Colonization Of Breast Cancer Cells Research Articles

MLLT6/ATF2 Axis Restrains Breast Cancer Progression by Driving DDIT3/4 Expression.

Epigenetic deregulation is strongly associated with tumor progression. The identification of natural tumor suppressors to overcome cancer metastasis is urgent for cancer therapy. We investigate whether myeloid/lymphoid or mixed-lineage leukemia translocated (MLLT) family members contribute to breast cancer progression and found that high MLLT6 expression predicted a better prognosis and that gradually decreased MLLT6 expression was accompanied by breast cancer malignancy. MLLT6 was downregulated by hypoxia-induced enrichment of DNMT1 at the MLLT6 promoter. The results of in vitro functional experiments indicated that MLLT6 depletion promoted colony formation and cell migration, probably by hampering apoptosis. RNA profiling revealed that the apoptotic pathway was downregulated following stable knockdown of MLLT6. DNA damage-inducible transcript 3/4 (DDIT3/4) were among the top 10 downregulated genes and may have expression patterns similar to that of MLLT6. Restoring DDIT3/4 expression in cells with MLLT6 depletion blocked colony formation and cell migration and attenuated the successful colonization of breast cancer cells in vivo. We also determined that the transcription factor activating transcription factor 2 is a binding partner of MLLT6 and participates in the MLLT6/ATF2 axis, which was reinforced by inhibition of AKT signaling, in turn inducing DDIT3/4 expression by establishing an active chromatin structure at the DDIT3/4 gene promoters. As MLLT6 promotes breast cancer cell apoptosis by inducing DDIT3/4 expression during metastasis, it could be a novel tumor suppressor. Implications: Control of MLLT6 expression via inhibition of PI3K/AKT kinase activity is a potential therapeutic approach for the management of metastatic breast cancer.

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Targeting EMT using low-dose Teniposide by downregulating ZEB2-driven activation of RNA polymerase I in breast cancer

Metastatic dissemination from the primary tumor is a complex process that requires crosstalk between tumor cells and the surrounding milieu and involves the interplay between numerous cellular-signaling programs. Epithelial–mesenchymal transition (EMT) remains at the forefront of orchestrating a shift in numerous cellular programs, such as stemness, drug resistance, and apoptosis that allow for successful metastasis. Till date, there is limited success in therapeutically targeting EMT. Utilizing a high throughput screen of FDA-approved compounds, we uncovered a novel role of the topoisomerase inhibitor, Teniposide, in reversing EMT. Here, we demonstrate Teniposide as a potent modulator of the EMT program, specifically through an IRF7–NMI mediated response. Furthermore, Teniposide significantly reduces the expression of the key EMT transcriptional regulator, Zinc Finger E-Box Binding Homeobox 2 (ZEB2). ZEB2 downregulation by Teniposide inhibited RNA polymerase I (Pol I) activity and rRNA biogenesis. Importantly, Teniposide treatment markedly reduced pulmonary colonization of breast cancer cells. We have uncovered a novel role of Teniposide, which when used at a very low concentration, mitigates mesenchymal-like invasive phenotype. Overall, its ability to target EMT and rRNA biogenesis makes Teniposide a viable candidate to be repurposed as a therapeutic option to restrict breast cancer metastases.

Boosting the Potential of Chemotherapy in Advanced Breast Cancer Lung Metastasis via Micro-Combinatorial Hydrogel Particles.

Breast cancer cell colonization of the lungs is associated with a dismal prognosis as the distributed nature of the disease and poor permeability of the metastatic foci challenge the therapeutic efficacy of small molecules, antibodies, and nanomedicines. Taking advantage of the unique physiology of the pulmonary circulation, here, micro-combinatorial hydrogel particles (µCGP) are realized via soft lithographic techniques to enhance the specific delivery of a cocktail of cytotoxic nanoparticles tometastatic foci. By cross-linking short poly(ethylene glycol) (PEG)chains with erodible linkers within a shape-defining template, a deformable and biodegradable polymeric skeleton is realized and loaded with a variety of therapeutic and imaging agents, including docetaxel-nanoparticles. In a model of advanced breast cancer lung metastasis, µCGP amplified the colocalization of docetaxel-nanoparticles with pulmonary metastatic foci, prolonged the retention of chemotoxic molecules at the diseased site, suppressed lesion growth, and boosted survival beyond 20 weeks post nodule engraftment. The flexible design and modular architecture of µCGP would allow the efficient deployment of complex combination therapies in other vascular districts too, possibly addressing metastatic diseases of different origins.

