Emerging Players in Prostate Cancer–Bone Niche Communication

Skeletal metastasis in advanced prostate cancer (PCa) is a major clinical problem that substantially reduces the quality of life and survival of patients.Extracellular vesicles have emerged as important messengers in the metastatic process of PCa to bone and have been recognized as critical mediators and potential targets in the communication between tumor cells and osteoblasts.Osteomimicry has been deciphered as an evasive strategy adopted by PCa cells to disguise themselves in bone and guarantee cancer cell survival in a privileged foreign microenvironment.The molecular events that initiate bone metastasis are potentially associated with the ability of PCa cells to shift normal osteoblasts into cancer-associated osteoblasts that promote PCa propagation. Patients with advanced prostate cancer (PCa) frequently develop skeletal metastases that are associated with fractures, disability, and increased mortality. Within the bone metastatic niche, mutual interactions between tumor cells and osteoblasts have been proposed as major contributors of osteotropism by PCa. Here, we highlight the emerging role of PCa-derived extracellular vesicles (EVs) in reprogramming osteoblasts and support of premetastatic niche formation. We also develop the concept of cancer-associated osteoblasts (CAOs) and outline the potential of PCa cells to acquire an osteoblastic phenotype, termed osteomimicry, as two strategies that PCa utilizes to create a favorable protected niche. Finally, we delineate future research that may help to deconstruct the complexity of PCa osteotropism. Patients with advanced prostate cancer (PCa) frequently develop skeletal metastases that are associated with fractures, disability, and increased mortality. Within the bone metastatic niche, mutual interactions between tumor cells and osteoblasts have been proposed as major contributors of osteotropism by PCa. Here, we highlight the emerging role of PCa-derived extracellular vesicles (EVs) in reprogramming osteoblasts and support of premetastatic niche formation. We also develop the concept of cancer-associated osteoblasts (CAOs) and outline the potential of PCa cells to acquire an osteoblastic phenotype, termed osteomimicry, as two strategies that PCa utilizes to create a favorable protected niche. Finally, we delineate future research that may help to deconstruct the complexity of PCa osteotropism. PCa is the most common male malignancy and the second leading cause of cancer-related death in men [1.Bray F. et al.Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J. Clin. 2018; 68: 394-424Crossref PubMed Scopus (45300) Google Scholar]. The insidious and indolent course of PCa is often associated with delayed detection of the tumor. Hence, older men with PCa exhibit a higher mortality rate compared with the younger population [2.Tay K.J. et al.Management of prostate cancer in the elderly.Clin. Geriatr. Med. 2016; 32: 113-132Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Although current therapeutic approaches can successfully control the primary tumor over a long period of time, most of the patients with late-stage disease display metastasis at distant sites. One of the major clinical challenges arising from PCa is its propensity to metastasize to bone. More than 90% of patients affected by advanced PCa show evidence of skeletal metastases, which are incurable and correlate with increased risk of fractures, bone pain, disability, and shorter survival. While certain solid tumors, such as breast and lung cancer, have the propensity to cause osteolytic lesions (see Glossary), metastatic PCa predominantly produces osteoblastic lesions with a concurrent osteolytic component [3.Roudier M.P. et al.Histological, immunophenotypic and histomorphometric characterization of prostate cancer bone metastases.Cancer Treat. Res. 2004; 118: 311-339Crossref PubMed Google Scholar]. The arrival of PCa cells in the skeleton typically leads to an activation of bone anabolic pathways [4.Wong S.K. et al.Prostate cancer and bone metastases: the underlying mechanisms.IJMS. 2019; 20: 2587Crossref Scopus (52) Google Scholar] with uncontrolled bone deposition of reduced quality. In fact, even though osteoblasts are more active, the spatial disposition of the bone matrix is disrupted, causing an impairment of bone microarchitecture and reduced mechanical resistance and bone stiffness [5.Sekita A. et al.Disruption of collagen/apatite alignment impairs bone mechanical function in osteoblastic metastasis induced by prostate cancer.Bone. 2017; 97: 83-93Crossref PubMed Scopus (46) Google Scholar]. The dynamic interactions between tumor cells and bone resident cells that result in the perturbation of bone homeostasis are classically described in the well-established ‘vicious cycle’ [6.Guise T.A. The vicious cycle of bone metastases.J. Musculoskelet. Neuronal Interact. 