LM2 Cells Research Articles

7076 Inhibiting Oxidosqualene Cyclase, An Enzyme In The Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis Of Triple-Negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 105 cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 105 MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

7076 Inhibiting Oxidosqualene Cyclase, an Enzyme in the Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis of Triple-negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 10[5] cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 10[5] MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

Cholesterol synthesis is essential for the growth of liver metastasis-prone colorectal cancer cells.

Metastasis to the liver is a leading cause of death in patients with colorectal cancer. To investigate the characteristics of cancer cells prone to metastasis, we utilized an isogenic model of BALB/c and colon tumor 26 (C26) cells carrying an active KRAS mutation. Liver metastatic (LM) 1 cells were isolated from mice following intrasplenic transplantation of C26 cells. Subsequent injections of LM1 cells generated LM2 cells, and after four cycles, LM4 cells were obtained. Invitro, using a perfusable capillary network system, we found comparable extravasation frequencies between C26 and LM4 cells. Both cell lines showed similar growth rates invitro. However, C26 cells showed higher glucose consumption, whereas LM4 cells incorporated more fluorescent fatty acids (FAs). Biochemical analysis revealed that LM4 cells had higher cholesterol levels than C26 cells. A correlation was observed between fluorescent FAs and cholesterol levels detected using filipin III. LM4 cells utilized FAs as a source for cholesterol synthesis through acetyl-CoA metabolism. In cellular analysis, cholesterol accumulated in punctate regions, and upregulation of NLRP3 and STING proteins, but not mTOR, was observed in LM4 cells. Treatment with a cholesterol synthesis inhibitor (statin) induced LM4 cell death invitro and suppressed LM4 cell growth in the livers of nude mice. These findings indicate that colorectal cancer cells prone to liver metastasis show cholesterol-dependent growth and that statin therapy could help treat liver metastasis in immunocompromised patients.

An Optimized Enzyme-Nucleobase Pair Enables In Vivo RNA Metabolic Labeling with Improved Cell-Specificity.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.

ALDH1A1 Gene Expression and Cellular Copper Levels between Low and Highly Metastatic Osteosarcoma Provide a Case for Novel Repurposing with Disulfiram and Copper.

Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis's ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.

Photosensitization of a subcutaneous tumour by the natural anthraquinone parietin and blue light

Photodynamic therapy (PDT) is an anticancer treatment involving administration of a tumour-localizing photosensitizer, followed by activation by light of a suitable wavelength. In previous work, we showed that the natural anthraquinone (AQ) Parietin (PTN), was a promising photosensitizer for photodynamic therapy of leukemic cells in vitro. The present work aimed to analyze the photosensitizing ability of PTN in the mammary carcinoma LM2 cells in vitro and in vivo in a model of subcutaneously implanted tumours. Photodynamic therapy mediated by parietin (PTN-PDT) (PTN 30 µM, 1 h and 1.78 J/cm2 of blue light) impaired cell growth and migration of LM2 cells in vitro. PTN per se induced a significant decrease in cell migration, and it was even more marked after illumination (migration index was 0.65 for PTN and 0.30 for PTN-PDT, *p < 0.0001, ANOVA test followed by Tukey’s multiple comparisons test), suggesting that both PTN and PTN-PDT would be potential inhibitors of metastasis. Fluorescence microscopy observation indicated cytoplasmic localization of the AQ and no fluorescence at all was recorded in the nuclei. When PTN (1.96 mg) dissolved in dimethyl sulfoxide was topically applied on the skin of mice subcutaneously implanted with LM2 cells, PTN orange fluorescence was strongly noticed in the stratum corneum and also in the inner layers of the tumour up to approximately 5 mm. After illumination with 12.74 J/cm2 of blue light, one PDT dose at day 1, induced a significant tumour growth delay at day 3, which was not maintained in time. Therefore, we administered a second PTN-PDT boost on day 3. Under these conditions, the delay of tumour growth was 28% both on days 3 and 4 of the experiment (*p < 0.05 control vs. PTN-PDT, two-way ANOVA, followed by Sidak’s multiple comparisons test). Histology of tumours revealed massive tumour necrosis up to 4 mm of depth. Intriguingly, a superficial area of viable tumour in the 1 mm superficial area, and a quite conserved intact skin was evidenced. We hypothesize that this may be due to PTN aggregation in contact with the skin and tumour milieu of the most superficial tumour layers, thus avoiding its photochemical properties. On the other hand, normal skin treated with PTN-PDT exhibited slight histological changes. These preliminary findings encourage further studies of natural AQs administered in different vehicles, for topical treatment of cutaneous malignancies.

