Prevention Of Breast Cancer Metastasis Research Articles

Abstract 2571: 1,25-dihydroxyvitamin D regulation of pyruvate carboxylase in MCF10A-ras cells decreases viability in extracellular matrix detached conditions

Abstract Metastasis is the main cause of most breast cancer related deaths. Throughout the process of metastasis, cancer cells strive to survive in conditions where they are detached from the extracellular matrix (ECM). Vitamin D may play a role in preventing breast cancer metastasis by modulating metabolism in breast cancer cells detached from ECM. The anaplerotic enzyme pyruvate carboxylase (PC) is essential for breast to lung metastasis and the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), downregulates PC expression through a vitamin D response element. PC replenishes the TCA cycle with the intermediate oxaloacetate (OAA), and lower expression of PC following 1,25D treatment may cause impaired energy metabolism via the TCA cycle. We hypothesize that the 1,25D mediated decrease in PC expression decreases breast cancer cell viability in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) treated with vehicle (ethanol) or 10 nM 1,25D and maintained in varying nutrient concentrations were utilized for these studies. Poly-HEMA coated plates were used to model ECM detachment paired with MTT assays to measure cell viability. mRNA expression of metabolic genes is quantified using qPCR. Results show that when compared to attached cells, detached cells have a 94% increase in PC mRNA expression, while 1,25D treatment decreases PC mRNA expression in both attached by 75% and detached conditions by 63%. In addition 1,25D treatment and PC knockdown decreases viability in ECM detached conditions significantly by 16% and 11%, with no additional effect when combined. Glutamine deprivation also decreases viability by 40% in detached conditions, however, the combination of 1,25D treatment and glutamine deprivation results in a 71% decrease in viability. These results suggest that while glutamine is required for ECM survival, regulation of glutamine metabolism is not the mechanism by which 1,25D affects ECM survival. Replenishing the TCA cycle with OAA rescues glutamine depleted cells by 84% as well as PC knockdown cells by 14%. Further, when stressed with low glutamine conditions, cells in attached conditions increase mRNA expression of PC by 311%, suggesting that changes in PC expression may be a mechanism that cancer cells use to adapt to energy related stress. These results suggest that 1,25D regulation of metabolism may sensitize breast cancer cells to ECM detachment death via downregulation of PC, reducing the required flux of substrates into the TCA cycle which are needed to survive ECM detached conditions during metastasis. Citation Format: Madeline Sheeley, Dorothy Teegarden, Katie Wong. 1,25-dihydroxyvitamin D regulation of pyruvate carboxylase in MCF10A-ras cells decreases viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2571.

Extracellular Acidosis Promotes Metastatic Potency via Decrease of the BMAL1 Circadian Clock Gene in Breast Cancer.

Circadian oscillation is an essential process that influences many physiological and biological mechanisms and a decrease of circadian genes is associated with many diseases such as cancer. Despite many efforts to identify the detailed mechanism for decreasing circadian genes and recovering reduced circadian genes in cancer, it is still largely unknown. We found that BMAL1 was reduced in tumor hypoxia-induced acidosis, and recovered by selectively targeting acidic pH in breast cancer cell lines. Surprisingly, BMAL1 was reduced by decrease of protein stability as well as inhibition of transcription under acidosis. In addition, melatonin significantly prevented acidosis-mediated decrease of BMAL1 by inhibiting lactate dehydrogenase-A during hypoxia. Remarkably, acidosis-mediated metastasis was significantly alleviated by BMAL1 overexpression in breast cancer cells. We therefore suggest that tumor hypoxia-induced acidosis promotes metastatic potency by decreasing BMAL1, and that tumor acidosis could be a target for preventing breast cancer metastasis by sustaining BMAL1.

Abstract P3-01-03: Modulating inflammation with nucleic acid scavengers to prevent breast cancer metastasis

