Metastatic Progression Of Breast Cancer Research Articles

Abstract 4526: Tumor invasion and escape from an engineered solid-like aggregate of human breast cancer cells into a cavity

Abstract The mechanical properties of the tumor microenvironment (TME) play a critical role on the progression of breast cancer metastasis. However, the complex architecture of the TME conceals the individual effects of different biophysical and biochemical factors on tumor invasion and intravasation. To investigate this question, we engineered a robust breast tumor model of solid-like 3D aggregate of human breast cancer cells with interstitial fluid pressure (IFP), and further integrated it with an empty cavity to emulate the presence of an impaired capillary vessel. In brief, we embed MDA-MB-231 human breast cancer cells in one of two neighboring collagen type I cavities that are molded within polydimethylsiloxane (PDMS) channels. This multicellular aggregate is subject to selected gradients of hydrostatic pressure through opposing reservoirs of culture media that are located at the base (Pbase) and the tip (Ptip) of the tumor. We found that breast cancer cells disseminate from the multicellular aggregate and escape into the proximal cavity under Ptip > Pbase. The separation distance between the aggregate and the cavity influences the features of tumor escape. Tumor models that were seeded within a distance of less than 150 μm from the cavity demonstrated significantly shorter time (t1/2~ 4 days) for the escape of 50% of the tumor population than those seeded between 150 and 300 μm. In contrast, less than 50% of the tumors that were seeded longer than 300 μm apart of the cavity successfully escaped after ~ 2 weeks under Ptip > Pbase. In addition, we found that cells escaped into the cavity through three major modes: a) single-cell migration, b) multicellular invasion, and c) tumor growth. Single-cell migration was the dominant route of escape in collagen gels of low concentration (2.5 mg/ml). In contrast, tumor growth and multicellular invasion were the dominant modes of escape in collagen gels of high concentration (4mg/ml). Moreover, the tumor invasions were found to be preferentially directed normal to the surface of the tumor, and to be drastically eliminated in effect of pharmacological inhibition of matrix metalloproteinases (MMPs). These preliminary findings will be put together with additional quantitative studies to correlate tumor-cavity separation with the different modes of tumor escape. Overall, our engineered breast tumor model composes a unique platform to investigate the biophysical and biochemical mechanisms of the tumor microenvironment that drive tumor invasion and intravasation into the circulatory system. Citation Format: Andreas P. Kourouklis, Usman Ghani, Siyang Han, Yoseph Dance, Allison K. Simi, Joe Tien, Celeste M. Nelson. Tumor invasion and escape from an engineered solid-like aggregate of human breast cancer cells into a cavity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4526.

Abstract 1112: CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1

Abstract Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown. Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot. Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS (P=0.009; P=0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis. Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1. Citation Format: Jing Si, Rong Guo. CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1112.

Abstract 4553: Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow

Abstract Systemic immunosuppression in cancer is a mechanism of immune escape allowing for cancer progression to metastasis and remains a major pathophysiologic barrier to treatment including immunotherapies. The mechanism of this phenomenon remains relatively obscured. Although immune suppressive humoral factors secreted by primary tumors are thought to be a major cause of immune escape, a major question remains whether factors critical for cancer metastasis also dually regulate systemic immunosuppression to prepare peripheral sites for the eventual encounter with metastasizing cancer cells. To that end, we report here that the master cancer epithelial-mesenchymal transition (EMT) initiating factor Snail1, whose expression is restricted to the primary tumor where it promotes the generation of invasive breast tumor-initiating cells, yet also has a direct role in creating a pro-tumor systemic immune environment to promote metastasis. These dual roles for Snail1 in breast cancer progression has not been elucidated. Using both spontaneous genetic murine model of breast cancer and a syngeneic orthotopic breast cancer model we demonstrate that Snail1 is uniquely required for metastatic spread of breast tumor cells while also directly modulates the bone marrow niche, skewing it towards a suppressive immune phenotype with an upregulation of myeloid derived suppressor cells (MDSCs) and a decrease in antigen presenting cell (APC) populations. Importantly, deletion of Snail1 specifically in primary tumors severely abrogated metastasis and simultaneously restored bone marrow immune populations to levels in non-tumor bearing animals. In vitro co-culture assays demonstrated that humoral factors from Snail1-repleted cancer cells promoted MDSC formation and suppressed APCs when compared to Snail1-deficient cancer cells. Mechanistically, Snail1 modulates systemic immunosuppression specifically through 1) upregulating the expression of GM-CSF, CXCL2, and CCL2, factors important for MDSC induction and recruitment, within the primary tumor to saturate the periphery with these factors; and 2) generating metastatic cells that may suppress immune functions within the metastatic niche. Taken together, these data demonstrate Snail1’s central role in metastatic progression in breast cancer through its direct generation of metastatic cells and suppressing the systemic immune system to increase the chance that these metastatic cells may survive the periphery. Note: This abstract was not presented at the meeting. Citation Format: Mathew Sebastian, Dongjiang Chen, Son Le, Duy Nguyen, Changwang Deng, Dan Jin, Nagheme Thomas, David Tran. Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4553.

