Breast Tumor Cell Invasion Research Articles

Adipsin-dependent adipocyte maturation induces cancer cell invasion in breast cancer

Adipocyte-cancer cell interactions promote tumor development and progression. Previously, we identified adipsin (CFD) and its downstream effector, hepatocyte growth factor (HGF), as adipokines that enhance adipocyte-breast cancer stem cell interactions. Here, we show that adipsin-dependent adipocyte maturation and the subsequent upregulation of HGF promote tumor invasion in breast cancers. Mature adipocytes, but not their precursors, significantly induced breast tumor cell migration and invasion in an adipsin expression-dependent manner. Promoters of tumor invasion, galectin 7 and matrix metalloproteinases, were significantly upregulated in cancer cells cocultured with mature adipocytes; meanwhile, their expression levels in cancer cells cocultured with adipocytes were reduced by adipsin knockout (Cfd KO) or a competitive inhibitor of CFD. Tumor growth and distant metastasis of mammary cancer cells were significantly suppressed when syngeneic mammary cancer cells were transplanted into Cfd KO mice. Histological analyses revealed reductions in capsular formation and tumor invasion at the cancer-adipocyte interface in the mammary tumors formed in Cfd KO mice. These findings indicate that adipsin-dependent adipocyte maturation may play an important role in adipocyte-cancer cell interaction and breast cancer progression.

Mammary adipocytes promote breast tumor cell invasion and angiogenesis in the context of menopause and obesity

The mechanism(s) underlying obesity-related postmenopausal (PM) breast cancer (BC) are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the BC microenvironment promotes the acquisition of an invasive and angiogenic BC cell phenotype and accelerates tumor proliferation and progression. BC cells, treated with primary mammary adipocyte secretome from premenopausal (Pre-M) and PM obese women (ObAdCM; obese adipocyte conditioned-media) upregulated the expression of several pro-tumorigenic factors including VEGF, lipocalin-2 and IL-6. Both Pre-M and PM ObAdCM stimulated endothelial cell recruitment and proliferation and significantly stimulated BC cell proliferation, migration and invasion. IL-6 and LCN2 induced STAT3/Akt signaling in BC cells and STAT3 inhibition abrogated the ObAdCM-stimulated BC cell proliferation and migration. Expression of proangiogenic regulators including VEGF, NRP1, NRP2, IL8RB, TGFβ2, and TSP-1 were found to be differentially regulated in mammary adipocytes from obese PM women. Comparative RNAseq indicated an upregulation of PI3K/Akt signaling, ECM-receptor interactions and lipid/fatty acid metabolism in PM versus Pre-M mammary adipocytes. Our results demonstrate that irrespective of menopausal status, cross-talk between obese mammary adipocytes and BC cells promotes tumor aggressiveness and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in obesity-driven breast tumorigenesis.

Effects of Garlic on Breast Tumor Cells with a Triple Negative Phenotype: Peculiar Subtype-Dependent Down-Modulation of Akt Signaling.

Breast cancer includes tumor subgroups with morphological, molecular, and clinical differences. Intrinsic heterogeneity especially characterizes breast tumors with a triple negative phenotype, often leading to the failure of even the most advanced therapeutic strategies. To improve breast cancer treatment, the use of natural agents to integrate conventional therapies is the subject of ever-increasing attention. In this context, garlic (Allium sativum) shows anti-cancerous potential, interfering with the proliferation, motility, and malignant progression of both non-invasive and invasive breast tumor cells. As heterogeneity could be at the basis of variable effects, the main objective of our study was to evaluate the anti-tumoral activity of a garlic extract in breast cancer cells with a triple negative phenotype. Established triple negative breast cancer (TNBC) cell lines from patient-derived xenografts (PDXs) were used, revealing subtype-dependent effects on morphology, cell cycle, and invasive potential, correlated with the peculiar down-modulation of Akt signaling, a crucial regulator in solid tumors. Our results first demonstrate that the effects of garlic on TNBC breast cancer are not unique and suggest that only more precise knowledge of the mechanisms activated by this natural compound in each tumor will allow for the inclusion of garlic in personalized therapeutic approaches to breast cancer.

