Inflammatory Breast Cancer Cells Research Articles

Experimental study on inhibiting invasion of inflammatory breast cancer cell lines SUM149 and SUM190 by silencing RhoC gene with the help of RNA interference

Objective In this study, we silence RhoC gene in inflammatory breast cancer (IBC) SUM149, SUM190 cell lines and explore its effects on cell invasion of IBC by RNA interference (RNAi) technology so as to provide experimental principal for target gene therapy of IBC. Methods Human IBC cells were divided blank group, control siRNA group, RhoC-siRNA group by balance randomization. RhoC-siRNA was used to transfect human IBC SUM149, SUM190 cell lines. Microscopy was applied to analyze transfection efficiency. After transfecting for 48 hours, reverse transcriptase-polymerase chain reaction (RT-PCR) was made to detect mRNA expression levels of RhoC. Western blotting was also used to analyze the expression level of RhoC protein. After transfecting for 48 hours, the ability to invasion was analyzed by transwell hole. Results We used siRNA to transfect human IBC SUM149, SUM190 cell lines and found that transfection efficiency can exceed to 80%. After we transfected for 48 hours, the expression levels of RhoC protein in IBC SUM149 cell lines 0.375±0.164 in RhoC siRNA group were significantly lower than those 0.897±0.376 in control siRNA group (t=6.274, P=0.045). The expression levels of RhoC protein in IBC SUM190 cell lines 0.381±0.166 in RhoC siRNA group were significantly lower than those 0.902±0.386 in control siRNA group (t=6.891, P=0.028). However, there were no obvious differences between in blank group and control siRNA group. After we transfected for 48 hours, in IBC SUM149 cell lines, the ability to invasion 85.6±12.7 in RhoC siRNA group was markedly lower than that 340.5±43.4 in control siRNA group (t=12.275, P=0.034). In IBC SUM190 cell lines, the ability to invasion 92.8±15.6 in RhoC siRNA group was markedly lower than that 345.9±47.7 in control siRNA group (t=11.769, P=0.022). Conclusion RhoC is known as an important role in the carcinogenesis of IBC. With the help of RNAi technology, we can greatly inhibit the invasion of IBC SUM149, SUM190 cell lines by silencing RhoC gene. RNAi technology perhaps becomes an important gene therapy tool for IBC. Key words: Inflammatory breast cancer; RhoC; RNA interference; Gene silence

Blocking Interleukin (IL)4- and IL13-Mediated Phosphorylation of STAT6 (Tyr641) Decreases M2 Polarization of Macrophages and Protects Against Macrophage-Mediated Radioresistance of Inflammatory Breast Cancer

To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "antitumor" (M1) or a "protumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24hours, then subjected to irradiation (0, 2, 4, or 6Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpinRNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

PO58 - Regulation of Stemness Properties by Ganoderma Lucidum Extract in Inflammatory Breast Cancer Cells via STAT3 Regulation

PO58 - Regulation of Stemness Properties by Ganoderma Lucidum Extract in Inflammatory Breast Cancer Cells via STAT3 Regulation

The Role of Interleukin-8 and Its Mechanism in Patients with Breast Cancer: Its Relation with Oxidative Stress and Estrogen Receptor

Context: Breast cancer is one of the leading causes of death among women all over the world. Prognostic markers can be used for patient survival. Likewise, these biomarkers are able to generate main information related to clinical manner of breast gland tumors. Interleukin-8 (IL-8) is a chemotactic cytokines caused initiation of an inflammatory response. Evidence Acquisition: We performed a computerized search in Medline/PubMed databases with key words: IL-8, breast cancer, and oxidative stress. Results: IL-8 secretion motivates the expression of adhesion molecule, including fibronectin in human inflammatory breast cancer cells. IL-8 activates PI3K/Akt pathway, which in turn activates NF-κB resulted up-regulation of integrin β3 expression and increases the invasion of breast cancer cells. Therefore, it seems that IL-8/PI3K/Akt/NF-κB/integrin β3 axis may be used for therapeutic intervention of breast cancer with metastasis status. NF-κB plays an important role in IL-8 gene transcription via several signals, such as LPS, TNF-alpha, and oxidative stress. Oxidative stress caused angiogenic factor production, such as vascular endothelial growth factor and IL-8 via cancer cells. Moreover, there is inverse relation between ER level and IL-8, but the mechanism of IL-8 action in estrogen receptor-negative breast cancer is largely unknown. Estrogen receptor and growth factor in breast cancer cells is influenced by mitogen activated protein kinase (MAPK) cascade, as a part of IL-8 signaling pathway. Conclusions: According to these studies, interleukin-8 may be as a main biomarker for breast cancer. It seems that signaling pathway of IL-8 may be used for therapeutic intervention of breast cancer. Also oxidative stress and breast cancer cells with lacking estrogen receptor generate more interleukin-8.

