Reduce Breast Cancer Metastasis Research Articles

Neutrophil Nanodecoys Inhibit Tumor Metastasis by Blocking the Interaction between Tumor Cells and Neutrophils.

Cancer metastasis is the main cause of cancer-related deaths and involves the interaction between tumor cells and neutrophils. In this study, we developed activated neutrophil membrane-coated nanoparticles (aNEM NPs) as nanodecoys to block neutrophil-mediated cancer metastasis. The aNEM NPs were fabricated by cloaking poly(lactic acid) nanoparticles with membranes derived from activated neutrophils and inherited the functional proteins of activated neutrophils. We demonstrated that aNEM NPs could interfere with the recruitment of neutrophils to the primary tumor and premetastatic niches, inhibit the adhesion of neutrophils to tumor vascular endothelium and circulating tumor cells (CTCs), and disrupt the formation of CTC-neutrophil clusters in vitro and in vivo. In 4T1-bearing mice, aNEM NPs could effectively reduce breast cancer metastasis to various organs in mice. Our results suggest that aNEM NPs are a promising nanomedicine for preventing or treating cancer metastasis by acting as neutrophil nanodecoys.

Role of cancer stem cell ecosystem on breast cancer metastasis and related mouse models.

Breast cancer metastasis is responsible for most breast cancer-related deaths and is influenced by many factors within the tumor ecosystem, including tumor cells and microenvironment. Breast cancer stem cells (BCSCs) constitute a small population of cancer cells with unique characteristics, including their capacity for self-renewal and differentiation. Studies have shown that BCSCs not only drive tumorigenesis but also play a crucial role in promoting metastasis in breast cancer. The tumor microenvironment (TME), composed of stromal cells, immune cells, blood vessel cells, fibroblasts, and microbes in proximity to cancer cells, is increasingly recognized for its crosstalk with BCSCs and role in BCSC survival, growth, and dissemination, thereby influencing metastatic ability. Hence, a thorough understanding of BCSCs and the TME is critical for unraveling the mechanisms underlying breast cancer metastasis. In this review, we summarize current knowledge on the roles of BCSCs and the TME in breast cancer metastasis, as well as the underlying regulatory mechanisms. Furthermore, we provide an overview of relevant mouse models used to study breast cancer metastasis, as well as treatment strategies and clinical trials addressing BCSC-TME interactions during metastasis. Overall, this study provides valuable insights for the development of effective therapeutic strategies to reduce breast cancer metastasis.

ASIC1a contributes to the epithelial-mesenchymal transformation of breast cancer by activating the Ca2+ /β-catenin pathway.

Breast cancer is the most common cancer in the world, with metastasis being one of the leading causes of death among patients. The acidic environment of breast cancer tissue promotes tumor cell invasion and migration by inducing epithelial-mesenchymal transformation (EMT) in tumor cells, but the exact mechanisms are not yet fully understood. This study investigated the expression of acid-sensitive ion channel 1a (ASIC1a) in breast cancer tissue samples and explored the mechanisms by which ASIC1a mediates the promotion of EMT in breast cancer cells in an acidic microenvironment through in vivo and in vitro experiments. The results showed that first, the expression of ASIC1a was significantly upregulated in breast cancer tissue and was correlated with the TNM (tumor node metastasis) staging of breast cancer. Furthermore, ASIC1a expression was higher in tumors with lymph node metastasis than in those without. Second, the acidic microenvironment promoted [Ca2+ ]i influx via ASIC1a activation and regulated the expression of β-catenin, Vimentin, and E-cadherin, thus promoting EMT in breast cancer cells. Inhibition of ASIC1a activation with PcTx-1 could suppress EMT in breast cancer cells. Finally, in vivo studies also showed that inhibition of ASIC1a could reduce breast cancer metastasis, invasion, and EMT. This study suggests that ASIC1a expression is associated with breast cancer staging and metastasis. Therefore, ASIC1a may become a new breast cancer biomarker, and the elucidation of the mechanism by which ASIC1a promotes EMT in breast cancer under acidic microenvironments provides evidence for the use of ASIC1a as a molecular target for breast cancer treatment.

