patterns of tumor metastases are dependent on the recipient organ microenvironment , and each component of the metastatic tumor microenvironment plays distinct and unique roles in disease progression . to better understand organ - specific metastases and to ultimately develop more effective therapeutics
, the biology of key cellular components in the microenvironment must be clearly determined . among many types of solid tumors , prostate ,
the normal physiology of the skeletal system requires a tightly - regulated balance between osteoblasts and osteoclasts .
briefly , the migrated breast cancer cells express bone - modulatory factors such as parathyroid hormone - related peptide ( pthrp ) , leading to up - regulation of receptor activator of nuclear factor b ( rankl ) and monocyte - colony stimulating factor ( m - csf ) in adjacent osteoblasts .
subsequently , osteoclastogenesis occurs , followed by osteolysis and the release of bone matrix - embedded growth factors ( e.g. , transforming growth factor  [ tgf- ] ) .
the homeostasis of the human skeletal system is maintained by the coupling of bone resorption ( by osteoclasts ) and new bone formation ( mainly by osteoblasts ) .
alterations in the bone homeostasis result in many types of bone diseases such as fractures and osteoporosis .
in addition to the vicious cycle hypothesis described above , increasing lines of evidence now clearly support that alterations in the bone homeostasis contribute to the progression of bone metastases . among the many cell types in the metastatic bone microenvironment , including osteocytes , osteoblasts , osteoclasts , mesenchymal stem cells , and hematopoietic bone marrow cells in diverse stages of differentiation ,
the majority of research data concentrate on osteoclasts , and thus the current therapeutic approaches targeting the metastatic bone microenvironment are mostly osteoclast inhibitors ( e.g. , bisphosphonates , denosumab , and cathepsin k inhibitors ) . on the other hand ,
numerous research groups have demonstrated that other types of cells , particularly osteoblasts , are also important in the metastatic bone microenvironment , and also that osteoblasts play distinct and essential roles in metastatic progression .
accordingly , this review paper will summarize and highlight the recent publications supporting the role of osteoblasts in bone metastasis .
hedgehog ( hh ) signaling is important in development , regulating cell growth , body pattern formation and organogenesis .
mammalians have three types of hh proteins , i.e. , sonic , indian , and desert hh with sonic hh ( shh ) being the most thoroughly investigated in development and pathology .
several lines of evidence suggest that hh is involved in organ - specific metastasis to bone , especially in breast and prostate cancers , as well as in bone tumors such as osteosarcomas .
.   demonstrated that shh up - regulates rankl expression in bone stromal cells and osteoblasts , leading to osteoclast formation .
subsequently , an increase in osteoclasts contributes to tumor - induced osteolysis and ultimatelys to increased tumor growth in bone .
recently , shimo et al .   analyzed human oral squamous cell carcinoma patient samples and identified that shh is expressed in bone - invasive tumor cells while patched receptor and gli2 ( a downstream transcription factor of hh signaling ) are expressed in osteoclast progenitor cells , indicating that tumor - derived shh stimulated osteoclast formation and bone resorption in tumor - induced jaw bone destruction .
the above examples clearly show that hh is a paracrine factor in the tumor microenvironment . accordingly , metastatic tumor - derived hh proteins stimulate the bone stromal cells to promote metastatic tumor growth . indeed
, hh proteins reregulate rankl expression in adjacent osteoblasts and bone marrow stromal cells , leading to osteoclastogenesis via activation of extracellular signal - regulated kinases ( erk ) , p38 mitogen - activated protein kinases ( mapk ) , increased expression of nuclear factor of activated t cells , cytoplasmic 1 ( nfatc1 ) .
chan et al .   demonstrated that osteoblast - specific hh expression increased osteosarcoma development in p53 mutant mice .
furthermore , inhibition of hh signaling reduced the expression of yes - associated protein ( yap1 ) , a potent oncogene , in osteosarcoma cells .
