The Role of α_vβ₃ in Prostate Cancer Progression

Abstract

Integrin α_vβ₃ is involved in varied cell biological activities, including angiogenesis, cell adhesion, and migration on several extracellular matrix components. Although α_vβ₃ is not typically expressed in epithelial cells, it is expressed in macrophages, activated leukocytes, cytokine-stimulated endothelial cells, osteoclasts, and certain invasive tumors. Interestingly, the adhesion and migration of breast cancer cells on bone matrix are mediated, in part, by α_vβ₃. Similar to breast cancer cells, prostate cancer cells preferentially metastasize to the bone. The biological events that mediate this metastatic pattern of prostate cancer are not well defined. This review discusses the role α_vβ₃ plays in prostate cancer progression, with specific emphasis on bone metastasis and on α_vβ₃ signaling in prostate cancer cells. The data suggest that α_vβ₃, in part, facilitates prostate cancer metastasis to bone by mediating prostate cancer cell adhesion to and migration on osteopontin and vitronectin, which are common proteins in the bone microenvironment. These biological events require the activation of focal adhesion kinase and the subsequent activation of PI-3 kinase/Akt signaling pathway.

Introduction

Integrins play a significant role in prostate tumor progression [1]. Schmelz et al. demonstrated that well-differentiated tumors had a higher expression of α₃ and α₆ integrins compared to anaplastic tumors. This study also reported that an increase in α₆β₁ integrin expression on prostate cancer cells was associated with invasion of the seminal vesicles. The expression of integrin α_vβ₃ was not monitored in this study [1]. Unfortunately, the information regarding the role of α_vβ₃ in prostate tumorigenesis and metastasis is limited. This review will infer from the literature the potential role this integrin plays in prostate tumor progression.

Integrin α_vβ₃ mediates cell adhesion and migration on a variety of extracellular matrix (ECM) proteins, including vitronectin, fibronectin, fibrinogen, laminin, collagen, osteopontin, and others [2]. It is involved in osteoclast adhesion to bone matrix components osteopontin and bone sialoprotein (BSP) and subsequent degradation of the bone [3,4]. This integrin also mediates osteoblast adhesion to osteopontin at the site of bone resorption [5]. The adhesion of breast cancer cells to bone matrix and the migration of breast cancer cells in BSP are mediated by α_vβ₃ [6,7]. Glioblastoma multiforme adhesion to fibronectin andvitronectin is also mediated by α_vβ₃ [8]. The expression of α_vβ₃ has been detected on macrophages, activated leukocytes, cytokine-stimulated endothelial cells, osteoclasts, and certain invasive tumors; however, it is not commonly expressed in epithelial cells [2]. Angiogenesis facilitates the growth and metastasis of solid tumors by, providing nutrients to the expanding mass and providing a pathway for tumor cell dissemination. The expression of α_vβ₃ on endothelial cells plays an important role in this process [2]. Endothelial cells stimulated by tumor-derived angiogenic factors enter the cell cycle and express the integrin α_vβ₃, which allows endothelial cells to interact with a wide variety of ECM proteins as they invade the tissue surrounding the tumor. Antagonists to this integrin induce apoptosis specifically in angiogenic endothelial cells, thereby facilitating regression in several tumors including breast [9–11].

One critical function of α_vβ₃ is to inhibit endothelial cell apoptosis during angiogenesis. The ligation of endothelial α_vβ₃ to osteopontin and vitronectin inhibits apoptosis in endothelial cell cultures upon serum withdrawal. This α_vβ₃-mediated activity requires the activation of NF-κB because nonphosphorylatable IκB completely blocks the survival effect of osteopontin and vitronectin [12]. Nonphosphorylated IκB binds NF-κB in the cytoplasm. When properly stimulated, IκB is phoshorylated, ubiquinated, and degraded, thereby releasing NF-κB to translocate to the nucleus and activate specific genes. In endothelial cells, NF-κB stimulates the expression of osteoprotegerin, which has been implicated as a cell survival factor due to its interaction with TNF-related apoptosis-inducing ligand. The mechanism used by osteoprotegerin to protect endothelial cells from apoptosis induced by serum withdrawal is not known [13].

