GAS6 Receptor Status Is Associated with Dormancy and Bone Metastatic Tumor Formation

Disseminated tumor cells (DTCs) are believed to lie dormant in the marrow before they can be activated to form metastases. How DTCs become dormant in the marrow and how dormant DTCs escape dormancy remains unclear. Recent work has shown that prostate cancer (PCa) cell lines express the growth-arrest specific 6 (GAS6) receptors Axl, Tyro3, and Mer, and become growth arrested in response to GAS6. We therefore hypothesized that GAS6 signaling regulates the proliferative activity of DTCs in the marrow. To explore this possibility, in vivo studies were performed where it was observed that when Tyro3 expression levels exceed Axl expression, the PCa cells exhibit rapid growth. When when Axl levels predominate, PCa cells remain largely quiescent. These findings suggest that a balance between the expression of Axl and Tyro3 is associated with a molecular switch between a dormant and a proliferative phenotype in PCa metastases.     

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.     

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.     

Secretome Analysis of an Osteogenic Prostate Tumor Identifies Complex Signaling Networks Mediating Cross-talk of Cancer and Stromal Cells Within the Tumor Microenvironment

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone-forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF-β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.A distinct feature of human prostate cancer (PCa)¹ with lethal potential is the development of metastases in bone with a bone-forming phenotype (1). This property of PCa bone metastasis suggests that PCa cells have unique interactions with cells in the bone microenvironment. Cells that are known to be present in the bone microenvironment include osteoblasts, osteoclasts, adipocytes, fibroblasts, and endothelial cells. Communication between PCa cells and each of these cells in the microenvironment is known to promote metastatic growth. This communication involves metastatic PCa cells that secrete factors to affect stromal cells in the bone microenvironment. The tumor-modified stromal cells may further alter the properties of the PCa cells to allow them to progress in the bone environment (1). Determining how secretory proteins from the metastatic PCa cells affect the PCa/stromal communication network will lead to the development of strategies to treat bone metastases.Although men with PCa and bone metastasis most frequently present with osteoblastic bone lesions, the commonly-used PCa cell lines to study metastatic properties, for example, PC3 and C4–2B, induce osteolytic or mixed osteoblastic/osteolytic lesions, respectively, when the cells are implanted into mouse femurs or tibia (2). In contrast, the PCa-118b patient-derived xenograft (PDX), generated from an osteoblastic bone lesion of a patient with PCa and bone metastasis, shows phenotypic characteristics similar to the tumor from which it was derived, including induction of a strong osteoblastic response when implanted into femurs (3). Interestingly, PCa-118b cells are also able to induce ectopic bone formation when implanted subcutaneously (3, 4). The capacity of PCa-118b cells to induce bone formation, in which human tumor cells interact with the murine stromal microenvironment, makes this PDX an ideal model system to study tumor-microenvironment signaling pathways that create a bone-like tumor microenvironment conducive to metastatic PCa growth.In this study, we identified secreted factors from the conditioned medium of isolated PCa-118b cells by mass spectrometry. A total of 26 secretory proteins, including cytokines and growth factors, were identified. Human- and mouse-specific PCR probes were used to identify the cells that expressed these factors. Analysis of the receptor for the corresponding secreted factor determined whether the factor exerted activities in a paracrine and/or autocrine manner. The effects of selected factors on PCa cells or stromal cells, including osteoblasts and endothelial cells, were also examined. Our studies showed that PCa-118b cells secreted multiple factors that establish an autocrine or paracrine signaling network that can mediate cross-talk among multiple cell types within the bone microenvironment.     

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context of prostate cancer bone metastasis

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic because of bone remodeling leading to the formation of new bone. However, recent studies have shown increased osteolytic activity in the beginning stages of PCa bone metastases, suggesting that targeting both osteolytic and osteoblastic mediators would likely inhibit bone remodeling and PCa bone metastasis. In this study, we found that PCa cells could stimulate differentiation of osteoclasts and osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting bone remodeling. Interestingly, we found that formulated isoflavone and 3,3'-diindolylmethane (BR-DIM) were able to inhibit the differentiation of osteoclasts and osteoblasts through the inhibition of cell signal transduction in RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone and BR-DIM down-regulated the expression of miR-92a, which is known to be associated with RANKL signaling, EMT and cancer progression. By pathway and network analysis, we also observed the regulatory effects of isoflavone and BR-DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc. Therefore, isoflavone and BR-DIM with their multi-targeted effects could be useful for the prevention of PCa progression, especially by attenuating bone metastasis mechanisms.     

