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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., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Thalia Jimenez et al.)

Unlabelled: The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment. Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1, and OPLAH) as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers was also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1(+) or Myf5(+) cells significantly reduced tumor development. There was increased COX2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX2 inhibitor (SC236) reduced tumor growth. In contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient tumor tissues were generated that expressed key brown adipose tissue-related markers and contained cells that morphologically resembled brown adipocytes., Implications: This is the first report demonstrating that beige/brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design., (©2015 American Association for Cancer Research.)

Background: Breast and cervical cancers are the most common causes of cancer mortality among women in India, but actually they are largely preventable diseases. Although early detection is the only way to reduce morbidity and mortality, there are limited data on breast and cervical cancer knowledge, safe practices and attitudes of teachers in India. The purpose of this study is to assess the level of awareness and impact of awareness programs in adoption of safe practices in prevention and early detection., Materials and Methods: This assessment was part of a pink chain campaign on cancer awareness. During cancer awareness events in 2011 at various women colleges in different parts in India, a pre-test related to cervical cancer and breast cancer was followed by an awareness program. Post-tests using the same questionnaire were conducted at the end of the interactive session, at 6 months and 1 year., Results: A total of 156 out of 182 teachers participated in the study (overall response rate was 85.7 %). Mean age of the study population was 42.4 years (range- 28-59 yrs). There was a significant increase in level of knowledge regarding cervical and breast cancer at 6 months and this was sustained at 1 year. Adoption of breast self examination (BSE) was significantly more frequent in comparison to CBE, mammography and the Pap test. Magazines and newspapers were sources for knowledge regarding screening tests for breast cancer in more than 60% of teachers where as more than 75% were educated by doctors regarding the Pap test. Post awareness at 6 months and 1 year, there was a significant change in alcohol and smoking habits. Major reasons for not doing screening test were found to be ignorance (50%), lethargic attitude (44.8%) and lack of time (34.6%)., Conclusions: Level of knowledge of breast cancer risk factors, symptoms and screening methods was high as compared to cervical cancer. There was a significant increase in level of knowledge regarding cervical and breast cancer at 6 months and this was sustained at 1 year. Adoption of BSE was significantly greater in comparison to CBE, mammography and the Pap test. To inculcate safe practices in lifestyle of people, awareness programmes such as pink chain campaign should be conducted more widely and frequently.

We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N(ω) hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA's action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.

Vitamin D signaling in mammary cancer stem cells (MCSCs), which are implicated in the initiation and progression of breast cancer, is poorly understood. In this study, we examined vitamin D signaling in mammospheres which are enriched in MCSCs from established breast cancer cell lines. Breast cancer cells positive for aldehyde dehydrogenase (ALDH(+)) had increased ability to form mammospheres compared to ALDH(-) cells. These mammospheres expressed MCSC-specific markers and generated transplantable xenografts in nude mice. Vitamin D receptor (VDR) was significantly down-regulated in mammospheres, as well as in ALDH(+) breast cancer cells. TN aggressive human breast tumors as well as transplantable xenografts obtained from SKBR3 expressed significantly lower levels of VDR but higher levels of CD44 expression. Snail was up-regulated in mammospheres isolated from breast cancer cells. Inhibition of VDR expression by siRNA led to a significant change in key EMT-specific transcription factors and increased the ability of these cells to form mammospheres. On the other hand, over-expression of VDR led to a down-regulation of Snail but increased expression of E-cad and significantly compromised the ability of cells to form mammospheres. Mammospheres were relatively insensitive to treatment with 1,25-dihydroxyvitamin D (1,25D), the active form of vitamin D, compared to more differentiated cancer cells grown in presence of serum. Treatment of H-Ras transformed HMLE(HRas) cells with DETA NONOate, a nitric oxide (NO)-donor led to induction of MAP-kinase phosphatase -1 (MKP-1) and dephosphorylation of ERK1/2 in the mammospheres. Combined treatment of these cells with 1,25D and a low-concentration of DETA NONOate led to a significant decrease in the overall size of mammospheres and reduced tumor volume in nude mice. Our findings therefore, suggest that combination therapy using 1,25D with drugs specifically targeting key survival pathways in MCSCs warrant testing in prospective clinical trial for treatment of aggressive breast cancer.

Nitric oxide is a pleiotropic ancestral molecule, which elicits beneficial effect in many physiological settings but is also tenaciously expressed in numerous pathological conditions, particularly breast tumors. Nitric oxide is particularly harmful in adipogenic milieu of the breast, where it initiates and promotes tumorigenesis. Epidemiological studies have associated populations at a greater risk for developing breast cancer, predominantly estrogen receptor positive tumors, to express specific polymorphic forms of endothelial nitric oxide synthase, that produce sustained low levels of nitric oxide. Low sustained nitric oxide generates oxidative stress and inflammatory conditions at susceptible sites in the heterogeneous microenvironment of the breast, where it promotes cancer related events in specific cell types. Inflammatory conditions also stimulate inducible nitric oxide synthase expression, which dependent on the microenvironment, could promote or inhibit mammary tumors. In this review we re-examine the mechanisms by which nitric oxide promotes initiation and progression of breast cancer and address some of the controversies in the field.

