Invasive Human Breast Cancer Research Articles

Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Å between the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

A Potent CD1d-binding Glycolipid for iNKT-Cell-based Therapy Against Human Breast Cancer.

Invariant natural killer T-cells (iNKT) stimulated by CD1d-binding glycolipids have been shown to exert antitumor effects by a number of studies in a mouse model. Breast cancer is a devastating disease, with different types of breast cancer recurring locally or distant as metastatic/advanced disease following initial treatment. The aim of this study was to examine the tumoricidal effect of a CD1d-binding glycolipid, called 7DW8-5, against a highly invasive human breast cancer cell line both in vitro and in vivo. Parental MDA-MB-231 cells and MDA-MB-231 cells transduced with human CD1d were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by loading with glycolipids. After co-culturing with human iNKT cells, the cells were permeabilized and stained with Alexa Flour 647-conjugated antibody to active caspase-3, and analyzed using a BD LSR II. For the in vivo tumoricidal effect, MDA-MB-231 cells transduced with human CD1d and luciferase genes were injected into the mammary fat pad of female NOD/SCID/IL2rγnull (NSG) mice, followed by the injection of human iNKT cells with or without 7DW8-5, and the levels of luminescence were analyzed with whole-body imaging. Human iNKT cells could kill CD1d-expressing human breast cancer cells in vitro in the presence of 7DW8-5, but not α-GalCer. As for in vivo, the adoptive transfer of human iNKT cells into tumor-challenged NSG mice significantly inhibited the growth of CD1d+ MDA-MB-231 human breast cancer cells in the presence of 7DW8-5. CD1d-binding, glycolipid-based iNKT-cell therapy is suggested as a potent and effective treatment against breast cancer in humans.

Cancer stem-like cells directly participate in vasculogenic mimicry channels in triple-negative breast cancer.

ObjectiveVasculogenic mimicry (VM) channels that are lined by tumor cells are a functional blood supply in malignant tumors. However, the role of VM-initiating cells remains poorly understood. Cancer stem-like cells (CSCs) are positively correlated with VM. In this study, triple-negative breast cancer (TNBC) enriched with CSCs was used to investigate the relationship between VM and CSCs.MethodsThe expression of several CSC markers was detected by immunohistochemistry in 100 human breast cancer samples. The clinical significance of CSC markers and the relationship between VM, CSCs, breast cancer subtypes, and VM-associated proteins were analyzed. CD133+ and ALDH+ human and mouse TNBC cells were isolated by FACS to examine the ability of VM formation and the spatial relationship between VM and CSCs.ResultsCSCs were associated with TNBC subtype and VM in human invasive breast cancer. CSCs in TNBC MDA-MB-231 cells formed more VM channels and expressed more molecules promoting VM than the non-TNBC MCF-7 cells in vitro. MDA-MB-231 cells that encircled VM channels on Matrigel expressed CD133. Moreover, CSCs were located near VM channels in the 3D reconstructed blood supply system in human TNBC grafts. The CD133+ and ALDH+ cells isolated from TA2 mouse breast cancer formed more VM channels in vivo. ConclusionsCSCs line VM channels directly. Additionally, CSCs provide more VM-related molecules to synergize VM formation. The signaling pathways that control CSC differentiation may also be potential treatment targets for TNBC.

Targeting PARP1 in XRCC1-Deficient Sporadic Invasive Breast Cancer or Preinvasive Ductal Carcinoma In Situ Induces Synthetic Lethality and Chemoprevention.

: Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS. SIGNIFICANCE: These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells.

MiR-4282 inhibits proliferation, invasion and metastasis of human breast cancer by targeting Myc.

To investigate the expression and role of MicroRNA 4282 (miR-4282) in the development of breast cancer. In situ hybridization was performed to investigate the expression of miR-4282 in human breast cancer tissues. Cell lines stably over-expressed miR-4282 were established by lentivirus transfection. The effects of miR-4282 on the biological behavior of breast cancer cells were then explored in vitro. The results showed that miR-4282 was down-regulated in human breast cancer tissues and was particularly in invasive and metastatic tumors. In addition, the expression of miR-4282 was related to the occurrence of metastasis and the clinical grade of breast cancer. Recovery of miR-4282 expression in the cell could inhibit the proliferation of breast cancer cells, blocked G1-S phase and promoted breast cancer cell apoptosis, inhibited breast cancer cell migration and invasion. Besides, miR-4282 also significantly enhanced the sensitivity of breast cancer cells to paclitaxel. The results of bioinformatics analysis combined with qRT-PCR and Western blot demonstrated that Myc might be the target gene of miR-4282 in breast cancer. miR-4282 inhibited the occurrence and development of breast cancer by regulating Myc, which made it a new target for clinical diagnosis and treatment of breast cancer.

