Cancer Skeletal Metastasis Research Articles

A novel mouse model of human breast cancer stem-like cells with high CD44+CD24-/lower phenotype metastasis to human bone.

Abstract Abstract #4155 Background: At present, our understanding of the development of bone metastasis is limited. Therefore, little progress has been made in preventing skeletal metastasis in the breast cancer patient. A satisfactory animal model that avoids the species-specific factor and simultaneously shares similarities to the clinical pathophysiological progression of breast cancer metastasizing to bone is unavailable. A subpopulation (CD44+/CD24-/lower) of breast cancer cells possesses stem/progenitor cell properties (cancer stem-like cell). CD44 potentiates the adherence of metastatic breast cancer cells to bone marrow endothelial cells. In the present study, we used hepatocyte growth factor to enhance the proportion of CD44+/CD24-/lower subpopulation in the human breast cancer cell line MDA-MB-231. We used these human breast cancer stem-like cells and implantation of human bone to build a novel human-source model of human breast cancer skeletal metastasis.
 Methods: The human breast cancer cell line MDA-MB-231 was cultured in serum-free DMEM-F12 supplemented with growth factors. Cells in different subpopulations were separated by Percoll gradient centrifugation and incubated in the presence of 50 ng/mL hepatocyte growth factor. The proportion of CD44+/CD24-/lower subpopulation in the human breast cancer cell line MDA-MB-231 was detected with flow cytometry. Before injection with human breast cancer stem-like cells, the experimental animals were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1 X 105, 1 X 106 human breast cancer stem-like cells, and 1 X 106 parental MDA-MB-231 cells, respectively. A positive control group (D) without implantation of human bone (non-human source model) was also injected with 1 X 106 MDA-MB-231 cells. A group of negative controls (E) with human bone implantation was injected with isotonic sodium chloride. For each group, Micro-SPECT was performed at weeks 4 and 7, and all animals were sacrificed at week 8. Immunohistochemistry was performed for CD34, CD105, SMA, CD44, CD24, CK, CXCR4, and OPN. mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction.
 Results: New vessels and connective tissue were found on the surface of implanted human bones and cells stained positive for antibodies against human CD105, SMA, and CD34, indicating that implanted human bones were viable and functional. Histologic and immunohistochemical analysis confirmed the metastases as cancer cells. Importantly, the results demonstrated that cells in implanted human bones of group B, which received 1 X 106 cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas that of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis (77.8%, P = 0.0230) and no accompaniment of other tissue metastasis. The real-time polymerase chain reaction (PCR) showed an increase of CD44 mRNA in metastatic bone tissues in group B compared with that of groups C and D (15.2- and 21.1-fold, respectively). The mRNA levels of CXCR4 and OPN (8.4- and 28.4-fold, respectively) in bone metastasis tissues of group B were all higher than that of groups C and D (4.8- and 11.6-fold; respectively). The levels of CD24 mRNA in group B were lowest, measuring only 30 percent of that in groups C and D.
 Conclusion: This study indicates that in the novel human source model of breast cancer, breast cancer stem-like cells demonstrate a higher human bone-seeking ability, which may contribute to increase metastasis incidence and attenuate species-specific influences. Its mechanism might be related to the higher expressions of CD44, CXCR4 and OPN, and the lower expression of CD24 in breast cancer stem-like cells. The model shares more similarities with clinical pathological features of bone metastatic patients. It will be helpful for further study of the mechanisms and subclinical diagnosis of bone metastasis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4155.

Receptor activator of NF-κB ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells: Odero-Marah VA, Wang R, Chu G, Zayzafoon M, Xu J, Shi C, Marshall FF, Zhau HE, Chung LW, Molecular Urology and Therapeutics Program, Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor beta1 (TGFβ1) and transcription factors including Snail and Slug. We utilized the ARCaP(E)/ARCaP(M) prostate cancer progression model and LNCaP clones stably overexpressing snail to identify novel markers associated with EMT. Compared with ARCaP(E) cells, the highly tumorigenic mesenchymal ARCaP(M) and ARCaP(M1) variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaP(M) and ARCaP(M1) expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-κB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFβ1 treatment and Snail overexpression induced EMT in ARCaP(E) and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Receptor activator of NF-κB Ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor beta1 (TGFbeta1) and transcription factors including Snail and Slug. We utilized the ARCaP(E)/ARCaP(M) prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaP(E) cells, the highly tumorigenic mesenchymal ARCaP(M) and ARCaP(M1) variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaP(M) and ARCaP(M1) expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-kappaB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFbeta1 treatment and Snail overexpression induced EMT in ARCaP(E) and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

CX3CR1 is expressed by prostate epithelial cells and androgens regulate the levels of CX3CL1/fractalkine in the bone marrow: potential role in prostate cancer bone tropism.