42 (PB032) - Utilizing Targeted Therapy to Block Triple-negative Breast Cancer Cell Colonization

Triple-negative (estrogen receptor [ER]/progesterone receptor [PR]/human epidermal growth factor receptor 2 [HER2]-negative) breast cancer (TNBC) is a highly aggressive subtype of breast cancer. Approximately 34% of patients will experience distant recurrence within 3 years and eventually develop the chemotherapy-resistant, incurable disease. Therefore, the development of novel treatments is necessary. Accumulating data demonstrate that microRNA (miR)-146a, which is part of the miR-146 family, inhibits cancer cell proliferation, invasion, and metastasis in human cancers, including breast cancer, suggesting that miR-146a functions as a tumor suppressor in tumor cells.

Elevated BCAA Suppresses the Development and Metastasis of Breast Cancer.

Branched-chain amino acids (BCAAs) are the three essential amino acids including leucine, isoleucine, and valine. BCAA metabolism has been linked with the development of a variety of tumors. However, the impact of dietary BCAA intake on breast tumor progression and metastasis remains to be fully explored. Here, we unexpectedly find that the elevated BCAA, either in the genetic model or via increasing dietary intake in mice, suppresses the tumor growth and lung metastasis of breast cancer. The survival analysis shows that BCAA catabolic gene expression is strongly associated with long-term oncological outcomes in patients with breast cancer. In Pp2cm knockout mice in which BCAAs accumulate due to the genetic defect of BCAA catabolism, the breast tumor growth is suppressed. Interestingly, while the cell proliferation and tumor vasculature remain unaffected, more cell death occurs in the tumor in Pp2cm knockout mice, accompanied with increased natural killer (NK) cells. Importantly, increasing BCAA dietary intake suppresses breast tumor growth in mice. On the other hand, there are fewer lung metastases from primary breast tumor in Pp2cm knockout mice and the high BCAA diet-fed mice, suggesting high BCAA also suppresses the lung metastasis of breast cancer. Furthermore, low BCAA diet promotes lung colonization of breast cancer cells in tail vein model. The migration and invasion abilities of breast cancer cells are impaired by high concentration of BCAA in culture medium. The suppressed tumor metastasis and cell migration/invasion abilities by elevated BCAA are accompanied with reduced N-cadherin expression. Together, these data show high BCAA suppresses both tumor growth and metastasis of breast cancer, demonstrating the potential benefits of increasing BCAA dietary intake in the treatment of breast cancer.

Abstract 6123: High-fat diets promote lung metastasis of breast cancer by activating lung fibroblasts

Abstract Metastasis is the main cause of patient death among many tumor types including breast cancer and breast cancer patients with obesity have higher risk of distant metastases during 10-year follow-up. Diet is one of the main and manageable factors that cause obesity and regular intake of high-fat diets can induce obesity. In addition, fibroblasts, as the most abundant stromal cell type in tumor microenvironment, are involved in cancer metastasis. However, the role of fibroblasts and the downstream mechanism in metastasis of patients with high-fat diet remains unclear. In this study, we fed MMTV-PyMT transgenic mice with high-fat diet to obtain a high-fat tumorigenesis model, and isolate primary lung fibroblasts and then coculture them with breast cancer cells to explore the interaction. We found that a high-fat diet can significantly promote progression of primary breast cancer and lung metastasis, and that before weight difference shows, there is a different chemotactic influence of lung fibroblasts on breast cancer cell between high-fat diet and normal diet group. And we observed increased chemotactic function of primary lung fibroblasts in high-fat diet group. Moreover, tumor cells treated with primary lung fibroblasts in high-fat diet group show increased ability of adhesion, suggesting that high-fat diet chemoattracts breast cancer cell colonization by activating lung fibroblasts. Collectively, we explored the functional differences of lung fibroblast and the key factors for the lung colonization of recruited breast cancer cells. Our findings suggest that dietary patterns are associated with the risk of breast cancer metastasis and targeting activated fibroblasts in pre-metastatic microenvironment may serve as potential early therapeutic strategy to reduce breast cancer lung metastasis. Citation Format: Huiping Chen, Penghan Huang, Jianing Chen, Erwei Song. High-fat diets promote lung metastasis of breast cancer by activating lung fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6123.