The activation of SIRT1 by resveratrol reduces breast cancer metastasis to lung through inhibiting neutrophil extracellular traps

Neutrophil extracellular traps (NETs) play a crucial role in breast cancer metastasis. However, the therapeutic target of NETs in breast cancer metastasis is still unknown. Using a natural metabolite library and single-cell sequencing data analysis, we identified resveratrol (RES), a polyphenolic natural phytoalexin, and agonist of silent information regulator-1 (SIRT1) that suppressed NETs formation after cathepsin C (CTSC) treatment. In vivo, RES significantly hindered breast cancer metastasis in a murine orthotopic 4T1 breast cancer model. Serum levels of myeloperoxidase-DNA and neutrophil elastase-DNA in mouse breast cancer model were significantly lower after RES treatment. Correspondingly, the tumour infiltrated CD8+T cells in the lungs increased after the treatment. Mechanistically, RES targets SIRT1 in neutrophils and significantly inhibits the citrullination of histones H3, which is essential for chromatin decondensation and NETs formation. Furthermore, we identified that the NETs were suppressed by RES in bone marrow neutrophils after CTSC treatment, while specific deficiency of SIRT1 in neutrophils promoted NETs formation and breast cancer to lung metastasis. Thus, our results revealed that RES could be potentially identified as a viable therapeutic drug to prevent neutrophil cell death and breast cancer metastasis.

TRAF6 as a potential target in advanced breast cancer: a systematic review, meta-analysis, and bioinformatics validation

TRAF6 has emerged as a key regulator of breast cancer (BCa). However, the TRAF family constitutes of seven members that exhibit distinct and overlapping functions. To explore which TRAF represents a potential druggable target for BCa treatment, we searched Medline, Web of Science and Scopus for relevant studies from inception to June 27, 2021. We identified 14 in vitro, 11 in vivo and 4 human articles. A meta-analysis of pharmacological studies showed that in vitro inhibition of TRAF2/4 (mean difference (MD): − 57.49, 95% CI: − 66.95, − 48.02, P < 0.00001) or TRAF6 (standard(Std.)MD: − 4.01, 95% CI: − 5.75, − 2.27, P < 0.00001) is associated with reduction in BCa cell migration. Consistently, inhibition of TRAF2/4 (MD: − 51.08, 95% CI: − 64.23, − 37.94, P < 0.00001) and TRAF6 (Std.MD: − 2.80, 95% CI: − 4.26, − 1.34, P = 0.0002) is associated with reduced BCa cell invasion, whereas TRAF2/4 inhibition (MD: − 40.54, 95% CI: − 52.83, − 28.26, P < 0.00001) is associated with reduced BCa cell adhesion. Interestingly, only inhibition of TRAF6 (MD: − 21.46, 95% CI: − 30.40, − 12.51, P < 0.00001) is associated with reduced cell growth. In animal models of BCa, administration of pharmacological inhibitors of TRAF2/4 (Std.MD: − 3.36, 95% CI: − 4.53, − 2.18, P < 0.00001) or TRAF6 (Std.MD: − 4.15, 95% CI: − 6.06, − 2.24, P < 0.0001) in mice is associated with reduction in tumour burden. In contrast, TRAF6 inhibitors (MD: − 2.42, 95% CI: − 3.70, − 1.14, P = 0.0002) reduced BCa metastasis. In BCa patients, high expression of TRAF6 (Hazard Ratio: 1.01, CI: 1.01, 1.01, P < 0.00001) is associated with poor survival rate. Bioinformatics validation of clinical and pathway and process enrichment analysis in BCa patients confirmed that gain/amplification of TRAF6 is associated with secondary BCa in bone (P = 0.0079), and poor survival rate (P < 0.05). Overall, TRAF6 inhibitors show promise in the treatment of metastatic BCa. However, low study number and scarcity of evidence from animal and human studies may limit the translation of present findings into clinical practice.

Identification of lysyl oxidase as an adipocyte-secreted mediator that promotes a partial mesenchymal-to-epithelial transition in MDA-MB-231 cells.

Breast cancer (BC) is the most common cancer in women worldwide, where adiposity has been linked to BC morbidity. In general, obese premenopausal women diagnosed with triple-negative BC (TNBC) tend to have larger tumours with more metastases, particularly to the bone marrow, and worse prognosis. Previous work using a 3-dimensional (3D) co-culture system consisting of TNBC cells, adipocytes and the laminin-rich extracellular matrix (ECM) trademarked as Matrigel, demonstrated that adipocytes and adipocyte-derived conditioned media (CM) caused a partial mesenchymal-to-epithelial transition (MET). Given that MET has been associated with secondary tumour formation, this study sought to identify molecular mediators responsible for this phenotypic change. Adipocytes were cultured with and without Matrigel, where semi-quantitative proteomics was used to identify proteins whose presence in the CM was induced or enhanced by Matrigel, which were referred to as adipocyte-secreted ECM-induced proteins (AEPs). The AEPs identified were assessed for association with prognosis in published proteomic datasets and prior literature. Of these, 4 were evaluated by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), followed by a functional and MET marker analysis of 1 AEP on MDA-MB-231 cells grown on Matrigel or as monolayers. The 4 AEPs showed a positive correlation between protein expression and poor prognosis. RT-qPCR analysis reported no significant change in AEPs mRNA expression. However, lysyl oxidase (LOX) was increased in CM of ECM-exposed adipocytes. Recombinant LOX (rLOX) caused the mesenchymal MDA-MB-231 TNBC cells to form less branched 3D structures and reduced the expression of vimentin. The data suggest that adipocyte-secreted LOX changes the mesenchymal phenotype of BC cells in a manner that could promote secondary tumour formation, particularly at sites high in adipocytes such as the bone marrow. Future efforts should focus on determining whether targeting LOX could reduce BC metastasis in obese individuals.

