Breast Cancer Metastasis Suppressor Research Articles

Additive effect of metastamiR-193b and breast cancer metastasis suppressor 1 as an anti-metastatic strategy.

It has been reported that enhancing the cellular levels of miR-193b as well as breast cancer-metastasis-suppressor-1 (BRMS1) protein is associated with diminished metastatic characteristics in breast cancer. In view of these facts, as a new therapeutic intervention, we employed a restoration-based strategy using both miR-193b-3p mimic and optimized BRMS1 in the context of a chimeric construct. miR-193b-3p and BRMS1 genes were cloned and the resulting plasmids were transfected into the MDA-MB231, MCF-7 and MCF-10A cell lines. microRNA expression levels were assessed by rea time PCR using LNA-primer and protein expression was confirmed by western blot method. Then, apoptosis, MTT, colony formation and invasion assays were carried out. The expression levels of miR-146a, miR-146b and miR-373 were up-regulated, while the miR-520c, miR-335 and miR-10b were down-regulated following the exogenous BRMS1 expression. The exogenous over-expression of BRMS1 was associated with higher amounts of endogenous miR-193b-3p expression and enabled more efficient targeting of the 3'UTR of uPA. Although, miR-193b-3p and BRMS1 are individually capable of suppressing breast cancer cell growth, migration and invasion abilities, their cistronic expression was capable of enhancing the ability to repress the breast cancer cells invasion. Our results collectively indicated the existence of an additive anti-metastatic effect between miR-193b-3p and BRMS1. Moreover, it has been hypothesized that the exogenous expression of a protein can effect endogenous expression of non-relevant microRNA. Our findings provide new grounds for miR-restoration therapy applications as an amenable anti-metastatic strategy.

Inhibitory Effect of Alisma canaliculatum Ethanolic Extract on NF-κB-Dependent CXCR3 and CXCL10 Expression in TNFα-Exposed MDA-MB-231 Breast Cancer Cells.

CXC motif chemokine ligand 10 (CXCL10) and its receptor CXC motif chemokine receptor 3 (CXCR3), play important roles in the motility of breast cancer cells. Alisma canaliculatum is a herb that has been used as a traditional medicine for thousands of years in Korea and China. Whether A. canaliculatum inhibits the motility of metastatic breast cancer cells is not clear yet. In this study, we show that A. canaliculatum ethanolic extract (ACE) prevented tumor necrosis factor-alpha (TNFα)-induced migration of MDA-MB-231 cells. ACE significantly attenuated TNFα-induced upregulation of CXCL10 and CXCR3 expression at the gene promoter level. Mechanistically, ACE inhibits TNFα-induced phosphorylation of inhibitor of κB (IκB) kinase (IKK), IκB and p65/RelA, leading to the suppression of nuclear translocation of p65/RelA nuclear factor kappa-B (NF-κB). Also, ACE inhibited NF-κB-dependent CXCR3 and CXCL10 promoter activities. These results suggest that ACE abrogates TNFα-induced migration of MDA-MB-231 breast cancer cells through down-regulation of IKK-NF-κB-dependent CXCR3 and CXCL10 expression. Our results suggest that ACE has potential as a herbal supplement for the inhibition of breast cancer metastasis.

GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

Abstract Breast cancer metastasis is the root cause of deaths from breast cancer. Currently, endocrine therapy resistance in estrogen receptor (ER)-positive (ER+) breast cancer remains a major clinical issue. Moreover, ER-negative (ER−) breast cancer is often associated with distant recurrence and death. G-protein-coupled ER (GPER1) participates in endocrine therapy resistance and is involved in the malignant progression of breast cancer. However, the underlying detailed mechanisms remain obscure. Here we investigated the role and mechanism of GPER1 in the activation of focal adhesion kinase (FAK) using ER+ or ER− breast cancer cell lines. In SK-Br-3 cells (ERα−/β−/GPER1+), both 17β-estradiol (E2) and the GPER1 agonist G1 resulted in rapid FAK phosphorylation. This action is due to GPER1 interaction with the non-receptor tyrosine kinase c-Src and subsequent activation of nuclear factor kappa B (NF-κB) signaling. Silencing of GPER1, c-Src or the nuclear factor kappa B p65 subunit blocked E2- or G1-induced SK-Br-3 cell migration and invasion. In MCF-7 cells (ERα+/β+/GPER1+), silencing of GPER1, but not ERα or ERβ, abolished FAK phosphorylation induced by E2 or G1. In MDA-MB-231 cells (ERα−/β+/GPER1−), E2 or G1 was also unable to stimulate E2-induced FAK phosphorylation. However, E2 and G1 regained the ability to induce FAK phosphorylation under conditions of overexpression of GPER1. In conclusion, we demonstrated that GPER1, but not ERα or ERβ, mediates FAK phosphorylation induced by E2 via the c-Src/p65 signaling pathway, which enhances cell migration and invasion. These findings may shed light on novel therapeutic strategies based on GPER1/FAK signaling pathways in suppression of breast cancer metastasis.