Mitochondria-Associated Gene SLC25A32 as a Novel Prognostic and Immunotherapy Biomarker: From Pan-Cancer Multiomics Analysis to Breast Cancer Validation

Background. Mutations in SLC25A32 in humans cause late-onset exercise intolerance, which is associated with various neurological and metabolic diseases. However, its specific mechanism of action in tumour development is poorly understood owing to the lack of multiomics integrated analysis of SLC25A32 in pan-cancer. Methods. We used various analytical tools to comprehensively investigate the transcription, protein level, and promoter methylation of SLC25A32. Furthermore, the GSCA and cBioPortal databases were used to evaluate the inheritance impact and epigenetic alterations of SLC25A32 in pan-cancer. SLC25A32 expression and the prognostic significance of copy number alterations in multiple cancers were compared using the UCSCXenaShiny and GEPIA2.0 platforms, and its specific function in breast cancer was experimentally verified. Results. SLC25A32 is abnormally expressed at the transcriptional and protein levels in most cancer types, with aberrant DNA promoter methylation and significant gene amplification in most tumours. SLC25A32 is significantly associated with the survival prognosis of some cancers, immune infiltrating cells, tumour stemness, and immune-related markers. SLC25A32 knockdown decreased breast tumour cell proliferation, invasion, and metastasis. Conclusions. This study aimed to reveal SLC25A32 as a novel prognostic biomarker for pan-cancer prediction and immunotherapy efficacy and specifically describes its underlying mechanism of action in breast cancer. SLC25A32 is widely differentially expressed in pan-cancer with prognostic significance and is correlated with immune infiltration. Additionally, it can affect breast cancer occurrence and development.

Matrix stiffening facilitates the collective invasion of breast cancer through the periostin-integrin mechanotransduction pathway

Matrix rigidity is a critical contributor to tumor progression; however, whether and how matrix stiffness modulates the collective invasion of tumor cells remain unknown. Here we demonstrate that increased matrix stiffness activates YAP to promote the secretion of periostin (POSTN) in cancer-associated fibroblasts, which in turn augments the matrix rigidity of mammary glands and breast tumor tissues by facilitating collagen crosslinking. Moreover, decreased tissue stiffening resulted from the POSTN deficiency impairs peritoneal metastatic potential of orthotopic breast tumors. Increased matrix stiffness also promotes three-dimensional (3D) collective breast tumor cell invasion via multicellular cytoskeleton remodeling. POSTN triggers the integrin/FAK/ERK/Cdc42/Rac1 mechanotransduction pathway during 3D collective invasion of breast tumor. Clinically, high POSTN expression correlates with high collagen levels in breast tumors and cooperatively determines the metastatic recurrence potential in breast cancer patients. Collectively, these findings indicate that matrix rigidity promotes 3D collective invasion of breast tumor cells via the YAP-POSTN-integrin mechanotransduction signaling.

Abstract 3135: Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis

Abstract Obesity is associated with a substantially increased risk (~50%) and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related BC are not clearly understood and, to date, most studies focus on the systemic effects of subcutaneous or visceral adipocytes on BC. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast microenvironment promotes the acquisition of an invasive and vascular breast tumor cell phenotype and thereby markedly accelerates tumor proliferation and progression. We first asked whether local interactions between mammary adipocytes and BC cells might promote tumor growth and if so, what the underlying mechanism(s) might be. BC cells (ER- and ER+), when treated with the secretome of mammary adipocytes from obese women (ObAdCM), upregulated potent angiogenic/inflammatory factors, including VEGF, bFGF, lipocalin-2 (LCN2), IL-6 and significantly suppressed the angiogenesis inhibitor Tsp-1. ObAdCM stimulated endothelial cell (EC) recruitment by BC cells and increased EC proliferation, indispensable steps in tumor neovascularization. PM ObAdCM significantly stimulated BC cell proliferation, migration and invasion. Both IL-6 and LCN2-neutralization resulted in significant reduction of the ObAdCM-stimulated BC cell migration and proliferation, suggesting that these factors play a role in obesity-mediated BC progression. ObAdCM induced pSTAT3/pAkt signaling in BC cells and IL-6 and LCN2 could independently stimulate pSTAT3 in BC cells. STAT3 inhibition abrogated the ObACDM-mediated effects on BC cell proliferation and migration, suggesting that the pro-tumorigenic effects of PM obese mammary adipocytes are mediated by LCN2/IL-6/STAT3 signaling. Comparative RNAseq analyses identified >30 differentially expressed genes (DEGs) in PM compared to pre-menopausal (Pre-M) obese mammary adipocytes. Furthermore, GO and KEGG enrichment analysis of DEGs suggested an upregulation of PI3K/Akt signaling, ECM-receptor interactions, focal adhesions and lipid/fatty acid metabolism in PM mammary adipocytes. Taken together, our results indicate that obese mammary adipocytes in the breast tumor microenvironment can promote migration, proliferation, invasion and the vascular phenotype of BC cells and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in the treatment of obesity-driven breast tumorigenesis. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Emily Man, Katherine Gonzalez, Lauren Merritt, Cassandra Daisy, Rama Aldakhallah, Liang Sun, Biju Isaac, Shira Rockowitz, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3135.