The Expression of α_vβ₃ on Prostate Cancer Cell Lines

The surface expression of α_vβ₃ is established in commonly used prostate cancer cell lines, including DU145, PC-3, and TSU [14,15]. The expression of α_vβ₃ on LNCaP cells is in dispute. Although Witkowski et al. [15] detected both α_v and β₃ integrin subunits in LNCaP cells, Zheng et al. [16] demonstrated that LNCaP cells did not express heterodimer α_vβ₃. These conflicting results may be due to the types of antibodies used in each study. The former study [15] used antibodies to the monomeric α_v and β₃ subunits, whereas the latter study [16] used antibodies that recognize the α_vβ₃ heterodimer. In addition to the previously mentioned prostate cancer cell lines, Putz et al. [17] established four prostate cancer cell lines that were derived from bone marrow aspirates and demonstrated that all four cell lines expressed α_v and β₃ integrin subunits. Interestingly, breast and lung cancer cell lines that were also derived from bone marrow aspirates also expressed α_vβ₃, suggesting that it may play a role in cancer cell metastasis to the bone marrow.

The Function of α_vβ₃ in Prostate Cancer Cells

The adhesion of cancer cells to the vascular endothelium is a critical step in the metastatic cascade and is mediated by integrins [18,19]. Romanov and Goligorsky [20] used phage display to identify integrins that are important mediators of prostate cancer cell adhesion to interleukin-1-stimulated human umbilical vein endothelial cells (HUVECs). Their data demonstrated that α_vβ₃, along with α₅β₁ and α₃β₁, mediated PC-3 and DU145 adhesion to HUVEC monolayers. Another study showed that murine melanoma cell adhesion to microvascular endothelium stimulated by eicosanoid 12(S)-hydroxyeicosatetraenoic acid was mediated solely by α_vβ₃ [21]. However, because Romanov and Goligorsky used HUVEC, the relevance of their study to clinical prostate cancer is questioned because prostate cancer frequently metastasizes to the bone. In addition, prostate cancer cell lines have been shown to adhere preferentially to human bone marrow endothelial cells over HUVEC [22]. While α_vβ₃ may be important for prostate cancer cell adhesion to HUVEC, the role this integrin plays in prostate cancer cell adhesion to bone marrow endothelium has not been determined.

Prostate cancer cell adhesion to and migration on components present in the bone matrix are mediated, in part, by α_vβ₃ [23]. A well-characterized antibody to α_vβ₃ (LM609) reduced DU145 cell adhesion to crude bone protein extract by 94%. The protein makeup of the crude bone protein extract was not determined; however, osteopontin and vitronectin are common proteins in mature bone and appropriate ligands for α_vβ₃ [16,24]. Zheng et al. [16] demonstrated that PC-3 cell adhesion and migration to vitronectin and osteopontin were α_vβ₃-dependent. LNCaP cells did not express this integrin and therefore did not adhere to nor migrate on vitronectin and osteopontin. Exogenous expression of α_vβ₃ in LNCaP cells mediated adhesion to vitronectin but not to osteopontin. Surprisingly, the LNCaP cells used in a study performed by Witkowski et al. [15] expressed α_vβ₃, yet these cells did not adhere to vitronectin, suggesting that the monomeric forms of this integrin were not functionally active. LNCaP cell adhesion to osteopontin was not evaluated in that study.

Osteopontin-induced growth of prostate cancer cells in the bone marrow is mediated by α_vβ₃ [25,26]. One study demonstrated that osteopontin stimulated anchorage-independent growth of cell lines LNCaP and C4-2, an androgenindependent subline of LNCaP. Interestingly, osteopontin was detected in greater amounts in androgen-independent prostate cancer cell lines such PC-3 and C4-2, suggesting that it contributes to androgen-independent growth in the bone [25]. Lecrone et al. [26] confirmed this observation by showing that bone-derived osteopontin triggered calcium [Ca²⁺]-dependent signalling in PC-3 cells. An antibody to α_vβ₃ inhibited these Ca²⁺ signals in PC-3 cells. Fluctuations in intracellular Ca²⁺ regulate signal transduction events that are often associated with cell growth. The fluctuations were not observed in DU145 and LNCaP in the presence of any bone-derived fractions. Interestingly, DU145 cells express α_v and β₃ integrin monomers, but the heterodimer expression of α_vβ₃ is not known. Moreover, secretory products from PC-3 cells but not LNCaP cells upregulated osteopontin expression in MC3T3-E1 cells, an osteoblastic cell line [27]. These observations suggest that prostate cancer cells entering the bone environment are surrounded by osteopontin from a few sources, including the bone matrix, osteoblasts, and the cancer cells themselves. The presence of osteopontin can mediate preferential adhesion, migration, and growth of prostate cancer cells expressing α_vβ₃.