Abstract Rationale: Chronic inflammation is a hallmark of cancer. Inflammation, whether it is an underlying baseline condition or induced/exacerbated due to treatment, is a major hurdle that ultimately impacts the efficacy of standard of care therapy. However, no strategy exists to effectively control endogenous or therapy-induced inflammation in women with breast cancer. Moreover, there is limited information on how endogenous or therapy-induced inflammation modulates immune cells in women with breast cancer. Nucleic acid-containing pro-inflammatory complexes, termed Nucleic Acid-containing Damage Associated Molecular Patterns (NA-DAMPs), are released from dead or dying cancer cells or activated immune cells. NA-DAMPs activate nucleic acid sensing toll-like receptors (TLR) and other pattern recognition receptors (PRR). NA-DAMPs promote tumor progression and metastasis by inducing pro-tumorigenic signaling in cancer cells. Excessive and persistent NA-DAMP-mediated PRR signaling in immune cells is associated with immune dysfunction. Nucleic acid binding polymers have been used as vehicles for DNA/RNA delivery. We have discovered that a subset of nucleic acid binding polymers, termed nucleic acid scavengers (NAS), act as anti-inflammatory agents by capturing cancer-promoting pro-inflammatory NA-DAMPs. Therefore, we investigated if a NAS could limit cancer progression and metastasis by capturing and mitigating the effects of NA-DAMPs in immunocompetent murine models of breast cancer. Methods: We tested the use of the NAS PAMAM-G3 in the 1] orthotopic surgical resection 4T1 breast cancer model; 2] intravenous experimental metastasis 4T1 breast cancer model; 3] MMTV-PyMT spontaneous breast cancer model. We also measured changes in inflammation/inflammatory cell profile in tumor and in blood. Next, we investigated the relationship between therapy, NA-DAMPs and immune cell profile in blood from women with breast cancer. We also examined if NA-DAMPs in patient serum induces tumor invasion and if the NAS PAMAM-G3 prevents tumor invasion using an in vitro assay. Results: We have determined that the NAS PAMAM-G3 controls lung metastasis in immunocompetent murine models of breast cancer and our data indicate that: 1] chronic inflammation and inflammatory cells in the tumor and periphery promote cancer invasion and metastasis; 2] modulating inflammation by removing excessive NA-DAMPs using the NAS PAMAM-G3 limits cancer metastasis; 3] scavenging inflammation-associated NA-DAMPs significantly alters immune cell and cytokine/chemokine profile in the tumor and periphery, promotes T cell infiltration in the tumor and induces antitumor CD8+ T cell responses. In experiments conducted using blood collected longitudinally from women undergoing standard of care therapy, we observed that: 1] NA-DAMPs and inflammatory monocytes are elevated in women undergoing standard of care therapy; 2] Post-chemotherapy breast cancer patient serum increases invasion/migration of human breast cancer cells in vitro; 3] PAMAM-G3 abrogates increase in invasion/migration of human breast cancer cells in vitro in response to post-chemotherapy breast cancer patient serum. Conclusions: These results highlight the potential utility of NAS to treat cancer associated inflammation and thereby inhibit cancer metastasis by 1) limiting extracellular NA-DAMP-induced persistent activation of pro-inflammatory signaling, and 2) promoting full engagement of both the innate and adaptive immune systems. These principles will aid in the development of a novel anti-metastatic therapy for women with breast cancer. This work is supported by Department of Defense Breast Cancer Research Program Awards W81XWH-16-1-0512 (Nair) and W81XWH-16-1-0513 (Sullenger). Citation Format: Smita Nair, Eda Holl, Karenia Landa, Victoria Frazier, Elias Eteshola, David Boczkowski, Rachel Rempel, Shelley Hwang, Bruce Sullenger. Modulating inflammation with nucleic acid scavengers to prevent breast cancer 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 P3-01-03.

Cancer stem cell property and gene signature in bone-metastatic Breast Cancer cells

The majority of the deaths from breast cancer is due to metastasis. Bone is the most common organ to which breast cancer cells metastasize. The mechanism regulating the bone-metastatic preference remains unclear; there is a lack of a gene signature to distinguish bone-metastatic breast cancer cells. Herein, florescence-labeled MDA-MB-231 cells were transplanted into the fat pads of of the mammary gland in nude mice to generate breast tumors. Tumor cells invaded into the circulation were tracked by in vivo flow cytometry system. Metastatic tumor cells in the bone were isolated using fluorescent-activated cell sorting technique, followed by assays of cell colony formation, migration and invasion, mammosphere formation in vitro, mammary gland tumorigenesis in vivo, and Next-Generation Sequencing analysis as well. Through tumor regeneration and cell sorting, two bone-metastatic cell sublines were derived from MDA-MB-231 cells; which showed higher abilities to proliferate, migrate, invade and epithelial-to-mesenchymal transit in vitro, and stronger ability to regenerate tumors and metastasize to the bone in vivo. Both cell sublines exhibited cancer stem cell-like characteristics including higher expression levels of stem cell markers and stronger ability for mommaspheres formation. Furthermore, a Normal Distribution-like pattern of the bone-metastatic cells invading into circulation was firstly identified. Deep-sequencing analysis indicated upregulation of multiple signaling pathways in regulating EMT, cell membrane budding and morphologic change, lipid metabolism, and protein translation, which are required to provide adequate metabolic enzymes, structural proteins, and energy for the cells undergoing metastasis. In conclusion, we established two bone-metastatic breast cancer cell sublines, carrying higher degree of stemness and malignancy. The gene signature distinguishing the bone-metastatic breast cancer cells holds therapeutic potentials in prevention of breast cancer metastasis to the bone.

Adipocyte-derived SFRP5 inhibits breast cancer cells migration and invasion through Wnt and epithelial-mesenchymal transition signaling pathways.