Abstract 972: Direct observation in living tumors shows macrophage-dependent induction and dissemination of cancer stem cells in breast cancer

Abstract Cancer stem cells (CSCs) play an important role during metastatic progression of breast cancer. However, the in vivo properties and dynamic behavior of CSCs are not well understood. Here, we employed high-resolution intravital multiphoton microscopy using a SOX2/OCT4 responsive fluorescent stem cell biosensor to directly observe CSC dynamics in the living animal using an orthotopic breast cancer xenograft model. We report that CSCs constitute a minority population (1-3%) in the primary tumors, and display the slow-migratory, invasive phenotype that is specifically associated with disseminating tumor cell population. We also report, for the first time, that CSCs are preferentially localized in direct contact with macrophages near and in tumor microenvironment of metastasis (TMEM) sites, the macrophage-containing intravasation doorway for tumor cells and that CSCs metastasize to lung and are strikingly enriched in early lung metastatic colonies. This is explained by our observation that, in vitro and in vivo, direct physical contact with macrophages induces stemness in non-stem cancer cells via juxtacrine Notch-Jagged1 signaling. These data indicate for the first time that macrophages play an actively inductive role in the CSC niche and promote TMEM-mediated CSC intravasation and early metastatic seeding. Citation Format: Ved P. Sharma, Yarong Wang, Binwu Tang, George S. Karagiannis, Emily A. Xue, David Entenberg, Lucia Borriello, Anouchka Coste, Chinmay R. Surve, Dominic Esposito, Maja H. Oktay, Lalage M. Wakefield, John S. Condeelis. Direct observation in living tumors shows macrophage-dependent induction and dissemination of cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 972.

Abstract 408: Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC)

Abstract Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs. Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics. Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs. Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis. Citation Format: Qiang Zhang, Lorezo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Richard G. Pestell, Firas Wehbe, Amir Behdad, Leonidas Platanias, William Gradishar, Massimo Cristofanilli. Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 408.

Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation

BackgroundVascular maturity and functionality are closely associated with tumor progression and chemosensitivity. The antidiabetic agent metformin has shown its ability to inhibit tumor angiogenesis in metastatic breast cancer models. However, it remains unclear if or how metformin remodels the abnormal vasculature of metastatic breast cancer, while inhibiting angiogenesis.MethodsMetastatic breast cancer models were constructed to compare microvessel density (MVD), vascular maturity and function, lung metastasis and chemosensitivity in metformin-treated or untreated mice. Protein array assay and transcriptome sequencing were performed for genetic screening. Lentiviral shRNA-PDGF-B transfection was used for observing the contribution of PDGF-B knockdown to metformin’s vascular effects.ResultsMetastatic breast cancers were characterized by an excessively angiogenic, immature and morphologically abnormal vasculature. Compared to control, metformin significantly reduced MVD, leakage and hypoxia, and increased vascular mural cells coverage and perfusion, namely, “vessel normalization”. Metformin at human blood concentrations had no direct effect on the migration and proliferation of cancer cells. Based on that, reduced lung metastasis of the primary tumor and improved chemosensitization by metformin were assumed to be mediated via metformin’s vascular effects. Further results of genetic screening and in vivo experiments showed that the downregulation of platelet-derived growth factor B (PDGF-B) greatly contributed to the metformin-induced vessel normalization.ConclusionsThese findings provide pre-clinical evidences for the vascular mechanism of metformin-induced metastasis inhibition and the chemosensitization of metastatic breast cancers.

IL-4/IL-13 Stimulated Macrophages Enhance Breast Cancer Invasion Via Rho-GTPase Regulation of Synergistic VEGF/CCL-18 Signaling.