A double-negative feedback loop between DEAD-box protein DDX21 and Snail regulates epithelial-mesenchymal transition and metastasis in breast cancer

DDX21, a DEAD-box protein, implicated in fundamental aspects of RNA metabolism such as gene transcription. DDX21 expression is dysregulated in cancer, but its specific contribution to tumor invasion and metastasis remains to be determined. Here, we demonstrate DDX21 down-regulation is associated with highly metastatic and poor prognosis human breast cancers. DDX21 overexpression inhibited, while DDX21 knockdown promoted epithelial-mesenchymal transition (EMT) in vitro and in vivo. Overexpression of Snail reversed DDX21 mediated inhibition of cell invasion. On the other hand, independent of its helicase activity, DDX21 suppressed Snail transcription by recruiting SUZ12 and EZH2, two core subunits of PRC2 (polycomb-repressive complex 2), to the Snail promoter. Furthermore, down-regulation of DDX21 is mediated by miR-218–5p. Surprisingly, Snail also modulates DDX21 transcription. Snail overexpression decreased DDX21 transcription, whereas Snail knockdown increased DDX21 expression. These novel observations demonstrate firstly, the antagonism/double negative feedback loop between DDX21 and Snail transcription, and secondly, the crucial role of miR-218–5p in promoting EMT, acting by decreasing the ratio of DDX21/Snail. Our results identify DDX21 as a breast cancer metastasis suppressor; blocking miR-218–5p will stabilize DDX21 and epigenetically suppress Snail expression and EMT.

Antrodia camphorata inhibits epithelial‐to‐mesenchymal transition by targeting multiple pathways in triple‐negative breast cancers

Antrodia camphorata (AC) exhibits potential for engendering cell-cycle arrest as well as prompting apoptosis and metastasis inhibition in triple-negative breast cancer (TNBC) cells. We performed the current study to explore the anti-epithelial-to-mesenchymal transition (EMT) properties of fermented AC broth in TNBC cells. Our results illustrated that noncytotoxic concentrations of AC (20-60 μg/ml) reversed the morphological changes (fibroblastic-to-epithelial phenotype) as well as the EMT by upregulating the observed E-cadherin expression. Furthermore, we discovered treatment with AC substantially inhibit the Twist expression in human TNBC (MDA-MB-231) cells as well as in those that were transfected with Twist. In addition, we determined AC to decrease the observed Wnt/β-catenin nuclear translocation through a pathway determined to be dependent on GSK3β. Notably, AC treatment consistently inhibited the EMT by downregulating mesenchymal marker proteins like N-cadherin, vimentin, Snail, ZEB-1, and fibronectin; at that same time upregulating epithelial marker proteins like occludin and ZO-1. Bioluminescence imaging that was executed in vivo demonstrated AC substantially suppressed breast cancer metastasis to the lungs. Notably, we found that western blot analysis confirmed that AC decreased lung metastasis as demonstrated by upregulation of E-cadherin expression in biopsied lung tissue. Together with our results support the anti-EMT activity of AC, indicating AC as having the potential for acting as an anticancer agent for the treatment of human TNBC treatment.

