Facilitate Breast Cancer Metastasis Research Articles

Baicalin reduces chronic stress-induced breast cancer metastasis via directly targeting β2-adrenergic receptor

Recent studies have shown that stress can substantially facilitate breast cancer metastasis, which can be reduced by nonselective β1/β2-adrenergic receptor (β1/β2-AR) blocker. However, several side effects were identified. Thus, it is extremely warranted to explore more effective and better-tolerated β2-AR blocker. Currently, we demonstrated that baicalin (BA), a major bioactive component of Scutellaria baicalensis Georgi, could significantly attenuate stress hormones especially epinephrine (Epi)-induced breast cancer cell migration and invasion in vitro. Mechanistically, we identified that β2-AR was a direct target of BA via the drug affinity responsive target stability (DARTS) combined with mass spectrum assay, and BA photoaffinity probe with pull-down assay, which was further confirmed by a couple of biophysical and biochemical assays. Furthermore, we demonstrated that BA could directly bind to the Phe-193 and Phe-289 of β2-AR, subsequently inhibit cyclic adenosine monophosphate-protein kinase A-focal adhesion kinase (cAMP-PKA-FAK) pathway, and thus impede epithelial-mesenchymal transition (EMT), thereby hindering the metastatic progression of the chronic stress coupled with syngeneic and xenograft in vivo orthotopic and tail vein mouse model. These findings firstly identify BA as a potential β2-AR inhibitor in the treatment of stress-induced breast cancer metastasis.

LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3

The focus of the study is to examine the function of TYMSOS in immune escape of breast cancer, which is the most frequently diagnosed malignancy among women globally. Our study demonstrated that upregulated TYMSOS was associated with unfavorable prognosis and immune escape in breast cancer. TYMSOS promoted the malignant phenotypes of breast cancer cells, and reduced the cytotoxicity of NK92 cells on these cells. CBX3 was a downstream effector in TYMSOS-induced malignant phenotypes in breast cancer cells. Mechanistic studies showed that TYMSOS facilitated CBX3-mediated transcriptional repression of ULBP3, and it also promoted SYVN1-mediated ubiquitin-proteasomal degradation of ULBP3. TYMSOS promoted cell growth, metastasis, and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis. The invivo studies further showed that silencing of TYMSOS repressed tumor growth and boosted NK cell cytotoxicity. In sum, TYMSOS boosted breast cancer metastasis and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis.

Stabilization of IGF2BP1 by USP10 promotes breast cancer metastasis via CPT1A in an m6A-dependent manner.

Metastasis leads to the vast majority of breast cancer mortality. Increasing evidence has shown that N6-methyladenosine (m6A) modification and its associated regulators play a pivotal role in breast cancer metastasis. Here, we showed that overexpression of the m6A reader IGF2BP1 was clinically correlated with metastasis in breast cancer patients. Moreover, IGF2BP1 promoted distant metastasis in vitro and in vivo. Mechanistically, we first identified USP10 as the IGF2BP1 deubiquitinase. USP10 can bind to, deubiquitinate, and stabilize IGF2BP1, resulting in its higher expression level in breast cancer. Furthermore, by MeRIP-seq and experimental verification, we found that IGF2BP1 directly recognized and bound to the m6A sites on CPT1A mRNA and enhanced its stability, which ultimately mediated IGF2BP1-induced breast cancer metastasis. In clinical samples, USP10 levels correlated with IGF2BP1 and CPT1A levels, and breast cancer patients with high levels of USP10, IGF2BP1, and CPT1A had the worst outcome. Therefore, these findings suggest that the USP10/IGF2BP1/CPT1A axis facilitates breast cancer metastasis, and this axis may be a promising prognostic biomarker and therapeutic target for breast cancer.

Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment

Primary tumors are drivers of pre-metastatic niche formation, but the coordination by the secondary organ toward metastatic dissemination is underappreciated. Here, by single-cell RNA sequencing and immunofluorescence, we identified a population of cyclooxygenase 2 (COX-2)-expressing adventitial fibroblasts that remodeled the lung immune microenvironment. At steady state, fibroblasts in the lungs produced prostaglandin E2 (PGE2), which drove dysfunctional dendritic cells (DCs) and suppressive monocytes. This lung-intrinsic stromal program was propagated by tumor-associated inflammation, particularly the pro-inflammatory cytokine interleukin-1β, supporting a pre-metastatic niche. Genetic ablation of Ptgs2 (encoding COX-2) in fibroblasts was sufficient to reverse the immune-suppressive phenotypes of lung-resident myeloid cells, resulting in heightened immune activation and diminished lung metastasis in multiple breast cancer models. Moreover, the anti-metastatic activity of DC-based therapy and PD-1 blockade was improved by fibroblast-specific Ptgs2 deletion or dual inhibition of PGE2 receptors EP2 and EP4. Collectively, lung-resident fibroblasts reshape the local immune landscape to facilitate breast cancer metastasis.

Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis.

Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF-cCAF homotypic clusters, and in heterotypic clusters with CTCs. In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I-IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells. We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.

Abstract 2848: Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer

Abstract Background: Hotspot estrogen receptor-α (ER/ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer and are associated with worse outcome. How these mutations facilitate metastasis remains ambiguous. It is necessary to identify a clear mechanism for therapeutic intervention. Methods: ESR1 mutations were detected by ddPCR. Transcriptome data were derived from cell lines and clinical samples. Y537S and D538G genome-edited MCF7 and T47D cell lines were used for in vitro phenotypic characterization. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and ibidi microfluidic assays. Altered cell-cell adhesion genes were validated using qRT-PCR, immunoblot and immunostaining. Desmosome blocking peptides and carbenoxolone were used for blockade of adhesion. ER cistromes were profiled by ChIP-seq. Tail vein injection was performed on athymic nude mice to evaluate metastasis in vivo. Circulating tumor cell (CTC) clustering propensity in vivo was assessed via intracardiac injection followed by CTC microfilter capture. CTC-cluster gene signatures were generated from ER+ breast cancer CTC RNA-seq data. Results: ESR1 mutations were significantly enriched in distant metastases (12/48) vs local (0/27) recurrences, confirming their critical role in promoting metastasis. Transcriptomic analysis revealed altered cell-cell interaction pathways in ESR1 mutant tumors. ESR1 mutant cells formed more compact spheroids in suspension culture, and exhibited stronger cell-cell adhesion in static condition. Under microfluidic condition with physiological shear stress, mutant ESR1 cells derived more and larger clusters, which were prominently preserved from pre-existing clusters. This effect was correlated with increased expression of desmosome and gap junction genes in mutant cells and pharmacological blockade significantly reduced the enhanced cell-cell adhesion. ER ChIP-seq revealed no de novo mutant ER binding sites at the loci of the target genes, suggesting indirect regulation by mutant ER. This was exemplified by a secondary regulation from cFos/AP1 signaling of GJA1 expression, and an epigenetic regulation at DSC1/DSG1 loci through enhanced H3K4me2 and H3K27ac modification. In vivo studies showed ESR1 mutant cells derived more distant metastases, and MCF7 Y537S cells formed larger CTC clusters with increased compactness compared to WT cells. Finally, ER+ CTC-cluster signatures were enriched in ESR1 mutant tumors, suggesting their unique dependence on this pathway during metastasis. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer enhanced cell-cell adhesion, which facilitates breast cancer metastasis via increased CTCs clustering propensity. These findings provide insights to the development of drugs targeting gap junction in ER mutant tumors. Citation Format: Yang Wu, Zheqi Li, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan M. Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben Ho Park, Jennifer M. Atkinson, Dorraya El-Ashry, Adrian V. Lee, Steffi Oesterreich. Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2848.