Pharmacological targeting of GLI1 inhibits proliferation, tumor emboli formation and in vivo tumor growth of inflammatory breast cancer cells

Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.

Histone Deacetylase Inhibitor Enhances the Efficacy of MEK Inhibitor through NOXA-Mediated MCL1 Degradation in Triple-Negative and Inflammatory Breast Cancer.

Purpose: Inflammatory breast cancer (IBC), diagnosed clinically, and triple-negative breast cancer (TNBC), diagnosed by molecular receptor status, are the two most aggressive forms of breast cancer, and both lack effective targeted therapies. We previously demonstrated involvement of histone deacetylase (HDAC) inhibitor entinostat in regulating apoptosis in IBC and TNBC cells; here, we aimed to identify novel combination therapy candidates.Experimental Design: Potential therapeutic targets were identified by mRNA expression profiling of TNBC and IBC cells treated with entinostat. Drug action and synergism were assessed by in vitro proliferation assays, tumor growth in vivo, and proteomic analyses. Gain/loss-of-expression studies were utilized to functionally validate the role of identified targets in sensitivity of TNBC and IBC cells to combination therapy.Results: Entinostat induced activity of the oncogenic ERK pathway and expression of proapoptotic NOXA. These are known to stabilize and degrade, respectively, MCL1, an antiapoptotic Bcl-2 protein. In breast cancer patients, high-MCL1/low-NOXA tumor expression correlated significantly with poor survival outcomes. Combination treatment of entinostat with MEK inhibitor pimasertib reduced the growth of TNBC and IBC cells in vitro and inhibited tumor growth in vivo The synergistic action of combination therapy was observed in TNBC and IBC cell lines in which NOXA expression was induced following entinostat treatment. The therapeutic activity depended on induction of mitochondrial cell death pathways initiated by NOXA-mediated MCL1 degradation.Conclusions: Our preclinical findings provide a rationale for the clinical testing of combination HDAC and MEK pathway inhibition for TNBC and IBC that exhibit elevated baseline tumor MCL1 expression. Clin Cancer Res; 23(16); 4780-92. ©2017 AACR.

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars.

Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%–30% in case of women with stage III–IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model.

EGFR signaling promotes inflammation and cancer stem-like activity in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer, with a strong proclivity to metastasize, and IBC-specific targeted therapies have not yet been developed. Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target in IBC. However, the mechanism behind the therapeutic effect of EGFR targeted therapy is not well defined. Here, we report that EGFR regulates the IBC cell population that expresses cancer stem-like cell (CSC) markers through COX-2, a key mediator of inflammation whose expression correlates with worse outcome in IBC. The COX-2 pathway promoted IBC cell migration and invasion and the CSC marker-bearing population in vitro, and the inhibition of this pathway reduced IBC tumor growth in vivo. Mechanistically, we identified Nodal, a member of the TGFβ superfamily, as a potential driver of COX-2-regulated invasive capacity and the CSC phenotype of IBC cells. Our data indicate that the EGFR pathway regulates the expression of COX-2, which in turn regulates the expression of Nodal and the activation of Nodal signaling. Together, our findings demonstrate a novel connection between the EGFR/COX-2/Nodal signaling axis and CSC regulation in IBC, which has potential implications for new combination approaches with EGFR targeted therapy for patients with IBC.