Abstract 2423: A Cdh3 - Lam332 signaling axis by a unique subset of leader cells controls breast tumor organoid protrusion dynamics and directed collective migration

Abstract Carcinoma dissemination can occur when heterogeneous tumor and tumor stromal cells clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and interact with the microenvironment to direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system that mimics the native breast tumor microenvironment, we developed 3D computational models. These hypothesize that for leader cells to polarize to the leading edge and lead directed collective migration they need to generate high protrusive forces and overcome extracellular matrix (ECM) resistance. From scRNAseq of migrating primary tumor organoids, we identify significant K14+ “leader” cell heterogeneity that differs depending upon the environmental signal. We isolate a unique Cadherin-3 (Cdh3) positive leader cell subpopulation that is necessary and sufficient to lead collective migration. Cdh3 controls leader cell protrusion dynamics through controlling the local production of Laminin-332 which is required for integrin/focal adhesion function. Loss of Cdh3 expression in mouse and human primary breast tumor organoids and invasive breast tumor cell lines significantly impairs directed collective migration. In the absence of leader cell Cdh-Cdh3 intercellular adhesions there is reduced cellular β-catenin levels and Laminin 332 production. In syngeneic, orthotopic transplant mouse models, Cdh3 depletion in tumor cells resulted in decreased laminin 332 production at the invasive edge of primary tumors and decreased lung metastasis. Chromatin Immunoprecipitation experiments revealed that in contacted cells β-catenin and TCF4 are present on Lam α3, β3, and γ2 promoter regions. Laminin 332 production by leader cells was required for optimal Integrin/FA function and cellular protrusion stability. These results indicated that in leader cells local Cdh3-Cdh3/β-catenin regulated Laminin 332 production controls protrusion dynamics and overcomes resistance to ECM to lead directed collective tumor migration. Our findings highlight how a unique subset of leader cells in breast tumors interact with the microenvironment to direct collective migration. Citation Format: Yanyang Cao, Priscilla Y. Hwang, Maria Clarke, Jose Almeida, Amit Pathak, Gregory D. Longmore. A Cdh3 - Lam332 signaling axis by a unique subset of leader cells controls breast tumor organoid protrusion dynamics and directed collective migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2423.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Tumor-Derived Exosomal Protein Tyrosine Phosphatase Receptor Type O Polarizes Macrophage to Suppress Breast Tumor Cell Invasion and Migration.

Tumor-derived exosomes, containing multiple nucleic acids and proteins, have been implicated to participate in the interaction between tumor cells and microenvironment. However, the functional involvement of phosphatases in tumor-derived exosomes is not fully understood. We and others previously demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) acts as a tumor suppressor in multiple cancer types. In addition, its role in tumor immune microenvironment remains elusive. Bioinformatical analyses revealed that PTPRO was closely associated with immune infiltration, and positively correlated to M1-like macrophages, but negatively correlated to M2-like macrophages in breast cancer tissues. Co-cultured with PTPRO-overexpressing breast cancer cells increased the proportion of M1-like tumor-associated macrophages (TAMs) while decreased that of M2-like TAMs. Further, we observed that tumor-derived exosomal PTPRO induced M1-like macrophage polarization, and regulated the corresponding functional phenotypes. Moreover, tumor cell-derived exosomal PTPRO inhibited breast cancer cell invasion and migration, and inactivated STAT signaling in macrophages. Our data suggested that exosomal PTPRO inhibited breast cancer invasion and migration by modulating macrophage polarization. Anti-tumoral effect of exosomal PTPRO was mediated by inactivating STAT family in macrophages. These findings highlight a novel mechanism of tumor invasion regulated by tumor-derived exosomal tyrosine phosphatase, which is of translational potential for the therapeutic strategy against breast cancer.

