BC Metastasis Research Articles

Abstract PS14-14: Distinct tumor microenvironments of breast cancer bone metastases in pre- and post-menopausal patients

Abstract Breast cancer (BC) patient prognosis has improved over the past 2 decades with a 99% 5 year survival rate for localized BC, yet metastatic BC continues to cause high mortality with a 5 year survival rate of 27%. Recent advances have been made in the treatment of primary BC, providing an opportunity to apply this positive momentum towards treating metastatic disease. Approximately 70% of BC metastases occur in the bone, with the majority of patients retaining estrogen receptor positive (ER+) disease. The BC tumor microenvironment (TME) in bone exhibits an osteolytic bone pathology of bone destruction and is composed of primarily four cellular groups including the cancer, immune, vascular and bone cells. These cellular constituents are uniquely responsive to estradiol (E2) and exhibit unique TMEs dependent on menopause status, when circulating E2 is lost. Both menopause and BC endocrine therapies induce an E2-deprived post-menopausal state in patients. However, studying the distinctions of pre- and post-menopausal BC patient TMEs is still necessary to understand how natural menopause status impacts non-treatment naïve ER+ BC that is already in a therapy induced menopausal state. In particular the full extent of the unique phenotypes harbored by pre- versus post-menopausal ER+ BC tumor induced bone disease (TIBD) remains unknown, and elucidation of these difference may identify novel mechanisms of TIBD. We investigated a cohort of demineralized formalin-fixed and paraffin-embedded (FFPE) pre- and post-menopausal lytic bone metastasis biopsies from non-treatment naïve ER+ BC patients. We utilized immunohistochemistry (IHC), NanoString nCounter gene expression panels and GeoMx Digital Spatial Profiling (DSP) to observe distinct TMEs in our archival biopsy cohort. IHC staining of pre- and post-menopausal samples displayed alterations to immune cell infiltration and composition. Differential gene expression analysis revealed enrichment of myeloid function in pre-menopausal patients compared to post-menopausal patients. Alterations to immune signaling pathways and cell profiles were also exhibited in pre-menopausal patients compared to post-menopausal patients. DSP analysis presented changes in both the tumor and microenvironment, and unique immune cell infiltration with checkpoint proteins in both pre-menopausal and post-menopausal ER+ BC bone metastases. The identification of these targetable markers unique to pre- and post-menopausal TIBD could provide new therapeutic strategies for ER+ BC patients with bone lesions. Thus, we propose that bone biopsies could be performed to guide selection for metastatic BC patients into appropriate therapeutic interventions, with pre- and post-menopausal patients receiving tailored treatments based on their distinct protein and RNA expression of available therapies. This personalized approach to treating metastatic disease could enhance patient quality of life and survival. Citation Format: Claire Ihle, Meredith Provera, Desiree Straign, Katy Zolman, Erin Smith, Adrie Van Bokhoven, Scott Lucia, Philip Owens. Distinct tumor microenvironments of breast cancer bone metastases in pre- and post-menopausal patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS14-14.

Abstract PS18-36: Proteomic profiling of specific tumor clones using spatially resolved mass spectrometry technologies for precision oncology

Abstract Background Breast cancer (BC) remains a leading cause of cancer-related death among women worldwide. The complexity of this disease, especially its heterogeneity, have prevented its eradication and driven resistance to treatments. To reach precision oncology to eradicate BC, therapy needs to be specific to each tumor clone. Yielding enough molecular information from tumor clones to identify new drug targets represents a technical challenge due to sample size limitation or loss of spatial resolution. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry combined with microproteomics enables a spatially-resolved unlabeled tumor imaging of its protein distribution, thus revealing proteomic clones. Our aims were to analyze the clonal proteome of luminal breast cancers, and explore its potential to expand new drug target discovery and drug repurposing. Methods A retrospective study at the Comprehensive Cancer Centre Oscar Lambret (Lille, France) was conducted to analyze 76 FFPE luminal HER2 negative tumors: 52 primary tumors from patients with early BC and 24 BC metastases. Patients gave their informed consent and the study was approved by the local institutional review board. MALDI mass spectrometry imaging and spatially-resolved on-tissue shotgun microproteomics were performed on FFPE slides of tumor tissue to determine the proteomic profile of selected clones using nanoLC-MS & MS/MS. Protein identification was performed using MaxQuant software against the Uniprot database. Functional annotation and characterization of the identified proteins were performed using Panther software. Candidate druggable targets were searched using DrugCentral druggable genome database, and their druggability level was assessed using the classification by the Illuminating the Druggable Genome Knowledge Management Center. The clonal proteome dataset was compared to publically available TCGA, BC360, and CDx datasets. Results The clonal proteome analysis identified a total of 2868 different proteins; 780 proteins were found in more than 50% of the patients. Panther analysis showed that 22% of the proteins were classified as enzymes, 15% were related to DNA processes, 6% were structural proteins, and less than 2% were related to immunity. Panther identified 139 pathways in the clonal proteome dataset. The clonal proteome analysis yielded the highest number of pathways compared to TCGA, BC360, and CDx datasets. 41 pathways (mainly metabolic pathways) were exclusive to the clonal proteome dataset. 1495 proteins of this dataset had an entry and were druggable in DrugCentral database, with 52% of them with known mechanisms of action and drug interaction. The main target classes were enzymes (60%), kinases (23%) and transporters (7%), whereas kinases were dominant in TCGA, BC360, and CDx datasets (46% to 77%). To explore the clonal proteome potential for repurposing anticancer drugs in luminal breast cancers, protein targets matching approved antineoplastic agents were searched using DrugCentral database. 97 approved anticancer drugs were identified, mostly chemotherapy (33%) or protein kinase inhibitors (28%), of whom only 17 were approved for breast cancer treatment. Compared to publically available TCGA, BC360, and CDx datasets, the clonal proteome analysis yielded the highest number of drug target candidate. Conclusion Mass spectrometry-based analysis of BC proteomic clones provides the technological means to access large functional molecular information at a clonal level to develop clone specific strategies for drug target discovery and drug repurposing in BC. Citation Format: Nawale Hajjaji, Soulaimane Aboulouard, Delphine Bertin, Yves Marie Robin, Isabelle Fournier, Michel Salzet. Proteomic profiling of specific tumor clones using spatially resolved mass spectrometry technologies for precision oncology [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-36.