Abstract 433: Glycogen accumulation in aggressive breast cancers during hypoxic exposure

Abstract Isolation of specific metabolic alterations in aggressive triple negative and inflammatory breast cancers represents a compelling avenue for the development of treatments for these diseases. The ability of cancer to utilize diverse metabolic pathways to modulate increased survival and proliferation is well established. Indeed, we have previously described a series of metabolic adaptations in the triple negative inflammatory breast cancer cell line SUM149 and demonstrated a role for the small GTPase RhoC in the metabolic phenotype of the cell line. In this work we seek to describe a survival mechanism for cancer cells that are subjected to hypoxic environments. Hypoxic survival or growth is often necessitated at the center of a growing tumor mass or if the tumor is growing in the lymphatic system, as is often the case for inflammatory breast cancers. Here we investigate the regulation of glycogen levels in breast cancer and normal-like cell lines in atmospheric and 1% oxygen environments. Reserves of glucose are typically stored in liver and muscle cells as the polysaccharide glycogen. Elevated levels of glycogen have previously been observed in various cancers, and recent studies have implicated the importance of glycogen metabolism in promoting cancer cell survival. We hypothesize that aggressive breast cancers utilize modifications of the glycogen synthesis and degradation pathways to provide nutritional flexibility as they proliferate and invade into diverse environments. MCF10A, SUM149, and SUM190 cell lines demonstrate a greater than 10-fold increase in glycogen accumulation when subjected to hypoxic growth conditions. MDA-MB-231 cells also increase glycogen storage, though at levels on the order of 2-3 times higher in hypoxia when compared to normoxic growth. Conditioned media for all cell lines in normoxic and hypoxic conditions were analyzed for glucose and lactate levels to determine changes in metabolite consumption. The data presented indicate an unexpected accumulation of glycogen in response to hypoxic conditions. Studies to determine the mechanisms through which these changes occur and why they are important for continued survival and growth of the cancer cells are ongoing and will be presented. Citation Format: Megan Altemus, Joel Yates, ZhiFen Wu, LiWei Bao, Sofia Merajver. Glycogen accumulation in aggressive breast cancers during hypoxic exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2017-433

Characterization and Targeting of Platelet-Derived Growth Factor Receptor alpha (PDGFRA) in Inflammatory Breast Cancer (IBC)

PURPOSE: Inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer due to its rapid onset and highly invasive nature. IBC carries 5- and 10-year disease-free survival rates of ~45% and <20%, respectively. Multiple studies demonstrate that in comparison with conventional breast cancer, IBC has a unique molecular identity. Here, we have identified platelet-derived growth factor receptor alpha (PDGFRA) as being uniquely expressed and active in IBC patient tumor cells. EXPERIMENTAL DESIGN: Here we focus on characterizing and targeting PDGFRA in IBC. Using gene expression, we analyzed IBC patient samples and compared them with non-IBC patient samples. Further, using IBC cells in culture, we determined the effect of small molecules inhibitors in both in vitro and in vivo assays. RESULTS: In IBC patients, we show more frequent PDGFRA activation signature than non-IBC samples. In addition, the PDGFRA activation signature is associated with shorter metastasis-free survival in both uni- and multivariate analyses. We also demonstrate that IBC cells express active PDGFRA. Finally, we show that PDGFRA targeting by crenolanib (CP-868-596), but not imatinib (STI571), two small molecule inhibitors, interferes with IBC cell growth and emboli formation in vitro and tumor growth in vivo. CONCLUSIONS: Our data suggest that PDGFRA may be a promising target for therapy in IBC.

In vitro and in vivo effect of flutamide on steroid hormone secretion in canine and human inflammatory breast cancer cell lines.

The aim was to study the effects of flutamide on cell proliferation, in vivo tumour growth and steroid production in canine and human IBC cell lines. IPC-366 and SUM149 cell cultures were exposed to flutamide concentrations for 72 hours. Additionally, IPC-366 and SUM149 xenotransplanted mice were treated subcutaneously with flutamide 3 times a week for 2 weeks. Steroid hormones determination in culture media, serum and tumour homogenates (pregnenolone, progesterone, androstenedione, testosterone, dihydrotestosterone, 17β-oestradiol and oestrone sulphate) were assayed by EIA. in vitro cell proliferation percentages showed a decrease in all flutamide dosages in IPC-366 and SUM149. in vivo flutamide reduced tumour size by 55% to 65%, and metastasis rates decreased. In treated groups, androgen levels in culture media, serum and tumour homogenates were increased as oestrogen levels decreased. These results suggest that flutamide treatment inhibits cell proliferation and promotes tumour reduction by increasing androgen levels and also support future therapy approaches.

