Breast Cancer Bone Metastasis Research Articles

Construction and Validation of New Nomograms to Predict Risk and Prognostic Factors of Breast Cancer Bone Metastasis in Asian Females: A Population-Based Retrospective Study.

PurposeTo construct a breast cancer bone-only metastasis (BCBM) risk and prognostic model for Asian females and provide a reference for treatment selection in breast cancer (BC) patients with bone-only metastasis (BM).Patients and MethodsThe data for newly diagnosed female patients of Asian Pacific Islander (API) ethnicity between 2010 and 2018 were obtained from the Surveillance, Epidemiology, and End Results database. A total of 16,972 patients were identified. Logistic regression analyses were used to establish a risk model for BCBM. Cox proportional hazards regression analyses were used to construct nomograms for the prognosis of BC and BCBM. Subsequently, the degree of discrimination of the nomogram was evaluated using the consistency index (C-index) and receiver operating curve.ResultsThe main independent risk factors of BM in Asian females with BC were primary site surgery (p<0.0001), ER (p=0.0015), and T-stage (p=0.0046). The C-index values in the training and validation cohorts were 0.933 and 0.941, respectively. The main independent risk factors of the prognosis of BC were age (p<0.001), summary stage (p<0.001), and grade (p=0.002). The C-index values of 5-year overall survival (OS) in the training and validation cohorts were 0.823 and 0.804, respectively. The risk factors of the prognosis of Asian females with BCBM were subtype (p<0.001), histology (p<0.001), and grade (p=0.033). The C-index values of 5-year OS in the training and validation cohorts were 0.793 and 0.723, respectively.ConclusionUsing population-based analysis, this study constructed a prediction model for the risk and prognosis of BM in Asian females with BC. Another newly constructed model was effective in predicting OS in BCBM patients. These models can help prevent skeletal-related events and weigh the risks and benefits of surgery for metastatic lesions in BCBM patients.

Regulatory roles of miRNAs 16, 133a, and 223 on osteoclastic bone destruction caused by breast cancer metastasis.

Osteolytic bone metastasis leads to skeletal-related events, resulting in a decline in the patient activities and survival; therefore, it is important to understand the mechanism underlying bone metastasis. Recent studies have suggested that microRNAs (miRNAs or miRs) are involved in osteoclast differentiation and/or osteolytic bone metastasis; however, the roles of miRNAs have not been elucidated. In the present study, the roles of miRNAs in bone destruction caused by breast cancer metastasis were investigated in vitro and in vivo. miR-16, miR-133a and miR-223 were transfected into a human breast cancer cell line, MDA-MB-231. The expression of osteolytic factors in conditioned medium (miR-CM) collected from the culture of transfected cells was assessed. To evaluate the effects of miRNAs on osteoclast differentiation and activities, tartrate-resistant acid phosphatase (TRAP) staining and bone resorptive assays were performed in osteoclasts following miR-CM treatment. To create in vivo bone metastasis models for histological and morphometric evaluation, miRNA-transfected MDA-MB-231 cells were transplanted into the proximal tibia of nude mice. Expression of osteolytic factors, including receptor activator for nuclear factor-κB ligand (RANKL), interleukin (IL)-1β, IL-6, parathyroid hormone-related protein (PTHrP), and tumor necrosis factor (TNF), was increased in miR-16-CM, whereas it was decreased in both miR-133a-CM and miR-223-CM. TRAP staining and bone resorptive assays revealed that osteoclast function and activities were promoted by miR-16-CM treatment, whereas they were suppressed by miR-133a-CM and miR-223-CM. Consistent with in vitro findings, in vivo experiments revealed that the overexpression of miR-16 increased osteoclast activities and bone destruction in MDA-MB-231 cells, whereas the opposite results were observed in both miR-133a- and miR-223-transfected MDA-MB-231 cells. Our results indicated that miR-16 promoted osteoclast activities and bone destruction caused by breast cancer metastasis in the bone microenvironment, whereas miR-133a and miR-223 suppressed them. These miRNAs could be potential biomarkers and therapeutic targets for breast cancer bone metastasis.

Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation.

From a mouse triple‐negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G‐protein–coupled receptor (ADGR) family, G‐protein–coupled receptor 56 (GPR56)/adhesion G‐protein–coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1‐transduced 4T1, TS/A (mouse breast cancer cell line), or MDA‐MB‐231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1‐induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

Immunization With RANKL Inhibits Osteolytic Bone Metastasis in Breast Cancer.

