Metastatic Potential Of Breast Cancer Research Articles

IL-9 Abrogates the Metastatic Potential of Breast Cancer by Controlling Extracellular Matrix Remodeling and Cellular Contractility.

IL-9 is produced by Th9 cells and is classically known as a growth-promoting cytokine. Although protumorigenic functions of IL-9 are described in T cell lymphoma, recently, we and others have reported anti-tumor activities of IL-9 in melanoma mediated by mast cells and CD8+ T cells. However, involvement of IL-9 in invasive breast and cervical cancer remains unexplored. In this study, we demonstrate IL-9-dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological three-dimensional invasion assays. To dissect underlying mechanisms of IL-9-mediated suppression of invasion, we analyzed IL-9-dependent pathways of cancer cell metastasis, including proteolysis, contractility, and focal adhesion dynamics. IL-9 markedly blocked tumor cell-collagen degradation, highlighting the effects of IL-9 on extracellular matrix remodeling. Moreover, IL-9 significantly reduced phosphorylation of myosin L chain and resultant actomyosin contractility and also increased focal adhesion formation. Finally, IL-9 suppressed IL-17- and IFN-γ-induced metastasis of both human breast (MDA-MB-231) and cervical (HeLa) cancer cells. In conclusion, IL-9 inhibits the metastatic potential of breast and cervical cancer cells by controlling extracellular matrix remodeling and cellular contractility.

The DNA methylation signature of EpCAM‐/CD49f‐ breast cancer stem cells correspond with worse clinical severity in basal‐like breast cancers

The metastatic potential in breast cancer can correspond with the uncontrolled expansion of mammary stem cells identified by loss of epithelial markers EpCAM and CD49f. These populations identified as breast cancer stem cells (CSCs) can be important from a prognostic standpoint, thus characterizing the CSC-specific epigenetic events would be of great interest with DNA methylation playing such a critical role in cell fate. To accomplish this characterization, we compared the 450K DNA methylation profile of EpCAM-/CD49f- cells from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine a CSC-specific DNA methylation signature. Additionally, we overlapped the profiles from 16 established breast cancer cell lines of varying aggressiveness to determine how these cells relate epigenetically with the isolated CSCs. Based on unsupervised PCA and matrix dissimilarity clustering, we identified distinct groups that cluster based on EpCAM-/CD49f- enrichment status, with a group of CSC-rich cells clearly clustering together. This grouping was interesting as these CSC-rich cell line all contained both a TP53 mutation and a PI3K/AKT pathway alteration. From the clustered groups, we performed differential DNA methylation analysis between varying genomic regions and discovered that DNA methylation changes varied by location between CSC-rich and CSC-poor cell lines (ANOVA FDR p-value <0.01). Specifically, CpG islands in promoter-associated regions were differentially hypermethylated in CSC-rich cells, yet non-CpG islands in promoters as well as gene body regions demonstrated extensive hypomethylation in CSCs. Additionally, when comparing CpG island vs non-CpG island promoter methylation, genes with CpG island promoter hypermethylation were enriched for cell differentiation genes (ex: CDH1, IRF6, CLDN7), whereas non-CpG island promoter hypomethylation were enriched for immune regulation and inflammation genes (ex: TLR9, IL32, IL27). Furthermore, correlation between differentially methylated regions and EpCAM-/CD49f- enrichment showed that hypomethylation significantly correlated with an increase in EpCAM-/CD49f- positive cells. Additionally, these hypomethylation events were present in the TCGA breast cohort with hypomethylation events being preferentially enriched in both basal-like tumors and tumors harboring TP53 mutations. Lastly, several genes with lower overall DNA methylation corresponded with worse progression-free survival including RBM47, PLEKHA7, DOK7, and OVOL1. In conclusion, we identified a DNA methylation signature in EpCAM-/CD49f- breast CSCs and demonstrated that these CSC-associated epigenetic events shed light on the clinically severe phenotype in basal-like breast cancers.

