Hypoxia and the Extracellular Matrix: Drivers of Tumour Metastasis

Home , Lysyl hydroxylase, Lysyl oxidase, Type IV collagen

Nature Reviews Cancer | AOP, published online 15 May 2014; doi:10.1038/nrc3726 PERSPECTIVES

of the total protein in humans, and they pro- OPINION vide the structural integrity and the tensile strength of human tissues and organs54. In Hypoxia and the extracellular matrix: the context of cancer biology, collagens regulate the physical and the biochemical properties of the tumour microenvironment, which drivers of tumour metastasis modulate cancer cell polarity, migration and signalling17,55–58. Collagen I deposition Daniele M. Gilkes, Gregg L. Semenza and Denis Wirtz and cancer metastasis have been causally linked using mice engineered to express a Of the deaths attributed to cancer, 90% are due to metastasis, and treatments that collagenase-resistant α1 chain of type I col- prevent or cure metastasis remain elusive. Emerging data indicate that hypoxia and lagen (Col1a1tm1jae mice)59. Col1a1tm1jae mice the extracellular matrix (ECM) might have crucial roles in metastasis. During tumour were crossed with mouse mammary tumour evolution, changes in the composition and the overall content of the ECM reflect virus promoter-driven polyoma middle T both its biophysical and biological properties and these strongly influence tumour antigen (MMTV-PyMT) transgenic mice and stromal cell properties, such as proliferation and motility. Originally thought of to model increased type I collagen deposition during the progression of human as independent contributors to metastatic spread, recent studies have established breast cancer59–61. Col1a1tm1jae;MMTV-PyMT a direct link between hypoxia and the composition and the organization of the ECM, bitransgenic mice had a threefold increase which suggests a new model in which multiple microenvironmental signals might in the incidence of tumour formation and converge to synergistically influence metastatic outcome. metastasis compared with their wild-type littermates60. Furthermore, histological studies of human breast carcinomas have shown Rapid cancer cell proliferation, combined Central to this emerging paradigm are three that fibrosis is localized to hypoxic regions with structural and functional abnormalities crucial findings: the ECM is a dynamic struc- within tumours and correlates with immuno in tumour blood vessels, results in regions ture that influences tumour progression13–18; staining of the HIF1 target gene product within solid tumours that have reduced oxy- multiple cell types, including cancer cells, carbonic anhydrase IX (CAIX)51,52. Highly gen availability1. Oxygen availability decreases contribute to ECM production19–23; and the fibrotic tumours also have the highest CAIX as the distance from the nearest blood vessel remodelled ECM within regions of intra immune reactivity, which can independently increases. Intratumoural hypoxia is associ- tumoural hypoxia could be a pathway rather predict patient relapse rate and shorter ated with disorganized vascular networks than an obstacle for cancer metastasis24–27. disease-free survival51,53. In this Opinion with intercapillary distances that are greater article, we discuss emerging data that has than the diffusion distance of oxygen (which The ECM and cancer provided experimental evidence linking the is ~100–200 μm, depending on the local The ECM is composed of approximately 300 mechanisms of hypoxia-induced collagen oxygen concentration in the blood and the proteins that regulate tissue homeostasis, deposition and remodelling to those of rates of oxygen consumption)2. The direct organ development, inflammation and dis- invasion and metastasis. measurement of the tumour partial pressure ease19. The major constituents of the ECM are Tumour ECM synthesis and degradation of oxygen (PO2) using Eppendorf microelec- fibrous proteins (such as collagens, elastins, trodes (which remains the gold standard for fibronectins and laminins) and proteoglycans The current view of tumour fibrosis suggests determining oxygen levels) has revealed that (such as chondroitin sulphate, heparan sul- that recruited and resident fibroblasts and patients whose primary tumours are poorly phate, keratan sulphate and hyaluronic acid) myofibroblasts within the primary tumour oxygenated (those with PO2 <10 mmHg) have that are locally secreted and assembled into are mediators of tumour fibrosis. These an increased risk of metastasis and mortal- an organized mesh, which forms the struc- cells are activated by proteins that are ity1,3,4. The best understood mechanism of tural framework for most tissues28. Molecular secreted by cancer cells, most notably by how cancer cells adapt to a hypoxic environ- approaches aiming to correlate clinical out- transforming growth factor‑β (TGFβ), which ment is through the transcriptional activity of comes with specific gene expression patterns stimulates the synthesis of ECM proteins