Development of improved SRC-3 inhibitors as breast cancer therapeutic agents.

Steroid receptor coactivators (SRCs) possess specific and distinct oncogenic roles in the initiation of cancer and in its progression to a more aggressive disease. These coactivators interact with nuclear receptors and other transcription factors to boost transcription of multiple genes, which potentiate cancer cell proliferation, migration, invasion, tumor angiogenesis and epithelial-mesenchymal transition (EMT). Targeting SRCs using small molecule inhibitors (SMIs) is a promising approach to control cancer progression and metastasis. By high-throughput screening analysis, we recently identified SI-2 as a potent SRC SMI. To develop therapeutic agents, SI-10 and SI-12, the SI-2 analogs are synthesized that incorporate the addition of F atoms to the SI-2 chemical structure. As a result, these analogs exhibit a significantly prolonged plasma half-life, minimal toxicity and improved hERG activity. Biological functional analysis showed that SI-10 and SI-12 treatment (5-50 nM) can significantly inhibit viability, migration and invasion of breast cancer cells in vitro and repress the growth of breast cancer PDX organoids. Treatment of mice with 10 mg/kg/day of either SI-10 or SI-12 was sufficient to repress the growth of xenograft tumors derived from MDA-MB-231 and LM2 cells. Furthermore, in spontaneous and experimental metastasis mouse models developed from MDA-MB-231 and LM2 cells, respectively, SI-10 and SI-12 effectively inhibited the progression of breast cancer lung metastasis. These results demonstrate that SI-10 and SI-12 are promising therapeutic agents and are specifically effective in blocking tumor metastasis, a key point in tumor progression to a more lethal state that results in patient mortality in the majority of cases.

Long non-coding RNA ARHGAP5-AS1 inhibits migration of breast cancer cell via stabilizing SMAD7 protein

PurposeTumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated.MethodsRNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-β signaling.ResultsWe analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-β signaling pathway due to its inhibitory role on SMAD7.ConclusionARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.

Abstract 5148: ADGRG1 promotes tumorigenesis, invasion/migration, and cell-cell adhesion in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with the highest risk of metastatic recurrence and shortest survival in patients. Chemotherapy, the current standard of treatment for systemic therapy in TNBC, has suboptimal efficacy but high toxicity. Therefore, drugs directed against new targets exhibiting superior efficacy and excellent safety profiles are urgently needed in the treatment of metastasis in TNBC. Recently, adhesion G protein-coupled receptors (GPCRs) have shown an important role in various cancer processes. Traditionally, GPCRs are considered excellent drug targets. In this study, we aimed to evaluate the role of ADGRG1, an adhesion GPCR, in TNBC. ADGRG1 was highly expressed in basal/TNBC compared to luminal subtypes in the Tumor Cancer Genome Atlas (TCGA) Provisional datasets. Higher expression of ADGRG1 predicted poor recurrence-free survival in TNBC patients as determined using the Kaplan-Meier plotter. ADGRG1 gene and protein expression was higher in LM2 derivative with higher lung metastatic potential compared to parental MDA-MB-231 cells. ADGRG1 knockdown using 2 independent target-specific validated siRNAs reduced cell growth by MTT assay in both MDA-MB-231 parental and LM2 cells. ADGRG1 knockdown decreased mammosphere formation in MDA-MB-231 Parental cells but not in LM2 cells. ADGRG1 knockdown significantly inhibited invasion and migration of both Parental MDA-MB-231 cells and their LM2 derivatives in Trans-well assay. In addition, ADGRG1 knockdown significantly decreased cell-cell interaction in the VybrantTM Cell Adhesion assay in both cell types. ADGRG1 was significantly overexpressed in circulating tumor cell (CTC) clusters compared to single CTCs from the LM2 xenograft models. Overall, our results indicate pro-tumorigenic and pro-metastatic role of ADGRG1 which needs further validation in animal studies. Understanding the role of ADGRG1 in cell-cell adhesion as well as its signaling in TNBC are crucial in discovering its potential as a novel and druggable target in TNBC. Citation Format: Raksha Bhat, Noor M. Abdulkareem, Hala Yasser, Hariprasad Thangavel, Meghana V. Trivedi. ADGRG1 promotes tumorigenesis, invasion/migration, and cell-cell adhesion in triple-negative breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5148.

