels of glucocorticoid receptors (GR) and androgen receptor (AR). We did not find any significant changes in any of the GR (GR1-3) or AR expression levels following NIC and Pal treatment alone or in combination (data not shown). Furthermore, immunoblot analysis showed no appreciable increase in the protein expression level of α-7 nAchR with the NIC + Pal treatment (~1.16-fold) group compared to the ethanol+ BSA-treated group ( Figure 5(b)). We further examined the effect of nAchR inhibition by MEC, on HCC70 cell proliferation/migration following treatment with NIC and Pal. We performed scratchwound assay with HCC70 cells grown to confluency followed by scratches and treatments with various combinations of NIC and or Pal with or without MEC (1 μM). Treatment with MEC significantly reduced NIC and Pal-induced cell proliferation/migration of HCC70 cells (Figure 5(c)). Although MEC reduced proliferation/migration, there was no change in levels of VEGF as shown by immunoblot analysis of breast cancer cells after various treatments (data not shown). On the other hand, simultaneous incubation of NIC + Pal-treated HCC70 cells with MEC resulted in decreased expression levels of vimentin (0.55-fold) and SOX2 (0.45-fold) expression compared to the NIC+ Pal-treated group ( Figure 5(d)).
These data indicate MEC treatment significantly attenuated NIC + Pal-mediated proliferation/migration of breast cancer cells as well as reduced levels of vimentin and SOX2 expression.

In Vivo Treatment with MEC and SB 203580 Inhibitors
Reduced Nicotine and HFD-Induced Xenograft Growth. Since xenografts exposed to HFD as well as NIC + HFD had upregulated pp38MAPK, we further examined whether inhibition of nAchRα and pp38 MAPK would influence xenograft development in vivo. Immunodeficient mice, fed HFD and exposed to IP injections of NIC (0.75 mg/kg/mice, twice a day) for two weeks, were implanted with HCC70 breast cancer cells (2×10 6 cell/mouse). We continued the treatment of nude mice to IP injection of MEC as well as subcutaneous injection of SB 203580 in addition to HFD and NIC. The xenografts were excised after growth for another 10 weeks, after which the tumor volume and weigh were measured (Figures 6(a)-6(c)). Compared to saline (455 ± 70 mm 3 ) at 10 weeks, the tumor volumes in NIC + HFD (899 ± 50 mm 3 ) was significantly higher (p ≤ 0:05). Simultaneous treatment of the NIC + HFD group with SB (634 ± 29 mm 3 ) and MEC (254 ± 18 mm 3 ) alone or in combination (169 ± 16 mm 3 ) resulted in significantly less tumor volume compared to the NIC plus HFD group (p ≤ 0:01) (Figure 6(b)). Tumor weight in NIC + HFD (1:05 ± 0:15 g) (p ≤ 0:01) was significantly higher compared to that in the control saline (0:5 ± 0:05 g) group (Figure 6(c)). Furthermore, cotreatment of NIC + HFD groups with either SB 203580 (0:8 ± 0:05 g; p ≤ 0:05) or MEC (0:28 ± 0:04 g; p ≤ 0:01) alone or in combination (0:19 ± 0:01 g; p ≤ 0:01) significantly reduced the tumor weight ( Figure 6(c)). We subjected pooled tumor samples from various treatment groups to immunoblot analysis for key genes known to contribute to xenograft growth in presence of NIC + HFD. We found appreciable changes in ALDH1 (that NIC + HFD increased ALDH1 (1.65-fold) as well as SOX2 (2.1-fold) protein    Figure 6: Effect of MEC and pp38MAPK inhibitor SB203580 on tumor growth in NIC and HFD-treated nude mice: HCC70 breast cancer cells (2×10 6 cell/mice) were implanted in nude mice prefed on HFD (2 weeks) and exposed to NIC (intraperitoneal injection twice/day for 2 weeks, 0.75 mg/kg/mice/injection). Xenografts were grown in mice continued on either HFD ± NIC treatment (0.75 mg/kg/mice/injection twice a day) along with treatment with MEC (0.8 mg/kg/mice/injection, twice a day) with or without SB203580 (0.2 μmols in 100 μl/mice once/day/every day) till the tumors were excised.

