The role of CIP4 in osteosarcoma: progress report
An ESUN Article
Editor's Note: The research study that Dr. Koshkina reports on here was co-funded by the Liddy Shriver Sarcoma Initiative and the FOSTER Foundation.
Osteosarcoma (OS) is the most frequent bone tumor that predominantly targets the adolescent age group. The lung is the most common and often the only site of metastasis for OS. This type of metastasis continues to confer a generally poor prognosis despite decades of trials using intensified dosing, different timing and variations in combinations of chemical agents. The response rate for patients who present with localized disease remains unchanged at approximately 65% for more than 20 years. The prognosis for OS patients who present with metastases is significantly worse, with few survivors. This indicates the urgency of developing novel treatment approaches for these patients.
Lung metastases in osteosarcoma patients indicate poor prognosis. These patients represent a real challenge for physicians, because metastatic disease is frequently resistant to chemotherapy. In the last 20 years, modifications to the standard treatments for these patients have not made any significant changes in prognosis. It is our goal to understand the mechanisms of metastases and, based on this knowledge, identify new targets that can be used for the development of new therapeutic approaches to treat metastatic osteosarcoma.
A recent breakthrough was achieved by adding liposomal muramyltripeptide (MTP) to the standard chemotherapy for patients with OS [1]. The use of this drug was recently approved by the European Medicines Agency. The idea of using this agent in OS came from the observation that many tumors, including OS, are immunologically "quiet", i.e. there is negligible presence of inflammatory cells in the tumor mass. Therefore, introduction of immonomodulators like MTP to patients with OS might activate immune cells and increase their infiltration into tumors. In preclinical and clinical studies it was proven that MTP was able to activate monocytes and macrophages to become tumoricidal for OS [2-4]. This is a bright example of how understanding the biology of tumors can contribute to the development of novel therapies.
We use similar approach in our studies. We recently found that one of the cytoskeleton molecules, Cdc-42 interacting protein 4 (CIP4), is expressed in different OS tumor cells and showed a trend for a higher expression pattern in metastatic sublines (unpublished data). The Liddy Shriver Sarcoma Initiative, together with the FOSTER Foundation, supported our proposal to study the role of CIP4 in OS tumorigenesis and metastogenesis.
Despite the fact that CIP4 was discovered in 1997 [5], most of the studies on its functional activity were done in macrophages, and very little is currently known about its role in tumor cells. It was shown that CIP4 is required to coordinate membrane tubulation with reorganization of the actin cytoskeleton during endocytosis. It binds to cell membrane lipids and promotes membrane invagination and formation of tubules (for details see review article Ref. 6). In order to understand the function of CIP4 in OS, we first transfected highly aggressive mouse OS cells, DLM8 with the plasmid that downregulated expression of CIP4 protein (DLM8/shCIP4). The behavior of these cells was initially studied in tissue culture. We found that the growth rate of DLM8/shCIP4 cells was reduced by 30% when compared with non-transfected DLM8 cells or DLM8 cells transfected with control plasmid (DLM8/sh). When DLM8/shCIP4 cells were injected into mice subcutaneously they formed tumors that grew significantly slower than DLM8 or DLM8/sh control cells (Fig. 1 , p<0.02). This is a new finding, which needs further investigation of the mechanism. We also found that inhibition of CIP4 expression in DLM8 cells decreased their mobility and invasion properties in vitro, which indicates on their ability to impair metastases growth.
Physiological function of CIP4 is associated with actin. In fact, it promotes Cdc42-induced actin polymerization [6]. Actin polymerization is required for the formation of podosomes, actin-rich adhesion structures specific to monocyte-derived cells [7]. Podosomes are necessary for directional movement of the cells, such as macrophages, and in osteoclasts they are thought to aid in the creation of sealing rings associated with the area of bone resorption [8]. In tumor cells podosome-similar structures are called invadopodia or “invasive feet” [9]. When we compared the actin filaments structures in DLM8/shCIP4 cells with wild-type DLM8 or DLM8/si control cells we observed lower perinuclear actin stress filaments formation and lower actin bridge to the lipid bilayer of the lipid cell membrane in them (Fig. 2). We also observed that downregulation of CIP4 changed cell membrane morphology in DLM8 cells – they had less protrusions and smoother cell surface than control cells. All these findings indicate that CIP4 may also play a role in metastatic behavior of these cells. We are currently working on studying the effect of CIP4 on the growth of OS metastases cells in vivo.
Summary and conclusions
OS remains a devastating disease, and better understanding of its biology is required for the development of novel therapeutic approaches. The information obtained in this study indicates that CIP4 may become a new target for OS treatment. We obtained the evidence that inhibition of CIP4 changes cytoskeleton arrangement of OS cells and alters the behavior of these cells in tissue culture changing its potential to impair growth of OS metastases. CIP4 caused the reduction of the primary tumor growth in vitro and in xenograft subcutaneous animal model. Work should be done in the future to better understand the mechanisms of CIP4 activity in OS tumors and to characterize the effect of CIP4 on the growth of OS metastases.
CIP4 may become a novel target for osteosarcoma treatment. Due to its effect on the cytoskeletal structure, it changes the proliferation growth of OS in vitro and in animal models. Our preliminary findings indicate its potential to affect metastatic osteosarcoma growth in the lungs. Currently the role and mechanisms of cytoskeletal remodeling during the metastatic process are receiving much attention, but such information specific to sarcomas is limited, and further study is needed.
References
1. Chou, A.J., et al., Addition of muramyl tripeptide to chemotherapy for patients with newly diagnosed metastatic osteosarcoma: a report from the Children's Oncology Group. Cancer, 2009.
2. Kleinerman, E.S., Biologic therapy for osteosarcoma using liposome-encapsulated muramyl tripeptide. Hematol Oncol Clin North Am, 1995. 9(4): p. 927-38.
3. Kleinerman, E.S., et al., Activation of tumoricidal properties in human blood monocytes by liposomes containing lipophilic muramyl tripeptide. Cancer Res, 1983. 43(5): p. 2010-4.
4. Kleinerman, E.S., et al., Activation of tumoricidal properties in monocytes from cancer patients following intravenous administration of liposomes containing muramyl tripeptide phosphatidylethanolamine. Cancer Res, 1989. 49(16): p. 4665-70.
5. Aspenstrom, P., A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton. Curr Biol, 1997. 7(7): p. 479-87.
6. Aspenstrom, P., Roles of F-BAR/PCH proteins in the regulation of membrane dynamics and actin reorganization. Int Rev Cell Mol Biol, 2009. 272: p. 1-31.
7. Linder, S., et al., Microtubule-dependent formation of podosomal adhesion structures in primary human macrophages. J Cell Sci, 2000. 113 Pt 23: p. 4165-76.
8. Jurdic, P., et al., Podosome and sealing zone: specificity of the osteoclast model. Eur J Cell Biol, 2006. 85(3-4): p. 195-202.
9. Caldieri, G., et al., Cell and molecular biology of invadopodia. Int Rev Cell Mol Biol, 2009. 275: p. 1-34.
V6N5 ESUN Copyright © 2009 Liddy Shriver Sarcoma Initiative.