ObjectiveObesity is closely associated with metastasis in breast cancer patients. Secreted frizzled-related protein 5 (SFRP5), one of the novel adipokines with anti-inflammatory properties, is associated with obesity. This study aims to study the role of SFRP5 in the crosstalk between obesity and breast cancer metastasis and identify the underlying mechanism.Methods3T3-L1 pre-adipocytes were differentiated to mature adipocytes and a hypertrophic adipocyte model was induced with palmitic acid (PA). Cell motility was measured in MDA-MB-231 and MCF-7 breast cancer cells co-cultured with adipocytes conditioned medium (CM) with or without SFRP5 protein. Wnt and epithelial-mesenchymal transition (EMT) signal pathways were investigated by western blot. Circulating SFRP5 level in 218 breast cancer patients and the association with clinicopathologic characteristics of breast cancer were further determined. Online databases ENCORI and PREDICT Plus were used to exam the link between SFRP5 and prognosis.ResultsReduced SFRP5 level was detected in the hypertrophic adipocyte model. Recombinant SFRP5 protein inhibited MDA-MB-231 and MCF-7 cells invasion and migration induced by PA-treated adipocyte CM, and SFRP5 inhibition by specific antibody reversed the effect of SFRP5. Furthermore, SFRP5 significantly inhibited Wnt and downstream EMT in breast cancer cells. Low circulating SFRP5 level correlated with body mass index (BMI), lymph node (LN) metastasis, TNM stage and high Ki67 expression in breast cancer patients. Increased SFRP5 level was associated with favorable predicted survival. Kaplan-Meier curves showed high SFRP5 level in tumor tissue was associated with better outcome of breast cancer patients.ConclusionsOur findings demonstrated SFRP5 is a vital adipokine that mediates the crosslink between obesity and the metastatic potential of breast cancer. Promotion of SFRP5 expression in the adipose microenvironment may represent a novel approach for preventing breast cancer metastasis.

1, 25-Dihydroxyvitamin D3 Altered Metabolism of MCF10A-ras Cells Decreases Viability in Extracellular Matrix Detached Conditions (FS13-05-19)

ObjectivesMetastasis is the main cause of most breast cancer related deaths. Throughout the process of metastasis, cancer cells strive to survive in conditions where they are detached from the extracellular matrix (ECM). Vitamin D may play a role in preventing breast cancer metastasis by modulating metabolism in breast cancer cells detached from ECM. Here we hypothesize that the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3(1,25D), regulates the metabolism of breast cancer cells, and decreases their viability in ECM detached conditions. MethodsThe Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. Poly-HEMA coated plates were used to model ECM detachment paired with MTT assays to measure cell viability. Viability is measured in cells treated with vehicle (ethanol) or 10 nM 1,25D and maintained in varying nutrient concentrations. mRNA expression of metabolic genes is quantified using qPCR and, triacylglycerol (TAG) is quantified using a Wako Diagnostics colorimetric assay. ResultsResults show that 1,25D treatment decreases viability in detached conditions by 23%. Glutamine deprivation also decreases viability in detached conditions by 40%. In addition, in combination 1,25D treatment and glutamine deprivation results in a 71% decrease in viability. These results imply that while glutamine is required for ECM survival, 1,25D’s modulation of metabolism is targeted elsewhere. Further, when compared to vehicle, 1,25D treated cells have a 45% increased TAG accumulation prior to plating in detached conditions. A 50% decrease was observed in mRNA expression of the enzyme pyruvate carboxylase (PC), and replenishing the product of PC and tricarboxylic acid (TCA) cycle intermediate oxaloacetate rescues detached cell viability in glutamine deprived conditions by 84%. ConclusionsThese results demonstrate the important role energy metabolism plays in cell survival during ECM detachment. Furthermore, our results suggest that 1,25D regulation of metabolism may sensitize breast cancer cells to ECM detachment death. We conclude that cells detached from ECM require flux into the TCA cycle in order to survive, and 1,25D’s downregulation of PC contributes to the observed decrease in viability following 1,25D treatment. Funding SourcesNational Institute of Health and USDA.

IL-4/IL-13 Stimulated Macrophages Enhance Breast Cancer Invasion Via Rho-GTPase Regulation of Synergistic VEGF/CCL-18 Signaling.

Tumor associated macrophages (TAMs) are increasingly recognized as major contributors to the metastatic progression of breast cancer and enriched levels of TAMs often correlate with poor prognosis. Despite our current advances it remains unclear which subset of M2-like macrophages have the highest capacity to enhance the metastatic program and which mechanisms regulate this process. Effective targeting of macrophages that aid cancer progression requires knowledge of the specific mechanisms underlying their pro-metastatic actions, as to avoid the anticipated toxicities from generalized targeting of macrophages. To this end, we set out to understand the relationship between the regulation of tumor secretions by Rho-GTPases, which were previously demonstrated to affect them, macrophage differentiation, and the converse influence of macrophages on cancer cell phenotype. Our data show that IL-4/IL-13 in vitro differentiated M2a macrophages significantly increase migratory and invasive potential of breast cancer cells at a greater rate than M2b or M2c macrophages. Our previous work demonstrated that the Rho-GTPases are potent regulators of macrophage-induced migratory responses; therefore, we examined M2a-mediated responses in RhoA or RhoC knockout breast cancer cell models. We find that both RhoA and RhoC regulate migration and invasion in MDA-MB-231 and SUM-149 cells following stimulation with M2a conditioned media. Secretome analysis of M2a conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Results from our functional assays reveal that M2a TAMs synergistically utilize VEGF and CCL-18 to promote migratory and invasive responses. Lastly, we show that pretreatment with ROCK inhibitors Y-276332 or GSK42986A attenuated VEGF/CCL-18 and M2a-induced migration and invasion. These results support Rho-GTPase signaling regulates downstream responses induced by TAMs, offering a novel approach for the prevention of breast cancer metastasis by anti-RhoA/C therapies.