MiR-142-3p as tumor suppressor miRNA in the regulation of tumorigenicity, invasion and migration of human breast cancer by targeting Bach-1 expression.

Breast cancer is the most common type of cancer among women, and despite improved treatments, it remains a major challenge. However, improved mechanistic insight may lead to novel therapeutic strategies. miR-142-3p belongs to the miR-142 family and is involved in pathogenesis and metastasis of various types of malignancies by targeting several important messenger RNAs (mRNAs) including Bach-1. This is especially true for breast cancer, where Bach-1 is involved in the metastatic spread by deregulation of metastasis-associated genes. In this study, we collected 24 breast cancer tissues with 24 adjusted normal tissues to measure the expression levels of miR-142-3p and Bach-1 mRNA using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and IHC. miR-142-3p targeting of Bach-1 expression in MCF-7 and MDA-MB-468 breast cancer cells was evaluated using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, invasion, and migration were assessed by MTT, transwell matrigel and wound healing assay and the EMT-associated proteins C-X-C chemokine receptor type 4 (CXCR-4), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor receptor (VEGFR) were analyzed by western blot analysis. Also, the expression levels of tumor suppressors including miR-330, miR-145, and miR-34a were estimated by qRT-PCR. Analysis of paired specimens of primary malignant and normal tissues showed that miR-142-3p was downregulated, while Bach-1 mRNA and protein both were overexpressed in the breast cancer tumors. This inverse relationship was confirmed by cell line experiments demonstrating that miR-142-3p expression reduced Bach-1 mRNA levels. Furthermore, replacement of miR-142-3p could inhibit the proliferation, invasion, and migration in breast cancer potentially by targeting of Bach-1 mRNA and subsequent inhibition of CXCR4, MMP9, and VEGFR protein expressions. In addition, induction of miR-142-3p could upregulate tumor suppressor miRNAs, including miR-330, miR-145, and miR34a. For the first time, our results revealed that miR-142-3p could target Bach-1in breast cancer cells leading to the reduction of EMT-related proteins and reduced cell proliferation, invasion, and migration. The results also demonstrated that miR-142-3p could regulate important tumor suppressor miRNAs in breast cancer cells. In conclusion, our results suggest that miR-142-3p could be a good candidate for the targeted therapy of breast cancer, especially for the invasive type.

SKA2 mediates invasion and metastasis in human breast cancer via EMT.

Spindle and kinetochore‑associated protein 2 (SKA2) is essential for regulating the progression of mitosis. In recent years, SKA2 upregulation has been detected in various human malignancies and the role of SKA2 in tumorigenesis has received increasing attention. However, the expression and functional significance of SKA2 in breast cancer are not completely understood. To study the effects of SKA2 on breast cancer, the expression levels of SKA2 in breast cancer tissues and cell lines were evaluated by western blotting, reverse transcription‑quantitative polymerase chain reaction and immunohistochemical staining. The results demonstrated that SKA2 expression was increased in breast cancer tissues and cells, and SKA2 overexpression was associated with clinical stage and lymph node metastasis. Functional investigations revealed that SKA2 knockdown in breast cancer cells significantly reduced migration and invasion, and resulted in the decreased expression levels of matrix metalloproteinase (MMP)2 and MMP9. Furthermore, the typical microtubule arrangement was altered in SKA2 small interfering RNA (siSKA2)‑transfected cells. Reduced levels of SKA2 also downregulated the expression of epithelial‑mesenchymal transition proteins, including fibronectin, N‑cadherin and vimentin, whereas there were no alterations in the protein expression levels of E‑cadherin. Conversely, upregulation of SKA2 decreased the expression levels of E‑cadherin, and increased N‑cadherin, fibronectin and vimentin levels. Notably, it was demonstrated that E‑cadherin was translocated from the cytoplasm to the nucleus in siSKA2‑transfected cells. These results demonstrated that SKA2 may be associated with breast cancer metastasis, and siSKA2 inhibited the invasion and metastasis of breast cancer via translocation of E‑cadherin from the cytoplasm to the nucleus.