Nitric oxide has varied effects on human breast cancer cells. At low concentration (micromolar range) it increases proliferation by increasing synthesis of some cells cycle protein and in higher concentration (nanomolar range) it leads to cytostasis or apoptosis by decreasing the translation of some cell cycle proteins.

Protein synthesis is a tightly controlled process, and its deregulation plays an important role in tumorigenesis. Protein synthesis remains poorly understood with very few well-identified validated targets for therapeutic purposes. In this study, we use nitric oxide (NO), which suppresses protein synthesis by inactivating eukaryotic initiation factor 2-alpha (eIF2-alpha), to examine the mechanism by which low and high oxidative stress inhibits protein synthesis. In breast cancer cells, low NO stress induced heme-regulated inhibitor (HRI) activation, which facilitated gradual decline in short half-life proteins. High NO stress induced HRI and protein kinase R (PKR) activation, leading to a sharp decline in protein synthesis as accessed by a decline in short and long half-life proteins and dramatic morphologic changes. In contrast, human mammary epithelial (HME) and Ras transfected untransformed HME (MCF-10A1 neo N) cells were less susceptible to NO-induced inhibition of protein synthesis and cytostasis. Our results suggest that NO-induced cytostasis in breast cancer cells was due to PKR activation and increased phosphorylation of eIF2-alpha, whereas the reduced susceptibility of normal mammary epithelial cells to NO could be due to the inaccessibility of PKR, which is bound to inhibitor p58.

Nitric oxide (NO) in nanomolar (nmol/L) concentrations is consistently detected in tumor microenvironment and has been found to promote tumorigenesis. The mechanism by which NO enhances tumor progression is largely unknown. In this study, we investigated the possible mechanisms and identified cellular targets by which NO increases proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7. DETA-NONOate, a long acting NO donor, with a half-life of 20 h, was used. We found that NO (nmol/L) dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 and also increased cell proliferation. NO specifically increased the translation of cyclin D1 and ornithine decarboxylase (ODC) without altering their mRNA levels or half-lives. Critical components in the translational machinery, such as phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were up-regulated following NO treatment, and inhibition of mTOR with rapamycin attenuated NO induced increase of cyclin D1 and ODC. Activation of translational machinery was mediated by NO-induced up-regulation of the Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK (Raf/MEK/ERK) and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt signaling pathways. Up-regulation of the Raf/MEK/ERK and PI-3 kinase/Akt pathways by NO was found to be mediated by activation of Ras, which was cyclic guanosine 3',5'-monophosphate independent. Furthermore, inactivation of Ras by farnesyl transferase inhibitor or K-Ras small interfering RNA attenuated NO-induced increase in proliferation signaling and cyclin D1 and ODC translation, further confirming the involvement of Ras activation during NO-induced cell proliferation.

Apoptosis is regulated by a series of biochemical events that commits a cell to death. We are interested in understanding and have been investigating the mechanisms by which nitric oxide (NO) induces apoptosis in human breast cancer cell lines. In this study, we investigated the possible interplay of extracellular signal-regulated kinase (ERK) and Akt pathways in NO-induced apoptosis. MKP-1 transcripts were induced in these cells as early as 4 h, peaking at 8 h leading to inactivation of ERK1/2 at 16-24 h after exposure to NO. We also found 50% decrease in the levels pAkt at 24 h of DETA-NONOate treatment. The inactivation of ERK1/2 preceded the dephosphorylation of Akt and apoptosis. NO was not able to inactivate ERK1/2 or Akt or to induce apoptosis in the presence of a phosphatase inhibitor, sodium orthovanadate, or antisense oligonucleotides, suggesting a cross-talk between the two pathways. NO also up-regulated MKP-1 in another breast cancer cell line, ZR 75-30, which led to inactivation of ERK1/2 and induced apoptosis. In MDA-MB-231, NO did not induce MKP-1, and there was no ERK inactivation or apoptosis. Our results indicate that expression of MKP-1 by NO leading to dephosphorylation of ERK1/2 is the initial essential event that commits the cells to the apoptotic pathway in breast cancer cells.

We have previously reported that nitric oxide (NO) induces apoptosis in MDA-MB-468 cells through its action on the mitochondria and the release of cytochrome c. In this study, we investigated the critical events that must occur after which these cells are committed to apoptosis. We used the long-acting NO donor DETA-NONOate, which, at a concentration of 1 mM, releases NO in the range produced by activated macrophages. Depolarization of mitochondrial membrane potential (MMP) occurred at 4 h of DETA-NONOate treatment, which returned to control values and which was followed by another wave of depolarization at 24 h. There was a 2-fold increase of cytochrome c in the cytosol at 6 h, but it was not until 36 h that the level of cytochrome c was increased by 15-fold. Although the initial release of cytochrome c from the mitochondria could be inhibited by cyclosporin A or by bongkrekic acid, the later release continued even in its presence. We observed that the later release of cytochrome c at 36 h was independent of MMP depolarization but was dependent on Bax integration into the mitochondrial membrane, which committed the cells to apoptosis. We also observed a decline in the levels of cytosolic phospho-Akt at 16-24 h of DETA-NONOate treatment. We also conclude that decrease in phospho-Akt is an essential event upstream from Bax integration in MDA-MB-468 cells.

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.