Human Inflammatory Breast Cancer Research Articles

RELT is overexpressed in human inflammatory bowel disease and breast cancer.

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR member that activates NF-kB and is implicated in several cancers. We therefore sought to characterize the association of RELT with inflammatory bowel disease (IBD) and breast cancer (BC). Additionally, we further characterized the association of RELT with Filamin A (FLNA), an actin-binding protein that can promote tumorigenesis, or counteract tumors, depending on its cellular location. Female CB-17 SCID mice were injected with anti-CD40 monoclonal antibody to induce colitis and euthanized seven days later. Immunohistochemistry (IHC) was utilized to compare RELT expression in the mouse colitis model with uninjected control mice. IHC was utilized to compare the expression of RELT in biopsies of human IBD and non-inflamed tissue, as well as human BC versus non-malignant tissue. Luciferase assays and an X-gal morphology assay were utilized to examine cell death in the BC cell line MDA-MB-231 (231). RELT immunostaining is significantly increased in both mouse colitis and human IBD biopsies in comparison to that of species-matched non-inflamed tissue. An apparent association between colonic histological damage and RELT immunostaining was observed in both mice and human specimens. RELT staining was also increased in human BC biopsies versus patient-matched non-malignant tissue. RELT overexpression induced death in 231 cells, an effect that was not abrogated by a RELT mutation in the binding site for the OXSR1 kinase. However, co-expression of a S2152A mutant of FLNA did abrogate the ability of RELT to induce death in 231 cells. Collectively, these results implicate RELT as being important for human disease and further our understanding of the interaction between RELT and FLNA.

Cell adhesion molecules E-cadherin and CADM1 are differently expressed in canine inflammatory mammary cancer

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and lethal types of mammary tumors with specific characteristics such as exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism. E-cadherin expression is another specific feature of IBC not previously studied in canine IMC. In this study, the expression of E-cadherin and CADM1 (Cell Adhesion molecule 1) and their possible role as key molecules involved in the pathogenesis of IMC were immunohistochemically analyzed in 19 canine IMC and 15 grade III non-IMC cases. E-cadherin and CADM1 expression was higher in IMC cases (p = 0.002, p = 0.008, respectively). In the IMC group, E-cadherin cytoplasmic immunolabeling was more frequent (p = 0.035) and it was associated to the expression of the angiogenic and lymphangiogenic factors COX-2 (p = 0.009), VEGF-A (p = 0.031) and VEGF-D (p = 0.008). The differential mRNA expression between IMC and non-IMC was studied by microarray analysis in 6 cases. E-cadherin gene (CDH1) was not up-regulated in IMC cases at a transcriptional level; interestingly CADM1 was 7-fold upregulated. The differential expression of E-cadherin protein in IMC suggests a possible role of E-cadherin in the characteristic exacerbated angiogenesis and lymphangiogenesis and further support IMC as a natural model for the study of human IBC. Future studies in IBC and IMC including a broad panel of adhesion molecules are necessary to elucidate their role in the metastatic process and angiogenesis.

Establishment of a new cell line of canine inflammatory mammary cancer: IMC-118.

Canine inflammatory mammary cancer (IMC) has long been regarded as an attractive animal model for research into human inflammatory breast cancer (IBC), Although some canine mammary tumour cell lines corresponding to human mammary cancer cell lines have been established, there is still a need to supplement the canine mammary tumour cell bank. The goal of this study was to create a new type of IMC cell line. The primary tumour, IMC-118, was identified as IMC by pathology examination. Immunohistochemistry analysis revealed negative immunoreactivity to oestrogen receptor (ER), but positive immunoreactivity to progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). Immunofluorescence (IF) analysis revealed that the IMC-118 cell line from this primary tumour was negative for ER but positive for PR and HER-2, and was also positive for epithelial and mesenchymal cell markers. This cell line was cultured stably for more than 50 passages and grew well after cryopreservation. In vivo, tumour masses and metastases in the lungs were discovered after inoculating the IMC-118 cells into the nude mice model. As a result, a novel canine IMC cell line, IMC-118, was effectively established, and could be employed as a promising model for immunotherapy and epithelial-mesenchymal transition mechanism of IMC research in both dogs and humans.