Targeting DAMP-induced inflammation to prevent breast cancer metastasis and improve anti-tumor immunity

Abstract Biomarkers associated with systemic inflammation correlate with poor prognosis in cancer patients. Circulating cell-free DNA (cfDNA) is released passively and actively by both tumor cells and host cells and cfDNA levels in blood correlate with tumor burden in many tumor types. Notably, studies in breast cancer have shown that inhibition of cfDNA has a significant effect in reducing invasion and migration in vitro and metastatic disease in vivo. We have previously demonstrated that nucleic acid binding polymers (NABPs) bind and inhibit the proinflammatory effects of cfDNA in a murine model of pancreatic cancer. We observed that levels of cfDNA in blood increase with tumor burden in an orthotopic immunocompetent murine model of breast cancer. Moreover, treatment of mice with the NABP, PAMAM-G3, following surgical removal of primary tumor results in decreased lung metastasis. This decrease in lung metastasis is associated with reduction in the levels of cfDNA in blood. In addition, treatment of mice with NABPs in an intravenous experimental metastasis breast cancer model results in reduced seeding of tumor cells in lungs and lung metastasis. Analysis of immune infiltrates in lungs of PAMAM-G3- and control-treated mice demonstrates NABP-mediated transition of immune cell composition in lungs from innate to adaptive immune cells. Collectively, these data suggest that NABPs can be used to target cfDNA and inhibit DAMPs associated pathological inflammation. The use of NABPs is an innovative approach to treat cancer progression and metastasis, particularly in a clinical setting following standard of care surgical tumor resection. This work is funded by the Department of Defense Breast Cancer Research Program award (PI, Smita Nair).

A clinical decision support system learned from data to personalize treatment recommendations towards preventing breast cancer metastasis.

ObjectiveA Clinical Decision Support System (CDSS) that can amass Electronic Health Record (EHR) and other patient data holds promise to provide accurate classification and guide treatment choices. Our objective is to develop the Decision Support System for Making Personalized Assessments and Recommendations Concerning Breast Cancer Patients (DPAC), which is a CDSS learned from data that recommends the optimal treatment decisions based on a patient’s features.MethodWe developed a Bayesian network architecture called Causal Modeling with Internal Layers (CAMIL), and an algorithm called Treatment Feature Interactions (TFI), which learns from data the interactions needed in a CAMIL model. Using the TFI algorithm, we learned interactions for six treatments from the LSDS-5YDM dataset. We created a CAMIL model using these interactions, resulting in a DPAC which recommends treatments towards preventing 5-year breast cancer metastasis.ResultsIn a 5-fold cross-validation analysis, we compared the probability of being metastasis free in 5 years for patients who made decisions recommended by DPAC to those who did not. These probabilities are (the probability for those making the decisions appears first): chemotherapy (.938, .872); breast/chest wall radiation (.939, .902); nodal field radiation (.940, .784); antihormone (.941, .906); HER2 inhibitors (.934, .880); neadjuvant therapy (.931, .837). In an application of DPAC to the independent METABRIC dataset, the probabilities for chemotherapy were (.845, .788).DiscussionPatients who took the advice of DPAC had, as a group, notably better outcomes than those who did not. We conclude that DPAC is effective at amassing and analyzing data towards treatment recommendations. Some of the findings in DPAC are controversial. For example, DPAC says that chemotherapy increases the chances of metastasis for many node negative patients. This controversy shows the importance of developing a conclusive version of DPAC to ensure we provide patients with the best patient-specific treatment recommendations.

Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression.

Curcumae radix is the dry root of Curcuma longa L. (turmeric) that can be used either as a spice or traditional medicine. The aim of this study was to investigate the survival benefits and the anti-metastatic activity of curcumae radix extract (CRE) in MCF7 cells and in MMTV-PyMT transgenic mice—a mouse model of breast cancer metastasis. In vitro wound scratch assay revealed that CRE treatment inhibited cell motility and cell migration in a dose-dependent manner. To investigate the effect of CRE in breast cancer metastasis, MMTV-PyMT transgenic female virgin mice were used and randomly divided into two groups. For survival curve analysis, CRE was administered in a dose of 50 mg/kg to 8–20-week-old mice. Interestingly, CRE treatment significantly increased the median and prolonged survival of MMTV-PyMT mice. Furthermore, CRE treatment decreased tumor burden and inhibited cell proliferation in primary breast tumor, and also suppressed mammary tumor-derived lung metastasis. The size of the lung metastases substantially decreased in the CRE-treated group compared with the ones in the control group. Curcumae radix extract showed anti-metastatic activity through regulating the expression of metastasis markers including C-C Chemokine Receptor Type 7, Matrix Metalloproteinase 9 and the proto-oncogenes c-fos and c-jun. We demonstrated that these metastatic regulators were decreased when CCR7 expression was suppressed in MCF7 cells transfected with CCR7 siRNA. The results of this study show that curcumae radix exerts antitumor and anti-metastatic activities, and we suggest that curcumae radix might be a potential supplement for the treatment and prevention of breast cancer metastasis.

