Induced Breast Tumors Research Articles

Adipsin-dependent adipocyte maturation induces cancer cell invasion in breast cancer

Adipocyte-cancer cell interactions promote tumor development and progression. Previously, we identified adipsin (CFD) and its downstream effector, hepatocyte growth factor (HGF), as adipokines that enhance adipocyte-breast cancer stem cell interactions. Here, we show that adipsin-dependent adipocyte maturation and the subsequent upregulation of HGF promote tumor invasion in breast cancers. Mature adipocytes, but not their precursors, significantly induced breast tumor cell migration and invasion in an adipsin expression-dependent manner. Promoters of tumor invasion, galectin 7 and matrix metalloproteinases, were significantly upregulated in cancer cells cocultured with mature adipocytes; meanwhile, their expression levels in cancer cells cocultured with adipocytes were reduced by adipsin knockout (Cfd KO) or a competitive inhibitor of CFD. Tumor growth and distant metastasis of mammary cancer cells were significantly suppressed when syngeneic mammary cancer cells were transplanted into Cfd KO mice. Histological analyses revealed reductions in capsular formation and tumor invasion at the cancer-adipocyte interface in the mammary tumors formed in Cfd KO mice. These findings indicate that adipsin-dependent adipocyte maturation may play an important role in adipocyte-cancer cell interaction and breast cancer progression.

Accumulation of CD56+ CD16- Natural Killer Cells in Response to Preoperative Chemotherapy for Breast Cancer.

The activation of the antitumor immune responses of T cells and natural killer (NK) cells is important to induce breast tumor shrinkage via preoperative chemotherapy. We evaluated how antitumor immune responses contribute to the effects of such therapy. Forty-three patients with stages I - IV breast cancer who underwent surgery between August 2018 and Jun 2023 after preoperative chemotherapy were enrolled. Peripheral natural killer (pNK) cell activity was assessed by 51Cr-release assay, and the counts and percentages of CD4+, CD8+, and NK cells and their subsets in peripheral blood were measured before and after chemotherapy by two-color flow cytometry. Associations of cell population changes with chemotherapy responses were analyzed. On univariate analysis, relative to grade (G) ≤ 1 effects, G ≥ 2 therapeutic effects were associated significantly with human epidermal growth factor receptor 2 (HER-2)+ breast cancer (P = 0.024) and post-chemotherapy CD56+ CD16- NK cell accumulation (8.4% vs. 5.5%, P = 0.042), and tended to be associated with increased pre-chemotherapy CD56+ CD16- NK cell percentages (5.4% vs. 3.3%, P = 0.054) and pNK cell activity (42.0% vs. 34.5%, P = 0.057). The accumulation and increased percentage of CD56+ CD16- NK cells in patients with G ≥ 2 effects were not associated with changes in pNK cell activity or the disappearance of axillary lymph-node metastases. On multivariate analysis, G ≥ 2 therapeutic effects tended to be associated with higher pre-chemotherapy pNK levels (odds ratio = 0.96; 95% confidence interval: 0.921 - 1.002; P = 0.067). The accumulation of the immunoregulatory CD56+ CD16- NK cell subset in the peripheral blood before and after chemotherapy may lead to the production of cytokines that induce an antitumor immune response. Activation of the immune response mediated by CD56+ CD16- pNK cells after chemotherapy and their high counts before chemotherapy may contribute to the improvement of therapeutic effects against breast cancer.

Non-Contact Electric Field May Induced Higher CD4, CD8, Caspase-8, and Caspase-9 Protein Expression in Breast Tumor Tissue of Rats (Rattus norvegicus Berkenhout, 1769)