Retracted: MicroRNA‐590‐5p functions as a tumor suppressor in breast cancer conferring inhibitory effects on cell migration, invasion, and epithelial–mesenchymal transition by downregulating the Wnt–β‐catenin signaling pathway

Breast cancer remains one of the foremost primary causes of female morbidity and mortality worldwide. During the current study, the effect of miR-590-5p and paired-like homeodomain transcription factor 2 (PITX2) on proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) of human breast cancer via the Wnt-β-catenin signaling pathway was investigated. Breast cancer-related genes and related signaling pathways were obtained from KEGG database. The PITX2 regulatory microRNA was predicted. To define the contributory role by which miR-590-5p influences the progression of breast cancer, the interaction between miR-590-5p and PITX2 was explored; the proliferation, invasion, and migration abilities as well as the tumor growth and metastasis in nude mice were detected following the overexpression or silencing of miR-590-5p. PITX2 was determined to share a correlation with breast cancer and miR-590-5p was selected for further analysis. PITX2, Wnt-1, β-catenin, N-cadherin, and vimentin all displayed higher levels, while miR-590-5p and E-cadherin expression were lower among breast cancer tissues than in the adjacent normal tissue. After overexpression of miR-590-5p or si-PITX2, the expression of E-cadherin was markedly increased, decreases in the expression of Wnt-1, β-catenin, N-cadherin, and vimentin, as well as inhibited cell proliferation, invasion, migration, metastasis, and EMT were observed. This study provides evidence suggesting that the transfection of overexpressed miR-590-5p can act to alleviate the effects of breast cancer demonstrating an ability to inhibit the processes of cell proliferation, migration, and invasion as well as EMT by suppressing the expression of PITX2 and activation of the Wnt-β-catenin pathway.

Elastin molecular aging promotes MDA-MB-231 breast cancer cell invasiveness.

Elastin is a long‐lived extracellular matrix protein responsible for the structural integrity and function of tissues. Breast cancer elastosis is a complex phenomenon resulting in both the deposition of elastotic masses and the local production of elastin fragments. In invasive human breast cancers, an increase in elastosis is correlated with severity of the disease and age of the patient. Elastin‐derived peptides (EDPs) are a hallmark of aging and are matrikines – matrix fragments having the ability to regulate cell physiology. They are known to promote processes linked to tumor progression, but their effects on breast cancer cells remain unexplored. Our data show that EDPs enhance the invasiveness of MDA‐MB‐231 breast cancer cells through the engagement of matrix metalloproteases 14 and 2. We therefore suggest that elastosis and/or an aged stroma could promote breast cancer cell invasiveness.

Coronin 1C promotes triple-negative breast cancer invasiveness through regulation of MT1-MMP traffic and invadopodia function.

Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.

PO-017 Is RECQL4 a novel player in glioblastoma pathogenesis?

IntroductionTargeting PARP1 for synthetic lethality is a novel strategy for BRCA germ-line mutated breast cancers. However, BRCA germ-line mutations are rare and reactivation of BRCA mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting the more common sporadic breast cancers and pre-invasive ductal carcinoma in situ (DCIS) are desirable.Material and methodsXRCC1 interacts with PARP1 and coordinates base excision repair. We investigated XRCC1- PARP1 expression in a large cohort of invasive breast cancers (n=1011 and pure DCIS (n=776). Pre-clinically, we monitored Olaparib effect on PARP1 and γH2AX cellular localisation by immunofluorescence. We investigated Olaparib sensitivity in a panel of XRCC1 proficient and deficient murine cell lines and human cancer cell lines. XRCC1 stable Knock out (KO) was generated by CRISPR-Cas9 system in invasive and DCIS model systems. We studied progression from epithelial to mesenchymal transition pheontype in XRCC1 deficient DCIS cells in a 3D culture model.Results and discussionsHere we show that XRCC1 downregulation is an early event in human breast cancer pathogenesis. XRCC1 deficient DCIS are aggressive and associated with increased risk of local recurrence. XRCC1 deficient human invasive breast cancers with high PARP1 levels also manifest aggressive features and poor outcome. At cell line levels,we demonstrate that Olaparib (PARP1 inhibitor) is synthetically lethal in XRCC1 deficient DCIS and invasive breast cancer cells. Cell cycle response to Olaparib treatment in XRCC1 deficient cells was influenced by p53 pathway.In 3D-culture model, XRCC1 KO increased MCF10DCIS invasiveness and promoted epithelial to mesenchymal transition phenotype which was evident with their genetic signature profiling.ConclusionWe conclude that PARP1 targeting is an attractive synthetic lethality and chemoprevention strategy in XRCC1 deficient breast cancers including pre-invasive DCIS.

