LT-IIc, A Bacterial Type II Heat-Labile Enterotoxin, Induces Specific Lethality in Triple Negative Breast Cancer Cells by Modulation of Autophagy and Induction of Apoptosis and Necroptosis.

Triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. To discover novel approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for cytotoxicity in TNBC cells. Only LT-IIc significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effect on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER⁺ cells, or SKBR-3 cells, HER2⁺ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as seen by accumulation of LC3B-II and p62. Morphologically, LT-IIc induced the formation of enlarged LAMP2+ autolysosomes, which was blocked by co-treatment with bafilomycin A1. LT-IIc induced apoptosis as demonstrated by the increase in caspase 3/7 activity and Annexin V staining. Co-treatment with necrostatin-1, however, demonstrated that the lethal response of LT-IIc is elicited, in part, by concomitant induction of necroptosis. Knockdown of ATG-5 failed to rescue LT-IIc-induced cytotoxicity, suggesting LT-IIc can exert its cytotoxic effects downstream or independently of autophagophore initiation. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy, while simultaneously blocking autolysosomal progression in TNBC cells, inducing a specific cytotoxicity in this breast cancer subtype.

Enhancement of humoral immunity by the type II heat-labile enterotoxin LT-IIb is dependent upon IL-6 and neutrophils.

LT-IIb, a type II heat-labile enterotoxin produced by Escherichia coli, is a potent intradermal adjuvant that enhances immune responses to coadministered antigens. Although the immune mechanisms that promote this augmented immune response have not been well defined, prior intradermal immunization experiments suggested that early cellular and immunomodulatory events at the site of immunization modulated the augmentation of antigen-specific immune responses by LT-IIb. To investigate that hypothesis, mice were intradermally immunized with a recombinant ricin vaccine, a prospective toxin subunit antigen, in the presence and absence of LT-IIb. Analysis of tissue-fluid collection, coupled with histologic sections from the site of intradermal immunization, revealed that a single dose of LT-IIb induced local production of interleukin 6 and promoted a regional infiltration of neutrophils. The adjuvant effects of LT-IIb were abrogated in interleukin 6-deficient mice and when mice were depleted of neutrophils by pretreatment with anti-Ly6G. Overall, these data firmly demonstrated that LT-IIb, when used as an intradermal adjuvant, recruits neutrophils and is a potent rapid inducer of interleukin 6.

Breast cancer risk factor associations differ for pure versus invasive carcinoma with an in situ component in case-control and case-case analyses.

PURPOSE: Invasive ductal carcinoma (IDC) is diagnosed with or without a ductal carcinoma in situ (DCIS) component. Previous analyses have found significant differences in tumor characteristics between pure IDC lacking DCIS and mixed IDC with DCIS. We will test our hypothesis that pure IDC represents a form of breast cancer with etiology and risk factors distinct from mixed IDC/DCIS. METHODS: We compared reproductive risk factors for breast cancer risk, as well as family and smoking history between 831 women with mixed IDC/DCIS (n = 650) or pure IDC (n = 181), and 1,620 controls, in the context of the Women's Circle of Health Study (WCHS), a case-control study of breast cancer in African-American and European-American women. Data on reproductive and lifestyle factors were collected during interviews, and tumor characteristics were abstracted from pathology reports. Case-control and case-case analyses were conducted using unconditional logistic regression. RESULTS: Most risk factors were similarly associated with pure IDC and mixed IDC/DCIS. However, among postmenopausal women, risk of pure IDC was lower in women with body mass index (BMI) 25 to <30 [odds ratio (OR) 0.66; 95 % confidence interval (CI) 0.35-1.23] and BMI ≥ 30 (OR 0.33; 95 % CI 0.18-0.67) compared to women with BMI < 25, with no associations with mixed IDC/DCIS. In case-case analyses, women who breastfed up to 12 months (OR 0.55; 95 % CI 0.32-0.94) or longer (OR 0.47; 95 % CI 0.26-0.87) showed decreased odds of pure IDC than mixed IDC/DCIS compared to those who did not breastfeed. CONCLUSIONS: Associations with some breast cancer risk factors differed between mixed IDC/DCIS and pure IDC, potentially suggesting differential developmental pathways. These findings, if confirmed in a larger study, will provide a better understanding of the developmental patterns of breast cancer and the influence of modifiable risk factors, which in turn could lead to better preventive measures for pure IDC, which have worse disease prognosis compared to mixed IDC/DCIS.