Tumor associated macrophages (TAMs) are increasingly recognized as major contributors to the metastatic progression of breast cancer and enriched levels of TAMs often correlate with poor prognosis. Despite our current advances it remains unclear which subset of M2-like macrophages have the highest capacity to enhance the metastatic program and which mechanisms regulate this process. Effective targeting of macrophages that aid cancer progression requires knowledge of the specific mechanisms underlying their pro-metastatic actions, as to avoid the anticipated toxicities from generalized targeting of macrophages. To this end, we set out to understand the relationship between the regulation of tumor secretions by Rho-GTPases, which were previously demonstrated to affect them, macrophage differentiation, and the converse influence of macrophages on cancer cell phenotype. Our data show that IL-4/IL-13 in vitro differentiated M2a macrophages significantly increase migratory and invasive potential of breast cancer cells at a greater rate than M2b or M2c macrophages. Our previous work demonstrated that the Rho-GTPases are potent regulators of macrophage-induced migratory responses; therefore, we examined M2a-mediated responses in RhoA or RhoC knockout breast cancer cell models. We find that both RhoA and RhoC regulate migration and invasion in MDA-MB-231 and SUM-149 cells following stimulation with M2a conditioned media. Secretome analysis of M2a conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Results from our functional assays reveal that M2a TAMs synergistically utilize VEGF and CCL-18 to promote migratory and invasive responses. Lastly, we show that pretreatment with ROCK inhibitors Y-276332 or GSK42986A attenuated VEGF/CCL-18 and M2a-induced migration and invasion. These results support Rho-GTPase signaling regulates downstream responses induced by TAMs, offering a novel approach for the prevention of breast cancer metastasis by anti-RhoA/C therapies.

Long non-coding RNA MAGI2-AS3 inhibits breast cancer cell migration and invasion via sponging microRNA-374a.

Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts that play important roles in tumorigenesis and tumor progression. Our study aimed to explore the role of lncRNA MAGI2-AS3 in breast cancer metastatic progression. In the present study, our results showed that MAGI2-AS3 can inhibit the migration and invasion of breast cancer cells. In addition, an increase in MAGI2-AS3 can inhibit microRNA-374a (miR-374a) expression in breast cancer cells. Bioinformatic analysis predicted the correlation between MAGI2-AS3 and miR-374a. Phosphatase and tensin homolog (PTEN) was found to be an novel mRNA target of miR-374a. MAGI2-AS3 upregulation inhibited breast cancer metastatic progression by decreasing miR-374a and enhancing PTEN expression. Together, our data revealed that lncRNA MAGI2-AS3 is involved in breast cancer cell progression by regulating the miR-374a-PTEN axis. These findings offer new insight into treatment strategies for breast cancer.

Abstract P4-08-08: Genomic progression, detected by circulating tumor DNA (ctDNA) sequencing, as an early predictor of disease progression in metastatic breast cancer (MBC)

Abstract Background: The availability of multiple therapies has transformed the landscape of MBC, but also brought the challenge of selecting the right therapy for an individual patient. Furthermore, in patients with Hormone Receptor positive (HR+) breast cancer who have bone metastases only it may be difficult to assess effectiveness of therapy via imaging. Peripheral ctDNA detection and analysis by next-generation sequencing (NGS) has gained popularity in cancer diagnosis and therapeutics due to its relative noninvasiveness, ease of use, and high sensitivity. Here, we explore the utility of ctDNA change as a predictor for disease progression in MBC. We hypothesized that genomic progression is a harbinger of subsequent radiologic progression in patients with MBC. Methods: We analyzed change from pre-treatment (baseline) to on-treatment ctDNA mutant allele fraction (MAF) among patients with MBC. Patients receiving standard-of-care therapies or investigational agents on clinical trials at our institution were included. All patients were followed from the date of baseline test until death or data cutoff (6/20/2018). All peripheral blood specimens were collected and analyzed between 1/7/2016 and 3/1/2018 via NGS (Guardant360®). Peripheral blood specimens were sequenced prior to initiation of a new therapeutic regimen (baseline) and subsequently at least once while on-treatment, on average 4-12 weeks later. All patients had a follow-up CT scan of chest, abdomen and pelvis 2-4 weeks after the on-treatment NGS. A priori, we defined genomic progression as increase in ctDNA total MAF of at least 20% from baseline. We utilized Cox regression analysis to identify whether genomic progression was a predictor of radiologic progression, adjusting for common prognostic variables. Results: All patients (N= 77) were female, predominantly White (83.1%), and median age was 57 (range 32 to 77). Fifty one out of 77 patients (66.2%) were ER+, 5 HER2+, and 9 had triple negative breast cancer. The median MAF at baseline was 2.2% (range 0% - 61.7%). Common genomic alterations in ctDNA included PIK3CA, TP53, ESR1, AKT1, NF1. 27 out of 77 (35%) patients showed disease progression on the first subsequent CT scan, while 59 out of 77 (76.6%) progressed during the follow up time. We found that an increase in ctDNA MAF of at least 20% was a strong predictor of disease progression (HR =2.46, CI [1.14-5.32], p=0.02), compared to those who had a MAF increase of less than 20% or a decrease in total MAF. In multi-variable analysis, adjusting for age, number of prior therapies, type of therapy, and visceral metastases, increase in ctDNA remained a significant predictor for subsequent disease progression (HR =3.84, CI [1.63-9.07], p=0.002). Subset results in patients with bone metastases only, and relative comparison of ctDNA with standard tumor markers will be presented at the meeting. Conclusions: Genomic progression, identified by an increase in ctDNA MAF, is potentially an early predictor of subsequent disease progression in patients with MBC. Further research is needed to prospectively evaluate the clinical utility of ctDNA change as a surrogate marker in guiding treatment decision-making for patients with MBC. Citation Format: Velimirovic M, Juric D, Niemierko A, Spring LM, Vidula N, Malvarosa G, Yuen M, Moy B, Isakoff SJ, Ellisen LW, Bardia A. Genomic progression, detected by circulating tumor DNA (ctDNA) sequencing, as an early predictor of disease progression in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-08.