MicroRNA‑320a suppresses tumor cell growth and invasion of human breast cancer by targeting insulin‑like growth factor1 receptor.

The present study was performed to investigate the biological functions of microRNA‑320a in human breast cancer and the underlying mechanisms. MicroRNA‑320a expression was downregulated in human breast cancer, compared with the normal control. Overexpression of microRNA‑320a induced apoptosis, and inhibited cell viability and invasion in MDA‑MB‑231cells while downregulation of microRNA‑320a reduced apoptosis, and increased cell viability and invasion in MDA‑MB‑231 cells. Then, overexpression of microRNA‑320a suppressed insulin‑like growth factor1 receptor (IGF‑1R), p‑AKt and cyclinD1 protein expression in MDA‑MB‑231cells. In addition, the downregulation of microRNA‑320a induced IGF‑1R, p‑Akt and cyclinD1 protein expression in MDA‑MB‑231cells. Furthermore, the IGF‑1R inhibitor increased the effects of microRNA‑320a on the apoptosis of MDA‑MB‑231 cells. The p‑Akt inhibitor (MK 2206 dihydrochloride, 2.5 nM) increased the effects of microRNA‑320a on the apoptosis of MDA‑MB‑231 cells. These results revealed that microRNA‑320a suppresses tumor cell growth and invasion of human breast cancer by targeting IGF‑1R.

RNA interference targeting CD147 inhibits metastasis and invasion of human breast cancer MCF-7 cells by downregulating MMP-9/VEGF expression.

Breast cancer is one of the most common malignancies. It is necessary to identify new markers for predicting tumor progression and therapeutic molecular targets. It has been reported that CD147 is one of the most commonly expressed proteins in primary tumors and in metastatic cells. In this study, we investigated the role of CD147 in human breast cancer metastasis and invasion, and examined its underlying molecular mechanisms. Immunohistochemistry results revealed high expression of CD147 in human breast tumor tissues, which was positively correlated with the malignancy of breast cancer. MCF-7 cells were transfected with CD147 siRNA eukaryotic expression vector, which resulted in significant knockdown of CD147. We found that CD147 siRNA dramatically inhibited cell proliferation, metastasis, and invasion. Furthermore, our results demonstrated that CD147 siRNA inhibited the synthesis of matrix metalloproteinase 9 (MMP-9) but had no significant effect on matrix metalloproteinase 2 (MMP-2). In addition, CD147 siRNA significantly inhibited the production of vascular endothelial growth factor (VEGF). Taken together, these data indicate that CD147 promotes breast cancer cell proliferation, metastasis, and invasion by modulating MMP-9 and VEGF expression. Thus, CD147 may be used as an important indicator for the judgment of malignant behavior of breast cancer, and may be a potential novel target for breast cancer therapy.

The Molecular Targets and Anti-Invasive Effects of 2,6-bis-(4-hydroxyl-3methoxybenzylidine) cyclohexanone or BHMC in MDA-MB-231 Human Breast Cancer Cells.