Paracrine IL-6 Signaling Confers Proliferation between Heterogeneous Inflammatory Breast Cancer Sub-Clones.

Simple SummaryThis study provides novel mechanistic insights into the capacity of the inflammatory cytokine IL-6 and its associated STAT3-dependent signaling pathway to stimulate proliferation in trans between individual sub-clones in a model of heterogeneity in inflammatory breast cancer. The clinical relevance of this discovery is provided by our observation that proliferation of the IL-6 responsive subclone is sensitive to inhibition with the clinically approved anti-IL-6 receptor humanized monoclonal antibody Tocilizumab. These findings therefore provide a rationale for potentially repurposing Tocilizumab for the treatment of a subset of inflammatory breast cancer patients.Inflammatory breast cancer (IBC) describes a highly aggressive form of breast cancer of diverse molecular subtypes and clonal heterogeneity across individual tumors. Accordingly, IBC is recognized by its clinical signs of inflammation, associated with expression of interleukin (IL)-6 and other inflammatory cytokines. Here, we investigate whether sub-clonal differences between expression of components of the IL-6 signaling cascade reveal a novel role for IL-6 to mediate a proliferative response in trans using two prototypical IBC cell lines. We find that SUM149 and SUM 190 cells faithfully replicate differential expression observed in a subset of human IBC specimens between IL-6, the activated form of the key downstream transcription factor STAT3, and of the HER2 receptor. Surprisingly, the high level of IL-6 produced by SUM149 cells activates STAT3 and stimulates proliferation in SUM190 cells, but not in SUM149 cells with low IL-6R expression. Importantly, SUM149 conditioned medium or co-culture with SUM149 cells induced growth of SUM190 cells, and this effect was abrogated by the IL-6R neutralizing antibody Tocilizumab. The results suggest a novel function for inter-clonal IL-6 signaling in IBC, whereby IL-6 promotes in trans proliferation of IL-6R and HER2-expressing responsive sub-clones and, therefore, may provide a vulnerability that can be exploited therapeutically by repurposing of a clinically approved antibody.

Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer

BackgroundInflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This...

High endogenous CCL2 expression promotes the aggressive phenotype of human inflammatory breast cancer

Inflammatory Breast Cancer (IBC) is a highly aggressive malignancy with distinct clinical and histopathological features whose molecular basis is unresolved. Here we describe a human IBC cell line, A3250, that recapitulates key IBC features in a mouse xenograft model, including skin erythema, diffuse tumor growth, dermal lymphatic invasion, and extensive metastases. A3250 cells express very high levels of the CCL2 chemokine and induce tumors enriched in macrophages. CCL2 knockdown leads to a striking reduction in macrophage densities, tumor proliferation, skin erythema, and metastasis. These results establish IBC-derived CCL2 as a key factor driving macrophage expansion, and indirectly tumor growth, with transcriptomic analysis demonstrating the activation of multiple inflammatory pathways. Finally, primary human IBCs exhibit macrophage infiltration and an enriched macrophage RNA signature. Thus, this human IBC model provides insight into the distinctive biology of IBC, and highlights potential therapeutic approaches to this deadly disease.

Blocking Estrogen Synthesis Leads to Different Hormonal Responses in Canine and Human Triple Negative Inflammatory Breast Cancer.