MiR-346 Promotes HCC Progression by Suppressing Breast Cancer Metastasis Suppressor 1 Expression.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. MicroRNA (miRNA), a class of noncoding single-stranded RNA molecules, is involved in regulating cancer cell proliferation, metastasis, migration, invasion, and apoptosis. We showed that the expression of miR-346 was significantly increased in HCC tissues and cell lines, compared with noncancerous controls, and was associated with poor prognosis. Overexpression of miR-346 promoted proliferation and inhibited apoptosis of SMMC-7721 cells, while knockdown of miR-346 significantly suppressed proliferation and induced apoptosis of HepG2 cells. Then we identified breast cancer metastasis suppressor 1 (BRMS1) as a direct target of miR-346 based on luciferase reporter assays. There was a negative correlation between miR-346 and BRMS1 expression at both the protein and mRNA levels. Furthermore, inhibition of BRMS1 expression reversed the tumor-suppression effects of miR-346 downregulation in HepG2 cells. These results indicate that miR-346 promotes HCC progression by regulating BRMS1 expression.

BRMS1L suppresses ovarian cancer metastasis via inhibition of the β-catenin-wnt pathway

A low level of breast cancer metastasis suppressor 1-like (BRMS1L) has been implicated in tumour metastasis involving breast cancer and other cancers. It remains unclear whether BRMS1L is involved in epithelial ovarian cancer (EOC) metastasis and what the molecular mechanism of BRMS1L is in suppressing EOC metastasis. In this study, we examined the mRNA expression and protein level of BRMS1L by screening EOC patients. Our results show that BRMS1L expression is downregulated in EOC patients compared to that in normal people and negatively correlated to pathological stages of EOC. We further explored examining epithelial to mesenchymal transition (EMT) as the molecular mechanism of BRMS1L in cancer cell metastasis. The overexpression of BRMS1L inhibits EOC cell migration and invasion, and this inhibition is correlated to the inactivation of EMT and Wnt/β-catenin signalling in vitro. Knockdown of BRMS1L by shRNA promotes EOC metastasis, enhances EMT process and activates Wnt/β-catenin signalling. These results suggest that BRMS1L plays a critical role in the suppression of ovarian cancer metastasis, and BRMS1L can be considered as a prognostic biomarker and potential therapeutic target for EOC patients.

Abstract 946: Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors

Abstract Over one-third of human breast tumors that become resistant to endocrine therapies have acquired mutations in the estrogen receptor (ER alpha) that result in constitutive receptor activity in the absence of estrogen, and these tumors are much less sensitive to suppression by current standard-of-care antiestrogens such as tamoxifen and fulvestrant. We reported recently that the novel adamantyl antiestrogen, K-07, was effective in inhibiting the proliferation of breast tumor xenografts containing the two most frequent constitutively active mutant forms of the ER, Y537S ER and D538G ER, and that this antiestrogen reduced ER levels and the expression of ER-regulated genes (Zhao et al., Cancer Research 77(20):5602-5613, 2017). Because mutant ERs are usually enriched in recurrent, metastatic breast cancers compared to the primary breast cancer, we have now examined the effectiveness of compound K-07 in preventing the growth of breast cancer metastases and in extending survival in a metastatic tumor model. MCF-7 breast cancer cells expressing luciferase and Y537S ER or D538G ER (ca. 50% mutant ER and 50% wild type ER) were injected i.v. by the tail vein into NOD-SCID-gamma (NSG) female mice. The constitutively active ER-containing breast cancer cells established metastases in liver, bone and brain that increased in number and size over time (day zero – 80 days) as monitored by IVIS imaging and immunohistochemical (IHC) analysis. Daily oral treatment with K-07 (80 mg/kg orally for 30 days and then 40 mg/kg) versus oral vehicle greatly reduced the metastasis of mutant ER-containing breast cancer cells. Notably, mice with mutant ER-containing metastases treated with K-07 survived much longer than mice given daily control vehicle. In fact, by day 60, only 25% of vehicle treated mice with mutant ER breast cancers were alive whereas all K-07 treated D538G mice and 80% of Y537S mice were still alive. Hence, the findings indicate that this antiestrogen can reduce the in vivo metastasis of breast cancers driven by constitutively active mutant ERs and extend host survival. The findings suggest that the antiestrogen K-07 may be suitable for further translational and clinical examination of its efficacy in suppression of metastasis in patients with breast cancers containing constitutively active mutant ERs. Citation Format: Mary J. Laws, Sung Hoon Kim, Jian Min, Yuechao Zhao, Yvonne Ziegler, David Chu, Ben H. Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen. Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors [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 946.