Dissecting RAS Oncogene-Induced Kinome Involved in Breast Cancer Metastasis

Background and Hypothesis:&#x0D; The RAS and PI3K-AKT-mTOR signaling pathways are often dysregulated in cancer. RAS pathway alterations, however, are more common in breast cancer metastasis. The laboratory’s recently developed model system demonstrated the ability of RAS but not PIK3CA-induced signals in promoting metastasis of breast cancer. Unbiased kinome analyses of isogenic RAS-transformed primary tumor and metastatic cells and PIK3CA-transformed primary tumor cells enabled identification of RAS-activated kinome, which included FER, PAK4, LIMK1, PIK3CD and Casein Kinase 2 (CK2). We hypothesized that therapeutic targeting of these kinases may reduce breast cancer metastasis. As a proof-of-principle, the effect of the CK2 inhibitor Silmitasertib, which is in clinical trial for COVID-19 and refractory multiple myeloma, was tested.&#x0D; Experimental Design:&#x0D; The study included four isogenic cell lines: “normal” (KTB34-hTERT), PIK3CA-transformed (TKTB34-PIK3CA), RAS-transformed (TKTB34-RAS), and RAS-transformed cells metastasized to lungs (MKTB34-RAS). Active kinomes in these cells were identified using phospho-proteomics and functional kinome profiling using multiplexed kinase inhibitor beads. Expression levels of FER, PAK4, LIMK1, and PIK3CD kinases were compared through Western Blot using the phospho-antibodies as an indicator of kinase activation. Sensitivity to Silmitasertib was measured using the BrdU Cell Proliferation Assay.&#x0D; Results:&#x0D; FER, PAK4, LIMK1, and PIK3CD were all overexpressed in the TKTB34-RAS and MKTB34-RAS cells compared to KTB34-hTERT and TKTB34-PIK3CA cells. The tested concentration range for Silmitasertib (500 nM to 5 µM) was ineffective in killing the RAS-transformed cells and was overly toxic to “normal” cells.&#x0D; Conclusion and Potential Impact:&#x0D; FER, PAK4, LIMK1, PIK3CD, and CK2 are potential therapeutic targets for breast cancer metastasis. However, Silmitasertib may not be a good candidate as it is more toxic to “normal” cells compared to cancer cells. The isogenic “normal” and transformed cell line model system described here may help to discover new targets and drugs that kill cancer but not normal cells.

Identification and characterization of potent, selective, and efficacious inhibitors of human arylamine N-acetyltransferase 1.

Arylamine N-acetyltransferase 1 (NAT1) plays a pivotal role in the metabolism of carcinogens and is a drug target for cancer prevention and/or treatment. A protein-ligand virtual screening of 2 million chemicals was ranked for predicted binding affinity towards the inhibition of human NAT1. Sixty of the five hundred top-ranked compounds were tested experimentally for inhibition of recombinant human NAT1 and N-acetyltransferase 2 (NAT2). The most promising compound 9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester (compound 10) was found to be a potent and selective NAT1 inhibitor with an in vitro IC50 of 0.75µM. Two structural analogs of this compound were selective but less potent for inhibition of NAT1 whereas a third structural analog 1,2-dihydroxyanthraquinone (a compound 10 hydrolysis product also known as Alizarin) showed comparable potency and efficacy for human NAT1 inhibition. Compound 10 inhibited N-acetylation of the arylamine carcinogen 4-aminobiphenyl (ABP) both in vitro and in DNA repair-deficient Chinese hamster ovary (CHO) cells in situ stably expressing human NAT1 and CYP1A1. Compound 10 and Alizarin effectively inhibited NAT1 in cryopreserved human hepatocytes whereas inhibition of NAT2 was not observed. Compound 10 caused concentration-dependent reductions in DNA adduct formation and DNA double-strand breaks following metabolism of aromatic amine carcinogens beta-naphthylamine and/or ABP in CHO cells. Compound 10 inhibited proliferation and invasion in human breast cancer cells and showed selectivity towards tumorigenic versus non-tumorigenic cells. In conclusion, our study identifies potent, selective, and efficacious inhibitors of human NAT1. Alizarin's ability to inhibit NAT1 could reduce breast cancer metastasis particularly to bone.