hypoxia-inducible factors (hypoxia-inducible within the primary tumour have highlighted and the remodelling of the ECM by pro- factor 1 (HIF1) and HIF2; see BOX 1)5. The genes that encode tumour-associated ECM teases produced by cancer-associated fibro- role of hypoxia and HIFs in reprogramming components19,29–34. An increased expression blasts62. Fibroblasts that are isolated from cancer cells by regulating the expression of of genes encoding proteins that mediate the site of a healing wound or from fibrotic multiple genes involved in angiogenesis, by ECM remodelling has been associated with tissues secrete higher levels of normal ECM regulating the metabolism of glucose and by increased mortality in patients with breast, constituents and proliferate more than regulating cancer cell invasion and metastasis lung and gastric cancers35,36. These studies their normal counterparts that are isolated has been extensively reviewed elsewhere6–10. corroborate histological findings that show from healthy organs, which is an explana- Recent reviews also highlight the importance an excessive ECM deposition (also termed tion for the increase in matrix deposition of hypoxia in recruiting the stromal cell com- fibrosis) within solid tumours37–43. that occurs within a tumour63. Although it ponents of the tumour microenvironment11,12. The most well-recognized ECM altera- is well accepted that invasive carcinoma is In this Opinion article, we focus on how tion that occurs in the tumour tissue is often associated with increased ECM depo- hypoxia affects extracellular matrix (ECM) increased collagen deposition44–53. Collagens sition in tumours64, there is also evidence deposition, remodelling and degradation, are the most abundant ECM components, for an increased deposition of ECM in which might potentiate cancer metastasis. constituting up to 90% of the ECM and 30% hypoxic tumour regions. Recent studies have

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uncovered mechanisms of tumour fibrosis folded, which leads to proteolytic degrada- required for cancer cell adhesion, spreading that specifically occur under hypoxic condi- tion and to reduced collagen deposition76,78. and invasion21,68. In contrast to P4HA1 and tions and that involve not only fibroblasts Three isoforms of the prolyl 4‑hydroxylase P4HA2, the depletion of PLOD2 in breast but also other cell types, including cancer α-subunit (P4HA) have been identified cancer cells did not suppress collagen depo- cells20,21,65–68. (P4HA isoform 1 (P4HA1), P4HA2 and sition in vitro or in vivo, but reduced tumour

P4HA3) that form A2B2 tetramers with stiffness by reducing fibrillar collagen con- Hypoxia induces increased collagen gene P4HB, which results in the generation of tent20. PLOD2 knockdown also significantly expression. Hypoxia and HIF1 have been P4H1 (from P4HA1), P4H2 (from P4HA2) impaired the invasion of cancer cells into implicated in renal, liver and adipose tissue and P4H3 (from P4HA3) holoenzymes79,80. the adjacent normal tissue of the mouse fibrosis69–71. Dermal, cardiac and renal fibro- Three procollagen-lysine 2‑oxyglutarate mammary fat pad, reduced the number of blasts cultured under hypoxic conditions 5‑dioxygenase genes (PLOD1, PLOD2 and circulating tumour cells and prevented the show increased type I procollagen α1 mRNA PLOD3) encode enzymes that mediate spontaneous metastasis of breast cancer levels72–74. Furthermore, increased levels collagen lysine hydroxylation. Collagen cells to the lungs and to the lymph nodes of of type I, II and IV procollagen mRNA are crosslinks that are derived from hydroxy- mice20. In murine models of sarcoma, abro- present in the peripheral lung parenchyma lated lysine residues compared with non- gating HIF1-dependent PLOD2 expres- and pulmonary artery of rats that have been hydroxylated lysine residues have increased sion disrupted collagen modification, cell exposed to hypoxia75. However, studies that stability, which leads to increased tissue migration, and pulmonary metastasis67. describe the regulation of collagen gene stiffness81. Thus, stiff tissues, such as bones, Taken together, the studies described above expression in hypoxic cancer cells in vitro cartilage and tendons, contain a higher indicate that hypoxia might regulate ECM and during cancer progression in vivo are percentage of hydroxylated lysine residues deposition by multiple cell types within the lacking. By contrast, the dramatic effect of in collagen compared with soft tissues, such tumour microenvironment20,21,67,68,82,83,86. In hypoxia on the post-translational modifica- as the skin81. addition to the marked effects of collagen tion of collagen is a matter of considerable HIF1 regulates the expression of P4HA1, prolyl and lysyl hydroxylase expression in investigation, as described below. P4HA2, PLOD1 and PLOD2 in cancer cells, experimental mouse models of metastasis, fibroblasts, chondrocytes and endothelial P4HA1, P4HA2 and PLOD2 