Non-contact electric field therapy has been studied as one of less invasive and safe alternative method in cancer therapy. Some studies reported that non-contact electric field therapy with low intensity (50 - 60 V/m) and medium frequency (150 kHz) could slower the rat’s breast tumor growth and did not cause significant damage on the rat's kidney and liver. However, further study needed to evaluate the potency of non-contact electric field to be developed into effective cancer therapy. Thus, we investigated the immune response of non-contact electric field therapy through the detection of CD4 and CD8 protein expression and the mechanism of apoptosis through the detection of caspase-8 and caspase-9 proteins on breast tumor tissue of rats. Twenty four rats were divided into 4 groups: 1. non-induction - non-therapy (NINT), 2. non-induction - therapy (NIT), 3. induction - non-therapy (INT), and 4. induction - therapy (IT). DMBA (7,12-dimethylbenz(α)anthracene) 20 mg/kg body weight was used to induced breast tumor formation. Rats with breast nodules that had reached 1 cm in size were exposed to an ECCT device with 150 kHz and 50 - 60 V/m electric fields. Further, the breast tissues were collected for routine histological and immunohistochemistry preparation for CD4, CD8, caspase 8, as well as caspase 9 detection. The data were statistically analyzed using Mann-Whitney U-Test method, with a significance level of p < 0.05 using the SPSS 16.0 version. The scoring results were compared between the INT and IT groups. Our results showed that non-contact electric field therapy could suppress breast tumor growth and improved its histological structure. Interestingly, the breast tissue of IT group qualitatively had slightly more necrotic and apoptotic cells than that of INT group. Moreover, the IT group showed by higher CD4 and CD8 as well as higher caspase 8 and caspase 9 expression. Collecting all the data together, we concluded that non-contact electric field therapy potent to improve histological structure of breast tumor.

Exploring the Protective Effect against 7,12-Dimethylbenz[a]anthracene-Induced Breast Tumors of Palmitoylethanolamide.

Breast cancer remains a global health burden, and the need for effective therapies is of chief importance. The current study explored the in vivo chemoprotective activity of palmitoylethanolamide (PEA) against 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumor in rats. Results of noninvasive photoacoustic imaging showed real-time progression in the tumor area and volume in DMBA-induced rats, while there was a reduction in tumor area and volume in PEA-treated tumor-bearing rats. The increase in the average oxygen saturation (sO2 %) and decrease in the average total hemoglobin (HbT %) indicated the PEA-mediated attenuation of hypoxia-induced neovascularization in DMBA-induced rats. Histopathological investigations confirmed the efficacy of PEA in mitigating breast carcinoma, hepatotoxicity and nephrotoxicity driven by DMBA. Moreover, PEA-mediated alterations in the metabolic activity of the tumor microenvironment were evidenced by decreased glucose and lactate dehydrogenase enzyme level in the blood plasma and mammary tissue. PEA also maintained the redox balance by inhibiting nitric oxide level, reducing malondialdehyde (a product of lipid peroxidation), and increasing the level of antioxidant enzyme reduced glutathione. PEA altered the expression of apoptosis-related genes (BAX, P53,BCL-XL, CASPASE-8, and CASPASE-9) and induced the activity of Caspase-3 protein in the mammary tissue of tumor-bearing rats, indicating its apoptosis inducing ability. Taken together, the findings of this study suggest that PEA may have a protective effect against DMBA-induced breast tumors.

Obesity Associated with Prediabetes Increases the Risk of Breast Cancer Development and Progression-A Study on an Obese Rat Model with Impaired Glucose Tolerance.

Patients with comorbidities of obesity and diabetes are recognized to be at high risk of breast cancer development and face worse breast cancer outcomes. Though several reports showed the reinforced link between obesity, diabetes, and prediabetes with breast cancer, the underlying molecular mechanisms are still unknown. The present study aimed to investigate the underlying molecular link between increased risks of breast cancer due to coincident diabetes or obesity using a spontaneous obese rat model with impaired glucose tolerance (WNIN/GR-Ob rat). A single dose of solubilized DMBA suspension (40 mg/kg body weight) was orally administered to the animals at the age of 60 days to induce breast tumors. The tumor incidence, latency period, tumor frequency, and tumor volume were measured. Histology, immunohistochemistry, and immunoblotting were performed to evaluate the tumor morphology and expression levels of signal molecules. The development of mammary tumors in GR-Ob rats was characterized by early onset and shorter latency periods compared to control lean rats. While 62% of obese rats developed breast tumors, tumor development in lean rats was only 21%. Overexpression of ER, PR, Ki67, and p53 markers was observed in tumor tissues of obese rats in comparison with lean rats. The levels of the hallmarks of cell proliferation and angiogenesis involved in IGF-1/PI3K/Akt/GSK3β/β-catenin signaling pathway molecules were upregulated in obese rat breast tumors compared to lean rats. Furthermore, obesity with prediabetes is associated with changes in IGF-1 signaling and acts on PI3K/Akt/GSK3β/β-catenin signaling, which results in rapid cell proliferation and development of breast tumors in obese rats than the lean rats. These results indicate that tumor onset and development were faster in spontaneous obese rat models with impaired glucose tolerance than in their lean counterparts.

