Breast cancer bone metastases: it's all about the neighborhood.

In order to establish themselves in distal sites, metastatic cancer cells need to acquire organ-specific traits. Zhang et al. provide evidence in breast cancer that a tumor cell's acquisition of properties for successful bone metastasis is influenced by signals from the stroma of the primary tumor.

Generalizability of a Machine Learning Model for Improving Utilization of Parathyroid Hormone-Related Peptide Testing across Multiple Clinical Centers.

BACKGROUND: Measuring parathyroid hormone-related peptide (PTHrP) helps diagnose the humoral hypercalcemia of malignancy, but is often ordered for patients with low pretest probability, resulting in poor test utilization. Manual review of results to identify inappropriate PTHrP orders is a cumbersome process. METHODS: Using a dataset of 1330 patients from a single institute, we developed a machine learning (ML) model to predict abnormal PTHrP results. We then evaluated the performance of the model on two external datasets. Different strategies (model transporting, retraining, rebuilding, and fine-tuning) were investigated to improve model generalizability. Maximum mean discrepancy (MMD) was adopted to quantify the shift of data distributions across different datasets. RESULTS: The model achieved an area under the receiver operating characteristic curve (AUROC) of 0.936, and a specificity of 0.842 at 0.900 sensitivity in the development cohort. Directly transporting this model to two external datasets resulted in a deterioration of AUROC to 0.838 and 0.737, with the latter having a larger MMD corresponding to a greater data shift compared to the original dataset. Model rebuilding using site-specific data improved AUROC to 0.891 and 0.837 on the two sites, respectively. When external data is insufficient for retraining, a fine-tuning strategy also improved model utility. CONCLUSIONS: ML offers promise to improve PTHrP test utilization while relieving the burden of manual review. Transporting a ready-made model to external datasets may lead to performance deterioration due to data distribution shift. Model retraining or rebuilding could improve generalizability when there are enough data, and model fine-tuning may be favorable when site-specific data is limited.

Parathyroidectomy for Normocalcemic Primary Hyperparathyroidism is Associated with Improved Bone Mineral Density Regardless of Postoperative Parathyroid Hormone Levels.

BACKGROUND: Biochemical cure in normocalcemic primary hyperparathyroidism (nPHPT) is defined as parathyroid hormone (PTH) level normalization 6 months after parathyroidectomy. However, recent studies show that a significant number of nPHPT patients have persistent PTH elevation postoperatively. We sought to correlate changes in PTH levels with skeletal outcomes after parathyroidectomy in nPHPT patients. METHODS: Adult patients who underwent parathyroidectomy at a tertiary referral center for sporadic PHPT between 2010 and 2020 were reviewed. Pre- and postoperative (6 months, 18 months, and last follow-up) laboratory and bone mineral densities (BMD) were recorded. Primary outcome was 18-month postoperative BMD change in the lumbar spine (LS), total hip (TH) and femoral neck (FN) in normocalcemic and hypercalcemic PHPT (hPHPT) patients. RESULTS: Of 661 patients included, 68 had nPHPT. nPHPT patients frequently had multigland disease (31% vs. 18%, p = 0.014), more bilateral cervical explorations (22% vs. 13%, p = 0.042), and fewer achieved biochemical cure (76% vs. 95%, p < 0.001) than hPHPT patients. Twenty-eight nPHPT patients had BMD data for comparison. Overall, nPHPT patients had improvement in the LS (1.84%, p = 0.002) and TH (1.64%, p = 0.014). When stratified by postoperative PTH levels, nPHPT patients with persistent PTH elevation had more BMD improvement at the TH than those who normalized PTH (3.73% vs. - 0.83%, p = 0.017). There was no difference in improvement at the LS or FN (p = NS). CONCLUSION: Parathyroidectomy is associated with improved BMD in nPHPT patients with bone disease. Although one in four nPHPT patients had elevated postoperative PTH levels persisting throughout the study, BMD improvement was still seen regardless of postoperative PTH level normalization.

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

The bone-muscle connection in breast cancer: implications and therapeutic strategies to preserve musculoskeletal health.

