**2.4 Osteosarcoma**

Osteosarcoma is an osteoid-producing malignancy of mesenchymal origin [12]. It is the third most frequent type of cancer in adolescence and represents more than 56% of all bone tumors [13]. It is the most common bone sarcoma and affects 60% of the patients below 25 years of age and 30% of the patients above 40 years [14, 15]. It is associated with extensive genomic disruptions and propensity of metastatic spread. Seventy-six percent of the osteosarcomas have been found to be associated with reduced expression of *HACE1* gene localized to human chromosome 6q21, thus resulting in poor survival [14]. Approximately, 30–40% children with pediatric osteosarcoma die due to metastasis to lungs [1, 16].

Environmental factors such as exposure to radiation, teriparatide (parathyroid hormone 1–34) usage, and consumption of fluorinated drinking water during

childhood increase the risk of osteosarcoma [12]. Its localization in regions that are largely cut off from the vasculature reduces the effectiveness of systemically administered chemotherapeutics [17]. The subtypes of osteosarcoma include osteoblastic, chondroblastic, fibroblastic, small cell, telangiectatic, high-grade surface, extraskeletal, and other lower-grade forms of tumors including parosteal and periosteal [12].

Osteosarcomas are one of the widely studied sarcomas of the skeletal system. Several investigations have reported the association of germline mutation disorders such as hereditary retinoblastoma, Rothmund-Thomson syndrome, Li-Fraumeni syndrome, and Bloom syndrome, with increased risk of osteosarcoma [12]. Various other pathways discussed below have been reported to be responsible for supporting growth and metastasis of osteosarcoma.


**17**

**Figure 1.**

*bone tissue.*

*Bone Tumors: Types and Treatments*

(**Figure 1**) [27].

*DOI: http://dx.doi.org/10.5772/intechopen.86550*

ties and enhanced invasive potential [30].

Additionally, FGF also regulates multiple signaling cascades in both autocrine and paracrine manner [4]. FGF receptor 1 was identified as a c-Fos-regulated gene playing an important role in lung metastases of osteosarcoma. The FGF receptors 1–4 belong to a family of receptor tyrosine kinase (RTK) that triggers intracellular signaling cascades through mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), STAT pathways, and signal transducers phospholipase Cγ (PLCγ) and casitas B-lineage lymphoma (CBL)

5.Majority of the osteosarcomas develop as a result of metastases of breast, prostate, or lung tumors into the bone. Breast cancer cells metastasize to the bone and secrete various factors in the bone microenvironment that enhance osteoclastogenesis and inhibit osteoblastogenesis, thus developing several skeletal-related events (SREs) such as pathological fracture, spinal cord compression, bone pain, and hypercalcemia [28]. Similarly, androgen signaling components such as androgen receptor, ARV7, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), cytochrome P450, and family 17 subfamily A polypeptide 1 (CYP17) and molecules such as phospho-Met, phospho-Src, glucocorticoid receptor, and Ki67 have been implicated to play a major role

in the metastasis of castration-resistant prostate cancer (CRPC) [29].

Some of the reports associate migration and invasion of breast cancer cells to the upregulation of micro RNAs, miR-10b, miR-373, and miR-520c. Out of these, miR-373 and miR-520c have been found to silence *CD44* gene that codes for hyaluronan receptor (plays an important in cellular adhesion). Similarly, miR-218 that is involved in osteoblast differentiation promotes breast cancer cell osteomimicry (ability to acquire bone cell phenotype for immune escape). miR-154 and miR-379 overexpression in bone metastatic cells was associated with mesenchymal proper-

*Cross talk of various signaling pathways in osteosarcoma. (i) JAK–STAT pathway confers apoptosis resistance, thus enhancing tumor cell survival. (ii) Inactivation of p53 favors cell cycle progression in cells with DNA damage and promotes tumorigenesis. (iii) Cross talk between GPCR-regulated PTHrP signaling and RTKmediated survival/growth factors. (iv) Role of RTK in supporting tumor cell survival and proliferation as a response to various growth factors. (v) Wnt signaling mechanism plays a major role in tumors associated with* 

#### *Bone Tumors: Types and Treatments DOI: http://dx.doi.org/10.5772/intechopen.86550*

*Hormone Therapy and Replacement in Cancer and Aging-Related Diseases*

and periosteal [12].