Mouse mammary gland is refractory to the effects of ethanol after natural lactation.

Ethanol is a dietary factor that dose-dependently increases breast cancer risk in women. We previously have shown that ethanol increases mammary epithelial density through increased branching after dietary exposure during puberty in CD2/F1 mice. To extend these studies to parous mice in a breast cancer model, we used a transgenic mouse model of human parity-associated breast cancer, the FVB-MMTV-Her2/Neu mouse, which overexpresses wildtype EGFR2, resulting in constitutive activation of growth signaling in the mammary epithelium. Here we describe the short-term effects of ethanol feeding on progression through involution. Mice were fed diets supplemented with 0%, 0.5%, 1%, or 2% ethanol for 4, 9, or 14 d starting on day 21 of lactation (that is, at the start of natural postlactational involution). Unlike peripubertal mice exposed to ethanol, postlactational dams showed no changes in body weight; liver, spleen, and kidney weights; and pathology. Ethanol exposure had no effect on mammary gland lobular density and adipocyte size throughout involution. Likewise, the infiltration of inflammatory cells and serum oxidized lipid species were unchanged by diet, suggesting that ethanol feeding had no effect on local inflammation (leukocyte infiltration) or systemic inflammation (oxidized lipids). In conclusion, ethanol exposure of parous dams had no effect on mammary gland structure or the regression of the lactating mammary gland to a resting state. The period of involution that follows natural lactation appears to be refractory to developmental effects of ethanol on mammary epithelium.

Chemopreventive and anti-angiogenic effects of dietary phenethyl isothiocyanate in an N-methyl nitrosourea-induced breast cancer animal model.

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen-dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N-methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor-free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC-treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC-treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC-treated animals. These studies indicate that supplementation with PEITC prolongs the tumor-free survival, reduces tumor incidence and burden, and is chemoprotective in NMU-induced estrogen-dependent breast cancer in rats. For the first time, it is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity.

Loss of IL-10 decreases mouse postpubertal mammary gland development in the absence of inflammation.

IL-10 is a pleiotrophic anti-inflammatory cytokine. Decreased IL-10 expression is associated with an increased breast cancer risk but the mechanism is not clear. This study was designed to test the hypothesis that the loss of IL-10 alters mammary development, even in the absence of inflammation. Wild-type and IL-10-/- mouse littermates were similar in growth, development, and breeding success. Using whole-mounts and paraffin sections, mammary glands from pre-pubertal mice (d21) were found to not be affected by the IL-10 null genotype. However, after the onset of estrous cycling, ductal structure, but not lymph nodes or adipocytes, of IL-10 knockout mice were found to moderately decrease at day 55, 80, and 150 of age. This phenotype was not rescued by lactogenesis. At day 2 of lactation, IL-10 null mice had reduced lobular complexity and glandular area with the retention of adipocytes. These results support the hypothesis that absence of IL-10 reduces glandular development during postnatal development, at maturity, and during the early stages of lactation. Although our study cannot distinguish between a direct IL-10 effect on the epithelial cells and an indirect systemic effect, epithelial cell responses to IL-10 should be considered in the therapeutic applications of cytokines or cytokine ablation.

Folate exacerbates the effects of ethanol on peripubertal mouse mammary gland development.

Alcohol consumption is linked with increased breast cancer risk in women, even at low levels of ingestion. The proposed mechanisms whereby ethanol exerts its effects include decreased folate levels resulting in diminished DNA synthesis and repair, and/or acetaldehyde-generated DNA damage. Based on these proposed mechanisms, we hypothesized that ethanol would have increased deleterious effects during periods of rapid mammary gland epithelial proliferation, such as peripuberty, and that folate deficiency alone might mimic and/or exacerbate the effects of ethanol. To test this hypothesis, weight-matched 28-35 day old CD2F1 female mice were pair-fed liquid diets ±3.2% ethanol, ±0.1% folate for 4 weeks. Folate status was confirmed by assay of liver and kidney tissues. In folate deficient mice, no significant ethanol-induced changes to the mammary gland were observed. Folate replete mice fed ethanol had an increased number of ducts per section, due to an increased number of terminal short branches. Serum estrogen levels were increased by ethanol, but only in folate replete mice. These results demonstrate that folate deficiency alone does not mimic the effects of ethanol, and that folate deficiency in the presence of ethanol blocks proliferative effects of ethanol on the mammary ductal tree.

Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling.

