Progression Of Bone Metastases Research Articles

204P - The effect of radium-223 therapy in agent orange related veterans with metastatic castrate resistance prostate carcinoma (CRPC)

BackgroundRa-223 radioisotope has been reported to increase median survival in bone metastatic prostate carcinoma. The addition of Ra-223 to abiraterone was associated with an increased risk of bone fractures. There is no comprehensive data for using Ra-223 in veteran population. MethodsWe present a retrospective study of patients (pts) with bone metastatic CRPC who received standard doses of Ra-223 and other sequential therapies at VAPHS from 2014 to 2018. Pts were divided into agent orange exposure (AO) and no exposure (Non-AO). Time to study was calculated from the initiation of Ra-223. Time to skeletal related events (SRE), progression of PSA, bone metastasis and alkaline phosphatase (ALP) were calculated in months (mos) using unpaired T-test with two-tailed p value. Median survival was calculated by Kaplan Meier R log rank test. ResultsThere were 34 pts with bone metastatic CRPC. 17 pts (50%) were AO, 17 pts (50%) Non-AO. The mean age of diagnosis of AO was 62 y/o and Non-AO was 69 y/o (p 0.005). The mean Gleason score for AO was 8.12 and Non-AO was 8.00 (p 0.7048). The median number of Ra-223 cycles was 6 (60%). 10 pts received Ra-223 as first line (29%) and 24 pts received Ra-223 later (71%). There were 12 SRE with median survival of 15 mos. There was no difference in mean time to SRE between AO (8 pts, 10.63 mos) and Non-AO (4 pts, 10.25 mos) with p 0.9306. The mean time to PSA progression for AO was 5.40 mos and Non-AO was 6.79 mos (p 0.2808). Mean time to bone progression for AO was 7.56 mos and Non-AO was 10.14 mos (p 0.1574). Mean time to ALP progression for AO and Non-AO was 6.25 mos and 8.71 mos respectively (p 0.0512). 20 patients (58%) had died. Median survival for Ra-223 first was 32 mos and for Ra-223 later was 15 mos with p 0.14 and HR 0.48 (95% CI, 0.17 to 1.3). Median survival between AO and Non-AO was 12 mos and 18 mos respectively with p 0.15 and HR 2.0 (95% CI, 0.77 to 5). ConclusionsThere is no statistical difference between AO and Non-AO in terms of time to SRE, PSA, bone and ALP progression even though there is a trend of shorter duration in AO veterans. There is no median survival difference between Ra-223 first versus Ra-223 later as well as between AO and Non-AO but there is a trend of worse survival in AO veterans. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Synergistic combination treatment to break cross talk between cancer cells and bone cells to inhibit progression of bone metastasis

Advanced-stage cancers often metastasize to bone, and is the major cause of cancer-related morbidity and mortality. Due to poor biodistribution of intravenously administered anticancer drugs within the bone, chemotherapy is not optimally effective in treating bone metastasis. Additionally, overexpression of receptor activator of nuclear factor κB ligand (RANKL) in the bone microenvironment drives the vicious, destructive cycle of progression of bone metastasis and bone resorption. We hypothesized that the combination treatment — with docetaxel (TXT), an anticancer drug encapsulated in sustained release biodegradable nanoparticles (TXT-NPs) that are designed to localize in bone marrow, and denosumab monoclonal antibody (DNmb), which binds to RANKL — could be more effective than either treatment alone. We tested our hypothesis in intraosseous prostate cancer (PC-3) cell-induced osteolytic mouse model of bone metastasis with treatments given intravenously. The results demonstrated better efficacy with TXT-NPs than with TXT-CrEL or saline control in inhibiting progression of metastasis and improving survival. TXT-NPs showed ~3-fold higher drug levels in metastasized bone tissue at 1 wk post-administration than TXT-CrEL, thus explaining their efficacy. However, the combination treatment (TXT-NPs + DNmb) given simultaneously was significantly more effective in inhibiting metastatic progression; it caused early tumor regression and improved survival, and caused no body weight loss or tumor relapse, even when the treatment was discontinued, whereas TXT-NPs or DNmb alone treatments showed tumor relapse after an initial regression. Micro-CT analysis of the bone from the combination treatment showed no bone loss and normal bone mineral content, bone density, and bone volume fraction, whereas TXT-NPs or DNmb alone treatments showed bone loss. Confirming the above results, histochemical analysis of the bone from the combination treatment demonstrated normal bone morphology, and osteoblast and osteoclast cell activities. In conclusion, TXT-NPs and DNmb in combination, because of their complementary roles in breaking the cross talk between cancer cells and bone cells, was significantly effective in treating bone metastasis.