Abstract P3-08-19: Proteomic tracking of breast cancer metastasis progression

Abstract Background Metastases from breast cancers have not yet reached curability mainly because of the continuous evolution of cancer cells leading to treatment resistance and tumor progression. Despite the identification of the main genomic drivers of breast cancer resistance, and the development of specific targeted therapies, resistance has not been eradicated. A deeper understanding of tumor biology underlying treatment escape and tumor progression is necessary to find new targets. The aim of this study was to determine whether mass spectrometry based imaging and microproteomics are able to capture changes in protein expression and pathways occurring during metastasis progression. Methods Ten tumor samples from five progressing metastatic breast cancer patients treated at Oscar Lambret Cancer Center (France) were selected based on the availability of paired metastasis biopsy performed at two time points during the evolution of the disease. The proteomic profiles of paired tumor samples were analyzed using MALDI mass spectrometry imaging combined with microproteomics, a spatially resolved proteomic technology. Results Comparison of paired samples showed gain and loss of proteins associated with tumor progression. However, few were shared between patients. The pathways and biological processes mainly represented during tumor progression, and which were found in at least two patients, were those involved in the negative regulation of leukocyte mediated cytotoxicity, the metabolism of carboxilic acid, peptides and nucleosides, in response to stress, oxidation-reduction process, endocytosis, catabolic processes, actin regulation, and extracellular matrix organization. During metastasis evolution, few shared proteins were identified in at least 3 patients such as SRPX2, CILP1, collagen alpha-2(V) chain, Ras-related C3 botulinum toxin substrate 1, filamin-C, PDZ and LIM domain protein 2 and chloride intracellular channel protein 4. The main proteins lost during progression and found in at least 2 patients were cell surface glycoprotein MUC18, collagen alpha-2(IV) chain, polyadenylate-binding protein 2, and latent-transforming growth factor beta-binding protein 4 (LTAGP4). Results from an expanded cohort will be presented at the meeting. Conclusion MALDI mass spectrometry proteomics identified private and shared changes in proteins and biological processes associated with breast cancer metastasis progression. Citation Format: Hajjaji N, Aboulouard S, Robin YM, Bertin D, Fournier I, Bonneterre J, Salzet M. Proteomic tracking of breast cancer metastasis progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-19.

Intravital imaging reveals systemic ezrin inhibition impedes cancer cell migration and lymph node metastasis in breast cancer

BackgroundLimited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The regional lymph nodes are the most common site of metastasis in most solid cancers and their involvement is a strong predictor of relapse in breast cancer (BC). We have previously shown that ezrin, a cytoskeletal–membrane linker protein, is associated with lymphovascular invasion and promotes metastatic progression in BC. However, the efficacy of pharmacological inhibition of ezrin in blocking cancer cell migration and metastasis remains unexplored in BC.MethodsWe quantified ezrin expression in a BC tissue microarray (n = 347) to assess its correlation with risk of relapse. Next, we developed a quantitative intravital microscopy (qIVM) approach, using a syngeneic lymphatic reporter mouse tumor model, to investigate the effect of systemic ezrin inhibition on cancer cell migration and metastasis.ResultsWe show that ezrin is expressed at significantly higher levels in lymph node metastases compared to matched primary tumors, and that a high tumor ezrin level is associated with increased risk of relapse in BC patients with regional disease. Using qIVM, we observe a subset of cancer cells that retain their invasive and migratory phenotype at the tumor-draining lymph node. We further show that systemic inhibition of ezrin, using a small molecule compound (NSC668394), impedes the migration of cancer cells in vivo. Furthermore, systemic ezrin inhibition leads to reductions in metastatic burden at the distal axillary lymph node and lungs.ConclusionsOur findings demonstrate that the tumor ezrin level act as an independent biomarker in predicting relapse and provide a rationale for therapeutic targeting of ezrin to reduce the metastatic capacity of cancer cells in high-risk BC patients with elevated ezrin expression.