Lung mesenchymal stromal cells influenced by Th2 cytokines mobilize neutrophils and facilitate metastasis by producing complement C3

Pre-metastatic niche formation is critical for the colonization of disseminated cancer cells in distant organs. Here we find that lung mesenchymal stromal cells (LMSCs) at pre-metastatic stage possess potent metastasis-promoting activity. RNA-seq reveals an upregulation of complement 3 (C3) in those LMSCs. C3 is found to promote neutrophil recruitment and the formation of neutrophil extracellular traps (NETs), which facilitate cancer cell metastasis to the lungs. C3 expression in LMSCs is induced and sustained by Th2 cytokines in a STAT6-dependent manner. LMSCs-driven lung metastasis is abolished in Th1-skewing Stat6-deficient mice. Blockade of IL-4 by antibody also attenuates LMSCs-driven cancer metastasis to the lungs. Consistently, metastasis is greatly enhanced in Th2-skewing T-bet-deficient mice or in nude mice adoptively transferred with T-bet-deficient T cells. Increased C3 levels are also detected in breast cancer patients. Our results suggest that targeting the Th2-STAT6-C3-NETs cascade may reduce breast cancer metastasis to the lungs.

Ruyiping formula inhibits metastasis via the microRNA-134-SLUG axis in breast cancer

BackgroundMetastasis is the leading cause of death among breast cancer patients. MicroRNA-134 has been reported to have a tumor-suppressive role in breast cancer. Ruyiping (RYP), a traditional Chinese formula, has been shown with the ability to reduce breast cancer metastasis in pre-clinical studies. This present study was designed to examine whether miR-134 was involved in RYP-inhibited breast cancer metastasis.MethodsThe expression of SLUG, E-Cadherin, N-Cadherin and miR-134 in MDA-MB-231 and 4 T1 cells treated with RYP or vehicle control were determined by quantitative realtime-PCR and western blot. Invasiveness determined by transwell assay as well as SLUG gene expression determined by qPCR were detected in cells transfected with chemically synthesized miR-134 mimics or inhibitors. BALB/c mice were injected with 4 T1 cells orthotopically and fed with RYP through gavage. Breast tumor growth, metastasis and tumor expression of EMT markers were detected.ResultsCompared with the control, Ruyiping formula significantly inhibited SLUG-regulated breast cancer cells invasion. MiR-134 was induced by RYP in vitro and in vivo and was able to suppress SLUG by targeting its 3’UTR. RYP suppressed SLUG expression and cell invasion through miR-134. In 4 T1 tumor-bearing mice, RYP significantly inhibited 4 T1 tumor growth and lung metastasis, increased the levels of miR-134 and epithelial marker while decreased the levels of SLUG and mesenchymal marker.ConclusionOur data uncovered that Ruyiping formula exerts an anti-metastatic activity against breast cancer cells by regulating SLUG through miR-134. MiR-134-SLUG axis might be a promising strategy in breast cancer therapy.

Time-restricted feeding normalizes hyperinsulinemia to inhibit breast cancer in obese postmenopausal mouse models

Accumulating evidence indicates that obesity with its associated metabolic dysregulation, including hyperinsulinemia and aberrant circadian rhythms, increases the risk for a variety of cancers including postmenopausal breast cancer. Caloric restriction can ameliorate the harmful metabolic effects of obesity and inhibit cancer progression but is difficult to implement and maintain outside of the clinic. In this study, we aim to test a time-restricted feeding (TRF) approach on mouse models of obesity-driven postmenopausal breast cancer. We show that TRF abrogates the obesity-enhanced mammary tumor growth in two orthotopic models in the absence of calorie restriction or weight loss. TRF also reduces breast cancer metastasis to the lung. Furthermore, TRF delays tumor initiation in a transgenic model of mammary tumorigenesis prior to the onset of obesity. Notably, TRF increases whole-body insulin sensitivity, reduces hyperinsulinemia, restores diurnal gene expression rhythms in the tumor, and attenuates tumor growth and insulin signaling. Importantly, inhibition of insulin secretion with diazoxide mimics TRF whereas artificial elevation of insulin through insulin pumps implantation reverses the effect of TRF, suggesting that TRF acts through modulating hyperinsulinemia. Our data suggest that TRF is likely to be effective in breast cancer prevention and therapy.