expression HIF1 regulates the expression of intracellular cells20,21,67,82–86. Abrogating the expression have also been suggested as biomarkers for collagen-modifying enzymes. Collagen of HIF1α, P4HA1 or P4HA2 through the human cancer progression in several biogenesis originates with gene transcrip- stable transfection of cells with short hair- independent studies (BOX 2). tion and is followed by the translation of pin RNA (shRNA) vectors inhibits collagen mRNA into procollagen (pro-α‑chains) deposition from both breast cancer cells and HIFs regulate extracellular collagen- (FIG. 1). At least 28 collagen subtypes, which fibroblasts in vitro21,82. Reducing the levels of modifying enzymes. Following enzymatic are encoded by 42 genes that generate 42 HIF1α, P4HA1 or P4HA2 in vivo results in modification of type I collagen by hydroxy- distinct pro-α‑chains, have been identi- decreased fibrosis and decreased tissue stiff- lation, two α1(I)-chains and one α2(I)- fied in vertebrates76. Within the endoplas- ness in orthotopic tumours that are formed chain associate to form a triple helix that is mic reticulum, the pro-α‑chains undergo by the injection of human breast cancer cells secreted into the extracellular space (FIG. 1). multiple post-translational modifications, into the mammary fat pads of immuno- Collagen peptidases cleave the carboxy- and which include the hydroxylation of proline deficient mice21,68. Decreasing the levels of amino‑terminal peptides, and type I collagen and lysine residues, followed by the glyco- HIF2α expression in breast cancer cells had fibrils form spontaneously, are covalently sylation of hydroxylysine residues76. The no effect21. Importantly, P4HA1 or P4HA2 crosslinked on hydroxylysine and lysine resi- modification of proline to 4‑hydroxyproline knockdown inhibited the spontaneous dues and form structurally stable collagen I is essential for the thermal stability of the metastasis of breast cancer cells to the lungs fibres76,87. Fibrillar collagens, such as type I collagen triple helix77. Procollagen α‑chains and to the lymph nodes of mice by reducing collagen, establish the interstitial matrix that are not hydroxylated are improperly the formation of collagen fibres, which are and contribute to tissue stiffness with exten- sive post-translational modifications that 54 Box 1 | Regulation of HIFs increase tensile strength . Non-fibrillar collagens, such as type IV collagen, constitute a Hypoxia-inducible factors (HIFs) are transcription factors that function as heterodimers, which key component of the basement membrane, consist of an oxygen-regulated HIF1α (or HIF2α) subunit and a constitutively expressed HIF1β which is a compact sheet-like structure that 178,179 subunit . HIFs bind to the consensus sequence 5ʹ-RCGTG‑3ʹ that is present within or near functions as a barrier to separate tumour HIF-regulated genes180. HIF1 protein levels are regulated by oxygen-dependent prolyl α cells from the adjacent stroma88. hydroxylation, which is required for binding of the von Hippel–Lindau (VHL) tumour suppressor Collagen crosslinking is extracellularly protein, leading to ubiquitylation and proteasomal degradation of HIF1α181. Hydroxylation of HIF1α residues Pro402 and Pro564 is catalysed by HIF prolyl hydroxylase domain-containing protein 1 initiated by the lysyl oxidase (LOX) family of (PHD1), PHD2 and PHD3 in a reaction that is dependent on the presence of cofactors, oxygen and secreted enzymes that oxidatively deaminate 87 α‑ketoglutarate (also known as 2‑oxoglutarate). Under low oxygen conditions, HIF1α lysine or hydroxylysine collagen residues . hydroxylation, ubiquitylation and degradation are inhibited153. HIF2α, which shares 48% amino Three LOX enzymes — LOX, LOX-like pro- acid sequence identity with HIF1α, is also oxygen-regulated and binds to HIF1β to form HIF2, tein 2 (LOXL2) and LOXL4 — are important which activates the transcription of some, but not all, HIF target genes182,183. Many oncogenic hypoxia-induced and HIF‑regulated target 184–186 alterations in cancers cells, including loss of function of VHL, PTEN and p53 , as well as gene products that are involved in collagen 187 activation of the PI3K–AKT pathway, also cause an increase in HIF activity. Data obtained from crosslinking and tumour fibrosis65,89–93. In many recent studies that use a range of approaches have revealed unique roles for HIF1 and α addition to collagen crosslinking within the HIF2α in both normal and cancer cells152. primary tumour, secreted LOX has been

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metastasis in experimental mouse models; Single however, βAPN might not inhibit the activ- procollagen ity of all LOX family members93, which sug- molecule gests that HIFs or pan-LOX inhibitors could a b P4HA1 PLOD1 P4HA2 PLOD2 represent broader targets than currently P4HA3 PLOD3 available drugs or antibodies that target only Procollagen α-chains OH OH OH OH a subset of LOX and LOXL proteins.