(**Figure 1**) [21].

microenvironment (**Figure 2**) [24].

ing growth and metastasis of osteosarcoma.

been observed in osteosarcoma (**Figure 1**) [19].

childhood increase the risk of osteosarcoma [12]. Its localization in regions that are largely cut off from the vasculature reduces the effectiveness of systemically administered chemotherapeutics [17]. The subtypes of osteosarcoma include osteoblastic, chondroblastic, fibroblastic, small cell, telangiectatic, high-grade surface, extraskeletal, and other lower-grade forms of tumors including parosteal

Osteosarcomas are one of the widely studied sarcomas of the skeletal system. Several investigations have reported the association of germline mutation disorders such as hereditary retinoblastoma, Rothmund-Thomson syndrome, Li-Fraumeni syndrome, and Bloom syndrome, with increased risk of osteosarcoma [12]. Various other pathways discussed below have been reported to be responsible for support-

1.Constitutively active signal transducers and activators of transcription 3 (STAT3) signaling have been found to be necessary for osteosarcoma survival and migration in vitro and tumor growth in vivo. STAT3 is a proto-oncogene that stimulates self-proliferation (due to expression of cyclin D1), mediates immune evasion, promotes angiogenesis, and confers apoptosis resistance (induced by conventional therapies, due to expression of BCL2) [18]. Similarly an upregulation in the expression of survivin (an oncogenic protein) has also

2.Attenuation of cell cycle arrest by p53 has been found to affect the upstream p53 signaling pathways [20]. Approximately 26.5% of non-hereditary osteosarcomas have been reported to be associated with somatic loss of p53, out of which 60% are high-grade osteosarcomas, whereas 1% are low-grade osteosarcomas [14]. Tumor suppressor genes such as p15 and p27 have been commonly found to be silenced due to methylation of promoter by DNA methyl transferase that adds methyl group to the fifth carbon position of cytosine ring in CpG islands, leading to heterochromatin and inhibition of gene expression

3.Sex steroid hormones have been found to play an important role in development and progression of bone tumors. Previous investigations have revealed the role of aromatase and sulfatase pathways in the in situ formation of active estrogen. Aromatase pathway involves aromatization of androgens to produce estrogen. In sulfatase pathway, estrone 3-sulfate is taken up by the cells and is activated by the removal of sulfate by steroid sulfatase, thus converting inactive estrogen to unconjugated and bioactive estrogen. Both these pathways allow generation of estrogen and androgen by the bone cells in the bone

4.Growth factors such as transforming growth factor (TGF)-β, insulin-like growth factors (IGFs), bone morphogenetic proteins (BMPs), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) released from degraded bone matrices also promote tumor cell proliferation by production of PTHrP that interacts with parathyroid hormone (PTH)/PTHrP receptors in the bone and kidney to cause hypercalcemia, osteoclast-mediated bone resorption, increased nephrogenous cyclic AMP, and phosphate excretion [4–6, 25]. PTH is known to regulate osteosarcoma cells, by inducing transcription of c-Fos that in turn targets calcium/cAMP-response element (CRE) through activation of protein kinase A (PKA) [26]. c-Fos is a member of activator protein-1 (AP-1) family of transcription factors containing c-Fos (FosB, Fra-1, Fra-2) (**Figure 1**) [27].

**16**

Additionally, FGF also regulates multiple signaling cascades in both autocrine and paracrine manner [4]. FGF receptor 1 was identified as a c-Fos-regulated gene playing an important role in lung metastases of osteosarcoma. The FGF receptors 1–4 belong to a family of receptor tyrosine kinase (RTK) that triggers intracellular signaling cascades through mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), STAT pathways, and signal transducers phospholipase Cγ (PLCγ) and casitas B-lineage lymphoma (CBL) (**Figure 1**) [27].

5.Majority of the osteosarcomas develop as a result of metastases of breast, prostate, or lung tumors into the bone. Breast cancer cells metastasize to the bone and secrete various factors in the bone microenvironment that enhance osteoclastogenesis and inhibit osteoblastogenesis, thus developing several skeletal-related events (SREs) such as pathological fracture, spinal cord compression, bone pain, and hypercalcemia [28]. Similarly, androgen signaling components such as androgen receptor, ARV7, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), cytochrome P450, and family 17 subfamily A polypeptide 1 (CYP17) and molecules such as phospho-Met, phospho-Src, glucocorticoid receptor, and Ki67 have been implicated to play a major role in the metastasis of castration-resistant prostate cancer (CRPC) [29].