Changes in Biomarker Status in Metastatic Breast Cancer and Their Prognostic Value.

PurposeThere is cumulative evidence that changes in biomarker status occur frequently during the metastatic progression of breast cancer and affect treatment response. The purpose of this study was to evaluate the frequency of biomarker changes in metastatic breast cancer (MBC) and its impact on prognosis.MethodsA total of 152 patients diagnosed with MBC at the time of initial diagnosis or during post-surgical follow-up were included. Changes in biomarker status in MBCs, their frequency according to various metastatic sites, tumor characteristics, and their association with patient survival were analyzed.ResultsEstrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 status changed in 9 (6.0%), 40 (26.3%), 12 (7.9%), and 29 (19.1%) patients, respectively. ER, PR, and HER2 mainly showed positive to negative conversion, whereas Ki-67 changed mostly from a low to high index. There were no differences in the frequencies of biomarker changes according to the metastatic sites. As for ER and HER2, cases with negative conversion showed low expression levels in the primary tumor. Survival analyses indicated that a positive to negative conversion of ER was an independent poor prognostic factor in patients with primary ER-positive breast cancer.ConclusionChanges in biomarker status are not rare, and usually occur in an unfavorable direction in breast cancer metastases. Negative conversion of ER status is a predictor of poor prognosis. Thus, it is beneficial to evaluate changes in biomarker status in MBC not only for the purpose of determining treatment options but also for prognostication of patients.

Plasma exosomes stimulate breast cancer metastasis through surface interactions and activation of FAK signaling

The interaction between malignant cells and surrounding healthy tissues is a critical factor in the metastatic progression of breast cancer (BC). Extracellular vesicles, especially exosomes, are known to be involved in inter-cellular communication during cancer progression. In the study presented herein, we aimed to evaluate the role of circulating plasma exosomes in the metastatic dissemination of BC and to investigate the underlying molecular mechanisms of this phenomenon. Exosomes isolated from plasma of healthy female donors were applied in various concentrations into the medium of MDA-MB-231 and MCF-7 cell lines. Motility and invasive properties of BC cells were examined by random migration and Transwell invasion assays, and the effect of plasma exosomes on the metastatic dissemination of BC cells was demonstrated in an in vivo zebrafish model. To reveal the molecular mechanism of interaction between plasma exosomes and BC cells, a comparison between un-treated and enzymatically modified exosomes was performed, followed by mass spectrometry, gene ontology, and pathway analysis. Plasma exosomes stimulated the adhesive properties, two-dimensional random migration, and transwell invasion of BC cells in vitro as well as their in vivo metastatic dissemination in a dose-dependent manner. This stimulatory effect was mediated by interactions of surface exosome proteins with BC cells and consequent activation of focal adhesion kinase (FAK) signaling in the tumor cells. Plasma exosomes have a potency to stimulate the metastasis-promoting properties of BC cells. This pro-metastatic property of normal plasma exosomes may have impact on the course of the disease and on its prognosis.

Sustained prognostic impact of circulating tumor cell status and kinetics upon further progression of metastatic breast cancer.

Serial longitudinal enumeration of circulating tumor cells (CTCs) has shown its prognostic value on progression-free survival and overall survival (OS) in patients with stage IV breast cancer. This study prospectively evaluated the role of CTCs as a prognostic marker during further progression of metastatic breast cancer (MBC). Among 476 MBC patients recruited between 2010 and 2015, the 103 patients with a known CTC status at baseline (CTCBL) and within 4weeks of tumor progression (CTCPD) were included. Progressive disease (PD) was defined according to the Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1). Using the CellSearch method, < 5 and ≥ 5 CTCs per 7.5ml blood were determined as negative and positive, respectively. A shift in CTC status from baseline to progression ([Formula: see text] to [Formula: see text] and vice versa) was considered as alternating KineticsBL-PD. Median follow-up was 29.9 [21.2, 40.0] months. CTCPD positivity (37%, n = 38) was associated with a significantly shorter OS than CTCPD negativity (8.0 [5.1, 10.9] vs 22.6 [15.3, 39.8] months; P < 0.001). Alternating KineticsBL-PD was observed in 24% of the patients. This significantly changed the OS prediction of [Formula: see text] patients ([Formula: see text] vs [Formula: see text], 11.4 [9.7, not available (NA)] vs. 7.6 [4.4, 11.5] months; P = 0.044) and [Formula: see text] patients ([Formula: see text] vs. [Formula: see text], 8.4 [4.0, NA] vs. 22.6 [18.9, NA] months, respectively; P < 0.001). Prediction of survival was significantly improved (P = 0.002) by adding CTCPD status to clinicopathological characteristics and CTCBL status. CTC status upon further disease progression is a prognostic factor that could significantly improve well-established models. Thus, it represents a potential additional instrument supporting treatment decision.