In order to metastasize, tumor cells need to migrate and invade the surrounding tissues. It is important to identify compound(s) capable of disrupting the metastasis of invasive cancer cells, especially for hindering invadopodia formation, so as to provide anti-metastasis targeted therapy. Invadopodia are thought to be specialized actin-rich protrusions formed by highly invasive cancer cells to degrade the extracellular matrix (ECM). A curcuminoid analogue known as 2,6-bis-(4-hydroxy-3-methoxybenzylidine)cyclohexanone or BHMC has shown good potential in inhibiting inflammation and hyperalgesia. It also possesses an anti-tumor effects on 4T1 murine breast cancer cells in vivo. However, there is still a lack of empirical evidence on how BHMC works in preventing human breast cancer invasion. In this study, we investigated the effect of BHMC on MDA-MB-231 breast cancer cells and its underlying mechanism of action to prevent breast cancer invasion, especially during the formation of invadopodia. All MDA-MB-231 cells, which were exposed to the non-cytotoxic concentrations of BHMC, expressed the proliferating cell nuclear antigen (PCNA), which indicate that the anti-proliferative effects of BHMC did not interfere in the subsequent experiments. By using a scratch migration assay, transwell migration and invasion assays, we determined that BHMC reduces the percentage of migration and invasion of MDA-MB-231 cells. The gelatin degradation assay showed that BHMC reduced the number of cells with invadopodia. Analysis of the proteins involved in the invasion showed that there is a significant reduction in the expressions of Rho guanine nucleotide exchange factor 7 (β-PIX), matrix metalloproteinase-9 (MMP-9), and membrane type 1 matrix metalloproteinase (MT1-MMP) in the presence of BHMC treatment at 12.5 µM. Therefore, it can be postulated that BHMC at 12.5 µM is the optimal concentration for preventing breast cancer invasion.

RNF126 as a Biomarker of a Poor Prognosis in Invasive Breast Cancer and CHEK1 Inhibitor Efficacy in Breast Cancer Cells.

Purpose: (i) To investigate the expression of the E3 ligase, RNF126, in human invasive breast cancer and its links with breast cancer outcomes; and (ii) to test the hypothesis that RNF126 determines the efficacy of inhibitors targeting the cell-cycle checkpoint kinase, CHEK1.Experimental Design: A retrospective analysis by immunohistochemistry (IHC) compared RNF126 staining in 110 invasive breast cancer and 78 paired adjacent normal tissues with clinicopathologic data. Whether RNF126 controls CHEK1 expression was determined by chromatin immunoprecipitation and a CHEK1 promoter driven luciferase reporter. Staining for these two proteins by IHC using tissue microarrays was also conducted. Cell killing/replication stress induced by CHEK1 inhibition was evaluated in cells, with or without RNF126 knockdown, by MTT/colony formation, replication stress biomarker immunostaining and DNA fiber assays.Results: RNF126 protein expression was elevated in breast cancer tissue samples. RNF126 was associated with a poor clinical outcome after multivariate analysis and was an independent predictor. RNF126 promotes CHEK1 transcript expression. Critically, a strong correlation between RNF126 and CHEK1 proteins was identified in breast cancer tissue and cell lines. The inhibition of CHEK1 induced a greater cell killing and a higher level of replication stress in breast cancer cells expressing RNF126 compared to RNF126 depleted cells.Conclusions: RNF126 protein is highly expressed in invasive breast cancer tissue. The high expression of RNF126 is an independent predictor of a poor prognosis in invasive breast cancer and is considered a potential biomarker of a cancer's responsiveness to CHEK1 inhibitors. CHEK1 inhibition targets breast cancer cells expressing higher levels of RNF126 by enhancing replication stress. Clin Cancer Res; 24(7); 1629-43. ©2018 AACR.

