Development Of Skeletal Metastases Research Articles

Abstract 520: Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines

Abstract Parathyroid Hormone-related Peptide (PTHrP)is a potent regulator of bone turnover and is thought to play a major role in the progression of skeletal metastasis through its activation of growth factors in the bone microenvironment. Our previous studies using genetically engineered mouse models in which PTHrP was ablated in the mammary epithelium have demonstrated that PTHrP also plays a direct role on tumor progression by enhancing tumor initiation, growth and metastasis outside the skeleton. We therefore proposed a novel mechanism of PTHrP driven bone metastasis by which PTHrP controls both the seed (tumor cells) and the soil (the bone microenvironment). We tested our hypothesis in triple negative breast cancer cell line (TNBC)models which are not responsive to either tamoxifen or Herceptin. Monoclonal antibodies against PTHrP were generated with strong in vitro anti-proliferative and anti-invasive potency in two human PTHrP expressing cell lines: the widely used TNBC MDAMB231 cell line and a patient derived TNBC cell line. Ablation of PTHrP or treatment with anti-PTHrP monoclonal antibodies induced major changes in the phenotype of these cells as determined by flow cytometry with stem cells and EMT markers, invasion assays and mammosphere assays indicating a reversal of their metastatic/invasive phenotype. Next we transplanted these cell lines into 8 weeks old female athymic nude mice to examine the efficacy of the therapeutic monoclonal antibodies on the progression of established bone metastasis.104 tumor cells were injected intra-tibially and animals examined at timed intervals(14, 21, 28, 25 and 42 days) with X-rays and high resolution CTs.At 14 days animals were administered intra-peritoneally with 200 micrograms of therapeutic anti-PTHrP antibodies or non-immune IgG(10 animals per group) and every 3 days thereafter til sacrifice(42 days). At sacrifice PET scan was performed to measure tumor volume and activity and bone were collected for histomorphometry analysis of tumor size and bone turnover activity. A significant reduction in bone lesions (P<0.05) was observed over time by imaging in animals treated with the therapeutic antibodies. At sacrifice PET scan indicates a strong reduction in tumor activity in treated animals (P<0.05). Histomorphometry analysis showed greater than 50% reduction in tumor volume associated with a significant inhibition of bone turnover in treated animals (P<0.05). In summary our data demonstrate that, in two TNBC models, PTHrP blockade can reprogram cancer cells to a reduced tumorigenicity and block the progression of established bone metastasis by targeting both the tumor cell and the bone microenvironment. Our data lend support to the “seed and soil” hypothesis proposed by Paget and the rational of anti-PTHrP therapeutic strategies alone or in combination with bone targeting agents in order to eradicate the development of skeletal metastasis. Citation Format: Jiarong Li, Anne Camirand, Mahvash Zakikhani, Karine Sellin, Richard Kremer. Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 520.