ER OH OH OH OH HIF1 and HIF2 can regulate ECM degradation. In addition to collagen deposition, OH OH OH OH collagen degradation also contributes to ECM remodelling and is mediated by several families of proteinases that have been suggested to promote cancer cell invasion; Triple procollagen molecule c for example, the matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that are divided into several subgroups (collagenases, gelatinases, stromelysins and cell membrane-bound LOX MMPs) with different substrate specifici- LOXL1 LOXL2 f ties. Hypoxia is associated with an increase in the expression and the activity of type IV d e LOXL3 Self-assembly LOXL4 f collagen-degrading enzymes (MMP2 and MMP9) in vitro94–96. MMP2 and MMP9 are upregulated by hypoxia in breast and Collagen ﬁbril Procollagen Procollagen Collagen ﬁbre colon cancer cells via a HIF1‑dependent N-terminal C-terminal mechanism94–96, whereas membrane-bound proteinase proteinase membrane-type 1 MMP (MT1‑MMP; also Figure 1 | Biosynthesis of fibrillar collagens. The biosynthesis of type I collagen and other fibrillar Nature Reviews | Cancer known as MMP14) is upregulated in a collagens can be divided into intracellular (parts a–c) and extracellular (parts d–f) steps. The first intra- HIF2‑dependent manner94,97. In addition cellular step involves the synthesis of procollagen polypeptides from any of 42 distinct collagen gene to collagen degradation by MMPs, hypoxic transcripts (part a). Procollagens are post-translationally modified within the cisternae of the endo- cancer cells also show increased proteo- plasmic reticulum (ER) by prolyl 4‑hydroxylase α-subunit isoform 1 (P4HA1), P4HA2 and P4HA3 and by procollagen-lysine 2‑oxyglutarate 5‑dioxygenase 1 (PLOD1), PLOD2 and PLOD3 lysyl hydroxylase lytic activity as a result of HIF-dependent enzymes (part b). Hydrolysine residues can be further modified to galactosyl hydroxylysine and to increases in their expression of urokinase glucosylgalactosyl hydroxylysine by collagen galactosyltransferase and glucosyltransferase, respec- plasminogen activator surface receptor98,99 tively. The carboxyl termini of three properly hydroxylated procollagen molecules will associate and (PLAUR). PLAUR promotes cell invasion spontaneously propagate a procollagen triple helix from the carboxyl terminus to the amino terminus. by altering the interactions between integ- The triple helical procollagen will be transported from the ER to the extracellular space via the Golgi rins and the ECM. When PLAUR expres- (part c). Two metalloproteinases, a procollagen N‑terminal proteinase and a procollagen C‑terminal sion levels are depleted by the expression proteinase, cleave the non-helical termini (part d) and the mature collagen proteins spontaneously of shRNAs, cells with reduced levels of aggregate to form a collagen fibril (part e). The final step, collagen fibre formation, is initiated by col- PLAUR are incapable of intravasation100. lagen crosslinking, which is catalysed by lysyl oxidase (LOX) family members and occurs via the lysine aldehyde- or hydroxylysine aldehyde-initiated pathway (part f). The number and the proportion of the Thus, HIFs activate a transcriptional pro- various crosslinks are tissue specific and are regulated by the steric relationship between localized gramme that results in the degradation of collagen molecules, the type of collagens co-polymerized and the glycosylation and the hydroxylation the basement membrane while simultane- of the participating amino acid residues. For example, lysine aldehyde-initiated crosslinks are found ously increasing the de novo synthesis of in soft connective tissue, in contrast to hydroxylysine aldehyde-initiated crosslinks, which are found in fibrillar collagens to function as a physical stiff connective tissues. Many non-fibrillar collagens retain a non-collagenous N- or C‑terminal, which pathway for tumour invasion (FIG. 2). prevents the spontaneous formation of collagen fibrils, and in these collagens cysteine crosslinks might be the only source of covalent intermolecular bonds. Enzymes highlighted in red are induced Growth factors and ECM deposition under hypoxic conditions. LOXL, LOX-like protein. Tumours have long been described as ‘wounds that won’t heal’ (REF. 101). Similarly, shown to localize within the lungs and to LOX family member expression in response hypoxia is known to have a role in both remodel existing collagen to establish a pre- to hypoxia, but in each case the expression is normal and pathological wound healing. metastatic niche containing bone marrow- HIF dependent89. Consideration of the spe- In normal cutaneous wounds, HIF1 is derived cells (BMDCs), which facilitates cific LOX family members that are induced important for appropriate angiogenic colonization of the niche by cancer cells in by hypoxia is therefore essential to prevent responses, for mobilization of circulating murine models of breast cancer90–91. LOX collagen remodelling, BMDC recruitment angiogenic cells, such as endothelial pre- family members are upregulated to vary- and metastasis in the lungs of tumour- cursor cells and mesenchymal stem cells ing levels and in different combinations in bearing mice89,90. The pharmacological (MSCs), and for normal wound contrac- human breast cancers93. Similarly, breast inhibition of LOX by β‑aminopropionitrile tion102. Partial reduction of HIF1α expres- cancer cell lines show different patterns of (βAPN) has been reported to inhibit sion is consequently sufficient to impair

