Role In Breast Cancer Invasion Research Articles

Mammalian enabled protein enhances tamoxifen sensitivity of the hormone receptor-positive breast cancer patients by suppressing the AKT signaling pathway

BackgroundMammalian enabled (MENA) protein is a member of the enabled/vasodilator stimulated phosphoprotein (Ena/VASP) protein family, which regulates cytoplasmic actin network assembly. It plays a significant role in breast cancer invasion, migration, and resistance against targeted therapy and chemotherapy. However, its role in the efficacy of endocrine therapy for the hormone receptor-positive (HR+) breast cancer patients is not known. This study investigated the role of MENA in the resistance against tamoxifen therapy in patients with HR+ breast cancer and the underlying mechanisms.MethodsMENA expression levels in the clinical HR+ breast cancer samples (n = 119) were estimated using immunohistochemistry (IHC) to determine its association with the clinicopathological features, tamoxifen resistance, and survival outcomes. Western blotting (WB) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was performed to estimate the MENA protein and mRNA levels in the tamoxifen-sensitive and -resistant HR+ breast cancer cell lines. Furthermore, CCK8, colony formation, and the transwell invasion and migration assays were used to analyze the effects of MENA knockdown on the biological behavior and tamoxifen sensitivity of the HR+ breast cancer cell lines. Xenograft tumor experiments were performed in the nude mice to determine the tumor growth rates and tamoxifen sensitivity of the control and MENA knockdown HR+ breast cancer cells in the presence and absence of tamoxifen treatment. Furthermore, we estimated the growth rates of organoids derived from the HR+ breast cancer patients (n = 10) with high and low MENA expression levels when treated with tamoxifen.ResultsHR+ breast cancer patients with low MENA expression demonstrated tamoxifen resistance and poorer prognosis compared to those with high MENA expression. Univariate and multivariate Cox regression analysis demonstrated that MENA expression was an independent predictor of tamoxifen resistance in patients with HR+ breast cancer. MENA knockdown HR+ breast cancer cells showed significantly reduced tamoxifen sensitivity in the in vitro experiments and the in vivo xenograft tumor mouse model compared with the corresponding controls. Furthermore, MENA knockdown increased the in vitro invasion and migration of the HR+ breast cancer cells. HR+ breast cancer organoids with low MENA expression demonstrated reduced tamoxifen sensitivity than those with higher MENA expression. Mechanistically, P-AKT levels were significantly upregulated in the MENA-knockdown HR + breast cancer cells treated with or without 4-OHT compared with the corresponding controls.ConclusionsThis study demonstrated that downregulation of MENA promoted tamoxifen resistance in the HR+ breast cancer tissues and cells by enhancing the AKT signaling pathway. Therefore, MENA is a promising prediction biomarker for determining tamoxifen sensitivity in patients with HR+ breast cancer.

The Association of Integrins β3, β4, and αVβ5 on Exosomes, CTCs and Tumor Cells with Localization of Distant Metastasis in Breast Cancer Patients.

Integrins are cell adhesion receptors, which play a role in breast cancer invasion, angiogenesis, and metastasis. Moreover, it has been shown that exosomal integrins provide organotropic metastasis in a mouse model. In our study, we aimed to investigate the expression of integrins β3, β4, and αVβ5 on exosomes and tumor cells (circulating tumor cells and primary tumor) and their association with the localization of distant metastasis. We confirmed the association of exosomal integrin β4 with lung metastasis in breast cancer patients. However, we were unable to evaluate the role of integrin β3 in brain metastasis due to the rarity of this localization. We established no association of exosomal integrin αVβ5 with liver metastasis in our cohort of breast cancer patients. The further evaluation of β3, β4, and αVβ5 integrin expression on CTCs revealed an association of integrin β4 and αVβ5 with liver, but not the lung metastases. Integrin β4 in the primary tumor was associated with liver metastasis. Furthermore, an in-depth analysis of phenotypic characteristics of β4+ tumor cells revealed a significantly increased proportion of E-cadherin+ and CD44+CD24- cells in patients with liver metastases compared to patients with lung or no distant metastases.