Hepatic lipids promote liver metastasis.

Obesity predisposes to cancer and a virtual universality of nonalcoholic fatty liver disease (NAFLD). However, the impact of hepatic steatosis on liver metastasis is enigmatic. We find that while control mice were relatively resistant to hepatic metastasis, those which were lipodystrophic or obese, with NAFLD, had a dramatic increase in breast cancer and melanoma liver metastases. NAFLD promotes liver metastasis by reciprocal activation initiated by tumor-induced triglyceride lipolysis in juxtaposed hepatocytes. The lipolytic products are transferred to cancer cells via fatty acid transporter protein 1, where they are metabolized by mitochondrial oxidation to promote tumor growth. The histology of human liver metastasis indicated the same occurs in humans. Furthermore, comparison of isolates of normal and fatty liver established that steatotic lipids had enhanced tumor-stimulating capacity. Normalization of glucose metabolism by metformin did not reduce steatosis-induced metastasis, establishing the process is not mediated by the metabolic syndrome. Alternatively, eradication of NAFLD in lipodystrophic mice by adipose tissue transplantation reduced breast cancer metastasis to that of control mice, indicating the steatosis-induced predisposition is reversible.

OR30-4 Parathyroid Hormone-related Peptide Antagonist and Inverse Agonist Reduce Breast Cancer Metastasis to Bone and Bone Loss

Bone is the most frequent site for breast cancer metastasis. Metastatic cancer cells secrete parathyroid-hormone related peptide (PTHrP) that stimulates bone turnover, releasing growth factors and creating a cavity for tumor growth. Current treatments include bisphosphonates and the RANK Ligand inhibitors that have limited efficacy. PTHrP antagonists have failed clinically due to rapid drug turnover and kidney side effects causing hypocalcemia. We synthesized two novel PTHrP antagonists that are fused to an inert bacterial collagen binding domain (CBD) of colG collagenase from Clostridium histolyticum. The collagen binding activity directs PTH analogs to the bone matrix where it blocks PTHrP action. PTH(7-33)-CBD is an amino truncated PTHrP antagonist fused to the CBD that we previously showed reduced metastatic tumor burden in bone and prevented bone destruction. We now compare PTH(7-33)-CBD to [W2]PTH(1-33)-CBD, an inverse agonist at the PTH/PTHrP receptor. In vitro neither drug induced PTH receptor activation in SaOS-2 osteosarcoma cells as measured by cAMP accumulation. Both compounds partially antagonized PTH(1-34) agonist-induced cAMP accumulation. Importantly, [W2]PTH(1-33)-CBD reduced agonist-stimulated cAMP accumulation by >95% (vs. 71% for PTH(7-33)-CBD) exceeding that reported previously for any PTH antagonist. Both compounds induced apoptosis of MDA-MB-231 breast cancer cells in vitro. In vivo testing was performed with an established mouse model of breast cancer bone metastasis using a bone-trophic variant of MDA-MB-231 breast cancer cells expressing luciferase (MDA-MB-231-BM/luc+). 24 hours after drug administration (1000 µg/kg administered IP), MDA-MB-231-BM/luc+ cells were injected intratibially into nude mice. PTH(7-33)-CBD significantly reduced tumor burden in bone (P<0.05) compared to both vehicle control (VC) (weeks 4-8), and to control PTH(7-34) antagonist that lacks the CBD (weeks 6-7). [W2]PTH(1-33)-CBD showed a trend towards decrease in tumor burden but did not reach statistical significance. Both PTH(7-33)-CBD and [W2]PTH(1-33)-CBD significantly reduced osteolytic lesions (P<0.05) compared to VC and PTH(7-34) weeks 3-8 and weeks 6-8, respectively. Drug treatments did not significantly affect animal weight nor alter serum calcium, alkaline phosphatase or N-terminal propeptide of Type 1 procollagen (PN1P) bone formation marker. Tartrate resistant acid phosphatase (TRAP), a marker of bone removal, was significantly reduced by treatments. In summary, a new PTHrP inverse agonist, [W2]PTH(1-33)-CBD, partially suppressed basal signaling of the PTH/PTHrP receptor and demonstrated a higher degree of antagonist activity in-vitro than any PTH antagonist published to date. It is expected that the superior antagonist activity of [W2]PTH(1-33)-CBD will provide significant tumor protection against breast cancer metastasis to bone.