CEST MRI pH Mapping of Tumor Acidity as a Predictor of Breast Cancer Metastatic Potential.

CEST MRI pH Mapping of Tumor Acidity as a Predictor of Breast Cancer Metastatic Potential.

Putting Warburg to work: how imaging of tumour acidosis could help predict metastatic potential in breast cancer

Putting Warburg to work: how imaging of tumour acidosis could help predict metastatic potential in breast cancer

Tumour acidosis evaluated in vivo by MRI-CEST pH imaging reveals breast cancer metastatic potential

BackgroundTumour acidosis is considered to play a central role in promoting cancer invasion and migration, but few studies have investigated in vivo how tumour pH correlates with cancer invasion. This study aims to determine in vivo whether tumour acidity is associated with cancer metastatic potential.MethodsBreast cancer cell lines with different metastatic potentials have been characterised for several markers of aggressiveness and invasiveness. Murine tumour models have been developed and assessed for lung metastases and tumour acidosis has been assessed in vivo by a magnetic resonance imaging-based chemical exchange saturation transfer (CEST) pH imaging approach.ResultsThe higher metastatic potential of 4T1 and TS/A primary tumours, in comparison to the less aggressive TUBO and BALB-neuT ones, was confirmed by the highest expression of cancer cell stem markers (CD44+CD24−), highlighting their propensity to migrate and invade, coinciding with the measurement obtained by in vitro assays. MRI-CEST pH imaging successfully discriminated the more aggressive 4T1 and TS/A tumours that displayed a more acidic pH. Moreover, the observed higher tumour acidity was significantly correlated with an increased number of lung metastases.ConclusionsThe findings of this study indicate that the extracellular acidification is associated with the metastatic potential.

Extracellular Matrix-Mediated Breast Cancer Cells Morphological Alterations, Invasiveness, and Microvesicles/Exosomes Release.

Breast cancer is a leading disease in women. Several studies are focused to evaluate the critical role of extracellular matrix (ECM) in various biochemical and molecular aspects but also in terms of its effect on cancer cell morphology and therefore on cancer cell invasion and metastatic potential. ECM fibrillar components, such as collagen and fibronectin, affect cell behavior and properties of mammary cancer cells. The aim of this study was to investigate using the scanning electron microscopy (SEM) how the highly invasive MDA-MB-231 breast cancer cells, interplaying with ECM substrates during cell migration/invasion, modify their morphological characteristics and cytoplasmic processes in relation to their invasive potential. In particular we reproduced and analyzed how natural structural barriers to cancer cell invasion, such as the basement membrane (Matrigel) and fibrillar components of dermis (fibronectin as well as the different concentrations/array of type I collagen), could induce morphological changes in 3D cultures. Interestingly, we demonstrate that, even with different effects, all collagen concentrations/arrays lead to morphological alterations of breast cancer cells. Intriguingly, the elongated mesenchymal shaped cells were more prominent in 3D cultures with a dense and thick substrate (thick Matrigel, high concentrated collagen network, and densely packed collagen fibers), even though cells with different shape produced and released microvesicles and exosomes as well. It is therefore evident that the peri-tumoral collagen network may act not only as a barrier but also as a dynamic scaffold which stimulates the morphological changes of cancer cells, and modulates tumor development and metastatic potential in breast cancer.

The Activity of KIF14, Mieap, and EZR in a New Type of the Invasive Component, Torpedo-Like Structures, Predetermines the Metastatic Potential of Breast Cancer.

