Inhibited Breast Cancer Invasion Research Articles

IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II-neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.

LINC00478-derived novel cytoplasmic lncRNA LacRNA stabilizes PHB2 and suppresses breast cancer metastasis via repressing MYC targets

BackgroundMetastasis is the predominant cause of mortality in patients with breast cancer. Long noncoding RNAs (lncRNAs) have been shown to drive important phenotypes in tumors, including invasion and metastasis. However, the lncRNAs involved in metastasis and their molecular and cellular mechanisms are still largely unknown.MethodsThe transcriptional and posttranscriptional processing of LINC00478-associated cytoplasmic RNA (LacRNA) was determined by RT-qPCR, semiquantitative PCR and 5′/3′ RACE. Paired-guide CRISPR/cas9 and CRISPR/dead-Cas9 systems was used to knock out or activate the expression of LacRNA. Cell migration and invasion assay was performed to confirm the phenotype of LacRNA. Tail vein model and mammary fat pad model were used for in vivo study. The LacRNA-PHB2-cMyc axis were screened and validated by RNA pulldown, mass spectrometry, RNA immunoprecipitation and RNA-seq assays.ResultsHere, we identified a novel cytoplasmic lncRNA, LacRNA (LINC00478-associated cytoplasmic RNA), derived from nucleus-located lncRNA LINC00478. The nascent transcript of LINC00478 full-length (LINC00478_FL) was cleaved and polyadenylated, simultaneously yielding 5′ ends stable expressing LacRNA, which is released into the cytoplasm, and long 3′ ends of nuclear-retained lncRNA. LINC00478_3′RNA was rapidly degraded. LacRNA significantly inhibited breast cancer invasion and metastasis in vitro and in vivo. Mechanistically, LacRNA physically interacted with the PHB domain of PHB2 through its 61–140-nt region. This specific binding affected the formation of the autophagy degradation complex of PHB2 and LC3, delaying the degradation of the PHB2 protein. Unexpectedly, LacRNA specifically interacted with PHB2, recruited c-Myc and promoted c-Myc ubiquitination and degradation. The negatively regulation of Myc signaling ultimately inhibited breast cancer metastasis. Furthermore, LacRNA and LacRNA-mediated c-Myc signaling downregulation are significantly associated with good clinical outcomes, take advantage of these factors we constructed a prognostic predict model.ConclusionTherefore, our findings propose LacRNA as a potential prognostic biomarker and a new therapeutic strategy.

KIF17 maintains the epithelial phenotype of breast cancer cells and curbs tumour metastasis

Kinesin superfamily motor protein 17 (KIF17) was previously identified downregulated in breast cancer and correlated with patient prognosis. However, its pathophysiological role in tumours remains unknown. Here, we confirmed that KIF17 was significantly under-expressed in breast cancer tissues and low KIF17 expression correlated with poor outcomes in patients with breast cancer. In vitro and in vivo experiments demonstrated that KIF17 overexpression in breast cancer cell lines significantly inhibited breast cancer invasion and metastasis. By establishing the lung metastatic MDA-MB-231 cell lines, we found a transient silence of KIF17 during the initiation of breast cancer metastasis. Further experiments revealed that KIF17 might suppress metastasis by regulating the level of acetylated tubulin to maintain cytoskeleton stability. Eventually, we found that the low expression of KIF17 in breast cancer is regulated by DNMT1-mediated 5-mC DNA methylation and epigenetic silencing. Decitabine can effectively improve the expression level of KIF17 in breast cancer cells. Our study demonstrates that KIF17 mediates microtubule acetylation to maintain the stability of microtubules, thereby inhibiting tumour invasion and metastasis.

Haematococcus pluvialis Microalgae Extract Inhibits Proliferation, Invasion, and Induces Apoptosis in Breast Cancer Cells.