Inhibition of the Stromal p38MAPK/MK2 Pathway Limits Breast Cancer Metastases and Chemotherapy-Induced Bone Loss.

The role of the stromal compartment in tumor progression is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and surrounding stromal cells, and its expression levels correlate with poor prognosis. This observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of p38MAPKα or its downstream kinase MK2 each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity that drastically affects quality of life for patients with cancer. These data underscore the vital role played by stromal-derived factors in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss.Significance: Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth, preserves bone quality, and extends survival. Cancer Res; 78(19); 5618-30. ©2018 AACR.

Abstract 5152: Cancer stem cell targeted exosomes for the treatment of metastatic breast cancer

Abstract Introduction: Therapy resistance and metastasis remain the two biggest challenges of breast cancer that result in a majority of cancer deaths. Cancer stem cells (CSCs) are a subpopulation of cancer cells that can initiate new tumors, remain refractory to conventional therapies, and spread throughout the body. In order to effectively block metastasis, the present study aims to develop novel therapeutics that specifically target breast CSCs and spare normal cells. While nanomedicine represents one of the promising future directions, it is necessary to overcome the shortcomings of synthetic nanoparticles, including bio-instability and off-target toxicity. Here, we aim to bio-engineer the naturally produced cell-secreted small vesicles, exosomes (~100 nm diameter), for a novel therapeutic delivery system. Methods: Exosomes from the human mesenchymal stem cells (MSCs) were isolated by differential ultracentrifugation, and characterized by multiple approaches including transmission EM, Immunoblotting and immunofluorescence assays to confirm the size, shape and presence of exosomal markers CD63, CD81 and LAMP2B. Exosomes were engineered to express a peptide ligand on its surface to specifically recognize and target CSCs, and the presence of the ligand was confirmed by western blot, flow cytometry (FCM) and a super resolution confocal laser scanning microscopy (CLSM, Leica SP8). The CSC targeting efficiency of the exosomes was evaluated in vitro by FCM and CLSM. Further, the exosomes were loaded with microRNAs (miR-200, miR-30c), as one of the key factors that regulate breast CSC functions, by following a novel transfection technology. Results: The bio-engineered exosomes specifically target and internalize into the CSCs compared to normal cells with absent expression of the ligand-binding protein. The developed exosomes can effectively deliver candidate miRNAs into the recipient cells and knock down the genes important for functional CSCs. The in vivo targeting of CSCs and therapeutic applications of the exosomes using patient-derived human-in-mouse breast tumor xenograft models are underway. Conclusion: It is expected that exosome-based miRNA therapeutics will have high biocompatibility, low toxicity, high specificity, and promising efficiency in preclinical and clinical applications to reduce breast cancer metastasis and improve patient survival. Citation Format: Golam Kibria, Katelyn E. Lee, Erika K. Ramos, Simo Huang, Clifford V. Harding, Jan Lötvall, Huiping Liu. Cancer stem cell targeted exosomes for the treatment of metastatic breast cancer [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 5152. doi:10.1158/1538-7445.AM2017-5152

Fibronectin induces epithelial-mesenchymal transition in human breast cancer MCF-7 cells via activation of calpain.

Fibronectin (FN) is a primary component of the mammary mesenchymal compartment and undergoes dramatic changes during breast cancer development. Increased FN expression is associated with an invasive and metastatic breast cancer phenotype. The present study demonstrated that FN causes an epithelial-mesenchymal transition (EMT)-like morphological change in MCF-7 breast cancer cells. FN stimulation caused the downregulation of epithelial markers E-cadherin and tight junction protein ZO-1, and the upregulation of mesenchymal markers N-cadherin and vimentin. Additionally, FN promoted cell migration and invasion in MCF-7 cells, with increased expression of calpain-2 and proteolysis of focal adhesion kinase 1 (FAK), indicating calpain activation. Notably, the FN induced changes in morphology and EMT markers were reversed with the treatment of calpain-specific inhibitors, calpain inhibitor I (N-acetyl-L-leucyl-L-leucyl-L-norleucinal), calpeptin and calpain inhibitor IV. Meanwhile, the effects of FN on cell migration and invasion, as well as FAK proteolysis were markedly suppressed by calpain inhibitors. Taken together, the results of the present study indicate that calpain plays an essential role in FN-induced EMT response, and that targeting calpain signaling may be a potential strategy to reduce breast cancer metastasis.