Anti-cancer effect of in vivo inhibition of nitric oxide synthase in a rat model of breast cancer.

Increased expression of nitric oxide synthase (NOS) is associated with different cancers such as cervical, breast, lung, brain, and spinal cord. Inhibition of NOS activity has been suggested as potential tool to prevent breast cancer. The anti-tumor therapeutic effect of L-nitro arginine methyl ester (L-NAME), NOS inhibitor, using in vivo models is currently under investigation. We hypothesized that L-NAME will show an anti-tumor effect by delaying a progression of breast cancer via a modulation of cell death and proliferation, and angiogenesis. We used a novel model of anti-cancer treatment by the administration of L-NAME (30mg/kg in a day, intraperitoneal) injected every third day for five weeks to rat model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumor. Concentrations of nitrite anions, polyamines, malondialdehyde, NH4+ levels, and arginase activity in the blood were decreased in DMBA + L-NAME-treated rats compared with DMBA rats. The mortality rates, tumor number, weight, and volume, as well as the histopathological grade of breast cancer were also significantly reduced. In addition, L-NAME treatment showed a delay in tumor formation, and in body weight compared with rats administrated only with DMBA. In conclusion, our data show that L-NAME is a promising anti-cancer agent to treat breast cancer, which can lead to development of anti-tumor therapeutic tools in future.

Therapeutic effects of Vernonia amygdalina on the expression of hormone and HER2 receptors in 7, 12-dimethylbenz(a)anthracene-induced breast tumours in obese and non-obese Wistar rats

BackgroundObesity, a risk factor for breast cancer has been shown to be associated with poor response to neoadjuvant chemotherapy in women with breast cancer. Medicinal plants have been used to treat chronic diseases like cancer with little or no side effects. Vernonia amygdalina (VA) is a medicinal plant with anticancer activity. Previous studies on the anticancer potential of V. amygdalina against breast cancer are however entirely in vitro studies. The efficacy of V. amygdalina on obesity-associated breast cancer has also not been investigated in previous studies. This study explored VA’s anti-cancer effects on obesity- and non-obesity-associated 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast tumours in Wistar rats (WRs). MethodThirty female obese and non-obese WRs were studied, each group divided into six subgroups of five. At the 7th postnatal week (p.w), three subgroups were given a subcutaneous injection of DMBA to the breast with two of these three subgroups also commenced on daily oral VA at 250 mg/kg and 500 mg/kg from the 9th p.w. Same doses of VA were administered to two of the subgroups that did not receive DMBA while the last subgroup was placed on placebo from the 9th p.w. Haematoxylin and eosin staining were carried out on the excised breast tumours to evaluate general histoarchitecture, while immunohistochemistry was done for oestrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2) expression. ANOVA and Mann-Whitney followed by posthoc tests were used to analyse the data. Alpha level was set at 0.05. ResultsWhile the DMBA-induced tumours were triple-negative in the obese WRs, they were ER, PR HER2-positive in the non-obese group. Vernonia amygdalina was associated with a reduction in cancer cell proliferation in both obese and non-obese groups despite the differences in immunohistochemical profiles of the tumours. Vernonia amygdalina was associated with reversal of the increased ER, PR and HER2 expressions in the breast tumours of the non-obese rats. ConclusionsConclusively, VA had more therapeutic effects on DMBA-induced breast tumours in the non-obese than obese WRs.

Abstract P4-02-16: Investigating the role of NRIP1 as a novel marker and therapeutic target for breast cancer