2002; 2: 570-572PubMed Google Scholar] (Figure 1, Key Figure). Briefly, PCa cancer cells that have migrated to the bone adapt to, and modify, the surrounding microenvironment by secreting active molecules that promote osteoblast differentiation and proliferation, including transforming growth factor beta (TGF-β) and endothelin-1 [7.Guise T.A. et al.Basic mechanisms responsible for osteolytic and osteoblastic bone metastases.Clin. Cancer Res. 2006; 12: 6213s-6216sCrossref PubMed Scopus (404) Google Scholar,8.Fizazi K. et al.Prostate cancer cells-osteoblast interaction shifts expression of growth/survival-related genes in prostate cancer and reduces expression of osteoprotegerin in osteoblasts.Clin. Cancer Res. 2003; 9: 2587-2597PubMed Google Scholar]. In turn, enhanced osteoblast activity drives tumor progression by releasing insulin-like growth factor (IGF-1), and interleukins 6 and 8 (IL-6 and IL-8) [9.Ottewell P.D. The role of osteoblasts in bone metastasis.J. 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Gene Expr. 2005; 15: 15-28Crossref PubMed Scopus (20) Google Scholar]. Although numerous mechanisms contributing to the survival of PCa cells within the bone have been deeply dissected, recent studies confirmed that interactions between neoplastic cells and bone cells are more complex than our traditional understanding. Advances in PCa research revealed that primary tumors can ‘prepare’ the site of future metastasis by educating bone marrow cells before the metastatic spread has even occurred [13.Liu Y. Cao X. Characteristics and significance of the pre-metastatic niche.Cancer Cell. 2016; 30: 668-681Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar]. This fosters the establishment of a conducive tumor milieu that supports invasion, immune escape, and metastasis formation [14.Shiao S.L. et al.Regulation of prostate cancer progression by the tumor microenvironment.Cancer Lett. 2016; 380: 340-348Crossref PubMed Scopus (110) Google Scholar]. Although multiple studies have focused mainly on the function of soluble factors in PCa progression, new evidence has reported the critical role of EVs in tumor metastasis and bone colonization [15.Willms E. et al.Extracellular vesicle heterogeneity: subpopulations, isolation techniques, and diverse functions in cancer progression.Front. Immunol. 2018; 9: 738Crossref PubMed Scopus (356) Google Scholar,16.Saber S.H. et al.Exosomes are the driving force in preparing the soil for the metastatic seeds: lessons from the prostate cancer.Cells. 2020; 9: 564Crossref Scopus (22) Google Scholar]. EVs represent a heterogeneous population of membrane-derived vesicles released by essentially all cell types. Their major characteristic is the transfer of active molecules to recipient cells, influencing the physiological properties [17.Yáñez-Mó M. et al.Biological properties of extracellular vesicles and their physiological functions.J. Extracell Vesicles. 2015; 427066Crossref PubMed Scopus (2605) Google Scholar]. Recent studies showed that PCa-derived EVs facilitate tumor progression by instructing osteoblasts toward a protumorigenic cell type. In this way, EVs ensure the formation of an attractive and supportive niche for tumor growth and survival [18.Vlaeminck-Guillem V. Extracellular vesicles in prostate cancer carcinogenesis, diagnosis, and management.Front. Oncol. 2018; 8: 222Crossref PubMed Scopus (55) Google Scholar]. Also, the presence of EVs in many body fluids could render them attractive biomarkers for the early detection of the disease [19.Huang T. Deng C.-X. Current progresses of exosomes as cancer diagnostic and prognostic biomarkers.Int. J. Biol. Sci. 2019; 15: 1-11Crossref PubMed Scopus (119) Google Scholar]. Thus, advances in this field will be useful to further deconstruct the complexity of the bone–PCa microenvironment. Another strategy adopted by PCa to fully exploit the bone metastatic niche relies on the ability of tumor cells to acquire an osteoblast-like phenotype, also known as osteomimicry [20.Scimeca M. et al.Prostate osteoblast-like cells: a reliable prognostic marker of bone metastasis in prostate cancer patients.Contrast Media Mol. Imaging. 