Another investigation demonstrated that α_vβ₃ was involved in bombesin stimulation of prostate cancer cell motility [28]. Neuroendocrine cells in prostate cancer express and secrete bombesin-like peptides, suggesting that these peptides are involved in prostate cancer progression. The study demonstrated that bombesin increased PC-3 cell invasion through matrigel, but did not alter its adhesion to ECM proteins including vitronectin. Bombesin also increased the tyrosine phosphorylation of a 95-kDa protein, which was coimmunoprecipitated with the α_v, β₃, and β₅ integrin subunits. In addition, bombesin treatment caused β₁, β₃, and β₅ integrin subunits to coimmunoprecipitate with focal adhesion kinase (FAK). This suggests that the bombesin signaling is mediated by α_vβ₃ and FAK activation.

The functions of α_vβ₃ in prostate cancer cells are mediated by FAK, which activates the phosphatidylinositol 3-kinase (PI-3 kinase)/Akt pathway [16,24] (Figure 1). Transfection of FAK-related nonkinase, which competes with FAK for its correct localization and phosphorylation, in LNCaP cells previously transfected with α_vβ₃ inhibited the migration of these cells on vitronectin. FAK was also activated in PC-3 cells adherent to vitronectin and α_vβ₃-transfected LNCaP adherent to osteopontin [16,24]. Although FAK phosphorylation was not evaluated in PC-3 cells adherent to osteopontin, one can speculate that FAK was phosphorylated because the PI-3 kinase/Akt pathway was activated in these cells as well [16].

Figure 1.

α_vβ₃ signaling pathway in prostate cancer cells. Ligation of α_vβ₃ with multiple ligands (i.e., osteopontin, BSP, or vitronectin) activates FAK, which interacts and activates PI-3 kinase [23]. The products of PI-3 kinase activity recruit PKB/Akt to the cell membrane where it is activated by phosphorylation and it phosphorylates several substrates to elicit a variety of biological responses, including cell survival, adhesion, and migration. Although NF-κB is potentially involved in the α_vβ₃ signal transduction pathway [8], its role in this signaling pathway is not known for prostate cancer cells.

The role of the PI-3 kinase/Akt pathway in prostate cancer cell adhesion and migration on both osteoponitin and vitronectin has been demonstrated by using wortmanin, a specific PI-3 kinase inhibitor. This compound inhibited PC-3 cell adhesion to and migration on both substrates [16]. The PI-3 kinase/Akt pathway may also be involved in androgen-independent growth of prostate cancer cells described earlier [25]. Once activated by an upstream kinase such as FAK, this pathway facilitates cell survival and proliferation by increasing cell cycle regulator E2F, which mediates progression through the cell cycle, and by preventing the proapoptotic activity of BAD. The phosphorylation of BAD by Akt-associated protein kinase B (PKB) prevents it from heterodimerizing with Bcl-2 or Bcl-X_L. These well-known antiapoptotic proteins may be able to provide survival signals that protect prostate cancer cells from apoptosis induced by various stresses like hormone ablation and chemotherapy [29,30]. FAK also activates NF-κB, which is known to regulate the transcription of antiapoptotic proteins [30].

Summary

Cell adhesion molecules (CAMs) are important for the preferential metastasis of prostate cancer to bone [19,22,31]. These CAMs mediate the initial adhesion to the human bone marrow endothelium [22,31] and then the underlying bone matrix [19]. Studies have shown that α_vβ₃ mediates prostate cancer cell adhesion to and migration on vitronectin and osteopontin, which are components of the bone microenvironment [16]. This integrin also mediates osteopontin-associated androgen-independent growth of prostate cancer cells, a common characteristic of advanced prostate cancer [25]. Ligation of α_vβ₃ with its respective ligands activates FAK and subsequently the PI-3 kinase/Akt pathway, resulting in a variety of biological effects, including suppression of apoptosis [29]. Although it is not known if direct engagement of α_vβ₃ in prostate cells prevents apoptosis through the PI-3 kinase/Akt pathway [16], the data presented suggest that α_vβ₃ through this signal pathway may be involved in prostate cancer metastasis to bone.