Dysregulation of tumour microenvironment driven by circ-TPGS2/miR-7/TRAF6/NF-κB axis facilitates breast cancer cell motility

Tumour microenvironment (TME) is frequently remodelled and deregulated in cancer development and progression. The underlying mechanisms are complex, multifactorial and largely unknown. Circular RNA (circRNA) is an endogenous RNA with a covalently closed loop that plays critical roles in the pathological and physiological processes of the organism. Here, we identified a TME-associated circRNA, circ-TPGS2, which promoted breast cancer (BC) cell dissemination via altering TME. High circ-TPGS2 was observed in metastatic BC tissues and cell lines in comparison to respective normal controls, which was linked to poor overall and recurrence-free survival. Overexpression of circ-TPGS2 notably promoted cell migration, while silencing of circ-TPGS2 resulted in an opposite trend. Moreover, circ-TPGS2 increased pro-inflammatory chemokine production and evoked tumour-associated inflammation by recruiting neutrophils via autocrine and paracrine manners. In terms of mechanism, circ-TPGS2 acted as a sponge of miR-7 and elevated TRAF6, leading to p65 phosphorylation and nuclear translocation, ultimately activating NF-κB signalling. In addition, p65 was abundantly occupied on circ-TPGS2 promoter, activating circ-TPGS2 transcription, thus, forming a positive feedback loop that amplified the pro-metastasis effect of circ-TPGS2. Taken together, our data for the first time reveal the biological implication of circ-TPGS2 in BC, it triggers TME reshaping that facilitates BC metastasis through the miR-7/TRAF6/NF-κB signalling cascade.

Hypoxia-Induced Suppression of Alternative Splicing of MBD2 Promotes Breast Cancer Metastasis via Activation of FZD1.

Metastasis is responsible for the majority of breast cancer-related deaths, however, the mechanisms underlying metastasis in this disease remain largely elusive. Here we report that under hypoxic conditions, alternative splicing of MBD2 is suppressed, favoring the production of MBD2a, which facilitates breast cancer metastasis. Specifically, MBD2a promoted, whereas its lesser known short form MBD2c suppressed metastasis. Activation of HIF1 under hypoxia facilitated MBD2a production via repression of SRSF2-mediated alternative splicing. As a result, elevated MBD2a outcompeted MBD2c for binding to promoter CpG islands to activate expression of FZD1, thereby promoting epithelial-to-mesenchymal transition and metastasis. Strikingly, clinical data reveal significantly correlated expression of MBD2a and MBD2c with the invasiveness of malignancy, indicating opposing roles for MBD2 splicing variants in regulating human breast cancer metastasis. Collectively, our findings establish a novel link between MBD2 switching and tumor metastasis and provide a promising therapeutic strategy and predictive biomarkers for hypoxia-driven breast cancer metastasis. SIGNIFICANCE: This study defines the opposing roles and clinical relevance of MBD2a and MBD2c, two MBD2 alternative splicing products, in hypoxia-driven breast cancer metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/5/1265/F1.large.jpg.

Abstract GS1-05: Hotspot ESR1 mutations rewire cell-cell adhesome to facilitate breast cancer metastasis