Hyaluronidases: A Therapeutic Enzyme.

Hyaluronidase is the most important enzyme found in the interstitial matrix, effectively degrading the hyaluronic acid. Hyaluronidases are extensively found in the venom of various animals such as snake, scorpion, spider and others. Up till now five venom hyaluronidases are identified with a defined three-dimensional structure. These enzymes are involved in different biochemical, physiological and pathological conditions like degradation of hyaluronic acid, embryogenesis, transmembrane diffusion of drugs and toxins, inflammatory and allergic response to antigens, healing of wounds, bacterial meningitis, bacteremia and pneumonia. These enzymes are used as an adjuvant therapy in cancer and to expedite the dispersion and absorption of drugs as well as to reduce the tissue edema. and to reduce the progression of metastatic breast cancer as well as used in ophthalmological procedures in combination with local anesthetics, reduction of dermatological aging, regulation of hyaluronan, as a spreading factor and with numerous clinical applications. In this review an attempt was made to summarize the physiological, adjuvaent and therapeutic significance of hyaluronidase enzymes from different animal sources.

Abstract B26: Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels

Abstract For the past four decades, dietary fat intake has composed a significant portion of the typical Western diet, at 33-35% of total kilocalories. Approximately 8% of this dietary fat is from consumption of deep fat fried foods. Oil has been demonstrated to be absorbed by food during frying and contributes between 8 to 40% of the food’s final weight. As oils are used and reused for frying, complex chemical reactions take place creating deleterious polar compounds (e.g., acrylamide, sterol derivatives, or other reactive oxidative species.) Although these compounds are associated with health risks, i.e., heart disease, diabetes mellitus, and cancer, there is no regulation in the United States for upper limits of polar material in frying oil. This raises an important question as to whether thermally abused fryer oil (TAFO) can alter metastatic breast cancer progression. We initially conducted an in vivo study in which a modest amount of dietary fat, 25% of kilocalories, came from fresh fryer oil or TAFO. Ovariectomized BALB/C mice fed the diet containing TAFO for 16 weeks and inoculated with luciferase-expressing 4T1 murine mammary cancer cells had significantly more metastasis to lungs than control-fed animals (p &amp;lt; 0.05, n=21). Serum metabolic analysis in these animals revealed significantly elevated levels of fumarate, a known onco-metabolite, in TAFO-fed compared with control-fed animals (p &amp;lt; 0.05, n=9). To confirm that fumarate was mechanistically involved in metastatic progression, we conducted in vitro studies. 4T1 cells exposed to 500 nM to 10 µM monomethyl fumarate (MMF), a cell-permeable form of fumarate, trended toward an increase in the healing rate over 24 hours in a wound healing (scratch) assay compared to control (n =2). This preliminary evidence supports the hypothesis that fumarate accumulation creates a protumorigenic and metastatic environment and needs confirmation in subsequent investigation. This information can help elucidate mechanistic details of the effects of TAFO on breast cancer metastasis. Further work will assess rates of 4T1 cellular proliferation and invasion induced by MMF. In summary, dietary intake of TAFO increased metastasis of tumor cells to lungs, and dysregulation of the metabolite fumarate may be mechanistically involved. Citation Format: Jennifer R. Hughes, Kaitlyn M. Joyce, Anthony Cam, William G. Helferich, Zeynep Madak-Erdogan. Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B26.