Breast cancer cells often metastasize to bone. Accumulating evidence suggests that inhibiting the receptor activator of nuclear factor-κB ligand (RANKL) not only leads to reduced bone metastasis of breast cancer but also has antitumoral effects. Here, we used mutant receptor activator of nuclear factor-κB ligand (RANKLM) as a vaccine for active immunization to induce antibodies for immunotherapy of bone metastatic cancer. We investigated whether anti-RANKL antibodies inhibit osteolytic bone metastasis in vitro and in a murine model. MC3T3 cells stimulated by MDA-MB-231 culture medium secreted growth differentiation factor-15 (GDF-15), which induced the nuclear factor-κB signaling cascade. In addition, RANKLM treatment-induced reduction of intraosseous growth of MDA-MB-231 cells correlated with decreased GDF-15 expression, a reduced number of osteolytic lesions, and slower tumor progression. In addition, vaccination with RANKLM led to significant improvement in overall survival and skeletal metastasis in tumor-bearing mice. Induction of anti-RANKL antibodies by RANKLM decreased GDF-15 production by deactivating nuclear factor-κB signaling, which in turn inhibited metastasis of MDA-MB-231 cells to bone. Taken together, the results demonstrate a role for RANKLM immunization in preventing bone metastasis of breast cancer.

Clinical Features and Serological Markers Risk Model Predicts Overall Survival in Patients Undergoing Breast Cancer and Bone Metastasis Surgeries.

BackgroundSurgical therapy of breast cancer and bone metastasis can effectively improve the prognosis of breast cancer. However, after the first operation, the relationship between preoperative indicators and outcomes in patients who underwent metastatic bone surgery remained to be studied. Purpose 1. Recognize clinical and laboratory prognosis factors available to clinical doctors before the operation for bone metastatic breast cancer patients. 2. Develop a risk prediction model for 3-year postoperative survival in patients with breast cancer bone metastasis.MethodsFrom 2014 to 2020, patients who suffered from breast cancer bone metastasis and received therapeutic procedures in our institution were included for analyses (n=145). For patients who underwent both breast cancer radical surgery and bone metastasis surgery, comprehensive datasets of the parameters of interest (clinical features, laboratory factors, and patient prognoses) were collected (n=69). We performed Multivariate Cox regression to identify factors that were associated with postoperative outcome. 3-year survival prediction model and nomograms were established by 100 bootstrapping. Its benefit was evaluated by calibration plot, C-index, and decision curve analysis. The Surveillance, Epidemiology, and End Results database was also used for external validation.ResultsRadiotherapy for primary cancer, pathological type of metastatic breast cancer, lymph node metastasis, elevated serum alkaline phosphatase, lactate dehydrogenase were associated with postoperative prognosis. Pathological types of metastatic breast cancer, multiple bone metastasis, organ metastases, and elevated serum lactate dehydrogenase were associated with 3-year survival. Then those significant variables and serum alkaline phosphatase counts were integrated to construct nomograms for 3-year survival. The C-statistic of the established predictive model was 0.83. The calibration plot presents a graphical representation of calibration. In the decision curve analysis, the benefits are higher than those of the extreme curve. The receiver operating characteristic of the external validation of the model was 0.82, indicating a favored fitting degree of the two models.ConclusionOur study suggests that several clinical features and serological markers can predict the overall survival among the patients who are about to receive bone metastasis surgery after breast cancer surgery. The model can guide the preoperative evaluation and clinical decision-making for patients. Level of evidence Level III, prognostic study.

Embracing Mechanobiology in Next Generation Organ-On-A-Chip Models of Bone Metastasis.

Bone metastasis in breast cancer is associated with high mortality. Biomechanical cues presented by the extracellular matrix play a vital role in driving cancer metastasis. The lack of in vitro models that recapitulate the mechanical aspects of the in vivo microenvironment hinders the development of novel targeted therapies. Organ-on-a-chip (OOAC) platforms have recently emerged as a new generation of in vitro models that can mimic cell-cell interactions, enable control over fluid flow and allow the introduction of mechanical cues. Biomaterials used within OOAC platforms can determine the physical microenvironment that cells reside in and affect their behavior, adhesion, and localization. Refining the design of OOAC platforms to recreate microenvironmental regulation of metastasis and probe cell-matrix interactions will advance our understanding of breast cancer metastasis and support the development of next-generation metastasis-on-a-chip platforms. In this mini-review, we discuss the role of mechanobiology on the behavior of breast cancer and bone-residing cells, summarize the current capabilities of OOAC platforms for modeling breast cancer metastasis to bone, and highlight design opportunities offered by the incorporation of mechanobiological cues in these platforms.