Abstract Background: Estrogen receptor alpha (ER/ESR1) mutations are found in 20-40% of endocrine resistant ER+ metastatic breast cancers, and they are associated with worse outcome. Preclinical studies have shown that they cause ligand-independent growth, resistance to endocrine therapy, and there is growing evidence for a role in metastasis. It is not known how ESR1 mutant cancer cells cause metastases and whether such a mechanism may indicate novel therapeutic options. Methods: ddPCR was used to detect hotspot ESR1 mutations. Transcriptome data were derived from cell line models and clinical samples. For in vitro phenotypic characterization, Y537S and D538G genome-edited MCF7 and T47D cell models from four different labs were used. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and microfluidic aggregation assays. Altered expression of cell-cell adhesion genes detected in RNA seq data was validated via qRT-PCR, immunoblot and immune staining. Functional contributions of desmosome and gap junctions were tested by blocking peptide and carbenoxolone respectively. Mutant ER cistromes were profiled using ChIP-sequencing. Tail vein injection was performed on nude mice to evaluate metastasis in vivo. Mouse lung micro-metastatic foci were quantified using human-specific CK19 staining. Circulating tumor cells (CTCs) clustering propensity in vivo was assessed via intracardiac injection of ESR1 WT and mutant cells into nude mice following CTC microfilter capture. CTCs enumeration from breast cancer patients’ blood samples was performed using CellSearchTM system. Results: We identified a significant enrichment of ESR1 mutations in distant (12/48) vs local (0/27) recurrences, confirming the strong association of mutant ER with metastasis. Transcriptomic analysis revealed altered cell-to-cell interaction pathways in ESR1 mutant tumors compared to ESR1 WT tumors, suggesting a previously undescribed role of ESR1 mutations in reprogramming cell-cell adhesome. ESR1 mutant cells grown in suspension culture revealed more compact multicellular spheroids compared to WT cells. This observation was confirmed under both static and microfluidic conditions, indicative of increased cell-cell interactions. The effect was more pronounced in MCF7 compared to T47D cells, and it correlated with increased expression of multiple desmosome and gap junction pathway genes, which were also significantly enriched in ESR1 mutant tumors. Pharmacological blockade of desmosome and gap junctions significantly rescued enhanced cell-cell adhesion in ESR1 mutant cells. Mechanistically, our ER ChIP-seq did not identify any gained mutant ER binding sites in proximity to cell-cell adhesion gene loci, indicating indirect regulation by mutant ER. Consistent with this, expression of Connexin 43, one of the top upregulated gap junction components, was induced by cFOS found to be highly upregulated in ESR1 mutant cells. Tail vein injection of ESR1-mutant cells derived more distant macro- (MCF7) and micro- (T47D) metastases. Given increasing evidence for role of cell-cell attachment in CTC phenotypes, we tested CTC formation for ESR1 WT and mutant cells. In vivo studies showed MCF7 Y537S ESR1 mutant cells formed larger multi-cellular CTC clusters with increased compactness compared to WT CTCs. These preclinical data translated to clinical observation, where we observed an enrichment of CTC clusters in patients with ESR1 mutant-metastatic breast cancers. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer increased cell-cell adhesion, which facilitate breast cancer metastasis via increased CTCs clustering propensity. These findings might guide approaches to test potential repurpose of drugs targeting gap junction in ER mutant tumors. Citation Format: Zheqi Li, Yang Wu, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben H. Park, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Jennifer M. Atkinson, Dorraya El-Ashry, Massimo Cristofanilli, Adrian V. Lee, Steffi Oesterreich. Hotspot ESR1 mutations rewire cell-cell adhesome to facilitate breast cancer metastasis [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 GS1-05.

Fibroblast-Derived IL33 Facilitates Breast Cancer Metastasis by Modifying the Immune Microenvironment and Driving Type 2 Immunity.

Lungs are one of the main sites of breast cancer metastasis. The metastatic microenvironment is essential to facilitate growth of disseminated tumor cells. Cancer-associated fibroblasts (CAF) are prominent players in the microenvironment of breast cancer. However, their role in the formation of a permissive metastatic niche is unresolved. Here we show that IL33 is upregulated in metastases-associated fibroblasts in mouse models of spontaneous breast cancer metastasis and in patients with breast cancer with lung metastasis. Upregulation of IL33 instigated type 2 inflammation in the metastatic microenvironment and mediated recruitment of eosinophils, neutrophils, and inflammatory monocytes to lung metastases. Importantly, targeting of IL33 in vivo resulted in inhibition of lung metastasis and significant attenuation of immune cell recruitment and type 2 immunity. These findings demonstrate a key function of IL33 in facilitating lung metastatic relapse by modulating the immune microenvironment. Our study shows a novel interaction axis between CAF and immune cells and reveals the central role of CAF in establishing a hospitable inflammatory niche in lung metastasis. SIGNIFICANCE: This study elucidates a novel role for fibroblast-derived IL33 in facilitating breast cancer lung metastasis by modifying the immune microenvironment at the metastatic niche toward type 2 inflammation.

RRM2 elicits the metastatic potential of breast cancer cells by regulating cell invasion, migration and VEGF expression via the PI3K/AKT signaling.