Breast cancer (BC) is the most common malignant cancer in females worldwide. Drug resistance, toxicity, and the failure of current therapies to completely cure BC has challenged conventional medicine. Consequently, complementary alternative medicine has become popular due to its safety and efficacy. Haematococcus pluvialis (H. pulvialis) is a green microalga living in fresh water, and its crude extract is rich of bioactives, including carotenoids, known to inhibit cancer cell growth. In the present study, we investigated the effects of a methanol crude extract called “T1” of H. pulvialis on cell growth and migration/invasion of the BC cell line MDA-MB-231 in comparison to the fibroblast control cells. TI significantly suppressed BC cell growth, inhibited migration and invasion and induced apoptosis. Interestingly, apoptosis was mediated by a significant loss of mutant p53 protein, and increased Bax/Bcl2 ratio. Our findings support our hypothesis that T1 exerts its anti-cancer effects by inhibiting BC invasion and inducing apoptosis mediated, at least, via the p53/Bax/Bcl2 pathway. Ongoing experiments aim to identify the molecular mechanisms underpinning T1-inhibited BC cell invasion using pre-designed metastasis gene-based array method.

Abstract P3-06-08: JAM2 inhibits breast cancer invasion and migration by inhibiting the EMT process and potentially affects the immune microenvironment by increasing CXCL9/CXCL10

Abstract Objectives: Large-scale epidemiological surveys have shown that patients with Down syndrome, which are caused by a chromosomal abnormality (an extra chromosome 21), are significantly less likely to develop solid tumors, including breast cancer than the normal population. This feature has prompted the search for oncogenes located on chromosome 21. Junctional Adhesion Molecule 2 (JAM2), which is located on chromosome 21, is lowly expressed in breast cancer and is associated with a good prognosis. These findings strongly suggest that JAM2 may be a potential oncogene for breast cancer. However, the role and function of JAM2 in breast cancer are not yet clear. Methods: Several databases such as CCLE, GTEx, METABRIC and TCGA were used to explore the expression of JAM2 in breast cancer and to analyze its diagnostic and prognostic value in breast cancer and to verify the stratification effect of JAM2 expression on the overall survival of breast cancer patients using Meta-analysis. Changes in relevant markers were examined at the genes and protein levels using RT-qPCR and Western blot techniques, cell migration and invasion ability by scratch assay and transwell assay, cell cycle and apoptosis by flow cytometry, and cell viability and chemotherapeutic drug sensitivity by CCK-8 assay. RNA-seq data of JAM2 overexpression were analyzed by bioinformatics methods, and we predicted drug sensitivity by machine learning algorithms, and finally the potential effect of JAM2 on the tumor microenvironment was investigated using liquid phase microarray technology. Results: JAM2 was found to be significantly downregulated in breast cancer tissues by our study, and the area under the curve (AUC) associated with JAM2 expression in Para-cancerous tissue and breast cancer tissues was analyzed by two algorithms, logistic regression and random forest algorithm, respectively, which calculated an AUC of 0.929 and 0.887 for the TCGA cohort, indicating that JAM2 has good clinical diagnostic value. Pearson's correlation analysis revealed that the expression of JAM2 and DNMT1 (r = -0.12, P < 0.01), DNMT3B (r = -0.14, P < 0.01) and EHMT2 (r = -0.19, P < 0.01) DNA methyltransferase expression showed a significant negative correlation, and after specific inhibition of DNA methyltransferase with 5-azacytidine, there was a significant increase in JAM2 mRNA levels in the JAM2-treated group compared with the control group, suggesting that the low expression of JAM2 in breast cancer is partly regulated by DNA methylation. By analyzing the database, we found that the expression of JAM2 was significantly lower in distant recurrent tumors than in primary focal tumors. Transwell assays also showed that JAM2 overexpression significantly inhibited the migration and invasion of breast cancer cells. Vimentin and EZH2 were decreased after JAM2 overexpression, and then the level of EMT process inhibitor protein E-cadherin was increased. Through the combination of RNA-seq sequencing and machine learning algorithms, we predicted the chemotherapeutic drugs that might be sensitized after JAM2 overexpression and verified that JAM2 overexpression could sensitize breast cancer cells to Doxorubicin, and we found in the follow-up experiments that JAM2 overexpression could increase BAX and inhibit BCL-2 thereby leading to apoptosis of breast cancer cells. Then we found that JAM2 could inhibit the process of EMT by increasing the expression of ATF3 and thus inhibiting FN1 through Western blot assay. Finally, by liquid-phase microarray technology, we found more chemokines of CXCL9/CXCL10 in the cell supernatant of JAM2 overexpressing cell lines compared to the null control, thus this could be a potential mechanism for JAM2 to affect the tumor immune microenvironment. Citation Format: Yang Peng. JAM2 inhibits breast cancer invasion and migration by inhibiting the EMT process and potentially affects the immune microenvironment by increasing CXCL9/CXCL10 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-08.