Abstract A14: A cholesterol metabolite acts on the host to increase breast cancer metastasis

Abstract With a woman's lifetime risk of developing invasive breast cancer at one in eight, breast cancer remains the most common cancer and the second leading cause of cancer death in women. Despite the success of frontline endocrine therapies, relapse with resistant metastatic disease is common, and triple negative disease remains without a targeted therapy. Importantly, breast cancer associated mortality is most often due to the metastatic spread of the disease. Therefore, there continues to be a need for studies that may lead to the development of new chemopreventive strategies and/or lifestyle changes that reduce breast cancer metastasis. In this regard, both obesity and elevated cholesterol are risk factors for metastatic relapse, while patients receiving cholesterol-lowering drugs such as HMG-CoA reductase inhibitors (statins) display an increased relapse-free survival time. Therefore, clinical observations strongly implicate cholesterol in the metastatic progression of, although the underlying mechanisms remain unknown. Thus, we initiated a series of studies to evaluate the mechanisms by which cholesterol promotes breast cancer metastasis. Using murine models of mammary cancer, we find that elevated cholesterol does indeed increase metastasis. Further, statins attenuated the metastatic effects of a high fat diet, implicating cholesterol as a primary mediator of this pathology. Importantly, the pro-metastatic effects of cholesterol required the presence of CYP27A1, the enzyme responsible for its conversion to 27-hydroxycholesterol (27HC). We have recently shown that 27HC is an endogenous ligand for the both the estrogen receptors (ERs) and liver X receptors (LXRs). Interestingly, however, the metastatic effects of 27HC were also prevalent in ER-negative models. While LXR activation did have a modest effect on epithelial to mesenchymal transition (EMT), we find that the robust pro-metastatic effects of 27HC are mediated in a cancer cell-extrinsic manner. Specifically, we find that 27HC alters myeloid cell recruitment and function at distal metastatic sites, and thereby facilitates cancer cell colonization and growth by modulating the host environment. In summary, our data strongly suggest that by altering the host immune system, 27HC is the down-stream mediator of the effects of obesity and cholesterol on breast cancer metastasis. These results provide additional support for development of specific CYP27A1 inhibitors and immune-modulatory therapies for the prevention and treatment of breast cancer metastasis. This work was supported by an NIH-NCI grant to ERN (4R00CA172357-03). Citation Format: Amy Eun Joo Baek, Erik Russell Nelson. A cholesterol metabolite acts on the host to increase breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A14.

Abstract P1-06-03: Epithelial to mesenchymal transition (EMT) regulates the spontaneous generation of GD2+ breast cancer stem-like cells through NFκB activation

Abstract Breast cancer recurrence may be a consequence of persistent breast cancer stem-like cells (BCSCs) that survive chemo- or hormonal therapy. Therefore, targeting BCSCs could complement standard chemotherapy. We discovered that the ganglioside GD2 is expressed on and defines BCSCs (Battula et al., JCI, 2012), as consequence of activation of the enzyme GD3 synthase (GD3S). Inhibition of GD3S expression inhibited breast cancer metastasis to lung. We also observed that GD2- breast cancer cells spontaneously generate GD2+ cells in vitro. As induction of EMT generates a stem cell–like phenotype, we hypothesized that EMT regulates the generation of GD2+ breast cancer cells. To test this hypothesis, MDA-MB-231and SUM159 cells were cultured in vitro and the percentage of GD2+ cells was measured over time. Interestingly, the percentage and absolute number of GD2+ cells increased in a time-dependent manner, suggesting the spontaneous generation of GD2+ cells. Concomitantly, mesenchymal-related markers including vimentin, N-cadherin, and twist increased 3 to 6 - fold. To further investigate whether this process is operational in vivo, GFP+ MDA-MB-231 cells were transplanted into mammary fat pads of NOD/SCID mice. Each week, a group of mice was sacrificed, tumors were extracted and the number of GFP+GD2+ cells was determined by flow cytometry. In line with our in vitro results, we observed significant increases in GD2+ BCSCs with increasing tumor volume from 15.1%±4.6% to 37%±8.7% over a 6 week period, suggesting that breast cancer cells spontaneously undergo EMT during tumor progression and generate GD2+ BCSCs. To identify possible targets to inhibit EMT in breast cancer cells, proteomic analysis using Kinexus® antibody arrays revealed activation of NFκB and focal adhesion kinase (FAK) signaling in GD2+ breast cancer cells. The activation of NFκB (phospho p65) in GD2+ cells was validated by CyTOF mass cytometry using metal tagged antibodies. These data suggest that inhibition of NFκB signaling may inhibit GD2+ BCSC growth. Indeed, the IKK inhibitor BMS345541 reduced GD2+ cells by &amp;gt;95% and inhibited GD3S expression (determined by qRT-PCR) in a dose- and time-dependent fashion. In contrast, treatment with doxorubicin increased the percentage of GD2+ cells, from 13.5±2.5% to 21±2.6% in MDA-MB-231 cells, suggesting that GD2+ cells are resistant to doxorubicin. In addition, treatment with BMS345541 inhibited the ability of breast cancer cells to form mammospheres by &amp;gt;90% in vitro. In-vivo tumorigenesis assay demonstrated that BMS345541 induced a significant decrease (p &amp;lt;0.01) in tumor volume, and increased survival of tumor bearing mice: median survival was 78 days for BMS345541-treated mice vs. 58 days for controls (p&amp;lt;0.002). Conclusion: GD2+ BCSCs are spontaneously produced during tumor progression by EMT and NFκB and FAK mediated signaling might regulate this process. Inhibition of NFκB and FAK signaling pathways may inhibit the spread of BCSCs and reduce breast cancer metastases. Citation Format: Battula VL, Sun J, Nguyen K, Hortobagyi G, Andreeff M. Epithelial to mesenchymal transition (EMT) regulates the spontaneous generation of GD2+ breast cancer stem-like cells through NFκB activation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-03.