Breast cancer and its therapies frequently result in significant musculoskeletal morbidity. Skeletal complications include bone metastases, pain, bone loss, osteoporosis, and fracture. In addition, muscle loss or weakness occurring in both the metastatic and curative setting is becoming increasingly recognized as systemic complications of disease and treatment, impacting quality of life, responsiveness to therapy, and survival. While the anatomical relationship between bone and muscle is well established, emerging research has led to new insights into the biochemical and molecular crosstalk between the skeletal and muscular systems. Here, we review the importance of both skeletal and muscular health in breast cancer, the significance of crosstalk between bone and muscle, and the influence of mechanical signals on this relationship. Therapeutic exploitation of signaling between bone and muscle has great potential to prevent the full spectrum of musculoskeletal complications across the continuum of breast cancer.

International Delphi consensus guidelines for follow-up after prophylactic total gastrectomy: the Life after Prophylactic Total Gastrectomy (LAP-TG) study.

BACKGROUND: Prophylactic total gastrectomy (PTG) remains the only means of preventing gastric cancer for people with genetic mutations predisposing to Hereditary Diffuse Gastric Cancer (HDGC), mainly in the CDH1 gene. The small but growing cohort of people undergoing PTG at a young age are expected to have a life-expectancy close to the general population, however, knowledge of the long-term effects of, and monitoring requirements after, PTG is limited. This study aims to define the standard of care for follow-up after PTG. METHODS: Through a combination of literature review and two-round Delphi consensus of major HDGC/PTG units and physicians, and patient advocates, we produced a set of recommendations for follow-up after PTG. RESULTS: There were 42 first round, and 62 second round, responses from clinicians, allied health professionals and patient advocates. The guidelines include recommendations for timing of assessments and specialties involved in providing follow-up, micronutrient supplementation and monitoring, bone health and the provision of written information. CONCLUSION: While the evidence supporting the guidelines is limited, expert consensus provides a framework to best manage people following PTG, and could support the collection of information on the long-term effects of PTG.

Increased S1P expression in osteoclasts enhances bone formation in an animal model of Paget's disease.

Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL-derived sphingosine-1-phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL-derived S1P can act as a coupling factor to increase normal bone formation via binding S1P-receptor-3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase-1 (SphK-1) compared with wild-type (WT) mouse and normal donor OCLs. SphK-1 production by MVNP-OCLs was interleukin-6 (IL-6)-dependent since OCLs from MVNP/IL-6-/- mice expressed lower levels of SphK-1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK-1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP-OCLs cocultured with OBs from MVNP or WT mice increased OB-S1PR3 expression and enhanced expression of OB differentiation markers in MVNP-OBs precursors compared with WT-OBs, which was mediated by IL-6 and insulin-like growth factor 1 secreted by MVNP-OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP-OBs treated with WT and MVNP-OCL-conditioned media (CM) blocked enhanced OB differentiation of MVNP-OBs treated with MVNP-OCL-CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col-1A expression in MVNP-OBs treated with MVNP-OCL-CM compared with WT-OBs treated with WT-OCL-CM. These results suggest that IL-6 produced by PD-OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD.

Differential stem- and progenitor-cell trafficking by prostaglandin E2.

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically.

Skeletal muscle Ca(2+) mishandling: Another effect of bone-to-muscle signaling.

Our appreciation of crosstalk between muscle and bone has recently expanded beyond mechanical force-driven events to encompass a variety of signaling factors originating in one tissue and communicating to the other. While the recent identification of new 'myokines' has shifted some focus to the role of muscle in this partnership, bone-derived factors and their effects on skeletal muscle should not be overlooked. This review summarizes some previously known mediators of bone-to-muscle signaling and also recent work identifying a new role for bone-derived TGF-ß as a cause of skeletal muscle weakness in the setting of cancer-induced bone destruction. Oxidation of the ryanodine receptor/calcium release channel (RyR1) in skeletal muscle occurs via a TGF-ß-Nox4-RyR1 axis and leads to calcium mishandling and decreased muscle function. Multiple points of potential therapeutic intervention were identified, from preventing the bone destruction to stabilizing the RYR1 calcium channel. This new data reinforces the concept that bone can be an important source of signaling factors in pathphysiological settings.