Intratumor morphological heterogeneity reflects patterns of invasive growth and is an indicator of the metastatic potential of breast cancer. In this study, we used this heterogeneity to identify molecules associated with breast cancer invasion and metastasis. The gene expression microarray data were used to identify genes differentially expressed between solid, trabecular, and other morphological arrangements of tumor cells. Immunohistochemistry was applied to evaluate the association of the selected proteins with metastasis. RNA-sequencing was performed to analyze the molecular makeup of metastatic tumor cells. High frequency of metastases and decreased metastasis-free survival were detected in patients either with positive expression of KIF14 or Mieap or negative expression of EZR at the tips of the torpedo-like structures in breast cancers. KIF14- and Mieap-positive and EZR-negative cells were mainly detected in the torpedo-like structures of the same breast tumors; however, their transcriptomic features differed. KIF14-positive cells showed a significant upregulation of genes involved in ether lipid metabolism. Mieap-positive cells were enriched in genes involved in mitophagy. EZR-negative cells displayed upregulated genes associated with phagocytosis and the chemokine-mediated signaling pathway. In conclusion, the positive expression of KIF14 and Mieap and negative expression of EZR at the tips of the torpedo-like structures are associated with breast cancer metastasis.

Enhancement of Breast Cancer Cell Aggressiveness by lncRNA H19 and its Mir-675 Derivative: Insight into Shared and Different Actions.

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19, the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

Aurora B induces epithelial-mesenchymal transition by stabilizing Snail1 to promote basal-like breast cancer metastasis.

Aurora B is a serine/threonine kinase that has been implicated in regulating cell proliferation in distinct cancers, including breast cancer. Here we show that Aurora B expression is elevated in basal-like breast cancer (BLBC) compared with other breast cancer subtypes. This high level of expression seems to correlate with poor metastasis-free survival and relapse-free survival in affected patients. Mechanistically, we show that elevated Aurora B expression in breast cancer cells activates AKT/GSK3β to stabilize Snail1 protein, a master regulator of epithelial-mesenchymal transition (EMT), leading to EMT induction in a kinase-dependent manner. Conversely, Aurora B knock down by short-hairpin RNAs (shRNAs) suppresses AKT/GSK3β/Snail1 signaling, reverses EMT and reduces breast cancer metastatic potential in vitro and in vivo. Finally, we identified a specific OCT4 phosphorylation site (T343) responsible for mediating Aurora B-induced AKT/GSK3β/Snail1 signaling and EMT that could be attenuated by Aurora B kinase inhibitor treatment. These findings support that Aurora B induces EMT to promote breast cancer metastasis via OCT4/AKT/GSK3β/Snail1 signaling. Pharmacologic Aurora B inhibition might be a potential effective treatment for breast cancer patients with metastatic disease.

Adipocyte-derived SFRP5 inhibits breast cancer cells migration and invasion through Wnt and epithelial-mesenchymal transition signaling pathways.

ObjectiveObesity is closely associated with metastasis in breast cancer patients. Secreted frizzled-related protein 5 (SFRP5), one of the novel adipokines with anti-inflammatory properties, is associated with obesity. This study aims to study the role of SFRP5 in the crosstalk between obesity and breast cancer metastasis and identify the underlying mechanism.Methods3T3-L1 pre-adipocytes were differentiated to mature adipocytes and a hypertrophic adipocyte model was induced with palmitic acid (PA). Cell motility was measured in MDA-MB-231 and MCF-7 breast cancer cells co-cultured with adipocytes conditioned medium (CM) with or without SFRP5 protein. Wnt and epithelial-mesenchymal transition (EMT) signal pathways were investigated by western blot. Circulating SFRP5 level in 218 breast cancer patients and the association with clinicopathologic characteristics of breast cancer were further determined. Online databases ENCORI and PREDICT Plus were used to exam the link between SFRP5 and prognosis.ResultsReduced SFRP5 level was detected in the hypertrophic adipocyte model. Recombinant SFRP5 protein inhibited MDA-MB-231 and MCF-7 cells invasion and migration induced by PA-treated adipocyte CM, and SFRP5 inhibition by specific antibody reversed the effect of SFRP5. Furthermore, SFRP5 significantly inhibited Wnt and downstream EMT in breast cancer cells. Low circulating SFRP5 level correlated with body mass index (BMI), lymph node (LN) metastasis, TNM stage and high Ki67 expression in breast cancer patients. Increased SFRP5 level was associated with favorable predicted survival. Kaplan-Meier curves showed high SFRP5 level in tumor tissue was associated with better outcome of breast cancer patients.ConclusionsOur findings demonstrated SFRP5 is a vital adipokine that mediates the crosslink between obesity and the metastatic potential of breast cancer. Promotion of SFRP5 expression in the adipose microenvironment may represent a novel approach for preventing breast cancer metastasis.