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

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

Luteolin, an aryl hydrocarbon receptor ligand, suppresses tumor metastasis invitro and invivo.

Estrogen receptor (ER)‑negative breast tumors are associated with low survival rates, which is related to their ability to grow and metastasize into distal organs. The aryl hydrocarbon receptor (AhR), a ligand‑activated transcription factor that is involved in several biological processes, is a promising anti‑metastatic target. Luteolin, a non‑toxic naturally occurring plant flavonoid with diverse biological activities, has been demonstrated to be effective against certain types of cancer, and has also been described as a ligand of AhR. In the present study, various cancer cell lines were first investigated following treatment with luteolin, and luteolin exhibited the lowest IC50 in MDA‑MB‑231 cells. Then, the efficiency of luteolin in suppressing the metastasis of ER‑negative breast cancer invitro was assessed. MDA‑MB‑231 cells were treated with luteolin invitro. Subsequently, MTT assay and flow cytometry were used to detect cell viability, the cell cycle and apoptosis, and a Transwell assay was used to evaluate cell invasion. In addition, reverse transcription‑semi‑quantitative PCR and western blot were performed to detect the mRNA and protein expression levels of matrix metalloproteinase (MMP)‑2 and MMP‑9. In addition, the number of surface tumor nodules was measured invivo, in mice bearing B16‑F10 tumors, following treatment with luteolin. Luteolin inhibited the viability and induced the apoptosis of MDA‑MB‑231 cells, which was accompanied by cell cycle arrest. This was associated with a decrease in the expression of the pro‑metastatic markers C‑X‑C chemokine receptor type 4 (CXCR4), MMP‑2 and MMP‑9, which was reversed by AhR inhibition. Furthermore, it was identified that luteolin could inhibit the metastasis in a B16F10 mouse xenograft model, and the levels of MMP‑9, MMP‑2 and CXCR4 were significantly decreased in the lung tissues isolated from tumor‑bearing nude mice following luteolin treatment. In conclusion, luteolin is a potential molecule for inhibiting breast cancer invasion and metastasis, which could have promising clinical applications.

Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is upregulated in breast cancer, and elevated cytoplasmic HuR correlates with high-grade tumors and poor clinical outcome of breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors, and cytokines that support major tumor hallmarks including invasion and metastasis. Here, we report a HuR inhibitor KH-3, which potently suppresses breast cancer cell growth and invasion. Furthermore, KH-3 inhibits breast cancer experimental lung metastasis, improves mouse survival, and reduces orthotopic tumor growth. Mechanistically, we identify FOXQ1 as a direct target of HuR. KH-3 disrupts HuR–FOXQ1 mRNA interaction, leading to inhibition of breast cancer invasion. Our study suggests that inhibiting HuR is a promising therapeutic strategy for lethal metastatic breast cancer.

