Prostate cancer bone metastases promote both osteolytic and osteoblastic activity

Advanced prostate cancer is frequently accompanied by the development of metastasis to bone. In the past, prostate cancer bone metastases were characterized as being osteoblastic (i.e., increasing bone density) based on radiographs. However, emerging evidence suggests that development of prostate cancer bone metastases requires osteoclastic activity in addition to osteoblastic activity. The complexities of how prostate tumor cells influence bone remodeling are just beginning to be elucidated. Prostate cancer cells produce a variety of pro-osteoblastic factors that promote bone mineralization. For example, both bone morphogenetic proteins and endothelin-1 have well recognized pro-osteoblastic activities and are produced by prostate cancer cells. In addition to factors that enhance bone mineralization prostate cancer cells produced factors that promote osteoclast activity. Perhaps the most critical pro-osteoclastogenic factor produced by prostate cancer cells is receptor activator of NFkappaB ligand (RANKL), which has been shown to be required for the development of osteoclasts. Blocking RANKL results in inhibiting prostate cancer-induced osteoclastogenesis and inhibits development and progression of prostate tumor growth in bone. These findings suggest that targeting osteoclast activity may be of therapeutic benefit. However, it remains to be defined how prostate cancer cells synchronize the combination of osteoclastic and osteoblastic activity. We propose that as the bone microenvironment is changed by the developing cancer, this in turn influences the prostate cancer cells' balance between pro-osteoclastic and pro-osteoblastic activity. Accordingly, the determination of how the prostate cancer cells and bone microenvironment crosstalk are important to elucidate how prostate cancer cells modulate bone remodeling.     

TGF-beta在前列腺癌骨转移中的研究进展

进展期前列腺癌多会发生骨转移,导致患者骨质破坏甚至死亡。前列腺癌发生骨转移的机制目前尚未研究清楚。既往多认 为是因为前列腺癌细胞表面携带者容易在骨环境中生长的表型。但是目前多认为是肿瘤细胞与骨骼微环境之间的相互作用导致的结果。它们之间是通过细胞因子来传递信息。在众多因子中,TGF-beta对前列腺癌骨转移灶中的各种细胞都起着重要作用。研究表明在体外实验中TGF-beta的作用极易受到细胞生长环境的影响,表现出不同的功能。这提示着TGF-beta信号通路和其他信号通路之间存在非常强的交互作用。本文的重点在于对TGF-beta在前列腺癌骨转移中的作用研究进展进行综述,阐述TGF-beta对转移灶中不同细胞的作用,为今后肿瘤的治疗研究寻找一个好的方向。     

TGF—β在前列腺癌骨转移中的研究进展

进展期前列腺癌多会发生骨转移,导致患者骨质破坏甚至死亡。前列腺癌发生骨转移的机制目前尚未研究清楚。既往多认为是因为前列腺癌细胞表面携带者容易在骨环境中生长的表型。但是目前多认为是肿瘤细胞与骨骼微环境之间的相互作用导致的结果。它们之间是通过细胞因子来传递信息。在众多因子中,TGF-β对前列腺癌骨转移灶中的各种细胞都起着重要作用。研究表明在体外实验中TGF-β的作用极易受到细胞生长环境的影响,表现出不同的功能。这提示着TGF-β信号通路和其他信号通路之间存在非常强的交互作用。本文的重点在于对TGF-β在前列腺癌骨转移中的作用研究进展进行综述,阐述TGF-β对转移灶中不同细胞的作用．为今后肿瘤的治疗研究寻找一个好的方向。     

Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone.     

Bone matrix osteonectin limits prostate cancer cell growth and survival

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.     