Abstract 21: p66ShcA is a contextual breast cancer metastasis promoter or suppressor depending on the tumor microenvironment

Abstract Introduction: Src homology and collagen A (ShcA) adaptor proteins are essential during breast cancer progression. However, the role of the largest isoform, p66ShcA, is conflicting and still poorly understood. Under high levels of stress p66ShcA is phosphorylated on serine36, within its CH2 domain, allowing it to translocate to the mitochondria and induce the formation of reactive oxygen species (ROS) to promote apoptosis. Previously, we provided the first in vivo evidence that p66ShcA can influence both pro and anti-tumorigenic functions in ErbB2+ luminal breast cancer. Stable overexpression of p66ShcA reduced tumor outgrowth while simultaneously elevating the expression of mesenchymal genes to promote tumor plasticity. In this study, we evaluated the role of p66ShcA in metastatic dissemination to the lung in a model of basal breast cancer, which typically is associated with poor outcome. Hypothesis: p66ShcA regulates basal breast cancer metastasis to the lung. Methods: Screening a panel of basal breast cancer cells in vivo selected to the lung, liver and bone, we found p66ShcA enriched in lung and liver metastatic variant cell lines relative to parental breast cancer cells and those in vivo selected through the mammary fatpad. Using CRISPR/Cas technology we genetically deleted p66ShcA and rescued with p66ShcA-WT or a p66ShcA-S36A mutant. Lung metastatic basal breast cancer cells were injected into the fourth gland of the mammary fatpad. Tumors were measured using calipers 3 times/week following first palpation (>50mm3) followed by surgical resection to monitor the lung metastatic burden. In addition, we performed tail vein injections to monitor lung metastatic burden following direct entry into the circulation. Results: Loss of p66ShcA significantly reduced the metastatic burden to the lung following surgical tumor resection and this reduction was partially rescued by stable overexpression of wild type (WT), but not S36A mutated p66ShcA from the primary site. These effects were not due to altered anti-oxidant expression levels, changes in oxidative DNA Damage or microvessel density. Intriguingly, we found that WT-rescue of p66ShcA significantly elevated the migratory speed of breast cancer cells in vitro and corroborates our in vivo metastatic burden data. However, this is in stark contrast to our tail vein data, where WT-rescue of p66ShcA significantly inhibited lung metastasis. Conclusion: p66ShcA is required for efficient metastasis to the lung in a mitrochondrial-ROS-dependent fashion from the primary site. Our data suggest that cues from the tumor microenvironement of the mammary fatpad are essential for successful colonization and outgrowth at oxygen rich sites, such as the lung, as breast cancer cells with elevated expression of p66ShcA directly entering the circulation suppressed lung metastatic burden. Citation Format: Jesse Hudson, Kyle Lewis, Julien Senécal, Alexander Kiepas, Sébastien Tabariès, Valérie Sabourin, Ryuhjin Ahn, Rachel La Selva, Peter Siegel, Giuseppina Ursini-Siegel. p66ShcA is a contextual breast cancer metastasis promoter or suppressor depending on the tumor microenvironment [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 21.

Abstract 2128: Autocrine fibronectin inhibits breast cancer metastasis

Abstract The processes of the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are strongly linked to breast cancer metastasis via their ability to generate cells of increased invasive capacity and enhanced tumor-initiating phenotypes. Numerous growth factors, cytokines and chemotherapies present in the tumor microenvironment are capable of inducing EMT, but the role of extracellular matrix (ECM) components in this process remains less well understood. Analysis of the METABRIC dataset strongly linked high-level FN expression to decreased patient survival. However, in vitro analysis of the MCF-10A progression series indicated that autocrine FN expression was associated with nonmetastatic cells. Therefore, we utilized differential bioluminescent imaging to track the metastasis of these isogenic epithelial and mesenchymal cells within heterogeneous primary tumors. Using this approach, we demonstrate that tumor cells expressing autocrine FN display a constitutively mesenchymal phenotype and are incapable of completing the metastatic process, even when grown within a tumor containing epithelial tumor cells. Importantly, depletion of FN allows tumor cells to exit their mesenchymal state, regain epithelial characteristics and initiate tumor growth in a metastatic microenvironment. Using a tessellated three-dimensional polymer scaffolding, we show that depletion of FN or pharmacologic inhibition of focal adhesion kinase prevents mesenchymal tumor cells from producing structural fibrils and thus supporting the growth and migration of metastatic-competent epithelial tumor cells. Finally, dynamic FN coating of this 3D growth scaffold resulted in formation of a functionalized fibrillar FN network that was sufficient to induce transient EMT events as metastatic cells migrated along FN fibrils. Taken together, our data indicate that autocrine expression of FN by tumor cells supports a stable mesenchymal phenotype limiting their metastatic potential. Instead, this tumor cell-derived production of fibrillar FN acts in a paracrine fashion to support transient EMT events in metastatic competent cells within a heterogeneous tumor microenvironment. Citation Format: Aparna Shinde, Ammara Abdullah, Luis Solorio, Michael Wendt. Autocrine fibronectin inhibits 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 2128.