Selective Targeting of Cancer Cells by Oxidative Vulnerabilities with Novel Curcumin Analogs

Recently, research has focused on targeting the oxidative and metabolic vulnerabilities in cancer cells. Natural compounds like curcumin that target such susceptibilities have failed further clinical advancements due to the poor stability and bioavailability as well as the need of high effective doses. We have synthesized and evaluated the anti-cancer activity of several monocarbonyl analogs of curcumin. Interestingly, two novel analogs (Compound A and I) in comparison to curcumin, have increased chemical stability and have greater anti-cancer activity in a variety of human cancer cells, including triple-negative, inflammatory breast cancer cells. In particular, the generation of reactive oxygen species was selective to cancer cells and occurred upstream of mitochondrial collapse and execution of apoptosis. Furthermore, Compound A in combination with another cancer-selective/pro-oxidant, piperlongumine, caused an enhanced anti-cancer effect. Most importantly, Compound A was well tolerated by mice and was effective in inhibiting the growth of human triple-negative breast cancer and leukemia xenografts in vivo when administered intraperitoneally. Thus, exploiting oxidative vulnerabilities in cancer cells could be a selective and efficacious means to eradicate malignant cells as demonstrated by the curcumin analogs presented in this report with high therapeutic potential.

Caveolin-1 Mediates Inflammatory Breast Cancer Cell Invasion via the Akt1 Pathway and RhoC GTPase.

With a propensity to invade the dermal lymphatic vessels of the skin overlying the breast and readily metastasize, inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer. We previously reported that caveolin-1 is overexpressed in IBC and that RhoC GTPase is a metastatic switch responsible for the invasive phenotype. RhoC-driven invasion requires phosphorylation by Akt1. Using a reliable IBC cell line we set out to determine if caveolin-1 expression affects RhoC-mediated IBC invasion. Caveolin-1 was down regulated by introduction of siRNA or a caveolin scaffolding domain. The ability of the cells to invade was tested and the status of Akt1 and RhoC GTPase examined. IBC cell invasion is significantly decreased when caveolin-1 is down regulated. Activation of Akt1 is decreased when caveolin-1 is down regulated, leading to decreased phosphorylation of RhoC GTPase. Thus, we report here that caveolin-1 overexpression mediates IBC cell invasion through activation Akt1, which phosphorylates RhoC GTPase.

Anti-RhoC siRNAs inhibit the proliferation and invasiveness of breast cancer cells via modulating the KAI1, MMP9, and CXCR4 expression.

Overexpression of RhoC in breast cancer cells indicates poor prognosis. In the present study, we aim to investigate the possible antitumor effects of anti-RhoC small-interfering RNA (siRNA) in inflammatory breast cancer cells. In this study, a specific anti-RhoC siRNA was used to inhibit RhoC synthesis. Transfection of anti-RhoC siRNA into two IBC cells SUM149 and SUM190 induced extensive degradation of target mRNA and led to significant decrease in the synthesis of protein. Anti-RhoC siRNA inhibited cell proliferation and invasion, increased cell apoptosis, and induced cell cycle arrest in vitro. Moreover, the transfection of siRNA increased the expression of KAI1 and decreased the expression of MMP9 and CXCR4 in both mRNA and protein levels. Furthermore, transplantation tumor experiments in BALB/c-nu mice showed that intratumoral injection of anti-RhoC siRNA inhibited tumor growth and increased survival rate. Our results suggested that RhoC gene silencing with specific anti-RhoC siRNA would be a potential therapeutic method for metastatic breast cancer.

Abstract P3-07-15: Treatment with a Jumonji demethylase inhibitor JIB-04 sensitizes resistant breast cancer cells to chemotherapeutic drugs in an in vitro model of intrinsic resistance