Suppression of αvβ6 downregulates P-glycoprotein and sensitizes multidrug-resistant breast cancer cells to anticancer drugs.

Multidrug resistance (MDR) in breast cancer treatment is the major cause leading to the failure of chemotherapy. P-glycoprotein (P-gp), the product of the human MDR1 gene, plays a key role in resistance to chemotherapy and confers cross-resistance to many structurally unrelated anticancer drugs. We have previously reported that integrin αvβ6 plays a critical role in breast cancer invasion and metastasis. However, whether and how αvβ6 is associated with P-gp and regulated by potential genetic mechanisms in breast cancer remains unclear. In the present study, we further investigated the reversal effect and underlying mechanisms of MDR in breast cancer. Two small interfering RNA constructs (pSUPER-β6shRNAs) targeting two different regions of the β6 gene have been designed to inhibit αvβ6 expression by transfecting them into adriamycin-resistant MCF-7/ADR cell lines. Suppression of αvβ6 dramatically downregulated the levels of MDR1 gene mRNA and P-gp. In particular, β6shRNA-mediated silencing of αvβ6 gene increased significantly the cellular accumulation of Rhodamine 123 and markedly decreased drug efflux ability, suggesting that β6shRNAs indeed inhibit P-gp mediated drug efflux and effectively overcome drug resistance. In addition, inhibition of integrin αvβ6 suppressed the expression of ERK1/2. Interestingly, our data demonstrate that suppression of integrin αvβ6 caused significant downregulation of Bcl-2, Bcl-xL and upregulation of caspase 3, Bad, accompanied by increasing activity of cytochrome C. A possible connection between αvβ6 and P-gp in drug resistance biology is suggested. Taken together, β6shRNA could efficiently inhibit αvβ6 and MDR1 expression in vitro and these findings may offer specifically useful means to reverse MDR in breast cancer therapy.

Laminin is over expressed in breast cancer and facilitate cancer cell metastasis.

Breast cancer invasion and metastasis is the main reason for the failure, and laminin is involved in it. This study intends to explore the expression of laminin in breast cancer and normal breast tissue and its clinical significance. We use immunohistochemical assay for the detection of breast infiltrating ductal cancer tissues and normal breast tissues of laminin expression and discuss their role in breast cancer invasion and metastasis. Our results showed that laminin was positive expressed in normal breast tissue, and strongly positive expressed but lost its' continuity in the breast cancer tissue. This results revealed laminin is involved in breast cancer invasion and metastasis, and we can use this to determine whether the integrity of a basement membrane for differential diagnosis of benign and malignant breast tumors.

Enterolactone Suppresses Proliferation, Migration and Metastasis of MDA-MB-231 Breast Cancer Cells Through Inhibition of uPA Induced Plasmin Activation and MMPs-Mediated ECM Remodeling

Background:To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin-MMPs system.Methods:MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values.Results:All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis.Conclusions:EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’.

Differential expression of matrix metalloproteinase-13 in association with invasion of breast cancer

Matrix metalloproteinase-13 (MMP-13) has a potential role in tumour invasion and metastasis. However, its relevance to the prognosis of human breast cancer is poorly understood. The aim of this study is to investigate the expression patterns of MMP-13 protein and to determine its prognostic value in breast cancer, and to define its relation to the clinicopathological features. Immunohistochemistry analysis of MMP-13 was performed on formalin-fixed, paraffin-embedded sections of cancerous breast tissue (n = 76) and normal breast tissue (n = 20), all of which had clinicopathological information available. Based on the principle of immunoreactivity, the detection of MMP-13 on breast tissue was conducted using monoclonal antibodies against MMP-13. A semi-quantitative scoring system was used to assess the presence of, as well as the cellular localisation of MMP-13. MMP-13 expression was significantly greater in the cancerous breast tissues in comparison to those of normal breast tissues. In addition, high levels of MMP-13 expression were also found to be related to the positive detection of breast cancer cells in lymph nodes-amongst breast cancer patients. The results of this study showed that MMP-13 was frequently present in breast tumours, especially when tumours were accompanied by positive breast cancer cell detection in lymph nodes. This suggests that MMP-13 plays a potentially significant role in breast cancer invasion and metastasis.