Regulation of OPN expression in MDA‐MB‐435: role of genistein

Breast cancer is the most common cause of cancer‐related mortality in women worldwide and metastatic spread is the leading cause of breast cancer deaths. Osteopontin (OPN) plays a functional role in normal mammary gland development, progression of breast cancer and in malignancy of breast cancer. High expression of OPN is observed in high‐grade metastatic malignant human gliomas and in the highly invasive and metastatic human breast cancer cell lines (e.g. MDA‐MB‐435). Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor‐β (ERβ), is known to have antitumoural properties and also induce apoptotic cell death of ERα‐negative breast cancer cells via p53‐dependent pathway. The isoflavone has been reported to exert antiproliferative activity in some breast cancer cell lines; however, its role with respect to OPN expression is not well understood. Therefore, to address this, the expression level of OPN was investigated upon genistein treatment in MDA‐MB‐435. The cells were treated with different concentrations of genistein for different time intervals; the expression of OPN, the signaling pathways and the promoter regions involved in the regulation of OPN were studied. Genistein displayed an inhibitory effect on OPN protein and mRNA levels in a dose dependent manner and treatment with 50 μM genistein for 24 h showed to be promising. Analysis of the signaling pathways showed no activation of Akt/PKB pathway, no alteration in p‐MEK½ expression whilst an increase in the expression of p‐ERK½ was observed in genistein treated group. These results indicate that genistein acts as an anti‐metastatic agent by inhibiting OPN expression through the activation of p‐ERK½ in MDA‐MB‐435 cells. The transcription factors responsible for this event are being studied in details.Support or Funding InformationDBT (GoI) No. BT/253/NE/TBP/2011 dated May 07, 2012This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting Androgen Receptor and TRAIL: A Novel Treatment Paradigm for Breast Cancer

ObjectiveTRAIL (TNF‐related apoptosis‐inducing ligand is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors. However, most breast cancer cells are resistant to TRAIL‐induced apoptosis. Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.MethodsTo identify modulators of TRAIL‐induced apoptosis, we carried out a genomewide siRNA screen. To validate the screening result, we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL‐induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model. Finally, we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL‐therapy.ResultsWe unexpectedly identified androgen receptor (AR) to be responsible for TRAIL resistance. While AR is classically viewed as the key factor in prostate cancer progression, we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple‐negative breast cancers (TNBC) that are highly aggressive with poor prognosis. Importantly, breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance. AR overexpression in MDA‐MB‐231 and MDA‐MB‐436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF‐7 and MDA‐MB‐453 cells sensitive to TRAIL‐induced apoptosis. AR overexpression also induced TRAIL resistance in breast tumors in vivo. Further, we observed an upregulation of the TRAIL receptor, Death Receptor 5 (DR5) in breast cancer cells, following the removal or inhibition of AR by its antagonists Casodex and MDV3100. Treatment with AR antagonists also enhanced TRAIL‐ induced breast cancer cell apoptosis.ConclusionAR signaling suppresses TRAIL‐induced breast cancer cell apoptosis, in part, by suppressing DR5 expression, and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.Support or Funding InformationThe project supported by the National Institutes of Health (Grants R21CA193271 and R01HL116849); the National Natural Science Foundation of China (Grants 31100595 and 31300683)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P5-10-01: Cellular senescence within HER2-amplified breast cancer: Potential implications for breast cancer immune surveillance and HER2-targeted therapy resistance

Abstract Background:Oncogene-induced senescence is considered as a barrier to tumor progression that arrests cells at risk for malignant transformation. Nevertheless, numerous findings demonstrate that senescent cells may also have the opposite function and promote tumor progression through the release of multiple factors called the senescence-associated secretory phenotype or senescence secretome. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities. Therefore, tumor initiation and progression of breast cancer subtypes is triggered by variable oncogenic stimuli, and differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Beside many studies concerning the role of senescence as a barrier to tumor progression using murine xenograft models very few investigations have been performed to elucidate how often senescent tumor cells appear within untreated human tumors, and if present whether these senescent tumor cells may play a role in disease progression, cancer immunosurveillance and therapy resistance. Methode: In the present study we analysed the appearance of senescent cells within invasive human breast cancers from 129 untreated patients. Cellular senescence was detected by the use of SAβ-gal staining and by immunohistochemical detection of p16, p21, p53, Ki67 and lamin B1. Results: Detection of cellular senescence by the use of SAβ-gal staining and detection of p16, p53, Ki67 and lamin B1 within invasive breast carcinomas indicate that senescent tumor cells varies strongly according to the breast cancer subtype. The highest percentages of senescent tumor cells exist within in the HER2-positive and luminal A breast carcinomas whereas no or very few senescent tumor cells were found in triple negative breast tumors. Based on these findings we suggest that the composition of secretomes released by senescent tumor cells from different breast cancer subtypes might be very distinct in respect to their ability to recruit immune cells, which can eliminate senescent tumor cells on one hand and regulate tumor growth, immune surveillance and therapy resistance on the other. Conclusion: Further characterization of senescent secretomes from HER2 and other breast cancer subtypes and their potential role in tumor progression, immune surveillance and therapy response might be warranted for the understanding of cancer biology as well as prognostic and therapeutic applications. Citation Format: Thaler S, Schad A, Kirkpatrick CJ, Sleeman JP, Springer E, Schmidt M, Cotarelo CL. Cellular senescence within HER2-amplified breast cancer: Potential implications for breast cancer immune surveillance and HER2-targeted therapy resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-10-01.

