THE ROLE OF HYPOXIA AND GROWTH FACTOR SIGNALING PATHWAYS IN PROMOTING SKELETAL METASTASIS
Tesutul osos reprezinta un micromediu hypoxic capabil sa potenteze metastazele tumorale si cresterea acestora. Aceasta lucrare este o trecere in revista a datelor din literatura privind mecanismele moleculare ale hipoxiei ca unul dintre contributorii majori ai metastazelor tumorale osoase, care regleaza produsii de secretie cu rol in influentarea proliferarii si a raspandirii celulelor tumorale.
Cuvinte-cheie: hypoxia, remodelarea osoasa, metastaze tumorale
Bone tissue is a hypoxic microenvironment capable of potentiating tumor metastasis and growth. This paper is a review of literature data on molecular mechanisms of hypoxia as one of the major contributors to tumor bone metastasis, regulating secreted products that drive tumor-cell proliferation and spread .
Keywords:hypoxia, bone remodeling,tumor metastasis
Solid tumors are particularly susceptible to hypoxia because they proliferate rapidly, outgrowing the malformed tumor vasculature, which is unable to meet the increasing metabolic demands of the expanding tumor. Hypoxia also contributes to resistance to radiation and chemotherapy in primary tumors; it regulates normal marrow hematopoiesis and chondrocyte differentiation. The medullary cavity oxygen pressure in humans is estimated to be 5% O2 (1). Cancer cells capable of surviving at low oxygen levels can thrive in the hypoxic bone microenvironment and participate in the vicious cycle of bone metastasis. Hypoxic signaling is mediated by hypoxia-inducible factor-1(HIF -1; ref. 2). This transcription factor is a heterodimer of HIF-1a and HIF-1h. HIF-1a expression is
regulated in response to oxygen levels, whereas HIF-1h is constitutively expressed.
Under normoxic conditions, oxygen-dependent prolyl hydroxylases modify HIF-1a at
specific residues within the oxygen-dependent degradation domain. Hydroxylated HIF-1a is recognized and targeted for proteosomal degradation by the von Hippel-Lindau
tumor suppressor, which is a component of an E32612 ubiquitin-protein ligase (3).
When oxygen levels are low,HIF-1a is no longer targeted for degradation by prolyl
hydroxylases and instead, heterodimerizes with HIF-1h.The HIF-1heterodimer enters the nucleus where it binds to hypoxia-response elements in DNA and mediates the
transcription of numerous hypoxia-response genes. Hypoxic signaling is increased in cancer cells exposed to low oxygen levels in the primary tumor. Hypoxia-response genes regulated by HIF-1 include glycolytic enzymes, glucose transporters, and vascular endothelial growth factor, which is important for angiogenesis. Other genes are expressed in a cell-type specific manner, including ones involved in tissue remodeling/migration/invasion, apoptosis,stress responses, proliferation/differentiation, and growth factor/cytokine function (4). Many are also prometastatic, suggesting a role for hypoxia signaling in the vicious cycle of bone metastasis.
In 13 different human cancers, including lung, breast,prostate, and colon, HIF-1a was overexpressed in two thirds of all the regional lymph node and bone metastases
examined, including 69% of metastases versus 29% of primary tumors among the breast cancers (5). HIF-1a overexpression was correlated with advanced tumor stage
(6), suggesting that increased HIF-1a is associated with a more aggressive and metastatic tumor phenotype. In vitro, HIF-1a overexpression correlated with increased invasive potential of human prostate cancer cells, as well as enhanced expression of vimentin, cathepsin D, and MMP-2,which are important for cell migration and invasion, and decreased levels of E-cadherin, which is responsible for maintenance of cell-cell contacts and adhesion (7). Vimentin and E-cadherin are involved in epithelial-mesenchymal transition early in metastastic progression. Through upregulation of these proteins, HIF-1alters the phenotype of tumor cells to increase their metastatic capability.
HIF-1a increases the transcription of factors that could accelerate the vicious cycle of skeletal metastases. MET, a receptor tyrosine kinase that binds hepatocyte growth
factor, is overexpressed in advanced breast cancer and is associated with invasion and metastasis. MET expression is mediated by HIF-1a under hypoxic conditions. HIF-1a and
MET cover expression in primary tumor samples from breast cancer patients who had undergone modified radical mastectomy was independently correlated with metastasis
and decreased 10-year disease-free survival (8). HIF-1 also regulates the expression of other factors, including adrenomedullin, chemokine receptor 4, and connective tissue
growth factor, with known roles in carcinogenesis and tumor metastasis (9,10).
Under normoxic conditions, HIF-1a stabilization is regulated by numerous growth factors and cytokines through the phosphatidylinositol-3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinase pathways(11). Growth factors, such as IGFs, fibroblast growth factor, epidermal growth factor (EGF), and tumor necrosis factor-a, have been shown to stabilize HIF-1a. Expression of these factors by tumor cells is associated with enhanced proliferation and tumor spread. Hypoxia and growth factor signaling pathways may synergistically promote the vicious cycle of skeletal metastasis.