Abstract 669: Combined inhibition of AKT & TBK1 for the treatment of breast cancer

Abstract Breast cancer (BC) is the most frequently diagnosed malignancy and the second leading cause of cancer mortality in Western women. In ~80% of BC cases, the PI3K-AKT pathway is aberrantly activated, due to the alterations in genes encoding the pathway components, such as Ras, Her2, PTEN, PIK3C and AKT. This pathway regulates multiple cellular processes to promote BC development, growth, metastasis, and drug resistance. Consequently, over 100 clinical trials are currently underway worldwide to evaluate the therapeutic efficacy of PI3K and AKT inhibitors in BC; however, initial data reported so far revealed that inhibition of this pathway is either not effective or often results in development of resistance and relapse of the disease. Thus, identification of additional targets and therapeutic combinations are urgently needed. We previously mapped TRAF2 phosphorylation sites (Ser-11 and Ser-55), identified PKC and TBK1 are the relevant kinases, and reported that TRAF2 phosphorylation enhances NF-κB activation to promote cancer cell survival under conditions of cellular stresses. Recently, we discovered that inhibition of AKT leads to increased phosphorylation of TRAF2 at Ser-11 by TBK1 in 4 out of 6 commonly used BC cell lines. TBK1 (aka T2K for TRAF2-associated kinase) and its close homologue IKKϵ are serine/threonine kinases overexpressed in ~70% of BC and play critical roles in BC cell survival. Interestingly, all 4 BC cell lines that exhibited enhanced TRAF2 phosphorylation were completely resistant to AKT inhibition-induced apoptosis in vitro, and combined inhibition of both AKT and TBK1/IKKϵ synergistically induced apoptosis in these BC lines. Importantly, combined inhibition of AKT and TBK1/IKKϵ also significantly suppresses xenograft BC tumor growth in vivo. Mechanistically, AKT did not directly inhibit TBK1, IKKϵ or TRAF2; it seems that an unknown kinase suppressed by AKT in cancer cells in normal culture conditions enhances TBK1-mediated TRAF2 Ser-11 phosphorylation in BC cells upon AKT inhibition. Nevertheless, our data suggest that inhibition of the PI3K-AKT pathway derepresses the pro-survival TBK1-TRAF2 pathway, which in turn confers resistance to PI3K-AKT inhibition in BC, and that combined inhibition of AKT and TBK1 effectively suppresses BC tumor growth. These findings also provide strong rationales for PDX and clinical investigation of combined AKT and TBK1 inhibition for the treatment of human BCs. Citation Format: Hasem Habelhah, Laiqun Zhnag. Combined inhibition of AKT & TBK1 for the treatment of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 669.

Effect of interleukins (IL-2, IL-15, IL-18) on receptors activation and cytotoxic activity of natural killer cells in breast cancer cell.

Breast cancer is one of the leading cause of cancer deaths in women. Metastasis in BC is caused by immunosurveillance deficiency, such NK cell maturation, low NK activity and decreasing cytotoxicity. This study was performed to improve activating receptors and cytotoxicity of NK cells using interleukins (ILs). Human recombinant IL-2, -15, and -18 were used to induce NK cells. We measured the activating and inhibiting receptors, proliferation activity of NK cells, and the cytotoxicity of NK cells on BC cells (MCF7). The effects of ILs were tested on the NK cell receptors CD314, CD158a and CD107a with flowcytometry, proliferation at various incubation times with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and concentrations of TNF-α and IFN-γ by NK cells with ELISA. ILs increased NK cell receptor levels (CD314, CD158a, and CD107a) at 24 hours of incubation. ILs increased NK cell viability, which increased with longer incubation. Moreover, ILs-induced NK cells inhibited proliferation in MCF7 cells, as well as increased TNF-α, IFN-γ, PRF1 and GzmB secretion. IL-2, IL-15, and IL-18 improved activating receptors and proliferation of NK cells. IL-induced NK cells increased TNF-α, IFN-γ, PRF1 and GzmB secretion and cytotoxic activity on BC cells. High NK cell numbers increased BC cell growth inhibition.

CCL5 protein level: influence on breast cancer staging and lymph nodes commitment.

Many tumor cells express chemokines and chemokine receptors, and these molecules can contribute to distinct modes of metastasis processes. It is known that they play a crucial role inbreast cancer (BC) tumorigenesis and progression. Considering this, it was investigated a possible role for C-Chemokine receptor type 5(CCR5) polymorphism (rs333/delta32) by conventional polymerase chain reaction (PCR) and CCL5 (C-C motif chemokine ligand 5) protein level by immunosorbent assay (ELISA) in 47 BC patients (resulting in 47 tumoral tissue samples and 47 adjacent normal tissue samples). There was a significant difference between CCL5 level in tumoral and adjacent normal tissues for the same BC patients (p < 0.0001). A significant association was also found for CCL5 level in relation to lymph nodes commitment (p = 0.03). Likewise, there was a significant difference in CCL5 level from tumor tissue of stage III in relation to stage I (p < 0.02). On the other hand, it was verified that CCR5-delta32 polymorphism presented no significant association in relation to CCL5 protein level. Considering the present findings, we suggest that CCL5 may be involved in BC staging and metastasis process.