Some of the reports associate migration and invasion of breast cancer cells to the upregulation of micro RNAs, miR-10b, miR-373, and miR-520c. Out of these, miR-373 and miR-520c have been found to silence *CD44* gene that codes for hyaluronan receptor (plays an important in cellular adhesion). Similarly, miR-218 that is involved in osteoblast differentiation promotes breast cancer cell osteomimicry (ability to acquire bone cell phenotype for immune escape). miR-154 and miR-379 overexpression in bone metastatic cells was associated with mesenchymal properties and enhanced invasive potential [30].

#### **Figure 1.**

*Cross talk of various signaling pathways in osteosarcoma. (i) JAK–STAT pathway confers apoptosis resistance, thus enhancing tumor cell survival. (ii) Inactivation of p53 favors cell cycle progression in cells with DNA damage and promotes tumorigenesis. (iii) Cross talk between GPCR-regulated PTHrP signaling and RTKmediated survival/growth factors. (iv) Role of RTK in supporting tumor cell survival and proliferation as a response to various growth factors. (v) Wnt signaling mechanism plays a major role in tumors associated with bone tissue.*

**Figure 2.**

*(A) Percentage of primary bone cancers in adult human population [22]. (B) Osteosarcoma cases per 100,000 population per year [23]. (C) Percentage of males and females diagnosed with primary bone tumor in the United States in the year 2017 [22].*

## **3. Pathophysiology of bone tumors**

General pathogenesis of bone tumors sequentially involves proliferation of primary neoplasm, local tissue invasion, intravasation into blood vessels, extravasation into bone marrow, tumor cell dormancy, proliferation in the bone, and modification of bone microenvironment. The site of metastases is governed by the "seed and soil" hypothesis by Paget which states that neoplastic cells grow or proliferate only in a suitable environment [3].

The initial step in metastasis involves escape of cancer cells from primary tumor into the systemic circulation, through epithelial–mesenchymal transition (EMT). EMT involves loss of cell surface intercellular adhesion proteins and epithelial polarization. EMT is followed by dissolution of the extracellular matrix by secreting certain proteolytic enzymes and migration into surrounding tissue to enter systemic circulation through intravasation. Such circulating tumor cells (CTCs) escape anoikis (cell apoptosis due to loss of cell-matrix or cell-cell interactions, preferably through overexpression of tyrosine kinase receptor, TrkB) resulting in activation of PI3K-AKT pro-survival pathways. These tumor cells also upregulate certain proteins on their surface (e.g., CD47) to escape destruction by macrophages [4]. The two major factors that govern the localization of CTCs are blood flow and molecular signaling. For example, the metastasis of breast cancer to thoracic spine is due to the venous drainage of the breast to thoracic region, whereas lung cancer shows general skeletal distribution due to the drainage of pulmonary veins into the left side of the heart, followed by eventual entry into systemic circulation. Alternatively, prostate cancer majorly

**19**

*Bone Tumors: Types and Treatments*

of AKT signaling [4].

*DOI: http://dx.doi.org/10.5772/intechopen.86550*

cells show a higher expression of ERβ [32].

displays metastasis to the axial skeletal in the lumbar spine, sacrum, and pelvis due to their drainage through pelvic plexus. As far as the signaling pathways are concerned, CXCL12-CXCR4 (CXC motif ligand [CXCL], CXC chemokine receptor [CXCR]) axis has been found to regulate CTC homing to the bone. CXCL12, also called as stromal cell-derived factor 1 (SDF1), is a chemokine factor secreted by bone marrow MSCs, endothelial cells, and osteoblast and primarily binds to G protein-coupled receptor, CXCR4, thus activating cell survival, chemotaxis, and expression of integrin αvβ3 on the surface of CTCs [4]. Another receptor involved in osteotropism is the calcium-sensing receptor (CaSR), expressed by advanced primary breast tumors that causes enhanced calcium-induced migration to the bone [30]. In addition to CXCL12-CXCR4 axis, non-receptor cytoplasmic tyrosine kinase, Src, has also been shown to mediate improved survival of breast cancer cells in the bone marrow by increasing the resistance to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and activation