Abstract 3909: Investigating the role of ANXA2R in metastatic prostate cancer

Abstract Skeletal metastasis is the most life-threatening complication in prostate cancer. The molecular mechanisms behind the interactions between prostate cancer and the bone marrow premetastatic niche remain unknown. Understanding and preventing such interactions could lead to new promising therapeutic strategies against lethal prostate cancer. Annexin 2 receptor (ANXA2R) was originally identified as a cell surface receptor for Annexin 2 (ANXA2) that mediates the stimulatory effects of ANXA2 during osteoclastic activation and osteoblast mineralization. Moreover, ANXA2R is able to induce apoptosis independent of its ligand, partially through activating caspases using non-conventional apoptotic pathways. Osteoblasts and marrow endothelial cells express ANXA2 which functions as a regulator of hematopoietic stem cell engraftment. Previous studies demonstrated blocking ANXA2 or its receptor prevents prostate cancer cells from establishing bone metastases in mouse models. It has been recently proven that ANXA2R does not bind to ANXA2 directly but to S100A10 present in the complex annexin A2 heterotetramer (AIIt), composed of 2 ANXA2 bound to a S100A10 dimer. Higher levels of ANXA2 and S100A10 are associated with proliferating and invasive cancers and correlate with poor prognosis. However, the loss of ANXA2 expression appears to be specific for prostate cancer. Previous work on prostate cancer has focused solely on ANXA2 as a ligand. In this study, we demonstrate that S100A10 and ANXA2 are co-localized in prostate cancer, and their expression and ANXA2R levels vary with grade in primary tumors as well as within the different metastatic sites. Given prostate cancer cells as well as bone marrow stroma can simultaneously express the ligand and receptor and levels of AIIt decrease with tumor stage in the primary site, we hypothesize prostate cancer cells require S100A10 and not only ANXA2 to spread to bone and other tissues. ANXA2R could act as a dependence receptor, mediating withdrawal-induced programed cell death in prostate cancer cells in the absence of AIIt. Furthermore, AIIt-ANXA2R interactions between prostate cancer cells and the premetastatic bone marrow niche might play an important role in the development of skeletal metastasis by inducing selective pressures on prostate cancer cells to metastasize to the bone marrow which is abundant of AIIt. Citation Format: Gonzalo Torga, Kenneth J. Pienta. Investigating the role of ANXA2R in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3909. doi:10.1158/1538-7445.AM2017-3909

Pharmacological methyl group donors block skeletal metastasis in vitro and in vivo.

DNA hypomethylation was previously implicated in metastasis. In the present study, we examined whether methyl supplementation with the universal methyl donor S-adenosylmethionine (SAM) inhibits prostate cancer associated skeletal metastasis. Highly invasive human prostate cancer cells PC-3 and DU-145 were treated with vehicle alone, S-adenosylhomocysteine (SAH) or SAM and their effects on tumour cell proliferation, invasion, migration and colony formation were monitored. For in vivo studies, control (SAH) and SAM-treated PC-3 cells were injected into the tibia of Fox chase SCID mice and skeletal lesions were determined by X-ray and μCT. To understand possible mechanisms involved, we delineated the effect of SAM on the genome-wide methylation profile of PC-3 cells. Treatment with SAM resulted in a dose-dependent inhibition of tumour cell proliferation, invasion, cell migration, colony formation and cell cycle characteristics. Animals injected with 250 μM SAM-treated cells developed significantly smaller skeletal lesions, which were associated with increases in bone volume to tumour volume ratio and connectivity density as well as decreased trabecular spacing. Genome-wide methylation analysis showed differential methylation in several key signalling pathways implicated in prostate cancer including the signal transducer and activator of transcription 3 (STAT3) pathway. A selective STAT3 inhibitor decreased tumour cell invasion, effects which were less pronounced as compared with SAM. These studies provide a possible mechanism for the role of DNA demethylation in the development of skeletal metastasis and a rationale for the use of hypermethylation pharmacological agents to impede the development and progression of skeletal metastasis.

Abstract 2614: Bone sialoprotein is an essential target in breast cancer skeletal metastasis