Abstract Background:. Tamoxifen and fulvestrant target estrogen receptor (ER), which is overexpressed in more than 80% of breast cancer (BC) cases, and have significantly improved the survival rate. However, in most metastatic cases, resistance is inevitable. Identifying gene targets for improving the efficiency of hormone therapy, delaying the progress of resistance development, and restoring the efficacy of hormone therapy has great significance. Epidemiological studies of human populations found that early age at menarche significantly associates with the risk of BC. Remarkably, a five-year delay in menarche has been shown to correspond to a 35% reduction of BC. This suggests that identifying genes that regulate female sexual maturation (FSM) might provide a novel approach for exploring the genetic and molecular mechanisms of BC, providing clues in the search for new therapeutic targets. We previously identified and verified that nuclear receptor interacting protein 1 (Nrip1, also named Rip140) is a regulator of female sexual maturation. NRIP1, a co-regulator of nuclear receptors, interacts with several known signaling pathways for BC, such as E2F and estrogen receptor (ER). Human genome-wide association studies suggested that polymorphisms of NRIP1 are significantly associated with the risk of BC. Furthermore, elevating NRIP1 can promote epithelial cells proliferation and increase the development rate and size of the mammary gland, while depleting NRIP1 expression has the opposite effect. Importantly, clinical studies suggested that breast cancer patients with higher level of NRIP1 have a relatively poorer prognosis. Experiments and results:. 1. To investigate the role of NRIP1 in BC, we first measured NRIP1 expression in cancers. We found that NRIP1 was overexpressed in the tissues of BC and the BC cell lines. Importantly, immunohistochemistry assay showed that that NRIP1 is elevated in BC cell lines and tissues from patients with BC. Interestingly, overexpressed NRIP1 accumulated in the cytoplasm of the stromal cells in the benign tumors; but, in the malignant tumors, there was more accumulation in the nuclei of epithelia. Further studies revealed that suppressing NRIP1 could increase apoptosis of cancer cells in vitro and suppress the initiation and growth of DMBA (7,12-Dimethylbenz(a)anthracene) induced breast tumors. 2. Our previous studies have shown that deletion of NRIP1, in a variety of cell types, could suppress the activity of NFκB, which promotes the development of hormone therapy resistance; therefore, indicating that the suppression of NRIP1 may also suppress hormone therapy resistance. To test this hypothesis, we induced tamoxifen resistance in MCF7, which is estrogen receptor positive. During the induction, we found that NRIP1 expression increased gradually with the development of tamoxifen resistance. Interestingly, along with the upregulation of NRIP1 expression, we found that NRIP1 was translocated from the cytosol into the nucleus. These results suggest that not only the level of NRIP1 expression, but also the location, are related to the development of tamoxifen resistant. Furthermore, we found that suppressing the expression of NRIP1 by siRNA in tamoxifen resistant MCF7 (MCF7-TR) could significantly reduce the percentage of cells in the S phase and increase apoptosis with or without tamoxifen treatment. Conclusion:. 1. Investigating the genetic regulation of FSM may provide clues for identifying novel BC related genes. 2. NRIP1 plays an important role in BC. 3. Inhibiting NRIP1 may suppress the initiation and growth of BC. 4. NRIP1 overexpression and nuclear translocation are involved in the development of hormone therapy resistance. 5. The deletion of NRIP1 can suppress the development tamoxifen resistance in vitro. Citation Format: Yun Zhu, Fawwad Zaidi, Krishna Rao, Rong Yuan. Investigating the role of NRIP1 as a novel marker and therapeutic target for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-16.

Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation

Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis.

Anti-breast cancer potential of Anonidium mannii (Oliv.) Engl. & Diels barks ethanolic extract: UPLC-ESI-QTOF-MS detection of anticancer alkaloids

Ethnopharmacological relevanceBreast cancer is a serious threat in low-income as well as developed countries. To face this, many herbal preparations are prescribed by traditional healers in Cameroon, among which is Anonidium mannii commonly called “wild soursop”. AimThis study was undertaken to assess the anti-tumor effect of A. mannii ethanolic extract on cancer cell growth and against DMBA-induced mammary tumors in rats. Materials and methodsThe well characterized MTT bioassay was used to assess the cytotoxic potential of A. mannii ethanolic extract in liver (HepG2), prostate (DU145 & PC3) and breast (MCF-7) cancer cell lines. Considering the fact that breast cells were the most sensitive to the extract, a 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast tumor rat model was used to assess the possible anticancer effect of A. mannii extract. Indeed, rats were treated with either tamoxifen (3.3 mg/kg BW) or A. mannii extract (16.5, 50 and 150 mg/kg BW) or vehicle (2% ethanol) for 20 weeks. Tumor incidence, tumor mass and volume, oxidative stress status in tumor as well as tumor histoarchitecture were evaluated. ResultsA 24 h incubation of tested cells with the A. mannii extract significantly slowed cell growth in a concentration-dependent manner with an interesting effect in breast cells (IC50 ~61.5 μg/mL). As compared to the DMBA rats, those treated with A. mannii extract (50 and 150 mg/kg) showed reduced breast tumor incidence (28%), tumor burden (95.34% at 50 mg/kg and 99.14% at 150 mg/kg) and tumor volume (~92%). A. mannii extract counteracted the high proliferation of terminal mammary ducts induced by DMBA, mainly at 50 mg/kg. Furthermore, the extract decreased MDA and nitrite levels but increased SOD activity in the mammary gland. High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS) analysis detected potential anticancer and antioxidant alkaloids in A. manni extract, which are close to those found in Annona muricata. ConclusionThese results provide evidence on the in vitro and in vivo anticancer effects of A. mannii, and therefore support its use in traditional medicine system to fight against cancer.