2018; 20189840962Crossref PubMed Scopus (16) Google Scholar]. As demonstrated, PCa cells in the bone can resemble osteoblasts by expressing bone matrix proteins and modulating bone cell crosstalk [20.Scimeca M. et al.Prostate osteoblast-like cells: a reliable prognostic marker of bone metastasis in prostate cancer patients.Contrast Media Mol. Imaging. 2018; 20189840962Crossref PubMed Scopus (16) Google Scholar]. This results in an alteration of physiological bone remodeling, which leads to enhanced tumor survival and proliferation. Growing understanding of skeletal metastasis also confirmed the ability of PCa to induce relevant phenotypic changes in bone marrow cells upon direct contact [21.Decker A.M. et al.Biochemical changes in the niche following tumor cell invasion.J. Cell. Biochem. 2017; 118: 1956-1964Crossref PubMed Scopus (4) Google Scholar]. In particular, the preferential distribution of PCa cells in osteoblast-rich areas together with an increased bone turnover [22.Wang N. et al.Prostate cancer cells preferentially home to osteoblast-rich areas in the early stages of bone metastasis: evidence from in vivo models.J. Bone Miner. Res. 2014; 29: 2688-2696Crossref PubMed Scopus (84) Google Scholar] suggests that physical contact of PCa cells shifts normal osteoblasts into CAOs to create a supportive niche within the hostile bone microenvironment. CAOs represent an important source of cytokines and growth factors that actively promote tumor cell homing and invasiveness. However, the genetic signatures, metabolic determinants, and functional characteristics that discriminate CAOs from healthy osteoblasts remain to be determined. In this review, we describe the regulatory function of PCa-derived EVs in osteotropism and osteomimicry properties of PCa cells during bone colonization, introduce the concept of CAOs, and summarize the current understanding of their role in PCa metastasis. Advances over the past decade confirmed that PCa cell colonization and homing to the bone is not a random event. Rather, tumor cells determine their own fate by releasing a multitude of active molecules that selectively modify organs of future metastasis and generate a conducive environment for the survival and outgrowth of the tumor [23.Costa-Silva B. et al.Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.Nat. Cell Biol. 2015; 17: 816-826Crossref PubMed Scopus (1527) Google Scholar,24.Peinado H. et al.Pre-metastatic niches: organ-specific homes for metastases.Nat. Rev. Cancer. 2017; 17: 302-317Crossref PubMed Scopus (840) Google Scholar]. This preconditioned microenvironment that occurs before tumor cells have settled is also termed the ‘premetastatic niche’. Since the existence of the premetastatic niche was demonstrated [25.Psaila B. Lyden D. The metastatic niche: adapting the foreign soil.Nat. Rev. Cancer. 2009; 9: 285-293Crossref PubMed Scopus (907) Google Scholar], many studies have focused on the role of soluble factors in modulating the metabolic and cellular changes in distant organs [23.Costa-Silva B. et al.Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.Nat. Cell Biol. 2015; 17: 816-826Crossref PubMed Scopus (1527) Google Scholar,26.Hoshino A. et al.Tumour exosome integrins determine organotropic metastasis.Nature. 2015; 527: 329-335Crossref PubMed Scopus (2637) Google Scholar]. However, the specific mechanisms by which cancer cells precondition the niche and home to these specific areas remain incompletely understood. More recently, accumulating evidence demonstrated the presence of an alternative and evolutionarily conserved communication system. Tumor cells are now known to regulate metastatic progression by releasing a heterogeneous group of membranous structures called EVs [27.Zaborowski M.P. et al.Extracellular vesicles: composition, biological relevance, and methods of study.Bioscience. 2015; 65: 783-797Crossref PubMed Scopus (436) Google Scholar,28.Turturici G. et al.Extracellular membrane vesicles as a mechanism of cell-to-cell communication: advantages and disadvantages.Am. J. Phys. Cell Phys. 2014; 306: C621-C633Crossref PubMed Scopus (311) Google Scholar]. Based on their size, biogenesis, cargo, and surface markers, EVs are usually classified into three subcategories: exosomes, microvesicles, and apoptotic bodies [29.Théry C. et al.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.J. Extracell Vesicles. 2018; 71535750Crossref PubMed Scopus (3701) Google Scholar,30.Zhang H. et al.Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.Nat. Cell Biol. 2018; 20: 332-343Crossref PubMed Scopus (671) Google Scholar]. One of the most important characteristics of PCa-shed EVs is their ability to modify recipient cells by transferring their cargo, which generally comprises nucleic acids, proteins, and lipids. PCa EVs can affect the proliferation, angiogenesis, and survival of cancer cells, as well as their ability to avoid immune surveillance [31.Lorenc T. et al.Exosomes in prostate cancer diagnosis, prognosis and therapy.Int. J. Mol. Sci. 2020; 21: 2118Crossref Scopus (45) Google Scholar]. Importantly, PCa exosomal molecules mediate cell–cell communication in osteoblastic metastasis by educating osteoblasts (EOs) toward a prometastatic phenotype (Figure 2). In vitro experiments showed that EVs derived from PC3 and DU-145 cell lines facilitate EO differentiation in murine preosteoblastic MC3T3 cells by releasing v-ets erythroblastosis virus E26 oncogene homolog 1 [32.Itoh T. et al.Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation.J. Mol. Histol. 2012; 43: 509-515Crossref PubMed Scopus (35) Google Scholar]. Additionally, it was demonstrated that the RNA cargo of PC3-derived EVs promotes EOs activity and creates a more favorable environment for tumor growth. In fact, using RNA-sequencing (RNA-seq) analysis, it was possible to identify potential candidates that might be involved in PCa bone metastasis, including osteoblastic factor colony-stimulating factor 1 and ephrin A3, required for osteoblast cell–cell interaction and osteoblastic bone formation [33.Probert C. et al.Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis.Oncogene. 2019; 38: 1751-1763Crossref PubMed Scopus (42) Google Scholar]. Among all the biomolecules carried by the EVs, miRNAs have an important regulatory role in cancer progression. Recent findings demonstrated that miR141-3p released by the humanized PCa cell line MDA-PCa 2b promotes osteoblastic activity in vitro and creates a favorable microenvironment for bone metastasis in vivo. From a mechanistic point of view, uptake of miR141-3p by EOs stimulates the expression of OPG by reducing the protein levels of its target gene, DLC1, and activating the p38-MAPK signaling pathway, thereby inhibiting osteoclast activity [34.Ye Y. et al.Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.Oncotarget. 2017; 8: 94834-94849Crossref PubMed Scopus (74) Google Scholar]. Another study reported a significant upregulation of exosomal miR-375, miR-21, and miR-let7c in urine samples of patients with PCa [35.Foj L. et al.Exosomal and non-exosomal urinary miRNAs in prostate cancer detection and prognosis.Prostate. 2017; 77: 573-583Crossref PubMed Scopus (100) Google Scholar]. In line with these observations, RNA-seq analysis of PCa-derived EVs revealed a significant expression of miR-375 in LNCaP cells. Furthermore, miR-375 mimic-transfected EOs exhibited increased expression levels of differentiation genes, thus suggesting the contribution of miRNA-375 in PCa bone metastasis formation [36.Li S.-L. et al.Exosomes from LNCaP cells promote osteoblast activity through miR-375 transfer.Oncol. Lett. 2019; 17: 4463-4473PubMed Google Scholar]. Further analyses also confirmed high expression of miR21 in PC3 cells and showed that exposure of human EOs to miR-21-depleted PC3-EVs resulted in a reduction in cell viability [33.Probert C. et al.Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis.Oncogene. 2019; 38: 1751-1763Crossref PubMed Scopus (42) Google Scholar]. Consistent with the ability of PCa-derived EVs to modify the bone tumor microenvironment, exosomal hs-miR-940 induces osteogenic differentiation of human mesenchymal stem cells by targeting ARHGAP1 and FAM13A, thereby promoting the onset of osteoblastic bone metastasis [37.Hashimoto K. et al.Cancer-secreted hsa-miR-940 induces an osteoblastic phenotype in the bone metastatic microenvironment via targeting ARHGAP1 and FAM134A.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 2204-2209Crossref PubMed Scopus (139) Google Scholar]. In contrast to miRNAs, proteins from signaling pathways important for osteoblasts, such as wingless Int-1 (Wnt) and bone morphogenetic protein (BMP) signaling, have barely been investigated in PCa. However, considering such molecules have already been identified in EVs from other cancer types [38.Scavo M.P. et al.Frizzled-10 extracellular vesicles plasma concentration is associated with tumoral progression in patients with colorectal and gastric cancer.J. Oncol. 2019; 20192715968Crossref PubMed Scopus (15) Google Scholar,39.Hu Y.-B. et al.Exosomal Wnt-induced dedifferentiation of colorectal cancer cells contributes to chemotherapy resistance.Oncogene. 