Abstract Breast cancer is a leading cause of cancer related death in women due to onset of metastasis. Bone metastasis is the most frequent complication occurring in patients with advanced breast cancer and bone sialoprotein (BSP) is related to this process. However, the underlying mechanisms are not clear yet. Therefore, the aim of the study was to analyze BSP functions in greater detail and to decipher its signaling pathways contributing to bone metastasis. To that purpose a combination of the tetracycline-controlled transcription activation system (“Tet-Off system”) and RNA interference was used to initiate and maintain the conditional knockdown of BSP for any intended period. This new technique was established in MDA-MB-231 subclones by recombinase-mediated cassette exchange. Additionally, the cell clones were equipped with the reporter genes mCherry and firefly luciferase for testing their regulative properties and following their fate. In absence of doxycycline, the expression of a miRNA targeting BSP was activated and after six days of BSP knockdown the ensuing cellular, metastatic or molecular properties were monitored by fluorescent microscopy, flow cytometry analysis, assays for proliferation, migration and colony formation, as well as expression profiling analysis. Furthermore, the cell clones were examined in a nude rat model for soft tissue and osteolytic lesions after 2 to 6 weeks of miRNA treatment. The clones revealed good regulative properties to doxycycline. Phenotypic changes indicating apoptosis were observed after 6 days of conditional knockdown which was characterized by up to 86% decreased BSP levels and resulted in significant anti-proliferative, anti-migratory and anti-clonogenic effects in vitro. Additionally, the effect of miRNA-mediated BSP knockdown was assessed in vivo. Significant decreases (p < 0.03) and even complete remissions of soft tissue and osteolytic lesions were found following 3 and 6 weeks of miRNA treatment by bioluminescence and magneting resonance imaging, as well as volume computed tomography. The microarray data showed modulated expression in 1.3% of all genes, thus hinting to specific effects in response to BSP knockdown. These genes included increased expression of endoplasmic reticulum stress and apoptosis related genes (ATF3, CHOP), of transcription factor c-FOS, of the gene related to breast epithelial differentiation (ID2) and the tumor suppressor gene EGR1. Conversely, there was suppression of metastasis associated genes (CD44, IL11). These findings were confirmed by western blot for induction of intrinsic and extrinsic apoptotic pathways as shown by cleavage of caspases 8, 9, 3 and 7, and of PARP, as well as the upregulation of ATF3, DDIT3 (CHOP), c-FOS, ID2 and CD44. In conclusion, the role of BSP in the development of skeletal metastasis has been defined more precisely and renders this protein an attractive target in the treatment of this disease. Citation Format: Marineta Kovacheva, Michael Zepp, Stefan Berger, Martin R. Berger. Bone sialoprotein is an essential target in breast cancer skeletal metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2614. doi:10.1158/1538-7445.AM2014-2614

Effect of oral bisphosphonates for osteoporosis on development of skeletal metastases in women with breast cancer: results from a pharmaco-epidemiological study.

Treatment with bisphosphonates in women with breast cancer and established bone metastasis delays further skeletal-related events. Evidence is emerging that bisphosphonates are beneficial for secondary prevention of bone metastasis. The study aimed to estimate the effect of oral bisphosphonates for treatment or prevention of osteoporosis on development of bone metastasis in a population of women with breast cancer. A historical cohort of 21664 women diagnosed with breast cancer was created from health administrative data in Quebec, Canada. The primary outcome was time to develop bone metastasis; exposure was bisphosphonate use prediagnosis, postdiagnosis, both, or neither and a cumulative index of drug exposure. The sample was stratified according to stage (0-II or III) at time of diagnosis. Cox proportional hazards tested the effect of bisphosphonate use on time to develop bone metastases. Taking bisphosphonates postdiagnosis of breast cancer only or continuing bisphosphonates started prior to diagnosis after diagnosis was associated with a reduction in risk of bone metastasis from 45% to 28% in women with local disease at diagnosis. In women with regional disease, postdiagnosis bisphosphonate use, with or without prediagnosis use, reduced risk by almost 50%. A statistically significant dose-response trend was observed relating increased use to lower risk (slope = 0.94, 95% confidence interval = 0.90 to 0.99). Bisphosphonates were also associated with a decreased risk of all-cause mortality similar to that of the development of bone metastasis. Low-dose oral bisphosphonates administered for prevention or treatment of postmenopausal osteoporosis were associated with lower risk of skeletal metastasis in patients with early- or more advanced-stage breast cancer.

LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo

Prostate cancer (PCa) is a common hormone-dependent malignancy associated with the development of skeletal metastases. This is due to the increased expression of a number of growth factors, cytokines, and proteases which collectively drive the metastatic cascade in general and increased propensity to develop skeletal metastasis in particular. While a number of signaling pathways have been implicated in PCa progression, the highly complex wnt/β-catenin pathway is unique due to its ability to regulate gene expression, cell invasion, migration, survival, proliferation, and differentiation to contribute in the initiation and progression of PCa. Members of the wnt family bind to the Frizzle proteins or lipoprotein-related receptor proteins 5, 6 (LRP5, -6) to activate this key pathway. In the current study, we have investigated the role of wnt/β-catenin pathway in PCa progression, skeletal metastasis, and gene expression using the dominant negative plasmid of LRP5 (DN-LRP5) and human PCa cells PC-3. Inactivation of LRP5 resulted in mesenchymal to epithelial shift, lack of translocation of β-catenin to cell surface, increased tumor cell proliferation, decreased colony formation, migration and invasion in vitro. These effects were attributed to decreased expression of pro-invasive and pro-metastatic genes. In in vivo studies, PC-3-DN-LRP5 cells developed significantly smaller tumors and a marked decrease in skeletal lesion area and number as determined by X-ray, micro (μ) CT and histological analysis. Collectively results from these studies demonstrate the dominant role of this key pathway in PCa growth and skeletal metastasis and its potential as a viable therapeutic target.

Effect of bisphosphonates on pain and quality of life in patients with bone metastases

Bone is the most common organ for tumor metastasis, especially in patients with cancers of the breast or prostate. Bone metastases disrupt skeletal metabolism and result in considerable skeletal morbidity, including intractable, chronic bone pain, hypercalcemia of malignancy, pathologic fracture and spinal-cord compression. In addition to the chronic pain caused by bone metastases, skeletal-related events (SREs) such as pathologic fractures and spinal-cord compression can result in acute increases in pain. These effects can severely impair mobility and contribute to a general decrease in quality of life. Palliative options to treat bone metastases include radiotherapy, analgesics, surgery and bisphosphonates. These drugs bind to the surface of the bone and impair osteoclast-mediated bone resorption, and reduce the tumor-associated osteolysis that is initiated by the development of skeletal metastases. In addition to preventing SREs, bisphosphonates can palliate bone pain caused by a variety of solid tumors. This Review summarizes the clinical trial data of bisphosphonates for the prevention of SREs and the palliation of bone pain. Among these agents, nitrogen-containing bisphosphonates are recognized as the most effective, and zoledronic acid has demonstrated the broadest clinical utility.

Role of Wnts in prostate cancer bone metastases: Hall CL, Kang S, MacDougald OA, Keller ET, Department of Urology, The University of Michigan, Ann Arbor, MI

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor-induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine-rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP-produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf-1 (DKK-1). It appears that DKK-1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK-1 expression decreases allowing for unmasking of Wnt's osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK-1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK-1 activity fits a model of CaP-induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone-related protein (PTHRP) and DKK-1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK-1); and an osteoblastic phase, in which tumor growth-associated hypoxia results in production of vascular endothelial growth factor and endothelin-1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases.

Paracrine inhibition of osteoblast differentiation induced by neuroblastoma cells