Exosomal long noncoding RNA HOXD-AS1 promotes prostate cancer metastasis via miR-361-5p/FOXM1 axis

Development of distant metastasis is the main cause of deaths in prostate cancer (PCa) patients. Understanding the mechanism of PCa metastasis is of utmost importance to improve its prognosis. The role of exosomal long noncoding RNA (lncRNA) has been reported not yet fully understood in the metastasis of PCa. Here, we discovered an exosomal lncRNA HOXD-AS1 is upregulated in castration resistant prostate cancer (CRPC) cell line derived exosomes and serum exosomes from metastatic PCa patients, which correlated with its tissue expression. Further investigation confirmed exosomal HOXD-AS1 promotes prostate cancer cell metastasis in vitro and in vivo by inducing metastasis associated phenotype. Mechanistically exosomal HOXD-AS1 was internalized directly by PCa cells, acting as competing endogenous RNA (ceRNA) to modulate the miR-361-5p/FOXM1 axis, therefore promoting PCa metastasis. In addition, we found that serum exosomal HOXD-AS1 was upregulated in metastatic PCa patients, especially those with high volume disease. And it is correlated closely with Gleason Score, distant and nodal metastasis, Prostatic specific antigen (PSA) recurrence free survival, and progression free survival (PFS). This sheds a new insight into the regulation of PCa distant metastasis by exosomal HOXD-AS1 mediated miR-361-5p/FOXM1 axis, and provided a promising liquid biopsy biomarker to guide the detection and treatment of metastatic PCa.Subject terms: Bone metastases, Prostate cancer     

A dormant state modulated by osmotic pressure controls clonogenicity of prostate cancer cells

Cell dormancy constitutes a limiting step of the metastatic process by preventing the proliferation of isolated cancer cells disseminated at distant sites from the primary tumor. The study of cancer cell dormancy is severely hampered by the lack of biological samples so that the mechanisms that regulate cell dormancy have not been extensively explored. In this work, we describe the rapid induction in vitro of a dormant state in prostate cancer cells by exposure to a slightly hypertonic growth medium. This quiescence is observed only when cells are seeded at low density and, once established, requires additional stimuli besides osmotic pressure to be reversed. Media conditioned by cells grown at high density can partially prevent or reverse dormancy, a phenomenon which can be reproduced with citric acid. In addition to this role of small metabolites, inactivation of the p53 and smad pathways also counters the entry into dormancy, whereas exposure to activin A induces it to some extent. Thus, this easily inducible dormancy reproduces several features associated with the dormancy of stem cells and cancer cells in vivo.     

The Mitogen-activated Protein (MAP) Kinases p38 and Extracellular Signal-regulated Kinase (ERK) Are Involved in Hepatocyte-mediated Phenotypic Switching in Prostate Cancer Cells

The greatest challenge for the seeding of cancer in metastatic sites is integration into the ectopic microenvironment despite the lack of an orthotopic supportive environment and presence of pro-death signals concomitant with a localized “foreign-body” inflammatory response. In this metastatic location, many carcinoma cells display a reversion of the epithelial-to-mesenchymal transition that marks dissemination in the primary tumor mass. This mesenchymal to epithelial reverting transition (MErT) is thought to help seeding and colonization by protecting against cell death. We have previously shown that hepatocyte coculture induces the re-expression of E-cadherin via abrogation of autocrine EGFR signaling pathway in prostate cancer (PCa) cells and that this confers a survival advantage. Herein, we show that hepatocytes educate PCa to undergo MErT by modulating the activity of p38 and ERK1/2. Hepatocytes inhibited p38 and ERK1/2 activity in prostate cancer cells, which allowed E-cadherin re-expression. Introduction of constitutively active MEK6 and MEK1 to DU145 cells cocultured with hepatocytes abrogated E-cadherin re-expression. At least a partial phenotypic reversion can be achieved by suppression of p38 and ERK1/2 activation in DU145 cells even in the absence of hepatocytes. Interestingly, these mitogen-activated protein kinase activities were also triggered by re-expressed E-cadherin leading to p38 and ERK1/2 activity in PCa cells; these signals provide protection to PCa cells upon challenge with chemotherapy and cell death-inducing cytokines. We propose that distinct p38/ERK pathways are related to E-cadherin levels and function downstream of E-cadherin allowing, respectively, for hepatocyte-mediated MErT and tumor cell survival in the face of death signals.     