Three photoinitiators induce breast tumor growth in mouse xenografts with MCF-7 breast cancer cells

Photoinitiators are utilized in the production of a wide range of commonly used products. However, some photoinitiators exert toxic effects. We previously demonstrated the endocrine-disrupting effects of photoinitiators in vitro. The present study investigated the estrogenic activities of three photoinitiators: 1-hydroxycyclohexyl phenyl ketone (1-HCHPK), methyl 2-benzoylbenzoate (MBB), and 2-methyl-4′-(methylthio)-2-morpholinopropiophenone (MTMP), which were subcutaneously injected into mouse xenografts with MCF-7 breast cancer cells. The results obtained showed that 1-HCHPK, MBB, and MTMP promoted breast tumor growth in these xenografts. A pretreatment with the estrogen receptor antagonist tamoxifen blocked the tumor growth-promoting effects of each photoinitiator. Collectively, the present results suggest that the three photoinitiators exhibit estrogenic agonist activities in vivo. Furthermore, as a factor for breast tumor growth, these photoinitiators potentially have estrogenic properties in vivo.

Pristane-induced mammary carcinomas.

Animal models for studying human breast cancer carcinogenesis and testing drug candidates on human breast cancer have extensively been proposed. Especially, chemically induced breast tumor models have been used because they can mimic the progression of clinical cancer from the beginning and can be generated with a facile procedure. Pristane is a hydrocarbon oil that is used as a chemical carcinogen to induce tumorigenesis in mice as well as arthritis and lupus nephritis in rats. In only a few studies, pristane-induced breast cancer models have been reported. This chapter is designated to describe pristane-induced mammary carcinoma models. Here, we provide a protocol for generating pristane-induced breast tumors in mice models for analyzing and for testing potential therapeutics on them. The given protocol can be applied to other animal models with some changes.

Aloe-Emodin Induces Breast Tumor Cell Apoptosis through Upregulation of miR-15a/miR-16-1 That Suppresses BCL2.

Purpose Aloe-emodin (AE) is a natural compound derived from aloe vera and palmatum rhubarb and shows anticancer activities in various cancers. Bcl-2 family is the main regulator of cell death or cell survival. This study describes the effects of AE on proliferation of breast tumor (BT) cells. Methods MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 cell lines were exposed to AE. Cell proliferation and apoptosis were assessed by CCK-8 and flow cytometry. Protein levels were measured by Western blotting. The levels of mRNA and miRNA were examined by RT-PCR. Bioinformatics was applied to screen miRNAs that bind to 3′-UTR of mRNA. Results The results showed that AE selective activity inhibited the proliferation and induced apoptosis of MCF-10AT and MCF-7 cells but exhibited no significant inhibition in MCF10A and MDA-MB-231 cells. Mechanistically, AE dose-dependently decreased the protein expression of Bcl-2 and Bcl-xl, while it increased Bax protein expression in MCF-10AT and MCF-7 cells. The levels of Bcl-xl and Bax mRNA were altered by AE treatment, which was consistent with the protein expression results. However, Bcl-2 mRNA levels were not affected in either cell line, suggesting that AE may modulate the protein translation of Bcl-2 through miRNAs. In all candidate miRNAs that bind to 3′-UTR of Bcl-2, miR-15a and miR-16-1 were dose-dependently downregulated by AE. Moreover, inhibition of miR-15a/16-1 could eliminate the inhibition of MCF-10AT and MCF-7 cells growth by AE and could reverse the downregulation of AE-induced Bcl-2 protein level. Conclusion Our research provides an important basis that AE induces BT cell apoptosis through upregulation of miR-15a/miR-16-1 that suppresses BCL2.

Development and characterization of a 99m Tc-labeled Neuropeptide Y short analog with potential application in breast cancer imaging.