The aim of our study was to investigate whether the defective function of osteogenic cells induced by neuroblastoma might play a role in the development of skeletal metastases. This mechanism has been extensively demonstrated for multiple myeloma, in which the blockage of osteoblast differentiation has been ascribed to the inhibitors of canonical Wingless pathway (Wnt), namely Dickkopf 1 (Dkk1). Our purpose was to verify if neuroblastoma cells derived from bone marrow metastases (SH-SY5Y, LAN1) or primaries (NB100, CHP212) hamper the differentiation of mesenchymal stem cells (hMSCs) into osteoblasts in a paracrine manner, and to test whether this ability depends on Dkk1 activity. We found that all neuroblastoma cells increased the proliferation of hMSCs collected from pediatric-aged donors, with a corresponding decrease in osteoblast differentiation markers, including alkaline phosphatase (ALP), analyzed as gene expression, enzymatic activity and number of ALP-positive colony forming units, osteoprotegerin (OPG) release, OPG and osteocalcin gene-expression. Dkk1 mRNA and protein were detectable in all cell lines, and the use of neutralizing anti-Dkk1 antibody reversed the effects induced by SH-SY5Y cells. Taken together, our results confirm that neuroblastoma hinders osteoblastogenesis, and that Dkk1 release seems to play a crucial role in blocking the differentiation of osteoprogenitor cells, though the ability to promote osteoclast activation remains an essential requirement for the development of skeletal metastases. Finally, our findings suggest that strategies regulating Wnt signaling and Dkk1 activity could be considered for adjuvant therapies in neuroblastoma metastasizing to the skeleton.

The role of Wnts in bone metastases

Wnts are a large family of secreted glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Autocrine Wnt signaling within tumor cells has been shown to promote tumorigenesis by enhancing tumor cell proliferation and survival. We recently demonstrated that prostate cancer cells (CaP) produce Wnts which act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. The ability of tumor-derived Wnts to influence bone development is regulated by multiple families of secreted antagonists including soluble frizzled related receptors (sFrp) and dickkopfs (DKK). CaP cells appear to produce DKK-1 early in the development of skeletal metastases, which masks osteogenic Wnts and thus favors an osteolytic environment at the metastatic site. As the metastases progresses, DKK-1 expression is lost allowing for a Wnt mediated osteoblastic response which predominates CaP boney lesions. Interestingly, blocking DKK-1 expression early in CaP metastasis prevents tumor establishment within the bone suggesting that osteolysis is a required first step in the development of CaP bone metastases. In this review, we discuss our data on the Wnt inhibitor DKK-1 in CaP bone metastasis in the context of current literature evidence that demonstrate that Wnt inhibitors can function as both tumor suppressors and tumor promoters. We provide a model that the affect of Wnt inhibitors on tumor development is dependent on the tumor micro-environment and suggest that DKK-1 is a switch which transitions CaP bone metastases from osteolytic to osteoblastic.

A non–RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo

Integrins are expressed by numerous tumor types including breast cancer, in which they play a crucial role in tumor growth and metastasis. In this study, we evaluated the ability of ATN-161 (Ac-PHSCN-NH2), a 5-mer capped peptide derived from the synergy region of fibronectin that binds to alpha5beta1 and alphavbeta3 in vitro, to block breast cancer growth and metastasis. MDA-MB-231 human breast cancer cells were inoculated s.c. in the right flank, or cells transfected with green fluorescent protein (MDA-MB-231-GFP) were inoculated into the left ventricle of female BALB/c nu/nu mice, resulting in the development of skeletal metastasis. Animals were treated with vehicle alone or by i.v. infusion with ATN-161 (0.05-1 mg/kg thrice a week) for 10 weeks. Tumor volume was determined at weekly intervals and tumor metastasis was evaluated by X-ray, microcomputed tomography, and histology. Tumors were harvested for histologic evaluation. Treatment with ATN-161 caused a significant dose-dependent decrease in tumor volume and either completely blocked or caused a marked decrease in the incidence and number of skeletal as well as soft tissue metastases. This was confirmed histologically as well as radiographically using X-ray and microcomputed tomography. Treatment with ATN-161 resulted in a significant decrease in the expression of phosphorylated mitogen-activated protein kinase, microvessel density, and cell proliferation in tumors grown in vivo. These studies show that ATN-161 can block breast cancer growth and metastasis, and provides a rationale for the clinical development of ATN-161 for the treatment of breast cancer.