Discussion
The combination of biological and social factors contributes to health disparity and high mortality rate among AA women with breast cancer. These factors include among others poor diet, obesity, and higher exposure to risk factors including smoking [1]. There is higher frequency of AA women with TNBC that is usually of a higher stage with lymph node metastases [20]. TNBC is an aggressive subtype of breast cancer that disproportionately occurs at a high rate among younger AA women and is more likely to metastasize to the brain as compared to non-NTBC types. TNBC also has a very high rate of relapse and a poor prognosis in patients that do not achieve a complete response with neoadjuvant chemotherapy [41]. Several studies have investigated the associations between obesity/and breast cancer risk [21,31] as well as cigarette smoking/and breast cancer risk [11,15,16]. However, the combined effect of the two common lifestyle risk factors, such as NIC and HFD, especially in TNBC, remains understudied. This report uses novel in vivo as well as ex vivo models to study cross-talk between the breast cancer cells and the microenvironment in the presence of nicotine and HFD. We found coexposures to both nicotine and HFD contributed to higher xenograft growth from AA TNBC cells when compared to saline, nicotine, or HFD alone. This study also elucidates some of the mechanisms that could have contributed to higher xenograft growth in the presence of NIC and HFD. In our study, xenografts from HFD as well as NIC + HFD showed higher expression of CD68, a pan-macrophage marker, when compared to only saline or NIC. We also found higher levels of proinflammatory or Th1 cytokines, IL12 and IL6, that favor classically activated or M1macrophages in xenografts grown in HFD, when compared to the other groups. Upregulation of these markers suggested that HFD produced adipose tissue characterized dysfunctional synthesis of several adipokines and immune cell infiltration that creates a state of sustained low-grade inflammation favoring tumor growth [42]. Although M1 macrophages can increase tumor growth, high density of another subset of macrophages called tumor-associated or M2 type is associated with poor prognosis in TNBC, particularly those from AA women [43]. An anti-inflammatory microenvironment with high levels of Th2 cytokines such as IL13, IL4, and IL10 favors macrophages with M2 phenotype. Nicotine increases the anti-inflammatory microenvironment in various conditions, and our study is first to show that xenografts exposed to NIC as well as NIC + HFD expressed high levels of the anti-inflammatory cytokines, IL13 and IL10, in addition to high levels of tissue-resident or M2 macrophages. Our in vitro experiments demonstrated that AA TNBC cells treated with NIC and NIC + Pal expressed higher protein levels of IL13 and IL10, as well as the tissue-resident macrophage marker CD163. IHC staining of paraffin embedded xenografts from various treatment groups also showed intense clusters of IL13-expressing cells only in those tumors exposed to NIC and NIC + Pal. Interestingly, our qPCR data with validated species-specific primers shows that most of the IL6 was of host origin, while IL13 was expressed by tumor cells.
Interestingly, we also found much higher protein levels of MCSF only in xenografts developed in the presence of both NIC and HFD. MCSF, in addition to stimulating the production of several cytokines, is a crucial factor in the development and maturation of tissue-resident macrophages, which are protumorigenic [43]. Overexpression of MCSF promotes angiogenesis and other aggressive behaviors in tumors where it is also associated with poor prognosis [44]. There are reports of MCSF-promoting metastases in breast cancer where the initial growth of tumors is not affected in its absence [45]. Our scratch-wound assay data supports this assumption since we found increased proliferation/migration or faster filling up the wound in HCC70 cells treated with NIC + Pal when compared to the other controls.
Browning or beige adipocytes was also increased in NIC as well NIC + Pal-treated cells as well in ex vivo experiments where cancer cells were coincubated with SAT in the presence of NIC. There are reports that NIC promoted beige cells in smokers where they burn energy and help lose weight [39]. We have reported that beige adipocytes are upregulated in xenografts and patient-derived xenografts (PDX) from AA TNBC cell lines as well as patient tumors, respectively, where they contributed to tumor growth [30]. One of the mechanisms by which beige cells could increase tumor growth is by increasing reactive oxygen species (ROS), which is high in AA TN breast tumors where it promotes its growth. Beige cells secrete cytokines like NRG4 and BMP8b, which contributes to adrenergic-induced remodeling of neuro-vascular network in adipose tissue [45]. These secretions by beige/brown adipocytes promoted sympathetic axon growth as well as proangiogenic transcriptional and secretory profile that increased vascular sprouting [45].