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Box 2 | Collagen hydroxylases and cancer fundamentally different from the ECM of normal tissues but are also continuously Procollagen-lysine 2‑oxyglutarate 5‑dioxygenase 2 (PLOD2) was included among the genes remodelled17,132, which reflects the dynamic 188 189 shown to be upregulated in gene expression screens of cervical cancer , glioblastoma and changes that occur in the tumour micro- gastric cancer190, and was 1 of 17 genes that predicted breast cancer metastasis to the brain191. environment, including changes in oxygen Gene expression studies also revealed increased PLOD2 mRNA expression in primary sarcoma samples from patients with metastatic compared to non-metastatic sarcomas67. Moreover, availability. human osteosarcoma samples have two to three times more hydroxylysine content than normal bone collagen, which indicates that PLOD activity is increased in these patients44. Increased Tumour Stiffness. Tumour stroma is typi- prolyl 4‑hydroxylase α-subunit isoform 1 (P4HA1) expression was revealed by a meta-analysis cally stiffer than normal stroma; for example, that was used to identify genes that are upregulated across many different cancer types192. breast tumours can be ten times stiffer than P4HA2 was determined to be a metastasis-associated protein in oral cavity squamous cell normal breast tissue57,133 and expression carcinoma using comparative tissue proteomics193. Increased P4HA2 expression levels also of collagen-modifying enzymes, such as discriminated papillary thyroid cancer from normal thyroid tissue194. Increased P4HA1, P4HA2 or P4HA1, P4HA2, PLOD2 and LOX, that can PLOD2 mRNA expression is predictive of breast cancer patient survival; the predictive power is be induced by hypoxia promote tumour stiff- improved when the expression of all three genes is evaluated and determined to be greater than ness20,21,58,65,66,82. Stiffening of the ECM causes the median expression level20,21. a reciprocal increase in the traction forces that are exerted by a cell134,135. Intracellular wound healing103. During wound healing, Hypoxia and macrophage recruitment contraction in response to ECM stiffening angiogenesis and ECM deposition occur Hypoxia-induced growth factor secretion results in an increase in the stiffness of the in parallel104; therefore, it is not surprising in the primary tumour also promotes the actin cytoskeleton and an increase in cell that some of the same factors that stimu- accumulation of macrophages, which rapidly migration57,58,136–138. Increased tumour stiffness late angiogenesis also promote fibrosis. respond to the hypoxic microenvironment by might regulate tumour progression in several Hypoxia-induced angiogenic growth factor altering their gene expression patterns124–126. ways; for example, increasing matrix stiffness production has been well established105. The importance of hypoxia in stimulat- increases RHO-generated cytoskeletal ten- HIF1 has been shown to bind to a cis-acting ing macrophage infiltration during wound sion to promote focal adhesion assembly and hypoxia-response element in the genes that healing has been shown in heterozygous to increase growth factor-dependent ERK encode vascular endothelial growth fac- HIF1α‑deficient mice, which show con- activation123,135. Moreover, matrix stiffness tor (VEGF), stromal cell-derived factor 1 siderable delays in myeloid cell infiltra- facilitates integrin clustering, leading to the (SDF1; also known as CXCL12), angio tion127. Recent studies highlight a potential activation of focal adhesion kinase 1 (FAK1), poietin 2 (ANG2), platelet-derived growth mechanism of macrophage recruitment into which in turn activates the MAP/ERK factor B (PDGFB), placental growth factor hypoxic regions involving the release of sem- kinase (MEK; also known as MAP2K)–ERK (PGF), connective tissue growth factor aphorin 3A by hypoxic cancer cells, which pathway and leads to increased cell survival, (CTGF) and stem cell factor (SCF)106–114 functions as an attractant for macrophages migration, invasion and proliferation57,138,139. and can also indirectly promote fibroblast that express neuropilin 1 (NRP1)128. Once in Depletion of FAK1 in mouse tumour models growth factor 2 (FGF2)115 production in a the region of hypoxia, macrophages stimulate inhibits local invasion and metastasis, which variety of cell types (TABLE 1). fibrosis by producing growth factors such as indicates that FAK1 activation might be an Although well-known for their influence TGFα, TGFβ1, VEGF, FGF, PDGF, tumour important mediator of stiffness-induced on tumour angiogenesis, many of these necrosis factor-α (TNFα), interleukin‑1 tumour metastasis140–142. Interestingly, matrix growth factors also contribute to fibro- (IL‑1) and IL‑8, which can attract additional stiffening can lead to a feed-forward signal- sis116–118, potentially by attracting fibroblasts macrophages and mesenchymal cells, such ling mechanism that further increases matrix to the primary tumour and/or by activating as fibroblasts and endothelial cells, and can stiffening; for example, YAP1 is required resident fibroblasts. Experimental evidence further activate stromal cells129. Macrophages for matrix stiffening by