Transfer of miRNA in tumor-derived exosomes suppresses breast tumor cell invasion and migration by inducing M1 polarization in macrophages

AimsMacrophage repolarization from M1 to M2 phenotype is one of the hallmarks of malignancy. M2 macrophages are the most represented population in the tumor microenvironment and play an active role in tumor progression. In recent years, microRNAs (miRNAs) have been identified as a regulator of macrophage polarization. Main methodsIn this study, miR-130 was delivered to M2 macrophages using tumor-derived exosomes. Then, we evaluated the macrophage polarization status by assessment of specific markers and cytokines for M1 and M2 phenotype. The phagocytosis ability of macrophages was also investigated. Additionally, we performed migration and invasion assays to detect the effect of macrophage reprogramming on breast cancer cells migration and invasion. Key findingsThe findings of the current study indicated that exosomes efficiently delivered miR-130 into macrophages. Delivery of miR-130 into macrophages resulted in upregulation of M1 specific markers and cytokines, including CD86, Irf5, Nos2, TNF-α, and IL-1β and downregulation of M2 specific markers and cytokines, including CD206, Ym1, Arg, TGF-β, and IL-10. The phagocytosis ability of macrophages also enhanced after treatment with miRNA-loaded exosomes. Furthermore, migration and invasion assays demonstrated reduced ability of 4T1 breast cancer cells for migration and invasion after macrophages reprogramming. SignificanceThese observations suggest that repolarization of M2 macrophages to M1 phenotype using miRNA-containing exosomes can be a therapeutic strategy against tumor invasion and metastasis in breast cancer.

Aspirin Inhibits Platelets from Reprogramming Breast Tumor Cells and Promoting Metastasis

Introduction: It is now recognized that tumor cells activate platelets, causing them to degranulate and release soluble factors that promote angiogenesis and metastasis. However, it is unknown if these released factors (‘platelet releasate‘) can also affect tumor cell signaling to further drive metastasis.Methods: We used in vitro arrays, ELISA, and invasion assays to study the interactions between platelets and tumor cells in culture. In addition we used an in vivo murine model of mice treated with aspirin and ex vivo patient data from breast cancer patients on aspirin therapy.Results: Breast tumor cells secrete up to 50-fold increased levels of interleukin 8 (IL-8, CXCL8) in response to platelet releasate in vitro, and this tumor-secreted IL-8 is necessary in driving platelet-mediated invasion. Upon characterizing the contents of the platelet releasate, we determined that chemokine (C-C motif) ligand 5 (CCL5) was the component of the platelet releasate that drove IL8 induction in tumor cell lines expressing the CCL5 receptor, CCR5. The mechanism of this platelet-mediated IL-8 induction was found to be activation of the AKT pathway in breast tumor cells; inhibition of this pathway eliminated IL-8 production.We therefore hypothesized that inhibiting platelet activation with aspirin could reverse the effects of platelets on pro-metastatic tumor cell signaling. Platelets treated ex vivo with 100 uM aspirin had up to 51% lower activation in response to tumor cells, released 40% less CCL5, did not activate the AKT pathway, resulted in lower levels of IL-8 production, and did not significantly increase breast tumor cell invasion. In a murine in vivo breast cancer model, immunohistochemistry of MCF-7ras tumor cells revealed that oral administration of aspirin resulted in less secretion of IL-8. In addition, samples were collected from breast cancer patients on aspirin therapy or not; patients on aspirin had 42% less circulating IL-8 and their platelets were less able to drive tumor cell invasion in vitro when compared to patients not on aspirin.Conclusions: Our data suggest that platelets drive breast tumor metastasis by releasing CCL5, activating AKT signaling, and stimulating tumor cells to upregulate and secrete IL-8. Furthermore, we propose aspirin acts as an anti-cancer agent by disrupting the communication between platelets and breast tumor cells. DisclosuresItaliano:Platelet BioGenesis: Equity Ownership; VCAN Bio: Membership on an entity's Board of Directors or advisory committees.