Simple SummaryEstrogen is responsible for tumor progression, and blocking its synthesis is effective in certain breast cancers. Therefore, the aim of this study is to determine the effect of letrozole (anti-aromatase) and STX-64 (anti-sulfatase) in canine and human inflammatory breast cancer cell lines and xenografts. The results reveal that letrozole blocks estrogen synthesis, reducing tumor progression. However, STX-64 increases estradiol synthesis, increasing tumor progression. In summary, letrozole may be an effective treatment for canine and human inflammatory breast cancer.Blocking estrogen synthesis by inhibitors of estrogen synthesis is a widely used therapy against estrogen receptor-positive tumors. However, these therapies are less effective in negative expression tumors. Therefore, this study determined the effectiveness of anti-aromatase and anti-sulfatase therapies in canine and human inflammatory breast cancer. Cell cultures and xenografts from IPC-366 and SUM149 were treated with different doses of letrozole (anti-aromatase) and STX-64 (anti-sulfatase), in order to observe their effectiveness in terms of cell proliferation, tumor progression, and the appearance of metastases and hormonal profiles. The results revealed that both treatments are effective in vitro since they reduce cell proliferation and decrease the secreted estrogen levels. In xenograft mice, while treatment with letrozole reduces tumor progression by 30–40%, STX-64 increases tumor progression by 20%. The hormonal results obtained determined that STX-64 produced an increase in circulating and intratumoral levels of estradiol, which led to an increase in tumor progression. However, letrozole was able to block estrogen synthesis by decreasing the levels of circulating and intratumoral estrogen and thus slowing down tumor progression. In conclusion, letrozole can be an effective treatment for canine and human inflammatory breast cancer. The knowledge of the hormonal profile of breast tumors reflects useful information on the effectiveness of different endocrine treatments.

Tumor Growth Progression in Ectopic and Orthotopic Xenografts from Inflammatory Breast Cancer Cell Lines.

Xenografts can grow in immunosuppressed hosts, such as SCID mice, and tumor material can be injected into hosts either ectopically or orthotopically. Choosing the correct model to use is a crucial step in animal research. The aim of this study was to report the differences between ectopic and orthotopic xenografts in tumor progression, metastasis capacity, histological features, and steroid hormone profiles in xenografts from the cIMC (canine inflammatory mammary cancer) cell line IPC-366 and hIBC (human inflammatory breast cancer) cell line SUM149. To achieve this purpose, 40 female mice 6–8 weeks old were inoculated with IPC-366 and SUM149 cells subcutaneously (ectopic models) or into mammary fat pad (orthotopic models). Mice were monitored for tumor progression and appearance of metastases, and generated tumors were analyzed in terms of histological examination and steroid hormone production. The results revealed differences in tumor appearance and percentage of metastasis between ectopic and orthotopic models, which were higher in the ectopic xenografts from both cell lines. However, both models had similar characteristics of tumor progression, histological features, and steroid hormone secretion profiles. We show that the ectopic model can be validated as a good and useful model of tumor development in addition to, not contrary to, the orthotopic model in breast cancer research.

Anti-Angiogenic Treatments Interact with Steroid Secretion in Inflammatory Breast Cancer Triple Negative Cell Lines

Simple SummaryInflammatory breast cancer (IBC) is the most aggressive breast cancer and is associated with poor prognosis. Exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism are hallmarks of this tumour. Current antiangiogenic therapies have minimal effects on overall survival in IBC patients. Furthermore, it is well established that steroid hormones are strongly related to tumour development and progression, angiogenesis regulation and metastasis. We investigated the effect of different antiangiogenic therapies on steroid and angiogenic growth factor production using two inflammatory breast cancer cell lines. We reported that sex steroid hormones could regulate the production of angiogenic factors, since after the results, P4 and E2 were involved in VEGF production and androgens in the formation of vascular-like structures. Moreover, we reported that elevated intratumoural concentrations of T and E1SO4 could be associated with decreased metastatic rates and the promotion of tumour progression, respectively, and thus the measurement of sex steroids and growth factors may be useful to develop preventive and individualised therapeutic strategies.Human inflammatory breast cancer (IBC) is a highly angiogenic disease for which antiangiogenic therapy has demonstrated only a modest response, and the reason for this remains unknown. Thus, the purpose of this study was to determine the influence of different antiangiogenic therapies on in vitro and in vivo steroid hormone and angiogenic growth factor production using canine and human inflammatory breast carcinoma cell lines as well as the possible involvement of sex steroid hormones in angiogenesis. IPC-366 and SUM149 cell lines and xenotransplanted mice were treated with different concentrations of VEGF, SU5416, bevacizumab and celecoxib. Steroid hormone (progesterone, dehydroepiandrostenedione, androstenedione, testosterone, dihydrotestosterone, estrone sulphate and 17β-oestradiol), angiogenic growth factors (VEGF-A, VEGF-C and VEGF-D) and IL-8 determinations in culture media, tumour homogenate and serum samples were assayed by EIA. In vitro, progesterone- and 17β-oestradiol-induced VEGF production promoting cell proliferation and androgens are involved in the formation of vascular-like structures. In vivo, intratumoural testosterone concentrations were augmented and possibly associated with decreased metastatic rates, whereas elevated E1SO4 concentrations could promote tumour progression after antiangiogenic therapies. In conclusion, sex steroid hormones could regulate the production of angiogenic factors. The intratumoural measurement of sex steroids and growth factors may be useful to develop preventive and individualized therapeutic strategies.

Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway.

The endoplasmic reticulum (ER) regulates protein folding, post-translational modifications, lipid synthesis, and calcium signaling to attenuate the accumulation of misfolded proteins causing ER stress and maintains cellular homeostasis. The tumor microenvironment is rich in soluble cytokines, chemokines, growth, and angiogenic factors and can drive the ER’s abnormal functioning in healthy cells. Cancer cells adapt well to the tumor microenvironment induced ER stress. We identified that the inflammatory breast cancer (IBC) cells abundantly express osteoprotegerin (OPG) and their tumor microenvironment is rich in OPG protein. OPG also called osteoclast differentiation factor/osteoclastogenesis inhibitory factor (OCIF) is a soluble decoy receptor for receptor activator of nuclear factor-kappa B ligand (RANKL). Employing mass spectrometry analysis, we identified a set of ER chaperones associated with OPG in IBC cell lysates (SUM149PT, SUM1315MO2) compared to healthy human mammary epithelial cells (HMEC). Proximity ligation assay (PLA) and immunoprecipitation assay validated the interaction between OPG and ER chaperone and master regulator of unfolded protein response (UPR) GRP78/BiP (glucose-regulated protein/Binding immunoglobulin protein). We detected remarkably high gene expression of CCAAT enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1α), protein disulfide-isomerase (PDI), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) in SUM149PT and SUM190PT cells when compared to HMEC. Similarly, tissue sections of human IBC expressed high levels of ER stress proteins. We evaluated cell death and apoptosis upon Salubrinal and phenylbutyrate treatment in healthy and IBC cells by caspase-3 activity and cleaved poly (ADP-ribose) polymerase (PARP) protein assay. IBC (SUM149PT and SUM190PT) cells were chemosensitive to Salubrinal treatment, possibly via inhibition in OPG secretion, upregulating ATF4, and CHOP, thus ultimately driving caspase-3 mediated IBC cell death. Salubrinal treatment upregulated PDI, which connects ER stress to oxidative stress. We observed increased ROS production and reduced cell proliferation of Salubrinal treated IBC cells. Treatment with antioxidants could rescue IBC cells from ROS and aborted cell proliferation. Our findings implicate that manipulating ER stress with Salubrinal may provide a safer and tailored strategy to target the growth of inflammatory and aggressive forms of breast cancer.

Exploration of the developmental process of vasculogenic mimicry in canine inflammatory mammary carcinoma

Canine inflammatory mammary carcinoma (CIMC) similar to human inflammatory breast cancer is a very aggressive, metastatic type of cancer. Previous studies have introduced a new type of tumor angiogenesis called vasculogenic mimicry that may play an important role in the progression of inflammatory mammary cancer. The purpose of this study is to investigate the development process of vessels by neoplastic cells in CIMC. Patient dog, 14-year old Shit-Tzu female, had a hard and somewhat movable dark-reddish mammary tumor, sized 6.2 cm in diameter. Bloody dark turbid exudate was released from the tumor. In addition to histological examination, immunohistochemistry for pancytokeratin, VCAM-1, MECA-32, TWIST-1, and Ki-67 was respectively performed using the ABC method. Histologically, the inflammatory mammary carcinoma was characterized by tubular solid tumor emboli within the lymphatic vessels surrounded by desmoplastic fibrous connective tissue. Some of the neoplastic cells were transforming into elongate or spindle shapes and forming small vessel-like structures in the solid tumor mass. The neoplastic cells were immunoreactive for VCAM-1 and MECA-32, but showed low immunoreactivity for Ki-67. Immunoreactive neoplastic cells for VCAM-1 and MECA-32 suggested the possibility that the neoplastic cells transform into endothelial cells of vessels by epithelial-mesenchymal transition, further supported by serial morphological changes identified by histological investigation and immunohistochemistry for TWIST-1. The high capacity of the neoplastic cells forming the vasculatures in CIMC explains the high ratio of metastasis to other regions, even though Ki-67 index was not so high.