Untreated Osteoporosis and Progression of Bone Metastases

Untreated Osteoporosis and Progression of Bone Metastases

Abstract 2042: Modeling estrogen receptor-positive breast cancer bone metastasis to query osteolytic effects of tumor ER signaling

Abstract Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming clinically evident bone metastases (BMETs). To query ERα’s role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. Female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, & 0.72 mg E2). Osteolytic BMET formation was assessed radiographically and E2effects on bone were determined by DXA and microCT at various time points. The BMET size and proliferation were assessed by immunohistochemical (IHC) analyses at the end of the experiment (42 days). The effect of E2 on tumoral secretion of the osteolytic factor, parathyroid hormone related protein (PTHrP), was determined using a commercially available radioimmunoassay. The incidence and size of osteolytic BMET, which were not evident in the absence of E2 supplementation, were E2 dose-dependent in young mice. In contrast, E2 effects on the bone microenvironment were not dose-dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV). In skeletally immature (young) mice vs skeletally mature mice treated with an identical E2 dose (0.72 mg), which caused significantly different effects on bone turnover, progression of E2-dependent BMET was identical. These results suggest that E2 effects on the tumor, rather than bone, were driving E2-dependency of BMET progression. IHC analysis demonstrated that neither the size of human cytokeratin-positive tumors nor the proportion of Ki67-positive proliferating tumor cells in bone were E2 dose-dependent, suggesting that proliferative effects of E2 could not explain E2-dose dependent differences in osteolytic BMET formation. PTHrP, an osteolytic factor expressed in most clinical BrCA BMET, was E2-inducible ex vivo and secreted in higher levels by tumor cells isolated from BMETs. These results suggest that during ER+ BrCA BMET progression, tumoral effects of E2 not only support ER+ BMET proliferation, but may also have bone-specific effects due to the induction of PTHrP, which may explain the marked osteolytic capacity of the ER+ tumor cells in this model despite a microenvironment where bone volume is markedly increased. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Lily A. Alvord, Janet L. Funk. Modeling estrogen receptor-positive breast cancer bone metastasis to query osteolytic effects of tumor ER signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2042.

Bioluminescence and near-infrared fluorescence imaging for detection of metastatic bone tumors.

Bioluminescence imaging is being increasingly utilized in biological research. However, since the most commonly used firefly luciferase generates relatively weak bioluminescent signals, detection of low numbers of luciferase-expressing cells in vivo is challenging. The weak signal makes it difficult to detect cells located in deep tissues, which is problematic for preclinical research in tumor metastasis. In this study, three different types of fluorophores such as D-luciferin, AkaLumine-HCl, and P800SO3 were compared to evaluate the progression of bone metastasis induced by MDA-MB-231 breast cancer cells in vivo. The fluorescent signals for D-luciferin, AkaLumine-HCl, and P800SO3 were differently detected in the chest and knee joint. In particular, the fluorescence signal of P800SO3 was clearly observed in a section of the ribs, where it pointed out fractured bone fragments by tumor mass. Moreover, the P800SO3 signal from the left knee joint also showed a small bone fragment in the distal femur and was highlighted in the proximal tibia. Using targeted NIR fluorophores, metastatic bone tumors were monitored under the NIR fluorescence imaging system in real time, which enabled the in vivo diagnosis of bone metastasis by providing the location of the metastatic bone tumors.

The PI3K inhibitor buparlisib suppresses osteoclast formation and tumour cell growth in bone metastasis of lung cancer, as evidenced by multimodality molecular imaging.