The aim of this work was to develop and evaluate a 99m Tc-labeled neuropeptide Y derivative with affinity toward Y1-receptor. The selected amino acid sequence included nine amino acids derived from the C-terminal portion of the NPY complemented with the addition of one cysteine-mercaptoacetic acid moiety to bind the radiometal. Labeling was achieved through the preparation of a 3+1 nitrido complex. Physicochemical evaluation, cell uptake, internalization and externalization studies, and competitive assays were performed. Biodistribution experiments were carried out in normal and tumor-bearing mice. A single product with radiochemical purity >90% and high stability was obtained. In vitro analysis showed specific cellular uptake, IC50 of 73.2nM, and a high internalization rate (80%). Biodistribution studies showed low blood and renal uptake and combined hepatobiliary and urinary elimination. Preliminary studies in mice bearing induced breast tumors rendered promising uptake values.

Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression.

The effects of autonomic innervation of tumors on tumor growth remain unclear. Here we developed a series of genetic techniques to manipulate autonomic innervation in a tumor- and fiber-type-specific manner in mice with human breast cancer xenografts and in rats with chemically induced breast tumors. Breast cancer growth and progression were accelerated following stimulation of sympathetic nerves in tumors, but were reduced following stimulation of parasympathetic nerves. Tumor-specific sympathetic denervation suppressed tumor growth and downregulated the expression of immune checkpoint molecules (programed death-1 (PD-1), programed death ligand-1 (PD-L1), and FOXP3) to a greater extent than with pharmacological α- or β-adrenergic receptor blockers. Genetically induced simulation of parasympathetic innervation of tumors decreased PD-1 and PD-L1 expression. In humans, a retrospective analysis of breast cancer specimens from 29 patients revealed that increased sympathetic and decreased parasympathetic nerve density in tumors were associated with poor clinical outcomes and correlated with higher expression of immune checkpoint molecules. These findings suggest that autonomic innervation of tumors regulates breast cancer progression.

Abstract B35: Class IIa HDAC inhibition promotes an antitumor macrophage phenotype that induces breast tumor regression and inhibits metastasis

Abstract Tumor macrophages generally promote tumorigenesis and are associated with increased vascular density, resistance to chemotherapy and a worse clinical outcome in the majority of human tumors. Therefore, significant pharmaceutical effort has focused on inhibiting the immunosuppressive effects of tumor macrophages by depleting or inhibiting them. However, macrophage types also exist that can promote tumor clearance. Shifting tumor macrophages to an anti-tumor phenotype offers great hope given their plasticity and high numbers in a variety of solid tumors. We recently reported that a novel, first in class, specific class IIa HDAC inhibitor, TMP195, significantly alters the gene expression signature of human monocytes and promotes their polarization to an anti-tumor phenotype in vitro. Here, we demonstrate that a first in class, class IIa histone deacetylase (HDAC) inhibitor, TMP195, can activate tumor macrophages in vivo to induce tumor regression and inhibit pulmonary metastases in a mouse model of breast cancer. We find that TMP195 induces macrophage recruitment and differentiation of highly phagocytic cells within the tumor, which increases tumor cell death while decreasing angiogenesis. Strikingly, we find that TMP195 enhances chemotherapy and immunotherapy to induce durable tumor reduction. These data reveal a novel role for harnessing the anti-tumor potential of macrophages to enhance cancer therapy. Citation Format: Jennifer L. Guerriero, Alaba Sotayo, Holly E. Ponichtera, Jessica A. Castrillon, Alexandra L. Pourzia, Sara Schad, Shawn F. Johnson, Ruben D. Carrasco, Susan B. Lazo, Roderick T. Bronson, Scott P. Davis, Mercedes X. Lobera, Michael A. Nolan, Anthony Letai. Class IIa HDAC inhibition promotes an antitumor macrophage phenotype that induces breast tumor regression and inhibits metastasis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B35.

Abstract P5-14-05: α-tocopherol succinate enhances the anti-tumor activity of pterostilbene against human breast cancer cells in vivo and in vitro