Role of miRNAs in Breast Cancer-induced Bone Disease

AbstractBone is the most common site of breast cancer recurrence. Despite the increasing knowledge about the metastatic process and treatment advances, the disease still remains incurable once the cancer cells actively proliferate in bone. Complex interactions between cancer cells and cells of the bone microenvironment (BME) regulate the initiation and progression of metastatic tumor growth in bone. In particular, breast cancer cells shift the otherwise tightly balanced bone remodeling towards increased bone resorption by osteoclasts. Cellular interactions in the metastatic BME are to a large extent regulated by secreted molecules. These include various cytokines as well as microRNAs (miRNAs), small non-coding RNAs that post transcriptionally regulate protein abundance in several cell types. Through this mechanism, miRNAs modulate physiological and pathological processes including bone remodeling, tumorigenesis and metastasis. Consequently, miRNAs have been identified as important regulators of cellular communication in the metastatic BME. Disruption of the crosstalk between cancer cells and the BME has emerged as a promising therapeutic target to prevent the establishment and progression of breast cancer bone metastasis. In this context, miRNA mimics or antagonists present innovative therapeutic approaches of high potential for interfering with pathological bone – cancer cell interactions. This review will discuss the role of miRNAs in the tumor-BME crosstalk in vivo and will emphasize how this could be targeted by miRNAs to improve therapeutic outcome for patients with breast cancer bone metastases.

ATP Inhibits Breast Cancer Migration and Bone Metastasis through Down-Regulation of CXCR4 and Purinergic Receptor P2Y11.

Simple SummaryThe skeleton is the most frequent metastatic site for advanced breast cancer, and complications resulting from breast cancer metastasis are a leading cause of death in patients. Therefore, the discovery of new targets for the treatment of breast cancer bone metastasis is of great significance. ATP released by bone osteocytes is shown to activate purinergic signaling and inhibit the metastasis of breast cancer cells in the bone. The aim of our study was to unveil the underlying molecular mechanism of ATP and purinergic signaling in inhibiting the bone metastasis of breast cancer cells. We demonstrated that CXCR4 and P2Y11 are key factors in regulating this process, and understanding of this important mechanism will aid in identifying new targets and developing first-in-class therapeutics.ATP released by bone osteocytes is shown to activate purinergic signaling and inhibit the metastasis of breast cancer cells into the bone. However, the underlying molecular mechanism is not well understood. Here, we demonstrate the important roles of the CXCR4 and P2Y11 purinergic receptors in mediating the inhibitory effect of ATP on breast cancer cell migration and bone metastasis. Wound-healing and transwell migration assays showed that non-hydrolysable ATP analogue, ATPγS, inhibited migration of bone-tropic human breast cancer cells in a dose-dependent manner. BzATP, an agonist for P2X7 and an inducer for P2Y11 internalization, had a similar dose-dependent inhibition on cell migration. Both ATPγS and BzATP suppressed the expression of CXCR4, a chemokine receptor known to promote breast cancer bone metastasis, and knocking down CXCR4 expression by siRNA attenuated the inhibitory effect of ATPγS on cancer cell migration. While a P2X7 antagonist A804598 had no effect on the impact of ATPγS on cell migration, antagonizing P2Y11 by NF157 ablated the effect of ATPγS. Moreover, the reduction in P2Y11 expression by siRNA decreased cancer cell migration and abolished the impact of ATPγS on cell migration and CXCR4 expression. Similar to the effect of ATPγS on cell migration, antagonizing P2Y11 inhibited bone-tropic breast cancer cell migration in a dose-dependent manner. An in vivo study using an intratibial bone metastatic model showed that ATPγS inhibited breast cancer growth in the bone. Taken together, these results suggest that ATP inhibits bone-tropic breast cancer cells by down-regulating the P2Y11 purinergic receptor and the down-regulation of CXCR4 expression.

Association between Bone Mineral Density and Bone Turnover Markers in Breast Cancer Patients and Bone-Only Metastasis.

