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Therapeutic Opportunities on the Horizon
Treatment options to extend the overall survival of patients diagnosed with mCRPC remains a major clinical challenge. Therefore, understanding the factors that drive the process of metastasis, the homing of the metastasis to organs (eg, bone), and how prostate cancer cells form life-threatening active metastases once in the bone warrants extensive research to generate new therapies to cure the disease. Although metastasis is classically thought of as a linear sequence of events beginning with the dissemination and invasion of tumor cells from the primary site and ending with proliferation at the metastatic site, recent evidence suggests that the first steps of metastasis can occur before a patient's tumor is diagnosed (Fig 2). This "step 0" of the metastatic cascade results in the nonrandom priming of future sites of metastasis, a concept known as the "premetastatic niche."
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Figure 2.
A–C Dormancy and the "vicious cycle" in bone marrow niches. (A) Disseminated tumor cells can home to the vascular niche and cluster on stable endothelium. Decreased expression of thrombospondin 1 combined with activation of transforming growth factor β and periostin in areas of "sprouting" vasculature can result in the outgrowth of tumor cells. (B) Cancer cells may also home to the endosteal niche via mechanisms such as chemokine motif 12/chemokine receptor 4 where they compete with quiescent hematopoietic stem cells for osteoblast interaction. Subsequently, the cancer cells can be maintained in a dormant state via interactions with GAS6- and ANXA2-expressing niche osteoblasts or proliferate into metastases. (C) A "vicious cycle" occurs between tumor cells and other cells of the bone microenvironment. Factors secreted by the tumor cells act on osteoblasts, leading to the increased production of RANKL. RANKL subsequently promotes the differentiation of osteoclast precursors into mature, bone-resorbing osteoclasts that degrade the bone and release additional factors into the microenvironment, providing positive feedback to the cancer cells. Matrix metalloproteinases 2, 7, and 9 contribute to the vicious cycle by regulating factors such as vascular endothelial growth factor A, RANKL, and transforming growth factor β, whereas myeloid-derived suppressor cells contribute by releasing protumorigenic factors, suppressing T cells, and differentiating into osteoclasts. RANKL = receptor activator of nuclear κB ligand.
Premetastatic Niche
Primary tumor-derived factors have been implicated in the development of premetastatic niches in distant organs. Through a series of in vivo experiments, it was illustrated that conditioned media derived from highly metastatic cancer cells lines, such as the B-16 melanoma cell line, could stimulate the mobilization of bone marrow–derived VEGF receptor 1 VLA4 Id3 hematopoietic precursor cells to develop premetastatic niche sites, including the lungs, liver, spleen, kidney, and testes. Cancer-derived exosomes have been implicated as the mechanism for facilitating long distance, tumor–stroma interactions and initiating the premetastatic niche. Exosomes are microvesicles measuring 30 nm to 100 nm that contain a variety of functional proteins and messenger/micro RNAs. In the context of premetastatic niche formation, B16-F10–derived exosomes have been labeled and shown to "home" to common sites of melanoma metastasis. Furthermore, in the premetastatic niche, exosomes can educate bone marrow–derived cells to support metastatic tumor growth via the horizontal transfer of the c-MET protein. c-MET inhibitors, such as cabozantinib, could be used to prevent the development of premetastatic niches and, thus, mitigate the ability of cancers to metastasize to new sites.
Exosome shedding has also been demonstrated in prostate cancer, and studies have shown the presence of microvesicles termed oncosomes (0.5–5 μm) in prostate cancer–conditioned media. Oncosomes contain a variety of signal transduction proteins, including Akt and Src, and can interact with tumor and stromal cells to elicit disease-promoting responses. In addition, a correlation exists between a Gleason score higher than 7 and the number of oncosomes present in patient plasma. Based on these findings, it is plausible that prostate cancer–derived exosomes can play a role in the formation of premetastatic niches in the bone microenvironment. Emerging evidence also suggests that prostate cancer cells homing to the bone microenvironment can occupy the endosteal niche, the vascular niche, or both.