Non-small cell lung cancer (NSCLC) metastasis commonly occurs in bone, which often results in pathological fractures. Sustained phosphoinositide-3-kinase (PI3K) signalling promotes the growth of PI3K-dependent NSCLC and elevates osteoclastogenic potential. The present study investigated the effects of a PI3K inhibitor on NSCLC growth in bone and osteoclast formation, and aimed to determine whether it could control symptoms associated with bone metastasis. A bone metastasis xenograft model was established by implanting NCI-H460-luc2 lung cancer cells, which contain a phosphatidylinositol- 4,5-bisphosphate 3-kinase catalytic subunit α mutation, into the right tibiae of mice. After 1 week, the tumours were challenged with a PI3K inhibitor (buparlisib) or blank control for 3 weeks. Tumour growth and burden were longitudinally assessed in vivo via reporter gene bioluminescence imaging (BLI), small animal positron emission tomography/computed tomography (CT) [18F-fluorodeoxyglucose (18F-FDG)] and single-photon emission computed tomography/CT [99mTc-methylene diphosphonate (99mTc-MDP)] imaging. Tibia sections of intraosseous NCI-H460 tumours were analysed by immunohistochemistry (IHC), western blotting and flow cytometry. Dynamic weight bearing (DWB) tests were further performed to examine the improvement of symptoms associated with bone metastasis during the entire study. Administration of buparlisib significantly inhibited the progression of bone metastasis of NSCLC, as evidenced by significantly reduced uptake of 18F-FDG, 99mTc-MDP and BLI signals in the treated lesions. In addition, buparlisib appeared to inhibit the expression of tartrate-resistant acid phosphatase and receptor activator of nuclear factor-κB ligand, as determined by IHC. Buparlisib also resulted in increased cell apoptosis, as determined by a higher percentage of Annexin V staining and increased caspase 3 expression. Furthermore, buparlisib significantly increased weight-bearing capacity, as revealed by DWB tests. The PI3K inhibitor, buparlisib, suppressed osteoclast formation in vivo, and exhibited antitumour activity, thus leading to increased weight-bearing ability in mice with bone metastasis of lung cancer. Therefore, targeting the PI3K pathway may be a potential therapeutic strategy that prevents the structural skeletal damage associated with bone metastasis of lung cancer.

Association of Bone Metastasis With Early-Stage Breast Cancer in Women With and Without Precancer Osteoporosis According to Osteoporosis Therapy Status

Deaths from cancer are attributed more to secondary than primary tumors, but the pathogenesis of organ-specific cancer metastasis has not been determined. To investigate whether precancer osteoporosis and osteoporosis therapy are associated with alteration of bone metastasis patterns. This nationwide retrospective cohort study was performed from January 1, 2002, to December 31, 2013, using 2 cohorts from the Taiwan National Health Insurance Research Database: a random sample of 1 million beneficiaries from the Longitudinal Health Insurance Database who were enrolled in 2005 (LHID2005) and a specific data set of all the patients with osteoporosis. Patients diagnosed with breast cancer and precancer osteoporosis from January 1, 2002, to December 31, 2011, were included in the study, and their records were examined through December 31, 2013. From LHID2005, we selected 9104 women with early-stage breast cancer, of whom 705 had precancer osteoporosis. We identified 29 183 patients from the cohort of patients with breast cancer and osteoporosis, 14 020 of whom had precancer osteoporosis. Data analysis was performed from December 31, 2016, to August 31, 2018. Precancer osteoporosis and osteoporosis therapy. The risk of bone metastasis in patients with and without precancer osteoporosis and patients receiving and not receiving osteoporosis therapy as well as time to bone metastasis development. Among 9104 patients with breast cancer from the Longitudinal Health Insurance Database (mean [SD] age, 46.7 [14.0] years), precancer osteoporosis was not associated with a difference in risk of bone metastasis (adjusted hazard ratio [aHR], 0.87; 95% CI, 0.58-1.30; P = .49). Among 14 020 patients with precancer osteoporosis from the other cohort (mean [SD] age, 58.9 [11.6] years), osteoporosis therapy had no association with the risk of bone metastasis (bisphosphonates: aHR, 1.47; 95% CI, 1.00-2.17; P = .05; nonbisphosphonate drugs: aHR, 1.00; 95% CI, 0.72-1.39; P > .99). Compared with those without precancer osteoporosis (median time to bone metastasis development, 2.87 years; interquartile range [IQR], 1.34-4.86 years), among those with precancer osteoporosis, the median time to develop bone metastasis was shorter in those who did not receive treatment (1.74 years; IQR, 0.58-3.60 years; P < .001), whereas this time was the same for those who received treatment (bisphosphonates: 2.34 years; IQR, 1.23-3.13 years; nonbisphosphonate drugs: 2.08 years; IQR, 0.92-4.95 years). Precancer osteoporosis was not associated with risk of bone metastasis, but untreated osteoporosis was associated with accelerated progression of bone metastasis when it occurred. Organ microenvironments interact with disseminated cancer mostly after the specific organ has been predetermined to be the designated location. Because recurrences and metastases are major obstacles to cancer treatments, determining which organs may be at risk for metastases may be crucial to treating the disease.