Abstract We have recently developed a usable model of panresistance in triple-negative breast cancer. We have shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. The MA cells can survive a variety of challenges in the metastasis process because of their embryo-like nature and high adaptability. Epigenetic state is a key player in cancer evolution. Therefore, a variety of compounds are being developed that would target epigenome for anticancer therapy. We believe that the identification of potentially effective epigenetic drugs can benefit from their evaluation in a usable system of panresistant cancer cells such as our model system of MA cells. This is particularly true for the compounds that may affect cellular adaptability upon a long treatment rather than affecting cell proliferation or apoptosis in a short period. In this study we evaluated JIB-04, a pan-inhibitor of Jumonji family of histone demethylases, for its ability to affect intrinsic resistance in MA cells. First, we found that MA cells are more resistant to JIB-04 treatment in a comparative evaluation with the parental SUM149 cells line. A treatment with 125-250 nM JIB-04 for 18-20 days killed all parental cells but not all MA cells; at least 20 MA cells survived and formed colonies after a 14-days recovery. Next, we asked whether JIB-04 treatment would affect the intrinsic resistance of MA cells, e.g., sensitize them to treatment with chemotherapeutic drugs. In order to assess the most resistant cells in population, these experiments involved treatment of MA cells with 125 nM JIB-04 for 10 days followed by a recovery in drug-free medium for 7 days; then we treated the cells with 5 nM paclitaxel or 100 nM doxorubicin for 6-8 days followed by a recovery for 1-12 days. We found that JIB-04 pre-treatment sensitized MA cells and the parental SUM149-Luc cell line to both these chemotherapeutic drugs as assessed by a dramatic drop in the number of colonies as compared to those obtained after treatment of control cells with the same chemotherapeutic drugs. To consider how our results may apply in the context of evolving disease in patients who are not taking chemotherapeutic drugs, we asked whether JIB-04 treatment would alter expression of surface molecules on resistant breast cancer cells such that they can be destroyed by immune system. In this regard, we specifically found that JIB-04 treatment increased the expression of PD-L1 in MA cells. Published results from a clinical study suggest that PD-L1 expression on breast cancer cells renders them responsive to various therapies, not just immune checkpoint blockade targeting PD-L1. In conclusion, our results suggest a novel approach for evaluating potential anticancer agents such as JIB-04 that would halt cancer evolution and prevent development of resistance to currently offered therapies. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Singh B, Washburn LJ, Kinne HE, Milligan RD, Lucci A. Treatment with a Jumonji demethylase inhibitor JIB-04 sensitizes resistant breast cancer cells to chemotherapeutic drugs in an in vitro model of intrinsic resistance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-15.

Abstract P3-16-02: Nanotheranostics using plasmonic gold nanostars to target inflammatory breast cancer cells and tumor emboli

Abstract Introduction: Due to the aggressive nature of inflammatory breast cancer (IBC) and insensitive imaging techniques, patients diagnosed with IBC have a poor prognosis. IBC's unique ability to form emboli leads to rapid growth and distant invasion. New therapies aimed at disrupting the formation of tumor emboli are needed. Gold nanostars (GNS) are unique nanoparticles that can be imaged in real time with high sensitivity, and are rapidly endocytosed into cancer cells. Due to their sharp spikes, GNS exhibit plasmonic properties, which strongly enhance the electromagnetic field upon laser excitation. In addition to their use as highly intense and stable fluorescent labels, these nanoparticles can be photothermally activated to trigger cellular ablation. The use of GNS as an imaging and therapeutic modality for IBC should be investigated. Methods: IBC (SUM-149/SUM190), non-IBC (BT474M1/MD-MBA-231), and drug resistant isotype variant (rSUM-149) cell lines were incubated with GNS, stained with Hoechst33342, and imaged with multiphoton microscopy (MPM). The dose and time dependent effects of GNS on SUM-149 proliferation was assessed with MTT assays. Photothermal treatment was performed on GNS-labeled IBC, non-IBC, and drug resistant cancer cell lines. The effects of photothermal therapy on cell viability were assessed using fluorescein diacetate and propidium iodide. Using a tumor emboli model, SUM-149, rSUM-149, and SUM-190 tumor emboli were labeled with GNS on the 3rd day of embolic maturation. Emboli were sectioned, stained with H/E, and imaged with MPM to demonstrate the depth of GNS penetrance. The potential for photothermal ablation of the GNS-labeled tumor emboli was assessed using various laser intensities. Propidium iodide was used to examine emboli viability following treatment. Results: In all cell lines, GNS displayed rapid cellular uptake without nuclear involvement. MTT assay showed that GNS concentrations of 0.15 and 0.20 nM caused decreases in cell proliferation at 6 and 12 hours. Proliferative capacity was unaltered at 24 hours for all concentrations, however this may be due to spectrophotometric interference with high intracellular GNS concentrations. Live/dead staining confirmed effective photothermal treatment in all cultures with a clear zone of cellular death. For tumor emboli studies, GNS allowed bright fluorescent monitoring of tumor emboli using MPM, and cross sectional imaging demonstrated GNS penetrance into the embolic core. Photothermal ablation of GNS-labeled tumor emboli was successfully demonstrated following laser irradiation. Cell death was confirmed with propidium iodide. Conclusion: Gold nanostars provide a highly fluorescent intracellular label for cancer cell lines, and tumor emboli without significantly altering cell proliferation. Furthermore, the inherent optical properties of the GNS allows for a combined therapeutic application following phototoactivation. This is the first study to demonstrate the nanothernostic application of GNS in IBC tumor emboli. Prior studies have shown the effectiveness of photothermal ablation in in vivo sarcoma models; we are currently extending our studies to an IBC mouse model. Supported by the P30 Cancer Center Support Grant (GRD) and Duke Exploratory Funds (TVD). Citation Format: Shammas RL, Fales AM, Crawford BM, Hollenbeck ST, Vo-Dinh T, Devi GR. Nanotheranostics using plasmonic gold nanostars to target inflammatory breast cancer cells and tumor emboli [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-16-02.