Specific Inhibition of Heparanase by a Glycopolymer with Well-Defined Sulfation Pattern Prevents Breast Cancer Metastasis in Mice.

Heparanase, the heparan sulfate polysaccharide degrading endoglycosidase enzyme, has been correlated with tumor angiogenesis and metastasis and therefore has become a potential target for anticancer drug development. In this systematic study, the sulfation pattern of the pendant disaccharide moiety on synthetic glycopolymers was synthetically manipulated to achieve optimal heparanase inhibition. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor of heparanase (IC50 = 0.10 ± 0.36 nM). This glycopolymer was further examined for cross-bioactivity using a solution-based competitive biolayer interferometry assay with other HS-binding proteins (growth factors, P-selectin, and platelet factor 4), which are responsible for mediating angiogenic activity, cell metastasis, and antibody-induced thrombocytopenia. The synthetic glycopolymer has low affinity for these HS-binding proteins in comparison to natural heparin. In addition, the glycopolymer possessed no proliferative properties toward human umbilical endothelial cells (HUVECs) and a potent antimetastatic effect against 4T1 mammary carcinoma cells. Thus, our study not only establishes a specific inhibitor of heparanase with high affinity but also illustrates the high effectiveness of this multivalent heparanase inhibitor in inhibiting experimental metastasis in vivo.

The pro-metastasis effect of circANKS1B in breast cancer

BackgroundRecent studies indicate that circular RNA (circRNA) plays a pivotal role in cancer progression. Here, we sought to investigate its role in breast cancer.MethodsCircANKS1B (a circRNA originated from exons 5 to 8 of the ANKS1B gene, hsa_circ_0007294) was identified by RNA-sequencing and validated by qRT-PCR and Sanger sequencing. Clinical breast cancer samples were used to evaluate the expression of circANKS1B and its associations with clinicopathological features and prognosis. Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to support clinical findings and elucidate the function and underlying mechanisms of circANKS1B in breast cancer.ResultsCircANKS1B was significantly up-regulated in triple-negative breast cancer (TNBC) compared with non-TNBC tissues and cell lines. Increased circANKS1B expression was closely associated with lymph node metastasis and advanced clinical stage and served as an independent risk factor for overall survival of breast cancer patients. Functional studies revealed that circANKS1B promoted breast cancer invasion and metastasis both in vitro and in vivo by inducing epithelial-to-mesenchymal transition (EMT), while had no effect on breast cancer growth. Mechanistically, circANKS1B abundantly sponged miR-148a-3p and miR-152-3p to increase the expression of transcription factor USF1, which could transcriptionally up-regulate TGF-β1 expression, resulting in activating TGF-β1/Smad signaling to promote EMT. Moreover, we found that circANKS1B biogenesis in breast cancer was promoted by splicing factor ESRP1, whose expression was also regulated by USF1.ConclusionsOur data uncover an essential role of the novel circular RNA circANKS1B in the metastasis of breast cancer, which demonstrate that therapeutic targeting of circANKS1B may better prevent breast cancer metastasis.

Melatonin inhibits breast cancer cell invasion through modulating DJ-1/KLF17/ID-1 signaling pathway.

Breast cancer is the most common neoplastic disorder diagnosed in women. The main goal of this study was to explore the effect of melatonin against breast cancer metastasis and compared this with the actions of taxol (a well-known chemotherapeutic drug), and the impact of their combination against breast cancer metastasis. Melatonin showed no cytotoxic effect while taxol showed antiproliferative and cytotoxic effects on MCF-7 and MDA-MB-231 cells. Furthermore, melatonin inhibited the generation of reactive oxygen species. Melatonin and taxol clearly decreased cell migration and invasion at low doses, especially those matching the normal physiological concentration at night. Melatonin and taxol markedly reduced DJ-1 and ID-1 and increased KLF17 messenger RNA and protein expression levels. The present results also showed that melatonin and taxol induced GSK3-β nuclear and Snail cytosolic localization. These changes were accompanied by a concurrent rise in E-cadherin expression. The above data show that normal levels of melatonin may help in preventing breast cancer metastasis through inhibiting DJ-1/KLF17/ID-1 signaling pathway. The combination of melatonin and taxol is a potent candidate against breast cancer metastasis, better than using melatonin or taxol as a single drug.