The Killer Cell Ig-like Receptor 2DL4 Expression in Human Mast Cells and Its Potential Role in Breast Cancer Invasion.

The killer-cell Ig-like receptor (KIR) 2DL4 (CD158d) acts as a receptor for human leukocyte antigen (HLA)-G and is expressed on almost all human natural killer (NK) cells. The expression and function of KIR2DL4 in other hematopoietic cells is poorly understood. Here, we focused on human mast cells, which exhibit cytotoxic activity similar to that of NK cells. KIR2DL4 was detected in all examined human cultured mast cells established from peripheral blood derived from healthy volunteers (PB-mast), the human mast cell line LAD2, and human nonneoplastic mast cells, including those on pathologic specimens. An agonistic antibody against KIR2DL4 decreased KIT-mediated and IgE-triggered responses, and enhanced the granzyme B production by PB-mast and LAD2 cells, by activating Src homology 2-containing protein tyrosine phosphatase (SHP-2). Next, we performed a coculture assay between LAD2 cells and the HLA-G(+) cancer cells, MCF-7 and JEG-3, and showed that KIR2DL4 on LAD2 cells enhanced MMP-9 production and the invasive activity of both cell lines via HLA-G. Immunohistochemical analysis revealed that the direct interaction between HLA-G(+) breast cancer cells and KIR2DL4(+) tissue mast cells (observed in 12 of 36 cases; 33.3%) was statistically correlated with the presence of lymph node metastasis or lymph-vascular invasion (observed in 11 of 12 cases; 91.7%; χ(2) = 7.439; P < 0.01; degrees of freedom, 1) in the clinical samples. These findings suggest that the KIR2DL4 on human mast cells facilitates HLA-G-expressing cancer invasion and the subsequent metastasis.

Inhibition of SDF-1/CXCR4-induced epithelial-mesenchymal transition by kisspeptin-10.

Recently we have shown that breast cancer cell invasion was dramatically increased when co-cultured with MG63 cells. In addition we have generated mesenchymal transformed MCF-7 breast cancer cells (MCF-7-EMT), showing significantly increased invasion in contrast to wild type MCF-7 cells (MCF-7 WT). In this study we have analyzed whether stromal derived factor-1 (SDF-1) is responsible for MCF-7 and T-47-D breast cancer cell invasion and epithelial-mesenchymal-transition (EMT). In addition we have analyzed whether kisspeptin-10 (KP-10) treatment affects SDF-1-induced invasion and EMT. Invasion was quantified by assessment of MCF-7 and T-47-D breast cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during co-culture with MG63 cells or after treatment with SDF-1α, SDF-1β or the combination of both isoforms. Induction of EMT was verified by analysis of protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2) and Vimentin (VIM). The role of SDF-1 for invasion and induction of EMT in breast cancer cells was analyzed by blocking SDF-1 secretion during co-culture with MG63 cells. In addition effects of KP-10 treatment on SDF-1-induced invasion and EMT were analyzed. Breast cancer cell invasion was significantly increased when co-cultured with MG63 cells. During co-culture SDF-1 protein expression of MG63 cells was significantly induced. The increased breast cancer cell invasion could be blocked by anti-SDF-1 antibodies. Treatment of breast cancer cells in monoculture (without MG63) with SDF-1α, SDF-1β or the combination of both isoforms resulted in a significant escalation of breast cancer cell invasion and induction of EMT. Protein expression of mesenchymal markers CDH2 and VIM was clearly elevated, whereas protein expression of epithelial marker CDH1 was clearly decreased. The SDF-1-induced increase of cell invasion was significantly reduced after treatment with KP-10. In addition, induction of EMT was inhibited. Furthermore, protein expression of the binding site of SDF-1, CXC-motive-chemokine receptor 4 (CXCR-4), was reduced by KP-10. Treatment of MCF-7-EMT cells with KP-10 resulted in a significant drop of cell invasion and CXCR-4 protein expression. Our findings suggest that SDF-1 plays a major role in breast cancer invasion and EMT. SDF-1-induced invasion and EMT can be inhibited by KP-10 treatment by down-regulating CXCR-4 expression.