AKT drives sustained motility following MEK inhibition via promoting SNAIL and AXL in MDA-MB-231 LM2

The adaptive activation of alternative signaling pathways contributes to acquired resistance against targeted cancer therapies. Our previous research has shown that blocking Ras/ERK signaling promotes PI3K/AKT signaling in the lung metastatic derivative of MDA-MB-231 (LM2). Because AKT activation was required to drive sustained cell motility following MEK suppression, we extend our research to elucidate how activation of the PI3K/AKT signaling drives sustained motility following MEK inhibition. Reverse phase protein array (RPPA) revealed that SNAIL (SNAI1) was upregulated in U0126 (MEK inhibitor)-treated LM2 cells. Importantly, LM2 cells simultaneously treated with U0126 and PI3K inhibitor LY294002 exhibited reduced expression of SNAIL. Furthermore, depletion of SNAIL led to reduced cell motility in U0126-treated LM2 cells. In addition, we identified AXL as another downstream effector of AKT. These results suggest that SNAIL and AXL are key factors mediating sustained motility of LM2 cells following MEK suppression. Because AKT mediates motile behavior under MEK suppression, our results suggest that AKT and AXL may be targeted to overcome resistance against drugs targeting the Ras/ERK pathway.

Specific chromatin landscapes and transcription factors couple breast cancer subtype with metastatic relapse to lung or brain

BackgroundFew somatic mutations have been linked to breast cancer metastasis, whereas transcriptomic differences among primary tumors correlate with incidence of metastasis, especially to the lungs and brain. However, the epigenomic alterations and transcription factors (TFs) which underlie these alterations remain unclear.MethodsTo identify these, we performed RNA-seq, Chromatin Immunoprecipitation and sequencing (ChIP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) of the MDA-MB-231 cell line and its brain (BrM2) and lung (LM2) metastatic sub-populations. We incorporated ATAC-seq data from TCGA to assess metastatic open chromatin signatures, and gene expression data from human metastatic datasets to nominate transcription factor biomarkers.ResultsOur integrated epigenomic analyses found that lung and brain metastatic cells exhibit both shared and distinctive signatures of active chromatin. Notably, metastatic sub-populations exhibit increased activation of both promoters and enhancers. We also integrated these data with chromosome conformation capture coupled with ChIP-seq (HiChIP) derived enhancer-promoter interactions to predict enhancer-controlled pathway alterations. We found that enhancer changes are associated with endothelial cell migration in LM2, and negative regulation of epithelial cell proliferation in BrM2. Promoter changes are associated with vasculature development in LM2 and homophilic cell adhesion in BrM2. Using ATAC-seq, we identified a metastasis open-chromatin signature that is elevated in basal-like and HER2-enriched breast cancer subtypes and associates with worse prognosis in human samples. We further uncovered TFs associated with the open chromatin landscapes of metastatic cells and whose expression correlates with risk for metastasis. While some of these TFs are associated with primary breast tumor subtypes, others more specifically correlate with lung or brain metastasis.ConclusionsWe identify distinctive epigenomic properties of breast cancer cells that metastasize to the lung and brain. We also demonstrate that signatures of active chromatin sites are partially linked to human breast cancer subtypes with poor prognosis, and that specific TFs can independently distinguish lung and brain relapse.