Prostate cancer cell survival pathways activated by bone metastasis microenvironment

The development of resistance to anti-cancer therapies in bones is a major hurdle preventing long-lasting clinical responses to anti-cancer therapies in hormone refractory prostate cancer. Herein, we present the major signal transduction pathways, which are activated in prostate cancer cells residing at bone metastasis microenvironment. These intracellular signal transduction pathways can inhibit anti-cancer therapy-induced apoptosis of metastatic prostate cancer cells, thereby optimizing their survival, locally. Employment of this knowledge in a clinical setting provides the conceptual framework for the development of bone-targeted therapies for advanced prostate cancer. Indeed, bone metastasis microenvironment-targeted therapies illustrate a novel paradigm in cancer treatment: anti-tumor treatment strategies may not only aim at directly inducing cancer cell apoptosis, but can also target the tumor metastasis microenvironment, and neutralize the protection it confers on metastatic cancer cells.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

前列腺癌骨转移：肿瘤细胞与骨微环境之间的相互作用

  肿瘤转移包括一系列复杂的过程,主要涉及肿瘤细胞由原发部位脱离开始,到肿瘤细胞在其它转移部位——比如骨骼,生长增殖的多个关键性步骤.“种子和土壤学说”预示骨微环境中表达许多因子,吸引多种肿瘤细胞的迁移以及促进肿瘤的增殖.通过肿瘤细胞与其所处生长环境中的双向和动态的作用,促进肿瘤在骨骼中的发展.因此,骨微环境中产生的因子对肿瘤骨转移具有重要意义.本综述以前列腺癌为例,总结了肿瘤转移的机理研究概况,特别强调了目前有关肿瘤细胞与骨微环境之间相互作用研究的重要性,并提出了未来的研究方向     

Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.     

Mechanisms of cancer metastasis to the bone

Some of the most common human cancers, including breast cancer, prostate cancer, and lung cancer, metastasize with avidity to bone. What is the basis for their preferential growth within the bone microenvironment? Bidirectional interactions between tumor cells and cells that make up bone result in a selective advantage for tumor growth and can lead to bone destruction or new bone matrix deposition. This review discusses our current understanding of the molecular components and mechanisms that are responsible for those interactions.     

Prostate cancer progression and surrounding microenvironment

Bidirectional cellular interactions between prostate cancer and prostate or bone stroma are needed for local tumor growth and distant metastasis. The genetics of cancer cells is affected by the host microenvironment and, reciprocally, permanent gene expression changes occur in the stroma surrounding epithelial cancer cells. The immune-mediated micromilieu also affects the progression of prostate cancer; the role of the immune system in controlling the growth of prostate cancer cells is complex, with immune escape mechanisms prevailing over effective antitumor response. Moreover, tumor stem cell models to explain the origin and progression of prostate cancer require appropriate environmental conditions. On the basis of a review of the literature, this article aims to outline the recent advances in the elucidation of the molecular mechanisms underlying the interactions between prostate cancer and its microenvironment.     

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

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 alpha(1), alpha(2) and alpha(3) collagen binding integrin subunits. Antibody function blocking studies reveal that PC3 cells can utilize alpha(2)beta(1) and alpha(3)beta(1) 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.     

A paradigm for the treatment of prostate cancer bone metastases based on an understanding of tumor cell-microenvironment interactions

The pliability of cancer cells to mutate into several different phenotypes in an attempt to find one that will survive and colonize at the metastatic site is a tremendous "hurdle" to overcome in designing novel cancer therapeutics. New targets of therapy are essential if we are to effectively overcome the evasiveness of cancer. The interaction between the tumor cell and the surrounding microenvironment creates a vicious cycle that perpetuates disease survival and progression. The future of cancer therapy resides in the ability to focus on the recruited and exploited relationships of the cancer cell with the host environment. These therapies target cancer cell growth early and interrupt the vicious cycle that is created by the tumor cells interacting with bone components by inhibiting osteoclasts, osteoblasts, stromal cells, and endothelial cells. They alter the bone microenvironment, creating a hostile "soil" that prevents the "seed" from developing into bone metastases and represent a potential new platform for the development of prostate cancer therapeutics.     