MicroRNA-6744-5p promotes anoikis in breast cancer and directly targets NAT1 enzyme.

Objective: Anoikis is apoptosis that is induced when cells detach from the extracellular matrix and neighboring cells. As anoikis serves as a regulatory barrier, cancer cells often acquire resistance towards anoikis during tumorigenesis to become metastatic. MicroRNAs (miRNAs) are short strand RNA molecules that regulate genes post-transcriptionally by binding to mRNAs and reducing the expression of its target genes. This study aimed to elucidate the role of a novel miRNA, miR-6744-5p, in regulating anoikis in breast cancer and identify its target gene.Methods: An anoikis resistant variant of the luminal A type breast cancer MCF-7 cell line (MCF-7-AR) was generated by selecting and amplifying surviving cells after repeated exposure to growth in suspension. MiRNA microarray analysis identified a list of dysregulated miRNAs from which miR-6744-5p was chosen for overexpression and knockdown studies in MCF-7. Additionally, the miRNA was also overexpressed in a triple-negative breast cancer cell line, MDA-MB-231, to evaluate its ability to impair the metastatic potential of breast cancer cells.Results: This study showed that overexpression and knockdown of miR-6744-5p in MCF-7 increased and decreased anoikis sensitivity, respectively. Similarly, overexpression of miR-6744-5p in MDA-MB-231 increased anoikis and also decreased tumor cell invasion in vitro and in vivo. Furthermore, NAT1 enzyme was identified and validated as the direct target of miR-6744-5p.Conclusions: This study has proven the ability of miR-6744-5p to increase anoikis sensitivity in both luminal A and triple negative breast cancer cell lines, highlighting its therapeutic potential in treating breast cancer.

Galectin-3 Regulates the Expression of Tumor Glycosaminoglycans and Increases the Metastatic Potential of Breast Cancer.

Galectin-3 (Gal-3) is a multifunctional β-galactoside-binding lectin that once synthesized is expressed in the nucleus, cytoplasm, cell surface, and extracellular environment. Gal-3 plays an important role in breast cancer tumors due to its ability to promote interactions between cell-cell and cell-extracellular matrix (ECM) elements, increasing tumor survival and metastatic dissemination. Still, the mechanism by which Gal-3 interferes with tumor cell migration and metastasis formation is complex and not fully understood. Here, we showed that Gal-3 knockdown increased the migration ability of 4T1 murine breast cancer cells in vitro. Using the 4T1 orthotopic breast cancer spontaneous metastasis mouse model, we demonstrated that 4T1-derived tumors were significantly larger in the presence of Gal-3 (scramble) in comparison with Gal-3 knockdown 4T1-derived tumors. Nevertheless, Gal-3 knockdown 4T1 cells were outnumbered in the bone marrow in comparison with scramble 4T1 cells. Finally, we reported here a decrease in the content of cell-surface syndecan-1 and an increase in the levels of chondroitin sulfate proteoglycans such as versican in Gal-3 knockdown 4T1 cells both in vitro and in vivo. Overall, our findings establish that Gal-3 downregulation during breast cancer progression regulates cell-associated and tumor microenvironment glycosaminoglycans (GAGs)/proteoglycans (PG), thus enhancing the metastatic potential of tumor cells.