Additionally, we present significant evidence that nicotine and HFD synergistically increase MCSCs, which are heterogeneous populations that have the potential to promote initiation and progression of breast tumors [11,13,21]. Others and we have reported that AATN breast cancer cells have higher ALDH1 expressing MCSCs when compared to those of other ethnic groups [35]. ALDH1 MCSCs contribute to initiation and engraftment of breast tumors as well as facilitate its interaction with the environment. In addition to ALDH1-expressing MCSCs, embryonic stem cells (ESCs) are a distinct subset of MCSCs that express embryonic transcription factors like SOX2/OCT4 and are upregulated in aggressive breast tumors [46]. In this study, we found that most ALDH1 MCSCs were of tumor cell origin with a relatively small contribution from the host. Higher protein expression of ALDH1 in xenografts exposed to NIC, HFD or coexposed to both, suggests an increase in ALDH1-MCSC population could be one of the mechanisms that influence larger growth of xenografts. We have further confirmed by FACS that treatment of breast cancer cells, in vitro, with NIC + Pal significantly upregulated cells that expressed ALDH1. Our in vitro studies where there was an increase in the number and size of mammospheres upon treatment of AA TNBC cells with NIC + Pal further suggested an increase in the MCSC population. Mammospheres propagated in low attachment plates under defined conditions enrich for MCSCs [35]. We found another subset of MCSCs, which are more embryonic become upregulated in xenografts developed in the presence of NIC as well as NIC + Pal. An increase in ESCs could be an additional mechanism that promoted larger tumor growth upon coexposure to NIC and HFD.
Stress kinase p38MAPK promotes aggressive breast tumor behaviors such as metastasis and angiogenesis [47]. Increased expression of key proteins in metastasis including urokinase plasminogen activator (UPA) and matrix metalloprotease-9 (MMP9) requires constitutive p38 alpha MAPK activity [48]. Aggressive and invasive breast tumors overexpress p38MAPK, whose increased activity has been associated with poor prognosis [49]. Treatment with SB 203580, which inhibits p38 MAPK activity, reduced proliferation and migration of highly invasive breast cancer cells, MDA-MB-231 [50]. In addition, inhibition of VEGF-C secretion in MDA-MB-231 cells by p38MAPK inhibitor, SB 203580, indicates a requirement of this kinase in angiogenesis [50]. In our study, treatment with SB 203580 alone did not have any effect on primary tumor growth in the presence of NIC plus HFD. Our data supports the findings from another study, where treatment with SB 203580 reduced metastatic potential of breast cancer cells but had no effect on its primary growth [51]. We found treatment with MEC, a nonselective nAchR antagonist, was effective in reducing the primary growth as well as decreasing the expression of vimentin and uPA in tumors developed in the presence of NIC plus HFD. However, cotreatment with both MEC plus SB 203580 was more effective in further reducing tumor volumes as well as attenuating protein expression of both SOX2 and vimentin, suggesting a casual role of both nicotine and p38 MAPK in NIC plus HFD-induced breast tumor progression. Combination therapy with both MEC plus SB 203580 may be effective in long-term suppression of NIC + HFD-exposed breast tumors as well as attenuating its metastatic potential. Understanding the signaling mechanisms that NIC + HFD upregulates in AATN breast cancer can facilitate identification of targets that may enable the development of pharmaceutical countermeasure to combat breast cancer initiation, progression, and metastasis in African American women.
Breast cancer is very heterogeneous where many key players interact and cross-talk to influence tumor growth. This study demonstrates that HFD induces inflammatory M1 macrophage-rich tumor-supporting microenvironment, which is a fertile ground for NIC to act upon to further increase additional protumorigenic factors like TAM and anti-inflammatory cytokines. While HFD and NIC individually increase various tumor-promoting factors that led to some tumor growth, their simultaneous presence and coordinated action appear to accelerate tumor growth. In addition, together, NIC and HFD upregulated important players among which was MCSF, which is a master regulator for macrophage maturation. Thus, it is apparent that the action of NIC and HFD in tumor microenvironment could be additive by bringing in various key elements together as well as synergistic by inducing novel players that contribute to tumor growth. Since both NIC and HFD had both independent and interdependent actions in a tumor microenvironment, to reduce tumor growth, we used specific inhibitors to target key mediators of NIC and HFD signaling, which was effective in reducing tumor growth.
The clinical/public health implications of our study are that a large number of women are exposed to both smoking and NRT (for the most part, available without a prescription), often for several years. While smoking leads to many cancers, including breast cancer, nicotine influences several cancers, including breast cancer. The mutagenic and tumor-promoting activities of nicotine may result from its ability to damage the genome, disrupt cellular metabolic processes, and facilitate growth and spreading of transformed cells. Our animal studies are able to understand the effects of nicotine per se on breast cancer proliferation. If the findings of our studies could be extrapolated to humans, the use of NRT may need to be reassessed.

Conclusion
This study demonstrates that the combination of nAchR antagonists with stress kinase inhibitors could significantly block xenograft growth in the presence of HFD plus nicotine, suggesting this combination therapy could potentially be used to treat obese breast cancer patients who smoke or use nicotine replacement therapy.