cancer-associated indicates that the recruitment of fibro- also directly promote the process of cancer fibroblasts during tumour progression143. blasts or myofibroblasts to sites of patho- cell intravasation into nearby blood vessels130. Conversely, stiff matrices and the contractile logical fibrosis is driven by hypoxia119–121. In addition, they contribute to ECM turn actin cytoskeleton further increase YAP1 Similarly, hypoxia increases the recruit- over by secreting MMPs, which suggests that activation143. ment of bone marrow-derived MSCs in the identification of a specific macrophage murine models of breast cancer, which subpopulation and/or soluble mediator that Tumour topography. In addition to the results in increased lymphatic and vascular preferentially promotes or degrades the ECM changes in matrix stiffness that occur during metastasis112,122; for example, VEGF — might be an important determinant of the tumour progression, the topography of the which is released by hypoxic cancer cells extent of fibrosis within a tumour129. Taken ECM is also highly dynamic. For example, but more often by endothelial cells, fibro- together, the studies described above suggest invasive breast cancers often contain type I blasts and inflammatory cells — has been that hypoxic signalling engages multiple cell collagen fibres that are oriented perpen- implicated in fibrosis because of its role in types that contribute to ECM remodelling dicular to the tumour margin at the invasive stromal cell activation and because it leads within the tumour microenvironment (FIG. 3). front, in contrast to the non-oriented fibrils to the production of an ECM that is rich that are often seen in less aggressive breast in fibronectin and type I collagen63. VEGF Physical properties of tumour ECM cancers25,26,144,145. Straightened and aligned also induces microvascular permeability, The physical properties of the tumour ECM collagen fibres are found at sites of breast which in turn mediates an influx of fibro- refer to its stiffness, topography, porosity cancer invasion — a histological pattern blasts, inflammatory cells and endothelial and solubility131. The physical properties of that is termed tumour-associated collagen cells to the primary tumour123. the tumour-associated ECM are not only signature 3 (REF. 146), which is associated

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with decreased patient survival25. Similarly than the mammary glands of control mice58. epithelial-to-mesenchymal transition (EMT), to breast cancer, in early melanomas, col- Taken together, these data suggest a model stem cell maintenance, resistance to radiation lagen is localized to the periphery of the in which hypoxic cells can generate and and chemotherapy, invasion and metastasis155. tumour144,145,147. By contrast, metastatic mela- organize an aligned ECM through multiple Although considerable work has been done to nomas have a less compact ECM structure mechanisms (FIG. 2). characterize the role of HIFs in experimental with no barrier between the cancer cells and cancers with regards to tumour incidence and the adjacent normal tissue. Highly aligned Biochemical properties of the ECM. The growth152, the direct requirement for HIFs in collagen fibres within a tumour might not just structure of the ECM influences the stability metastasis has only recently been shown in be predictive of the metastatic potency of the and the bioavailability of growth factors and both orthotopic models and autochthonous tumour but may also be causative given the cytokines14, many of which are generated breast tumour models156,157. Conditional finding that cancer cells preferentially invade under hypoxic conditions (TABLE 1). The bal- knockout models of HIFs have also aided our along straightened and aligned collagen ance between ECM-mediated confinement understanding of how the hypoxic tumour bundles24,26,148. or distribution of growth factors and their environment affects different cell types to The ECM isolated from wild-type fibro- concentration will determine their availabil- drive cancer progression; for example, loss blasts that have been exposed to hypoxia is ity to cell surface receptors and consequently of either HIF1α or HIF2α in mouse vascular more highly aligned than the ECM deposited will regulate intracellular signalling14. endothelial cells has been shown to reduce by fibroblasts that have been cultured under A highly aligned ECM might more readily tumour growth because of impaired angio- ambient conditions, and collagen fibre align- establish a chemoattractive gradient that genesis158,159. Conversely, haplodeficiency ment under hypoxic conditions is abrogated potentiates hypoxic signalling. Conversely, a of prolyl hydroxylase domain-containing in fibroblasts transfected with shRNA against dense collagen network could function as protein 2 (PHD2; also known as EGLN1) HIF1α82. Breast cancer cells that have been a sink for growth factors and thereby could increases the HIF-driven upregulation of plated on ECM produced by hypoxic cells are reduce their rate of diffusion. Future stud- expression of VEGF receptor 1 (VEGFR1) in highly aligned and migrate with directional ies to determine the ECM arrangement and endothelial cells and decreases intratumoural persistence along ECM fibres, in contrast to composition that supports the optimal dis- hypoxia, resulting in