Abstract BS1-3: New insights into early-stage bone colonization of disseminated breast cancer cells

Abstract The fate of disseminated tumor cells is heavily influenced by their microenvironment niche. The osteogenic niche promotes cancer cell proliferation and bone metastasis progression. We investigated the underlying mechanisms using a variety of pre-clinical models and bioinformatics analyses of clinical data. We find that disseminated tumor cells may follow a “migration-by-tethering” mechanism to reach the osteogenic niche and establish tight direct interaction with the osteogenic cells. This interaction in turn profoundly alters signaling cascades in bone micrometastases. Heterotypic adherens junctions between cancer cells and osteogenic cells induce activation of the mTOR signaling. Gap junctions mediate calcium flux from osteogenic cells to cancer cells, and activate the calcium signaling. Upon the development of these junctions, ER signaling in ER+ breast cancer cells is dramatically suppressed, conferring endocrine independence. These alterations lead to unexpected therapeutic resistance and sensitivity, which were elucidated in part by a novel ex vivo bone-in-culture array system. Our findings, together with others in the literature, demonstrate the importance of considering microenvironment effects in metastatic therapies, and suggest novel therapeutic strategies to eliminate micrometastases. Citation Format: Zhang X. New insights into early-stage bone colonization of disseminated breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr BS1-3.

Abstract P2-01-08: The potential role of CTNND1 (catenin (cadherin-associated protein), Delta 1) in breast cancer bone metastasis

Abstract Background: The bone is a frequently visited site by breast cancer cells. Most women who die of metastatic breast cancer would already have bone metastases, whether they are micro- or macro-metastases. Metastatic bone metastasis from breast cancer is mostly osteolytic, with reasons unclear and little in vitro and in vivo studies exploring the osteolytic nature of bone metastasis. In the present study, we investigated the potential role of CTNND1, Catenin (Cadherin-Associated Protein) Delta 1, in the context of bone metastasis of breast cancer. Materials and Methods: In order to identify potential genes involved in bone metastasis, we established a novel in vitro model named Bone Matrix Extract (BME) which was extracted from human femur and used to mimic the bone environment. Full profile of gene expression in response to BME was conducted using Ampliseq technology. Potential genes associated with bone metastasis was examined in a clinical breast cohort containing both cancer and normal tissues (n = 103), collected immediately following surgery. Gene transcript levels were quantified using QPCR and analysed against patient's pathological information and clinical outcome. We generated a series of cell models by knocking down and over-expressing one of the most relevant genes, CTNND1, using siRNA, sh-RNA, ribozyme transgenes and insertion of full coding sequence containing plasmids. Function assays including Matrigel based-adhesion, cancer cell-osteobalstic cell contact, proliferation, transwell invasion and migration were used to investigate the changes of biological features after interfering with CTNND1 expression in relation to BME / co-culture models. Results: CTNND1 was down regulated in all breast epithelial cells following BME treatment at both mRNA and protein level. From the clinical cohort, we found that compared with benign tissue, breast cancer tissues had significantly decreased CTNND1 transcript expression. Reduced CTNND1 was associated with advanced TNM stage and poor distant metastasis, local recurrence and bone metastasis. We went on to knockdown CTNND1 by siRNA, ribozyme as well as lenti-shCTNND1 transfection in MCF-10A and MDA-231 cells and overexpressed CTNND1 in MCF-7 cells. In vitro study demonstrated that knockdown of CTNND1 expression led to decreased capacity of Matrigel-adhesion, migration and invasion but increased cancer cell-osteobalstic cell adhesion. No effects were observed on cell proliferation after altering CTNND1 expression, in the presence or absence of BME. Conclusions: In this initial study on CTNND1 in breast cancer, our current data suggests that lower transcript expression of CTNND1 associates with a poorer patient prognosis. CTNND1 reduction may play a role in the progression of breast cancer bone metastasis. Citation Format: Gong C, Andrew A, Feng Y, Owen S, Liang G, Davies E, Song E, Jiang W. The potential role of CTNND1 (catenin (cadherin-associated protein), Delta 1) in breast cancer bone metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-08.

Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

Bone is the most frequent site of prostate cancer (PCa) metastasis; however, little is known about the role of the most common cell in bone, the osteocyte (OCy), in cancer biology. In this study we explored the crosstalk between PCa cells and OCys to determine if it contributes to PCa progression. PCa cells induced OCys to promote PCa proliferation, migration and invasion. A chemokine screen revealed that PCa cell induced OCys to produce growth-derived factor 15 (GDF15). Knockdown of GDF15 in OCys demonstrated that PCa cells conferred the ability on OCys to promote PCa proliferation, migration and invasion through GDF15. Consistent with this finding was the observation that the GDF15 receptor, GFRAL, was expressed on multiple PCa cell lines. Transcription factor array screening of PCa cells exposed to OCys with or without knockdown of GDF15 revealed that GDF15 in OCys promoted early growth response 1 (EGR1) expression in the PCa cells. Knockdown of EGR1 expression in PCa cells revealed it was required for the OCy-derived GDF15-mediated induction of in vitro PCa cell proliferation, migration and invasion. Subcutaneous co-injection of PCa cells and OCys into mice revealed that OCys promoted tumor growth in vivo, which was diminished by knockdown of GDF15 in the OCys. Knockdown of GDF15 in the tibiae diminished growth of PCa cancer cells injected into the tibiae, which was accompanied by decreased tumor cell proliferation and EGR1 expression. These results shed light on a novel mechanism through which PCa cells educate OCys to promote progression of PCa bone metastasis. They also suggest that targeting of GDF15-based and EGR1-based signaling pathways should be further explored for their potential to diminish progression of PCa bone metastasis.

Targeting the Hepatocyte Growth Factor and c-Met Signaling Axis in Bone Metastases.

Bone metastasis is the terminal stage disease of prostate, breast, renal, and lung cancers, and currently no therapeutic approach effectively cures or prevents its progression to bone metastasis. One of the hurdles to the development of new drugs for bone metastasis is the complexity and heterogeneity of the cellular components in the metastatic bone microenvironment. For example, bone cells, including osteoblasts, osteoclasts, and osteocytes, and the bone marrow cells of diverse hematopoietic lineages interact with each other via numerous cytokines and receptors. c-Met tyrosine kinase receptor and its sole ligand hepatocyte growth factor (HGF) are enriched in the bone microenvironment, and their expression correlates with the progression of bone metastasis. However, no drugs or antibodies targeting the c-Met/HGF signaling axis are currently available in bone metastatic patients. This significant discrepancy should be overcome by further investigation of the roles and regulation of c-Met and HGF in the metastatic bone microenvironment. This review paper summarizes the key findings of c-Met and HGF in the development of novel therapeutic approaches for bone metastasis.

The function of OPG/RANKL/RANK system in tumor bone metastasis

Tumor bone metastasis is one of the main causes of treatment failure in tumor. Since tumor bone metastasis is a complex process, the exact etiopathogenesis of tumor bone metastasis has not been clarified. Recent studies demonstrated that osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) system plays an important role during the development and progression of tumor bone metastasis. In this review, the research progress of OPG/RANKL/RANK system, including biological functions,the bone microenvironment regulation, the detection method,the diagnosis and treatment of tumor will be summerized. Key words: Neoplasm metastasis; Bone neoplasms; Osteoprotegerin; RANK Ligand; Receptor activator of nuclear factor-kappa B

A “Connexin” Responsible for the Fatal Attraction of Cancer to Bone

Tumor cell interactions with the bone microenvironment are vital for the establishment and progression of bone metastases. Recently in Cancer Cell, Wang etal. (2018) showed that cells of the osteoblast lineage are critical for the delivery of calcium to tumor cells through gap junctions, pointing toward potential therapies for bone metastases.