After tumor cells colonize the bone, they induce the expression of receptor activator of nuclear factor-κB ligand (RANKL) via production of PTHrP, prostaglandin E2 (PGE2), interleukin 6 (IL-6), IL-1β, TNF, and epidermal growth factor (EGF), which promote osteoclast differentiation and activation. RANKL induces osteoclastic bone resorption resulting in growth of osteolytic tumor that causes hypercalcemia, and it also acts as a chemoattractant to the bone for tumor cells [6]. In relation to this, hypoxia-inducible factor (HIF)-1α has been reported to act as an upstream master switch to many of the osteolytic factors such as IL-11 and IL-8 and angiogenic factors such as PDGF and vascular endothelial growth factor (VEGF) [4, 31]. TGF-β plays a central role in the pathogenesis of osteolytic bone metastasis from breast carcinoma via potentiation of estrogen receptor (ER)-α-mediated transcription induced by constitutively active ERα [25]. A higher expression of ERα in the cortical bone suggests its role in bone formation, whereas trabecular bone

Osteolytic bone metastases are most often caused by breast cancer and multiple myeloma [6, 33], whereas osteoblastic metastases are mostly observed in bone metastasis of prostate cancer, which is due to osteoblast stimulation by cancer cells [6, 34]. Factors that are locally produced by cancer cells, such as bone BMP, IGF, FGF, TGF-β, and endothelin-1, also promote osteoblast proliferation and bone formation [6]. Another important cellular component of bone microenvironment that is involved in tumor metastases is osteocytes. Osteocytes present in the bone regulate osteoclast development through expression of RANKL, macrophage colonystimulating factor (M-CSF), and osteoprotegerin (OPG) and inhibit osteoblast differentiation by the expression of sclerostin. Osteocytes have an interesting ability to respond to mechanical stress and pressure. An increase in pressure due to prostate cancer metastasis thus results in upregulation of matrix metalloproteinases (MMPs) and chemokine (C-C motif) ligand 5 (CCL-5). Additionally, apoptotic osteocytes have been shown to release IL-11 that enhances osteoclast differentiation. Endothelial cells are yet another important component of the bone marrow that contributes to the bone metastatic process. Endothelial cells in the metaphysis of the long bone aid CTC adhesion due to constitutive expression of P-selectin, E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule A (ICAM-1). Decrease in shear forces due to reduced blood flow velocity

in the large volume of sinusoids also favors CTC attachment [4].

The bone marrow is also a major reservoir for dendritic cells, macrophages, myeloid-derived cells, and different subsets of T cells. T and B cells are known to produce RANKL and impact osteoclastogenesis, whereas production of IL-6, IL-23, and IL-1 by dendritic cells in the bone microenvironment of multiple

#### *Bone Tumors: Types and Treatments DOI: http://dx.doi.org/10.5772/intechopen.86550*

*Hormone Therapy and Replacement in Cancer and Aging-Related Diseases*

**3. Pathophysiology of bone tumors**

in a suitable environment [3].

*United States in the year 2017 [22].*

**Figure 2.**

General pathogenesis of bone tumors sequentially involves proliferation of primary neoplasm, local tissue invasion, intravasation into blood vessels, extravasation into bone marrow, tumor cell dormancy, proliferation in the bone, and modification of bone microenvironment. The site of metastases is governed by the "seed and soil" hypothesis by Paget which states that neoplastic cells grow or proliferate only

*(A) Percentage of primary bone cancers in adult human population [22]. (B) Osteosarcoma cases per 100,000 population per year [23]. (C) Percentage of males and females diagnosed with primary bone tumor in the* 

The initial step in metastasis involves escape of cancer cells from primary tumor into the systemic circulation, through epithelial–mesenchymal transition (EMT). EMT involves loss of cell surface intercellular adhesion proteins and epithelial polarization. EMT is followed by dissolution of the extracellular matrix by secreting certain proteolytic enzymes and migration into surrounding tissue to enter systemic circulation through intravasation. Such circulating tumor cells (CTCs) escape anoikis (cell apoptosis due to loss of cell-matrix or cell-cell interactions, preferably through overexpression of tyrosine kinase receptor, TrkB) resulting in activation of PI3K-AKT pro-survival pathways. These tumor cells also upregulate certain proteins on their surface (e.g., CD47) to escape destruction by macrophages [4]. The two major factors that govern the localization of CTCs are blood flow and molecular signaling. For example, the metastasis of breast cancer to thoracic spine is due to the venous drainage of the breast to thoracic region, whereas lung cancer shows general skeletal distribution due to the drainage of pulmonary veins into the left side of the heart, followed by eventual entry into systemic circulation. Alternatively, prostate cancer majorly