Defining Factors Controlling the Homing of Bone Metastatic Castration-resistant Prostate Cancer
An unsolved question regarding metastasis is why prostate cancer has such a predilection for the bone microenvironment. More than a century ago, Paget formulated the "seed and soil" hypothesis to address this question. His hypothesis suggested that metastasis is a challenging process that requires "fertile soil" for outgrowth but begins long before the "seed" meets the "soil." Ewing challenged Paget's hypothesis in the 1920s, proposing that metastasis was instead dependent on anatomy, vasculature, and lymphatics. Metastasis by anatomy would become the accepted model until the 1970s when modern experiments rekindled interest in the "seed and soil" hypothesis, notably observing that circulating tumor cells reach the vasculature of all organs, but only certain organs are receptive for metastasis. In reality, prostate to bone metastasis occurs by a blend of both hypotheses: It metastasizes first to the pelvic lymph node and then to sites in the bone, including iliac crests, sacrum wings, L1 to L5 vertebrae, T8 to T12 vertebrae, ribs, manubrium, humeral heads, and femoral necks. Although 15% to 30% of prostate to bone metastases are due to cells traveling through the Batson plexus to the lumbar spine, it is clear that molecular factors, such as chemokines and integrins, underpin the propensity for prostate cancer cells to metastasize to the skeleton. Elucidating those factors could help identify new therapies to prevent bone metastatic CRPC.
Bone is the home of regulatory sites for hematopoietic stem cells (HSCs), which are cells localized to the vascular and endosteal niches where they either await hematopoietic demand or reside in a quiescent state. One well-defined signaling axis implicated in metastasis is that between stromal cell–derived factor 1/CXCL12 and its receptor CXCR4, a system normally utilized by HSCs homing to the niche. CXCL12 expression is increased in the premetastatic niche, and studies in prostate cancer have demonstrated that tumor cells with high bone-homing capacity express CXCR4 and CXCR7 to parasitize the HSC niche. Furthermore, CXCR4 expression correlates with poor prognosis. Additional axes, including MCP-1/CCR2 and CXCL16/CXCR6, have also been found to contribute to the progression of prostate cancer through increases in proliferation, migration, and invasion.
Disseminated Tumor Cells and Dormancy
Evidence suggests that tumor cells disseminated from the prostate localize to the bone marrow niche, displace HSCs, and either proliferate to form a metastatic mass or enter a state of dormancy. Dissemination from the primary site to reside in distant environments is an early event seen in prostate cancer, as patients who undergo prostatectomy may present with metastases many years later. Disseminated tumor cells (DTCs) reside in the bone marrow niche where they can remain dormant and resistant to chemotherapy for long periods of time (> 10 years) before emerging to form metastatic outgrowths. Although most patients with prostate cancer harbor DTCs, not all will develop metastases, suggesting that mechanisms exist to maintain DTC dormancy as well as to promote awakening.
Several bone marrow–dependent mechanisms have been identified as modulators of prostate cancer DTC dormancy. In the endosteal niche, the osteoblast expression of Anxa2 combined with the expression of the Anxa2 receptor (Anxa2R) by HSCs is important in regulating HSC homing to the niche. Anxa2R expression is elevated in metastatic prostate tumor cells and, as such, the Anxa2/Anxa2R axis can be hijacked to promote the homing of prostate tumor cells to the niche. Interrupting the interaction between Anxa2 and Anxa2R is sufficient to reduce tumor burden in the niche. Evidence has revealed that the ligation of Anxa2 with Anxa2R stimulates the expression of the Axl receptor tyrosine kinase. Axl, along with Tyro3 and Mer, are receptors for osteoblast-expressed growth arrest-specific 6 (GAS6). As was the case with Anxa2/Anxa2R, the GAS6/Axl interaction typically occurs between HSCs and osteoblasts and is one mechanism of controlling HSC dormancy. Engaging osteoblast-expressed GAS6 and tumor cell–expressed Axl yields a similar result that includes growth arrest and enhanced drug resistance in prostate cancer cells. Following-up on these observations, data show that these activities may be specific to the Axl receptor compared with other GAS6 receptors. A high ratio of Axl to Tyro3 expression encourages maintenance of a dormant state, whereas reducing the expression of Axl and increasing the expression of Tyro3 has been shown to promote outgrowth.