Carcinoma of uterine cervix with isolated metastasis to fibula and its unusual behavior: Report of a case and review of literature

Bone metastasis from carcinoma cervix is uncommon, especially in the distal appendicular skeleton. A 36 year old lady presented with carcinoma of uterine cervix, FIGO, stage IIb. She was treated with radical radiotherapy. Nine months later, she developed an isolated lytic lesion in right fibula, which turned out to be a metastatic lesion. The patient is doing well, 3 years after the surgical excision of metastasis. This is one of the few documented cases of metastasis to fibula, arising from carcinoma of uterine cervix and probably the first with isolated metastasis of this site. Unlike the dismal outcome commonly seen in patients with bone secondaries, she continues to be disease free and alive at 39 months of follow up, after the development of skeletal metastasis.

Role of wnts in prostate cancer bone metastases

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor-induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine-rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP-produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf-1 (DKK-1). It appears that DKK-1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK-1 expression decreases allowing for unmasking of Wnt's osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK-1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK-1 activity fits a model of CaP-induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone-related protein (PTHRP) and DKK-1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK-1); and an osteoblastic phase, in which tumor growth-associated hypoxia results in production of vascular endothelial growth factor and endothelin-1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases.

A synthetic 15-mer peptide (PCK3145) derived from prostate secretory protein can reduce tumor growth, experimental skeletal metastases, and malignancy-associated hypercalcemia.

In previous studies, we have shown that prostate secretory protein (PSP-94) can reduce prostate cancer growth in vivo. In the current study, we identified the amino acid sequence of PSP-94 that is required for eliciting this response. For these studies, we used rat prostate cancer Mat Ly Lu cells overexpressing parathyroid hormone-related protein (PTHrP), which is the main pathogenetic factor responsible for hypercalcemia of malignancy. Synthetic peptides corresponding to amino acids 7-21 (PCK721), 31-45 (PCK3145), and 76-94 (PCK7694) of PSP-94 were synthesized. Only PCK3145 showed a significant reduction in tumor cell proliferation. For in vivo studies, syngenic male Copenhagen rats were inoculated s.c. with Mat Ly Lu cells overexpressing PTHrP into the right flank or into the left ventricle via intracardiac injection, which results in experimental metastases to the lumbar vertebrae causing hind-limb paralysis. Animals were infused with different doses (1, 10, and 100 microg/kg/day) of peptides for 15 days, and the effect of these treatments on tumor volume, skeletal metastases, or development of hind-limb paralysis was determined. Treatment with PCK3145 resulted in a dose-dependent decrease in tumor volume and delay in the development of skeletal metastases. Bone histomorphometry showed that after intracardiac inoculation of tumor cells, the highest dose of PCK3145 (100 microg/kg/day) resulted in reducing skeletal tumor burden, which delayed the development of hind-limb paralysis. Treatment with PCK3145 led to reduction of plasma calcium and PTHrP levels and a significant decrease in PTHrP levels in the primary tumors and in vertebrae of experimental animals. These effects of PCK3145 were due to its ability to promote tumor cell apoptosis. Collectively, the results of these studies have demonstrated the ability of a small peptide derived from PSP-94 to reduce tumor volume and experimental skeletal metastases-results that will be highly beneficial in the continued development of this peptide as a novel therapeutic agent for patients with hormone refractory, late-stage prostate cancer.

The role of bisphosphonates in the management of metastatic prostate cancer.

Most men with advanced prostate cancer have primarily skeletal metastases, which are responsible for most of the morbidity and mortality of prostate cancer. Although bone lesions are osteoblastic in radiographic appearance, recent evidence has demonstrated that anti-osteoclast therapy with bisphosphonates reduces skeletal-related complications in hormone-refractory prostate cancer. This observation may be explained by the demonstration that osteoclast activity is greatly upregulated even in osteoblastic metastases. Furthermore, androgen deprivation therapy, the mainstay of treatment for advanced prostate cancer, leads to increased bone resorption and reduces bone mineral density. These effects can be ameliorated, and potentially completely prevented, by coadministration of bisphosphonates. These findings may point to a role for bisphosphonates in men with prostate cancer even prior to the development of skeletal metastases. Determination of whether such early therapy ultimately results in delayed time to skeletal progression or in improved survival will require large randomized studies.