Abstract 162: ASB13 inhibits breast cancer progression and metastasis through SNAI2 degradation and transcriptional regulation of YAP

Abstract Metastasis is responsible for the majority of cancer-related deaths. The transcription factor SNAI2 promotes metastasis by facilitating tumor cell invasion and tumor initiating activity. However, its post-translational regulation is less studied. We performed a dual luciferase-based, genome-wide E3 ligase siRNA library screening and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells leads to increased cell migration and decreased F-actin polymerization; while overexpression of ASB13 suppresses lung metastasis formed by LM2 cells. Furthermore, we discovered that ASB13 knockout decreases YAP expression, which is dependent on increased SNAI2 protein level. YAP functions as a tumor suppressor gene in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, and cell migration. Clinical data analysis reveals that ASB13 expression is positively correlated with overall survival in breast cancer patients. These findings establish the ASB13-SNAI2-YAP axis as a regulatory mechanism for breast cancer migration and metastasis. Citation Format: Huijuan Fan, Xuxiang Wang, Wenyang Li, Minhong Shen, Yong Wei, Hanqiu Zheng, Yibin Kang. ASB13 inhibits breast cancer progression and metastasis through SNAI2 degradation and transcriptional regulation of YAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 162.

Abstract P6-05-01: A novel cleaved cytoplasmic lncRNA LacRNA interacts with PHB2 and suppresses breast cancer metastasis via repressing MYC targets

Abstract Long noncoding RNAs (lncRNAs) have been implicated in breast cancer metastases through largely unknown mechanisms. In this study, we used microarray analysis to compare lncRNAs expression levels between matched pairs of breast lymph node metastatic tissues and primary tumors. We discovered that lncRNA LINC00478 was substantially downregulated in the metastatic tumor samples. Interestingly, we found that LINC00478 could be cleaved by RNase to simultaneously generates the mature 5' ends of cytoplasmic RNA and 3' ends of nuclear RNA by polyadenylation. We named 5' ends 791-nt RNA as LacRNA (LINC00478-assciated cytoplasmic RNA). Over expression of full-length LINC00478 and LacRNA, but not LINC00478 3' RNA, significantly inhibited breast cancer proliferation, invasion and metastasis in vitro and in vivo.We used CRISPR-dCas9 complex to mediate efficient transcriptional activation of LacRNA at endogenous genomic loci followed by RNA-seq analyses. Gene set enrichment analysis (GSEA) showed that the MYC pathway/targets were prominent gene sets negatively enriched in LacRNA-activated cells. Further study revealed that LacRNA exerted its tumor suppressive activity by directly binding with prohibitin2(PHB2) to enhance its protein stability, which promoted PHB2 competing with MYC for transcriptionally suppressing the MYC target genes (e.g., CDC20, CDC45, CCNA2 and MAD2L1). Mechanistically, LacRNA inhibits breast cancer invasion and metastasis by interacting with PHB2 through LacRNA's 1-300nt region. In addition, taking advantage of CRISPR system to knock-out and activate the expression of LacRNA, as well as rescue experiment, we uncovered the positive correlation between LacRNA and PHB2 and their role in suppressing MYC target genes and cancer metastasis. At the same time, LacRNA can attenuated the MYC induced activation of MYC targets through binding with PHB2, indicating that LacRNA plays a central role in the suppression of MYC target genes. We further explored the role of LacRNA in inhibiting lung metastasis by implanting LacRNA-activated LM2 cells into the mammary fat pads of NOD-SCID mice. Luciferase imaging and histological analysis were used to detect lung metastasis and found that LacRNA significantly suppressed lung metastasis. Immunohistochemistry were used to detect the expression of PHB2 and MYC targets in both orthotopic tumors and lung metastasis and verified their correlation in vivo. Extensive analyses of clinical data indicated that LacRNA level was substantially downregulated in metastases tumors accompanied by enrichment of MYC targets. The robustness value of LacRNA expression was further verified in two independent patient cohorts, including 530 invasive breast cancer tumors in Fudan University Shanghai Cancer Center (FUSCC) and 819 breast patients' data from TCGA. High LacRNA expression level had a significantly better clinical outcome in both cohorts and represented an independent prognostic predictor for DFS (HR=0.48, P=0.006, multivariate analysis) and OS (HR=0.32, P=0.009, multivariate analysis) in FUSCC cohort. Collectively, LacRNA functions as a tumor suppressor lncRNA that inhibits breast cancer invasion-metastasis cascade. Citation Format: Guo R, Su Y-H, Xue J-y, Si J, Chi Y-y, Wu J. A novel cleaved cytoplasmic lncRNA LacRNA interacts with PHB2 and suppresses breast cancer metastasis via repressing MYC targets [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 P6-05-01.