The Effect of Piperine on MMP-9, VEGF, and E-cadherin Expression in Breast Cancer MCF-7 Cell Line

Background: Vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and E-cadherin play a vital role in the behavior of angiogenesis, metastasis, and invasion of breast tumor cells. Piperine, the main component of Piper Nigrum, has shown anti-cancer properties in various malignancies. This Study investigates the potential effect of piperine on MMP-9, E-cadherin, and VEGF expression in breast cancer MCF-7 cell line. Methods: MTT assay was applied to assess the viability of MCF-7 cells. The mRNA levels of MMP-9, VEGF, and E-cadherin were assayed by qRT-PCR. Western blot was performed to identify the protein level of MMP-9. Results: MTT assay results showed that piperine treatment (5, 10, 25, 50, 75, and 100 μM) for 24 hours effectively inhibited cell viability of MCF-7 cells as compared with the control group. Furthermore, the gene expression of VEGF, MMP-9, and E-cadherin was dose-dependently suppressed by piperine treatment (5, 10 and 25 μM) (P<0.05; P<0.01). The results also indicated that piperine (5, 10, and 25 μM) significantly suppressed MMP-9 protein expression after 24 hours of piperine treatment (P<0.01). Conclusion: These results suggest that piperine may prevent angiogenesis, migration, and invasion of MCF-7 cells by suppressing MMP-9 and VEGF, and by inducing E-cadherin expression. Hence, it may be a suitable candidate for designing new drugs in cancer therapy.

Receptor-type protein tyrosine phosphatase alpha (PTPα) mediates MMP14 localization and facilitates triple-negative breast cancer cell invasion.

The ability of cancer cells to invade surrounding tissues requires degradation of the extracellular matrix (ECM). Invasive structures, such as invadopodia, form on the plasma membranes of cancer cells and secrete ECM-degrading proteases that play crucial roles in cancer cell invasion. We have previously shown that the protein tyrosine phosphatase alpha (PTPα) regulates focal adhesion formation and migration of normal cells. Here we report a novel role for PTPα in promoting triple-negative breast cancer cell invasion in vitro and in vivo. We show that PTPα knockdown reduces ECM degradation and cellular invasion of MDA-MB-231 cells through Matrigel. PTPα is not a component of TKS5-positive structures resembling invadopodia; rather, PTPα localizes with endosomal structures positive for MMP14, caveolin-1, and early endosome antigen 1. Furthermore, PTPα regulates MMP14 localization to plasma membrane protrusions, suggesting a role for PTPα in intracellular trafficking of MMP14. Importantly, we show that orthotopic MDA-MB-231 tumors depleted in PTPα exhibit reduced invasion into the surrounding mammary fat pad. These findings suggest a novel role for PTPα in regulating the invasion of triple-negative breast cancer cells.

KYNU, a novel potential target that underpins CD44-promoted breast tumour cell invasion.

Using a validated tetracycline‐off‐inducible CD44 expression system in mouse model, we have previously demonstrated that the hyaluronan (HA) receptor CD44 promotes breast cancer (BC) metastasis to the liver. To unravel the mechanisms that underpin CD44‐promoted BC cell invasion, RNA samples were isolated from two cell models: (a) a tetracycline (Tet)‐Off‐regulated expression system of the CD44s in MCF‐7 cells and; (b) as a complementary approach, the highly metastatic BC cells, MDA‐MB‐231, were cultured in the presence and absence of 50 µg/mL of HA. Kynureninase (KYNU), identified by Microarray analysis, was up‐regulated by 3‐fold upon induction and activation of CD44 by HA; this finding suggests that KYNU is a potential novel transcriptional target of CD44‐downtstream signalling. KYNU is a pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of NAD cofactors from tryptophan that has been associated with the onset and development of BC. This review will attempt to identify and discuss the findings supporting this hypothesis and the mechanisms linking KYNU cell invasion via CD44.

A novel ruthenium(ii) gallic acid complex disrupts the actin cytoskeleton and inhibits migration, invasion and adhesion of triple negative breast tumor cells.

This work describes the synthesis of three new ruthenium(ii) complexes with gallic acid and derivatives of the general formula [Ru(L)(dppb)(bipy)]PF6, where L = gallate (GAC), benzoate (BAC), and esterified-gallate (EGA), bipy = 2,2'-bipyridine and dppb = 1,4-bis(diphenylphosphino)butane. The complexes were characterized by elemental analysis, molar conductivity, NMR, cyclic voltammetry, UV-vis and IR spectroscopy, and two of them by X-ray crystallography. Cell viability assays show promising results, indicating higher cytotoxicity of the complexes in MDA-MB-231 cells, a triple-negative breast cancer (TNBC) cell line, compared with the hormone-dependent MCF-7 cell line. Studies in vitro with the MDA-MB-231 cell line showed that only Ru(BAC) and Ru(GAC) interacted with BSA. Besides that, the Ru(GAC) complex, which has a polyphenolic acid, interacted in an apo-Tf structure and function dependent manner and it was able to inhibit the formation of reactive oxygen species. Ru(GAC) was able to cause damage to the cellular cytoskeleton leading to inhibition of some cellular processes of TNBC cells, such as invasion, migration, and adhesion.