they also showed that the long non - coding rna h19 , a highly relevant candidate for osteosarcoma development , is aberrantly expressed and induced by up - regulated hh signaling and yap1 overexpression , suggesting a molecular mechanism of hh - dependent osteosarcoma progression from osteoblasts .
briefly , in the absence of hh ligands , the patched ( ptch1 ) receptors accumulate and inhibit the function of smoothened ( smo ) protein , leading to degradation of the gli transcription factor by proteasome . upon activation ,
the ptch1 receptors are degraded and thus gli is activated , leading to its nuclear translocation and function as a transcriptional activator .
johnson et al .   demonstrated that bone matrix - derived tgf- increased the expression of gli2 and pthrp in metastatic breast cancer cells , leading to further bone destruction .
furthermore , the authors showed that gli activity was not dependent on canonical hh signaling , because mda - mb-231 breast cancer cells do not express smo .
.   demonstrated that hh pathway is important in osteoblast maturation , particularly in the presence of breast cancer cells .
hh - mediated gli2 stimulated secretion of bone morphogenetic protein ( bmp2 ) , a potent osteogenic differentiation factor , as well as cyclin d , osteopontin , and insulin - like growth factor , suggesting that hh is an important factor of osteoblastic functions .
collectively , the hh proteins are important mediators between tumor and bone , and the signaling is bi - directional ( i.e. , tumor - to - stroma and stroma - to - tumor ) .
runt - related transcription factor 2 ( runx2 ) is also known as core - binding factor subunit- 1 ( cbf-1 ) and serves as a key transcription factor in osteoblast differentiation and gene expression .
runx2 binds to cognate dna elements with the consensus nucleotide sequence 5-accaca in the promoters / enhancers of target genes .
franceschi and xiao   extensively summarized the data supporting that runx2 is important in the responsiveness to multiple signal transduction pathways in osteoblasts .
indeed , the franceschi group demonstrated that functional runx2 is essential to fibroblast growth factor 2 ( fgf2)- and parathyroid hormone - induced osteocalcin expression in osteoblasts , and bmp2-stimulated osteoblast differentiation . in this context
, runx2 was shown to be a critical regulator of osteoblasts in the bone metastatic tumor microenvironment .
for example , zong et al .   showed that the runx2-phtrp - rankl pathway contributes to the progression of an experimental breast cancer bone metastases .
furthermore , li et al .   demonstrated that runx 2 increased integrin -like 1 expression , leading to up - regulation of tgf- in the bone metastatic breast cancer stroma .
runx2 has been classically considered an osteoblast - specific transcription factor , but more recently , increasing evidence supports that runx2 is not only important in osteoblasts but also in tumor cells in the bone microenvironment .
for example , li et al .   recently showed that runx2 promotes breast cancer bone metastasis by increasing integrin 5-mediated colonization .
in addition , ge et al .   provided pivotal evidence that phospho - runx2 is proportionately expressed during the progression of prostate cancer bone metastasis with the highest expression in metastatic lesions using tissue microarray and in vitro prostate cancer cell line models .
the authors also concluded that phospho - runx2 has a prognostic value for prostate cancer patients .
runx2 is also involved in tumor - induced osteolysis by transcriptional suppression of tssc1 via the bmp - tgf- , wnt , pthrp , and src pathways .
more specifically , expression of runx2 is associated with osteolysis , and up - regulated runx2 mediated direct interactions between the tgf-/bmp and smad signaling pathways , contributing to the formation of osteolytic- and osteoblastic - mixed bone lesions that are related with prostate cancer cells .
moreover , tumor growth and distant metastasis of prostate cancer occur via the runx2-smad complex .
the runx2-smad complex has functional activity that mediated tgf- and bmp signaling through smid ( i.e. , smad interaction domain ) .
for example , vascular endothelial growth factor ( vegf ) , matrix metalloproteinases , interleukin 8 ( il-8 ) , and pthrp are up - regulated by runx2 in tumor cells , and mediated tumor growth and organ - specific metastasis to bone . vegf and il-8 are both potent angiogenic factors and strong pro - tumorigenic factors .