Alkaline phosphatase in metastatic castration-resistant prostate cancer: reassessment of an older biomarker.

Since most patients with metastatic castration-resistant prostate cancer (mCRPC) have bone metastases, it is important to understand the potential impact of therapies on prognostic biomarkers, such as ALP. Clinical studies involving mCRPC life-prolonging agents (i.e., sipuleucel-T, abiraterone, enzalutamide, docetaxel, cabazitaxel, and radium-223) have shown that baseline ALP level is prognostic for overall survival, and may be a better prognostic marker for overall survival than prostate-specific antigen in patients with bone-dominant mCRPC. Mechanism of action differences between therapies may partly explain ALP dynamics during treatment. ALP changes can be interpreted within the context of other parameters while monitoring disease activity to better understand the underlying pathology. This review evaluates the current role of ALP in mCRPC.

The Role of TGFß in Bone-Muscle Crosstalk.

PURPOSE OF REVIEW: The role of bone-derived factors in regulation of skeletal muscle function is an important emerging aspect of research into bone-muscle crosstalk. Implications for this area of research are far reaching and include understanding skeletal muscle weakness in cancer, osteoporosis, cachexia, rare diseases of bone, and aging. RECENT FINDINGS: Recent research shows that bone-derived factors can lead to changes in the skeletal muscle. These changes can either be anabolic or catabolic, and we focus this review on the role of TGFß in driving oxidative stress and skeletal muscle weakness in the setting of osteolytic cancer in the bone. The bone is a preferred site for breast cancer metastasis and leads to pathological bone loss. Osteolytic cancer in the bone leads to release of TGFß from the bone via osteoclast-mediated bone destruction. Our appreciation of crosstalk between the muscle and bone has recently expanded beyond mechanical force-driven events to encompass a variety of signaling factors originating in one tissue and communicating to the other. This review summarizes some previously known mediators of bone-to-muscle signaling and also recent work identifying a new role for bone-derived TGFß as a cause of skeletal muscle weakness in the setting of osteolytic cancer in the bone. Multiple points of potential therapeutic intervention are discussed.

Breaking down bone: new insight into site-specific mechanisms of breast cancer osteolysis mediated by metalloproteinases.

Bone metastases are the most common skeletal complication of malignancy. Tumor cells disrupt normal bone remodeling to promote bone destruction and its associated morbidity. In the August 15, 2009, issue of Genes & Development, Lu and colleagues (pp. 1882-1894) propose a novel molecular mechanism by which tumor-produced metalloproteinases release epidermal growth factor (EGF) ligands to activate the central osteoclastogenic pathway receptor activator of NF-kappaB ligand (RANKL) to promote breast cancer osteolysis. This work has important therapeutic applications that may quickly translate to more effective treatment for bone metastases.

Spontaneous bone metastases in a preclinical orthotopic model of invasive lobular carcinoma; the effect of pharmacological targeting TGFß receptor I kinase.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80-90% and 10-15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E-cadherin and p53 in a conditional K14cre;Cdh1((F/F));Trp53((F/F)) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD-208 (60 mg/kg per day), an orally available TGFß receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD-208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD-208 treatment reduced TGFß signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti-tumour effects of TGFß. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events.

Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.

INTRODUCTION: Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases-a major site of treatment-refractory tumor growth in patients with advanced disease. METHODS: The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR. RESULTS: Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity. CONCLUSIONS: The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo.

Effect of Age at Time of Irradiation, Sex, Genetic Diversity, and Granulopoietic Cytokine Radiomitigation on Lifespan and Lymphoma Development in Murine H-ARS Survivors.