Pamidronate prevents the development of skeletal metastasis in nude mice transplanted with human breast cancer cells by reducing tumor burden within bone

Pamidronate is used routinely in the treatment of established bone metastasis. However, pamidronate has not yet been assessed in the prevention of osteolytic bone metastasis and its precise mechanism of action in this disorder remains to be established. In the present study, pamidronate or vehicle alone was administered subcutaneously to nude mice either simultaneously or as post intracardiac injection of the human breast cancer MDA-MB-231 cells. Radiographs were used first to assess the presence of osteolytic bone metastases. Kaplan-Meier analysis demonstrated that animals treated with pamidronate early, but not late, showed a slower progression of bone metastases and hind limb paralysis than did vehicle-treated animals. Mann-Whitney analysis showed that only 44.4% of mice treated with pamidronate at the time of tumor cell inoculation developed bone metastases as compared to over 80% (p<0.05) of mice receiving vehicle alone. We then analyzed the number of bone lesions and their volume at time of sacrifice by bone histomorphometry. In contrast to X-ray analysis, morphometric analysis indicates that the number of lesions within bone was similar in pamidronate and vehicle-treated mice but that the lesions were significantly smaller and therefore, often not visible on radiographs. These results demonstrate that pamidronate is effective in reducing tumor burden in breast cancer metastatic to bone and is most effective as a preventative agent when administered closest in time to implantation of tumor cells. Our data also suggest that pamidronate acts mainly by inhibiting the growth of established bone metastatic lesions but has no effect on the metastatic spread itself.

Differences in the cytokine profiles associated with prostate cancer cell induced osteoblastic and osteolytic lesions in bone

Prostate adenocarcinoma is associated with the formation of osteoblastic metastases in bone. It is hypothesized that osteoclastogenesis is a critical component in the development of skeletal metastases. These findings, however, were generally noted in predominantly osteolytic lesions. The pathophysiology of osteoblastic lesions remains unknown but the type of bone lesion formed may be influenced by the cytokines produced by prostate tumors. To test this theory, we implanted PC-3 and LAPC-9 cells into the tibias of SCID mice. These mice were sacrificed at 1, 2, 4, 6, and 8 weeks after implantation and histologic analysis was performed on these tibias. PCR analysis was also performed on bulk tumors. The results showed that the PC-3 implanted tibias developed pure osteolytic lesions while the LAPC-9 implanted tibias developed pure osteoblastic lesions on radiographs. Analysis of tibias after injection with PC-3 cells revealed progressive osteolytic lesions with abundant osteoclast activity at 2 weeks and destruction of the proximal tibia at 6 weeks after cell implantation. In contrast, the LAPC-9 cells formed osteoblastic lesions six weeks after cell injection. There were rare osteoclasts prior to the establishment of the osteoblastic lesions but greater osteoclast activity was noted with remodeling of the osteoblastic lesion 8 weeks after implantation of the tumor cells. PCR analysis revealed that PC-3 cells produced RANKL, IL-1, and TNF-α, which are associated with osteoclastogenesis. In contrast, LAPC-9 cells produced osteoprotegerin, which blocks osteoclast production and no detectable levels of RANKL or IL-1 and only minimal amounts of TNF-α were noted. These cells secreted BMP-2, -4, -6, and IL-6, which are associated with bone formation. These results suggest that the role of the osteoclast in the development of a metastatic lesion is variable depending on the phenotype of the prostate cancer cells, and that tumor-induced osteolysis may not be required for osteoblastic metastases.