Background and Objectives: The aim of this study was to determine the influence of bone turnover markers, namely the N-terminal cross-linking telopeptide (NTx) and alpha C-terminal cross-linking telopeptide of type I collagen (α-CTx), in detecting bone metastasis (bone-only) in breast cancer (BC) patients, as well as to determine whether this effect is related to changes in bone mineral density (BMD). Materials and Methods: The participants in this study comprised 30 postmenopausal BC patients with bone metastases (age range: 59.56 ± 9.02), 20 postmenopausal BC patients without bone metastases (age range: 55.30 ± 11.55), and 20 healthy postmenopausal female controls (age range: 55.55 ± 5.85). Bone turnover markers (serum NTx and urine α-CTx) were measured using the ELISA method. A densitometer using dual-energy X-ray absorptiometry (DEXA) was used to analyze the BMD, and tumor markers were measured using the chemiluminescent immunometric assay. Results: The corresponding levels of serum NTx (p = 0.004), parathyroid hormone (PTH) (p = 0.001), and urine α-CTx (p < 0.001) of BC patients were found to be higher than the standard levels. After the BC patients were divided into subgroups on the basis of the presence of metastasis, the urine α-CTx levels (p = 0.001) were seen to be at critically high levels in those patients suffering from BC with metastasis. Though the BMD values in the lumbar spine (p < 0.001) and femoral neck (p = 0.001) were found to be significantly low in BC patients, no statistically substantial difference in the BMD levels of BC patients suffering from metastasis was observed. It was observed that urine α-CTx (specificity: 70%; sensitivity: 85%) values are critical factors that differentiate BC patients with metastasis from BC patients without metastasis. Conclusions: We found that alterations in bone turnover could be detected by using the values of urine α-CTx while differentiating BC patients with metastasis from BC patients without metastasis. Using the biochemical markers of bone turnover and BMD together would be pertinent for determining the level of metastasis present and examining the efficiency of bone density preservation therapy. Ideally, BMD measurement would be evaluated together with biochemical markers.

Fam20C Regulates Bone Resorption and Breast Cancer Bone Metastasis through Osteopontin and BMP4.

Fam20C is a kinase that generates the majority of secreted phosphoproteins and regulates biomineralization. However, its potential roles in bone resorption and breast cancer bone metastasis are unknown. Here we show that Fam20C in the myeloid lineage suppresses osteoclastogenesis and bone resorption, during which, osteopontin (OPN) is the most abundant phosphoprotein secreted in a Fam20C-dependent manner. OPN phosphorylation by Fam20C decreased OPN secretion, and OPN neutralization reduced Fam20C deficiency-induced osteoclast differentiation and bone metastasis. In contrast, Fam20C in breast cancer cells promoted bone metastasis by facilitating the phosphorylation and secretion of BMP4, which in turn enhanced osteoclastogenesis. Mutation of the BMP4 phosphorylation site elevated BMP4 lysosomal degradation and reduced BMP4 secretion. In breast cancer cells, BMP4 depletion or treatment with a BMP4 signaling inhibitor diminished osteoclast differentiation and bone metastasis and abolished Fam20C-mediated regulation of these processes. Collectively, this study discovers distinct roles for Fam20C in myeloid cells and breast cancer cells and highlights OPN and BMP4 as potential therapeutic targets for breast cancer bone metastasis. SIGNIFICANCE: Osteoclastogenesis and bone metastasis are suppressed by myeloid-derived Fam20C, but enhanced by breast cancer-associated Fam20C, uncovering novel Fam20C functions and new therapeutic strategies via targeting Fam20C substrates OPN and BMP4.

Circular RNA circIKBKB promotes breast cancer bone metastasis through sustaining NF-\u03baB/bone remodeling factors signaling