BACKGROUND: The mammary epithelium undergoes proliferation and regression accompanied by remodeling of the fibrocellular and vascular stroma. Mast cells are abundant in these compartments and have been implicated in remodeling during wound healing and cancer progression. The purpose of this study was to test the hypothesis that mast cell abundance correlates with physiologic mammary tissue remodeling during estrous cycling, lactogenesis (pregnancy and lactation) and involution. RESULTS: Mast cell and capillary frequency were quantified in the stroma surrounding ducts and lobules from mammary glands of rats. During estrous cycling, periductal mast cell numbers were unchanged, but lobule-associated mast cells significantly increased in the regressive phase of diestrus II. During lactogenesis, lobular stroma mast cells peaked early in pregnancy, at D2, followed by a significant decrease throughout lactation. Involution was associated with a rapid return in mast cell numbers, similar to diestrus II. Lobular vascularization peaked during the state of metestrus, when limited secretory differentiation occurs. Lobular angiogenesis peaked at D7 of pregnancy, regressed, and then returned to high levels during lactation and early involution, when secretory differentiation is high. CONCLUSIONS: These results suggest mast cells are predominantly associated with regressive lobular remodeling during cycling and involution, whereas angiogenesis is predominantly associated with secretory differentiation.

Direct effects of conjugated linoleic acid isomers on P815 mast cells in vitro.

Conjugated linoleic acid (CLA) is a dietary fatty acid which causes extensive remodeling and mast cell recruitment in the mouse mammary gland. Two CLA isomers, 9,11- and 10,12-CLA, have differing effects in vivo, with only 10,12-CLA increasing mast cell number. The purpose of this project is to test the hypothesis that CLA acts directly on the mast cell. The P815 mastocytoma cell line was assayed for the effects of CLA on mast cell number, proliferation, apoptosis, and differentiation. Both CLA isomers decreased viable mast cell number, with no effect on membrane integrity, or cell cycle distribution. 10,12-CLA induced an increase in apoptosis, assessed by Annexin-FITC binding. Both isomers increased mast cell granularity, and secretion of MMP-9. The complex effects of CLA isomers on mast cells in the mammary gland are distinct from direct effects on mast cells in vitro, and may require interactions between multiple cell types present in vivo.

TLR2-dependent modulation of dendritic cells by LT-IIa-B5, a novel mucosal adjuvant derived from a type II heat-labile enterotoxin.

A host of human pathogens invades the body at mucosal surfaces. Yet, strong, protective mucosal immune responses directed against those pathogens routinely cannot be induced without the use of adjuvants. Although the strongest mucosal adjuvants are members of the family of HLTs, the inherent toxicities of HLT holotoxins preclude their clinical use. Herein, it is shown that LT-IIa-B(5) enhances mucosal immune responses by modulating activities of DCs. i.n. immunization of mice with OVA in the presence of LT-IIa-B(5) recruited DCs to the NALT and significantly increased uptake of OVA by those DCs. Furthermore, LT-IIa-B(5) increased expression of CCR7 by DCs, which mediated enhanced migration of the cells from the NALT to the draining CLNs. LT-IIa-B(5) also enhanced maturation of DCs, as revealed by increased surface expression of CD40, CD80, and CD86. Ag-specific CD4(+) T cell proliferation was augmented in the CLNs of mice that had received i.n. LT-IIa-B(5). Finally, when used as an i.n. adjuvant, LT-IIa-B(5) dramatically increased the levels of OVA-specific salivary IgA and OVA-specific serum IgG. Strikingly, each of the activities induced by LT-IIa-B(5) was strictly TLR2-dependent. The data strongly suggest that the immunomodulatory properties of LT-IIa-B(5) depend on the productive modulation of mucosal DCs. Notably, this is the first report for any HLT to demonstrate in vivo the elicitation of strong, TLR2-dependent modulatory effects on DCs with respect to adjuvanticity.

Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.