2019; 38: 1951-1965Crossref PubMed Scopus (93) Google Scholar], it is likely that future studies will also identify Wnt and BMP molecules in PCa-derived EVs, which might affect osteoblast fate. Recent advances also showed that tumor cells secrete a higher amount of EVs compared with normal proliferating cells, and their cargo differs in terms of composition and effects on recipient cells [40.Isola A.L. Chen S. Exosomes: the messengers of health and disease.Curr. Neuropharmacol. 2017; 15: 157-165Crossref PubMed Scopus (104) Google Scholar]. Defining molecular mechanisms through which PCa cells release prometastatic EVs will help to reduce tumor aggressiveness and eventually identify potential therapeutic targets. Of note, expression of cavin-1 in PC3 cells has been shown to selectively alter EV cargo by reducing tumor-associated growth factors and cytokines, including IL-6 and TGF-β2 [41.Inder K.L. et al.Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation.J. Extracell Vesicles. 2014; 323784Crossref Scopus (68) Google Scholar]. These changes go in line with a reduction in tumor growth as well as attenuation of osteoclast differentiation and osteoblast proliferation [41.Inder K.L. et al.Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation.J. Extracell Vesicles. 2014; 323784Crossref Scopus (68) Google Scholar,42.Moon H. et al.PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer.Oncogene. 2014; 33: 3561-3570Crossref PubMed Scopus (53) Google Scholar]. Additionally, it was demonstrated that inhibition of exosome biogenesis following GW4869 treatment is associated with a significant reduction in both the survival and colony-forming ability of PCa cancer [43.Panigrahi G.K. et al.Hypoxia-induced exosome secretion promotes survival of African-American and Caucasian prostate cancer cells.Sci. Rep. 2018; 8: 3853Crossref PubMed Scopus (66) Google Scholar]. In accordance with that, manumycin-A was identified as a potential adjuvant therapeutic drug in patients with castration-resistant PCa. The ability of manumycin-A to inhibit exosome secretion by targeting the Ras signaling pathway in PCa cells might help to control the growth of the tumor and eventually reduce metastatic progression [44.Datta A. et al.Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras/Raf/ERK1/2 signaling and hnRNP H1 in castration-resistant prostate cancer cells.Cancer Lett. 2017; 408: 73-81Crossref PubMed Scopus (101) Google Scholar]. Although the effects of EVs in PCa progression are usually evaluated by considering tumor cells as the only relevant source of vesicles, there is now strong evidence that EOs can also release EVs, which in turn modulate PCa cells. In vitro experiments showed that cellular uptake of human osteoblast-derived EVs induced a twofold increase in PC3 cell growth compared with EV-free medium controls [45.Morhayim J. et al.Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth.FASEB J. 2015; 29: 274-285Crossref PubMed Scopus (47) Google Scholar]. Additionally, proteomic profiling of vesicles isolated from murine primary osteoblasts revealed a wide range of molecules that might be involved in both physiological and pathophysiological processes. Among all the proteins identified, expression of the adhesion molecule Cadherin-11 appears to mediate successful EV uptake into PCa cells, contributing to the metastatic potential of tumor cells [46.Bilen M.A. et al.Proteomics profiling of exosomes from primary mouse osteoblasts under proliferation versus mineralization conditions and characterization of their uptake into prostate cancer cells.J. Proteome Res. 2017; 16: 2709-2728Crossref PubMed Scopus (29) Google Scholar]. Taken together, these observations confirm that the functions of EVs in bone metastasis are dependent on the interaction between osteoblasts and tumor cells. However, because of the complexity and heterogeneity of EV-mediated communication, further analyses need to be performed to better understand the synergic interaction of PCa with the tumor microenvironment and decipherer the molecular changes of EOs before metastasis formation. Osteomimicry represents a hallmark of osteotropic cancer entities and comprises the ability of malignant cells to resemble resident bone cells, thereby interfering with the physiology of the skeleton and evading attack by the immune system. In the PCa context, tumor cells boost their survival within the bone microenvironment by acquiring an osteoblastic phenotype [20.Scimeca M. et al.Prostate osteoblast-like cells: a reliable prognostic marker of bone metastasis in prostate cancer patients.Contrast Media Mol. Imaging. 