Dissection of major cancer gene variants in subsets of circulating tumor cells in advanced breast cancer

Enumeration of circulating tumor cells (CTCs) may reflect the metastatic potential of breast cancer (BC). By using the DEPArray, we investigated CTCs with respect to their epithelial-to-mesenchymal transition phenotype and compared their genomic heterogeneity with tissue biopsies. Seventeen stage IV BC patients were enrolled. Pre-enriched CTC suspensions were stained with fluorescent-labeled antibodies to epithelial (E) and mesenchymal (M) markers. CTC samples were processed by DEPArray system and clustered in relation to their markers. DNA from CTCs, as well as from primary tumor samples, was sequenced by next generation sequencing to assess the mutational state of 50 major cancer-related genes. We identified four different CTC subsets that harbored different gene variants. The most heterogenous CTC subsets included the M+/E− phenotype, which, however, expressed only 7 repeatedly mutated genes, while in the M−/E+ subset multiple mutations affected only 2 out of 50 genes. When matching all gene variants among CTC subsets, a small number of mutations was shared by only 4 genes, namely ATM, FGFR3, PIK3CA, and TP53 that, however, were absent in primary tumors. Our results postulate that the detected mutations in all CTC subsets may be considered as genomic markers of metastatic dissemination to be investigated during early stages of BC.

OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition.

BackgroundHormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT).MethodsThe relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells.ResultsER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don’t detach at significant rates.ConclusionOSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.

MEST induces Twist-1-mediated EMT through STAT3 activation in breast cancers.

The loss of imprinting of MEST has been linked to certain types of cancer by promoter switching. However, MEST-mediated regulation of tumorigenicity and metastasis are yet to be understood. Herein, we reported that MEST is a key regulator of IL-6/JAK/STAT3/Twist-1 signal pathway-mediated tumor metastasis. Enhanced MEST expression is significantly associated with pathogenesis of breast cancer patients. Also, MEST induces metastatic potential of breast cancer through induction of the EMT-TFs-mediated EMT program. Moreover, MEST leads to Twist-1 induction by STAT3 activation and subsequently enables the induction of activation of the EMT program via the induction of STAT3 nuclear translocation. Furthermore, the c-terminal region of MEST was essential for STAT3 activation via the induction of JAK2/STAT3 complex formation. Finally, MEST is required for metastasis in an experimental metastasis model. These observations suggest that MEST is a promising target for intervention to prevent tumor metastasis.

Abstract PD9-09: Proteomic analysis of extracellular matrix helps define drivers of metastatic progression

Abstract Tumor microenvironment (TME) plays a central role in the development of distant metastasis. However, given its complexity, successful targeting of TME will require a detailed understanding of its composition. Cancer-associated fibroblasts represent a large component of TME and are a major contributor to the extracellular matrix (ECM). Previously, we demonstrated the presence of two fibroblast sub-populations (CDCP1pos vs CD146pos fibroblasts) in breast cancer TME, which determine therapeutic response in estrogen receptor (ER) positive breast cancer. We hypothesized that the same fibroblast subtypes would also influence ECM composition and alter the metastatic potential of breast cancer cells. Here, we present the development of a novel fibroblast driven orthotopic model of ER+ breast cancer metastasis, which we combined with an innovative proteomics approach to precisely quantify extracellular matrix proteins. Results: 1.CDCP1pos fibroblasts promote increased breast cancer cell mobility, invasion and metastasis compared to CD146pos fibroblasts.We demonstrate that CDCP1pos fibroblasts significantly increase the invasion potential of breast cancer cells, when compared to CD146pos fibroblasts. Orthotopic co-implantation of ER+ tumor cells with CDCP1pos fibroblasts into the mammary fat pad of mice more frequently drives distant organ metastases to lung and brain when compared with tumors implanted with CD146pos fibroblasts. 2. Proteomic analysis of ER+ tumors influenced by CDCP1pos fibroblasts revealed known and novel drivers of breast cancer metastasis. Breast cancer cells mixed with CDCP1pos fibroblasts produce a non-uniform collagen orientation to the tumor boarder. Our novel proteomic analysis of TME specific proteins revealed that tumors influenced by CDCP1pos fibroblasts have high expression of many ECM proteins linked to increased risk of breast cancer metastasis, including TNC, FN1, COL5A3, and FBN1 among others. Derived proteomic TME signature accurately predicted lymph node involvement in patients who presented with early stage (T0 and T1) tumors in a cohort of 1,009 breast cancer patients from Cancer Genome Atlas Database. 3. Inhibiting fibroblast production of Tenascin C (TNC) results in decreased breast cancer cell invasion. Our in vitro mixed co-culture models, which contain ER+ breast cancer cells with both fibroblast subtypes, demonstrate that only CDCP1pos fibroblasts produce TNC. Furthermore, in spheroid assays with CDCP1posfibroblasts and breast cancer tumor cells, invasion is inhibited by TNC knockdown. The invasion phenotype can be rescued by addition of EGF, which suggests TNC promotes invasion via EGFR signaling. Conclusion: Metastatic spread of cancer cells relies heavily on TME alterations and makeup of the extracellular matrix. Our data suggest that fibroblast composition directly influences ECM properties and metastatic potential in breast cancer. Taken together, we believe that a better understanding of ECM composition will lead to a more personalized approach to breast cancer treatment. Citation Format: Brechbuhl HM, Barrett AS, Kopin E, Gillen A, Hagen J, Johnson L, Finaly-Shultz J, Sartorius CA, Hansen KC, Kabos P. Proteomic analysis of extracellular matrix helps define drivers of metastatic progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-09.