Breast cancer metastasis suppressor gene, breast cancer metastasis suppressor 1, may be associated with clinicopathological features of breast cancer.

We aim to investigate whether the breast cancer metastasis suppressor gene, breast cancer metastasis suppressor 1 (BRMS1), is correlated with clinicopathological features of breast cancer or not. Following a stringent inclusion and exclusion criteria, case-control studies related to the association between BRMS1 and breast cancer were selected from articles retrieved by electronic database searches. All statistical analyses were performed by Stata version 12.0 (Stata Corp, College Station, TX, USA). A total of 12 studies were ultimately included in this meta-analysis. Results of our meta-analysis suggested that BRMS1 protein in breast cancer tissues was significantly lower compared with normal breast tissues (odds ratio [OR] =0.08, 95% confidence interval [CI] =0.04-0.15, P < 0.001). The BRMS1 protein in metastatic breast cancer tissue was lower than that in nonmetastatic breast cancer tissue (OR = 0.20, 95% CI = 0.13-0.29, P < 0.001), and BRMS1 protein in tumor-node-metastasis (TNM) stages 1, 2 was found to be higher than TNM stages 3, 4 (OR = 4.62, 95% CI = 2.77-7.70, P < 0.001). With respect to breast cancer types, BRMS1 protein in all the three major types of breast cancer was lower than the normal tissues. We also found strong correlations between BRMS1 mRNA levels and TNM stage and tumor size. Our meta-analysis results showed that reduced BRMS1 expression level was significantly associated with clinicopathological features of breast cancer, suggesting that loss of expression or reduced levels of BRMS1 might be a strong indicator of the metastatic capacity of breast cancer, with poor prognosis.

Mechanism of metastasis suppression by luteolin in breast cancer.

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

Transketolase Regulates the Metabolic Switch to Control Breast Cancer Cell Metastasis via the α-Ketoglutarate Signaling Pathway.

Although metabolic reprogramming is recognized as a hallmark of tumorigenesis and progression, little is known about metabolic enzymes and oncometabolites that regulate breast cancer metastasis, and very few metabolic molecules have been identified as potential therapeutic targets. In this study, the transketolase (TKT) expression correlated with tumor size in the 4T1/BALB/c syngeneic model. In addition, TKT expression was higher in lymph node metastases compared with primary tumor or normal tissues of patients, and high TKT levels were associated with poor survival. Depletion of TKT or addition of alpha-ketoglutarate (αKG) enhanced the levels of tumor suppressors succinate dehydrogenase and fumarate hydratase (FH), decreasing oncometabolites succinate and fumarate, and further stabilizing HIF prolyl hydroxylase 2 (PHD2) and decreasing HIF1α, ultimately suppressing breast cancer metastasis. Reduced TKT or addition of αKG mediated a dynamic switch of glucose metabolism from glycolysis to oxidative phosphorylation. Various combinations of the TKT inhibitor oxythiamine, docetaxel, and doxorubicin enhanced cell death in triple-negative breast cancer (TNBC) cells. Furthermore, oxythiamine treatment led to increased levels of αKG in TNBC cells. Together, our study has identified a novel TKT-mediated αKG signaling pathway that regulates breast cancer oncogenesis and can be exploited as a modality for improving therapy.Significance: These findings uncover the clinical significance of TKT in breast cancer progression and metastasis and demonstrate effective therapy by inhibiting TKT or by adding αKG. Cancer Res; 78(11); 2799-812. ©2018 AACR.

Low doses of Paclitaxel repress breast cancer invasion through DJ-1/KLF17 signalling pathway.