Interactions between osteoblasts and tumor cells may be important to DTC dormancy. Prostate cancer cells that bind with osteoblasts also upregulate the expression of TANK-binding kinase 1 (TBK1). In vitro and in vivo knockdown of TBK1 resulted in decreased drug resistance, suggesting that TBK1 may also play a role in dormancy and drug resistance. A high p38:ERK ratio has been shown to maintain dormancy of squamous carcinoma cells, whereas interactions with the microenvironment can stimulate a switch to high ERK:p38 and reverse dormancy. Bone marrow–derived transforming growth factor (TGF) β2 has been implicated in maintaining the dormancy of DTCs by p38 activation, and inhibiting either the TGF-β receptor 1 or p38 leads to the proliferation and metastasis of DTCs. Similarly, bone morphogenetic protein 7 triggers prostate cancer DTC dormancy in part by activating p38.
Although much focus has been on the endosteal niche, the vascular niche also has implications for DTC dormancy. Through the use of advanced imaging techniques, dormant DTCs have been shown to home to perivascular niches in the bone marrow and the lungs. These niches promote dormancy through the expression of TSP-1; however, dormancy is lost in regions of sprouting vasculature due to a loss of TSP-1 and the activation of TGF-β and periostin.
In vivo experiments in mice receiving bone marrow transplantation revealed that fewer HSCs successfully engraft in tumor-bearing mice, suggesting that the tumor cells occupying the niche outcompete HSCs for residence. In addition, expanding the endosteal osteoblast niche with parathyroid hormone (PTH) promoted metastasis, whereas decreasing the size of the niche using conditional osteoblast knockout models reduced dissemination. Tumor cells can also be forced out of the niche using methods to mobilize HSCs, perhaps offering an opportunity for therapeutic intervention. Filgrastim is an agent that mobilizes HSCs out of the niche, and plerixafor blocks the interaction with stromal cell–derived factor 1 by acting as a CXCR4 antagonist to mobilize HSCs. Both agents have been approved by the FDA and may serve as a method of awakening and forcing the DTCs into circulation where they would become vulnerable to chemotherapy. A small molecule inhibitor specific to CXCR6 but not other chemokine receptors was developed for investigating the CXCL16/CXCR6 axis. Although the clinical utility of such an inhibitor must be investigated, the selectivity of small molecule antagonists could aid in the targeting of dormant tumor cells.
Therapeutic Opportunities for "Active" mCRPC
Although therapies to prevent the homing and establishment of mCRPC in the bone microenvironment are important clinical tactics, many patients in the clinical setting present with "active" bone metastases that cause extensive bone remodeling. Defining the mechanisms that control cell–cell communication between the metastases and the microenvironment are also likely to reveal important therapeutic targets.
Osteomimicry. A recurring theme in bone metastasis is the hijacking of normal bone mechanisms by tumor cells. The concept of osteomimicry is that bone metastatic prostate cells acquire the ability to produce proteins typically restricted to bone cells, such as osteoblasts, to survive and proliferate in the otherwise restrictive bone microenvironment. Select genes normally expressed in bone have been detected in prostate cells, including osteocalcin, osteopontin, bone sialoprotein, osteonectin, RANK, RANKL, and PTH-related protein. The expression of these genes appears to be associated with the metastatic capacity of the cells. Studies in both the PC3 and LNCaP cell lines have shown that the expression of osteonectin is highest in the more invasive and metastatic sublines, including the LNCaP metastatic variant C4-2B. Analysis of patient samples support these findings, showing that osteonectin staining in prostate to bone metastases was more intense than from soft-tissue metastases. In addition to changes in gene expression, prostate tumor cells may adopt biological activities usually specific to bone cells. In vitro studies indicate that human C4-2B prostate tumor cells are capable of depositing hydroxyapatite and contributing to mineralization, a common feature of the sclerotic lesions observed in vivo.