Novel role of BRCA1 interacting C-terminal helicase 1 (BRIP1) in breast tumour cell invasion.

Breast cancer (BC) is the most common malignancy and the leading cause of death in women worldwide. Only 5%‐10% of mutations in BRCA genes are associated with familial breast tumours in Eastern countries, suggesting the contribution of other genes. Using a microarray gene expression profiling study of BC, we have recently identified BRIP1 (fivefold up‐regulation) as a potential gene associated with BC progression in the Omani population. Although BRIP1 regulates DNA repair and cell proliferation, the precise role of BRIP1 in BC cell invasion/metastasis has not been explored yet; this prompted us to test the hypothesis that BRIP1 promotes BC cell proliferation and invasion. Using a combination of cellular and molecular approaches, our results revealed differential overexpression of BRIP1 in different BC cell lines. Functional assays validated further the physiological relevance of BRIP1 in tumour malignancy, and siRNA‐mediated BRIP1 knockdown significantly reduced BC cell motility by targeting key motility‐associated genes. Moreover, down‐regulation of BRIP1 expression significantly attenuated cell proliferation via cell cycle arrest. Our study is the first to show the novel function of BRIP1 in promoting BC cell invasion by regulating expression of various downstream target genes. Furthermore, these findings provide us with a unique opportunity to identify BRIP1‐induced pro‐invasive genes that could serve as biomarkers and/or targets to guide the design of appropriate BC targeted therapies.

Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides.

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.

Abstract 5323: Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis

Abstract Purpose: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme over-expressed by tumor cells. Our group and others have shown that QSOX1 is involved in invasion and metastasis. We previously reported that a small molecule, SBI-183, inhibited the enzymatic activity of QSOX1 and suppressed tumor cell invasion in vitro and metastasis of MDA-MB-231 breast tumor cells in vivo. In our current work we tested SPX-009, an analog of SBI-183 with increased potency, to suppress invasion and metastasis in vitro and in vivo. Methods: Commercially available analogs for SBI-183 were screened in a cell-free fluorescent screening assay for the ability to inhibit QSOX1. Analog compounds that inhibited QSOX1 in a concentration-dependent manner were then tested for the ability to inhibit growth and invasion of triple negative breast cancer (TNBC) cells (MDA-MB-231), sarcomatoid kidney cancer cells (RCJ-41T2) and pancreatic adenocarcinoma cells (MIAPaCa2). MTT assays were used to measure the effect of the compounds on tumor growth. 2D invasion assays and 3D tumor spheroid assays were employed to measure the effect of the analogs on invasion. One potent analog (SPX-009) was evaluated for the ability to suppress tumor growth and metastasis in mice bearing MDA-MB-231-luc breast cancer orthotopic xenografts. Results: Three of 53 analogs inhibited the enzymatic activity of QSOX1 at levels similar to the parent compound. However, one of those compounds, SPX-009, suppressed invasion of breast, kidney and pancreas tumor cells in 2D and 3D invasion assays at >10-fold lower concentrations than the parent compound (SBI-183). In a non-metastatic xenograft of kidney cancer SBI-183 suppressed primary kidney tumor growth by 51%, but in a metastatic xenograft breast cancer model (MDA-MB-231-luc TNBC), SBI-183 reduced lung metastasis by 76% compared to control. Human-mouse xenograft studies are ongoing with the more potent analog (SPX-009) and will also be presented. Conclusions: Chemical inhibitors of QSOX1 have a minor effect on tumor cell viability, but a major effect on tumor cell invasion in vitro and metastasis in vivo. This finding suggests that combining a QSOX1 inhibitor with a cytotoxic agent might provide a means to inhibit metastasis of cells that are resistant to cytotoxic agents. Citation Format: Amber Fifield, Thai H. Ho, Douglas O. Faigel, Sergei Svarovsky, Eduard Sergienko, Thomas C. Chung, Douglas F. Lake. Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis [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 5323.