pthrp is an important mediator of the vicious cycle of bone metastasis and also plays diverse roles in tumor progression .
runx2 physically and functionally interacts with the receptors for androgens and estrogens , which are the targets for many prostate cancer and breast cancer drugs . in short ,
overexpression of runx2 is observed in the stromal compartment as well as in tumor cells and its phosphorylation is important when the tumor cells metastasize to bone .
the runx2-smad complex regulates the activation of tgf-/bmp signaling , stimulating osteolysis and bone destruction .
lastly , runx2 inhibits the expression of tssc1 , a tumor suppressor that inhibits bone metastasis by blocking the promoter of the tssc1 gene .
micrornas ( mirnas ) are non - coding rnas composed of very short nucleotides sequences up to 22 nucleotides .
mirnas down - regulate mrna - dependent translation of target genes by producing short hairpin rnas .
one mirna can affects dozens of mrnas , and mirnas have an important role as post - transcription regulators in bone formation .
mirnas potentially contribute to the proliferation and metastasis of tumor cells by regulating target gene expression .
many mirna function as tumor suppressors ; however , some mirnas aid in tumor growth and metastasis .
for example , mir103/107 contributes to tumor progression by down - regulating dicer , which has an important role in processing mirna precursors .
up - regulated dicer promotes the expression of mirnas that inhibit tumor progression and metastasis , and therefore mir103/107 inhibits the expression of dicer , contributing to tumor progression .
mir-154 , mir-379 , and mir-409 - 3p/5p work as oncogenes in bone metastasis of prostate cancer .
mir-154 , mir379 and mir-409 - 3p/5p are members of -like 1 homolog - deiodinase , iodothryonine3 ( dlk1-dio3 ) , and are activated in the epithelial to mesenchymal transition ( emt ) of prostate cancer cells .
the mirnas of the dlk1-dio3 cluster are up - regulated in bone metastatic prostate and breast cancer cells . among those , mir-379 and mir-154 located in the dlk1-doi3 cluster are specifically up - regulated in prostate cancer cells with the mesenchymal phenotype ( i.e. , more metastatic ) .
mir-379 and mir-154 are expressed by embryonic stem cells and pluripotent stem cells , and these mirnas regulate bone - specific prostate cancer cells by inducing emt .
second , mir-154 regulates the expression of emt- and stemness - related genes to mediate downstream convergent signal pathways .
when mir-154 is inhibited , e - cadherin expression is up - regulated , suggesting decreased invasion and metastasis .
in addition , mir-379 is located in the upstream of the mir-154 gene , and has a similar role as mir-154 . in bone metastatic prostate cancer cells ,
mir-409 - 3p/5p inhibits tumor suppressor genes in prostate cancer cells , for example stromal antigen 2 ( stag2 ) , ras suppressor protein 1 ( rsu1 ) , retinoblastoma - like 2 ( rbl2 ) , and nitrogen permease regulator - like 2 ( nprl2 ) .
mir-409 - 3p is predicted to activate the ras signaling pathway and the hypoxia inducible factor-1 pathway , as well as regulate polycomb group proteins and osteoblastic pathways .
mir-409 - 5p is predicted to activate the e2f pathway , ras signaling pathway , akt pathway , and aneuploidy .
taken together , multiple lines of evidence demonstrate that mir-409 - 3p/5p is elevated in the bone metastatic emt cell models and this microrna functions by repressing several tumor suppressor genes .
osteoblasts are derived from mesenchymal stem cells in bone marrow , and serves as the major cell type essential to bone anabolic processes .
differentiation of osteoblasts is determined by multiple steps via many growth factors , receptors , and transcriptional factors such as hh / ptch , bmp , fibroblast growth fators ( fgfs ) , parathyroid hormone ( pth ) , runx2 , wnt , and tgf--smad .
the pathways have a high degree of cross - talk among them , with runx2 being a major point of convergence .