Abstract 2044: CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts

Abstract Background: Transport of tumor cells via the circulation (CTCs) is a key step in metastasis. CTC clusters and in particular, cancer stem cell-like cell (cCSC) clusters exhibit enhanced metastatic capacity over individual CTCs or cCSCs. Cancer associated fibroblasts (CAFs) are critical players in the breast tumor microenvironment (TME) and are also found in metastases. In clinical and pre-clinical studies, we have identified circulating CAFs (cCAFs) and cCAF/CTC co-clusters as potential biomarkers for breast cancer progression and metastasis. Our data suggest that the ability of breast cancer cells (BCCs) to cluster with CAFs depends on their intrinsic metastatic capacity, and specifically, their CSC-like phenotype, characterized by expression of the CD44 cell surface marker: both in vivo and in in vitro cluster assays, only metastatic BCCs could form co-clusters while non-metastatic BCCs could not. Further, both in vivo and in vitro, co-clustering potential was marked by a stem cell-like phenotype, including increased CD44 expression. We hypothesize that cCAF/cCSC co-clusters are the functional units of metastasis. The goal of this current study is to identify mechanisms that influence co-clustering between CTCs and cCAFs and specifically to investigate the role of CD44 in co-clustering. Methods: Co- and mono-clusters of CAFs and BCCs were established in vitro by plating equal numbers of CAFs/BCCs into ultra-low attachment plates. After 24 hrs, clusters were collected and injected into female NSG mice via tail vein. Metastases was monitored by IVIS and confirmed by IHC. Mice were sacrificed at specific time points and blood collected by cardiac puncture. In vitro-generated clusters and blood were filtered using the faCTCheker microfluidic filtration instrument (Circulogix) to capture circulating cells with an 8-micron pore filter. Filters were stained by immunofluorescence for tumor cells, CAFs, and CD44. Hyaluronin (HA) was visualized with biotinylated HA binding protein. anti-CD44 siRNA (Dharmacon smart pool) was used to knockdown CD44 in BCCs or in CAFs Results: Injection of CAF/BCC co-clusters resulted in accelerated metastases compared to mono-cluster injection. In both co-clusters captured from mouse blood and established in vitro, HA, a CD44 ligand secreted by CAFs as well as produced by cancer cells, was observed. Elimination of HA in CAFs and BCCs reduced co-cluster formation. Knockdown of CD44 expression in BCCs via siRNA abrogated BCC co-clustering ability. Conclusion: In vitro and in vivo models of induced and conditional knockout of CD44 are defining the role of cancer cell-autonomous or CAF-autonomous CD44 in cCAF/CTC co-clustering and BC metastasis. These studies implicate CD44 as a driver of co-clustering between BCCs and CAFs. Inactivation of CD44 may abrogate co-clustering as well as impair the CSC phenotype, thus impacting critical pathways in BCC metastasis. Citation Format: Benjamin Troness, Angela Spartz, Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Marc Lippman, James McCarthy, Dorraya El-Ashry. CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts [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 2044.

Abstract 2520: Analysis of the targeted sequencing with RNA Seq. results between primary breast cancer and metastatic sites: Matched paired analysis

Abstract Background and Purpose In several breast cancer analyses of targeted gene panels, there was considerable concordance of mutations observed between primary tumors and matched metastases. We are to investigate to gain an understanding of the underlying genetics leading to BC metastasis to compare genomics data using paired biopsies between primary breast and several metastatic sites. Methods We performed CancerSCANTM of forty-eight patients with paired primary and metastatic tumors, which is 380-gene targeted panel sequencing using HiSeq 2500 sequencing platform (Illumina). We made variant calls using Mutect for SNV, Pindel for INDEL, Contra for CNV, and in-house software for Fusion with default parameters. Tumor mutational burden (TMB) was measured by number of mutations per megabase (mb), and TMB was divided into two groups: high TMB (&amp;gt;=20mutations/mb) and low TMB (&amp;lt;20mutations/mb). Mutational signatures were estimated using R deconstructSigs package, and divided into high and low group by each signature’s median value. Twelve patients among this cohort were performed whole-transcriptome sequencing with TruSeq RNA prep kit (Illumina) on primary and/or metastatic fresh-frozen tumors. Results Seven among 48 patients (14.6%) showed discordant subtypes between primary and metastatic tumors, discordant patterns were diverse. Lung and pleural was the most frequent organ in metastatic tumors, and liver and lymph node were followed. There were 15 shared, 6 primary-specific, 4 metastatic-specific mutations per patient on average, and no significant difference in overall survival (OS) with respect to mutation-sharing patterns. TMB and number of mutations related to mismatch-repair (MMR) genes such as NF1 (P=0.0398) and PMS2 (P=0.1020) tended to be reduced in metastatic tumors compared to paired primary tumors. Mutational signature 3 (BRCA1/2) was increased in metastatic tumors, and signature 5 (General Cancer) was decreased (P&amp;gt;0.05). defect in MMR genes (P=0.0445) and germline BRCA mutation (P=0.0186) were significantly associated to OS, but TMB status, and mutational signature related to MMR respectively were not significant variables for OS according to the molecular profiles in primary tumors. Patients with PMS2 (P=0.156) and NF1 (P=0.0734) multi-hit mutations were better prognosis. Combining molecular profiles in primary tumors such as TMB and mutational signature 6 (MMR) and signature 13 (APOBEC) showed significant differences in OS: low TMB with high signature 6 plus 13 as worse, high TMB as intermediate, and low TMB with low signature 6 plus 13 as better prognosis (P=0.0228). Conclusions With the combination of TMB and MMR-related molecular profiles, we could stratify patients into three groups according to OS. Citation Format: Kyung Hee Park, Eunjin Lee, Seri Park, Se Kyung Lee, Seok Won Kim, Jeong Eon Lee, Seok Jin Nam, Ji-Yeon Kim, Hee Kyung Ahn, Woong-Yang Park, Yeon Hee Park. Analysis of the targeted sequencing with RNA Seq. results between primary breast cancer and metastatic sites: Matched paired analysis [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 2520.