Abstract P3-01-11: Targeting RAGE inhibits breast cancer invasion and metastasis

Abstract Background: Breast cancer (BC) is treatable with early detection, but once metastasis occurs, there is no cure. Therefore, it is of upmost importance to find targetable biomarkers that are involved in modulating the multi-step metastatic process. The Receptor for Advanced Glycation Endproducts (RAGE) and its ligands are an inflammatory pathway that are involved in modulating breast cancer progression and metastasis. We recently demonstrated that genetic or pharmacological interruption of RAGE signaling affected tumor progression and metastasis. However, no studies have dissected the role of RAGE in tumor growth from the metastatic cascade. Here, we show for the first time in multiple metastatic breast cancer models that targeting RAGE impairs BC metastasis. Methods: We tested the anti-metastatic effect of RAGE in vitro using the RAGE inhibitor FPS-ZM1 or RAGE shRNA knockdown in cell invasion, proliferation, migration and sphere formation assays (with 4T1, Py8119 and E0771 mouse BC cells). For in vivo assays we used orthotopic BC models (4T1/BALB/c and E0771 & Py8119/C57BL6) and experimental metastasis assays (tail vein injection of 4T1/BALB/c and Py8119/C57BL6). To target RAGE, we used genetic (shRNA in 4T1 cells and RAGE knockout in C57BL6 mice) and pharmacological approaches (I.P. injection of FPS-ZM1). To investigate the synergistic effects of RAGE inhibition on progression and metastasis, we tested combination therapy of low-dose doxorubicin and FPS-ZM1. Results: Inhibition of RAGE with FPS-ZM1 (1-5uM) impaired tumor cell invasion of 4T1, E0771 and Py8119 cells. Similarly, in spheroid assays, treatment of cells with FPS-ZM1 resulted in fewer and smaller colonies. However, FPS-ZM1 treatment did not affect cell proliferation. In vivo studies revealed that FPS-ZM1 (1mg/kg) has a modest effect on tumor growth in 4T1/BALB/c injected mice but displayed a dramatic inhibitory effect on metastasis to the lungs. In experimental metastasis assays, tail-vein injection of 4T1 cells in BALB/c mice demonstrated that FPS-ZM1 treatment strongly impaired metastatic disease in mice compared to controls. To dissect the genetic role of RAGE in the tumor versus host, we test the effect of RAGE knockdown in 4T1 cells in experimental metastasis assays. RAGE shRNA impaired metastasis to the lungs, albeit to a lower degree seen with the RAGE inhibitor. To test the role of the host, we injected Py8119 cells into wild-type and RAGE knockout (RKO) mice. RKO mice displayed fewer metastatic burden compared to wild-type mice. Finally, in our combination treatment experiments, treatment of 4T1-injected BALB/c mice with Doxorubicin and FPS-ZM1 (alone and in combination), demonstrated that drug combination was more effective in inhibiting lung metastasis than either reagent alone. We are currently assessing how RAGE mechanistically drives these metastatic changes. Conclusion: Our data strongly suggests RAGE plays an important role in breast cancer metastasis, with less of an effect on tumor growth. Ongoing studies in our lab are testing which stage of the metastatic cascade RAGE is involved in, and the underlying mechanisms driving these processes. In conclusion, the use of RAGE inhibitors could represent a novel therapeutic approach for metastatic breast cancer. Citation Format: Gyong Ha Hwang, Melinda Magna, Barbara Mera, Toni Yeasky, Taekyoung Kwak, Lucas Outcault, Masaru Takabatake, Thuy-Mai Le, Marc E. Lippman, Barry I. Hudson. Targeting RAGE inhibits breast cancer invasion and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-11.

The Effect of Metformin on the Clinicopathological Features of Breast Cancer With Type 2 Diabetes.