Identification and functional validation of RAD23B as a potential protein in human breast cancer progression.

Identification of protein targets that play a role in breast cancer invasion may help to understand the rapid progression of cancer and may lead to the development of new biomarkers for the disease. In this study, we compared two highly invasive and two poorly invasive breast cancer cell lines using comparative label-free LC-MS profiling in order to identify differentially expressed proteins that may be linked to the invasive phenotype in vitro. Forty-five proteins were found to be upregulated, and 34 proteins, downregulated. UV excision repair protein RAD23 homologue B (RAD23B) was found among the downregulated proteins in highly invasive breast cancer cell lines. In poorly invasive breast cancer cell lines, siRNA-mediated downregulation of RAD23B subsequently led to an increase in invasion and adhesion in vitro. Immunohistochemistry analysis of 164 specimens of invasive breast cancer showed that having a high percentage (>80%) of RAD23B positive nuclei was significantly associated with histopathological grades 1 and 2 breast cancer and with low mitotic activity. In addition, a high staining intensity for RAD23B in the cytoplasm was significantly associated with histopathological grade 3 breast cancer. This study suggests a potential role of RAD23B in breast cancer progression and may further imply a tumor suppressor role of nuclear RAD23B in breast cancer.

The difference in miR-21 expression levels between invasive and non-invasive breast cancers emphasizes its role in breast cancer invasion

MicroRNA-21 (miR-21) overexpression is characteristic for various types of tumors, but it is still unknown whether its expression levels differ between invasive and non-invasive breast carcinomas. The main goal of the study was to determine the difference in miR-21 expression among normal tissue, non-invasive, invasive with non-invasive component, and pure invasive breast cancer samples, to explain its potential role and significance in breast cancer invasiveness. The second goal was to propose miR-21 as molecular marker of breast cancer invasiveness and potential target for future anti-miR therapies for the prevention of invasion and metastasis. In order to reveal the role of miR-21 in breast cancer invasiveness, we measured miR-21 expression levels in 44 breast cancer and four normal samples by stem-loop real-time RT-PCR using TaqMan technology. Relative expression levels of miR-21 were significantly higher in invasive than in other groups (P=0.002) and significantly higher in invasive compared with invasive with non-invasive component group in histological (P=0.043) and nuclear grade 2 (P=0.036), estrogen-receptor-positive (ER+) (P=0.006), progesterone-receptor-positive (PR+) (P=0.008), ER+PR+ (P=0.007), and proliferation index (Ki-67)≤20% (P=0.036) tumors. Our findings suggest that miR-21 could be independent molecular marker of breast cancer invasiveness and potential target for future anti-miR therapies for the prevention of invasion and metastasis.

Epithelial-mesenchymal transition increases during the progression of in situ to invasive basal-like breast cancer

Epithelial-mesenchymal transition (EMT) is known to play an important role in breast cancer invasion and metastatic progression. However, the pattern of expression of EMT markers in the progression from in situ to invasive breast carcinoma is not clear. To investigate this, we performed immunohistochemical analyses of EMT markers (expression of vimentin, smooth muscle actin, osteonectin, and N-cadherin; loss of E-cadherin; alteration of β-catenin), breast cancer stem cell (CSC) markers (CD44(+)/CD24(-), ALDH1), and CD146, an EMT inducer, in invasive carcinomas and ductal carcinoma in situ (DCIS) of the breast. Expression of EMT markers was closely associated with the basal-like subtype and CSC phenotype in invasive carcinoma but not in pure DCIS, except for vimentin. The expression of smooth muscle actin and N-cadherin, loss of E-cadherin, and alteration of β-catenin were significantly higher in invasive carcinomas than in pure DCIS (P = .015, P = .029, P = .001, and P = .007, respectively). Subgroup analyses revealed greater loss of E-cadherin and alteration of β-catenin in invasive carcinoma than in pure DCIS in basal-like subtype (P = .001) but not in non-basal-like subtypes. Moreover, expression of EMT markers and CD146 was higher in the invasive than in the DCIS component of basal-like cancers. Our study confirmed that EMT is an intrinsic characteristic of basal-like subtype and is associated with CSC phenotype. Furthermore, we showed higher expression of EMT markers in invasive carcinomas than in pure DCIS, especially in basal-like subtype, and in the invasive component of basal-like breast cancers, suggesting that EMT may be involved in the progression from in situ to invasive basal-like breast cancers.