Breast cancer is the second leading primary cause for cancer-related mortality among women and metastasis to the brain is a disastrous event for patients with increasing incidence. A previous study confirmed the critical function of RRM2 in breast cancer cell growth. Unfortunately, the role and fundamental molecular mechanism of RRM2 in breast cancer metastasis remains elusive. In the current study, higher RRM2 expression was validated in breast cancer tissues, especially in the brain metastasis group. Simultaneously, the expression of RRM2 was increased in breast cancer cells relative to the normal breast epithelial cell line MCF-10A, concomitant with higher levels of RRM2 in the highly metastatic MDA-MB-231 cell line relative to the weakly metastatic MCF-7 cell line. Knockdown of RRM2 by small interfering-RRM2 transfection notably suppressed the malignant metastatic behavior of breast cancer cells, including invasion and migration. Simultaneously, RRM2 downregulation also restrained the transcription and release of vascular endothelial growth factor (VEGF) in breast cancer cells. Moreover, inhibition of RRM2 dampened the activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling by decreasing phosphorylated-AKT and downstream matrix metalloproteinases-2 expression. Intriguingly, reactivation of the PI3K/AKT pathway with its agonist insulin-like growth factor-1 reversed the adverse effects of RRM2 suppression on cancer cell invasion, migration and VEGF expression. Together, these findings suggest that RRM2 may act as a pro-metastatic factor to facilitate breast cancer metastasis by evoking cell invasion, migration and VEGF expression through the PI3K/AKT signaling pathway. This study may provide an attractive target for metastatic intervention in breast cancer.

Abstract 2019A: TLR3 facilitate breast cancer metastasis to lymph node

Abstract Tumor cells can only enter the lymphatic circulation at the interface between the invasive edge of the tumor and the adjacent host stroma that contains the lymphatic circulation. The existence of this distance barrier means that it would be unlikely for tumor cells to passively enter the lymph drainage. Instead, Toll-like receptors (TLRs) are important mediators for active migration and chemotaxis for tumor cells entry into the lymphatic drainage from the primary tumor. Here we use a microsurgical technique to collect lymph circulating tumor cells (LCTC) prior to its entry into sentinel lymph node in rat models and determine if the TLR expression induces the migration and tumor progression. In our results, we observed high levels of TLR3 expression in LCTC when cells were treated with Poly (I:C), a synthetic ligand for TLR3 induction. NFkB activation was also observed in LCTCs, after TLR3 activation this pathway can express genes involved in cancer progression. In addition, when looking at the expression of cytokines and chemokines factors present in the lymph or secreted by the tumor cells, CXCL10 had a high expression on induced TLR3 of LCTC and its ligand CXCR3 was also expressed in the lymph. CXCL10 is involved in chemotaxis, induction of apoptosis, and regulation of cell growth. The CXCL10/CXCR3 signaling mediates paracrine interactions between tumor and stromal cells that govern leukocyte trafficking and angiogenesis. Suppression of CXCR3 or receptors signaling limits metastasis suggesting CXCR3 contributes to the metastatic process. TLR3 Knockdown prevented tumor growth and metastasis in rat. Our data suggest that for breast cancer metastasis, TLR3 might play a role in active migration of tumor cells and tumor progression. Activation of TLRs in tumor cells can dampen the anti-tumor functions of infiltrating immune cells, altering the inflammatory response and promoting cancer progression. Citation Format: Odalys J. Torres-Luquis, Sulma Mohammed. TLR3 facilitate breast cancer metastasis to lymph node [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 2019A.

Low mutation and neoantigen burden and fewer effector tumor infiltrating lymphocytes correlate with breast cancer metastasization to lymph nodes