Abstract 3347: SOX9 is a critical regulator of triple-negative breast cancer growth and invasion

Abstract Background: Triple-negative breast cancers (TNBCs) have the worst prognosis of all breast cancers, and have few available therapies other than non-specific and toxic chemotherapy. To identify novel targets for TNBCs, we investigated expression levels of transcription factors (TFs) in TNBCs compared to those in Non-TNBCs. Our previous analyses identified the TFs highly expressed in TNBCs. Of these, six SRY (Sex Determining Region Y)-related HMG-box (SOX) TFs were highly expressed in TNBCs as compared to non-TNBCs. SOX genes belong to a superfamily of gene. There are approximately 20 SOX genes in humans and mice. Hypothesis: SOX TFs are important regulators of TNBC cell growth and metastasis. Material and Methods: We measured breast cancer cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell assays in non-TNBC (MCF7 and ZR75-1) and TNBC (MDA231 and MDA468) cells. DOX-inducible SOX9-knockout (KO) cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs were used to knockdown (KD) SOXs in the different breast cancer cells. Cell cycle phase and apoptosis were detected using flow cytometric analysis. Protein and mRNA levels of SOX9 in cell lines were examined by western blotting and qRT-PCR assays. SOX9 RNA expression data were obtained from the Oncomine Database. The Curtis dataset was used to analyze survival according to SOX9 expression using Kaplan-Meier survival curves and the statistical significance was determined using the log-rank test. Results: We performed a screen using specific siRNA targeting the 6 SOX transcription factors (SOX4, 6, 8, 9, 10 and 11) that are high-expressed in TNBC, and measured the effect of loss on SOX expression on TNBC cell growth and invasion. SOX4, 6, 8, 9, and 10-KD using siRNA caused decreased cell proliferation (by 30% or more) of MDA231 and MDA468 TNBC cells, but not of MCF7 and T47D cell lines (non-TNBC). SOX11 or 17-KD had no effect on breast cancer growth. SOX9-KD and SOX9-KO decreased cell migration and invasion of MDA231 and MDA468 cells. Reduced expression of SOX9 also inhibited the in vivo growth and metastasis of MDA MB-231 and LM2 cells in mice. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. Our studies also demonstrated that loss of SOX9 induced a G1 to S cell cycle arrest and apoptosis. In addition, high expression of SOX9 was correlated with worse overall survival and 5-year disease-free survival in patients with TNBC breast cancer. Conclusion: Our results demonstrate that the SOX9 acts as an essential molecule regulating TNBC growth and invasion. In the future, it may be possible to target SOX9 and its downstream genes to treat TNBC and prevent its metastasis. Grant Support: These studies were supported by a Susan G. Komen for the Cure Promise Grant (PB). Citation Format: Yanxia Ma, Jonathan Shepherd, Dekuang Zhao, Lakshmi Bollu, Jamal Hill, Yun Zhang, Abhijit Mazumdar, Powel H. Brown. SOX9 is a critical regulator of triple-negative breast cancer growth and invasion [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 3347.

Abstract 26: MiR-205 targets integrin-α5 and inhibits triple-negative breast cancer metastasis