Abstract 4037: Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells

Abstract Evidence suggests that growth of cancer stem cells (CSCs) drives intratumor heterogeneity, a major clinical problem that results in drug resistance, recurrence of tumors, and metastatic progression of breast cancer. Therapeutic strategies to eliminate CSCs require identification of signaling pathways that facilitate the emergence and/or expansion of CSCs. These signaling pathways are implicated in altering cell behavior, as well as promoting the expression of genes involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Previous work demonstrated that a cooperative STAT3/SMAD3 gene transcription program increased the expression of Hyaluronan Synthase 2 (HAS2), an enzyme that produces Hyaluronic Acid (HA) which serves as a CD44 ligand and facilitates EMT. We hypothesize that STAT3 is a critical regulator of HA synthesis and signaling which enhances EMT and stemness of human mammary epithelial cells, driving tumor progression. We tested this hypothesis in a TGFβ triggered model of EMT. Immortalized human mammary epithelial cells (HMLE) expressing either a control vector (pWZL-GFP) or a vector containing TGFβ1 (pWZL-TGFβ1-GFP) were characterized with respect to STAT3 signaling, HA synthesis, and mammosphere formation. Our data revealed that HMLE-pWZL-TGFβ1-GFP cells have significantly higher expression of active STAT3 (Y705) compared to control cells, but total STAT3 gene and protein expression were similar in the two cell lines. HMLE-pWZL-TGFβ1-GFP cells exhibited a significant increase in expression of HAS2 and HAS2-AS1, a long non-coding RNA that enhances the transcription of HAS2. Preliminary results show that overexpression of TGFβ1 increased HA secretion in media compared to control cells. HMLE-pWZL-TGFβ1-GFP cells demonstrated significantly higher mammosphere forming ability compared to control cells, suggesting that TGFβ increases the percentage of stem cells in the population. Initial results demonstrated changes in mammosphere size and morphology of HMLE-pWZL-TGFβ1-GFP cells treated with the HA synthesis inhibitor 4-Methylumbelliferone (4MU). Together, these results suggest STAT3 and TGFβ1 co-regulate HAS2 expression and HA production in a model of EMT supporting the use of the HMLE-pWZL cell series as a promising model to study STAT3 regulation of the HA-CD44 signaling pathway. Citation Format: Brenda G. Trevizo-Barresi, JoEllen Welsh. Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4037.

Abstract 997: The cholesterol metabolite 27-hydroxycholesterol promotes breast cancer progression by affecting immune responses

Abstract With upwards of 90% of mortalities associated with breast cancer being attributed to metastasis, there is an urgent need to better understand what drives this stage of disease. In this regard, it has been reported that an elevated concentration of circulating cholesterol is an independent risk factor for breast cancer recurrence. Supporting the relationship between cholesterol and breast cancer progression, the use of cholesterol lowering drugs (statins) has been demonstrated to improve recurrence-free survival. These clinical observations suggest that cholesterol influences the metastatic progression of breast cancer. Therefore, in this study we directly tested the impact of cholesterol on breast cancer metastasis, and interrogated the downstream mechanisms by which it may be doing so. In strong support of our hypothesis, increased metastasis was observed when mice were placed on a high cholesterol diet. These findings highlighted recent work by us and others demonstrating that the primary metabolite, 27-hydroxycholesterol (27HC), was able to modulate the activities of two hormone receptors: the estrogen and the liver X receptors. Thus, we pursued a series of experiments to determine whether 27HC was responsible for the metastatic actions of cholesterol. 27HC is synthesized from cholesterol by the enzyme CYP27A1. The genetic ablation of CYP27A1 completely attenuated the effects of a high cholesterol diet, implicating 27HC as a primary mediator of cholesterol. Treatment with exogenous 27HC robustly increased metastasis. Importantly, genetic or pharmacologic inhibition of CYP27A1 also reduced the basal metastatic burden, indicating that this enzyme may be a suitable target for the prevention and/or treatment of metastatic breast cancer. Intriguingly, we found that the pro-metastatic effects of 27HC also required the presence of myeloid cells, which was demonstrated by their ablation using clodronate-loaded liposomes. In the absence of myeloid cells, the ability of 27HC to promote metastasis was dramatically reduced. Whilst interrogating the distal metastatic site, we found that this oxysterol enriched Ly6G+/CD11b+ polymorphonuclear-neutrophils (PMNs) and γδ T cells. The induction of metastasis by 27HC was lost in models where either PMNs or γδ T cells were ablated. We further demonstrate that 27HC treatment results in a decrease in cytotoxic CD8+ T cells, suggesting that the net result of the 27HC enriched γδ T cells and PMNs is local immune suppression. Collectively, our results demonstrate that cholesterol increases metastasis via the actions of its metabolite 27HC, which exerts its effects through γδ T cells and PMNs to suppress acquired immunity. Since 27HC acts on the host environment to promote breast cancer progression, these results strongly support the immediate translational potential of targeting the 27HC pathway for the prevention and treatment of metastasis. Citation Format: Amy E. Baek, Sisi He, Hannah B. McDowell, Erik R. Nelson. The cholesterol metabolite 27-hydroxycholesterol promotes breast cancer progression by affecting immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 997.