Palbociclib inhibits epithelial-mesenchymal transition and metastasis in breast cancer via c-Jun/COX-2 signaling pathway.

Palbociclib, a highly selective CDK4/6 inhibitor, has been shown to be a novel anti-tumor agent that suppresses breast cancer cell proliferation. However, its anti-metastasis activity remains controversial. In the present study, we evaluated whether palbociclib prevented breast cancer cell metastasis and revealed its regulatory mechanism. We found that palbociclib inhibited migration and invasion in the breast cancer cells MDA-MB-231 and T47D. The epithelial-mesenchymal transition (EMT) markers, vimentin and Snail, were down-regulated with palbociclib treatment. Moreover, we revealed that this inhibition was mediated by the c-Jun/COX-2 pathway. COX-2 was decreased after palbociclib treatment. The production of PGE2 was also reduced along with COX-2. Additionally, our data showed that c-Jun, a crucial transcriptional regulator of COX-2, was down-regulated by palbociclib. We found that palbociclib weakened the COX-2 promoter binding activity of c-Jun and prevented its translocation from the cytoplasm to cell nuclei. Bioluminescence imaging and tail intravenous injection were used to evaluate the anti-metastasis effect of palbociclib in vivo. The data demonstrated that palbociclib reduced breast cancer metastasis to the lung. These results therefore demonstrated that the anti-metastasis activity of palbociclib is mediated via the c-Jun/COX-2 signaling pathway by inhibiting EMT in breast cancer cells.

Abstract A82: The cholesterol metabolite, 27-hydroxycholesterol, promotes breast cancer metastasis

Abstract Breast cancer remains the most common cancer in women and is the second leading cause of cancer death in women. It is important to note that the majority of patients eventually succumb to the metastatic spread of their disease, not to the primary tumor itself. The magnitude of this problem provides strong rationale for studies that may lead to the development of new chemopreventative strategies and/or lifestyle changes that reduce breast cancer metastasis. It is of significance, therefore, that patients taking inhibitors of 2-hydroxy-3-methylglutaryl coenzyme A (statins) experience a significantly increased relapse free survival time. Therefore, our recent finding that 27-Hydroxycholesterol (27HC), a primary metabolite of cholesterol, exerts partial agonist activity on both the estrogen receptors (ERs) and liver X receptors (LXRs), suggests a potential mechanistic link between hypercholesterolemia and breast cancer pathology. We demonstrate that 27HC (1) increases in vivo tumor growth and lung metastases, (2) promotes an epithelial to mesenchymal like transition, and (3) in a myeloid cell dependent manner, alters the metastatic microenvironment to promote cancer cell colonization. Our ongoing work is aimed at identifying the specific mechanisms by which 27HC induces these changes and thereby increases metastasis. In summary, our data thus far strongly indicate a role for 27HC in breast cancer pathophysiology and metastasis. These results provide additional support for the exploration of potential chemopreventative benefits of lower cholesterol diets, pharmacological inhibitors of HMG-CoA reductase or the enzyme responsible for the synthesis of 27HC (CYP27A1), and macrophage ablative strategies. This work was supported by an NIH-NCI grant to ERN (4R00CA172357-03). Citation Format: Erik Russell Nelson, Suzanne E. Wardell, Donald P. McDonnell. The cholesterol metabolite, 27-hydroxycholesterol, promotes breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A82.