We have previously shown that the chemokine fractalkine promotes the adhesion of human prostate cancer cells to bone marrow endothelial cells as well as their migration toward human osteoblasts in vitro. Thus, the interaction of fractalkine with its receptor CX3CR1 could play a crucial role in vivo by directing circulating prostate cancer cells to the bone. We found that although CX3CR1 is minimally detectable in epithelial cells of normal prostate glands, it is overexpressed upon malignant transformation. Interestingly, osteoblasts, stromal and mesenchymal cells derived from human bone marrow aspirates express the cell-bound form of fractalkine, whereas the soluble form of the chemokine is detected in bone marrow supernatants. To investigate the mechanisms regulating the levels of soluble fractalkine in the bone marrow, we focused on androgens, which play a critical role in both prostate cancer progression and skeletal metastasis. Here, we show that dihydrotestosterone dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide--an antagonist of the androgen receptor--as well as the wide-spectrum inhibitor of matrix metalloproteases, GM6001. However, dihydrotestosterone was unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells on a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. In conclusion, our results indicate that CX3CR1, fractalkine, and the enzymes responsible for its cleavage might represent suitable targets for therapies aiming to counteract skeletal secondary tumors from prostate adenocarcinoma.

Monocyte Chemotactic Protein-1 Mediates Prostate Cancer–Induced Bone Resorption

Prostate cancer preferentially metastasizes to bone, resulting in high mortality. Strategies to inhibit prostate cancer metastasis include targeting both tumor-induced osteoblastic lesions and underlying osteoclastic activities. We and others have previously shown that blocking receptor activator of nuclear factor-kappaB ligand (RANKL) partially blocks tumor establishment and progression in bone in murine models. However, levels of RANKL in the cell lines used in these studies were very low, suggesting that soluble factors other than RANKL may mediate the cancer-induced osteoclast activity. To identify these factors, a human cytokine antibody array was used to measure cytokine expression in conditioned medium collected from primary prostate epithelial cells (PrEC), prostate cancer LNCaP and its derivative C4-2B, and PC3 cells. All prostate cancer cells produced high amounts of monocyte chemotactic protein-1 (MCP-1) compared with PrEC cells. Furthermore, levels of interleukin (IL)-6, IL-8, GROalpha, ENA-78, and CXCL-16 were higher in PC3 than LNCaP. These results were confirmed by ELISA. Finally, human bone marrow mononuclear cells (HBMC) were cultured with PC3 conditioned medium. Although both recombinant human MCP-1 and IL-8 directly stimulated HBMC differentiation into osteoclast-like cells, IL-8, but not MCP-1, induced bone resorption on dentin slices with 21 days of culture in the absence of RANKL. However, the conditioned medium-induced bone resorption was inhibited by MCP-1 neutralizing antibody and was further synergistically inhibited with IL-8 antibody, indicating that MCP-1, in addition to IL-8, mediates tumor-induced osteoclastogenesis and bone resorption. MCP-1 may promote preosteoclast cell fusion, forming multinucleated tartrate-resistant acid phosphatase-positive osteoclast-like cells. This study may provide novel therapeutic targets for treatment of prostate cancer skeletal metastasis.

Extracellular Calcium as a Candidate Mediator of Prostate Cancer Skeletal Metastasis

Prostate cancer almost exclusively metastasizes to skeletal sites, indicating that the bone provides a favorable microenvironment for its localization and progression. A natural yet understudied factor in bone that could facilitate tumor localization is elevated extracellular calcium ([Ca2+]o). The present study found that elevated [Ca2+]o (2.5 mmol/L) enhanced proliferation of skeletal metastatic prostate cell lines (PC-3 and C4-2B), but not the nonskeletal metastatic, epithelial-derived prostate cell line LNCaP. The proliferative effect of elevated [Ca2+]o was associated with higher expression of the calcium-sensing receptor (CaSR), a heterotrimeric G-protein-coupled receptor that is the predominant cell-surface sensor for [Ca2+]o. Knockdown of the CaSR via RNA interference reduced cell proliferation in vitro and metastatic progression in vivo. CaSR signaling in PC-3 cells was evaluated by measuring the elevated [Ca2+]o-dependent inhibition of cyclic AMP accumulation, induced by either prostaglandin E2 or forskolin. Elevated [Ca2+]o stabilized expression of cyclin D1, a protein required for cell cycle transition. Furthermore, elevated [Ca2+]o triggered activation of the Akt signaling pathway and enhanced PC-3 cell attachment. Both pertussis toxin (a G-protein inhibitor) and LY294002 (an inhibitor of Akt signaling) reduced cell attachment. These data suggest that elevated [Ca2+]o following increased bone remodeling could facilitate metastatic localization of prostate cancer via the CaSR and the Akt signaling pathway. Taken together, [Ca2+]o is a candidate mediator of prostate cancer bone metastasis.