Vasculogenic mimicry-associated ultrastructural findings in human and canine inflammatory breast cancer cell lines

BackgroundHuman inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most lethal mammary cancers. An exacerbated angiogenesis and the existence of vasculogenic mimicry (VM) are hallmarks of these tumors. The information regarding VM and ultrastructural characteristics of mammary cell lines is scant.MethodsIn this study, IBC cell line SUM149 and IMC cell line IPC-366 in adherent (2D) and non-adherent (3D) (mammospheres, cancer stem cells) conditions were analyzed by transmission and scanning electron microscopy (TEM and SEM, respectively).ResultsThe TEM revealed round to oval shape cells with microvilli on the surface, high numbers of peroxisomes in close apposition to lipid droplets and some extracellular derived vesicles. The TEM and the SEM mammospheres revealed group of cells clumping together with a central lumen (resembling a mammary acini). The cells joint are tight junctions and zonula adherens. By SEM two cell morphologies were observed: spherical and flattened cells. There was evidence endothelial-like cells (ELCs), which is characteristic for this disease, showing several or unique cytoplasmic empty space. ELCs were more frequent in 3D than in 2D culture conditions and contained Weibel-Palade cytoplasmic bodies, which are exclusive structures of endothelial cells.ConclusionsBoth cell lines, IPC-366 and SUM-149, shared ultrastructural characteristics, further supporting canine IMC as a model for the human disease. To the best of our knowledge, this is the first study that demonstrate the morphological differentiation of cultured cancer stem cells from cancer epithelial cell lines into endothelial-like cells, confirming the vasculogenic mimicry phenomenon from an ultrastructural point of view.

Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-β-catenin signalling, the stem cell phenotype and disease progression

The aim of the present study was to evaluate the expression levels of the aryl hydrocarbon receptor (AHR) and its target gene CYP1B1 and to correlate their expression with Wnt5a/b-β-catenin, the CD44+/CD24(−/low) cancer stem cell (CSC) subset and factors associated with poor prognosis in inflammatory breast cancer (IBC) and non-IBC patients. The methods of analysis used were quantitative real-time PCR, western blotting, immunohistochemistry and flow cytometry. Compared to non-IBC tissues, IBC tissues exhibited the overexpression of AHR and its target gene/protein CYP1B1. AHR and CYP1B1 mRNA levels were associated with the poor clinical prognosis markers tumour grade, lymphovascular invasion, cell proliferation and lymph node metastasis. Furthermore, AHR expression correlated with the expression of Wnt5a/b and β-catenin signalling molecules, and Wnt5a mRNA expression was downregulated in the SUM149 human IBC cell line and the MDA-MB-231 non-IBC cell line upon inhibition of AHR. AHR gene knockout (CRISPR-Cas9) inhibits CYP1B1 and Wnt5a expression in the IBC cell line. The CD44+/CD24(−/low) subset was significantly correlated with the expression of AHR, CYP1B1, Wnt5a/b and β-catenin in IBC tissues. The overexpression of AHR and its target CYP1B1 correlated with the expression of Wnt5a/b and β-catenin, CSCs, and poor clinical prognostic factors of IBC. Thus, targeting AHR and/or its downstream target molecules CYP1B1 and Wnt5a/b may represent a therapeutic approach for IBC.

Homeoprotein DLX4 expression is increased in inflammatory breast cancer cases from an urban African-American population.