Tumor–stroma co-evolution in prostate cancer progression and metastasis

Cancer development is complex and involves several layers of interactions and pleotropic signaling mechanisms leading to progression. Cancer cells associate with resident stromal fibroblasts, smooth muscle cells, macrophages, endothelium, neurons and migrating cells at metastatic sites and phenotypically and genotypically activate them. These become an integral part of the cancer cell community through activated cell signaling mechanisms. During this process, the cancer cells and cells in the cancer microenvironment “co-evolve” in part due to oxidative stress, and acquire the ability to mimic other cell types (which can be termed osteomimicry, vasculomimicry, neuromimicry and stem cell mimicry), and undergo transition from epithelium to mesenchyme with definitive morphologic and behavioral modifications. In our laboratory, we demonstrated that prostate cancer cells co-evolve in their genotypic and phenotypic characters with stroma and acquire osteomimetic properties allowing them to proliferate and survive in the skeleton as bone metastasis. Several signaling interactions in the bone microenvironment, mediated by reactive oxygen species, soluble and membrane bound factors, such as superoxide, β2-microglobulin and RANKL have been described. Targeting the signaling pathways in the cancer-associated stromal microenvironment in combination with known conventional therapeutic modalities could have a synergistic effect on cancer treatment. Since cancer cells are constantly interacting and acquiring adaptive and survival changes primarily directed by their microenvironment, it is imperative to delineate these interactions and co-target both cancer and stroma to improve the treatment and overall survival of cancer patients.     

Hypoxia stabilizes GAS6/Axl signaling in metastatic prostate cancer

The receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our laboratory indicate that Axl and its ligand growth arrest-specific 6 (GAS6) may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial-mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were downregulated. Moreover, CoCl(2), a hypoxia mimicking agent, prevented GAS6-mediated downregulation of Axl in these cell lines. Immunochemical staining of human prostate cancer tissue microarrays showed that Axl, GAS6, and hypoxia-inducible factor-1α (Hif-1α; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.     

Interleukin‐27 expression modifies prostate cancer cell crosstalk with bone and immune cells in vitro

Prostate cancer is frequently associated with bone metastases, where the crosstalk between tumor cells and key cells of the bone microenvironment (osteoblasts, osteoclasts, immune cells) amplifies tumor growth. We have explored the potential of a novel cytokine, interleukin‐27 (IL‐27), for inhibiting this malignant crosstalk, and have examined the effect of autocrine IL‐27 on prostate cancer cell gene expression, as well as the effect of paracrine IL‐27 on gene expression in bone and T cells. In prostate tumor cells, IL‐27 upregulated genes related to its signaling pathway while downregulating malignancy‐related receptors and cytokine genes involved in gp130 signaling, as well as several protease genes. In both undifferentiated and differentiated osteoblasts, IL‐27 modulated upregulation of genes related to its own signaling pathway as well as pro‐osteogenic genes. In osteoclasts, IL‐27 downregulated several genes typically involved in malignancy and also downregulated osteoclastogenesis‐related genes. Furthermore, an osteogenesis‐focused real‐time PCR array revealed a more extensive profile of pro‐osteogenic gene changes in both osteoblasts and osteoclasts. In T‐lymphocyte cells, IL‐27 upregulated several activation‐related genes and also genes related to the IL‐27 signaling pathway and downregulated several genes that could modulate osteoclastogenesis. Overall, our results suggest that IL‐27 may be able to modify interactions between prostate tumor and bone microenvironment cells and thus could be used as a multifunctional therapeutic for restoring bone homeostasis while treating metastatic prostate tumors. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Loss of TGF-β responsiveness in prostate stromal cells alters chemokine levels and facilitates the development of mixed osteoblastic/osteolytic bone lesions