BackgroundBreast cancer (BC) has a marked tendency to spread to the bone, resulting in significant skeletal complications and mortality. Recently, circular RNAs (circRNAs) have been reported to contribute to cancer initiation and progression. However, the function and mechanism of circRNAs in BC bone metastasis (BC-BM) remain largely unknown.MethodsBone-metastatic circRNAs were screened using circRNAs deep sequencing and validated using in situ hybridization in BC tissues with or without bone metastasis. The role of circIKBKB in inducing bone pre-metastatic niche formation and bone metastasis was determined using osteoclastogenesis, immunofluorescence and bone resorption pit assays. The mechanism underlying circIKBKB-mediated activation of NF-κB/bone remodeling factors signaling and EIF4A3-induced circIKBKB were investigated using RNA pull-down, luciferase reporter, chromatin isolation by RNA purification and enzyme-linked immunosorbent assays.ResultsWe identified that a novel circRNA, circIKBKB, was upregulated significantly in bone-metastatic BC tissues. Overexpressing circIKBKB enhanced the capability of BC cells to induce formation of bone pre-metastatic niche dramatically by promoting osteoclastogenesis in vivo and in vitro. Mechanically, circIKBKB activated NF-κB pathway via promoting IKKβ-mediated IκBα phosphorylation, inhibiting IκBα feedback loop and facilitating NF-κB to the promoters of multiple bone remodeling factors. Moreover, EIF4A3, acted acting as a pre-mRNA splicing factor, promoted cyclization of circIKBKB by directly binding to the circIKBKB flanking region. Importantly, treatment with inhibitor eIF4A3-IN-2 reduced circIKBKB expression and inhibited breast cancer bone metastasis effectively.ConclusionWe revealed a plausible mechanism for circIKBKB-mediated NF-κB hyperactivation in bone-metastatic BC, which might represent a potential strategy to treat breast cancer bone metastasis.

IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

Breast cancer bone metastasis is currently incurable, ~75% of patients with late-stage breast cancer develop disease recurrence in bone and available treatments are only palliative. We have previously shown that production of the pro-inflammatory cytokine interleukin-1B (IL-1B) by breast cancer cells drives bone metastasis in patients and in preclinical in vivo models. In the current study, we have investigated how IL-1B from tumour cells and the microenvironment interact to affect primary tumour growth and bone metastasis through regulation of the immune system, and whether targeting IL-1 driven changes to the immune response improves standard of care therapy for breast cancer bone metastasis. Using syngeneic IL-1B/IL1R1 knock out mouse models in combination with genetic manipulation of tumour cells to overexpress IL-1B/IL1R1, we found that IL-1B signalling elicited an opposite response in primary tumours compared with bone metastases. In primary tumours, IL-1B inhibited growth, by impairing the infiltration of innate immune cell subsets with potential anti-cancer functions but promoted enhanced tumour cell migration. In bone, IL-1B stimulated the development of osteolytic metastases. In syngeneic models of breast cancer, combining standard of care treatments (Doxorubicin and Zoledronic acid) with the IL-1 receptor antagonist Anakinra inhibited both primary tumour growth and metastasis. Anakinra had opposite effects on the immune response compared to standard of care treatment, and its anti-inflammatory signature was maintained in the combination therapy. These data suggest that targeting IL-1B signalling may provide a useful therapeutic approach to inhibit bone metastasis and improve efficacy of current treatments for breast cancer patients.

New insight into long non-coding RNAs associated with bone metastasis of breast cancer based on an integrated analysis

BackgroundBone is the most common site of metastatic breast cancer, and it is a leading cause of breast cancer-related death. This study aimed to explore bone metastasis-related long non-coding RNAs (lncRNAs) in breast cancer.MethodsFour mRNA datasets and two lncRNA datasets of bone metastasis, lung metastasis and liver metastasis of breast cancer were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) in group of bone metastasis vs lung metastasis and bone metastasis vs liver metastasis, as well as the overlap of the two groups, were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein–protein interaction (PPI) network construction of DEmRNAs were conducted. The cis nearby-targeted DEmRNAs of DElncRNAs were obtained. Quantitative real-time polymerase chain reactions (qRT-PCR) was used to detect the expression levels of selected DEmRNAs and DElncRNAs. LOC641518-lymphoid enhancer-binding factor 1 (LEF1) pair was selected to verify its role in migration and invasion capability of breast cancer cells by wounding healing assay and transwell invasion assay.ResultsA total of 237 DEmRNAs were obtained in bone metastasis compared with both lung metastasis and liver metastasis. A total of three DElncRNAs in bone metastasis compared with both lung metastasis and liver metastasis were obtained. A total of seven DElncRNA-nearby-targeted DEmRNA pairs and 15 DElncRNA-nearby-targeted DEmRNA pairs in group of bone metastasis vs lung metastasis and bone metastasis vs liver metastasis, were detected, respectively. Four cis LncRNA-mRNA interaction pairs were identified, which are LOC641518-LEF1, FLJ35024-Very Low Density Lipoprotein Receptor (VLDLR), LOC285972-Retinoic Acid Receptor Responder 2 (RARRES2) and LOC254896-TNF receptor superfamily member 10c (TNFRSF10C). qRT-PCR using clinical samples from our hospital confirms the bioinformatics prediction. siRNA knocking down LOC641518 down-regulates LEF1 mRNA expression, and reduces the migration and invasion capability of breast cancer cells.ConclusionsWe concluded that four LncRNA-mRNA pairs, including LOC641518-LEF1, may play a central role in breast cancer bone metastasis.