Abstract Background: The cancer preventive activity of vitamin E (α-tocopherol, α-TP) has been suggested by many epidemiologic studies. Our previous study showed that Tocopherol-Associated Protein (TAP), a major tocopherol–binding protein, is preferentially detected in normal breast tissues than in tumor regions among 93 clinical breast cancer patients, which postulate its tumor suppressive role in breast cells. Pterostilbene (3,5-dimethoxi-49-hydroxystilbene, PS) is a natural dimethylated analogue of Resveratrol found in different plants. Previous studies have shown that PS has the ability to induce apoptosis in a range of cancer cell lines. The purpose of our study was to assess the effect of TAP and Pterostilbene in tumor suppression of breast cancer cell. Methods: Cell growth, proliferation, and viability were determined using the MTT assay. TAP protein expression was ablated in MDA-MB-231 breast cancer cell with nine independent siRNA sequences. MDA-MB-231 cells were transfected with pcDNA5 plasmids expressing the firefly luciferase gene. TAP protein expressions for each plasmid transfection were measured by western-blot analysis. Synergic effects of tumor growth and metastasis inhibitions in MDA-MB-231 xenograft animal model by PS and α-tocopherol treatment were evaluated. Results: TAP served as a key molecular in responding to α-tocopheryl succinate (α-TOS) induced cell cycle regulation. PS has demonstrated the synergic decrease on breast cancer cell growth effects by the combination of α-TOS exposure. In animal model, treatment of PS and high level vitamin E diet not only induced breast tumor growth inhibition, but also suppressed tumor cell invasion by measuring circulation tumor cells in mouse blood. Conclusions: Upon the evidences showed in this study, we suggest taking vitamin E and PS as supplements may provide both breast tumor preventative and therapeutic benefits in clinic. Citation Format: Tam K-W, Lee C-H. α-tocopherol succinate enhances the anti-tumor activity of pterostilbene against human breast cancer cells in vivo and in vitro [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-05.

Abstract P3-05-05: Class IIa HDAC inhibition promotes an anti-tumor macrophage phenotype that induces breast tumor regression and inhibits metastasis

Abstract While tumor-associated macrophages (TAMs) often have net pro-tumor effects, their embedded location and their untapped potential provide impetus to the discovery of strategies to turn them against tumors. We recently reported that a first in class selective class IIa HDAC inhibitor (TMP195) influenced human monocyte responses to colony stimulating factors CSF-1 and CSF-2 in vitro. Here, we utilize a macrophage-dependent autochthonous mouse model of breast cancer to demonstrate that in vivo TMP195 treatment alters the tumor microenvironment and reduces tumor burden and pulmonary metastases through macrophage modulation. TMP195 induces recruitment and differentiation of highly phagocytic and stimulatory macrophages within tumors. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade in this model significantly enhances the durability of tumor reduction. These data introduce class IIa HDAC inhibition as a novel means to harness the anti-tumor potential of macrophages to enhance cancer therapy. Citation Format: Guerriero JL, Sotayo A, Ponichtera HE, Castrillon JA, Pourzia AL, Schad S, Johnson SF, Carrasco RD, Lazo S, Bronson RT, Davis SP, Lobera M, Nolan MA, Letai A. Class IIa HDAC inhibition promotes an anti-tumor macrophage phenotype that induces breast tumor regression and inhibits metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-05.

Abyssinone V-4' Methyl Ether Isolated from Erythrina droogmansiana (Leguminosae) Inhibits Cell Growth and Mammary Glands Hyperplasia Induced in Swiss Mice by the 7,12-Dimethylbenz(a)anthracene.

There is a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. The present study was designed to evaluate the in vitro and in vivo antiproliferative effects of Abyssinone V-4' methyl ether (AVME) on breast tissue of mice. The cytotoxicity of AVME was evaluated using MTT assay in four cancer cell lines (DU145, PC3, HepG2, and MCF-7). Further, a protective effect of AVME was evaluated on 7,12-dimethylbenz(a)anthracene- (DMBA-) induced breast tumor in Swiss mice. Incidence, burden, volume, and histological analysis of mammary tumors were measured. As a result, AVME inhibits DU145, PC3, HepG2, and MCF-7 cells growth. In vivo, no tumor was detected in mice from the normal group as compared to those of DMBA group. Moreover, AVME inhibits the DMBA-induced mammary glands hyperplasia in mice at the dose of 10 mg/kg, evidenced by a decrease of tumor incidence, tumor weight, and volume as well as a protective effect against the lobular alveolar hyperplasia. Taken all together, these results suggest that Abyssinone V-4' methyl ether is endowed with antitumor properties and could be a source of traditional medicine which deserves to be more elucidated and explored in the foreseeable future.

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1\u03b1/VEGF pathway in the breast tumor microenvironment

BackgroundThe G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells.MethodsWe analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1.ResultsWe first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation.ConclusionsThese findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies.