Renal Cell Carcinoma Bone Metastasis Research Articles

149 CALCIUM PROMOTES THE FORMATION OF BONE METASTASES VIA THE CALCIUM SENSING RECEPTOR IN PATIENTS WITH RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Although a number of molecules have been implicated in the process of formation of cancer metastases, the organ-selective nature of cancer cells is still poorly understood. Renal cell carcinoma (RCC) often metastasize into bones, a calcium enriched tissue. We analyzed the influence of the calcium sensing receptor (CaSR) in RCC and an enhanced extracellular calcium concentration in processes of metastatic spread. METHODS: In 33 matched RCC specimens, 11 non-metastasized, 11 metastasized into the lung and 11 metastasized into bones five years after nephrectomy, CaSR was quantified by RT-PCR and Western blot analyses. To investigate the influence of enhanced extracellular calcium on the formation of metastases, primary RCC cells were isolated from patients developing bone, lung and no metastasis five years after nephrectomy (each 3 cell lines). Cell proliferation after calcium treatment (2.5, 5 and 10 M) was quantified by BrdU incorporation. Cell migration was determined in a Boyden chemotaxis assay with calcium as chemotactical agent (10 M). Expression and activity of intracellular signal transduction mediators were analyzed by a human phospho kinase array and Western blot analyses. RESULTS: Compared to non-metastasizing RCC, CaSR mRNA expression was 10.8-fold increased and CaSR protein level was 1.5-fold increased in tumor specimens of patients developing bone metastases. In primary RCC cells with a high bone metastastic potential CaSR expression was 3.7-fold higher compared to non-metastasizing RCC cells. Calcium treatment of bone-metastasizing RCC cells, but not of non-metastasizing cells, induced a concentration dependent enhancement of proliferation. In a Boyden chamber experiment with calcium as chemo attractant, RCC cells from patients with bone metastases showed a 9.4-fold higher migratory potential compared to cells from patients without metastases. In lung metastatic RCC cells migration was only 1.7-fold enhanced. The enhanced proliferation and migration of cells with high potential to build bone metastases seem to be caused by an activation of Akt and a highly increased integrin alpha 5 expression. Additionally, the tumor suppressor PTEN was markedly reduced in tumors metastasizing into bones and after calcium treatment, promoting tumor progression. CONCLUSIONS: Our results indicate that formation of bone metastases of RCC is caused by an enhanced expression of the CaSR. Extracellular calcium has a promoting effect on cell migration and proliferation of bone metastasizing RCC cells via Akt signalling and integrin alpha 5.

A bone-seeking clone exhibits different biological properties from the ACHN parental human renal cell carcinoma in vivo and in vitro

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently. Very little is currently known about the mechanism of preferential metastasis of RCC to bone. We hypothesize that RCCs that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (ACHN-BO) clones of the human RCC cell line ACHN by repeated four passages in nude mice and in vitro of metastatic cells obtained from bone. These clones were examined for distinguishing biological characteristics and compared with the ACHN parental cells (ACHN-P) in vivo and in vitro. Our results showed that the ACHN-BO cell line could be successfully obtained by in vivo selection through the lateral tail vein. This approach results in the development of multiple osteolytic lesions in the distal femora and proximal tibiae within four weeks after inoculation, with a success rate of 85–100% and no additional comorbidity. ACHN-P cells developed metastases in lung, bone, brain, ovary and adrenal glands. Conversely, ACHN-BO cells exclusively metastasized to bones with larger osteolytic lesions. Compared with the ACHN-P cell line, the proliferation ability in ACHN-BO6 was increased by 9.68 and 6.42%, respectively (P<0.05), while the apoptotic ratio decreased significantly (P<0.05) and cells were blocked in the S phase with suppressed migration and invasion capacities. The ACHN-BO6 cell line produced greater amounts of the pro-angiogenic factors VEGF and TGF-β than ACHN-P. Our data suggest that these phenotypic changes allow RCC cells to promote osteoclastic bone resorption, survive and proliferate in bone, which consequently leads to the establishment of bone metastases. This model provides a reliable reproduction of the clinical situation and, therefore, is suitable for designing and evaluating more effective treatments for RCC bone metastasis.

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Background: The prognoses of patients with bone metastases, a common type of metastasis for renal cell carcinoma (RCC), is poor when treated with cytokines; average life expectancy is 8 to 16 months. Sunitinib is a newly approved, multitargeted, small-molecule tyrosine kinase inhibitor for the treatment of metastatic RCC (mRCC). In this study, we show that sunitinib has anticancer activity as well as inhibitory activity against osteolysis in an experimental mouse model of bone metastasis of RCC cells. Methods: In vitro cell proliferation was determined using the MTS assay. Mice, which had RCC cells transplanted into the left ventricle for the establishment of bone metastases, were treated orally with sunitinib (40 mg/kg/day) or placebo for 3 weeks. Growth of the cancer cells was monitored using an in vivo imaging system. In addition, a total of 16 patients with mRCC were treated with sunitinib; serum and urine levels of amino-terminal telopeptide (NTx) were measured as markers of bone resorption. Results: Sunitinib did not inhibit the growth of RCC cells in vitro at clinically or experimentally achievable serum levels (100 nM – 1 mM). To investigate the inhibitory effect of sunitinib in vivo, we established luciferase-labeled human RCC cells (ACHN Luc ). Metastatic bone lesions of ACHN Luc cells in the control group progressed during the 3 weeks. On the other hand, photon emissions were significantly suppressed in the sunitinib treatment group (P ,.001). The mean body weights of mice did not differ significantly between the 2 groups. The mean number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in mice treated with sunitinib was significantly lower than that in mice treated with vehicle control (23.1+ 4.7 versus 33.2+ 7.9 osteoclasts/100 high-power fields, respectively; P ¼ .0137). In addition, serum and urine levels of NTx in patients with mRCC declined significantly during the first 4 weeks of sunitinib treatment (P ¼ .027). Conclusion: In an animal model, we demonstrated that oral administration of a clinically achievable dose of sunitinib prevented the growth of RCC bone metastases in vivo. Because RCC cell lines are resistant to clinically and preclinically achievable plasma concentrations of sunitinib in vitro, prevention of osteoclast activity and/or maturation are possible mechanisms of sunitinib mediated growth inhibition in metastatic bone lesions in vivo. Our study supports the use of sunitinib as an initial treatment for RCC patients with bone metastasis.

Skeletal complications and survival in renal cancer patients with bone metastases

Skeletal metastases occur in around one third of patients with advanced or metastatic renal cell carcinoma (RCC). Skeletal involvement is commonly an aggressive, lytic process which causes substantial morbidity through skeletal complications and occurrence of skeletal related events (SREs). However, compared with bone metastases in breast and prostate cancer, there is a paucity of data relating to the demographics of bone metastases in RCC and their sequelae in terms of SREs and survival. The study population included all patients ( N = 803) with advanced or metastatic RCC treated in a tertiary centre serving a regional population of 2.6 million between 1998 and 2007. Demographic and survival data and information relating to metastatic disease were extracted from electronic records. Thirty-two percent ( N = 254) of the study population presented with ( N = 131) or later developed ( N = 123) bone metastases and 83% of these ( N = 210) also developed metastases elsewhere. The mean number of SREs experienced by the bone metastatic patients over the course of their disease was 2.4 and only 37 patients experienced no SRE. A high proportion of patients (80%) received radiotherapy for bone pain and there was a surprising and strikingly high incidence of spinal cord/nerve root compression, which was experienced by 28% patients. Although bisphosphonate use increased following the availability of zoledronic acid in 2004, approximately 50% patients with bone metastases did not receive bisphosphonate treatment. The skeletal morbidity rate (number of SREs per patient years at risk) was 1.0 and 1.4 for patients who received or did not receive bisphosphonates, respectively. The median survival following diagnosis of RCC was similar in patients who developed bone metastases (20.4 months) and those who did not (20.9 months). Median survival from diagnosis of metastases was 13.3 months for those who never developed bone metastases, 10.6 months for those who presented with them, 19.6 months for those who developed them later and 22.6 months for patients who had bone only metastases. This is the largest study to date focusing specifically on skeletal complications in RCC. A striking finding was the high incidence of spinal cord/nerve root compression and more research into this area is needed. Clearer, internationally accepted guidelines are recommended for the management of this patient group.

Identification of a novel molecular signature associated with renal carcinoma bone metastasis.

e15089 Background: Although recent advancements have been made in the understanding of breast and prostate bone metastasis, there has been less knowledge in the area of metastatic renal cell carcinoma (RCC) to bone, a frequent site of systemic spread of renal cancer. This particular cancer in bone remains relatively resistant to standard forms of treatment such as radiation and chemotherapy. A better understanding of the biology of RCC bone metastasis and the identification of predicting biomarkers of bone metastasis is critically needed and could be useful to identify sub- clinical disease, may improve staging and guide adjuvant therapeutic decisions. Methods: The purpose of this study was to investigate in a multiparametric manner the expression of a panel of biomarkers involved in angiogenesis and tumor invasion, in primary tumors and normal tissues both obtained from clear-cell RCC patients with and without bone metastases. Results: Using a multiplex real-time RT-PCR assay we identified a novel combination of biomarkers involved in angiogenesis and tumor invasion, associated with the risk of bone metastasis. Indeed, among the 25 transcripts of genes studies: VEGF-R1, VEGF-R2, Hif-1α, uPA and PAI-1 overexpression in tumor tissues was significantly associated with the presence of bone metastasis (p = 0.02, 0.02, <10-4, 0.04 and 0.03 respectively). No differences where found of these transcripts expression in corresponding normal tissues. Conclusions: Our results suggest that this novel signature which discriminate the RCC primary cancers with a significantly higher risk of bone metastasis can improve risk stratification independently of known standard clinical variables. A validation in a larger series will help to incorporate this molecular signature with clinical variables to refine prognosis and prediction in metastatic clear-cell RCC patients treated with novel tyrosine kinase-targeted agents. No significant financial relationships to disclose.

Characterization of a new renal cell carcinoma bone metastasis mouse model

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently and becomes more and more prevalent presumably because survival times among patients with disseminated cancers are increasing. Patients with bone metastases from renal cell carcinoma suffer from severe pain, nerve compression syndromes and pathologic fractures. Very little is known about the mechanisms of skeletal metastases of RCC. Thus, to better understand the molecular mechanism of renal cell cancer (RCC) bone metastasis, it is crucial to develop new animal models. We have established a new animal model of RCC metastasis to bone by inoculation of human 786-O/luciferase cells into the left cardiac ventricle of athymic nude mice. The animals developed aggressive osteolytic bone destruction as monitored by radiography and micro-CT-scans with the mean endpoint at 62 +/- 8 days. The extensive bone destruction observed was comparable to the clinical setting and mainly occurred in hind limbs, forelimbs and the spine. The tumors were primarily located within the bone and resulted in destruction of cortical bone. No soft tissue metastases were detected by BLI or histomorphometry. To increase the bone-metastatic potential of the 786-O cell line, an in vivo selection was done yielding a subpopulation causing osteolytic lesions with the mean endpoint of 47 +/- 3 days. The selected subline secreted more proangiogenic factors VEGF and bFGF in vitro compared to the parental cell line suggesting that these tumors are highly vascular. This model provides a reliable reproduction of the clinical situation and therefore, is suitable for designing and evaluating more effective treatments for RCC bone metastasis.

Identification of a Novel Biomarker Signature Associated with Risk for Bone Metastasis in Patients with Renal Cell Carcinoma

When renal cell carcinoma (RCC) metastasizes to bone (a frequent site of systemic spread of this cancer) it becomes highly resistant to radiation therapy and chemotherapy. A better understanding of the biology of bone metastasis in RCC may permit to identify biomarkers for early detection of subclinical disease and better stratification of patients prior to treatment. We therefore investigated in this study, using a multiplex real-time RT-PCR assay, the expression of a panel of 16 biomarkers involved in angiogenesis and tumor invasion; the panel was applied to primary tumors and normal tissues obtained from clear-cell RCC patients with and without bone metastases. We identified a novel combination of biomarkers associated with the risk of bone metastasis. Among the transcripts of the genes studied, VEGFR-1, VEGFR-2, HIF-1alpha, uPA , and PA I-1 overexpression in tumor tissues was significantly associated with the presence of bone metastasis (p=0.02, p=0.02, p<0.0001, p=0.04, and p=0.03, respectively). No differences were found in the expression of these transcripts in the corresponding normal tissues. This preliminary study provides a promising tool that may help in the management of RCC patients with bone metastasis. Indeed, these predictive markers could be useful to identify subclinical disease, improve staging, and guide treatment decisions.

Efficacy of targeted therapy in patients with renal cell carcinoma with pre-existing or new bone metastases

This single-centre retrospective analysis of data from three randomised studies and two expanded-access studies compared the effect of interferon (IFN)-alfa, sunitinib, and sorafenib on the occurrence and progression of metastatic bone lesions in patients with renal cell carcinoma (RCC). The analysis included 292 patients: 107 received sunitinib 50 mg/day in 6-week cycles (Schedule 4/2), 147 received sorafenib 800 mg/day, and 38 received placebo or IFN-alfa 9 MU t.i.w. Pre-existing metastatic bone lesions were reported in 82 patients, of which 30 experienced progression. Twenty-three of 210 patients developed new bone lesions. Overall, sunitinib appeared slightly more effective than sorafenib or IFN-alfa at extending mean time to progression of pre-existing bone lesions (P = 0.057). Compared with sorafenib, sunitinib significantly decreased formation (P = 0.034) and prolonged time to occurrence of new bone lesions (P = 0.047). Further evaluation of the effect of these therapies on bone metastases in RCC is warranted.

MMP-13 is over-expressed in renal cell carcinoma bone metastasis and is induced by TGF-β1

Bone metastasis occurs frequently in renal cell carcinoma (RCC) patients causing significant morbidity by stimulating excessive osteolysis, yet the mechanisms responsible have been little studied. Matrix metalloproteinases (MMPs) are over-expressed in many cancer types and are believed to play a role in bone metastasis, however, the expression of MMPs in RCC bone metastasis (RBM) has not been investigated. Due to their ability to degrade the main component of organic bone matrix, type I collagen, we investigated the expression of MMP-1, -2, -8, -9, and -13 in RBM. By quantitative (q)RT-PCR, expression of MMP-13 was significantly increased in RBM tissues relative to that in RCC and adjacent normal kidney while no differences in the expression of MMP-1, -2, -8, or -9 mRNA were observed. Correspondingly, increased expression of MMP-13 protein was also observed in RBM relative to RCC by immunohistochemical analysis. Intriguingly, the expression of MMP-13 in the human RBM cell line RBM1-IT4 was stimulated by TGF-beta1, a growth factor abundant in the bone microenvironment and known to promote RBM-induced osteolysis in animals. Exposure of RBM1-IT4 cells to TGF-beta1 increased MMP-13 mRNA levels as well as the latent and active forms of MMP-13 protein. Further, stable expression of a dominant-negative TGF-beta type II receptor in RBM1-IT4 cells inhibited MMP-13 expression following TGF-beta1 exposure. These data suggest that MMP-13 expression is elevated in RBM relative to primary RCC and adjacent normal kidney, and is regulated at the cellular level by TGF-beta1.

Macrophage Inflammatory Protein–1δ: A Novel Osteoclast Stimulating Factor Secreted by Renal Cell Carcinoma Bone Metastasis

Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1 delta (MIP-1 delta), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1 delta expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1 delta, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1 delta stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1 delta treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1 delta significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1 delta expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.

P14. Development and characterization of a new renal cell carcinoma (RCC) bone metastasis mouse model

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently and is becoming more and more prevalent presumably because survival times among patients with disseminated cancers are increasing. Patients with bone metastases from renal cell carcinoma suffer from severe pain, nerve compression syndromes and pathological fractures. Very little is known about the mechanisms of skeletal metastases of renal cell carcinoma. Therefore, it is important to develop animal models of RCC bone disease to better understand the biology of RCC bone metastasis and improve therapy options.

Renal cell carcinoma bone metastasis—elucidating the molecular targets

The development of bone metastasis from renal cell carcinoma (RCC) signals a transition to a terminal state for the patient with previously isolated disease. These patients may suffer the morbidity of severe, persistent pain, pathologic fractures, and spinal compression from vertebral metastasis before they succumb to their cancer. Although recent advancements have been made in the understanding of breast and prostate bone metastasis, there has been less knowledge in the area of metastatic RCC to the skeleton. This particular cancer in bone remains relatively resistant to standard forms of treatment such as radiation and chemotherapy. A better understanding of the biology of RCC bone metastasis is critically needed in order to improve treatment. Bone-derived cell lines and an experimental animal model have been developed in order to explore the relevant mechanisms of how RCC cells survive within and destroy the bone. This review will focus on the growth factor signaling pathways most important for the RCC-stimulated osteoclast-mediated bone destruction, namely the epidermal growth factor receptor (EGF-R) and transforming growth factor-beta receptor (TGF-betaR) pathways. By inhibiting these receptors, growth of RCC within the bone is decreased which, directly or indirectly, decreases bone destruction.