Overexpression of Lrp5 enhanced the anti-breast cancer effects of osteocytes in bone

Osteocytes are the most abundant cells in bone, which is a frequent site of breast cancer metastasis. Here, we focused on Wnt signaling and evaluated tumor-osteocyte interactions. In animal experiments, mammary tumor cells were inoculated into the mammary fat pad and tibia. The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model. The results revealed that administration of osteocytes or their conditioned medium (CM) inhibited tumor progression and osteolysis. Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity, accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53. The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound. Notably, silencing Lrp5 in tumors inhibited tumor progression, while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression. Osteocytes exhibited elevated Lrp5 expression in response to tumor cells, implying that osteocytes protect bone through canonical Wnt signaling. Thus, our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes. We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.

Bioinformatics analysis for the identification of key genes and long non-coding RNAs related to bone metastasis in breast cancer.

The molecular mechanism of bone metastasis in breast cancer is largely unknown. Herein, we aimed to identify the key genes and long non-coding RNAs (lncRNAs) related to the bone metastasis of breast cancer using a bioinformatics approach. We screened differentially expressed genes and lncRNAs between normal breast and breast cancer bone metastasis samples using the GSE66206 dataset from the Gene Expression Omnibus. We also constructed a differentially expressed lncRNA-mRNA interaction network and analyzed the node degrees to identify the driving genes. After finding potential pathogenic modules of breast cancer bone metastasis, we identified breast cancer bone metastasis-related modules and functional enrichment analysis of the genes and lncRNAs in the modules. Based on the above analysis, we constructed a differentially expressed lncRNA-mRNA network related to bone metastasis in breast cancer and identified core driver genes, including BNIP3 and the lncRNA RP11-317-J19.1. The role of core driver genes and lncRNAs in the network implies their biological functions in regulating bone development and remodeling. Thus, targeting the core driver genes and lncRNAs in the network may be a promising therapeutic strategy to manage bone metastasis.

Abstract 3154: Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis

Abstract Prostate and breast cancers are the most prevalent and the second leading causes of cancer-deaths for men and women in the United States. Cancer survival rates generally worsen significantly with advanced and disseminated disease with nearly 90% of all cancer deaths resulting from metastasis. For breast and prostate cancers, bone is the most common site of metastasis. A key signaling pathway involved in metastatic dissemination of tumor cells is the receptor tyrosine kinase Axl. Axl belongs to the Tyro3, Axl, and MerTK (TAM) subfamily, is expressed in a variety of tumor types, including breast and prostate, and is associated with poor prognosis, metastasis, and outcome. The goal of the present study was to characterize the role of Axl signaling in prostate and breast cancer cell dissemination to bone. This was accomplished using Axl knockdown neoplastic cells, a selective small molecule Axl inhibitor (BGB324), and osteoclast progenitor cells in vitro, as well as intracardiac injection of Axl knockdown tumor cells in vivo. The results showed that Axl inhibition significantly decreases tumor cell migration and invasion in vitro, and suppression of Axl signaling in osteoclast precursor cells reduces the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation of neoplastic bone lesions. Taken together our findings indicate that therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis [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 3154.

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&amp;lt;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.

Correlation between ER, PR, HER-2, and Ki-67 with the risk of bone metastases detected by bone scintigraphy in breast cancer patients: A cross sectional study