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, lacking effective target therapy compared to ER/PR+ or HER2+ breast cancer. Micro-RNAs are small non-coding RNAs playing a significant role in cancer stemness maintenance, tumor growth, and metastasis. Studies showed that miR-205 plays an important role in regulating EMT, cell proliferation and migration, tumor growth and metastasis, and drug-resistance of cancers, such as lung, renal, prostate and breast cancer. However, whether miR-205 plays a role in TNBC tumor growth and metastasis remains to be determined. We found that higher miR-205 expression level was strongly correlated with breast cancer patient better overall survival. The expression level of miR-205 was prominently downregulated in basal mesenchymal-like highly migratory and invasive TNBC cell lines, compared to basal epithelial-like TNBC and other breast cancer cell lines. Bioinformatics analysis identified that integrin- α5 (ITGA5) is a potential target of miR-205. Western blot analysis revealed that the expression level of integrin-α5 is significantly higher in basal mesenchymal-like TNBC cell lines than other breast cancer cell lines. Two miR-205 overexpression cell lines were established by transfecting TNBC LM2 and SUM-159 cell lines with miR-205 precursor expressing lentivirus. The expression level of ITGA5 was downregulated in miR-205 overexpressing LM2 and SUM-159 cells. Wound healing assay and transwell cell migration assay revealed that miR-205 overexpression reduced migration of TNBC cells, although no difference of cell proliferation was observed between miR-205 overexpression and vectors control cells as determined by the MTT assay. The cancer stem cell-like property of TNBC cells was inhibited by miR-205 overexpression as demonstrated by the mammary sphere formation assay. In in vivo experiments, mammary tumor growth and lung metastasis were inhibited by miR-205 overexpression using mammary fat pad xenograft tumor models. When ITGA5 was re-expressed in miR-205 overexpression cell lines, the migratory ability of TNBCs was rescued. The metastatic capability of TNBC mammary xenograft tumors was also restored. Further mechanistic studies proved that miR-205 can reduce the formation of lamellipodium, a hallmark of Rac1/cdc42 activation and cell migration, by targeting ITGA5. Rac1/cdc42 pull-down assay showed that miR-205 inhibited Rac1/cdc42 activation, while ITGA5 overexpression restored the activation of Rac1/cdc42. For uncovering the upstream regulator of Rac1/cdc42, several Rac inhibitors were scanned and it was found that inhibition of vav2 by EHop-016 reduced the downstream effects from ITGA5 signaling. CoIP revealed that Src/Vav2 pathway was activated by ITGA5. In conclusion, miR-205 suppresses TNBC cell migration and tumor metastasis by targeting ITGA5 and inhibits Src/vav-2/Rac1/cdc42 pathway. Citation Format: Yajuan Xiao, Zhishan Wang, Yunfei Li, Jianjun Wu, Hua Tao, Aimin Li, Rongcheng Luo, Chengfeng Yang. MiR-205 targets integrin-α5 and inhibits triple-negative breast cancer metastasis [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 26.

Abstract 4914: Luteolin inhibits metastasis of triple-negative breast cancer cells to the lungs

Abstract Triple-negative breast cancers (TNBC) are basal-like tumors which lack the traditional pharmaceutical targets ER, PR and Her2neu. Most TNBC related deaths occur following metastasis of cancer cells, and development of tumors at secondary sites such as the lungs and bones. Since TNBC lacks ER, PR and Her2neu, toxic non-specific chemotherapeutic agents are administered to these women, who generally present with early metastatic lesions that originate from drug-resistant residual cells, and have poor prognosis. Consequently, novel therapeutic strategies are sought. Luteolin (LU) is a naturally occurring, non-toxic plant compound that has proven effective against several types of cancer, though its effects against cancers of the breast remain unknown. With this in mind we conducted studies, both in vivo and in vitro, to determine whether LU might suppress metastasis of TNBC. In vivo studies were performed using MDA-MB-231 (4175) LM2 cells, a subpopulation of clinically relevant cells (kindly provided by Dr. J. Massagué; Minn et al. Nature. 2005, 436:518-24). MDA-MB-231 (4175) LM2 is a particularly aggressive TNBC breast cancer cell line with a molecular signature preferential for lung metastasis. Herein we report that LU effectively suppressed metastasis of MDA-MB-231 (4175) LM2 cells to lung. Compared with animals given MDA-MB-231 (4175) LM2 cells alone, which developed 67.6 ± 27.1 metastatic lung colonies, treatment with 40 mg/kg LU reduced colony formation to 22.8 ± 3.6 (P = 0.035). Confirmation of the anti-metastatic effect of LU was achieved by inoculating animals with an alternative, less aggressive TNBC cell line, MDA-MB-435. Administration of 20 mg/kg LU to animals treated with MDA-MB-435 cells reduced the number of lung colonies from 14.1 ± 1.6 to 5.3 ± 0.5; P &amp;lt; 0.05. When tumor cells were incubated in vitro with different concentrations (0-50 µM) of LU, we observed a significant dose-dependent reduction in cell viability, induction of apoptosis (P = &amp;lt;0.001), and decreased tumor cell migration. These data suggest that LU suppresses several steps of the metastatic process. Furthermore, relatively low levels (10 μM) of LU significantly inhibited VEGF secretion from tumor cells (P = &amp;lt;0.001), suggesting that the flavonoid has the ability to suppress a potent angiogenic and cell survival factor. In addition, when tumor cells were exposed to either LU or VEGF receptor (KDR) antibody, a similar reduction in TNBC migration potential was observed. This suggests that the anti-tumor actions of LU may, in part, be due to its ability to reduce VEGF levels and block KDR receptor-mediated activity, thereby inhibiting tumor cell migration. These studies demonstrate that the use of LU, a non-toxic, plant derived compound, deserves further investigation as a treatment option for women with TNBC.Supported by a COR award from the College of Veterinary Medicine, and in part by funds from generous donors to the Ellis Fischel Cancer Center, University of Missouri. Citation Format: Matthew T. Cook, Yayun Liang, Cynthia Besch-Williford, Salman Hyder. Luteolin inhibits metastasis of triple-negative breast cancer cells to the lungs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4914. doi:10.1158/1538-7445.AM2017-4914