TGF-β Promotes the Establishment of Renal Cell Carcinoma Bone Metastasis

Bone metastases develop in approximately 30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF-beta1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone. Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-beta1. TGF-beta1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-beta1 in the growth of RCC bone metastasis (RBM). To inhibit TGF-beta1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-betaRII cDNA were generated. The in vivo effect of TGF-beta1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-beta1 on RBM cell growth was determined after TGF-beta1 treatment by MTT assay. TGF-beta1 and the TGF-beta receptors I and II (TGF-betaRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-beta1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-beta1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-beta1 may promote RBM tumor growth indirectly in vivo. TGF-beta1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-beta1 signaling may be useful in the treatment of RBM.

Renal cell carcinoma bone metastasis: epidermal growth factor receptor targeting.

Renal cell carcinoma frequently metastasizes to the skeleton in the later stages of the disease. Patients with bone metastasis from renal cell carcinoma experience severe pain, neurologic compromise, and frequent pathologic fractures. These tumors are relatively resistant to chemotherapy and radiation, and the 5-year survival of patients is less than 10%. The epidermal growth factor receptor is overexpressed in human renal cell carcinoma and hypothesized to be a potential target in the treatment of bone metastasis. Using in vitro studies, it was shown that blockade of the epidermal growth factor receptor was effective in decreasing proliferation and receptor autophosphorylation of a human bone-derived renal cell carcinoma cell line. In an experimental nude mouse model, treatment with Taxol and protein tyrosine kinase inhibitor 166, a small molecule receptor tyrosine kinase inhibitor, blocked the growth of renal cell carcinoma in the tibia and resulted in decreased bone destruction. The use of protein tyrosine kinase inhibitor 166 and Taxol was cytostatic and nontoxic in long-term animal experiments. Epidermal growth factor receptor blockade is an exciting potential therapy for renal cell carcinoma bone metastasis in humans, but because it is cytostatic rather than cytotoxic, its optimal role may be as a supplement to cytotoxic chemotherapy. It ultimate role and its relationship to other therapeutic interventions remains to be elucidated and validated.

Characterization of a Renal Cell Carcinoma Cell Line Derived From a Human Bone Metastasis and Establishment of an Experimental Nude Mouse Model

We provide the necessary tools to study the biology of bone metastasis using renal cell carcinoma as a model. A relevant renal cell carcinoma cell line developed from a human bone metastasis is described and an experimental model in the nude mouse was established. The novel cell line RBM1 was developed from bone metastasis from a patient with renal cell carcinoma. In vitro methods used to study the cell line included karyotype analysis, reverse-transcriptase polymerase chain reaction, ribonuclease protection assay and Western blot analysis. An intratibial injection model in the nude mouse resulted in bone lesions similar to those in human patients. Chromosomal analysis of this cell line revealed characteristic cytogenetic abnormalities common in renal cell carcinoma. RBM1 cells expressed parathyroid hormone parathyroid hormone related peptide, interleukin-6 and macrophage colony-stimulating factor, which are cytokines involved in osteoclast activation and subsequent bone resorption. Western blot analysis revealed the presence of high levels of epidermal growth factor receptor and c-MET. Reproducible osteolytic lesions developed in the nude mouse after injecting 1 x 106 cells into the tibia. This cell line and animal model may allow further study of the biology and mechanism of renal cell carcinoma bone metastasis.

Immunoperoxidase staining of fine-needle aspiration biopsies of renal cell carcinoma using tumor-specific monoclonal antibody.

In 30 nephrectomy specimens, fine needle aspiration biopsies (FNAB) were performed both in the tumor and in the macroscopically normal part of the kidney: 18 were well differentiated renal cell carcinoma (RCC), 6 were moderately differentiated RCC, 4 were poorly differentiated RCC, and 2 were oncocytomas. FNAB was also performed in a bone metastasis of RCC. FNAB materials were stained using the immunoperoxidase method with RCC-specific monoclonal antibody and were compared with the staining of frozen sections. In all cases where tumor-antigen expression could be demonstrated in the frozen sections, a FNAB had already proven positive. There were only 2 false-negative cases where sufficient cellular material could not be obtained due to extensive tumor necrosis. There was no false-positive case. Oncocytomas showed no expression of antigen. The use of immunoperoxidase staining of FNAB with tumor-specific monoclonal antibody is discussed.