Myocardin-related transcription factor's interaction with serum-response factor is critical for outgrowth initiation, progression, and metastatic colonization of breast cancer cells.

Breast cancer (BC)‐related mortality primarily results from metastatic colonization of disseminated cells. Actin polymerization plays an important role in driving post‐extravasation metastatic outgrowth of tumor cells. This study examines the role of myocardin‐related transcription factor (MRTF)/serum‐response (SRF), a transcription system well known for regulation of cytoskeletal genes, in metastatic colonization of BC cells. We demonstrated that co‐depletion of MRTF isoforms (MRTF‐A and MRTF‐B) dramatically impairs single‐cell outgrowth ability of BC cells as well as retards growth progression of pre‐established BC cell colonies in three‐dimensional (3D) cultures. Conversely, overexpression of MRTF‐A promotes initiation and progression of tumor‐cell outgrowth in vitro, primary tumor formation, and metastatic outgrowth of seeded BC cells in vivo, and these changes can be dramatically blocked by molecular disruption of MRTF‐A's interaction with SRF. Correlated with the outgrowth phenotypes, we further demonstrate MRTF's ability to augment the intrinsic cellular ability to polymerize actin and formation of F‐actin‐based protrusive structures requiring SRF's interaction. Pharmacological proof‐of‐concept studies show that small molecules capable of interfering with MRTF/SRF signaling robustly suppresses single‐cell outgrowth and progression of pre‐established outgrowth of BC cells in vitro as well as experimental metastatic burden of BC cells in vivo. Based on these data, we conclude that MRTF activity potentiates metastatic colonization of BC cells and therefore, targeting MRTF may be a promising strategy to diminish metastatic burden in BC.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Extracellular Vesicle-Packaged CDH11 and ITGA5 Induce the Premetastatic Niche for Bone Colonization of Breast Cancer Cells.

This study provides mechanistic insights into the generation of an osteogenic premetastatic niche by breast cancer-derived EVs and identifies potential EV-derived diagnostic biomarkers and targets for breast cancer bone metastasis.

Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis

The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20–30% decreased nucleolar abundance per cell and a 30–60% downregulation of RNA Pol I transcription activity, as well as a 50–70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.Fisetin, a natural flavanol, can localize to the nucleolus and decrease rRNA biogenesis. It preferentially targets tumorigenic cells and cells with cancer stem-like properties. In combination with the RNA Pol I inhibitor BMH-21, it synergistically inhibits pulmonary metastasis, making this combination an exciting potential therapeutic strategy.

A novel inhibitor of N6-methyladenosine demethylase FTO induces mRNA methylation and shows anti-cancer activities

N6-methyladenosine (m6A) modification is critical for mRNA splicing, nuclear export, stability and translation. Fat mass and obesity-associated protein (FTO), the first identified m6A demethylase, is critical for cancer progression. Herein, we developed small-molecule inhibitors of FTO by virtual screening, structural optimization, and bioassay. As a result, two FTO inhibitors namely 18077 and 18097 were identified, which can selectively inhibit demethylase activity of FTO. Specifically, 18097 bound to the active site of FTO and then inhibited cell cycle process and migration of cancer cells. In addition, 18097 reprogrammed the epi-transcriptome of breast cancer cells, particularly for genes related to P53 pathway. 18097 increased the abundance of m6A modification of suppressor of cytokine signaling 1 (SOCS1) mRNA, which recruited IGF2BP1 to increase mRNA stability of SOCS1 and subsequently activated the P53 signaling pathway. Further, 18097 suppressed cellular lipogenesis via downregulation of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and C/EBPβ. Animal studies confirmed that 18097 can significantly suppress in vivo growth and lung colonization of breast cancer cells. Collectively, we identified that FTO can work as a potential drug target and the small-molecule inhibitor 18097 can serve as a potential agent against breast cancer.