BackgroundBreast cancer is one of the most common cancers in women. About 30%–85% of breast cancers will metastasize to the bone during the course of the illness. Many studies have shown that molecular marker/subtypes can be useful in determining incidence of different and inconsistent bone metastases. This study aimed to determine the correlation of the risk of bone metastases in breast cancer based on the expression of molecular markers. MethodsThe research was conducted retrospectively by searching patients' medical record data. The target population of this study was all patients diagnosed with breast cancer who came to our tertiary hospital in the Nuclear Medicine and Molecular Imaging Department from January 2012 to December 2016. ResultsOne hundred and thirty patients (n = 130) were enrolled during the study period with characteristics of sex, age, and immunohistochemical/molecular subtype examination that underwent bone scintigraphy. Mean of age was 50.2 (23–79) years. There were no significant correlations between ER, PR, and HER-2 expressions with bone metastases in breast cancer patients. Ki-67 was showed to be correlated with bone metastases in breast cancer patients in our bivariate analysis. Molecular subtype/markers had no statistically significant correlation with bone metastases in patients with breast cancer. ConclusionKi-67 with high proliferation index was the most powerful molecular marker to determine the risk of bone metastases. The prevalence of bone metastases in the group with Ki-67 expression with high proliferation (≥20) was 1.8 times greater than the prevalence of bone metastases in the weakest HER-2 group.

The osteogenic niche-targeted arsenic nanoparticles prevent colonization of disseminated breast tumor cells in the bone

Up to 70% of patients with late-stage breast cancer have bone metastasis. Current treatment regimens for breast cancer bone metastasis are palliative with no therapeutic cure. Disseminated tumor cells (DTCs) colonize inside the osteogenic niches in the early stage of bone metastasis. Drug delivery into osteogenic niches to inhibit DTC colonization can prevent bone metastasis from entering its late stage and therefore cure bone metastasis. Here, we constructed a 50% DSS6 peptide conjugated nanoparticle to target the osteogenic niche. The osteogenic niche was always located at the endosteum with immature hydroxyapatite. Arsenic-manganese nanocrystals (around 14 nm) were loaded in osteogenic niche-targeted PEG-PLGA nanoparticles with an acidic environment-triggered arsenic release. Arsenic formulations greatly reduced 4T1 cell adhesion to mesenchymal stem cells (MSCs)/preosteoblasts (pre-OBs) and osteogenic differentiation of osteoblastic cells. Arsenic formulations also prevented tumor cell colonization and dormancy via altering the direct interaction between 4T1 cells and MSCs/pre-OBs. The chemotactic migration of 4T1 cells toward osteogenic cells was blocked by arsenic in mimic 3D osteogenic niche. Systemic administration of osteogenic niche-targeted arsenic nanoparticles significantly extended the survival of mice with 4T1 syngeneic bone metastasis. Our findings provide an effective approach for osteogenic niche-specific drug delivery and suggest that bone metastasis can be effectively inhibited by blockage of tumor cell colonization in the bone microenvironment.

Heterogeneity of bone metastases as an important prognostic factor in patients affected by oestrogen receptor-positive breast cancer. The role of combined [18F]Fluoroestradiol PET/CT and [18F]Fluorodeoxyglucose PET/CT

PurposeTo assess the prognostic role of different inter and intralesional expression (heterogeneity) of oestrogen receptor (ER) in bone metastases, as identified by the combined use of [18F]FES PET/CT and [18F]FDG PET/CT in patients with oestrogen receptor-positive (ER+) metastatic breast cancer (BC). MethodsWe analysed patients with a new diagnosis of bone metastases who were candidates for first-line systemic endocrine therapy. Before starting therapy, patients underwent baseline [18F]FES PET/CT and [18]FDG PET/CT. Semi-quantitative evaluation of whole-body bone metabolic burden (WB-B-MB) was performed on [18F]FES and [18F]FDG PET/CT in order to evaluate disease extent, tumour metabolism and ER heterogeneity. We used time-to-event analyses (Kaplan-Meier and Cox proportional-hazards methods) to estimate progression-free (PFS) and overall survival (OS), in order to assess the independent prognostic value of [18F]FES PET/CT and [18F]FDG PET/CT, alone and in combination. ResultsAccording to our criteria, we enrolled 49 patients. Over a median follow-up of 44.7 months, 35 patients suffered disease progression (71.4 %) and 15 died of disease (30.6 %). When the risk of disease progression was calculated by means of the Cox model, only [18F]FDG WB-B-MB was independently and directly associated to PFS (p = 0.02).On analysing the association between all prognostic parameters and survival, the Cox model showed that the only parameter associated with OS was the WB-B-MB FES/FDG ratio (p = 0.01). ConclusionThe combined use of [18F]FES-PET/CT and [18F]FDG-PET/CT can identify ER heterogeneity in BC bone metastases. This heterogeneity is significantly associated with survival. Moreover, the extension of the FDG-avid component correlates with the risk of disease progression.