MicroRNA-3648 Is Upregulated to Suppress TCF21, Resulting in Promotion of Invasion and Metastasis of Human Bladder Cancer

Although microRNAs (miRNAs) are well-known for their potential in cancer, the function and mechanisms of miR-3648 have barely been explored in any type of cancer. We show here that miR-3648 is upregulated in human BC tissues in comparison with adjacent non-tumor tissues. Functional studies showed that inhibition of miR-3648 expression in the human invasive BC UMUC3 and T24T cell lines decreased migration and invasion in vitro and suppressed lung metastasis in vivo, whereas miR-3648 overexpression promoted BC cell migration and invasion. A bioinformatics screen and mRNA 3′ UTR luciferase reporter assay showed that transcription factor 21 (TCF21) was a direct target of miR-3648, and the results obtained from using a miR-3648 inhibitor revealed that miR-3648 inhibited TCF21 protein expression by reduction of its mRNA stability. Further, Kisspeptin 1 (KISS1) was identified as a TCF21 downstream effector responsible for miR-3648-mediated BC invasion and lung metastasis. Collectively, the present results suggest that miR-3648 is overexpressed and plays an oncogenic role in mediation of BC invasion and metastasis through directing the TCF21/KISS1 axis, revealing miR-3648 as a potential biomarker for BC prognosis and a target for BC therapy.

Abstract P3-01-23: Targeting of endocrine therapy-induced estrogen-receptor/HER2-cross-talk in circulating tumor cells from metastatic ER+/HER2- breast cancer: Implications for treatment of ER+/HER2- breast cancer

Abstract Background: Transient induction of HER2 expression upon inhibition of the estrogen-receptor (ERα) might be an underestimated rescue mechanism that enables ER+/HER2- breast cancer cells to survive and to grow in the presence of endocrine therapies. Besides protecting ER+/HER2- BC cells from endocrine treatment, transient HER2 expression is also considered to save circulating, disseminated and dormant ER+/HER2- BC cells from apoptotic cell death in hostile environments and might therefore be of importance for ER+ BC progression, metastasis formation and recurrence. Thus therapeutic strategies that efficiently target both ERα and HER2 simultaneously might improve treatment of patient with ER+/HER2- BCs. Methods: Circulating tumor cells (CTCs) were originally isolated from a patient with bilateral ER+/HER2- metastatic BC. These cells have been treated with either the proteasome inhibitor (PI) carfilzomib or fulvestrant alone or with both drugs in combination. The consequences of these drugs upon ERα and HER2 expression were monitored by western blotting or qPCR. Induction of cell death upon treatment was measured by PARP and caspase cleavage or by quantification of SubG1 cells using propidium iodide staining or by the use of colony forming assays. Results: Fulvestrant treatment of CTCs decreases the amount of ERα but immediately increases HER2 expression whereas carfilzomib markedly inhibits the expression of both ERα and HER2 simultaneously. Combined treatment of CTCs with carfilzomib + fulvestrant cause reduced expression of HER2 and lead to a much stronger decrease of ERα than carfilzomib or fulvestrant alone. Fulvestrant causes no significant reduction of proliferation and no induction of cell death. Conversely the combination of carfilzomib and fulvestrant causes a significant induction of apoptotic cell death and a massive reduction of colonies in colony forming assays. Conclusion: These findings suggest that rapid and transient up-regulation of HER2 expression following endocrine treatment might be an important so far underestimated adaptive mechanism which enables ER+/HER2- BC cells to sustain proliferation in the presence of ERα-inhibitory drugs and to stay alive during the metastatic process. Furthermore these data also lead to the assumption that PIs such as carfilzomib in combination with ERα degraders could be a potential therapeutic strategy for efficient targeting of metastatic ER+/HER2- BC cells. Citation Format: Thaler S, Roßwag S, Pantel K, Sleeman JP, Schmidt M, Cotarelo CL. Targeting of endocrine therapy-induced estrogen-receptor/HER2-cross-talk in circulating tumor cells from metastatic ER+/HER2- breast cancer: Implications for treatment of ER+/HER2- breast cancer [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 P3-01-23.

Ursolic acid a promising candidate in the therapeutics of breast cancer: Current status and future implications.

Breast cancer [BC] is the deadliest neoplasm in women globally and the second leading cause of cancer associated deaths. Current treatment methods include chemotherapy, hormonal therapy, radiation therapy and surgery. However, BC has shown resistance to these therapies and are often associated with side effects, multidrug resistance, recurrence are the major issues in BC treatment. Currently, dietary phytocompounds have emerged as beneficial agents for the prevention and treatment of cancer because of their safe and cost effective nature. Ursolic acid [UA] is widely spread in fruits and vegetables having the ability to inhibit BC proliferation, angiogenesis and metastasis, arrest cell cycle, induced apoptosis, scavenge free radicals and regulate several anti-apoptotic and pro-apoptotic proteins. UA has also shown potential anticancer, anti-inflammatory and antioxidant activities in several human BC cells. This review paper encompasses the role of UA against BC and their mechanism of action in vitro and in vivo studies.

Abstract 2598: In silico gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 10-20% of all BC cases, and is characterized by an aggressive clinical course with high risk of metastasis and lack of targeted therapy. Gene expression profiling revealed the heterogeneity of TNBC enabling its classification into distinct molecular subtypes with distinct susceptibilities to chemotherapies; including basal-like (BL), mesenchymal (M), and luminal androgen receptor (LAR) subtypes. Overexpression of the parathyroid hormone-related protein (PTHrP) in breast cancer has been extensively linked to its progression with increased propensity for bone metastasis. However, its expression and implication in organ-specific metastasis in the TNBC subtype remains largely unknown. In this study, we conducted in silico analyses to examine the association of PTHrP with various molecular BC subgroups and with organ-specific metastasis in TNBC. Breast Cancer Gene-Expression Miner Version 4.0 (bc-GenExMiner v4.0) online database was used to evaluate the relative mRNA expression levels of the PTHLH gene (which encodes PTHrP) with respect to other markers. This microarray-based tool uses 36 public datasets and 5861 patients, and allows the expression, correlation, and prognostic analyses of genes in BC. Using the gene expression correlation analysis module of bc-GenExMiner v4.0, we assessed Pearson's correlation coefficient between PTHLH expression and gene signatures characteristic of TNBC molecular subtypes or representative of organ-specific metastasis in BC. We found that PTHLH expression displays significant positive correlations with components of signalling pathways enriched in the mesenchymal subtype, and with key luminal markers and AR signalling genes characteristic of the LAR subtype. While PTHLH expression presents significant positive correlations with signature genes involved in bone and lung metastases in all BC subtypes, we identified for the first time a correlation between PTHLH expression and brain metastasis specifically in TNBC patients. Interestingly, PTHLH correlates with the brain metastatic genes HBEGF (Heparin-binding EGF-like growth factor) and ANGPTL4 (Angiopoietin-like 4) selectively in both TNBC and BL subtypes as opposed to other BC subtypes, which is in line with studies reporting their common morphological and genetic features and increased rate of brain metastasis. In conclusion, our in silico analysis reveals for the first time a strong association between PTHrP expression and specific TNBC molecular subtypes' markers, as well as its potential role in the mechanisms of TNBC brain metastasis and the identification of a novel gene signature with a prognostic value for brain progression in TNBC. Funding: Department of Defense (DoD, USA) Award No. W81XWH-15-1-0723 Citation Format: Gloria Assaker, Anne Camirand, Siham Sabri, Richard Kremer. In silico gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer [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 2598.