BackgroundThe present study aimed to review the use of hypoglycemic drugs and clinicopathological data in breast cancer patients with type 2 diabetes mellitus (T2DM), and to investigate the effect of metformin on the clinicopathological features of breast cancer in patient with T2DM.MethodsEighty-nine patients with breast cancer hospitalized in the Second Affiliated Hospital of Xi’an Jiaotong University from January 2012 to December 2014 were included. Thirty-three patients were on metformin (metformin group) and 56 patients were on control group. Streptavidin-peroxidase (SP) method was used to quantify protein expression of molecular markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)), molecular markers of proliferation (Ki-67 and epidermal growth factor receptor (EGFR)) and epithelial-mesenchymal transition (EMT) molecular markers (matrix metalloproteinase-2 (MMP-2), E-cadherin and downstream N-cadherin). Fluorescence in situ hybridization was used to detect HER-2 (+ and ++).ResultsThe rate of lymph node metastasis and the level of Ki-67/MMP-2 in the metformin group were significantly lower than those in the control group (P < 0.05). The ratio of luminal pattern in metformin group was higher than that in the control group (P < 0.05). However, there were no differences in the parameters of age, duration of diabetes, body mass index, tumor size, histological grade of cancer and clinical pathological features between the two groups. No significant difference was observed in the expressions of ER, PR, HER-2, EGFR, E-cadherin, N-cadherin and the recurrence rate between two groups.ConclusionsMetformin is associated with luminal breast cancer and can inhibit breast cancer invasion and metastasis in some cases. It may be associated with EMT and is beneficial to the prognosis of breast cancer.

Effects of HOXD10 on the mechanism of cordycepin action in different types of human breast cancer cells

Objective We investigate the molecular mechanism underlying inhibitory effects of cordycepin on proliferation, apoptosis, migration and invasion of breast cancer. We focus on the role of HOXD10 in inhibitory effects of cordycepin. Methods Two individual breast cancer cell lines, MCF-7 and MDA-MB-231, were used in this study to investigate the effects of cordycepin on proliferation, apoptosis, migration and invasion of breast cancer, by cell counting kit-8 (CCK-8) assays, flow cytometry and Transwell assays. The small interfering RNAs (siRNAs) targeted HOXD10 were transfected into MCF-7 and MDA-MB-231 cells to knock down HOXD10. We investigate the role of HOXD10 by comparing the difference between group NC and group siRNAs. Results The A values of cordycepin treated MCF-7 and MDA-MB-231 cells were significantly lower than those of control group (DMSO group) (MCF-7cells: 0.665±0.004 vs. 0.733±0.005, t=10.450, and MDA-MB-231cells: 0.632±0.005 vs. 0.722±0.005, t=13.330, P<0.05), which means the proliferation of breast cancer cells was significantly inhibited, The apoptosis rateof cordycepin treated MCF-7 and MDA-MB-231 cells were significantly higher than those of control group (MCF-7cells: 20.200±0.322 vs. 5.500±0.000, t=45.730, MDA-MB-231cells: 21.800±1.493 vs. 5.367±0.318, t=10.760, P<0.05). There were significantly fewer migrated MCF-7 and MDA-MB-231 cells in cordycepin group than in control group(MCF-7 cells: 28.670±1.764 vs. 83.330±2.186, t=19.460, MDA-MB-231cells: 29.000±2.646 vs. 114.700±3.180, t=20.710, P<0.05). There were significantly fewer invasive MCF-7 and MDA-MB-231 cells in cordycepin group than control group(MCF-7cells: 24.670±2.603 vs. 49.000±1.528, t=8.0620, MDA-MB-231cells: 12.330±1.453 vs. 36.670±2.728, t=7.872, P<0.05). After transfection of MCF-7 and MDA-MB-231 cells with siRNA and intervention with cordycepin, the proliferation of breast cancer cells was inhibited (MCF-7cells: 0.627±0.004 vs. 0.648±0.006, t=2.951, MDA-MB-23 cells: 0.620±0.006 vs. 0.635±0.004, t=2.087, P<0.05). The apoptosis rate of the treatment group was significantly higher than the control group (MCF-7 cells: 20.470±0.260 vs. 16.300±0.153, t=13.800, MDA-MB-23 cells: 19.170±0.167 vs. 17.030±0.186, t=8.5520, P<0.05). There were significantly fewer migrated MCF-7 and MDA-MB-231 cells in siRNA group than in control group(MCF-7cells: 11.000±2.082 vs. 30.330±2.028, t=6.653, MDA-MB-23cells: 11.330±1.4530 vs. 23.000±1.528, t=5.534, P<0.05). There were significantly fewer invasive MCF-7 and MDA-MB-231cells in siRNA group than control group(MCF-7 cells: 16.330±1.764 vs. 23.670±1.760, t=2.940, MDA-MB-2 cells: 9.333±1.453 vs. 19.670±2.333, t=3.759, P<0.05). Those values above are statistically significant. Conclusion Cordycepin induces apoptosis and inhibits proliferation, migration and invasion of breast cancer.2. Suppression of HOXD10 promptes the effects of cordycepin on proliferation, apoptosis, migration and invasion of breast cancer. Key words: Cordycepin; HOXD10; Breast cancer; Signaling pathway