Abstract 2904: Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone

Abstract Breast cancer is the most commonly diagnosed cancer and frequently metastasizes to bone. Previous evidence has suggested that cancer cells target the osteoblastic lineage cells to establish colonization of bone. It is well established that mechanical loading (e.g. load bearing exercise) elevates bone remodeling via stimulating osteoblastic activities. Whether mechanical loading facilitates the colonization of breast cancer cells in bone is not fully understood. To test this, we have firstly applied a murine tibial mechanical loading model and determined the effects of mechanical loading on bone remodeling in vivo. Six 10-week-old BALB/c nude female mice were subjected to a right tibial dynamic load (9N) onto a 2N pre-load using the ElectroForce 5500 Test Instrument, 40 load cycles with 10 sec intervals, 3 times a week for 1 week. Mice were then euthanized with additional 4 mice euthanized before mechanical loading as baseline controls. Quantitative micro-CT analysis suggested that bone mass related parameters significantly increased in the loaded tibias compared to contra-lateral non-loaded controls, while data in the baseline group showed no differences between left and right tibias. As previously published 2-week loading regimens, one week of loading was enough to induce a 24.3% increase in trabecular bone volume (P=0.001, paired t-test), a 9.0% increase in trabecular thickness (P= 0.01), and a 17.9% increase in cortical bone volume (P<0.0001). Using this model, we tested whether mechanical loading affects the arrival of breast cancer cells in bone. Human and mouse breast cancer cells (MDA-MB-231 and 4T1) were firstly labelled with vital fluorescent lipophilic dyes (Vybrant DiD) to allow tracking of individual tumor cells arriving in bone ex vivo. Cells were injected into the circulation (intracardiac) after mice (BALB/c nude and BALB/c) subjected to 1-week of mechanical loading. Mice were then loaded 3 times more to maintain bone mass before euthanized 7 days post-injection. DiD labelled cancer cells were examined and quantified in tibial bone marrow using multiphoton microscopy ex vivo. In both xenograft and syngeneic models, there were no significant differences found in the number of breast cancer cells arriving in bone marrow (MDA: P=0.32; 4T1: P=0.67) and their relative location to bone surfaces (MDA: P= 0.45; 4T1: P= 0.91) in loaded vs non-loaded bones. There was no significant difference in size of objectives (indicating size of micro tumors) detected in the MDA model (P= 0.21), comparing loaded to non-loaded control tibias but a significant 17.7% reduction in the 4T1 model in the loaded tibias (P=0.02). These pre-clinical data demonstrate that mechanical loading induced osteoblastic bone formation does not affect the initial colonization of breast cancer cells in bone, and suggest that load bearing exercise will not increase the risk of breast cancer bone metastasis in patients. Citation Format: Alexandria R. Sprules, Norain B. Latif, Janet E. Brown, Alison Gartland, Ning Wang. Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2904.

Centchroman prevents metastatic colonization of breast cancer cells and disrupts angiogenesis via inhibition of RAC1/PAK1/β-catenin signaling axis

AimsWe have previously reported that Centchroman (CC), an oral contraceptive drug, inhibits breast cancer progression and metastasis. In this study, we investigated whether CC inhibits local invasion of tumor cells and/or their metastatic colonization with detailed underlying mechanisms. Main methodsThe effect of CC on the experimental metastasis and spontaneous metastasis was demonstrated by using tail-vein and orthotopic 4T1-syngeneic mouse tumor models, respectively. The anti-angiogenic potential of CC was evaluated using well established in vitro and in vivo models. The role of RAC1/PAK1/β-catenin signaling axis in the metastasis was investigated and validated using siRNA-mediated knockdown of PAK1 as well as by pharmacological PAK1-inhibitor. Key findingsThe oral administration of CC significantly suppressed the formation of metastatic lung nodules in the 4T1-syngeneic orthotopic as well as experimental metastatic models. More importantly, CC treatment suppressed the tube formation and migration capacities of human umbilical vein endothelial cells (HUVEC) and inhibited pre-existing vasculature as well as the formation of neovasculature. The suppression of migration and invasion capacities of metastatic breast cancer cells upon CC treatment was associated with the inhibition of small GTPases (Rac1 and Cdc42) concomitant with the downregulation of PAK1 and downstream β-catenin signaling. In addition, CC upregulated the expression of miR-145, which is known to target PAK1. SignificanceThis study warrants the repurposing of CC as a potential therapeutic agent against metastatic breast cancer.