Paclitaxel (taxol) is an important agent against many tumours, including breast cancer. Ample data documents that paclitaxel inhibits breast cancer metastasis while others prove that paclitaxel enhances breast cancer metastasis. The mechanisms by which paclitaxel exerts its action are not well established. This study focuses on the effect of paclitaxel, particularly the low doses on breast cancer metastasis and the mechanisms that regulate it. Current results show that, paclitaxel exerts significant cytotoxicity even at low doses in both MCF-7 and MDA-MB-231 cells. Interestingly, paclitaxel significantly inhibits cell invasion and migration, decreases Snail and increases E-cadherin mRNA expression levels at the indicated low doses. Furthermore, paclitaxel-inhibiting breast cancer metastasis is associated with down-regulation of DJ-1 and ID-1 mRNA expression level with a concurrent increase in KLF17 expression. Under the same experimental conditions, paclitaxel induces KLF17 and concurrently represses ID-1 protein levels. Our results show for the first time that paclitaxel inhibits breast cancer metastasis through regulating DJ-1/KLF17/ID-1 signalling pathway; repressed DJ-1 and ID-1 and enhanced KLF17 expression.

Wild-type p53 upregulates an early onset breast cancer-associated gene GAS7 to suppress metastasis via GAS7\u2013CYFIP1-mediated signaling pathway

The early onset breast cancer patients (age ≤ 40) often display higher incidence of axillary lymph node metastasis, and poorer five-year survival than the late-onset patients. To identify the genes and molecules associated with poor prognosis of early onset breast cancer, we examined gene expression profiles from paired breast normal/tumor tissues, and coupled with Gene Ontology and public data base analysis. Our data showed that the expression of GAS7b gene was lower in the early onset breast cancer patients as compared to the elder patients. We found that GAS7 was associated with CYFIP1 and WAVE2 complex to suppress breast cancer metastasis via blocking CYFIP1 and Rac1 protein interaction, actin polymerization, and β1-integrin/FAK/Src signaling. We further demonstrated that p53 directly regulated GAS7 gene expression, which was inversely correlated with p53 mutations in breast cancer specimens. Our study uncover a novel regulatory mechanism of p53 in early onset breast cancer progression through GAS7–CYFIP1-mediated signaling pathways.

MicroRNA-708-3p mediates metastasis and chemoresistance through inhibition of epithelial-to-mesenchymal transition in breast cancer.

Metastasis and chemoresistance remain major challenges in the clinical treatment of breast cancer. Recent studies show that dysregulated microRNAs (miRNAs) play an important role in metastasis and chemoresistance development in breast cancer. Herein, we identified downregulated expression of miR‐708‐3p in breast cancers. In particular, miR‐708‐3p expression was significantly decreased in specimens from breast cancer patients with metastasis compared to that in specimens from patients with no metastasis. Consistent with clinical data, our in vitro data show that miR‐708‐3p was more significantly decreased in invasive breast cancer cell lines. In addition, our data show that inhibition of miR‐708‐3p significantly stimulated breast cancer cell metastasis and induced chemoresistance both in vitro and in vivo. In contrast, overexpression of miR‐708‐3p dramatically inhibited breast cancer cell metastasis and enhanced the sensitivity of breast cancer cells to chemotherapy both in vitro and in vivo. Furthermore, we identified that miR‐708‐3p inhibits breast cancer cell epithelial‐to‐mesenchymal transition (EMT) by directly targeting EMT activators, including ZEB1, CDH2 and vimentin. Taken together, our findings suggest that miR‐708‐3p acts as a cancer suppressor miRNA and carries out its anticancer function by inhibiting EMT in breast cancer. In addition, our findings suggest that restoration of miR‐708‐3p may be a novel strategy for inhibiting breast cancer metastasis and overcoming the chemoresistance of breast cancer cells.

Long non-coding RNA-CTD-2108O9.1 represses breast cancer metastasis by influencing leukemia inhibitory factor receptor.

Breast cancer (BC) is an aggressive malignant disease in women worldwide with a high tendency to metastasize. However, important biomarkers for BC metastasis remain largely undefined. In the present study, we identified that long non‐coding RNA‐CTD‐2108O9.1 is downregulated in BC tissues and cells and acts as a metastatic inhibitor of BC. Mechanistic investigation determined that lncRNA‐CTD‐2108O9.1 represses metastasis by targeting leukemia inhibitory factor receptor (LIFR), which is designated as a metastasis suppressor in BC. Our study characterizes a significant tumor suppressor active in BC metastasis repression through the known metastasis inhibitor LIFR.