decreased HIF1α expres- cells that have been plated on ECM produced tribution of growth factors to mediate metas- sion in cancer cells, which reduces pulmonary by non-hypoxic cells, which migrate in a tasis might lead to a better understanding of metastasis160,161. The studies described above random manner21. Similarly, in an ortho- how the ECM influences cancer cell motility suggest that clinical trials are warranted for topic mouse model of breast cancer, aligned and dissemination. HIF inhibitors that show efficacy in preclini- ECM fibres are present in the perinecrotic cal models. It will also be crucial to deter- (hypoxic) region of control tumours; by con- Potential therapeutic interventions mine how the activity of each HIFα subunit trast, tumours that are derived from breast HIF inhibitors. Increased expression of is affected by the potential inhibitor, given cancer cells expressing shRNA against HIF1α HIF1α and HIF2α has been observed in a the reported functional differences between have a disorganized ECM comprised of broad range of human cancers and has been HIF1α and HIF2α162,163. almost no fibrillar collagen21. associated (in most but not all cases) with Two inhibitors of HIF1α accumulation One potential mechanism of collagen a poor prognosis152–154, which suggests that that have shown anticancer effects in pre- alignment in hypoxia might involve the use of HIF inhibitors has the potential to clinical models are the topoisomerase I activity of the small GTPase RHOA, which improve patient survival not only by blocking inhibitor topotecan151 and the cardiac glyco- interacts with RHO-associated protein ECM deposition but also by blocking dozens side digoxin90,106,157. In addition to reducing kinase 1 (ROCK1) to mediate myosin II of other HIF target genes that encode pro- the expression of many HIF target genes, phosphorylation, resulting in cell contraction. teins involved in cell survival, angiogenesis, treatment of tumour-bearing mice with RHOA-mediated ROCK1 activity is required metabolic reprogramming, immortalization, digoxin reduces tumour fibrosis, as well as for caveolin 1-induced cell contraction, which enables cancer cells to align with and poten- a Carcinoma in situ b Local invasion c Intravasation tially to migrate along the pre-existing col- Protease Cancer cell Aligned lagen matrix in vitro149. Experiments in vivo Hypoxia collagen also indicate that caveolin 1‑dependent regu- ﬁbre lation of RHOA is required for fibroblasts to Basement Growth membrane factor produce an aligned matrix149. In renal clear cell carcinoma, caveolin 1 is a direct tran- ECM scriptional target of HIF1 and HIF2 (REF. 150). Moreover, hypoxia coordinately regulates the Fibroblast expression of RHOA and ROCK1 through HIF1- and HIF2‑dependent transcription in Blood vessel breast cancer cells, which results in increased Figure 2 | Hypoxia promotes ECM remodelling to facilitate metastasis. Extracellular matrix cell-induced matrix contraction151. An alter- Nature Reviews | Cancer (ECM) remodelling is tightly controlled to maintain tissue integrity. Cancer cells and associated stromal native or an additional mechanism of collagen cells that have been exposed to hypoxia are transcriptionally reprogrammed to produce: matrix alignment could involve LOX expression. metalloproteinases (MMPs) and other proteases, which degrade the basement membrane surrounding Second harmonic generation (SHG) imaging a tumour (part a); aligned collagen fibres within the interstitial matrix, which function as a highway for of mammary glands that have been precondi- local invasion, intravasation and metastasis (part b); and growth factors, which might be retained in tioned with LOX-expressing fibroblasts shows the fibrotic microenvironment and function as chemotactic signals that recruit and activate stromal that they contain more linearized collagen cells to further promote cancer progression (part c).

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lymph node and lung metastasis90,106,157. A pyridine 2,4‑dicarboxylate and pyridine AB0024 (also known as simtuzumab) Phase II clinical trial for digoxin is currently 2,5‑dicarboxylate, coumalic acid and has been tested in Phase I dose escala- being carried out for men with recurrent 3,4‑ethyl dihydroxybenzoate (EDHB)166. As tion trials in patients with advanced solid prostate cancer (ClinicalTrials.gov, num- these agents are not selective for collagen tumours171 (ClinicalTrials.gov, number: ber: NCT01162135). In addition, a pilot hydroxylases, it is probable that they will also NCT01323933) and with idiopathic clinical trial of topotecan (ClinicalTrials.gov, inhibit the HIF PHDs and will potentially pulmonary fibrosis (ClinicalTrials.gov, number: NCT00182676) in patients with promote HIF expression. Preclinical testing number: NCT01362231). Enrolment for a advanced cancer and HIF1α overexpres- will have to be carried out to determine their Phase II clinical trial in patients with idi- sion shown on tumour biopsy was recently potential usefulness in preventing metas opathic pulmonary fibrosis (ClinicalTrials. reported in which HIF1α protein levels tasis. Minoxidil has been shown to decrease gov, number: NCT01759511) has begun. were undetectable in the post-treatment the expression of PLOD mRNAs and the Additional non-selective inhibitors of lysyl biopsy samples from four of seven patients