The commensal fungus Candida albicans causes oropharyngeal candidiasis (OPC; thrush) in settings of immunodeficiency. Although disseminated, vaginal, and oral candidiasis are all caused by C. albicans species, host defense against C. albicans varies by anatomical location. T helper 1 (Th1) cells have long been implicated in defense against candidiasis, whereas the role of Th17 cells remains controversial. IL-17 mediates inflammatory pathology in a gastric model of mucosal candidiasis, but is host protective in disseminated disease. Here, we directly compared Th1 and Th17 function in a model of OPC. Th17-deficient (IL-23p19(-/-)) and IL-17R-deficient (IL-17RA(-/-)) mice experienced severe OPC, whereas Th1-deficient (IL-12p35(-/-)) mice showed low fungal burdens and no overt disease. Neutrophil recruitment was impaired in IL-23p19(-/-) and IL-17RA(-/-), but not IL-12(-/-), mice, and TCR-alphabeta cells were more important than TCR-gammadelta cells. Surprisingly, mice deficient in the Th17 cytokine IL-22 were only mildly susceptible to OPC, indicating that IL-17 rather than IL-22 is vital in defense against oral candidiasis. Gene profiling of oral mucosal tissue showed strong induction of Th17 signature genes, including CXC chemokines and beta defensin-3. Saliva from Th17-deficient, but not Th1-deficient, mice exhibited reduced candidacidal activity. Thus, the Th17 lineage, acting largely through IL-17, confers the dominant response to oral candidiasis through neutrophils and antimicrobial factors.

Conjugated linoleic acid induces mast cell recruitment during mouse mammary gland stromal remodeling.

Conjugated linoleic acid (CLA) is a dietary chemopreventive agent that induces apoptosis in the mammary adipose vascular endothelium and decreases mammary brown adipose tissue (BAT) and white adipose tissue (WAT). To determine onset and extent of stromal remodeling, we fed CD2F1/Cr mice diets supplemented with 1 or 2 g/100 g mixed CLA isomers for 1-7 wk. BAT loss, collagen deposition, and leukocyte recruitment occurred in the mouse mammary fat pad, coincident with an increase in parenchymal-associated mast cells in mice fed both levels of CLA. Feeding experiments with purified isomers (0.5 g/100 g diet) demonstrated that these changes were induced by trans-10, cis-12 CLA (10,12-CLA), but not by cis-9, trans-11 CLA (9,11-CLA). This stromal remodeling did not require tumor necrosis factor (TNF)-alpha, a major cytokine in mast cells, as TNF-alpha null mice demonstrated collagen deposition, increased leukocytes, and BAT loss in the mammary fat pad in response to 10,12-CLA. To test the hypothesis that mast cells recruited in response to 10,12-CLA were required for stromal remodeling, Steel mice (WBB6F1/J-kit(W)/kit(W-V)), which lack functional mast cells, were examined for their stromal response to 10,12-CLA. Both wild-type and Steel mice showed a significantly increased leukocytic adipose infiltrate, collagen deposition, and decreased adipocyte size, although BAT was maintained in Steel mice. These results demonstrate that 10,12-CLA induces an inflammatory and fibrotic phenotype in the mouse mammary gland stroma that is independent of TNF-alpha or mast cells and suggest caution in the use of 10,12-CLA for breast cancer chemoprevention.

The t10,c12 isomer of conjugated linoleic acid stimulates mammary tumorigenesis in transgenic mice over-expressing erbB2 in the mammary epithelium.

Conjugated linoleic acid (CLA), a family of isomers of octadecadienoic acid, inhibits rat mammary carcinogenesis, angiogenesis, and lung metastasis from a transplantable mammary tumor. c9,t11-CLA, the predominant isomer in dairy products, and t10,c12-CLA, a component of CLA supplements, are equally effective. The objective of the current studies was to test the efficacy of these two CLA isomers in a clinically relevant breast cancer model. Transgenic mice over-expressing erbB2 in the mammary epithelium were fed control or 0.5% CLA-supplemented diets continuously from weaning. Unexpectedly, t10,c12-CLA stimulated lobular hyperplasia of the mammary epithelium and accelerated mammary tumor development, decreasing median tumor latency to 168 days of age compared with 256 and 270 days in the c9,t11-CLA and control groups, respectively. Metastasis was also increased by t10,c12-CLA, with percentage of tumor-bearing mice with lung metastasis 73, 14 and 31% in the t10,c12-CLA, c9,t11-CLA and control groups, respectively. A second study, in which CLA administration was initiated after puberty, confirmed the stimulatory effect of t10,c12-CLA on mammary tumor development and metastasis. Additionally, t10,c12-CLA, but not c9,t11-CLA, increased the size of the liver, heart, spleen and mammary lymph node. The effects of t10,c12-CLA were not specific to erbB2 transgenic mice, as t10,c12-CLA supplementation increased proliferation in the mammary epithelium of both wild-type FVB and FVB/erbB2 mice. Moreover, the number of terminal end buds, the mammary epithelial structures most sensitive to a carcinogenic insult, was increased 30-fold in FVB wild-type mice fed t10,c12-CLA. These data suggest that it would be prudent to avoid CLA supplements containing the t10,c12-CLA isomer. However, even though c9,t11-CLA was not efficacious in the erbB2 model, its ability to inhibit mammary tumor development in rat models suggests that it may have activity for prevention of some types of breast cancer.

Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adipose vasculature.

Dietary conjugated linoleic acid (CLA) is a cancer chemopreventive agent that has been shown to inhibit angiogenesis in vivo and in vitro, and to decrease vascular endothelial growth factor (VEGF) and Flk-1 concentrations in the mouse mammary gland. To determine which isomer mediates the antiangiogenic effects of CLA in vivo, the effects of diets supplemented with 5 or 10 g/kg c9,t11- or t10,c12-CLA isomers were compared in CD2F1Cr mice. Both isomers inhibited functional vascularization of a matrigel pellet in vivo and decreased serum VEGF concentrations; the t10,c12 isomer also decreased the proangiogenic hormone leptin (P < 0.05). Additionally, the t10,c12 isomer, but not c9,t11-CLA, rapidly induced apoptosis of the white and brown adipocytes as well as the preexisting supporting vasculature of the mammary fat pad (P < 0.05). Independent of this isomer-specific adipose apoptotic effect, both isomers induced a rapid and reversible decrease in the diameter of the unilocular adipocytes (P < 0.05). The ability of both CLA isomers to inhibit angiogenesis in vivo may contribute to their ability to inhibit carcinogenesis. Moreover, we propose that each CLA isomer uniquely modifies the mammary stromal "soil" in a manner that is useful for chemoprevention of breast cancer.

Prevention of mammary cancer with conjugated linoleic acid: role of the stroma and the epithelium.

Conjugated linoleic acid (CLA), found naturally in dairy products and ruminant meats, refers to isomers of octadecadienoic acid with conjugated double bonds. CLA inhibits both DMBA- and NMU-induced rat mammary carcinogenesis, and its antitumor efficacy is similar whether it is fed only during puberty, or continuously during promotion. Pubertal feeding is associated with a reduced proliferation of the epithelial cells within the terminal end buds (TEBs) and lobular epithelium, and results in a decrease in the epithelial density, suggesting a reduction in the carcinogen-sensitive target population. During promotion, CLA feeding induces apoptosis of preneoplastic lesions. The effects of CLA are mediated by a direct action on the epithelium, as well as by an indirect effect through the stroma. CLA is incorporated into the neutral lipids of mammary adipocytes, where it can serve as a local reservoir of CLA. Additionally, CLA induces the adipogenic differentiation of multipotent mammary stromal cells in vitro, and inhibits their development into three-dimensional capillary networks. This suggested that CLA might inhibit angiogenesis in vivo, a hypothesis that was subsequently confirmed. The antiangiogenic effect is mediated, in part, through a CLA-induced decrease in serum VEGF (vascular endothelial growth factor) and mammary gland VEGF and flk-1. Together, the data suggest that CLA may be an excellent candidate for prevention of breast cancer.

Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid.

Dietary conjugated linoleic acid (CLA) has been shown previously to inhibit rat mammary carcinogenesis. In addition to direct effects on mammary epithelial cells,including decreased proliferation and induction of apoptosis, CLA may exert its effects indirectly by inhibiting the differentiation of mammary stromal cells to an endothelial cell type. Specifically, CLA was found to decrease the ability of mammary stromal cells to form complex anastomosing microcapillary networks in vitro on Engelbreth-Holm-Swarm-derived reconstituted basement membrane. This suggested that CLA might inhibit angiogenesis in vivo. To test this possibility, CD2/F(1) mice were placed on synthetic diets containing 0, 1, or 2% CLA for 6 weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate (Matrigel pellet assay). After 7 days, the pellets from animals fed the control diet were infiltrated by abundant branching networks of blood vessels with patent lumen-containing RBCs. In contrast, pellets from the CLA-fed animals contained fewer infiltrating cells, which formed limited branching cellular networks, the majority of which had collapsed lumen and no RBCs. Both levels of dietary CLA showed similar effects, with the number of RBC-containing vessels per 20x field decreased to a third of that seen in control. Dietary CLA decreased serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers were effective in inhibiting angiogenesis in vitro in a dose-dependent fashion. The ability of CLA to inhibit angiogenesis may contribute to its efficacy as a chemopreventive agent.