The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex.

While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, β-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.

LINC01133 inhibits breast cancer invasion and metastasis by negatively regulating SOX4 expression through EZH2.

Mounting evidence highlights long non‐coding RNAs (lncRNAs) as crucial regulators in multiple types of biological processes and contributing to tumourigenesis. LINC01133, located in chromosome 1q23.2, was a recently identified novel lncRNA with a length of 1154nt. It was involved in the development of colorectal cancer and non‐small cell lung cancer. However, its clinical relevance, biological functions and potential molecular mechanism in breast cancer are still unclear. In this study, we found that the LINC01133 expression was significantly down‐regulated in breast cancer samples and was associated with progression and poor prognosis of breast cancer. Further experiments demonstrated that overexpression of LINC01133 inhibited invasion and metastasis in breast cancer both in vitro and in vivo. Mechanistic investigations revealed that LINC01133 repressed SOX4 expression by recruiting EZH2 to SOX4 promoter. Moreover, rescue experiments further confirmed that LINC01133 functional acted as an anti‐oncogene, at least partly, via repressing SOX4 in breast cancer. Taken together, these findings imply that LINC01133 could serve as a novel prognostic biomarker and potential therapeutic target for breast cancer.

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.

MiRNA-30 Family Members Inhibit Breast Cancer Invasion, Osteomimicry, and Bone Destruction by Directly Targeting Multiple Bone Metastasis-Associated Genes.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivoIn vitro, miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell-conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation (IL8, IL11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11), and invasiveness (CTGF, ITGA5, ITGB3) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention.Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259-73. ©2018 AACR.

Abstract 486: MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes

Abstract MicroRNAs (miRNAs), which are master regulators of gene expression, have emerged as key players in cancer metastasis. Here, we established miR-30 family members (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) as breast cancer bone metastasis suppressor genes. Low expression levels of miR-30s in primary tumors of breast cancer patients were associated with poor relapse-free survival. Additionally, miR-30s levels were significantly lower in estrogen receptor (ER)-negative/progesterone receptor (PR)-negative tumors than in their hormone-responsive counterparts. Overexpression of miR-30s in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. MiR-30s overexpression also reduced tumor outgrowth, when tumor cells were implanted subcutaneously in animals. In vitro, miR-30s did not affect tumor cell proliferation, but inhibited tumor cell invasion. Furthermore, miR-30s restored bone homeostasis by reversing the in vitro effects of the tumor cell conditioned medium on stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. We identified a number of genes associated with osteoclastogenesis stimulation (IL-8, IL-11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11) and invasiveness (CTGF, ITGA5, ITGB3) that were direct and/or indirect targets for repression by miR-30s. Among these genes, integrin ITGA5 was a previously unknown miR-30 target. By silencing ITGA5 in ER-/PR-negative breast cancer cells, colonization of the bone marrow by tumor cells was drastically reduced in vivo. Overall, our data indicate that miR-30s employ multiple mechanisms to impede breast cancer bone metastasis. These findings may pave the way to a new field of therapeutic interventions in breast cancer patients with bone metastasis. Citation Format: Martine Croset, Francesco Pantano, Casina Kan, Edith Bonnelye, Francoise Descotes, Catherine Alix-Panabières, Charles Lecellier, Richard Bachelier, Nathalie Allioli, Saw See Hong, Kai Bartkowiak, Klaus Pantel, Philippe Clézardin. MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes [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 486.