Targeting CXCR4–CXCL12 Axis for Visualizing, Predicting, and Inhibiting Breast Cancer Metastasis with Theranostic AMD3100–Ag2S Quantum Dot Probe

AbstractBreast cancer metastasis remains the primary cause of death and efforts to predict and reduce metastatic risk are particularly appealing. CXC chemokine receptor 4 (CXCR4) is reported as a specific metastasis due to its chemotactic homing to CXCL12. Herein, conjugation of a CXCR4 antagonist, AMD3100, to a fluorescent silver sulfide quantum dot (Ag2S) core (QD‐AMD) allows accurate detection of CXCR4 expression in tumor. Particularly, the probe precisely distinguishes highly metastatic breast cancer cells from those of lower metastatic ability. Longitudinal in vivo imaging predicts at early stages that the high CXCR4 expressing orthotopic 4T1 tumor would subsequently metastasize to lungs 14 d after tumor inoculation, while no metastasis forms from the low CXCR4 expressing MCF‐7 tumor. Correlative measurements find that the CXCL12 levels in lung increase with tumor progression. Perturbations of either CXCR4 on tumor cells by QD‐AMD or CXCL12 in the lungs by antibody successfully inhibit cancer metastasis. Intravenous injection of QD‐AMD in primary 4T1 tumor model effectively reduces lung metastasis. More importantly, due to the intrinsic photothermal effect, the metastatic spread is more thoroughly abrogated along with substantial shrinkage of primary tumor. Altogether, the probe is promising to detect, predict, and inhibit the metastatic spread of breast tumor.

Discovery and evaluation of nNav1.5 sodium channel blockers with potent cell invasion inhibitory activity in breast cancer cells

Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Nav1.5 (nNav1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-231. The activity of nNav1.5 is known to promote the breast cancer cell invasion in vitro and metastasis in vivo, and its expression in primary mammary tumors has been associated with metastasis and patient death. Metastasis development is responsible for the high mortality of breast cancer and currently there is no treatment available to specifically prevent or inhibit breast cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of low micromolar small molecule VGSC blockers. Using this model, we have designed, synthesized and evaluated five small molecule compounds as blockers of nNav1.5-dependent inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active compound identified from these studies blocked sodium currents by 34.9 ± 6.6% at 1 μM. This compound also inhibited the invasion of MDA-MB-231 cells by 30.3 ± 4.5% at 1 μM concentration without affecting the cell viability. The potent small molecule compounds presented here have the potential to be developed as drugs for breast cancer metastasis treatment.

Inhibition of breast cancer metastasis by co-transfection of miR-31/193b-mimics.

Objective(s):Various studies have been conducted to reduce the metastatic behavior of cancerous cells. In this regard, ectopic expression of anti-metastatic microRNAs by miR-mimic and miR-restoration-based therapies could bring new insights to the field. In the present study, the consequences of co-transfecting breast cancer cell lines with miR-193b and miR-31 were investigated via invasion and migration assays.Materials and Methods:Double stranded oligonucleotide of mature miR-193b-3p and miR-31-5p were cloned into pcDNA 6.2gw/EmGFP plasmid. The resulting plasmids were used for transfection. Real time-PCR was performed to assess the expression of miR-193b and miR-31 as well as Ras homolog gene family member A (RhoA) and urokinase-type plasminogen activator (uPA) as miR targets. Scratch, Transwell migration and Matrigel invasion assays were carried out to assess the extent of migration and invasion of cell lines.Results:The most significant increase in expression of miRs belonged to the single transfection of mimic-miRs in MDA-MB231. Although the co-transfection was not as successful as single transfection in miR expression, it was significantly more effective in inhibition of the cells invasive potential.Conclusion:Although the miR-restoration therapy based on co-transfection of two miRs could be less effective in expression of each miRNA, the resulting decrease in metastatic behavior of the cells is more significant due to collective effect of co-transfection to decrease target gene expression. Our results revealed that employing this sort of combinatorial strategies could lead to more efficient reduction in metastatic behavior. It seems that using this strategy would bring about more successful therapeutic outcomes.