Elsevier

Matrix Biology

Volume 20, Issue 7, November 2001, Pages 429-437
Matrix Biology

Type I collagen-mediated proliferation of PC3 prostate carcinoma cell line: implications for enhanced growth in the bone microenvironment

https://doi.org/10.1016/S0945-053X(01)00159-7Get rights and content

Abstract

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to bone. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as, extracelluar matrix components, have been implicated in the spread and propagation of prostatic carcinoma. The prostate cell line, PC3, adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-l-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. Antibody function blocking studies reveal that PC3 cells can utilize α2β1 and α3β1 integrins to adhere to collagen I. Cells plated on collagen I exhibit increased rates of proliferation over cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of cyclin D1, a molecule associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), map kinase (MAPK) and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. Type I collagen may facilitate the colonization and growth of metastatic prostate tumor cells in the bone microenvironment.

Introduction

The American Cancer Society estimates that in the year 2000, 180,400 cases of prostate cancer will be diagnosed, and 31,900 American men will succumb to the disease, making prostate cancer the second leading cause of male-related cancer deaths in the United States (Greenlee et al., 2000). While prostate specific antigen (PSA) screening has assisted clinicians to diagnose prostate cancer in its early stages, some patients still present with metastatic lesions in the bone. Additionally, more than 70% of patients with advanced disease exhibit bone metastases (Chiarodo, 1991). These patients experience significant pain due to spinal cord compression and pathological bone fractures. Most significantly, metastatic prostate cancer is often refractory to most therapeutic modalities, representing a major obstacle in treating patients with advanced disease. Often, palliative treatment is the sole option in these cases. Since a prominent feature of prostate cancer is its ability to colonize and thrive in skeletal tissue, better understanding of the complex biology of prostate cancer bone metastasis will allow for the development of novel and effective treatments for patients with advanced metastatic disease.

Bone is a dynamic tissue that is constantly being remodeled under the direction of various systemic hormones and locally derived cytokines and growth factors. Remodeling is the result of the coupled action of two bone-specific cell types. The hematopoietic-derived osteoclasts degrade bone, while the stromally derived osteoblast is responsible for directing new bone formation (Canalis et al., 1991, Mundy, 1999). Osteoblast activity results in the deposition of numerous growth factors and extracellular matrix molecules (ECM) in mineralized bone tissue, and remodeling cycles liberate a variety of chemotactic substances that may influence the homing to and initial seeding of bone by metastatic prostate cells (Hullinger et al., 1998, Lang et al., 1995). Additionally, remodeling activity may make available various mitogens, such as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), that possibly potentiate the proliferation and survival of prostate cancer cells in bone (Gleave et al., 1992, Iwamura et al., 1993). Clearly, the skeletal microenvironment is an inviting target for prostate cancer cell metastasis.

In addition to the supply of growth factors, the ECM of skeletal tissue also can impact skeletal metastasis of prostate cancer. The organic component of the bone ECM consists primarily (>95%) of type I collagen which is uniquely post-translationally modified to provide a scaffold for hypertrophic growth (Termine, 1990). The remaining non-collagenous component consists of molecules such as bone sialoprotein, osteopontin, thrombospondin, vitronectin, fibronectin, osteocalcin and osteonectin. Previous studies show that these non-collagenous proteins either by themselves or in various combinations can promote prostate cancer cell adhesion and growth (Jacob et al., 1999, Koeneman et al., 1999, Lecrone et al., 2000, Thalmann et al., 1999). However, given its abundance in the bone matrix, the role collagen I may play in prostate cancer bone metastasis has yet to be elucidated. Previous studies demonstrate that collagen I serves as an adhesive substrate for numerous cancer cells and may influence adhesion and retention of various metastatic cells in skeletal tissue (Klein et al., 1991, Kostenuik et al., 1996, Kostenuik et al., 1997, Ridley et al., 1993). Type I collagen, therefore, is an attractive candidate molecule capable of influencing homing and adherence of prostate cells to bone.

Prostate cancer bone metastasis involves the complex interplay of prostate tumor cells and the skeletal microenvironment. Knowledge of this interplay is important in designing effective therapies to treat patients with advanced prostatic skeletal disease. In the present study, we provide evidence that the major component of bone ECM, type I collagen, may serve as a mediator of prostate tumor bone metastasis. Employing the human prostate carcinoma cell line, PC3, we identified type I collagen as a major adhesive substrate for PC3 cells and demonstrate that type I collagen serves as a permissive substrate for enhanced PC3 cell proliferation. Inhibitor studies suggest that the phosphatidylinositol 3-kinase (PI3K) pathway is the major signaling molecule responsible for PC3 cell proliferation on a type I collagen matrix. These results suggest a role for collagen I in facilitating prostate cancer cell bone metastasis by enhancing both cellular attachment and proliferation.

Section snippets

Cell lines and reagents

PC3 cells, a human prostatic carcinoma cell line derived from a bone metastasis, were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum (FCS) (Atlanta Biologicals, Norcross, VA) and 100 U/ml of penicillin and 100 μg/ml of streptomycin (Life Technologies, Rockville, MD). The inhibitors LY29002, rapamycin and PD98059 were obtained from Calbiochem (La Jolla, CA). Blocking antibodies

PC3 cell adhesion to type I collagen

Initial experiments were carried out to investigate the adhesive properties of PC3 cells to the predominant bone component, type I collagen. Fibronectin (FN), a major integrin adhesive substrate for numerous cell types, was used as a comparison matrix for PC3 cell adhesion. Adhesion results were normalized to poly-l-lysine (PLL), a receptor independent adhesive substrate. Adhesion of cells to plates coated with BSA served as the negative control. Serial dilutions were performed with the test

Discussion

Skeletal tissue represents an inviting target for the metastatic spread of prostate cancer cells. The rich supply of growth factors located in the bone microenvironment potentiates the metastatic spread and growth of prostate tumor cells. The skeletal ECM also may influence prostate metastasis by promoting attachment, growth and subsequent survival of prostate cancer cells in bone. The major component of the skeletal ECM is type I collagen. In agreement with a report by Kostenuik and colleagues

Acknowledgements

We wish to express our thanks to Ms Sharron Kingston for her secretarial assistance in the preparation of this manuscript. This work was supported by Department of Defense Prostate Research Grant DAMD17-98-1-8655 (M.C.F.-C.).

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