Lymph node metastasis is of major prognostic significance for breast cancer. Lymph node metastasis arises at a very early stage in some patients. Using the data downloaded from the TCGA database, we studied the differences between primary tumors with and without lymph node metastasis at the multi-omics level using bioinformatics approaches. Our study found that low mutation and neoantigen burdens correlated with lymph node metastazation of breast cancer. All three conserved domains in TP53 were mutated in lymph node-negative breast cancers, whereas only one domain was mutated in lymph node-positive samples. Mutations in microtubule-related proteins appear to help immune cells recognize tumors and inhibit their lymph node metastasis. Destroying microtubule-related proteins is a potential therapeutic strategy to inhibit lymph node metastasis of breast cancer. As the neoantigens specifically present in lymph node-positive breast cancers, MAPK10, BC9L, TRIM65, CD93, KITLG, CNPPD1, CPED1, CCDC146, TMEM185A, INO80D, and PSMD11 are potential targets for vaccine design. In the tumor microenvironment, reduced numbers of effector immune cells, especially activated memory CD4+ T cells and activated mast cells, facilitate breast cancer metastasis to the lymph nodes. According to transcriptome data, lymph node metastasis was mostly driven by gene mutation rather than by gene expression. Although differential gene expression analysis was based on lymph node metastasis status, many genes were shown to be differentially expressed based on estrogen receptor status.

Abstract ES8-3: Circulating complexes of stromal and tumor cells in breast cancer metastasis

Abstract The establishment of breast cancer metastases at sites distant from the primary lesion site is predicated on the ability of breast cancer cells to mobilize from the primary tumor, survive in circulation, and colonize in a suitable and permissive metastatic niche. While quantitation of individual CTCs has prognostic value in breast cancer patients with metastatic disease, and elevated counts of CTCs may associate with poor outcome in early breast cancer, recent evidence from mouse models indicates that CTCs in clusters have a greater capacity to produce metastases than individual CTCs and new studies in breast cancer suggest that the presence of CTC clusters in patients can be prognostic. We have found in breast cancer patients, clusters of circulating cells that include CTCs, immune cells, and stromal cells found in the tumor microenvironment, specifically cancer associated fibroblasts (CAFs). Like CTC and circulating CAF (cCAF) counts, the incidence and number of CTC/cCAF clusters increases with disease stage. While the clinical significance of circulating complexes of CTCs and stromal cells remains to be fully elucidated, the recent identification of cCAFs in the peripheral blood of patients with metastatic breast cancer, traveling individually or in clusters with CTCs emphasizes the potential importance of stromal cells as circulating mediators of cancer metastasis. Preclinical murine models of breast cancer metastasis provide opportunities to observe the composition, dynamics, and behavior of these circulating CTC/stromal cell complexes, and to interrogate mechanisms driving their mobilization and clustering. Identifying the cellular components in these circulating complexes, and understanding the mechanisms by which they facilitate breast cancer metastasis, may provide novel therapeutic avenues for the treatment or prevention of metastatic breast cancer. Citation Format: El-Ashry D. Circulating complexes of stromal and tumor cells in breast cancer 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 ES8-3.

The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells

Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8+T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia.

Abstract P5-05-02: NEDD9 promotes breast cancer metastasis by regulating mitochondrial functions

Abstract NEDD9 has been characterized as a metastasis-promoting gene in various cancer cells including breast. We previously reported that NEDD9 promotes malignant phenotypes of breast cancer cells through distinct and non-overlapped domains. For example, the FAT (Focal Adhesion Targeting) domain of NEDD9 promotes cancer cell growth, while the SH-domain facilitates cell migration. These results suggest that NEDD9 promotes tumor metastasis by enhancing dissemination and growth in the tumor-host microenvironments through distinct and non-overlapped domains. Thus, targeting functions of NEDD9 is a promising approach for breast cancer therapies. In order to further characterize NEDD9-mediated breast cancer growth, we performed yeast-two hybrid (Y2H) screening to identify proteins that associate with the FAT domain of NEDD9. Using the FAT domain constructed in pGBKKT7 (Clonetech, CA) as a bait to screen library of human fibroblast (Clonetech, CA), we identified several proteins that associate with the domain. They are small GTPases (i.e. RAB11a and ARF4), cytoskeletal proteins (i.e. Nexilin), and cytosolic proteins (i.e. HAX-1). Among of these potential partner proteins, we focused on the interaction between NEDD9 and HAX-1 in breast cancer cells. Co-immunoprecipitation assays confirm the molecular complex of NEDD9-HAX-1 in both SK-Br3 and SUM149 cells. Importantly, p130cas, which harbors similar domain structures with NEDD9, was not precipitated with NEDD9, suggesting a specific interaction between NEDD9 and HAX-1. Given the fact of NEDD9 as a key metastasis promoting gene, these results suggest that NEDD9-HAX-1 plays a key role breast cancer metastasis by facilitating growth in microenvironments. While the biological function are not clear at present, previous studies demonstrated that HAX-1 localizes in mitochondria in breast cancer cells, Indeed, we demonstrated that NEDD9 was found in both cytosol and mitochondria fractions in malignant breast cancer cell MDA-MB-231, but not non-metastatic HCC38. These results suggest the presence of NEDD9-HAX1 complex in mitochondria and this complex may facilitate breast cancer metastasis. In addition to HAX-1, several mitochondrial proteins such as EFG1, DCTN6, and MMADHC were found in the Y2H screening system as described above. These results suggest that NEDD9 facilitates breast cancer metastasis through regulating multiple pathways including signaling pathways and mitochondrial functions, thus serving as a promising therapeutic target for cancer patients including breast. The view expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S.Government. Citation Format: Iida J, Dorchak J, Slavik J, Clancy R, Cutler ML, Shriver CD. NEDD9 promotes breast cancer metastasis by regulating mitochondrial functions [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-02.

Abstract 4396: Circulating CAFs and CAF-secreted factors may be indicative of breast cancer metastasis

Abstract Tumor metastasis is the main cause of breast cancer mortality. Increasing evidence demonstrates stromal cells play pivotal roles in promoting breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that promote breast cancer progression and metastasis; one of these factors is SDF-1 (CXCL12), a soluble chemokine that is promotes chemotaxis and motility, and facilitates cancer cell motility and angiogenesis. CAFs also secrete soluble factors that activate ERK 1/2 MAPK signaling in breast cancer cells, which has been shown to promote loss of estrogen receptor (ER) in luminal breast cancer cells. Hyperactivation of MAPK signaling (hMAPK) also associates with aggressive, basal-like and HER2-positive breast cancer and poor prognosis. Recently, we identified a patient-derived hMAPK-microRNA signature indicative of poor clinical outcome that contains microRNAs known to regulate breast cancer associated genes. The vast majority of ER- breast cancers display this microRNA signature, as do a subset of ER+ breast cancers with poorer clinical outcome. We also discovered that the breast cancers that exhibit this microRNA signature display high stromal and immune infiltrate scores, suggesting that breast cancer stroma provides important contributions to this microRNA signature and the poor clinical outcomes associated with it. To study the role of CAFs and CAF-secreted factors in breast cancer progression and metastasis, we established primary breast CAF lines from ‘indolent’ breast cancers (Luminal A), and from ‘aggressive’ breast cancers (ER-/HER2 amplified; triple negative). We have demonstrated that these CAFs differentially express several members of the hMAPK-microRNA signature compared to cultured primary breast cancer cells, supporting the contribution of stroma to the signature. Importantly hMAPK-microRNAs secreted from “aggressive” CAFs can be taken up by breast cancer cells, whereupon they repress their targets. Normal human mammary fibroblasts (HMFs) and ‘indolent’ CAFs do not secrete these microRNAs. We identified a novel class of circulating cells in the blood of breast cancer patients with metastases -CAFs (cCAFs). Patients without metastases did not have these cCAFs - suggesting cCAFs may be “aggressive” CAFs that facilitate breast cancer metastasis. Patients with overt metastasis and elevated counts of cCAFs have significantly higher levels of circulating SDF-1 in their plasma, as well as differential circulating microRNAs, and specifically hMAPK-microRNAs also found secreted by “aggressive” CAFs. Our results suggest there is a hierarchy of CAFs whereby “aggressive” tumors establish “aggressive” CAFs to facilitate metastasis. We also establish a clear link between circulating CAFs and CAF-secreted factors such as SDF-1 and microRNAs with breast cancer metastasis. Citation Format: Sanket H. Shah, Phil Miller, Leah Machlin, Kelsie Medina-Saenz, Ritesh Parajuli, Marc E. Lippman, Dorraya El-Ashry. Circulating CAFs and CAF-secreted factors may be indicative of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4396.