Models of Prostate Cancer Bone Metastasis.

More than 80% of patients with advanced prostate cancer (PCa) experience bone metastasis, which negatively impacts overall survival and patient quality of life. Various mouse models have been used to study the mechanisms of bone metastasis over the years; however, there is currently no model that fully recapitulates what happens in humans because bone metastasis rarely occurs in spontaneous PCa mouse models. Nevertheless, animal models of bone metastasis using several different tumor inoculation routes have been developed to help study bone metastatic progression, which occurs particularly in late-stage PCa patients. This chapter describes the protocols commonly used to develop models of bone metastatic cancer in mice using different percutaneous injection methods (Intracardiac and Intraosseous). These models are useful for understanding the molecular mechanisms of bone metastatic progression, including tumor tissue tropism and tumor growth within the bone marrow microenvironment. Betterunderstanding of the mechanisms involved in these processes will clearly lead to the development of new therapeutic strategies for PCa patients with bone metastases.

A source of calcium

Wang et al. demonstrate that increased flux of calcium derived from osteogenic cells into cancer cells promotes early-stage bone colonization. Calcium signalling in cancer cells can be targeted by arsenic trioxide, thereby reducing bone metastasis progression.

Hypoxic Microenvironment and Metastatic Bone Disease.

Hypoxia is a common feature of solid tumors and is associated with an increased risk of metastasis and a poor prognosis. Recent imaging techniques revealed that bone marrow contains a quite hypoxic microenvironment. Low oxygen levels activate hypoxia signaling pathways such as hypoxia-inducible factors, which play critical roles in the key stages of metastatic dissemination including angiogenesis, epithelial-mesenchymal transition, invasion, maintenance of cancer stem cells, tumor cell dormancy, release of extracellular vesicles, and generation of pre-metastatic niches. Hypoxia also affects bone cells, such as osteoblasts and osteoclasts, and immune cells, which also act to support the development and progression of bone metastases. Paradoxically, hypoxia and related signaling molecules are recognized as high-priority therapeutic targets and many candidate drugs are currently under preclinical and clinical investigation. The present review focuses on our current knowledge of the potential roles of hypoxia in cancer metastasis to bone by considering the interaction between metastatic cancer cells and the bone microenvironment. Current therapeutic approaches targeting hypoxia are also described.

The Osteogenic Niche Is a Calcium Reservoir of Bone Micrometastases and Confers Unexpected Therapeutic Vulnerability

The fate of disseminated tumor cells is largely determined by microenvironment (ME) niche. The osteogenic niche promotes cancer cell proliferation and bone metastasis progression. We investigated the underlying mechanisms using pre-clinical models and analyses of clinical data. We discovered that the osteogenic niche serves as a calcium (Ca) reservoir for cancer cells through gap junctions. Cancer cells cannot efficiently absorb Ca from ME, but depend on osteogenic cells to increase intracellular Ca concentration. The Ca signaling, together with previously identified mammalian target of rapamycin signaling, promotes bone metastasis progression. Interestingly, effective inhibition of these pathways can be achieved by danusertib, or a combination of everolimus and arsenic trioxide, which provide possibilities of eliminating bone micrometastases using clinically established drugs.

Curcumin, but not curcumin-glucuronide, inhibits Smad signaling in TGFβ-dependent bone metastatic breast cancer cells and is enriched in bone compared to other tissues