Loss of TGF-β type II receptor (TβRII, encoded by Tgfbr2) expression in the prostate stroma contributes to prostate cancer initiation, progression, and invasion. We evaluated whether TβRII loss also affected prostate cancer bone metastatic growth. Immunohistologic analysis revealed that TβRII expression was lost in cancer-associated fibroblasts in human prostate cancer bone metastatic tissues. We recapitulated the human situation with a conditional stromal Tgfbr2 knockout (Tgfbr2-KO) mouse model. Conditioned media from primary cultured Tgfbr2-KO or control Tgfbr2-flox prostatic fibroblasts (koPFCM or wtPFCM, respectively) were applied to C4-2B prostate cancer cells before grafting the cells tibially. We found that koPFCM promoted prostate cancer cell growth in the bone and development of early mixed osteoblastic/osteolytic bone lesions. Furthermore, the koPFCM promoted greater C4-2B adhesion to type-I collagen, the major component of bone matrix, compared to wtPFCM-treated C4-2B. Cytokine antibody array analysis revealed that koPFCM had more than two-fold elevation in granulocyte colony-stimulating factor and CXCL1, CXCL16, and CXCL5 expression relative to wtPFCM. Interestingly, neutralizing antibodies of CXCL16 or CXCL1 were able to reduce koPFCM-associated C4-2B type-I collagen adhesion to that comparable with wtPFCM-mediated adhesion. Collectively, our data indicate that loss of TGF-β responsiveness in prostatic fibroblasts results in upregulation of CXCL16 and CXCL1 and that these paracrine signals increase prostate cancer cell adhesion in the bone matrix. These microenvironment changes at the primary tumor site can mediate early establishment of prostate cancer cells in the bone and support subsequent tumor development at the metastatic site.     

Bone marrow mesenchymal stem cells increase motility of prostate cancer cells via production of stromal cell‐derived factor‐1α

Prostate cancer frequently metastasizes to the bone, and the interaction between cancer cells and bone microenvironment has proven to be crucial in the establishment of new metastases. Bone marrow mesenchymal stem cells (BM‐MSCs) secrete various cytokines that can regulate the behaviour of neighbouring cell. However, little is known about the role of BM‐MSCs in influencing the migration and the invasion of prostate cancer cells. We hypothesize that the stromal cell‐derived factor‐1α released by BM‐MSCs may play a pivotal role in these processes. To study the interaction between factors secreted by BM‐MSCs and prostate cancer cells we established an in vitro model of transwell co‐culture of BM‐MSCs and prostate cancer cells DU145. Using this model, we have shown that BM‐MSCs produce soluble factors which increase the motility of prostate cancer cells DU145. Neutralization of stromal cell‐derived factor‐1α (SDF1α) via a blocking antibody significantly limits the chemoattractive effect of bone marrow MSCs. Moreover, soluble factors produced by BM‐MSCs greatly activate prosurvival kinases, namely AKT and ERK 1/2. We provide further evidence that SDF1α is involved in the interaction between prostate cancer cells and BM‐MSCs. Such interaction may play an important role in the migration and the invasion of prostate cancer cells within bone.     

Dynamic process of prostate cancer metastasis to bone

Prostate cancer metastasis to the bone occurs at high frequency in patients with advanced disease, causing significant morbidity and mortality. Over a century ago, the "seed and soil" theory was proposed to explain organ-specific patterns of metastases. Today, this theory continues to be relevant as we continue to discover factors involved in the attraction and subsequent growth of prostate cancer cells to the bone. These include the accumulation of genetic changes within cancer cells, the preferential binding of cancer cells to bone marrow endothelial cells, and the release of cancer cell chemoattractants from bone elements. A key mediator throughout this metastatic process is the integrin family of proteins. Alterations in integrin expression and function promote dissociation of cancer cells from the primary tumor mass and migration into the blood stream. Once in circulation, integrins facilitate cancer cell survival through interactions between other cancer cells, platelets, and endothelial cells of the target bone. Furthermore, dynamic changes in integrins and in integrin-associated signal transduction aid in the extravasation of cancer cells into the bone and in expansion to a clinically relevant metastasis. Thus, we will review the critical roles of integrins in the process of prostate cancer bone metastasis, from the escape of cancer cells from the primary tumor, to their survival in the harsh "third microenvironment" of the circulation, and ultimately to their attachment and growth at distant bone sites.