Abstract SY38-02: Postpartum tissue remodeling drives breast cancer metastasis

Abstract Women diagnosed with breast cancer within 10 years of a completed pregnancy are at ~2-3 fold increased risk for developing metastatic disease (1,2). This increased risk for metastasis is independent of tumor stage and biologic subtype, implicating a tumor-extrinsic biology unique to the postpartum woman (3,4). Further, 30-50% of all young women diagnosed with breast cancer have had a completed pregnancy within the last 10 years (4), making a postpartum diagnosis an independent poor prognostic indicator for a significant number of patients. One postpartum event that may drive tumor progression in rodents (5-8) and women (9) is weaning-induced mammary gland involution, a physiologic tissue remodeling process that shares numerous attributes with protumorigenic wound healing. We have developed rodent models designed to mimic this highly metastatic subset of young women's breast cancer and find that weaning-induced mammary gland involution supports breast cancer growth, dissemination, and escape to secondary organs in a COX-2 dependent manner (6,10,11). Here we extend these studies by investigating the mammary gland from a mucosal organ perspective, as identifying links with mucosal biology and its associated immune suppression may provide insight into the poor prognosis of postpartum breast cancers. While the mammary gland is not classically considered a mucosal organ, bacteria routinely interface with luminal epithelium during lactation, necessitating barrier function. Similar barrier defenses are anticipated during weaning-induced mammary gland involution, as milk stasis increases risk for mastitis. One key immunologic hallmark of a mucosal organ is the presence of a distinct CD4+ helper T-cell subset, Th17 cells. Th17 cells stimulate epithelial cell junction integrity and epithelial secretion of mucins and defensins, mechanisms that barricade against bacterial activation of antitumor Th1 immune cells. Within the mammary gland we find a baseline mucosal program of Th17 T cells, which is elevated within lactating and involuting mammary glands. Mucosal features are further expanded during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, alternatively activated Th2 CD4+ cells, and immunosuppressive Treg CD4+T cells. Further, during involution, we find suppression of mammary-derived antigen-dependent CD4+ T-cell activation, data consistent with immune tolerance. We also find antigen-independent accumulation of memory Th17- Treg CD4+ T cells, data consistent with weaning-induced mammary inflammation. Overall, these data elucidate strong mucosal immune programs within the lactating gland and immune-suppressive programs within the involuting gland. Tumor cells within the involution immunologic microenvironment are anticipated to avoid immune detection and readily escape the mammary gland, a mechanism that may contribute to increased metastasis observed in postpartum breast cancer patients. Importantly, to successfully metastasize, a tumor cell must not only escape from the primary tumor, but also seed and grow within the secondary site. Thus, an additional potential mechanism by which a postpartum diagnosis may confer increased risk of metastasis is through involution-specific changes at metastatic sites. We report evidence for such a metastatic advantage within the liver of postpartum hosts. Using both intracardiac and portal vein liver metastasis models, we find that the post-weaning liver supports increased seeding of murine mammary tumor cells compared to livers of nulliparous control mice (12). Relevance to women is suggested by data obtained from a cohort of young women's breast cancer patients, where liver metastatic tropism is observed specifically in postpartum patients. In rodents, the biology driving this metastatic advantage is weaning-induced liver involution, a previously unrecognized tissue remodeling process. Post weaning, we find a 50% reduction in liver volume, hepatocyte cell death, deposition of fibrillar collagen and tenascin-c, increased matrix metalloproteinase activity, and influxes of immune cell populations with immune-suppressive phenotypes (12,13). These tissue-level changes are consistent with the establishment of a prometastatic niche during liver involution. Combined, our studies are supportive of the mammary gland being a hormone-responsive, immune-suppressed mucosal organ, and suggest the liver as a hormone-responsive organ that is functionally coordinated with the mammary gland during the cycle of pregnancy, lactation, and weaning. Further, we identify stromal remodeling in the mammary gland and liver as a mediator of breast cancer metastasis in the postpartum window. These findings shed light on how normal reproductive physiology, specifically mucosal biology interfaced with tissue remodeling, alters site-specific metastasis. These findings provide the framework necessary to investigate postpartum involution as a target for the prevention of breast cancer metastasis in young women.

Abstract 1311: Integrating network biology and polypharmacology to reveal TAMS/CXCL-1 as key mediator of XIAOPI formula preventing breast cancer metastasis

Abstract Purpose: Network pharmacology has become a powerful means of understanding the mechanisms underlying the action of Chinese herbs in cancer treatment. This study aims to validate the preventive effects and molecular mechanisms of a clinical prescription XIAOPI formula against breast cancer. Experimental Design: The anti-metastasis ability of XIAOPI was validated by MMTV-PyMT mice and in vitro cell models. A network pharmacology-based method, which combined large-scale text-mining, drug-likeness filtering, target prediction and network analysis, was conducted to predict TAMS/CXCL-1 as key mediator of XIAOPI formula preventing breast cancer metastasis, further supported by cytokine array analysis in vitro and in vivo. Results: In vivo breast cancer xenograft data showed that XIAOPI delayed breast cancer development and efficiently inhibited lung metastasis, accompanied by prolonged survival benefits and decreased cancer stem cell subpopulations. However, similar phenomenon were not observed in a cell model. The herb-ingredient-target network analysis further identified a total of 81 genes closely correlated with the breast cancer chemoprevention effects of XIAOPI. Cytokine array analysis further validated CXCL-1 as the key target of XIAOPI both in vitro and in vivo. Evaluation of the mechanism demonstrated that CXCL-1 administration significantly abrogated the metastatic inhibition effects of XIAOPI on breast cancer migration, invasion, stem cells subpopulations, epithelial-mesenchymal transition(EMT), or mammosphere formation abilities. Conclusions: Overall, our study not only provides experimental evidence and molecular mechanisms that may facilitate the safe and effective use of herbal medicine for the prevention of breast cancer growth or metastasis, and may lead to CXCL-1-based therapeutic strategies for mammary malignancies. Citation Format: Neng Wang, Zhiyu Wang, Yifeng Zheng, Shengqi Wang, Fengxue Zhang, Yi Lin. Integrating network biology and polypharmacology to reveal TAMS/CXCL-1 as key mediator of XIAOPI formula preventing 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 1311.

OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

BackgroundSystemic and chronic inflammatory conditions in patients with breast cancer have been associated with reduced patient survival and increased breast cancer aggressiveness. This paper characterizes the role of an inflammatory cytokine, oncostatin M (OSM), in the preintravasation aspects of breast cancer metastasis.MethodsOSM expression levels in human breast cancer tissue samples were assessed using tissue microarrays, and expression patterns based on clinical stage were assessed. To determine the in vivo role of OSM in breast cancer metastasis to the lung, we used three orthotopic breast cancer mouse models, including a syngeneic 4T1.2 mouse mammary cancer model, the MDA-MB-231 human breast cancer xenograft model, and an OSM-knockout (OSM-KO) mouse model. Progression of metastatic disease was tracked by magnetic resonance imaging and bioluminescence imaging. Endpoint analysis included circulating tumor cell (CTC) counts, lung metastatic burden analysis by qPCR, and ex vivo bioluminescence imaging.ResultsUsing tissue microarrays, we found that tumor cell OSM was expressed at the highest levels in ductal carcinoma in situ. This finding suggests that OSM may function during the earlier steps of breast cancer metastasis. In mice bearing MDA-MB-231-Luc2 xenograft tumors, peritumoral injection of recombinant human OSM not only increased metastases to the lung and decreased survival but also increased CTC numbers. To our knowledge, this is the first time that a gp130 family inflammatory cytokine has been shown to directly affect CTC numbers. Using a 4T1.2 syngeneic mouse model of breast cancer, we found that mice bearing 4T1.2-shOSM tumors with knocked down tumor expression of OSM had reduced CTCs, decreased lung metastatic burden, and increased survival compared with mice bearing control tumors. CTC numbers were further reduced in OSM-KO mice bearing the same tumors, demonstrating the importance of both paracrine- and autocrine-produced OSM in this process. In vitro studies further supported the hypothesis that OSM promotes preintravasation aspects of cancer metastasis, because OSM induced both 4T1.2 tumor cell detachment and migration.ConclusionsCollectively, our findings suggest that OSM plays a crucial role in the early steps of metastatic breast cancer progression, resulting in increased CTCs and lung metastases as well as reduced survival. Therefore, early therapeutic inhibition of OSM in patients with breast cancer may prevent breast cancer metastasis.

Targeting inflammation to prevent breast cancer metastasis

Abstract Biomarkers associated with systemic inflammation correlate with poor prognosis in cancer patients. Circulating cell-free DNA (cfDNA) is released passively and actively by both tumor cells and host cells and cfDNA levels in blood correlate with tumor burden in many tumor types. Notably, studies in breast cancer have shown that inhibition of cfDNA has a significant effect in reducing invasion and migration in vitro. We have previously demonstrated the ability of nucleic acid binding polymers (NABPs) to bind and inhibit the pro-inflammatory effects of cfDNA and RNA in models of systemic lupus erythematosus and influenza. Herein, we demonstrate that in tumor bearing mice, levels of cfDNA increase with tumor burden and decline post NABP in vivo administration. Using an orthotopic breast cancer murine model we demonstrate that NABP treatment, following surgical resection, results in reduced or completely absent lung metastasis. In addition, intravenous delivery of breast cancer cells in the presence of NABPs also results in decreased tumor cell lung seeding and lung metastasis. Taken together, our data suggest that NABPs can be used to target cfDNA and inhibit such DAMPs from activating pathological inflammation, an innovative approach to treat cancer progression and metastasis. In particular, NABPs may be useful in the clinical setting following standard of care surgical tumor resection.

Telocinobufagin inhibits the epithelial-mesenchymal transition of breast cancer cells through the phosphoinositide 3-kinase/protein kinase B/extracellular signal-regulated kinase/Snail signaling pathway.