Inhibition of Dipeptidyl Peptidase-4 Accelerates Epithelial-Mesenchymal Transition and Breast Cancer Metastasis via the CXCL12/CXCR4/mTOR Axis.

Dipeptidyl peptidase (DPP)-4 is a multifunctional glycoprotein involved in various biological and pathologic processes. DPP-4 has been widely recognized as a therapeutic target for type 2 diabetes mellitus but is also implicated in the development of human malignancies. Here, we show that inhibition of DPP-4 accelerates breast cancer metastasis via induction of CXCL12/CXCR4, which activates mTOR to promote epithelial-mesenchymal transition (EMT). In cultured cells, DPP-4 knockdown induced EMT and cell migration. Treatment with the DPP-4 inhibitor KR62436 (KR) promoted primary tumor growth and lung metastasis in a 4T1 tumor allograft mouse model; DPP-4 knockdown in 4T1 cells displayed similar phenotypes in vivo and in vitro. KR treatment enhanced the levels of CXCL12/CXCR4 and phosphorylated mTOR, which were associated with the induction of EMT in metastatic cancer cells. KR-induced EMT in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 or the mTOR inhibitor rapamycin, and AMD3100 suppressed KR-induced metastasis in vivo. Our findings suggest that DPP-4 plays a significant role in cancer biology and that inhibition of DPP-4 promotes cancer metastasis via induction of the CXCL12/CXCR4/mTOR/EMT axis. SIGNIFICANCE: These findings reveal that inhibition of DPP-4 increases the metastatic potential of breast cancer. This is especially important given the potential use of DPP-4 inhibition as a therapeutic strategy for type 2 diabetes.

MiR-133b targets Sox9 to control pathogenesis and metastasis of breast cancer

The miR-133b, a commonly recognized muscle-specific miRNA, was reported to be deregulated in many kinds of cancers. However, its potential roles in tumorigenesis remain greatly elusive. Herein, we demonstrate that miR-133b is significantly suppressed in human breast cancer specimens, which is reversely correlated to histological grade of the cancer. Ectopic expression of miR-133b suppresses clonogenic ability and metastasis-relevant traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies have identified Sox9, c-MET, and WAVE2 as direct targets of miR-133b, in which Sox9 contributes to all miR-133b-endowed effects including cell proliferation, colony formation, as well as cell migration and invasion in vitro. Moreover, re-expression of Sox9 reverses miR-133b-mediated metastasis suppression in vivo. Taken together, these findings highlight an important role for miR-133b in the regulation of tumorigenesis and metastatic potential of breast cancer and suggest a potential application of miR-133b in cancer treatment.