A synthetic peptide, PCK3145, for the treatment of hormone refractory prostate cancer

4654 Background: PCK3145 is a 15-mer synthetic peptide derived from Prostate Secretory Protein (PSP94). PSP94 is down regulated in advanced prostate cancer suggesting a role as survival mechanism for prostate cancer cells. PCK3145, as PSP94, demonstrated anti-tumor activities both in vivo and in vitro. Its underlying mechanism of action is thought to be by activation of receptor-mediated signal transduction pathways that cause apoptosis and normalize matrix metalloproteinase-9 (MMP-9) levels in the treated animals. Methods: Given the pre-clinical data in animal models suggesting that PCK3145 reduces prostate tumor growth, a dose-escalating study was conducted in 16 patients (pts) with metastatic hormone refractory prostate cancer (HRPC). Four cohorts of 4 pts received doses of 5, 20, 40 or 80 mg/m2. One cycle consisted of i.v. administration of PCK3145, three times per week for 4 weeks followed by a 7-day post-treatment period. Results: On the average, pts received two cycles although one pt received 5 cycles without adverse toxicity. The mean elimination half-life ranged from 0.354 to 1.45 hours with doses from 20 to 80 mg/m2. Mean Clearance appeared to increase with increasing dose level and the mean AUC values did not increase proportionally over the dose ranges studied. Pts remain under continued observations for PSA assessment and radiographic progression with 3 pts having stable disease for 2 cycles. Overall, 7 pts had stable disease and 7 pts had progressive disease at the time of finishing the study. One pt had a unconfirmed partial response. The biological activity of PCK3145 was monitored by assessing in patient blood samples, the level of MMP-9, MMP-2, TIMP-1, in addition to PSA, PAP, CEA, NSE, hCG. A substantial reduction in MMP-9 level was observed in 7 pts with elevated MMP-9 levels at baseline; 6 pts with low baseline MMP-9 levels (<100 μg/L) had no change after the first cycle. Conclusions: The mechanism of action of PCK3145 and its effect as a single agent and in combination with other therapeutic drugs to block cancer growth and skeletal metastasis is currently being evaluated. A Phase II trial is currently under development to explore the impact of PC3145 on MMP levels and in delaying time to radiographic progression. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Procyon Biopharma Procyon Biopharma Procyon Biopharma Procyon Biopharma

New Prospectives of Prostate Cancer Gene Therapy: Molecular Targets and Animal Models

Prostate cancer is the most common cancer and the second leading cause of cancer-related death among North American men. The low cure rate for prostate cancer is associated with the fact that many patients have metastatic disease at the time of disease presentation. Currently available therapeutic modalities for prostate cancer, such as surgery, radiation, hormone therapy, and chemotherapy, have failed to cure patients because these therapies are not sufficiently tumor-specific, resulting in dose-limiting toxicity. Therefore, gene therapy may offer great promise in this regard. In this article, we summarize current advances in gene therapy technologies for the treatment of cancer in general, and future prospects for treatment of human prostate cancer metastasis. We specifically emphasize current studies for improvement, both in the efficiency and the specificity of viral and nonviral vectors, and restricted transgene expression in tumors, to achieve selective targeting with minimized host organ toxicity, based on the molecular understanding of potential regulatory differences between normal and tumor cells. Cell and animal models used to study prostate cancer growth, invasion, and metastasis, and their usefulness in preclinical evaluation of therapeutic vectors in the treatment of prostate cancer skeletal metastasis are also discussed.

Osteomimetic properties of prostate cancer cells: a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment.

Unlike most other malignancies, prostate cancer metastasizes preferentially to the skeleton and elicits osteoblastic reactions. We present a hypothesis, based upon results obtained from our laboratory and others, on the nature of progression of prostate cancer cells and their predilection to growth and metastasis in the bone microenvironment. We propose the hypothesis that osseous metastatic prostate cancer cells must be osteomimetic in order to metastasize, grow, and survive in the skeleton. The reciprocal interaction between prostate cancer and bone stromal growth factors, including basic fibroblast growth factor (bFGF), hepatocyte growth factor/scatter factor (HGF/SF), and especially the insulin growth factor (IGF) axis initiates bone tropism, and is enhanced by prostate secreted endothelin-1 (ET-1) and urokinase-type plasminogen activator (uPA). Growth factors and peptides that have differentiating activity, such as transforming growth factor beta (TGF-beta), parathyroid hormone-related protein (PTH-rp), and the bone morphogenetic proteins (BMPs), can shift local homeostasis to produce the characteristic blastic phenotype, via interaction with prostate-secreted human kalikrein 2 (hK2), and prostate-specific antigen (PSA). This proposal asserts that altering the expression of certain critical transcription factors, such as Cbfa and MSX in prostate cancer cells, which presumably are under the inductive influences of prostate or bone stromal cells, can confer profiles of gene expression, such as osteopontin (OPN), osteocalcin (OC), and bone sialoprotein (BSP), that mimic that of osteoblasts. Elucidation of common proteins, presumably driven by the same promoters, expressed by both prostate cancer and bone stromal cells, could result in the development of novel preventive and therapeutic strategies for the treatment of prostate cancer skeletal metastasis. Agents developed using these strategies could have the potential advantage of interfering with growth and enhancing apoptosis in both prostate cancer and bone stromal compartments. The selective application of gene therapy strategy, driven by tissue-specific and tumor-restricted promoters for the safe delivery and expression of therapeutic genes in experimental models of prostate cancer metastasis, is discussed.

BRONŞ KARSİNOMUNA BAĞLI İSKELET KASI METASTAZI

SUMMARY Skeletal muscle metastasis from Lung Cancer Skeletal muscle metastasis from Lung Cancer is very rare. We present two cases with the cases reported in the literature and the features of this type of metastasis.