Protein expression of Distal-less homeobox 4 (DLX4) was analyzed in inflammatory breast cancer (IBC) cases from an African-American (AA) population to determine if a) DLX4 gene over expression exists in this cohort and b) if the overexpression is associated with breast cancer clinicopathological characteristics (ER, PR, HER2, triple-negative). Twenty-nine blocks of formalin-fixed paraffin-embedded (FFPE) tissue from well-characterized human IBC cases were used for immunohistochemical staining (IHC). IHC results were assigned an intensity and percentage score. Percentage scores were assigned as 0, 1, 2, 3, or 4 and intensity scores were assigned 0, 1+, 2+ or 3+. For the analysis of the IHC, a percentage score of 3 or 4 and an intensity score of 2+ or 3+ were categorized as high. Chi-square or Fisher's exact tests were used to compare the high and low groups.In this cohort, 89.7% (26 out of 29) of IBC cases showed high percentages of positive cells staining for the DLX4 protein, while 40.0% (12 out of 30) of normal breast tissue from reduction mammoplasty cases demonstrated DLX4 expression (p < 0.01). In IBC patients, 65.5% of cases showed a high level of staining intensity, compared to 20.0% of normal breast tissues (test, p = 0.001). Intensity to DLX4 was higher in the HER2 negative status (78.3%) than the HER2 positive status (16.7%) (test, p = 0.011).DLX4 expression is higher in the IBC cases in this study of an urban AA population than in normal breast tissue cases. HER2 negative status is positively associated with high intensity of DLX4.

Abstract B47: Efficacy of GLI antagonists in triple-negative inflammatory breast cancer 2D and 3D models

Abstract Our objective was to investigate the differential effects of specific GLI1 antagonists in triple-negative inflammatory breast cancer 2D and 3D cell culture systems. Inflammatory breast cancer (IBC) accounts for 15% of all breast cancer deaths. IBC is a unique form of locally advanced breast cancer, an aggressive and highly invasive breast cancer having one of the worst clinical outcomes among breast cancers. African-American, American-Indian, and Arab-American women are disproportionately affected by IBC (1-3). Compared to non-IBC, IBC has a higher incidence of the triple-negative (TN) phenotype for which there are few treatment options. There are currently limited strategies for targeting IBC; thus, IBC patients continue to have worse survival outcomes than non-IBC breast cancer patients. GLI1 (glioma-associated oncogene homolog 1), the terminal effector of the hedgehog (Hh) pathway, has emerged as a potential therapeutic target for TN breast cancers including IBC (4). There is overwhelming evidence for GLI1 activation across many tumor types, including aggressive breast cancers, and its importance for tumor progression. Nuclear expression of GLI1 has been shown to be a strong predictor of poor prognosis in pancreatic cancer and brain gliomas and correlates with aggressiveness in basal-like breast cancer. In our studies, we utilized various automated phenotypic and functional assays (5) to examine the efficacy of GLI antagonists with varying mechanism of action for effects on IBC cell proliferation and growth in 2D and 3D cell culture systems. We are also assessing by high-throughput transcriptome sequencing (RNA-seq) gene expression differences in IBC cell lines after treatment with GLI antagonists. In both our 2D and 3D cell culture systems, TN-IBC cell lines were highly sensitive to two GLI antagonists, JK184 and GANT61. JK184 and GANT61 showed significant but differential effects on TN-IBC cell proliferation, colony formation, cell motility, and spheroid formation. This study was supported in part by Department of Defense award W81WXH-13-1-0141, NIH award P20CA202924, Komen Graduate Training in Disparities Research award GTDR16377604, Developmental Funds from Duke School of Medicine and Duke Cancer Institute (as part of the P30CA014236 and Cancer and Environment Program supported by Mr and Mrs Fred and Alice Stanback P3917733).

Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of canine inflammatory mammarycancer

BackgroundIndole-3-carbinol, derived from Cruciferous vegetables is an estrogen receptor antagonist considered a preventive agent that is naturally present in diet. There are no previous studies on its effects in human inflammatory breast cancer or canine inflammatory mammary cancer that is the most aggressive type of breast cancer.MethodsThe aim of this study was to analyze the effect of indole-3-carbinol on a SCID mice xenograft model of canine inflammatory mammary cancer, using equivalent human oral dose as a preventive therapy in humans for 3 weeks.ResultsIndole-3-carbinol treatment decreased tumor proliferation and increased apoptosis, although tumor embolization and liver metastasis were observed in some animals. There was a characteristic subpopulation of lipid-rich cells and increased contents of select steroid hormones in tumor homogenates and serum.ConclusionsOur data reveal for the first time that the ingestion of indole-3-carbinol, as administered, diminishes proliferation and increases apoptosis of tumor cells in an experimental model of inflammatory breast cancer, although this effect could not be enough to avoid the appearance of tumor embolization and metastasis. Future clinical trials will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved.

Abstract OT3-06-04: A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC)

Abstract Primary Objective: To determine pathological complete response (pCR) rate in patients with primary TNIBC treated with PaCT in comparison with CT, followed by AC. To explore if the pCR rate correlates with reduced nodal expression status; and with arginine methylation status of epidermal growth factor receptor (EGFR). We will identify molecular biomarkers predictive of the pCR rate by analysis of multiplexed immunohistochemical (IHC) staining, identify molecular biomarkers predictive of the pCR rate by genomic and proteomic analysis, and determine whether the inhibition of the EGFR pathway down regulates the COX-2 pathway and mesenchymal marker. Background: EGFR is overexpressed in triple negative breast cancer (TNBC) and inflammatory breast cancer (IBC). Therefore, EGFR targeted therapy may have a promising role in TNBC and IBC. A study showed that EGFR-targeted therapy may enhance the initial chemosensitivity of TNBC cells. Panitumumab blocks epidermal growth factor ligands and transforming growth factor EGFá (TGFá) binding to EGFR, inhibits tumor growth, and elicits both tumor regression and eradication of established tumors in murine xenograft tumor models. Panitumumab, a fully humanized anti-EGFR antibody, has been shown to be active in a breast cancer preclinical model using human breast cancer cell line MDA-MB-468, which has been shown to overexpress EGFR by both IHC and fluorescence in situ hybridization (FISH). Furthermore, EGFR tyrosine kinase inhibitors such as erlotinib have antitumor activity against human IBC cell lines. Thus, EGFR targeted therapy may have a promising role in TNBC and IBC. Study Design: In this open label randomized phase II trial, up to 72 patients with primary IBC, have no HER2 overexpression, and have &amp;lt;10% expression of ER and PgR, who also meet other criteria will be randomized to PaCT arm - receiving panitumumab single agent in window study and 4 cycles PaCT, or CT arm - receiving 4 cycles of CT. All patients will receive 4 cycles of AC before surgery. Statistical Considerations: A sample size of 36 patients per arm will achieve 84% power to detect a difference of 0.24 in pCR rate between 0.2 in the CT arm and 0.44 in the PaCT arm with a type I error rate of 10% using one-sided Z test. Based on historical data, we expect that the pCR rate of a PaCT regimen to achieve 24% additional efficacy compared with the CT regimen. Status of the study: Activation date: Oct. 2016. So far 6 patients have been enrolled. Enrollment continues. Sponsor: Amgen. State of Texas appropriation for rare and aggressive breast cancer research. NIH grant 1R01CA205043-01A1 Citation Format: Willey JS, Parker CA, Lim B, Valero V, Le-Petross HT, Krishnamurthy S, Woodward WA, Lucci A, Wood AL, Sun H, Babiera GV, Song J, Shen Y, Valero V, Wang X, Ueno NT. A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC) [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 OT3-06-04.

P-glycoprotein is Overexpressed in Human and Canine Inflammatory Breast Cancer Cell Lines But Not in Xenotransplanted Mouse Tumours

P-glycoprotein is Overexpressed in Human and Canine Inflammatory Breast Cancer Cell Lines But Not in Xenotransplanted Mouse Tumours

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

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.