Abstract P1-08-04: SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion

Abstract Background: SRY (Sex Determining Region Y)-related HMG-box (SOX) genes belong to a super-family of genes, which is characterized by a homologous sequence called the HMG-box residing on the Y-chromosome. There are 20 SOX genes present in humans and mice. We performed a siRNA screen of SOX transcription factors, and found that SOX9 was essential for breast cancer cell growth. The SOX9 protein recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins and has been shown to be required for development, differentiation and lineage commitment. Moreover, SOX9 is expressed in adenocarcinomas, and is highly expressed in the most aggressive cancers. Our previous data shows SOX9 is highly expressed in “triple negative breast cancer” (TNBC) than in non-TNBC. Thus, we hypothesized that the SOX9 transcription factor acts as an essential molecule regulating TNBC growth and invasion. To test the hypothesis, we used SOX9-overexpressed, or SOX9-knockdown/knockout breast cancer cell models to determine whether SOX9 is necessary and/or sufficient to regulate TNBC cell proliferation, migration and invasion. Methods: We measured the cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell migration &amp; invasion assays in ER-positive (MCF7 and ZR75-1) and ER-negative (MDA231 and MDA468) breast cancer cells. DOX-inducible SOX9-knockout cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs was used to knockdown SOXs in the different breast cancer cells. Protein and mRNA levels of SOX9 in TNBC, non-TNBC, immortalized human breast epithelial cell lines were examined by western blotting and qRT-PCR assay. Results: Knockdown of SOXs by siRNA caused decreased cell proliferation of MDA231 by ≥50% and of MDA468 by 30%-50% in siSOX4, siSOX6, siSOX9, siSOX10 and siSOX11 treatment groups (but not in siSOX8 and siSOX17 treatment groups). However, in MCF7 and T47D cell lines, treatment with siRNA to these SOX factors did not cause significant cell growth reduction. We demonstrated that SOX9 is more highly expressed in TNBC cells at both the mRNA and protein levels. Knockdown of SOX9 decreased cell migration and invasion of MDA231 to 25% and 50% respectively. The same effect also was observed in MDA468 cells, with approximately a 50% decline in migration and invasion. In SOX9-knockout MDA231, MDA468, and LM2 cells, cell proliferation, migration, and invasion were significantly reduced. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. We are now conducting in vivo studies to determine the effect of SOX9 on breast cancer cell metastasis. Conclusion: SOX9 is a critical regulator of TNBC cell proliferation, migration and invasion. These studies suggest that regulating SOX9 transcription factor and its signaling pathway will be a promising therapeutic strategy to treat TNBC and prevent metastasis. This work was supported by a Susan G. Komen Scientific Advisory Board Grant, SAB1600006 (PB), and a grant from the Breast Cancer Research Foundation 2015-2016 BCRF grant(PB), and by the Charles Cain Endowment (PB). Citation Format: Ma Y, Shepherd J, Mazumdar A, Zhao D, Bollu L, Hill J, Zhang Y, Brown P. SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-04.

Tissue factor-specific ultra-bright SERRS nanostars for Raman detection of pulmonary micrometastases.