Abstract P6-01-20: Effects of local and radiation enhanced TGFβ on the invasive nature of inflammatory breast cancer cells

Abstract Inflammatory Breast Cancer (IBC) is the deadliest form of epithelial breast cancer, accounting for ~10% of breast cancer deaths in the United States. A hallmark of IBC is the formation intralymphatic emboli that are known to be chemotherapy and radiation resistant and contribute to rapid metastasis. This form of breast cancer progresses very quickly, expressing aggressive behavior. IBC and melanoma share a number of similarities in disease presentation and progression. Both spread via dermal lymphatics, form intralymphatic emboli and have a propensity to form cutaneous metastases. Melanoma can also present as “inflammatory melanoma”, which resembles IBC phenotypically. Thus, new leads for studying cutaneous metastasis can be gathered from the melanoma literature. Studies demonstrate a role for transforming growth factor beta (TGFβ) in the etiology of melanoma cutaneous metastasis. TGFβ promotes tumor cell invasion and its expression can be induced in the stroma by radiation treatment. Recently, our lab has demonstrated low expression of TGFβ in IBC patients, which we believe promotes cohesive invasion of IBC cells. Stimulation of IBC cells with 2ng/ml of TGFβ causes altered tumor cell behavior such as stimulating single cell invasion. The invasion of KPL4, SUM149 and MDA-MB231 cell lines were significantly higher in TGFβ stimulated cells compared to non-stimulated cells. As in melanoma cells we hypothesize that radiation enhanced local TGFβ production in the stroma. We radiated normal human epidermal fibroblasts cells with 0gy, 0.5gy, 1.0gy, 2.5gy and 5.0gy intensity and observed that the invasion was significantly higher in 1.0gy, 2.5gy and 5.0gy. We have also looked at the levels of TGFβ-1 in different conditioned medium. ELISA results don't show significant level of increase in TGFβ-1. Since there are two more TGFβ receptors in the TGFβ pathway (TGFβ -2 TGFβ -3), next I will look at the level of TGFβ-2 and 3 on the different intensity of radiation-conditioned medium. Our prediction is that the increase in the invasion of IBC cells is because of TGFβ, which alters the cohesive nature of IBC cells, and enhance single cell invasion. Moreover, radiation increases TGFβ levels in the stroma, which is responsible for rapid metastasis of IBC cells to the skin. Citation Format: Barkataki S, van Golen K. Effects of local and radiation enhanced TGFβ on the invasive nature of inflammatory breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-20.

Abstract P3-05-07: Flutamide reduced tumor progression and altered steroid hormone secretion in human and canine inflammatory breast cancer cell lines