2018; 20189840962Crossref PubMed Scopus (16) Google Scholar]. Of note, interactions with osteoblasts cause alterations of PCa cell behavior and promote a shift toward an osteoblast-like gene signature. As a result, PCa cells release molecules that are typical for bone cell differentiation and maintenance, such as osteocalcin, osteopontin (OPN), alkaline phosphatase, and BMPs [47.Knerr K. et al.Bone metastasis: osteoblasts affect growth and adhesion regulons in prostate tumor cells and provoke osteomimicry.Int. J. Cancer. 2004; 111: 152-159Crossref PubMed Scopus (39) Google Scholar,48.Hagberg Thulin M. et al.Osteoblasts stimulate the osteogenic and metastatic progression of castration-resistant prostate cancer in a novel model for in vitro and in vivo studies.Clin. Exp. Metastasis. 2014; 31: 269-283Crossref PubMed Scopus (25) Google Scholar]. Interestingly, most of these proteins are highly expressed in bone-metastatic prostate tumors compared with tumors from the primary site [49.Gardner T.A. et al.Differential expression of osteocalcin during the metastatic progression of prostate cancer.Oncol. Rep. 2009; 21: 903-908PubMed Google Scholar], confirming the ability of PCa cells to ensure long-term tumor growth by adapting to, and disguising themselves within, a foreign microenvironment. Among all the osteoblast-derived molecules released by tumor cells, runt-related transcription factor 2 (Runx2) has a decisive role in the establishment of skeletal metastasis. Endogenous expression of Runx2 has shown to support PCa cell growth and tumor progression by enhancing TGFβ and androgen-responsive pathways [50.van der Deen M. et al.The cancer-related Runx2 protein enhances cell growth and responses to androgen and TGFbeta in prostate cancer cells.J. Cell. Biochem. 2010; 109: 828-837PubMed Google Scholar]. Also, it was demonstrated that upregulation of connective tissue growth factor in the PC3 cell line enhances Runx2 stability by reducing ubiquitination-dependent degradation. This leads to an overexpression of metalloproteinases and RANKL, which, in turn, promote tumor invasiveness [51.Kim B. et al.A CTGF-RUNX2-RANKL axis in breast and prostate cancer cells promotes tumor progression in bone.J. Bone Miner. Res. 2020; 35: 155-166Crossref PubMed Scopus (34) Google Scholar]. Furthermore, a functional connection between Runx2 and expression of a metastatic phenotype in PCa cells was demonstrated by Runx2 overexpression and knockdown studies that showed an increase and reduction, respectively of PCa growth and osteolytic disease [52.Akech J. et al.Runx2 association with progression of prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic metastatic lesions.Oncogene. 2010; 29: 811-821Crossref PubMed Scopus (207) Google Scholar]. Specifically, overexpression of Runx2 in PC3 cells promotes the activation of VEGF, OPN, and MMPs, underlining the involvement of Runx2 in tumor proliferation and invasiveness [52.Akech J. et al.Runx2 association with progression of prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic metastatic lesions.Oncogene. 2010; 29: 811-821Crossref PubMed Scopus (207) Google Scholar]. Interestingly, expression of the transcription factor TWIST in human PCa cells mediates PCa bone metastasis by regulating Dickkopf-1 expression and enhancing PCa osteomimicry via activation of Runx2 [53.Yuen H.-F. et al.TWIST modulates prostate cancer cell-mediated bone cell activity and is upregulated by osteogenic induction.Carcinogenesis. 2008; 29: 1509-1518Crossref PubMed Scopus (46) Google Scholar]. Another important and translationally relevant group of osteoblastic factors involved in bone-related malignancies are the Wnt proteins. While the role of Wnts in bone development and homeostasis is well defined [54.Regard J.B. et al.Wnt signaling in bone development and disease: making stronger bone with Wnts.Cold Spring Harb. Perspect. Biol. 2012; 4a007997Crossref PubMed Scopus (136) Google Scholar, 55.Houschyar K.S. et al.Wnt pathway in bone repair and regeneration – what do we know so far.Front. Cell Dev. Biol. 2019; 6: 170Crossref PubMed Scopus (109) Google Scholar, 56.Steinhart Z. Angers S. Wnt signaling in development and tissue homeostasis.Development. 2018; 145dev146589Crossref PubMed Scopus (209) Google Scholar], the function of Wnt signaling in carcinogenesis is intriguingly complex and remains only rudimentarily understood. 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