Telocinobufagin (TBG), an active ingredient of Venenumbufonis, exhibits an immunomodulatory activity. However, its antimetastatic activity in breast cancer remains unknown. The present study investigated whether TBG prevents breast cancer metastasis and evaluated its regulatory mechanism. TBG inhibited the migration and invasion of 4T1 breast cancer cells. Furthermore, TBG triggered the collapse of F-actin filaments in breast cancer. The epithelial-mesenchymal transition (EMT) markers, vimentin and fibronectin, were downregulated following TBG treatment. However, E-cadherin was upregulated following TBG treatment. Snail, a crucial transcriptional factor of EMT, was downregulated following TBG treatment. Signaling pathway markers, including phosphorylated protein kinase B (P-Akt), p-mechanistic target of rapamycin (mTOR) and p-extracellular signal-regulated kinase (ERK), were decreased following TBG treatment. The same results were obtained from in vivo experiments. In conclusion, in vitro and in vivo experiments reveal that TBG inhibited migration, invasion and EMT via the phosphoinositide 3-kinase (PI3K)/Akt/ERK/Snail signaling pathway in breast cancer.

Abstract P2-01-10: Circulating CAF/CTC complexes and breast cancer metastasis

Abstract Background: Metastatic disease in breast cancer (BC) is the leading cause of cancer-related mortality among women worldwide. Synergy between cancer cells and non-cancer cells of the tumor microenvironment (TME) are vital for disease progression. Cancer associated fibroblasts (CAFs) are the major cell type in the stroma of BC and are critical mediators of tumor progression and metastasis. Transport of circulating tumor cells (CTCs) and CTC clusters through the vasculature seeds metastasis and clinical and preclinical studies demonstrate that CTC clusters have a higher metastatic potential than individual CTCs. More recently, circulating cancer stem cells (cCSCs) have been implicated as more metastatic than non-CSC CTCs. In our lab, we have demonstrated that CAFs also circulate (cCAFs). We have observed cCAFs in peripheral blood from breast cancer patients and in murine models of breast cancer. Furthermore, we have observed that cCAFs are present in circulation as both individual cells and as well as in complexes with CTCs. Given the integral role of CAFs in BC metastasis, we hypothesize that cCAFs complex with CTCs/cCSCs to bolster BC metastasis. Methods: cCAF/CTC clusters were identified and enumerated from peripheral blood of patients with BC, and associations with clinical features and disease outcomes were evaluated. Blood was collected by cardiac puncture from PyMT mice from 4 weeks through to the presence of metastases (10 weeks) and cCAF/CTC clusters enumerated. We co-injected CAFs with MCF-7 cellsl into NSG mice, blood collected by cardiac puncture, and cCAF/CTC clusters were enumerated. At time of final sacrifice, tumors were removed and assessed for presence of CSCs. Using our established model of cCAF/CTC clustering in vitro we interrogated cCAF/CTC complexing with both metastatic and poorly metastatic BC cells. Results: Circulating cCAFs/CTCs clusters are significantly increased in the blood of patients with advanced stage BC and associate not only with severity of disease but also with poorer clinical outcomes. In the spontaneous PyMT mouse model, the appearance of circulating cCAF/CTC clusters increased significantly as tumors grew but prior to metastasis. We demonstrate that metastatic BC cells form clusters with CAFs in vitro while non-metastatic BC cells do not form complexes with CAFs in vitro. Enriching for stem cells from MCF7 mammospheres, resulted in CAF/CSC clusters in vitro. In mice that were co-injected with non-metastatic MCF7 cells and CAFs from a TNBC/Basal-like BC (CAF23) we observed disease metastasis, an enrichment for cancer stem cell (CSC)-like CTCs, and the presence of circulating cCAF/MCF7-CSC clusters. Conclusions: Circulating clusters of CTCs and cCAFs are characteristic, and potentially causative, of BC metastasis. Observations of cCAF/CTC clusters from preclinical and clinical samples are corroborated by our determination that the ability of BC cells to form complexes with CAFs in vitro is related to the intrinsic metastatic ability of the breast cancer cells. Both in vitro and in circulation, the BC cells in cCAF/cBC clusters are CSCs, so cCAF/cCSC clusters. Disrupting the formation of cCAF/CTC complexes may be a potential strategy to reduce treat or prevent breast cancer metastasis. Citation Format: Miller P, Sharma U, Medina-Saenz K, Yeasky T, Picon-Ruiz M, Morata-Tarifa C, Seagroves T, Slingerland J, Lippman M, El-Ashry D. Circulating CAF/CTC complexes and breast cancer metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-10.

The role of katanin p60 in breast cancer bone metastasis.

p60 is a subunit of katanin involved in microtubule-severing. Previous studies of p60 were primarily focused on microtubule regulation and cell cycle regulation. More recent research has demonstrated that katanin p60 possesses a function in prostate cancer bone metastasis; however, its role in breast cancer bone metastasis remains unclear. In the present study, immunohistochemistry was used to analyze the expression of katanin p60 in primary and bone metastatic breast cancer. The role of up- and downregulated katanin p60 was investigated using cell proliferation, and migration experiments. Overall, katanin p60 was highly expressed in breast cancer bone metastatic tissue compared with primary tumor tissue. In breast cancer cells, overexpression of katanin p60 inhibited cell proliferation, but promoted cell migration, whereas silencing katanin p60 expression promoted cell proliferation but inhibited cell migration. Overall, the present study indicated that katanin p60 serves a role in cell proliferation and migration, and thus may be a novel therapeutic target for prevention of breast cancer metastasis.