Abstract 189: Examining how the extracellular environment alters gene expression in breast cancer

Abstract Breast cancer represents 25% of all cancers diagnosed in women globally with almost 1.7 million new cases annually. The average survival rate for patients with early stage breast cancer has increased, however the survival rate for those with metastatic breast cancer has not improved. Metastasis is the number one cause of death in breast cancer patients; it remains a scientific enigma, with no available early detection screen or targeted therapies available to patients. Therefore, understanding how and why cancer cells migrate is of significant biological and clinical interest. Accumulating evidence suggests the metastatic potential of breast cancer is influenced by the composition of the extracellular matrix (ECM). The ECM is composed of fibrous proteins and other non-cellular biomolecules and has a well-documented role in influencing tumour structure and behaviour. We are examining genes that code for several categories of proteins involved in regulating the composition of the ECM. These genes code for proteins that are involved in signal transduction from the cell membrane or for proteins that are secreted into the ECM. We are also examining genes that code for fibrous proteins, glycoproteins, transmembrane proteins, growth factors and proteases. To refine our panel, we used STRING [1], an online database, to examine the relationships between the proteins coded by our genes based on the experimental determination of protein co-expression and protein homology. Using qRT-PCR, we are examining the expression of our refined gene set in three different cell lines, (representing the major sub-categories of breast cancer) which have been cultured on collagen, fibronectin, laminin and stimulated by various growth factors. We extended our study to examine the expression pattern in cells that were maintained in 3-Dimensional culture over a period of 10 days in an attempt to mimic more closely the physiological process. We have determined that there is significant differential gene expression of integrins and growth factor binding proteins as we change the composition of the cells culture environment. This work is helping us to refine a signature set by identifying genes whose expression is dysregulated in response to changes in the stromal environment. We are moving now to determining whether dysregulation of the expression of our gene panel correlates with disease progression by examining matched normal and diseased patient tissue. This could provide the opportunity to generate a novel gene signature that may be used as a prognostic tool to predict invasive cancers. It may also provide information on the design of novel treatment therapies for patients with metastatic cancers. 1. Szklarczyk, D., et al., The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res, 2017. 45(D1): p. D362-d368. Citation Format: Joanne Nolan, Maeve Kiely, Colum P. Dunne, Aoife J. Lowery, Patrick A. Kiely. Examining how the extracellular environment alters gene expression in 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 189.

Mechanism of metastasis suppression by luteolin in breast cancer.

Metastatic breast cancer is typically an extremely aggressive cancer with poor prognosis. Metastasis requires the orchestration of homeostatic factors and cellular programs, many of which are potential therapeutic targets. Luteolin (2-[3,4-dihydroxyphenyl]-5,7-dihydroxy-4-chromenone), is a naturally occurring flavonoid found in fruits and vegetables that exhibits many anticancer properties. Luteolin obstructs metastasis through both direct and indirect mechanisms. For instance, luteolin may suppress breast cancer invasion by acting as an antiangiogenic therapeutic inhibiting VEGF production and its receptor’s activity. Furthermore, luteolin decreases epithelial–mesenchymal transition markers and metastatic proclivity. Luteolin also acts as an antiproliferative by suppressing receptor tyrosine-kinase activity and apoptosis, both of which could prevent incipient colonization of breast cancer. Many of these antimetastatic characteristics accredited to luteolin are likely functionally related. For instance, the PI3K/Akt pathway, which is impeded by luteolin, has several downstream programs involved in increased proliferation, survival, and metastatic potential in breast cancer. In this review, luteolin’s ability to ameliorate breast cancer is summarized. The paper also offers insight into the molecular mechanisms by which luteolin may suppress breast cancer metastasis.