Several studies have shown crosstalk between hypoxia and growth factor signaling pathways. In normoxic conditions, the EGF receptor (EGFR) signaling pathway
activates HIF-1a–mediated transcription of survivin, a protein which increases apoptotic resistance of human breast cancer cells, thus contributing to a more aggressive cancer phenotype (12). Crosstalk also occurs between the HIF-1a and TGF-h signaling pathways: TGF-h increases hypoxic signaling by selectively inhibiting prolyl hydroxylase 2 and decreasing HIF-1a degradation (13). As discussed previously, TGF-h is important in osteolytic bone metastases, and these results show that TGF-h
potentiates HIF-1signaling within the hypoxic bone microenvironment. As a regulator of tumor progression and metastasis, the hypoxia signaling pathway is an important chemotherapeutic target. Inhibiting this pathway may prevent the development of HIF-mediated resistance to chemotherapy and radiation therapy. A number of small molecule inhibitors of hypoxia signaling are under development. One such inhibitor is 2-methoxyestradiol, a poorly estrogenic estrogen metabolite and microtubule-depolymerizing agent with antiangiogenic and antitumorigenic properties
(14). 2-Methoxyestradiol decreases HIF-1a levels and vascular endothelial growth factor mRNA expression in vitro and induces apoptosis of tumor cells (15,16). 2-Methoxyestradiol is currently being evaluated in phases I and II clinical trials for the treatment of multiple types of cancer, and more potent analogues with improved antiangiogenic and antitumor effects are being developed (17).
Other small molecule antihypoxic agents include inhibitors of topoisomerase I and II, such as camptothecin and GL331,and inhibitors of phosphatidylinositol-3-kinase, such as
LY294002 — all of which have been shown to inhibit HIF mediated gene transcription (10). Because HIF-1crosstalks with multiple signaling pathways, inhibiting hypoxia
signaling alone may be inadequate to halt tumor growth and spread (18). However, small molecule inhibitors could be useful in combination with other therapies to halt the
vicious cycle of metastasis. Acidic pH Acidosis of the bone microenvironment also potentiates the vicious cycle of bone metastasis. Extracellular pH is tightly regulated within bone and has significant effects on osteoblast and osteoclast function. Extracellular acidification results in increased osteoclast resorbtion pit formation,with osteoclasts being maximally stimulated at pH levels of<6.9 (19). Osteoblast mineralization and bone formation is significantly impaired by acid (18). The combined effect on osteoclasts and osteoblasts is the release of alkaline bone mineral from the skeleton, compensating for systemic acidosis. Tumor metastasis leads to localized regions of acidosis within the skeleton (19). Increased glycolysis and lactic acid production by proliferating cancer cells and decreased buffering capacity of the interstitial fluid contribute to the acidic microenvironment within primary tumors (20). The acid-mediated tumor invasion hypothesis states that altered glucose metabolism in cancer cells stimulates cancer cell proliferation and results in a more invasive tumor phenotype (21). Acidosis alters cellular dynamics at the interface between the tumor and normal tissue, promoting apoptosis in adjacent normal cells and facilitating extracellular matrix degradation through the release of proteolytic enzymes. Unlike normal cells, cancer cells have compensatory mechanisms to allow proliferation and metastasis even at low extracellular pH and thus are not susceptible to acid-induced apoptosis. Hypoxia further promotes acidosis within tumor cells through HIF-mediated overexpression of glycolytic enzymes and increased lactic acid production (22). Together, hypoxia and pH regulatory mechanisms control survival and proliferation of tumor cells. Apoptosis of E1a/Rastransformed mouse embryo fibroblasts is mediated by hypoxia-induced acidosis rather than as a direct effect of hypoxia exposure (23). Tumor acidosis promotes the release and activation of proteins, such as cathepsins B, D, and L and MMPs, which degrade the extracellular matrix and facilitate metastasis (21). Cathepsin B is a cysteine proteases expressed by tumor cells, which is activated in an acidic microenvironment and could participate in the vicious cycle of bone metastasis (24). It is expressed at low levels in primary prostate tumors; however, bone metastatic lesions express high levels of activated cathepsin B, suggesting that protease activity is modulated by interactions between tumor cells and the bone microenvironment (25). Hypoxia-mediated acidosis also activates numerous stress signaling cascades within tumor cells, including the nuclear factor-nB and activator protein-1pathways, which in turn regulate the transcription of prometastatic factors, such as IL-8, a cytokine important for cell motility, proliferation, and angiogenesis (26). IL-8 expression is induced by prolonged hypoxia and decreased intracellular pH in pancreatic and prostate cancer cells (27). Its overexpression correlates with increasing tumor grade and metastasis in many cancers, including breast and prostate. Both hypoxia and acidosis have been implicated in resistance of cancer cells to radiation and chemotherapy. Extracellular acidity contributes to chemotherapeutic resistance via a pH gradient that prevents the intracellular accumulation of weakly basic drugs, such as Adriamycin (22).
The bone microenvironment contains numerous physical factors, such as hypoxia, acidosis, and extracellular calcium, and growth factors, like TGF-h, which have
been implicated in this vicious cycle. These factors activate signaling pathways in cancer cells, promoting a more aggressive tumor phenotype. Tumor acidosis is a direct consequence of hypoxia exposure. Thus, therapeutic approaches, which target hypoxia signaling may exert their beneficial effects by correcting pH in cancer cells, making them more susceptible to conventional radiation and chemotherapy.
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