Hypoxia-inducible Factor 1 (HIF-1) Promotes Extracellular Matrix Remodeling under Hypoxic Conditions by Inducing P4HA1, P4HA2, and PLOD2 Expression in Fibroblasts

Extracellular matrix (ECM) composition, organization, and compliance provide both architectural and chemical cues that modulate tissue structure and function. ECM produced by stromal fibroblasts plays a key role in breast cancer invasion and metastasis, which are also stimulated by intratumoral hypoxia. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of ECM remodeling by fibroblasts under hypoxic conditions. HIF-1 activates expression of genes encoding collagen prolyl (P4HA1 and P4HA2) and lysyl (PLOD2) hydroxylases. P4HA1 and P4HA2 are required for collagen deposition, whereas PLOD2 is required for ECM stiffening and collagen fiber alignment. Together P4HA1, P4HA2, and PLOD2 mediate remodeling of ECM composition, alignment, and mechanical properties in response to hypoxia. HIF-1-dependent ECM remodeling by hypoxic fibroblasts induces changes in breast cancer cell morphology, adhesion, and motility that promote invasion and metastasis.

MicroRNA miR-24 Enhances Tumor Invasion and Metastasis by Targeting PTPN9 and PTPRF to Promote EGF Signaling

MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

Abstract P1-05-06: A novel mutation in the tyrosine kinase domain of ErbB2: molecular and proteomic investigation of its role in breast cancer invasion

Abstract Introduction: A landmark genomic study revealed 17 somatic coding mutations in a lobular metastatic breast tumor, none of which were present in the patient's primary tumor. Among these was a novel mutation in the tyrosine kinase domain of ErbB2, a gene often amplified in breast cancer. Other mutations in this domain have been shown to stimulate transformation, growth and invasion in breast cancer cells. We hypothesize that an accumulation of mutations in the primary tumor over time preceded the development of an invasive clone, which allowed it to metastasise. Therefore, we investigated the specific effects of ErbB2 on cellular signalling and invasion, in conjunction with proteomic analyses to show its effects on global protein expression. Methods: Wild-type and mutant ErbB2 cDNAs were cloned into V180 pLP 3X FLAG-tagged expression vectors. T47D breast cancer cells were transfected with one of these constructs. Protein was detected by immunoblotting with an anti-ErbB2 antibody. Membranes were then immunoblotted with an anti-phospho-ERK1/2 antibody. Transfected cells were seeded onto Matrigel; cells that migrated through the matrix were stained and counted after 48h. To identify global proteomic changes caused by mutant ErbB2, T47D cells transfected with mutant/wild-type ErbB2 or vector control, were differentially labeled by growing them in medium containing various lysine/arginine isotopes (known as stable isotope labeling of amino acids in cell culture; SILAC). Protein lysates were mixed and subjected to LC-MS/MS mass spectrometry. Proteomic changes were analysed using MaxQuant and DAVID software. Results: Overexpression of the wild-type or mutant ErbB2 genes produced protein expression in T47D, a non-invasive breast cancer cell line, confirmed by western blotting. To measure signalling downstream of ErbB2, we measured levels of the MAP kinase phospho-ERK1/2. Increased phosphorylation of ERK1/2 was demonstrated in cells expressing mutated ErbB2, compared with cells transfected with wild-type. This indicates increased activation of mutant ErbB2, demonstrating distinct phenotypic effects between wild-type and mutant ErbB2 in breast cancer cells. To analyze whether increased activation was accompanied by enhanced invasive properties, invasion assays were performed using Matrigel. A greater number of cells (1.5-fold increase) expressing mutant ErbB2 migrated across this matrix compared with wild-type-expressing cells. Global proteomic analysis of T47D cells overexpressing wild-type or mutant ErbB2 yielded 1,357 total proteins, of which 543 could be quantified. Two subsets of proteins were identified, whose levels were either increased or decreased, in the mutant versus wild-type-transfected lysates (compared to control ratios). Proteins elevated in the mutant-expressing cells included candidates involved in cell migration, inhibition of apoptosis and promotion of cellular survival. Proteins showing reduced expression were involved in pathways including adhesion and DNA damage response. Conclusion: This previously undescribed mutation in the tyrosine kinase domain of ErbB2 differentially effects downstream signalling pathways and may play a role in invasion of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-06.