Due to the shared expression of specific bone genes between tumor and stroma cells, these common proteins could be used to simultaneously target both compartments. Understanding that soluble factors like bone morphogenetic protein 2, RANKL, TGF-β, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor are partially responsible for inducing osteomimetic genes may also provide options to specifically target osteomimicry and establish bone outgrowths. It has been suggested that promoters for the common genes between the tumor and stroma cells could be utilized to drive the expression of therapeutic genes, thus targeting both the stroma and tumor cells.
Halting the Vicious Cycle of Bone Metastases. Once the DTCs awaken and establish micrometastases, continued outgrowth arises through the interaction with multiple stromal cell types, growth factors, and enzymes in a process known as the vicious cycle model. Prostate to bone metastases are characterized by areas of mixed osteogenesis and osteolysis that give rise to painful lesions. A number of tumor-derived factors, including PTH-related protein, interleukin (IL) 1, IL-6, and IL-11, have been shown to interact with osteoblasts and stimulate the production of RANKL. RANKL is a crucial molecule for osteoclast differentiation; therefore, it contributes to the extensive bone remodeling seen in bone metastasis. In addition to bone destruction, osteoclast-mediated bone resorption also releases a multitude of bone-derived factors such as TGF-β, insulin growth factor, platelet-derived growth factor, and fibroblast growth factor. These factors provide positive feedback via interaction with their respective receptors on the surface of tumor cells, thus promoting the proliferation and continued production of tumor-derived factors. The vicious cycle is continually evolving to include other cell types, cytokines, proteases, and therapeutics. Several studies have shown contributory roles for highly expressed host matrix metalloproteinases (MMPs) in the vicious cycle, including the regulation of latent TGF-β and VEGF-A bioavailability by MMP-2 and MMP-9, and the generation of a soluble form of RANKL by MMP-7, which promotes osteoclastogenesis and mammary tumor–induced osteolysis in vivo. In recent years, the interactions with immune cells have become an integral part of the vicious cycle. For example, T cells stimulate and inhibit the formation of osteoclasts, and the recruitment of regulatory T cells to bone marrow may inhibit osteoclastogenesis. Myeloid-derived suppressor cells suppress T cells and release angiogenic, tumor-promoting factors. Recruited myeloid-derived suppressor cells have also been shown to differentiate into osteoclasts.
Although the need for therapies aimed at the early stages of metastasis has been emphasized, patients will still present in the later stages of the disease; therefore, improving therapies for these patients must still remain a priority. The interactions between tumor and stromal cells in the vicious cycle model offer many opportunities to intervene. Therapies such as zoledronic acid and denosumab interfere with the osteolytic component of the vicious cycle; however, therapies to inhibit the unique osteosclerotic component of prostate to bone metastases are lacking. Many roles for specific MMPs have been elucidated in the vicious cycle, and the development of MMP inhibitors with improved specificity is perhaps a promising method to modulate the vicious cycle.
From these discoveries, it is becoming evident that the metastasis of prostate cancer is not a linear, stepwise procedure. Defining the mechanisms that control CRPC metastasis may help elucidate new therapeutic targets that directly impact the cancer cells and the processes that facilitate the formation of a premetastatic niche, niche seeding, dormancy, and the vicious cycle. Such new discoveries are highly likely to impact the clinical treatment of patients with mCRPC.
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