Cancer and bone repair mechanism: clinical applications for hormone refractory prostate cancer

It is a long-standing clinical observation that the bone corresponds to the prevalent site for metastatic growth of prostate cancer. In addition, bone metastases of this malignancy produce a potent blastic reaction, in contrast to the overwhelming majority of other osteotropic neoplasms, whose metastases are generally associated with an osteolytic reaction. Osteoblastic metastases represent almost always the first and, frequently, the exclusive site of disease progression to hormone refractory stage, stage D3. Moreover, the number of skeletal metastatic foci is the most powerful independent prognostic factor associated with a limited response to hormone ablation therapy and poor survival of advanced prostate cancer. It is noteworthy that disease progression to hormone refractory stage occurs almost always in osteoblastic metastases. These clinical observations suggested that the osteoblastic reaction is possibly not an innocent bystander of the metastatic prostate tumour growth, simply suffering its consequences, but it may in fact facilitate the efforts of metastatic cells to expand their population. An extensive line of research in the pathophysiology of osteoblastic metastases has established that the local blastic reaction involves the uPA/plasmin/IGF/IGFBP-3/TGFbs bioregulation system which can stimulate both the growth of osteoblasts and prostate cancer cells. Furthermore, we were the first to characterize osteoblast-derived 'survival factors' able to rescue metastatic prostate cancer cells from chemotherapy-induced apoptosis. These data resulted in the development of a novel concept of an anti-survival factor therapy, namely an anti-IGF-1 therapy, which has provided encouraging preliminary data in a phase II clinical trial with terminally-ill hormone/chemotherapy-resistant prostate cancer patients.     

Murine mammary tumor cells with a claudin-low genotype

In adults, bone is the preferential target site for metastases from primary cancers of prostate, breast, lungs and thyroid. The tendency of these cancers to metastasize to bone is determined by the anatomical distribution of the blood vessels, by the genetic profile of the cancer cells and by the biological characteristics of the bone microenvironment that favour the growth of metastatic cells of certain cancers. Metastases to bone may have either an osteolytic or an ostoblastic phenotype. The interaction in the bone microenvironment between biological factors secreted by metastatic cells, and by osteoblasts and osteoclasts, and the osteolytic and osteoblastic factors released from the organic matrix mediate a vicious cycle characterized by metastatic growth and by ongoing progressive bone destruction. This interaction determines the phenotype of the metastatic bone disease.     

HER2 as a potential therapeutic target on quiescent prostate cancer cells

Quiescent prostate cancer (PCa) cells are common in tumors but are often resistant to chemotherapy. Quiescent PCa cells are also enriched for a stem-like tumor initiating population, and can lead to recurrence after dormancy. Unfortunately, quiescent PCa cells are difficult to identify and / or target with treatment in part because the relevant markers are intracellular and regulated by protein stability. We addressed this problem by utilizing PCa cells expressing fluorescent markers for CDKN1B (p27) and CDT1, which can separate viable PCa cells into G₀, G₁, or combined S/G₂/M populations. We used FACS to collect G₁ and G₀ PC3 PCa cells, isolated membrane proteins, and analyzed protein abundance in G₀ vs G₁ cells by gas chromatography mass spectrometry. Enrichment analysis identified nucleocytoplasmic transport as the most significantly different pathway. To identify cell surface proteins potentially identifying quiescent PCa cells for future patient samples or for antibody based therapeutic research, we focused on differentially abundant plasma membrane proteins, and identified ERBB2 (HER2) as a cell surface protein enriched on G₀ PCa cells. High HER2 on the cell membrane is associated with quiescence in PCa cells and likely induced by the bone microenvironment. Using a drug conjugated anti-HER2 antibody (trastuzumab emtansine) in a mouse PCa xenograft model delayed metastatic tumor growth, suggesting approaches that target HER2-high cells may be beneficial in treating PCa. We propose that HER2 is deserving of further study in PCa as a target on quiescent cells to prevent recurrence, decrease chemotherapy resistance, or eradicate minimal residual disease.     

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.     

Interleukin-6: a bone marrow stromal cell paracrine signal that induces neuroendocrine differentiation and modulates autophagy in bone metastatic PCa cells

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.     

Dynamic interaction between breast cancer cells and osteoblastic tissue: comparison of two- and three-dimensional cultures

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.     

Glypican-3 (GPC3) inhibits metastasis development promoting dormancy in breast cancer cells by p38 MAPK pathway activation

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to several tumor types. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast cancer progression and its molecular and cellular operational machineries are not fully understood.In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic spread and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through p38 pathway activation. The in vivo inhibition of p38 induced an increase in cell invasion of GPC3 reexpressing orthotropic tumors as well as in spontaneous and experimental metastatic dissemination.In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairs in vivo metastasis and induces tumor dormancy through p38 MAPK signaling activation. These results help to identify genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.