Here we demonstrate a novel application of 'surface enhanced resonance Raman scattering nanoparticles' (SERRS NPs) for imaging breast cancer lung metastases with much higher precision than currently feasible. A breast cancer lung metastasis mouse model was established by intravenous injection of LM2 cells. These mice were intravenously administered SERRS NPs conjugated with ALT-836, an anti-tissue factor (TF) monoclonal antibody, and subjected to Raman imaging to visualize the expression of TF both in vivo and ex vivo. Raman imaging indicated marked uptake of αTF-SERRS-NPs by the lung metastases compared to isotype and blocking controls. Conversely, little uptake of αTF-SERRS-NPs was observed in the lungs of healthy mice. Successful detection and delineation of pulmonary micrometastatic lesions as small as 200 μm, corroborated by histology, immunohistochemistry, and bioluminescence imaging confirmed the suitability of both TF as a target and αTF-SERRS-NPs as an effective contrast agent for imaging breast cancer lung metastases. Further advancements of this technique in the form of Raman endoscopes coupled with ultrabright SERRS NPs developed in this work could lead to minimally invasive detection and resection of lung metastases.

Luteolin inhibits lung metastasis, cell migration, and viability of triple-negative breast cancer cells.

Most breast cancer-related deaths from triple-negative breast cancer (TNBC) occur following metastasis of cancer cells and development of tumors at secondary sites. Because TNBCs lack the three receptors targeted by current chemotherapeutic regimens, they are typically treated with extremely aggressive and highly toxic non-targeted treatment strategies. Women with TNBC frequently develop metastatic lesions originating from drug-resistant residual cells and have poor prognosis. For this reason, novel therapeutic strategies that are safer and more effective are sought. Luteolin (LU) is a naturally occurring, non-toxic plant compound that has proven effective against several types of cancer. With this in mind, we conducted in vivo and in vitro studies to determine whether LU might suppress metastasis of TNBC. In an in vivo mouse metastasis model, LU suppressed metastasis of human MDA-MB-435 and MDA-MB-231 (4175) LM2 TNBC cells to the lungs. In in vitro assays, LU inhibited cell migration and viability of MDA-MB-435 and MDA-MB-231 (4175) LM2 cells. Further, LU induced apoptosis in MDA-MB-231 (4175) LM2 cells. Relatively low levels (10 µM) of LU significantly inhibited vascular endothelial growth factor (VEGF) secretion in MDA-MB-231 (4175) LM2 cells, suggesting that it has the ability to suppress a potent angiogenic and cell survival factor. In addition, migration of MDA-MB-231 (4175) LM2 cells was inhibited upon exposure to an antibody against the VEGF receptor, KDR, but not by exposure to a VEGF165 antibody. Collectively, these data suggest that the anti-metastatic properties of LU may, in part, be due to its ability to block VEGF production and KDR-mediated activity, thereby inhibiting tumor cell migration. These studies suggest that LU deserves further investigation as a potential treatment option for women with TNBC.

Integrin β1, myosin light chain kinase and myosin IIA are required for activation of PI3K-AKT signaling following MEK inhibition in metastatic triple negative breast cancer.

The effectiveness of targeted therapies against the Ras-ERK signaling pathway are limited due to adaptive resistance of tumor cells. Inhibition of the Ras-ERK pathway can result in activation of the PI3K-AKT pathway, thereby diminishing the therapeutic effects of targeting ERK signaling. Here we investigated the crosstalk between the Ras-ERK and PI3K-AKT pathways in MDA-MB-231 breast cancer cell lines that have a preference to metastasize to lung (LM2), brain (BrM2) or bone (BoM2). Inhibition of the Ras-ERK pathway reduced motility in both parental and BoM2 cells. In contrast, inhibition of the Ras-ERK pathway in BrM2 and LM2 cells resulted in activation of PI3K-AKT signaling that was responsible for continued cell motility. Analysis of the cross talk between Ras-ERK and PI3K-AKT signaling pathways revealed integrin β1, myosin light chain kinase (MLCK) and myosin IIA are required for the activation of PI3K-AKT following inhibition of the Ras-ERK pathway. Furthermore, feedback activation of the PI3K-AKT pathway following MEK suppression was independent of the epidermal growth factor receptor. Thus, integrin β1, MLCK, and myosin IIA are factors in the development of resistance to MEK inhibitors. These proteins could provide an opportunity to develop markers and therapeutic targets in a subgroup of triple negative breast cancer (TNBC) that exhibit resistance against MEK inhibition.