Abstract P5-04-03: Epithelial-mesenchymal transition is associated with in situ to invasive transition of basal-like breast cancer

Abstract BACKGROUND: Epithelial-mesenchymal transition (EMT) is known to play an important role in breast cancer invasion and metastatic progression and to be associated with cancer stem cells and basal-like subtype. However, its role in the progression of in situ to invasive breast carcinoma is uncertain. To investigate this question, we evaluated the difference in the expression of EMT-related markers between pure ductal carcinoma in situ (DCIS) and invasive carcinomas according to molecular subtype. METHODS: We performed immunohistochemical analyses of EMT-related markers [expression of vimentin, smooth muscle actin (SMA), osteonectin and N-cadherin, translocation of β-catenin and loss of E-cadherin] and breast cancer stem cell markers (CD44+/CD24−, ALDH1) in 320 invasive carcinomas and 179 pure DCIS of breast using tissue microarrays. We also analyzed 39 basal-like invasive cancers with adjacent DCIS component to determine the difference in the expression of EMT-related markers in the invasive and DICS component within individual cases. RESULTS: In invasive carcinomas, vimentin, SMA and osteonectin were highly expressed in basal-like subtype. In addition, loss of E-cadherin and translocation of β-catenin were most frequently found in basal-like subtype. Furthermore, there were positive correlations between the expression of EMT-related markers and stem cell markers (CD44+/CD24- and ALDH1). However, there was no significant difference in the expression of EMT-related markers according to molecular subtype in pure DCIS. When comparing invasive carcinoma with pure DCIS, expression of EMT-related markers was significantly higher in invasive carcinoma than in pure DCIS. Subgroup analysis revealed higher expression of EMT-related markers in invasive carcinoma than in DCIS in basal-like subtype, but not in non-basal-like subtypes. Moreover, in 39 basal-like invasive cancers with adjacent DCIS, expression of mesenchymal markers was increased in invasive component compared to DCIS component. CONCLUSIONS: Our study confirmed that EMT is an intrinsic characteristic of basal-like subtype and is associated with breast cancer with stem cell phenotype. However, increased expression of EMT-related markers in invasive carcinoma compared to pure DCIS, especially in basal-like subtype, and in the invasive component of basal-like invasive carcinoma with DCIS component also suggests a role of EMT in the transition of in situ- to invasive carcinoma in basal-like breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-03.

Crosstalk between Chemokine Receptor CXCR4 and Cannabinoid Receptor CB2 in Modulating Breast Cancer Growth and Invasion

BackgroundCannabinoids bind to cannabinoid receptors CB1 and CB2 and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB2 may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.Methodology/Principal FindingsWe observed high expression of both CB2 and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB2-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.Conclusions/SignificanceThis study provides novel insights into the crosstalk between CB2 and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB2 receptors could be used for developing innovative therapeutic strategies against breast cancer.

Role of chemokine receptor CXCR2 expression in mammary tumor growth, angiogenesis and metastasis