Abstract A09: Development of epigenetic-based strategies for blocking breast cancer growth, invasion, and metastasis in vitro and in vivo

Abstract DNA hypomethylation has been implicated in the coordinated targeting of various signaling pathways involved in tumor growth and metastasis. In the current study, through various in vitro and in vivo assays, we have examined the plausibility of using universal methyl donor S-adenosyl methionine (SAM) to block breast cancer development, growth, and metastasis in our xenograft and transgenic models of breast cancer. Treatment of highly invasive human triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs578T with SAM resulted in a significant dose-dependent decrease in cell proliferation, invasion, migration, colony formation, and increased apoptosis in vitro. Affymetrix gene expression array and real-time PCR (qPCR) validated the ability of SAM to decrease the expression of several genes implicated in cancer progression in MDA-MB-231 cells. For the in vivo studies, MDA-MB-231 cells expressing green fluorescent protein (MDA-MB-231-GFP) were inoculated into female CD-1 nude mice via mammary fat pad. From day three post tumor cell inoculation, animals were treated with SAM (0.8-1.6 mg/day) or vehicle alone as control via daily oral gavage, and tumor volume was determined at weekly intervals for 10 weeks. SAM treatment caused a significant dose-dependent decrease in tumor volume and GFP-positive metastasis to lungs, liver, and spleen in experimental animals compared to vehicle-treated controls. Analysis of RNA from primary tumors by qPCR showed the ability of SAM to cause a marked decrease in the expression of several prometastatic and EMT pathway genes. Pyrosequencing of tumoral DNA from control and experimental animals showed that SAM treatment causes a significant increase in the percentage of CpG methylation at the promoter region of several cancer-related genes which were seen to be downregulated in the qPCR assay. We next determined the effect of SAM in MMTV-PyMT transgenic mouse model of breast cancer where SAM treatment resulted in a significant delay in the development of mammary tumors and decreased tumor growth in experimental animals as compared to vehicle-treated controls. SAM was found to bioavailable in the serum of experimental animals as determined by mass spectrometry and no notable adverse side effects were seen, including any change in animal behavior. Results from these studies provide compelling evidence for the therapeutic potential of SAM in breast cancer to provide the rationale for initiating clinical trials with SAM in patients with breast and other common cancers as monotherapy or in combination setting with current therapeutic agents to reduce cancer-associated morbidity and mortality. Note: This abstract was not presented at the conference. Citation Format: Niaz Mahmood, David Cheishvili, Ani Arakelian, William J. Muller, Imrana Tanveer, Haseeb Khan, Moshe Szyf, Shafaat A. Rabbani. Development of epigenetic-based strategies for blocking breast cancer growth, invasion, and metastasis in vitro and in vivo [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A09.

Long noncoding RNA SNHG15 promotes human breast cancer proliferation, migration and invasion by sponging miR-211-3p

Long non-coding RNAs (lncRNA) have been demonstrated to act as essential regulators in the development and progression of breast cancer. In our study, we found that long noncoding RNA SNHG15 was highly expressed in breast cancer tissues and cell lines. And the expression of SNHG15 was correlated with TNM stage, lymphnode metastasis and survival in breast cancer patients. SNHG15 knockdown significantly inhibited the proliferation and induced apoptosis in breast cancer cells in vitro and in vivo. Besides, SNHG15 downregulation suppressed cell migration and invasion in MCF-7 and BT-20 cells, and inhibited epithelial-mesenchymal transition (EMT). In mechanism, we found that SNHG15 acted as a competing endogenous RNA to sponge miR-211-3p, which was downregulated in breast cancers and inhibited cell proliferation and migration. Our results showed that there was a negative correlation between SNHG15 and miR-211-3p expression in breast cancer patients. Collectively, we, for the first time, revealed the functions of SNHG15 and miR-211-3p in breast cancer.