Abstract 5079: Role of syntaxin-1A, a synaptic-related protein, in HER2-enriched and luminal B breast cancer progression

Abstract Breast cancer (BC) is the most prevalent cancer in women. It can be classified into 4 subtypes according to their gene expression signature, each one characterized by its aggressiveness and treatment approach. Mortality has declined as a result of earlier diagnoses and the availability of adjuvant therapies. However, a significant number of patients do not respond to these therapies or develop resistance after an initial response. The therapies currently available fail at curing or preventing BC tumor progression and metastasis. Therefore, there is an urgent need to develop novel therapeutic tools to improve BCs patients´ survival rates. The interaction of cancer cells with the stroma has been well established and has led to exciting new therapies. However, the presence and relevance of nerve axons and their secreted neurotransmitters and neuropeptides in the tumor microenvironment has been acknowledged only very recently. We have identified 6 nervous system related genes differentially expressed in BC subtypes. Among them, Syntaxin-1A (STX1A) is up-regulated in luminal B and HER2-enriched tumors, and its expression significantly correlates with a shorter overall survival and metastasis free survival. Therefore, we hypothesized that STX1A regulates luminal B and HER2 enriched tumors progression and metastasis development. STX1A belongs to the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins superfamily that regulate membrane fusion events, including exocytosis and neurotransmission, intracellular trafficking, and cell proliferation. To test its role in breast cancer we have characterized STX1A pathway in a panel of BC cells. Our results showed that STX1A is overexpressed in luminal B and HER2-enriched cell lines at protein and mRNA level, as we have seen in patient's database. Moreover we have analyzed STX1A expression in cell lines that were derived either from lung or brain metastases respectively and we have discovered that the cells that have migrated to the brain or to the lung have increased STX1A expression in comparison to the parentals. Moreover, downregulation of STX1A using siRNA results in inhibition of epithelial to mesenchymal markers, such as Twist-1, indicating a possible role of STX1A in tumor cell migration and metastasis. Furthermore, downregulation of STX1A also represses FGFR2 activation, suggesting that STX1A might regulate tyrosine kinase receptors membrane trafficking. In conclusion we propose STXIA and SNARE proteins as potential biomarkers and targets for novel BC therapy. Citation Format: Aleix Noguera-Castells, Leire Recalde-Percaz, Núria Moragas, Patricia Fernández-Nogueira, Gemma Fuster, Anna López-Plana, Patricia Jauregui, Pere Gascón, Paloma Bragado, Mario Mancino. Role of syntaxin-1A, a synaptic-related protein, in HER2-enriched and luminal B breast cancer progression [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 5079.

A novel long non-coding RNA linc-ZNF469-3 promotes lung metastasis through miR-574-5p-ZEB1 axis in triple negative breast cancer.

Triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. The major sites for TNBC metastasis include the lungs, brain, liver, and bone. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides and have been reported as important regulators in BC metastasis. However, the underlying mechanisms for lncRNAs regulating TNBC metastasis are not fully understood. Here we found that linc-ZNF469-3 was highly expressed in lung-metastatic LM2-4175 TNBC cells and overexpression of linc-ZNF469-3 enhanced invasion ability and stemness properties in vitro and lung metastasis in vivo. Furthermore, we found linc-ZNF469-3 physically interacted with miR-574-5p and overexpression of miR-574-5p attenuated ZEB1 expression. Importantly, endogenous high expressions of linc-ZNF469-3 and ZEB1 were correlated with tumor recurrence in TNBC patients with lung metastasis. Taken together, our findings suggested that linc-ZNF469-3 promotes lung metastasis of TNBC through miR-574-5p-ZEB1 signaling axis and may be used as potential prognostic marker for TNBC patients.

Abstract P5-03-11: Targeted disruption of transcriptional effector GLI2 attenuates breast tumor growth and metastasis