Background:Chemokines and their receptors have long been known to regulate metastasis in various cancers. Previous studies have shown that CXCR2 expression is upregulated in malignant breast cancer tissues but not in benign ductal epithelial samples. The functional role of CXCR2 in the metastatic phenotype of breast cancer still remains unclear. We hypothesize that the chemokine receptor, CXCR2, mediates tumor cell invasion and migration and promotes metastasis in breast cancer. The objective of this study is to investigate the potential role of CXCR2 in the metastatic phenotype of mouse mammary tumor cells.Materials and Methods:We evaluated the functional role of CXCR2 in breast cancer by stably downregulating the expression of CXCR2 in metastatic mammary tumor cell lines Cl66 and 4T1, using short hairpin RNA (shRNA). The effects of CXCR2 downregulation on tumor growth, invasion and metastatic potential were analyzed in vitro and in vivo.Results:We demonstrated knock down of CXCR2 in Cl66 and 4T1 cells (Cl66-shCXCR2 and 4T1-shCXCR2) cells by reverse transcriptase polymerase chain reaction (RT-PCR) at the transcriptional level and by immunohistochemistry at the protein level. We did not observe a significant difference in in vitro cell proliferation between vector control and CXCR2 knock-down Cl66 or 4T1 cells. Next, we examined the invasive potential of Cl66-shCXCR2 cells by in vitro Matrigel invasion assay. We observed a significantly lower number (52 ± 5) of Cl66-shCXCR2 cells invading through Matrigel compared to control cells (Cl66-control) (182 ± 3) (P < 0.05). We analyzed the in vivo metastatic potential of Cl66-shCXCR2 using a spontaneous metastasis model by orthotopically implanting cells into the mammary fat pad of female BALB/c mice. Animals were sacrificed 12 weeks post tumor implantation and tissue samples were analyzed for metastatic nodules. CXCR2 downregulation significantly inhibited tumor cell metastasis. All the mice (n = 10) implanted with control Cl66 cells spontaneously developed lung metastasis, whereas a significantly lower number of mice (40%) implanted with Cl66-shCXCR2 cells exhibited lung metastases.Conclusions:Together, these results suggest that CXCR2 may play a critical role in breast cancer invasion and metastasis.

Targeted Next-Generation Sequencing and Genome-Wide High-Resolution Copy Number DNA Arrays Allow the Identification of Five Novel RUNX1 Fusions In Hematological Malignancies.

AbstractAbstract 1193RUNX1 is a crucial transcription factor involved in cell lineage differentiation during hematopoiesis. It contains a “Runt homology domain” (RHD; exons 3–5, amino acids 50–177) and a transactivation domain (TAD; exon 8, amino acids 291–371). RUNX1 can act as an activator or repressor of target gene expression and thus far two different mechanisms of somatically acquired alterations have been recognized: intragenic mutations and translocations. Most of the translocations involving RUNX1 lead to the formation of a fusion gene consisting of the 5' part of RUNX1 fused to sequences on partner chromosomes. We here present data on 5 cases, 4 acute myeloid leukemias (AML) and 1 chronic myelomonocytic leukemia (CMML) patient, respectively, where previous cytogenetic and FISH analyses revealed reciprocal translocations involving RUNX1. However, even sophisticated molecular diagnostic work-up failed to identify the corresponding RUNX1 fusion partners. Therefore, we used a combination of 454 shotgun pyrosequencing and long-oligonucleotide sequence capture microarrays to reveal these unknown RUNX1 partner genes in four cases. In detail, we performed DNA sequence enrichment using microarrays containing capture probes that were covering a contiguous region on chr. 21 (36,160,098 – 36,421,641), thereby allowing a specific enrichment by hybridization for genomic DNA where the RUNX1 gene is located (Roche NimbleGen 385K chip, Penzberg, Germany). This targeted next-generation sequencing (NGS) assay enabled to capture and sequence single reads mapping to both RUNX1 and other genomic regions (Burrows-Wheeler Aligner's Smith-Waterman algorithm). In median, 324 bp per patient (170,000 reads) with an 18-fold coverage were sequenced and in all cases chimeric reads were detectable, thereby confirming the presence of RUNX1 translocations and, moreover, identifying and characterizing 4 novel fusions on a molecular level. In one AML case, KCNMA1 was fused to RUNX1. KCNMA1, a potassium large conductance calcium-activated channel family member on chromosome 10q22.3, had recently been described to play a role in breast cancer invasion and metastasis to brain. In our case, as confirmed by RT-PCR and Sanger sequencing, the chimeric RUNX1-KCNMA1 fusion led to the disruption of the RHD of RUNX1. In the three additional cases, RUNX1 was fused to genomic regions on chromosomes 10q22, 17q21, and 5q13.3, respectively. The RUNX1-10q22 and the reciprocal 10q22-RUNX1 fusion were confirmed by PCR from genomic DNA and subsequent Sanger sequencing. According to its genomic structure the translocation RUNX1-chr.10q22 will result into the translation of a truncated RUNX1 protein with an intact RHD, but without TAD. Notably, in the remaining two cases, chr.17q21-RUNX1 and chr.5q13.3-RUNX1, only the reciprocal fusion events were detectable by PCR. In case chr.17q21-RUNX1 the translocation would disrupt RUNX1 after the RHD. In chr.5q13.3-RUNX1 the predicted fusion would not impact the RHD and TAD domains because the breakpoint is located before exon 1. In the fifth patient, we performed an analysis using a high-resolution genome-wide cytogenetic copy number DNA microarray to resolve a novel t(X;21)(p11;q22). In this case, the derivative chromosome × was duplicated, leading to a partial trisomy 21q and a partial trisomy X. On chr. 21 the breakpoint was mapped to be located in intron 6–7 within the RUNX1 gene. The breakpoint on the X-chromosome mapped to Xp11.23, thus leading to a truncated RUNX1 protein without the TAD domain. In summary, RUNX1 rearrangements either led to RUNX1 with an intact RHD and TAD (n=1), RUNX1 with an intact RHD but without TAD (n=3, dominant negative effect; similar to RUNX1-RUNX1T1), or to RUNX1 with a disrupted RHD and without TAD domains, leading to haploinsufficiency (n=1). In conclusion, the RUNX1 recombinome is an interesting target to understand pathogenetic heterogeneity in hematological malignancies. Here, we demonstrated that NGS and copy number DNA microarrays allow the identification of novel RUNX1 fusion partners not detectable by standard molecular techniques and reveals that cytogenetic reciprocal translocations lead to different types of RUNX1 alterations. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.