Abstract Background: Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate and accounts for 6% of diagnosed breast cancers. The role of androgens on breast cancer is on rise in research, trying to propose anti-androgen therapeutic strategies. The aim of this study was to determine the effects in vivo and in vitro of flutamide (anti-androgen drug) on cell proliferation, tumor progression and steroid production in two cancer IBC triple negative cell lines (SUM-149 and IPC-366, human and canine, respectively). Material and Methods: IPC-366 was cultured in Dulbecco's modified Eagle medium nutrient mixture F-12 Ham (DMEM/F12) and SUM149 was maintained in Ham's F-12 media. Flutamide concentrations added to the culture media were: 5 µm, 10 µm, and 15 µm for 72 hours. Additionally, IPC-366 and SUM149 xenotrasplanted mice were used for in vivo assays with the same flutamide concentrations administrated subcutaneously. Steroid hormones determination in culture media and tumor homogenates (pregnenolone (P5), progesterone (P4), dihydroepiandrostenedione (DHEA), androstenedione (A4), testosterone (T), dihydritestosterone (DHT), 17β-estradiol (E2) and estrone sulphate (SO4E1)) were assayed by EIA previously validated. Immunohistochemical (IHC) analysis of the steroidogenic enzymes CYP11A1, 3β-HSD, CYP19A1, 17β-HSD and 5α-reductase were assayed. Results: Percentage of cell proliferation showed a decrease in all treatments in IPC-366 and SUM149. In vivo tumor progression was reduced in around 65% in IPC-366 and SUM149 xenotrasplanted mice. Regarding hormonal secretion assayed in pellets and homogenates. in treated groups there was an increased in steroid secretion as showed the high levels found in P5, P4 and A4. T and DHT concentrations were higher in treated groups, in contrast to E2 levels that decreased. 17β-HSD and 5α-reductase by IHC showed a high expression in treated groups. Conclusion: IPC-366 and SUM149 treated with flutamide reduced the proliferation of neoplastic cells, reduced tumor progression in xenotrasplanted mice and altered steroid hormone secretion by increasing T production and decreasing in E2 levels. These results open a future approach for IBC and triple negative breast cancer. Citation Format: Caceres S, Pena L, Silvan G, Illera MJ, Monsalve B, Woodward WA, Reuben J, Illera JC. Flutamide reduced tumor progression and altered steroid hormone secretion in human and canine inflammatory breast cancer cell lines [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-07.

Abstract B49: Profiling of GLI antagonists in phenotypic models for effects on inflammatory breast cancer cell growth

Abstract Our objective was to assess GLI pathway antagonists for activity in inflammatory breast cancer (IBC) phenotypic models. Introduction: IBC is one of the most lethal forms of breast cancer and accounts for ~15% of all breast cancer deaths. IBC affects younger patients and is diagnosed in minorities at higher rates than in other racial and ethnic groups1. African-American women with IBC appear to have worse survival regardless of inflammatory status, stage, socioeconomic position, tumor, or treatment characteristics2,3. IBC is characterized by rapid progression, is highly invasive and is not usually detected by mammograms or ultrasounds. The presence of tumor emboli (TE) is a hallmark of the disease in which distinctive clusters of tumor cells migrate collectively and drive lymphatic invasion and metastasis. Activation of GLI1, the terminal effector of hedgehog (Hh) signaling, via gene amplification, has been linked to tumorigenesis, and invasiveness in many cancers including our studies on IBC4. Targeting at the level of GLI1 with small molecules has been effective with some demonstrating activity in vivo. Experimental procedures: In our studies we have used a tiered approach of phenotypic and functional assays to evaluate a panel of GLI antagonists for effects on IBC cell growth, in particular assessing their effects in models that more closely model or predict tumor growth in vivo including; migration, anchorage independent growth (AIG), mammosphere formation and TE assays. Results: Using our previously described quantitative cell proliferation assay5 we first identified a subset of GLI antagonists that effected IBC proliferation and down-regulated GLI1 transcriptional activity. We have now used high content imaging to simultaneously measure nuclear count, morphology, cell integrity, and mitochondrial health in live cells. We have identified GLI antagonists that show an anti-proliferative response but have no effect on nuclear size suggesting a lack of general cytotoxicity. In contrast, other antagonists had dramatic effects on nuclear area and nuclear texture indicative of cytotoxicity. We further examined their effects on colony formation using the AIG assay. SUM149 cells were treated with GLI inhibitors and effects on colony size and number noted. All three GLI antagonists tested reduced the size of the colonies with one also giving a significant reduction in number of colonies formed. Recently we have optimized an in vitro TE assay and are using this to screen GLI antagonists for effects on TE formation. Conclusion: we have identified a subset of GLI antagonists by phenotypic profiling for those with effects in cancer-specific functional assays that will be prioritized for testing in vivo. Funded in part by DOD/CDMRP IDEA (BC121850) (KPW). 1 Anderson, W. F., Schairer, C., Chen, B. E., Hance, K. W. &amp; Levine, P. H. Epidemiology of inflammatory breast cancer (IBC). Breast disease 22, 9-23 (2006). 2 Schlichting, J. A., Soliman, A. S., Schairer, C., Schottenfeld, D. &amp; Merajver, S. D. Inflammatory and non-inflammatory breast cancer survival by socioeconomic position in the Surveillance, Epidemiology, and End Results database, 1990-2008. Breast Cancer Research and Treatment 134, 1257-1268 (2012). 3 Yang, R. et al. A comprehensive evaluation of outcomes for inflammatory breast cancer. Breast cancer research and treatment 117, 631-641 (2009). 4 Thomas, Z. et al. Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104, 1575-1586, doi:10.1038/bjc.2011.133 (2011). 5 Williams, K. P. et al. Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Letters 337, 77-89 (2013). Citation Format: Helen Oladapo, Scott Sauer, Michael Tarpley, Gayathri Devi, Kevin P. Williams. Profiling of GLI antagonists in phenotypic models for effects on inflammatory breast cancer cell growth. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B49.