activity of PLOD proteins and thereby to oxidases also include p‑halobenzylamines, who were studied, and decreased tumour inhibit fibrosis167. In a mouse model of sar- ethylenediamine and homocysteine blood flow was observed in 70% of patients coma, minoxidil treatment reduced tumour thiolactone93,172. by contrast-enhanced dynamic magnetic fibrosis and suppressed lung metastasis67. resonance imaging164. Neither trial assessed Studies that target LOX family members Conclusions treatment-induced changes to the tumour have focused on blocking the enzymatic Although tumours from two different ECM. However, preclinical use of the HIF1α activity of these proteins using competitive patients might have similar genetic altera- inhibitor PX‑478 or overexpression of a inhibitors such as βAPN168 or using neutral- tions, these tumours will develop in dif- dominant-negative HIF1α mutant showed izing antibodies, which abrogate lung and ferent microenvironmental contexts13, that the increased fibrotic response identi- liver metastases in xenograft and transgenic which suggests that hypoxia and the ECM fied in fat pads from mice that were fed a mouse models93. d‑penicillamine (DPEN), are important in contributing to tumour high-fat diet could be effectively prevented which is a LOXL2 inhibitor, was developed heterogeneity, which might influence by treatment with PX‑478. The preclinical and used to treat rheumatoid arthritis and metastatic outcome. Hypoxic regions effectiveness of PX‑478 has previously been biliary cirrhosis but it does have some within the tumour microenvironment can established in tumour models where treat- unintended side effects169. A more selective simultaneously relay signals to cancer cells ment reduces tumour growth165 and it will be inhibitory monoclonal antibody (AB0023) and cells that have been recruited to the interesting to determine the effect of PX‑478 against LOXL2 has been developed and local environment directly (for example, by on tumour fibrosis. was effective in reducing fibrosis in pri- transcriptional reprogramming), through mary and metastatic xenografts as well as paracrine signalling events and, as high- Targeting fibrosis. Blocking collagen in liver and lung fibrosis models in mice170. lighted in this Opinion article, by establish- hydroxylases or lysyl hydroxylases might The ECM of tumours from mice that ing a hypoxia-induced ECM that is fibrotic, also provide a strategy to reduce tumour had been treated with AB0023 showed a stiff and aligned, which are all properties fibrosis. P4Hs have been regarded as attrac- marked reduction in crosslinked collagen that promote metastatic dissemination11,173. tive targets for the pharmacological inhibi- compared with results in mice that had Further studies are needed to investigate tion of collagen accumulation in fibrotic been treated with the lysyl oxidase inhibi- the mechanisms by which the hypoxia- diseases and severe scarring. P4Hs belong tor βAPN170. Treatment with AB0023 also induced ECM might have a role in dynami- to a superfamily of dioxygenases that use resulted in a marked reduction in the num- cally maintaining and distributing growth oxygen and α‑ketoglutarate (also known ber of activated fibroblasts and endothelial factors that provide chemotactic signals as 2‑oxoglutarate) as substrates. P4Hs are cells and led to a decreased production of to recruit cells to the primary tumour and competitively inhibited by α‑ketoglutarate growth factors and cytokines170. The safety that promote the intravasation of cancer analogues, including N‑oxalylglycine, of the humanized version of AB0023, cells for dissemination to distant organs. Advances in imaging techniques, such as intravital microscopy, have the potential Table 1 | Factors induced by HIFs and their role in fibrosis to shed light on this issue and might direct Factor induced by HIFs Role in fibrosis our research to appropriate targeting strat- Platelet-derived growth factor Stimulates the replication, the survival and the migration egies that will be most beneficial to prevent of myofibroblasts118 metastasis174. Connective tissue growth factor Promotes collagen deposition by myofibroblasts195 It is also important to consider that the collagen-modifying enzymes discussed in Fibroblast growth factor 2 Promotes the proliferation and the differentiation of endothelial cells, smooth muscle cells and fibroblasts, and this Opinion article might have alternative stimulates collagen deposition196 roles in cancer progression that are not lim- ited to fibrosis93. For example, LOX has a role Endothelin Promotes fibroblast activation, proliferation and differentiation into myofibroblasts197 in PDGF and insulin growth factor 1 (IGF1) signalling, but its precise mechanisms of 198 Angiotensin Stimulates TGFβ production and promotes collagen I 175 and collagen III deposition199 action remain to be elucidated . Additional regulators of the collagen hydroxylases and Insulin growth factor 2 Increases connective tissue growth factor-stimulated lysyl oxidases remain to be determined; collagen deposition200 for example, TGFβ1 has been shown to CXC-chemokine ligand 2 Promotes fibrocyte recruitment119 influence LOX expression93. Furthermore, HIFs, hypoxia-inducible factors; TGFβ, transforming growth factor-β. the role of collagen in the regulation of

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