Abstract 5153: RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression

Abstract Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers. Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression. Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells. Citation Format: Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression [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 5153.

TP53INP1 inhibits hypoxia-induced vasculogenic mimicry formation via the ROS/snail signalling axis in breast cancer.

Tumour protein p53‐inducible nuclear protein 1 (TP53INP1) is a tumour suppressor associated with malignant tumour metastasis. Vasculogenic mimicry (VM) is a new tumour vascular supply pattern that significantly influences tumour metastasis and contributes to a poor prognosis. However, the molecular mechanism of the relationship between TP53INP1 and breast cancer VM formation is unknown. Here, we explored the underlying mechanism by which TP53INP1 regulates VM formation in vitro and in vivo. High TP53INP1 expression was not only negatively correlated with a poor prognosis but also had a negative relationship with VE‐cadherin, HIF‐1α and Snail expression. TP53INP1 overexpression inhibited breast cancer invasion, migration, epithelial‐mesenchymal transition (EMT) and VM formation; conversely, TP53INP1 down‐regulation promoted these processes in vitro by functional experiments and Western blot analysis. We established a hypoxia model induced by CoCl2 and assessed the effects of TP53INP1 on hypoxia‐induced EMT and VM formation. In addition, we confirmed that a reactive oxygen species (ROS)‐mediated signalling pathway participated in TP53INP1‐mediated VM formation. Together, our results show that TP53INP1 inhibits hypoxia‐induced EMT and VM formation via the ROS/GSK‐3β/Snail pathway in breast cancer, which offers new insights into breast cancer clinical therapy.

Loss of fibulin-2 expression is involved in the inhibition of breast cancer invasion and forms a new barrier in addition to the basement membrane.

Previous studies have demonstrated that fibulin-2 may facilitate cancer cell invasion and metastasis during tumor progression. In the present study, immunohistochemical analyses of fibulin-2 and collagen IV expression in 35 patients with breast cancer were performed to define their localization and association with breast cancer tissue. Fibulin-2 was revealed to be expressed in all tissues surrounding the breast ducts and blood vessels in normal breast tissue, while its expression was not integrated in invasive ductal carcinoma or terminal duct-lobular unit. In malignant breast tissue, collagen IV was integrated around the duct, while fibulin-2 was expressed around collagen IV and was incomplete. These results demonstrated that fibulin-2 was associated with breast cancer invasion. Fibulin-2 expression decreased prior to basement membrane (BM) degradation, indicating that fibulin-2 forms an additional barrier around the BM. Therefore, it was proposed that fibulin-2 composes the general BM, which differs from the traditional BM. These results provide insight into extracellular matrix components and the involvement of fibulin-2 in tumor invasion and metastasis. Fibulin-2 was involved in the process of breast cancer development. It performed an important role in prevention of cancer cell penetration and metastasis.

OVOL2 antagonizes TGF-β signaling to regulate epithelial to mesenchymal transition during mammary tumor metastasis.

Great progress has been achieved in the study of the role of TGF-β signaling in triggering epithelial-mesenchymal transition (EMT) in a variety of cancers; however, the regulation of TGF-β signaling during EMT in mammary tumor metastasis has not been completely defined. In the present study, we demonstrated that OVOL2, a zinc finger transcription factor, inhibits TGF-β signaling-induced EMT in mouse and human mammary tumor cells, as well as in mouse tumor models. Data from the Oncomine databases indicated a strong negative relationship between OVOL2 expression and breast cancer progression. Moreover, our experiments revealed that OVOL2 inhibits TGF-β signaling at multiple levels, including inhibiting Smad4 mRNA expression and inducing Smad7 mRNA expression, blocking the binding between Smad4 and target DNA, and interfering with complex formation between Smad4 and Smad2/3. These findings reveal a novel mechanism that controls the TGF-β signaling output level in vitro and in vivo. The modulation of these molecular processes may represent a strategy for inhibiting breast cancer invasion by restoring OVOL2 expression.