Abstract B44: Immune pathway dysregulations in inflammatory breast cancer health disparity

Abstract Background: Inflammatory breast cancer (IBC) is a distinct, aggressive and the most lethal form of breast cancer. Furthermore, data from population-based registries, when stratified by race and compared to other locally advanced breast cancers, suggest that IBC has a disproportionately high incidence, prevalence &amp; shorter median survival time in African Americans (AA) compared to Caucasian American (CA) patients. IBC health disparities that are independent of external factors like differences in income, screening rates, and access to health care suggest a biological component to poor outcomes. IBC pathobiology is also unique wherein instead of a solid tumor, tightly packed tumor cell clusters/emboli are formed with a propensity for lymphatic dissemination. The underlying mechanisms for this extremely aggressive breast cancer phenotype are largely unknown and, therefore, present a major impediment to identifying the molecular underpinnings for health disparities in IBC patients. Methods and Results: In order to understand how IBC cells evade cell death signals in the host microenvironment, we conducted comparative analysis of the largest collection of untreated primary tumor samples of IBC (N=137), along with stage- and subtype-matched non-IBC (N=252) and normal breast tissue. Results reveal that pretreatment tumor tissue samples from IBC patients have a heightened expression of adaptive stress response genes comprised of high levels of the nuclear transcription factor, NFkappaB, and its target genes in the anti-apoptotic and immune/inflammatory pathways that correlate with cell survival. In addition, guided by analysis based on presence of tumor infiltrating leukocytes in IBC (N=69) and non-IBC (N=62) patient samples, we have identified a set of pathways and genes that have the ability to distinguish the immune responses in IBC and non-IBC patients in a subtype independent manner. In particular, interferon alpha/beta and NFκB signaling pathways were differentially regulated in IBC vs. non-IBC. Gene expression driven analysis of enrichment of leukocyte subsets reveal specific differences in plasma cells and effector memory CD8 cells in IBC bulk tumor samples. Preliminary data analysis of gene expression data sets from laser microdissected epithelium and stromal compartments of AA and CA breast cancer patients also show enrichment of plasma cells, effector memory CD4+, and M1 macrophages. Studies are ongoing to characterize these immune profiles that are unique and distinguishable in IBC patients and further identify race-related differences. Most importantly, we discovered that the constitutive NFκB hyperactivation observed in IBC tumor cells is driven by stress-mediated translational upregulation of the most potent anti-apoptotic protein, XIAP. Interestingly, XIAP:NFκB overexpressing cells are highly resistant to immunotherapy mediated cell death. These data strongly suggest that in IBC, host immune phenotype promotes outgrowth of cell death resistant population and specific tumor cell pro-survival signaling mechanisms that enhances invasion and lymphatic dissemination. We have developed novel in vitro IBC tumor emboli culture model and a transgenic mouse bearing red fluorescent lymphatic vasculature that allow recapitulating host factors to define tumor cell signaling mechanisms on immune effector function and the resultant invasive phenotype. Conclusions: As the incidence is rising and targets for therapy are scant, IBC has the potential to become a major public health disparity concern. This study reveals that the immune factors in the host microenvironment may interact with underlying IBC genetics to promote the aggressive nature of the tumor, and more specifically, these factors may be one of the sources of biologic variation between patients of different ethnicities leading to IBC disparity outcomes. Supported in part by Duke IBC Consortium, DCI NIH CAO14236 development funds (GRD), DoD W81XWH-13-1-0047 (GRD). Citation Format: Gayathri R. Devi, Eun-Sil Shelley Hwang, John Stewart, Michael A. Morse, Steven Van Laere. Immune pathway dysregulations in inflammatory breast cancer health disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B44.