Abstract 121: Mammary adipocytes drive breast tumor progression and angiogenesis via the VEGF/IL6/STAT3 signaling axis

Abstract Obesity is associated with a substantially increased risk (~50%) and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related BC are not clearly understood and, to date, most studies focus on the systemic effects of subcutaneous or visceral adipocytes on BC. We hypothesize that the increased local presence of ‘obese' mammary adipocytes within the breast microenvironment promotes the acquisition of an angiogenic and invasive breast tumor cell phenotype and thereby markedly accelerates tumor proliferation and progression. We first asked whether local interactions between mammary adipocytes and BC cells might promote tumor growth and if so, what the underlying mechanism(s) might be. Preadipocytes isolated from mammary adipose tissues of overweight/obese pre- (Pre-M; n=7) and post-menopausal (PM; n=9) women were differentiated into mature adipocytes using standard protocols. The effects of both normal (NA) and cancer-associated (CAA) mammary adipocyte secretome from Pre-M and PM obese women were tested on human and mouse BC cell lines respectively. BC cells (MDA-MB-436, MCF-7, M-Wnt), when treated with the secretome of mammary adipocytes from obese women (ObAd-CM), upregulated potent angiogenic factors, including VEGF-A, Angiopoietin-1, Jagged 1, HIF1-α and bFGF and significantly suppressed the angiogenesis inhibitor Tsp-1. ObAd-CM from both Pre-M and PM women significantly stimulated BC cell proliferation, migration and invasion. Importantly, compared to Pre-M patients, CAA Ad-CM from PM patients promoted higher levels of BC cell proliferation, migration and invasion. Pre-treatment with ObAd-CM resulted in increased endothelial cell (EC) recruitment by BC cells, an indispensable step in tumor angiogenesis. This effect was reversed by VEGF neutralization. In addition, ObAd-CM treatment significantly increased EC proliferation and capillary tube formation. Adipokines such as IL-6, IL-8, MCP-1, adiponectin and leptin were highly expressed in ObAd-CM. Of these, IL-6 neutralization alone inhibited the ObAd-CM-induced migratory and invasive phenotype of BC cells. Treatment with ObAd-CM also resulted in significant activation of pSTAT3, pAkt and pErk in BC cells. STAT3 inhibition reversed the ObAd-CM-mediated effects on BC cell proliferation and migration, suggesting that the pro-tumorigenic effects of Ob mammary adipocytes are mediated via IL-6/STAT3 signaling. Taken together, our results indicate that Ob mammary adipocytes promote migration, proliferation, invasion and the angiogenic phenotype in BC cells and suggest that targeting the VEGF/IL-6/STAT3 signaling axis may be a useful strategy in obesity-driven breast tumor growth and metastasis. [Supported by NIH RO1CA185530, the Karp Family Foundation and the Breast Cancer Research Foundation] Citation Format: Roopali Roy, Takaya Shimura, Lauren Merritt, Katherine Gonzalez, Emily Man, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Mammary adipocytes drive breast tumor progression and angiogenesis via the VEGF/IL6/STAT3 signaling axis [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 121.

Postpartum breast cancer progression is driven by semaphorin 7a-mediated invasion and survival.

Young women diagnosed with breast cancer (BC) have poor prognosis due to increased rates of metastasis. Additionally, women within 10 years of most recent childbirth at diagnosis are ~3 times more likely to develop metastasis than age and stage matched nulliparous women. We define these cases as postpartum BC (PPBC) and propose that the unique biology of the postpartum mammary gland drives tumor progression. Our published results revealed roles for SEMA7A in breast tumor cell growth, motility, invasion, and tumor associated-lymphangiogenesis, all of which are also increased in pre-clinical models of PPBC. However, whether SEMA7A drives progression in PPBC remains largely unexplored. Our results presented herein show that silencing of SEMA7A decreases tumor growth in a model of PPBC while overexpression is sufficient to increase growth in nulliparous hosts. Further, we show that SEMA7A promotes multiple known drivers of PPBC progression including tumor associated COX-2 expression and fibroblast-mediated collagen deposition in the tumor microenvironment. Additionally, we show for the first time that SEMA7A expressing cells deposit fibronectin to promote tumor cell survival. Finally, we show that co-expression of SEMA7A/COX-2/FN predicts for poor prognosis in breast cancer patient cohorts. These studies suggest SEMA7A as a key mediator of BC progression and that targeting SEMA7A may open avenues for novel therapeutic strategies.