Abstract We reported that transcriptional regulation by the major Hedgehog (HH) pathway effector GLI2 in mammary gland stromal cells coordinates a hormone-responsive niche signaling program that directs epithelial stem cell activity during puberty [Zhao, et al., Science. 2017 Apr 21;356(6335)]. HH signaling is a key growth pathway in human carcinogenesis. The first targeted therapies aimed at G-protein coupled receptor Smoothened (SMO) resulted in rapid acquired resistance, underscoring the need for developing new HH pathway-targeting therapies downstream of SMO. In BC GLI-dependent transcription may involve a non-receptor-based mechanism of GLI2 activation involving cross-talk from other signaling pathways such as TGFß54, PI3K55,56, Wnt, or NF-κB. Accordingly, we investigated the role of HH pathway and its transcriptional effectors using the MMTV-PyMT BC mouse model. Development of mammary tumors arising proceeds through neoplastic lesions ranging from carcinoma in situ (10-12 weeks) to highly invasive ductal carcinoma with high incidence of pulmonary metastasis at 16-18 weeks. Epithelial ablation of GLI2 dramatically reduced tumor progression. Cre recombinase under control of the Ck14 promoter to genetically ablate GLI2 in basal cells of the mammary gland thus dramatically attenuated metastasis despite continued (albeit reduced) formation of primary tumors. In human BC, we applied Bayesian methods to gene expression data to identify metastatic BC with HH pathway activation. Kaplan-Meier analysis demonstrated significantly worse progression-free survival in patients with HH pathway activity (Log-rank p = 0.0013). Next, we analyzed 1294 BC samples, stratified according to HH activity, using the HH probability as a continuous score ranging between 0 and 1, univariate Cox regression analysis supports the hypothesis that HH activity in BC is a risk factor for relapse after surgery, HR = 2.45 (95% CI: 1.67 – 3.61, p = 2.72e-6). There is also a significant difference in survival between HH-actives and HH-inactives (Log-rank p = 3.91e-4), suggesting a pathogenic role of GLI activation in BC progression and metastasis. Arsenic trioxide (ATO), inhibits HH pathway activity by de-stabilizing the GLI transcriptional effectors of HH signaling, likely due to arsenic displacement of zinc within the DNA-binding zinc fingers of GLI proteins. ATO treatment of BC cell lines resulted in dose-dependent cell growth inhibition (Alamar Blue) and induction of apoptosis (PARP cleavage and Annexin V expression) at clinically achievable concentrations. In NSG mice with orthotopic transplant of 3e5 SU151 human BC PDX, with strongly positive GLI-active signature, daily IP injection of 10mg/kg ATO resulted in marked tumor growth inhibition in vivo (2.75-fold smaller tumor diameter vs. PBS vehicle controls, P= 0.006; 55d vs. 17d to reach 1.5 cm criterion for ATO vs. vehicle). We conclude the major HH pathway transcriptional effector GLI2 coordinates a transcriptional program with a central role in BC growth and metastasis in a significant subset of BC, and that systemic treatment with ATO (new oral formulation in commercial development), will reduce metastatic progression and improve clinical outcomes in patients whose tumors harbor a GLI-active transcript signature. Citation Format: Beachy P, Cai S, Ma Y, Hatakeyama J, Zhao C, Stoffels M, Verhaegh W, van de Stolpe A, Pegram M. Targeted disruption of transcriptional effector GLI2 attenuates breast tumor growth and metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-11.

Abstract GS6-07: EMBRACA: A phase 3 trial comparing talazoparib, an oral PARP inhibitor, to physician's choice of therapy in patients with advanced breast cancer and a germline BRCA mutation

Abstract Background: Talazoparib (TALA) is a highly potent, dual-mechanism PARP inhibitor that inhibits the PARP enzyme and effectively traps PARP on single-stranded DNA breaks, preventing DNA damage repair and causing cell death in BRCA1/2-mutated cells. Methods: EMBRACA is an open-label, randomized, 2-arm, phase 3 trial comparing the efficacy and safety of TALA (1 mg/day) with standard single-agent physician's choice of therapy (PCT) (capecitabine, eribulin, gemcitabine, or vinorelbine) in patients with advanced breast cancer (aBC) and a germline BRCA1/2 mutation (gBRCAmut). The primary objective was PFS assessed by blinded independent central review (BICR). Secondary objectives: OS, ORR, CBR at 24 weeks (CBR24), and safety. Exploratory objectives: patient-reported QoL and DOR. Eligibility criteria: age ≥ 18 years; HER2-negative aBC; deleterious or suspected deleterious gBRCAmut; ≤ 3 prior cytotoxic regimens for aBC; and ECOG PS ≤ 2. Prior platinum was allowed. Patients were randomized 2:1 and stratified by receptor status, extent of prior therapy, and CNS metastases (NCT01945775). Results: 431 patients were randomized (median age 46 years; 54% hormone-receptor [HR]+ BC; 45% BRCA1+ and 55% BRCA2+; 55% ECOG PS = 0; 38% chemo-naïve for aBC; 18% prior platinum; 15% CNS metastases); 287 were assigned to TALA and 144 to PCT (1 TALA, 18 PCT patients were not treated). Median duration of exposure was 6.1 and 3.9 months, respectively; TALA had a relative dose intensity of 87%. At 62% PFS data maturity: TALAPCTHazard Ratio/Odds Ratio (P value)PFS by BICR, mo (95% CI)8.6 (7.2-9.3); n = 2875.6 (4.2-6.7); n = 1440.542 (&amp;lt; 0.0001)OS [interim], mo (95% CI)22.3 (18.1-26.2); n = 28719.5 (16.3-22.4); n = 1440.761 (0.105)ORR by INV, % (95% CI)62.6% (55.8-69.0); n = 21927.2% (19.3-36.3); n = 1144.99 (&amp;lt; 0.0001)DOR by INV, mo (IQR)5.4 (2.8-11.2); n = 1373.1 (2.4-6.7); n = 310.431 (0.0005)*CBR24 by INV, % (95% CI)68.6% (62.9-74.0); n = 28736.1% (28.3-44.5); n = 1444.28 (≤0.0001) INV, investigator. *Not a randomized subset. Improved clinical benefit was seen in all subsets including those with HR+ BC (HR 0.47; 95% CI 0.32-0.71) and CNS metastasis (HR 0.32; 95% CI 0.15-0.88). There was a significant delay in the time to deterioration in global health status (GHS)/QoL for TALA vs PCT (HR 0.38; 95% CI 0.26-0.55; P &amp;lt; 0.0001]. Grade 3-4 hematologic adverse events (AEs) occurred in 55% TALA (mainly anemia)/39% PCT (mainly neutropenia). Grade 3-4 non-hematologic AEs were seen in 32% TALA/38% PCT; TALA was associated with fewer gastrointestinal disorders (5.6% vs 11.9%) and skin/subcutaneous tissue disorders (0.7% vs 5.6%) than PCT. Grade 3-4 serious AEs were observed in 26% TALA/25% PCT. AEs associated with permanent study drug discontinuation occurred in 8% TALA/10% PCT. AE resulting in death occurred in 2.1% TALA/3.2% PCT.Conclusions: Single-agent TALA significantly prolonged PFS by BICR in HER2-negative aBC patients with a gBRCAmut compared to PCT; all key secondary efficacy endpoints demonstrated benefit with TALA, with a significant delay in time to deterioration in GHS/QoL. TALA was generally well tolerated with minimal non-hematologic toxicity and few AEs associated with treatment discontinuations. Citation Format: Litton J, Rugo HS, Ettl J, Hurvitz S, Gonçalves A, Lee K-H, Fehrenbacher L, Yerushalmi R, Mina LA, Martin M, Roché H, Im Y-H, Quek RGW, Tudor IC, Hannah AL, Eiermann W, Blum JL. EMBRACA: A phase 3 trial comparing talazoparib, an oral PARP inhibitor, to physician's choice of therapy in patients with advanced breast cancer and a germline BRCA mutation [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 GS6-07.