Epidermal growth factor promotes breast cancer cell chemotaxis in CXCL12 gradients

The chemokine receptor CXCR4 and its ligand CXCL12 play an important role in breast cancer invasion and metastasis, and induce the chemotaxis of various types of cancer cells. Previous studies of CXCL12-induced chemotaxis have, for the most part, relied on endpoint assays (e.g., transwell assays) that provide poor control over the cell microenvironment. Specifically, these assays lacked the ability to dissect the role that autocrine and paracrine growth factors play in chemokine-induced cancer cell chemotaxis. Here, we employ a microfluidic chemotaxis chamber that allows the effects of specific exogenous factors on cell migration to be directly characterized, without the interference of autocrine/paracrine signaling. Using this approach, we investigated the migration of MDA-MB-231 breast cancer cells in well-defined CXCL12 gradients. We found that CXCL12 alone failed to stimulate chemotaxis of these cells; however, when the CXCL12 gradient was supplemented with a uniform stimulus of either EGF or conditioned media, a directional response was induced. This dependence on growth factor signaling points to the importance of autocrine and paracrine factors in determining the migratory response of the cells, and may play an important role in cancer metastasis.

Calcitonin inhibits invasion of breast cancer cells: Involvement of urokinase-type plasminogen activator (uPA) and uPA receptor

There is a growing body of evidence indicating that calcitonin (CT) and calcitonin receptor (CTR) are involved in the regulation of cell growth, differentiation, and survival and in tissue development. However, the precise functional role of CT/CTR in breast cancer is still unknown. It is well established that the urokinase plasminogen activator (uPA) system plays an important role in breast cancer invasion and metastasis. The goal of this study was to investigate the effects of CT on regulation of the uPA system and invasive capacity of breast cancer cells. In the highly invasive MDA-MB-231 cell line, 10(-8) M CT decreased both uPA and uPAR mRNA and protein expression which was associated with inhibition of the extracellular signal-regulated kinase (ERK) 1/2 pathway. Furthermore, two weeks of CT administration to nude mice inhibited the expression of uPA mRNA in primary tumors by 25% (P<0.05), as compared to control, untreated animals. CT also inhibited the invasiveness of MDA-MB-231 cells by 37% (10(-8) M CT, P<0.05), as determined by a Matrigel invasion assay. To the best of our knowledge, this is the first report describing a direct effect of CT on breast cancer cell invasion. Our data might suggest a close link between CT signaling, the uPA-mediated pathway, and breast cancer invasion.