Abstract 1073: Breast cancer intravasation signature from patient fine-needle aspirates

Abstract A key step in the progression of metastatic breast cancer is the process of blood vessel intravasation. Understanding the molecular mechanisms that underlie this crucial step is essential in designing strategies to block the same with an aim to prevent metastatic disease. Recently our group has developed and published an in vitro intravasation assay (1) that recapitulates the process of intravasation and allows the use of patient derived breast cancer cells. Previously our group had identified a breast cancer invasion signature that helps facilitate clinical decision making in breast cancer patients (2). Here using fine needle aspirates from breast cancer resections we collected viable cancer cells which were labeled green using vital dyes and mixed with a macrophage cell line labeled red and layered on the top compartment of a transwell. The bottom of the transwell membrane contains a layer of extracellular matrix and a tight layer of HUVECs. Green cancer cells were separated one at a time using a florescent microscope and a micromanipulator. The cancer cells capable of intravasation are divided into two groups, one group that has crossed the entire layer and fallen to the bottom of the well, and a second group that crossed the endothelial cells but are still bound to the bottom of the endothelial layer. Cancer cells from top of the transwell which did not intravasate were also collected. RNA was extracted from the few cells collected by micromanipulation from all the three groups, amplified and converted to Cy3 labeled cDNA. The labeled cDNA was hybridized to whole genome microarrays. Gene expression was compared between the three groups of cells. Both unsupervised and supervised data analysis was performed on the three groups of cells from five patients. Significance Analysis of Microarrays, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis was performed on these datasets. Supervised analysis and validation of the altered genes was performed using qRT-PCR. Preliminary data analysis indicates the upregulation of the genes associated with Breast Cancer Stem cells and DNA repair. This is the first report of whole genome gene expression analysis from cells collected by FNA from patients, separated in vitro into intravasation competent and intravasation incompetent and unsupervised whole genome analysis performed to identify intravasation specific genes and pathways. Once unique targets and pathways are identified in intrvasation competent cells, a number of additional function blocking steps will be designed and undertaken. (1) Pignatelli J., Goswami S., et. al. Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration. Sci Signal. 2014 Nov 25;7(353) (2) Karagiannis GS., Goswami S., Jones JG., Oktay MH., and Condeelis JS. Signatures of breast cancer metastasis at a glance. J Cell Sci. 2016 May 1;129(9):1751-8. Citation Format: Gargi Bandyopadhyaya, Srinjoy Goswami, John S. Condeelis, Maja H. Oktay, Sumanta Goswami. Breast cancer intravasation signature from patient fine-needle aspirates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1073.

OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

BackgroundSystemic and chronic inflammatory conditions in patients with breast cancer have been associated with reduced patient survival and increased breast cancer aggressiveness. This paper characterizes the role of an inflammatory cytokine, oncostatin M (OSM), in the preintravasation aspects of breast cancer metastasis.MethodsOSM expression levels in human breast cancer tissue samples were assessed using tissue microarrays, and expression patterns based on clinical stage were assessed. To determine the in vivo role of OSM in breast cancer metastasis to the lung, we used three orthotopic breast cancer mouse models, including a syngeneic 4T1.2 mouse mammary cancer model, the MDA-MB-231 human breast cancer xenograft model, and an OSM-knockout (OSM-KO) mouse model. Progression of metastatic disease was tracked by magnetic resonance imaging and bioluminescence imaging. Endpoint analysis included circulating tumor cell (CTC) counts, lung metastatic burden analysis by qPCR, and ex vivo bioluminescence imaging.ResultsUsing tissue microarrays, we found that tumor cell OSM was expressed at the highest levels in ductal carcinoma in situ. This finding suggests that OSM may function during the earlier steps of breast cancer metastasis. In mice bearing MDA-MB-231-Luc2 xenograft tumors, peritumoral injection of recombinant human OSM not only increased metastases to the lung and decreased survival but also increased CTC numbers. To our knowledge, this is the first time that a gp130 family inflammatory cytokine has been shown to directly affect CTC numbers. Using a 4T1.2 syngeneic mouse model of breast cancer, we found that mice bearing 4T1.2-shOSM tumors with knocked down tumor expression of OSM had reduced CTCs, decreased lung metastatic burden, and increased survival compared with mice bearing control tumors. CTC numbers were further reduced in OSM-KO mice bearing the same tumors, demonstrating the importance of both paracrine- and autocrine-produced OSM in this process. In vitro studies further supported the hypothesis that OSM promotes preintravasation aspects of cancer metastasis, because OSM induced both 4T1.2 tumor cell detachment and migration.ConclusionsCollectively, our findings suggest that OSM plays a crucial role in the early steps of metastatic breast cancer progression, resulting in increased CTCs and lung metastases as well as reduced survival. Therefore, early therapeutic inhibition of OSM in patients with breast cancer may prevent breast cancer metastasis.