Significance of hypoxia inducible factor-1 expression in liver metastasis of colorectal cancer.

464 Background: In colorectal liver metastasis (CRLM), the difference of tumor malignancy between primary and metastatic lesion has been elucidated. Hypoxia inducible factor-1 (HIF-1) represent tumor malignancy including angiogenesis, tumor growth and epithelial mesenchymal transition (EMT). The aim of this study was to investigate the difference of tumor malignancy between metastatic and primary lesion of CRLM and its impact for patient’s prognosis. Methods: In an initial curative hepatectomy of 75 cases in CRLM, HIF-1 expression in primary and metastatic lesion was evaluated by immunostaining method (Sigma-Aldrich, HPA 001275). Staining score was classified as follows, staining intensity (0: negative, 1: low, 2: medium, 3: high) and staining area (0: 0%, 1: -25%, 2: 26-50%, 3: ≥ 51%), and defined more than 4 points as positive expression. We evaluated the clinicopathological features according to HIF-1 expression. Results: Regarding HIF-1 expression of metastatic site, we divided into the positive group (n = 54) and the negative group (n = 21). There was no difference between metastatic HIF-1 expression and clinicopathological factors. Nevertheless, in overall survival, multivariate analysis revealed that HIF-1 positive in metastasis (HR: 2.850, p = 0.042) and poor differentiation type of primary lesion (HR: 20.873, p = 0.001) were independent prognostic factors. HIF-1 positive and negative patients were 3 year survival of 95.2% and 58.6%, respectively. Also, in disease free survival, HIF-1 positive in metastasis (HR: 2.608, p = 0.004), Synchronous (HR: 1.794, p = 0.049), Grade BC (HR: 2.145, p = 0.008), and lymph node metastasis in primary lesion (HR: 2.070, p = 0.016) was identified. Regarding HIF-1 expression of primary site, we divided into 51 cases of positive group and 24 cases of negative group. There was no relationship to clinicopathological factors as well as HIF-1 expression in metastasis, besides HIF-1 expression of prognosis was not associated. Conclusions: In CRLM, HIF-1 expression in the metastatic lesion is not associated with the primary lesion and may be useful as prognostic marker.

Abstract 2933: Involvement of CXCL12-CXCR7 axis in adipocyte-induced TNBC metastasis

Abstract Breast cancer (BC) is the most frequent tumor in women worldwide. Although its mortality has significantly decreased owing to advanced therapies, triple negative breast cancer (TNBC) is still difficult to treat because of lack of estrogen receptor, progesterone receptor and HER-2. TNBC accounts for 12-24% of total BC and contributes to the aggressiveness and poorer outcomes. The risk of BC increases significantly in obese women. Obesity is also associated with the worse clinical prognosis of BC. But the underlying mechanisms between obesity and BC, particularly TNBC, remain unclear. Therefore, we compare the molecular mechanisms by which human adipocyte conditioned media (CM) affect TNBC cells (BT549) and non-TNBC cells (MCF7A). Adipocyte CM had no effect on the proliferation of both TNBC and non-TNBC cells. However, adipocyte CM enhanced significantly migration in TNBC cells compared to non-TNBC cells. Next, we examined the signaling pathway by which adipocyte CM promote migration of TNBC cell. AKT and ERK were activated in both TNBC cells and non-TNBC cells. However, phospho-STAT3 was significantly increased in TNBC cells. Also, N-cadherin, a marker for epithelial to mesenchymal transition (EMT), was increased at the late time point in TNBC cells. Furthermore, CXCR7 was specifically increased in TNBC cells after treatment with adipocyte CM using a PCR array. CXCR7 is chemoattractive to CXCL12 which is highly expressed in the lung, the bone marrow, and the liver, common sites of BC metastasis. Taken together, the results indicate that adipocyte CM may promote metastasis of TNBC cells through the CXCL12-CXCR7 axis by activating STAT3. Citation Format: Hyeongjwa Choi, Rosa Mistica C. Ignacio, Carla R. Gibbs, Eunsook Lee, Samuel E. Adunyah, Deok-Soo Son. Involvement of CXCL12-CXCR7 axis in adipocyte-induced TNBC metastasis [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 2933. doi:10.1158/1538-7445.AM2017-2933

Characteristics of HER2 3+ metastatic breast cancer patients with long-term benefit of trastuzumab.