**18**

displays metastasis to the axial skeletal in the lumbar spine, sacrum, and pelvis due to their drainage through pelvic plexus. As far as the signaling pathways are concerned, CXCL12-CXCR4 (CXC motif ligand [CXCL], CXC chemokine receptor [CXCR]) axis has been found to regulate CTC homing to the bone. CXCL12, also called as stromal cell-derived factor 1 (SDF1), is a chemokine factor secreted by bone marrow MSCs, endothelial cells, and osteoblast and primarily binds to G protein-coupled receptor, CXCR4, thus activating cell survival, chemotaxis, and expression of integrin αvβ3 on the surface of CTCs [4]. Another receptor involved in osteotropism is the calcium-sensing receptor (CaSR), expressed by advanced primary breast tumors that causes enhanced calcium-induced migration to the bone [30]. In addition to CXCL12-CXCR4 axis, non-receptor cytoplasmic tyrosine kinase, Src, has also been shown to mediate improved survival of breast cancer cells in the bone marrow by increasing the resistance to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and activation of AKT signaling [4].

After tumor cells colonize the bone, they induce the expression of receptor activator of nuclear factor-κB ligand (RANKL) via production of PTHrP, prostaglandin E2 (PGE2), interleukin 6 (IL-6), IL-1β, TNF, and epidermal growth factor (EGF), which promote osteoclast differentiation and activation. RANKL induces osteoclastic bone resorption resulting in growth of osteolytic tumor that causes hypercalcemia, and it also acts as a chemoattractant to the bone for tumor cells [6]. In relation to this, hypoxia-inducible factor (HIF)-1α has been reported to act as an upstream master switch to many of the osteolytic factors such as IL-11 and IL-8 and angiogenic factors such as PDGF and vascular endothelial growth factor (VEGF) [4, 31]. TGF-β plays a central role in the pathogenesis of osteolytic bone metastasis from breast carcinoma via potentiation of estrogen receptor (ER)-α-mediated transcription induced by constitutively active ERα [25]. A higher expression of ERα in the cortical bone suggests its role in bone formation, whereas trabecular bone cells show a higher expression of ERβ [32].

Osteolytic bone metastases are most often caused by breast cancer and multiple myeloma [6, 33], whereas osteoblastic metastases are mostly observed in bone metastasis of prostate cancer, which is due to osteoblast stimulation by cancer cells [6, 34]. Factors that are locally produced by cancer cells, such as bone BMP, IGF, FGF, TGF-β, and endothelin-1, also promote osteoblast proliferation and bone formation [6].

Another important cellular component of bone microenvironment that is involved in tumor metastases is osteocytes. Osteocytes present in the bone regulate osteoclast development through expression of RANKL, macrophage colonystimulating factor (M-CSF), and osteoprotegerin (OPG) and inhibit osteoblast differentiation by the expression of sclerostin. Osteocytes have an interesting ability to respond to mechanical stress and pressure. An increase in pressure due to prostate cancer metastasis thus results in upregulation of matrix metalloproteinases (MMPs) and chemokine (C-C motif) ligand 5 (CCL-5). Additionally, apoptotic osteocytes have been shown to release IL-11 that enhances osteoclast differentiation. Endothelial cells are yet another important component of the bone marrow that contributes to the bone metastatic process. Endothelial cells in the metaphysis of the long bone aid CTC adhesion due to constitutive expression of P-selectin, E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule A (ICAM-1). Decrease in shear forces due to reduced blood flow velocity in the large volume of sinusoids also favors CTC attachment [4].

The bone marrow is also a major reservoir for dendritic cells, macrophages, myeloid-derived cells, and different subsets of T cells. T and B cells are known to produce RANKL and impact osteoclastogenesis, whereas production of IL-6, IL-23, and IL-1 by dendritic cells in the bone microenvironment of multiple

myeloma patients causes an increase in Th17 cells that in turn increase IL-17. IL-17 is an important cytokine that promotes osteoclast and myeloma proliferation and also mediates interactions between T cells and bone metastatic environment. Additionally, myeloid-derived suppressor cells from the bone marrow suppress innate and adaptive immune responses by impairing T-cell antigen recognition and promotion of regulatory T cells [4].

However, as an individual ages, the hematopoietic red bone marrow gets converted to adipose tissue-rich yellow bone marrow that has a significant impact on the development of bone metastasis. These bone marrow adipocytes not only serve as an energy source but also secrete several pro-inflammatory mediators such as IL-1β, IL-6, leptin, adiponectin, VCAM-1, TNF-α, and CXCL12 that increase cancer cell survival and proliferation [4].