Acute, high-dose radiation exposure results in life-threatening acute radiation syndrome (ARS) and debilitating delayed effects of acute radiation exposure (DEARE). The DEARE are a set of chronic multi-organ illnesses that can result in early death due to malignancy and other diseases. Animal models have proven essential in understanding the natural history of ARS and DEARE and licensure of medical countermeasures (MCM) according to the FDA Animal Rule. Our lab has developed models of hematopoietic (H)-ARS and DEARE in inbred C57BL/6J and Jackson Diversity Outbred (JDO) mice of both sexes and various ages and have used these models to identify mechanisms of radiation damage and effective MCMs. Herein, aggregate data from studies conducted over decades in our lab, consisting of 3,250 total-body lethally irradiated C57BL/6J young adult mice and 1,188 H-ARS survivors from these studies, along with smaller datasets in C57BL/6J pediatric and geriatric mice and JDO mice, were examined for lifespan and development of thymic lymphoma in survivors up to 3 years of age. Lifespan was found to be significantly shortened in H-ARS survivors compared to age-matched nonirradiated controls in all four models. Males and females exhibited similar lifespans except in the young adult C57BL/6J model where males survived longer than females after 16 months of age. The incidence of thymic lymphoma was increased in H-ARS survivors from the young adult and pediatric C57BL/6J models. Consistent with our findings in H-ARS, geriatric mice appeared more radioresistant than other models, with a lifespan and thymic lymphoma incidence more similar to nonirradiated controls than other models. Increased levels of multiple pro-inflammatory cytokines in DEARE bone marrow and serum correlated with shortened lifespan and malignancy, consistent with other animal models and human data. Of interest, G-CSF levels in bone marrow and serum 8-11 months after irradiation were significantly increased in females. Importantly, treatment with granulopoietic cytokine MCM for radiomitigation of H-ARS did not influence the long-term survival rate or incidence of thymic lymphoma in any model. Taken together, these findings indicate that the lifespan of H-ARS survivors was significantly decreased regardless of age at time of exposure or genetic diversity, and was unaffected by earlier treatment with granulopoietic cytokines for radiomitigation of H-ARS.

Molecular and clinical effects of aromatase inhibitor therapy on skeletal muscle function in early-stage breast cancer.

We evaluated biochemical changes in skeletal muscle of women with breast cancer initiating aromatase inhibitors (AI), including oxidation of ryanodine receptor RyR1 and loss of stabilizing protein calstabin1, and detailed measures of muscle function. Fifteen postmenopausal women with stage I-III breast cancer planning to initiate AI enrolled. Quadriceps muscle biopsy, dual-energy x-ray absorptiometry, isokinetic dynamometry, Short Physical Performance Battery, grip strength, 6-min walk, patient-reported outcomes, and serologic measures of bone turnover were assessed before and after 6 months of AI. Post-AI exposure, oxidation of RyR1 significantly increased (0.23 ± 0.37 vs. 0.88 ± 0.80, p < 0.001) and RyR1-bound calstabin1 significantly decreased (1.69 ± 1.53 vs. 0.74 ± 0.85, p < 0.001), consistent with dysfunctional calcium channels in skeletal muscle. Grip strength significantly decreased at 6 months. No significant differences were seen in isokinetic dynamometry measures of muscle contractility, fatigue resistance, or muscle recovery post-AI exposure. However, there was significant correlation between oxidation of RyR1 with muscle power (r = 0.60, p = 0.02) and muscle fatigue (r = 0.57, p = 0.03). Estrogen deprivation therapy for breast cancer resulted in maladaptive changes in skeletal muscle, consistent with the biochemical signature of dysfunctional RyR1 calcium channels. Future studies will evaluate longer trajectories of muscle function change and include other high bone turnover states, such as bone metastases.

Curcuminoids block TGF-ß signaling in human breast cancer cells and limit osteolysis in a murine model of breast cancer bone metastasis.

Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated. In vitro, transforming growth factor (TGF)-ß-stimulated PTHrP secretion was inhibited by curcuminoids (IC50 = 24 µM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition. Effects on TGF-ß signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-ß signaling. In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days. Osteolytic bone lesion area was reduced up to 51% (p < 0.01). Consistent with specific effects on bone osteolysis, osteoclast number at the bone-tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered. In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment. These data suggest that curcuminoids prevent TGF-ß induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.