Breast cancer (BCa) bone metastases (BMETs) drive osteolysis via a feed-forward loop involving tumoral secretion of osteolytic factors (e.g., PTHrP) induced by bone-matrix-derived growth factors (e.g., TGFβ). In prior experiments, turmeric-derived curcumin inhibited in vivo BMET progression and in vitro TGFβ/Smad-signaling in a TGFβ-stimulated PTHrP-dependent human xenograft BCa BMET model (MDA-SA cells). However, it is unclear whether curcumin or curcumin-glucuronide mediates in vivo protection since curcumin-glucuronide is the primary circulating metabolite in rodents and in humans. Thus, effects of curcumin vs. curcumin-glucuronide on Smad-dependent TGFβ signaling were compared in a series of BCa cell lines forming TGFβ-dependent BMET in murine models, and tissue-specific metabolism of curcumin in mice was examined by LC–MS. While curcumin inhibited TGFβ-receptor-mediated Smad2/3 phosphorylation in all BCa cells studied (human MDA-SA, MDA-1833, MDA-2287 and murine 4T1 cells), curcumin-glucuronide did not. Similarly, curcumin, but not curcumin-glucuronide, blocked TGFβ-stimulated secretion of PTHrP from MDA-SA and 4T1 cells. Because the predominant serum metabolite, curcumin-glucuronide, lacked bioactivity, we examined tissue-specific metabolism of curcumin in mice. Compared to serum and other organs, free curcumin (both absolute and percentage of total) was significantly increased in bone, which was also a rich source of enzymatic deglucuronidation activity. Thus, curcumin, and not curcumin-glucuronide, appears to inhibit bone-tropic BCa cell TGFβ-signaling and to undergo site-specific activation (deconjugation) within the bone microenvironment. These findings suggest that circulating curcumin-glucuronide may act as a prodrug that preferentially targets bone, a process that may contribute to the bone-protective effects of curcumin and other highly glucuronidated dietary polyphenols.

Fourier transform infrared spectroscopy based spectral biomarkers of metastasized breast cancer progression.

Breast cancer is a global health issue and the second leading cause of cancer death in women. Breast cancer tends to migrate to bone and causes bone metastases which is ultimately the cause of death. Here, we report the use of FTIR to identify spectral biomarkers of cancer progression on 3D in vitro model of breast cancer bone metastasis. Our results indicate that the following spectral biomarkers can monitor cancer progression, for example, lipids (CH2 asymmetric/CH2 symmetric stretch), Amide I/Amide II, and RNA/DNA. Principal component analysis also confirmed the involvement of protein, lipids and nucleic acids in cancer progression on sequential culture. The collective observations from this study suggest successful application of FTIR as a non-invasive and accurate method to identify biochemical changes in cancer cells during the progression of breast cancer bone metastasis.

Mechanically-Loaded Breast Cancer Cells Modify Osteocyte Mechanosensitivity by Secreting Factors That Increase Osteocyte Dendrite Formation and Downstream Resorption.

Advanced breast cancer predominantly metastasizes to the skeleton, at which point patient prognosis significantly declines concomitant with bone loss, pain, and heightened fracture risk. Given the skeleton's sensitivity to mechanical signals, increased mechanical loading is well-documented to increase bone mass, and it also inhibited bone metastatic tumor formation and progression in vivo, though the underlying mechanisms remain under investigation. Here, we focus on the role of the osteocyte because it is the primary skeletal mechanosensor and in turn directs the remodeling balance between formation and resoprtion. In particular, osteocytic dendrites are important for mechanosensing, but how this function is altered during bone metastatic breast cancer is unknown. To examine how breast cancer cells modulate dendrite formation and function, we exposed osteocytes (MLO-Y4) to medium conditioned by breast cancer cells (MDA-MB231) and to applied fluid flow (2 h per day for 3 days, shear stress 1.1 Pa). When loading was applied to MLOs, dendrite formation increased despite the presence of tumor-derived factors while overall MLO cell number was reduced. We then exposed MLOs to fluid flow as well as media conditioned by MDAs that had been similarly loaded. When nonloaded MLOs were treated with conditioned media from loaded MDAs, their dendrite formation increased in a manner similar to that observed due to loading alone. When MLOs simultaneously underwent loading and treatment with loaded conditioned media, dendrite formation was greatest. To understand potential molecular mechanisms, we then investigated expression of genes related to osteocyte maturation and dendrite formation (E11) and remodeling (RANKL, OPG) as well as osteocyte apoptosis. E11 expression increased with loading, consistent with increased dendrite formation. Though loaded conditioned media decreased MLO cell number, apoptosis was not detected via TUNEL staining, suggesting an inhibition of growth instead. OPG expression was inhibited while RANKL expression was unaffected, leading to an overall increase in the RANKL/OPG ratio with conditioned media from loaded breast cancer cells. Taken together, our results suggest that skeletal mechanical loading stimulates breast cancer cells to alter osteocyte mechanosensing by increasing dendrite formation and downstream resorption.