ITGA2 promotes expression of ACLY and CCND1 in enhancing breast cancer stemness and metastasis

Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Here we report that the integrin receptor CD49b-encoding ITGA2, a direct target of miR-206, promotes breast cancer stemness and metastasis. ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. ITGA2 overexpression reversed miR-206-caused cell cycle arrest in G1. RNA sequencing analyses revealed that ITGA2 knockdown inhibits genes related to cell cycle regulation and lipid metabolism, including CCND1 and ACLY as representative targets, respectively. Knockdown of CCND1 or ACLY inhibits mammosphere formation of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. ACLY-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels. CCND1 knockdown further mimics ITGA2 knockdown in abolishing lung colonization of breast cancer cells. We identified that the low levels of miR-206 as well as high expression levels of ITGA2, ACLY and CCND1 are associated with an unfavorable relapse-free survival of the patients with estrogen receptor-negative or high grade breast cancer, especially basal-like or TNBC, possibly serving as potential biomarkers of cancer stemness and therapeutic targets of breast cancer metastasis.

Parathyroid Hormone (PTH) Increases Skeletal Tumour Growth and Alters Tumour Distribution in an In Vivo Model of Breast Cancer.

Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.

Abstract 5168: Understanding the role of integrins in breast cancer

Abstract Breast cancer is a highly prevalent disease and the second-leading cancer related death in women. Mortality rates have declined a 38% in recent years thanks to early detection and development of treatments. However, the five year survival rate of patients with a distant metastasis is only 26%. Hence, it is important to identify key players that mediate or promote the migration of cells to other organs. Integrins are cell surface receptors that mediate cell adhesion to the extracellular matrix. They are involved in roles such as cell migration and proliferation. In this project we seek to understand the role of integrin α2 in breast cancer metastasis. It has been previously reported that integrin α2 is a metastasis suppressor of a Her2+ model of breast cancer. However, its role in triple negative breast cancer is unknown. We found the integrin α2 gene (ITGA2) a target of has-miR-206 which controls metastasis and stemness of breast cancer. We hypothesize integrin α2 has a metastasis regulating role in triple negative breast cancer. The triple negative breast cancer cell line, MDA-MB-231 has been used to characterize the role of integrin α2 in migration, cell viability and lung colonization of mice injected though tail vein injection. Lung colonization of breast cancer cells in NSG mice is significantly altered by the knockdown expression of integrin α2. Consistent with the phenotypes in migration and invasion. Taken together, these results suggest integrin α2 regulates breast cancer metastasis through modified cell migration and lung colonization. Citation Format: Valery Adorno-Cruz, Xia Liu, Huiping Liu. Understanding the role of integrins in breast cancer [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 5168.