Chemotherapy-induced uridine diphosphate release promotes breast cancer metastasis through P2Y6 activation.

Although purinergic signaling is important in regulation of immune responses, the therapeutic potential of it in the tumor microenvironment is little defined. In this study, we demonstrate that UDP/P2Y6 signaling facilitates breast cancer metastasis both in vitro and in vivo. We found that P2Y6 is not only aberrantly expressed and mutated in most tumor types, but also highly correlated with poor prognosis in breast cancer patients. Furthermore, the migration and invasion of breast cancer cells was obviously increased by UDP and blocked by P2Y6 specific inhibitor MRS2578 and P2Y6 shRNA. Similar results was also found in breast cancer cell metastasis mouse model. Interestingly, the endogenous agonist UDP was released significantly by doxorubicin treated cells. In addition, the expression and enzyme activity of MMP-9 were both promoted by UDP and inhibited by MRS2578 or P2Y6 shRNA. Furthermore, UDP-induced cell invasion was blocked by an MMP-9 inhibitor. Mechanistically, the MAPKs and NF-κB signaling pathways, known to be involved in regulation of MMP-9 expression, were both activated by UDP. Taken together, our study reveals a relationship between extracellular danger signals and breast cancer metastasis, which suggests the potential therapeutic significance of UDP/P2Y6 signaling in cancer therapy.

Abstract IA04: Cancer-associated systemic inflammation facilitates breast cancer metastasis

Abstract Over 90% of breast cancer deaths are due to complications as a consequence of metastasis formation. Despite its devastating effects, metastatic disease is still poorly understood. Accumulating evidence indicates that cells and mediators of the immune system influence metastasis formation. In our lab, we use preclinical mouse models for metastatic breast cancer to dissect the impact of the immune system on the different steps of the metastatic cascade. Neutrophils make up a significant proportion of the inflammatory infiltrate in many tumors and their systemic accumulation in cancer patients has been associated with metastasis formation. Also in our recently developed spontaneous breast cancer metastasis mouse model that accurately mimics each step of the metastatic cascade in humans, metastasis formation is accompanied by pronounced systemic accumulation of neutrophils. Antibody-mediated depletion of neutrophils did not affect primary breast cancer outgrowth, but profoundly decreased spontaneous metastasis formation in lungs and lymph nodes. Using biological and genetic approaches, we have uncovered a novel mammary tumor-induced systemic communication network between gamma delta T cells and neutrophils that is critical for breast cancer metastasis. Our data indicate that targeting this novel cancer cell-initiated systemic inflammatory cascade represents a viable strategy to inhibit metastatic disease. Citation Format: Seth B. Coffelt, Kelly Kersten, Max Wellenstein, Chris W. Doornebal, Camilla Salvagno, Kim Vrijland, Cheei-Sing Hau, Jos Jonkers, Karin E. de Visser. Cancer-associated systemic inflammation facilitates breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA04.

Abstract 154: Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis

Abstract Background: Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. TGFβ signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Methods: Genome-wide lncRNA profile was performed using NCode™ Mouse Noncoding Microarray chip from Life Technologies, which contains 10,802 lncRNAs and 25,178 protein-coding genes. Total RNA was isolated from mouse mammary epithelial NMuMG cells, which had been treated with TGFβ for 0, 12 and 24 hours, and was used to hybridize the microarray. The role of a top TGFβ-upregulated lncRNA, lncRNA-HIT, in EMT and metastasis was further characterized in cell culture and orthotopic breast cancer models Results: Among 10,802 lnRNAs profiled, 680 were up-regulated and 633 were down-regulated during TGFβ-induced EMT, respectively. Further, we identify that lncRNA-HIT (HOXA antisense transcript induced by TGFβ) mediates TGFβ function, i.e., depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth and metastasis. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Conclusions: These data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers. Citation Format: Edward Richards, Gu Zhang, Jennifer Permuth-Wey, Zhu-Peng Li, Sridevi Challa, William Kong, Dan Su, Domenico Coppola, Wei-min Mao, Thomas Sellers, Jin Q. Cheng. Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2015-154