e12523 Background: Because there is no cumulative dose, trastuzumab could be given until disease progression. However, characteristics of patients with long term benefit of trastuzumab treatment are seldom reported. Methods: Trastuzumab become available for HER2 3+ MBC at November 2005 at the Institute for Oncology and Radiology of Serbia. Since then, we have registered 42 patients with HER2 3+ MBC and/or inoperable loco-regional recurrent BC treated with first line trastuzumab for at least 24 months (range 24-123+). Results: Median age was 53 years (range 35-76). 30/42 patients (71%) had ER/PR positive BC. MBC was initial presentation in 14 patients (33%), and as relapse in 28 (67%) patients. Only 7/28 (25%) patients received neoadjuvant/adjuvant trastuzumab, therefore majority have been trastuzumab naive (83.3%). Metastases were present in the liver (9), lungs (9), bones (16), soft tissue (6), CNS (1), while 20 (48%) patients had multiple metastatic sites. After antracyclines based chemotherapy, patients have been treated with first line weekly taxane concurrently with three weekly trastuzumab. After taxanes (up to 24 doses), trastuzumab was continued +/-hormonal therapy. Objective response was achieved in 29 (69%) patients with 17 patients (40%) achieving complete response. Median duration of response was 32+ months; 11 patients are still in remission 5 years after trastuzumab initiation. The longest complete remission duration is 123+ months, in patient with initially inoperable BC and solitary cerebellar metastasis. During observational period 10 patients stopped trastuzumab predominantly because of disease progression (8/42 19%) and 2 (4.8%) because of decreased ejection fraction (EF≤ 50%) that occurred as a rather late effect, after 34 and 49 months of continuous trastuzumab treatment. Three patients are lost from follow-up (7%); 39 (93%) patients included in this analysis are alive, still receiving trastuzumab for median 37 cycles (range 29-172+). Conclusions: A subgroup of patient with HER2 3+ MBC has a long-term benefit of trastuzumab treatment. In this analysis majority (71%) had also ER/PR positive MBC and majority have not been treated with neoadjuvant/adjuvant trastuzumab (83.3%).

Targeting Cellular Signaling Pathways in Breast Cancer Stem Cells and its Implication for Cancer Treatment.

Breast cancer is a public health problem both in developing and developed countries. The breast cancer stem cell (BCSC) hypothesis has grown in the cancer research community. These BCSCs comprise of a small subpopulation of cells within the tumor mass which exhibit stem cell-like characteristics and have emerged as being responsible for tumor development, recurrence and metastasis in BC. The complexity of control of gene expression in BCSC is commonly driven by a myriad of signaling pathways triggered by extracellular signals, mutations and epigenetic control. Thus, some signaling pathways have been highlighted in BC, especially those linked to stem cell phenotype, such as nuclear factor-kappa B, signal transducer and activator of transcription 3, wingless-type MMTV integration site family (Wnt)/β-catenin, Hedgehog and NOTCH. Moreover, these BCSCs can also be influenced by the tumor microenvironment, for instance, hypoxic areas. Given the importance of signaling pathways and tumor microenvironment for breast cancer, this review focuses on the relationship between cellular signaling and BCSCs and its therapeutic implications.

Abstract P2-05-09: Vascular mimicry in metastatic breast cancer patients: Molecular insight to vascular mimicry using in vitro and in vivo models

Abstract Background: Vascular Mimicry (VM) is an endothelium-independent matrix-embedded, blood-perfusion phenomenon exhibited by highly plastic and aggressive tumor cells in patients with solid tumors including breast. VM is characterized by PAS+ de novo formations of micro-vascular networks which are essentially CD31- and CD34-. VM is a micro-circulatory phenomenon which perfuses rapidly growing tumors, transporting fluid from leaky vessels and/or connects with the constituent endothelial-lined vasculature. One of the hallmarks of metastasis in BC is the heterogeneity observed between primary tumors and metastases (&amp;amp; among metastases) (Marino etal., 2013). Recently a model of BC heterogeneity revealed VM as a driver of metastasis which in turn has been associated with disease heterogeneity (Wagenblast etal., 2015). Aim: Since BC is an aggressive and heterogeneous disease and VM is associated with the aggressiveness / poor outcome in BC, we sought to understand the functional relationship of VM with metastasis. Method: Tumors from our BC patients and TMAs were stained for CD31/PAS and CD34/PAS to identify VM. Genomic and proteomic data from these patients were obtained from re-biopsied (after consultation) samples (IHC for ER, PR, and HER2; FFPE samples for genomic [Foundation Medicine] and proteomic [Theralink] analyses). Result: VM was identified in metastatic tumors from ER+ and TNBC patients as CD31-/PAS+ and CD34-/PAS+ structures in contrast to the CD31+/PAS+ and CD34+/PAS+ angiogenic compartment of the individual tumor(s). Metastatic tumors exhibiting VM were characterized by pathological features like metaplastic lesions, positive lympho-vascular invasion and were found to be poorly differentiated. Predominant genetic alterations in these patients included (1) PI3K-mTOR pathway genes, (2) p53 (R273P, D281V), (3) BRCA1 E1683* (for TNBC), (4) MYC amplification, (5) aurora kinase, (6) CCND1 &amp;amp; CDK4 amplifications and (7) loss of CDH1 exons1-3. Amplifications of cell surface TKIs/ligands included EGFR, FGFR and several FGFs. Proteomic data indicated an overexpression/activation of HER family (HER1/HER2/HER3), mTOR activation (p∼S2448), MEK1/2 activation (p∼S217-221) and JAK2 activation. In order study VM, we standardized VM formation both in 2D and 3D configurations in multiple BC cell lines which exhibited VM at differing times. The earliest response was observed around 2-3 hours in BT20, Hs578t and MDA-MB231. By 24 hours BT474, BT474HerR, SUM149, DKAT, MDA-MB231BR, Hs578t and MDA-MB468 cells demonstrated 2D VM. The BT20 cell line showed the most pronounced 3D-VM at 24 hours while MDA-MB468 was least sensitive to VM. Typical cord formation in HUVEC cells stained with hematoxylin and PAS were used for comparison. Using 2D and 3D models of VM we demonstrated the involvement of PI3K and Wnt-beta-catenin pathways in VM. Considering the involvement of VM in mediating the aggressive/metastatic nature of TNBC, we also tested VM in in vivo xenograft models using brain metastasic specific MDA-MB231BR cells, results of which will be presented in the meeting. Significance: To our knowledge this is the first report to identify genetic alterations and proteomic changes associated with VM in metastatic BC. Citation Format: Dey N, Carlson JH, Lin X, Sun Y, Theel S, Krie A, Sulaiman RA, Williams C, De PK, Leyland-Jones BR. Vascular mimicry in metastatic breast cancer patients: Molecular insight to vascular mimicry using in vitro and in vivo models. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-09.