Abstract 29: RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation

Abstract Denosumab is a fully human monoclonal antibody to human RANKL, currently approved in the management of established bone metastases, mainly due to its anti-resorptive activity which blocks tumor-induced osteoclastogenesis and osteolysis. Our aims are to determine the role of RANK expression in breast cancer (BC) bone metastasis and the efficacy of RANKL blocking by AMG161 (IgG1 equivalent to Denosumab). AMG161 does not recognize murine RANKL, so we use a BalB/c nude knock-in (KI) mouse model, which expresses a chimeric murine/human RANKL. Moreover, non-invasive in vivo imaging techniques such as bioluminescence and µCT were used to monitor metastatic tumor burden and bone degradation, respectively. To evaluate bone marrow early colonization by BC cells, DiD labelling and ex vivo laser scanning multiphoton confocal microscopy were used. Long-term studies showed that intra-arterial (i.a.) injection of MDA-MB231 RANK versus MDA-MB231 leads to a poorer overall survival (OS), with the most striking differences seen in the earlier time-points. In this respect, the extent of radiographic osteolytic lesions was higher in the RANK overexpressing group than in the control group. Similar findings were observed following intra-tibial inoculation of RANK-expressing MCF-7 cells, when compared to that observed with parental MCF-7 cells. However, at later time points, bone and lung metastatic burden was similar between the two groups, suggesting that the poor OS of the RANK overexpressing group was due to a faster tumor progression owing to enhanced bone homing and colonization. Indeed, using short-term studies (5 and 14 days), i.a. injection of MDA-MB231 RANK led to higher incidence and extension of bone marrow micro-metastases than MDA-MB231, whereas there was no difference for lung micro-metastases. Daily subcutaneous injections of 1,5mg/kg AMG161 antibody to MDA-MB231RANK tumor-bearing animals, decreased bone micro-metastases and early bone marrow colonization without affecting lung micro-metastasis. We therefore hypothesized that RANK overexpressing BC cells interact with RANKL produced by osteocytes, osteoblasts and other cells in the osteogenic niche, favoring survival and colonization of BC cells in bone. To test this hypothesis, we made use of heterotypic mammosphere assays in which BC cells were cultured with or without cells of the osteogenic niche (e.g. pre-osteoblasts, mesenchymal stem cells), recombinant RANKL and anti-RANK antibody or AMG161. Co-culture with recombinant RANKL or a cellular source of RANKL increased BC cell mammosphere formation, and blocking RANK/RANKL signaling impaired it. In conclusion, RANK/RANKL signaling promotes the early engraftment of BC cells in the bone marrow. Blocking this signaling with AMG161 decreases bone marrow micro-metastasis formation in vivo, suggesting that adjuvant treatment of early-stage breast cancer patients with denosumab may prevent bone relapse. Citation Format: Sofia Sousa, Evelyne Gineyts, Sandra Geraci, Martine Croset, Philippe Clézardin. RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation [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 29.

Myeloid-derived suppressor cell and macrophage exert distinct angiogenic and immunosuppressive effects in breast cancer.

The immunosuppressive tumor microenvironment is a key obstacle to hinder a cancer immunotherapy. Myeloid-derived suppressor cells (MDSCs) have been considered as a major player in immunosuppression. In this study, we find that tumor-infiltrating MDSCs (tiMDSCs) are less immunosuppressive than tumor-associated macrophages (TAMs) in multiple murine orthotopic breast tumor models. Compared to TAMs, tiMDSCs produce higher levels of pro-inflammatory factors and lower levels of anti-inflammatory factors. Furthermore, tiMDSCs are preferentially located in hypoxic areas and are more pro-angiogenic than TAMs. Consistent with these functional disparities, a shift from tiMDSCs to TAMs is observed during the progression of breast cancer. Moreover, infiltration of tiMDSCs is also noted in distal colonization of breast cancer cells in the lung. Taken together, our findings indicate that tiMDSCs are more pro-angiogenic and promote tumor initiation, while TAMs are more immunosuppressive and facilitate tumor immune evasion. This study suggests that selectively targeting on TAMs could alleviate the immunosuppressive tumor microenvironment and potentiate cancer immunotherapy.

TMEM2 Is a SOX4-Regulated Gene That Mediates Metastatic Migration and Invasion in Breast Cancer.

The developmental transcription factor SOX4 contributes to the metastatic spread of multiple solid cancer types, but its direct target genes that mediate cancer progression are not well defined. Using a systematic molecular and genomic approach, we identified the TMEM2 transmembrane protein gene as a direct transcriptional target of SOX4. TMEM2 was transcriptionally activated by SOX4 in breast cancer cells where, like SOX4, TMEM2 was found to mediate proinvasive and promigratory effects. Similarly, TMEM2 was sufficient to promote metastatic colonization of breast cancer cells and its expression in primary breast tumors associated with a higher likelihood of metastatic relapse. Given earlier evidence that genetic inactivation of SOX4 or TMEM2 yield similar defects in cardiac development, our findings lead us to propose that TMEM2 may not only mediate the pathologic effects of SOX4 on cancer progression but also potentially its contributions to embryonic development. Cancer Res; 76(17); 4994-5005. ©2016 AACR.