osteons , a functional unit of osteoblasts , mediate the bone anabolic reaction by producing organic matrices of bone tissue including cross - linked collagen , and osteocalcin , osteopontin .
subsequently , the matrices become mineralized , forming hard tissue . the roles of osteoblasts in the tumor microenvironment
have been a focus of extensive research efforts thus far , with the vicious cycle hypothesis being the most well investigated by dr .
the hypothesis was later clinically and experimentally proven to be legitimate and provided a solid scientific rationale for the development of multiple bone - targeted agents .
however , the over - simplified cycle comprised of tumor , osteoblasts , osteoclasts , and tgf- , does not perfectly explain the complex interactions among the extremely diverse stromal cells in the bone microenvironment or the pathologic progression of bone metastasis .
for example , we previously demonstrated that megakaryocytes , previously considered to have no clear roles in bone metastasis , are the first cell type that encounter extravasating tumor cells in the bone sinusoidal vessel architecture , and induce apoptosis of tumor cells thus protecting from bone metastasis .
provided pivotal evidence supporting that the osteoblastic hematopoietic stem cell niches are the main sites of tumor cell localization , and metastatic tumor cells compete with stem cells for the occupancy of the stem cell niche .
most notably , we demonstrated that primary tumor - derived pthrp circulates to stimulate osteoblasts , leading to up - regulation of myeloid - derived suppressor cells ( mdsc ) .
these pre - activated mdscs in the tumor hosts then enter the systemic circulation to increase primary tumor growth and angiogenesis .
1 demonstrates that osteoblasts are stimulated to grow ( bracket ) and form new woven bones ( solid arrow ) , indicating that osteoblasts are one of the first responder cells in bone metastasis , potentially playing critical functions other than expressing osteoclastic factors ( rankl and m - csf ) .
this review paper summarizs the current evidence supporting how osteoblasts are regulated and what roles they play in the progression of bone metastasis .
in particular , we examined the three most significant mediators including hh and patched receptor signaling ( i.e. , an extracellular stimulant and the cell surface receptor ) , the runx2 transcription factor and mirnas ( i.e. , post - transcriptional level regulation ) .
all of this evidence clearly warrants extensive further research , potentially providing an important foundation for novel and advanced therapeutics specific to bone metastasis .
beyond the conventional concept of tumor - initiative and bone - responsive process , recent data are focused on how the cells in the bone environment ( i.e. , osteocytes , osteoblasts , and osteoclasts ) initiate the dormant disseminates tumor cells in bone .
the current evidence clearly demonstrates that tumor cells engage with osteoblastic lineage cells on the endosteal surface and enter the long - term dormancy .
on the other hand , osteoclast - mediated bone remodeling that is activated by estrogen withdrawal can release dormant tumor cells from the active control of the endosteal niche , facilitating reactivation and tumor growth . instead of osteoclasts passively responsing to tumor - derived products
in this point of view , the osteoblastic niche can be a potential therapeutic target for the treatment of bone metastasis .
further intensive studies focusing on bone cell responding to tumor cells are required to overcome bone metastasis , one of the leading causes of cancer mortality .
 the tumor microenvironment is comprised of diverse stromal cell populations in addition to tumor cells . increasing evidence 
 now clearly supports the role of microenvironment stromal cells in tumor progression and metastasis , yet the regulatory mechanisms and interactions among tumor and stromal cells remain to be elucidated . 
 bone metastasis is the major problem in many types of human malignancies including prostate , breast and lung cancers , and the biological basis of bone metastasis let alone curative approaches are largely undetermined . among the many types of stromal cells in bone , 
 osteoblasts are shown to be an important player . in this 
 regard , osteoblasts are a key target cell type in the development of bone metastasis , but there are currently no drugs or therapeutic approaches are available that specifically target osteoblasts . 
 this review paper summarizes the current knowledge on osteoblasts in the metastatic tumor microenvironment , aiming to provide clues and directions for future research endeavor . 