The Role of Bisphosphonates in Breast Cancer Management: Review Article

SummaryBone metastases are a common problem in the management of breast cancer and are associated with considerable morbidity. Bone pain, hypercalcaemia, fractures and cord compression all occur requiring interventions such as analgesia, radiotherapy and surgery. Bisphosphonates are drugs that are active in the bone microenvironment. Their effects on osteoclasts are well described: they potently inhibit osteoclast mediated bone resorption by delaying the maturation of immature osteoclasts and by directly inducing osteoclast apoptosis. It has been known for some time that bisphosphonates, in combination with intravenous rehydration, effectively treat hypercalcaemia associated with solid malignancies. It has now been demonstrated in clinical trials in breast cancer patients that regular bisphosphonate administration reduces the morbidity associated with osteolytic skeletal metastases. There is an emerging suggestion from clinical trial work that bisphosphonates may be able to reduce or delay the development of skeletal metastases although this remains controversial as the three published trials present conflicting results. The more potent third-generation bisphosphonates, such as zoledronate, are now being tested for each of these indications with promising results and may replace other bisphosphonates in the future. Laboratory studies have recently demonstrated that bisphosphonates have direct cytotoxic effects against breast cancer cells in vitro, inducing apoptosis and preventing adhesion to bone. This adds support to the hypothesis that bisphosphonates may have a genuine beneficial effect in the adjuvant setting.

The palliative management of skeletal metastases in prostate cancer: Use of bone-seeking radionuclides and bisphosphonates

In prostate cancer, the development of skeletal metastases is associated with a significant increase in morbidity, mainly because of severe bone pain, which eventually becomes refractory to conventional analgesia. Androgen ablation is the treatment of choice, but the majority of patients relapse within 2 to 3 years from initiation of treatment. After failure of hormone therapy, external-beam irradiation therapy is effective in the palliation of pain, but radionuclides represent an attractive and cost-effective alternative. Strontium 89 is currently the most commonly used radionuclide in the palliative management of prostate cancer metastatic to the skeleton. The rationale for the use of bisphosphonates in metastatic prostate cancer is not immediately obvious, given the predominantly osteoblastic nature of the metastatic process. The clinical use of these agents rests on a number of basic and clinical observations that provide ample evidence that, in prostate cancer, the metastatic process is associated with increased bone resorption. Evidence regarding the beneficial effects of bisphosphonates in reducing morbidity from metastatic prostate cancer is reasonably solid, although the choice of optimal bisphosphonate, mode of administration, dose, and duration of treatment must be determined in large, controlled studies before their widespread clinical use can be advocated. Available therapeutic modalities that use either radionuclides or bisphosphonates can effectively and safely be used in the palliative management of metastatic prostate cancer. Neither radionuclides nor bisphosphonates have been shown to prolong survival, but the potential of both agents to beneficially alter the metastatic process in prostate cancer is intriguing.

Prostatic tumor (R3327) skeletal metastasis

Male Copenhagen rats were inoculated with monodispersed R3327-MatLyLu prostate tumor cells via the tail vein under concomitant temporal occlusion of the inferior vena cava to develop an animal model for skeletal metastasis of prostate cancer. This procedure reproducibly resulted in metastatic tumor growth in the lumbar region of the vertebral column. Microscopically, tumor growth became visible in the fifth and sixth lumbar vertebrae within 4 days after inoculation. Clinical signs of nerve function disablement (hind leg paresis and paralysis) followed within 14 days of such a procedure. Cell culture technique confirmed the presence of a viable, proliferating tumor cell population within the spinal canal of the fifth and sixth lumbar vertebrae. Histologically, a clear response of osteoclastic and concomitant osteoblastic activities was observed in the lumbar spinal column. In the serum, a transient phase of hypercalcemia could be demonstrated. The development of skeletal metastases in these animals was not reflected by significant alteration in serum levels of acid phosphatase, prostatic-specific antigen, or osteocalcin. These observations support the concept of the vertebral venous plexus being involved in the dissemination of prostate tumor cells. The surgical procedures described permit experimental investigations of bone metastasis of prostatic cancer.