Precancerous niche (PCN), a product of fibrosis with remodeling by incessant chronic inflammation

This is anOp Abstract -Fibroblasts are actively involved in the creation of the stroma and the extracellular matrix which are important for cell adhesion, cell–cell communication, and tissue metabolism. The role of fibrosis in carcinogenesis can be examined by analogy to tissues of various cancers. The orchestration of letters in the interplay of manifold components with signaling and crosstalk is incompletely understood but available evidence suggests a hitherto underappreciated role for fibrosis in carcinogenesis. Complex signaling and crosstalk by pathogenic stimuli evoke persistent subclinical inflammation, which in turn, results in a cascade of different cell types, ubiquitous proteins and their corresponding enzymes, cytokine releases, and multiple signaling pathways promoting the onset of fibrosis. There is considerable evidence that the body’s attempt to resolve such a modified extracellular environment leads to further disruption of homeostasis and the genesis of the precancerous niche as part of the six-step process that describes carcinogenesis. The precancerous niche is formed and can be understood to develop as a result of (1) pathogenic stimulus, (2) chronic inflammation, and (3) fibrosis with alterations of the extracellular matrix, stromal rigidity, and mechano-transduction. This is why carcinogenesis is not just a process of aberrant cell growth with damaged genetic material but the role of the PCN in its entirety reveals how carcinogenesis can occur without invoking the need for somatic mutations.


Introduction
Fibroblasts are actively involved in the creation of the stroma and the extracellular matrix (ECM), which is important for cell adhesion, cell-cell communication, and tissue metabolism. The orchestration of letters in the interplay of manifold components with fibroblasts along with signaling and crosstalk with varied constituents are incompletely understood but suggest a hitherto underappreciated role in carcinogenesis.
The importance of fibrosis in carcinogenesis can be seen by analogy in that "atrophic gastric mucosa is not just a case of simple atrophy but can be in some respects compared to a cirrhotic organ" [1].
Helicobacter pylori (H. pylori) promotes hepatic fibrosis in animal models [2] by sensitizing transforming growth factor-b1 (TGF-b1) resulting in inflammatory signaling [3]. H. pylori lysates promote the translocation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB) into the nucleus in the presence of TGF-b1, as hydrogen peroxide (H 2 O 2 ) treatment induces proliferation of hepatic stellate cell (HSC) lines from rats [3]. The prevalence of H. pylori is elevated in Opisthorchis viverrini associated with cholangiocellular carcinoma (CCC), with correlation to biliary periductal fibrosis [4]. Co-infection of hepatitis C virus (HCV) with H. pylori was common in up to 62% and with higher rates of lymphocytic infiltration. Fibrosis and cirrhosis were observed in coinfected groups compared to HCV infection alone, revealing that infections associated with both HCV and H. pylori result in an exacerbation of inflammation and fibrosis [5]. Furthermore, Opisthorchis felineus (O. felineus) was recently shown in a rodent model to be associated with chronic inflammation, fibrosis with all its changes, and consequent intrapeithlial neoplasia to directly induce a precancerous niche (PCN) facilitating malignancy [6].
The interplay of inflammation-triggered lesions, oral submucosal fibrosis (OSMF) and/or ubiqouitous proteins such as vimentin, are important in carcinogenesis. Investigating paraffin-embedded tissue sections of n = 208 OSMF and n = 222 oral squamous cell carcinoma (OSCC) yielded positive results for the presence of human papilloma virus  in 25.96% and 1.92%, respectively and of human papilloma virus 18  in OSMF in 32.43% of HPV-16 positive cases and 12% in HPV-18 positive cases in OSCC [7]. Overall, the staining intensity of vimentin was greater in precancerous OSMF compared to a control group of normal buccal mucosa and this phenomenon was also demonstrated in fibroblasts [8].
Fibroblasts are instrumental in fueling the process of subclinical inflammation [16].
Fibrosis is a complex series of events and involves, among other things, an imbalance of fibronectin, decorin, and vimentin. Fibronectin activates Toll-like receptor 4 (TLR-4) on macrophages, a pathway usually used by lipopolysaccharides [17]. Decorin has two opposing effects: one repressing TGF-b1 effects and the other influencing cell behavior by auto-and paracrine pathways there is growing evidence that the signaling pathways for EGF receptors and decorin become desensitized through a decrease in the number of receptor molecules [18 reviewed in 19]. Despite the regulatory effects of decorin on collagen fibrillogenesis, decorin can reverse the effect of TGF-b1 with a net result of increasing "IL-1b, IL6, TNFa, inducible nitric oxide synthetase (NOS), and the expression of major histocompatibility complex class II genes" [20 reviewed in 21]. "Overexpression of certain extracellular matrix proteins (such as decorin) during the ongoing desmoplastic reaction in chronic pancreatitis may be the cause of the altered behaviour of mononuclear cells, thus continuously maintaining the inflammatory process" [21]. However, increased decorin levels in transgenic mice do not repress TGF-b1 in the liver [22]. Furthermore, decorin specifically promotes monocyte chemoattractant protein-1 (MCP-1) in mononuclear cells and appears to be necessary for "continuous MCP-1 mediated recruitment of non-optimally activated macrophages and lymphocytes" [21].
The glycogen serine-threonine kinase and negative regulator of the oncogenic Wnt/b-catenin signaling pathway, glycogen synthase kinase 3b (GSK-3b) is increased in chronic inflammation. Inhibition of GSK-3b in vitro results in a shift from NF-kB activity toward cAMP response element-binding protein (CREB) activity [29]. GSK-3b regulates cyclooxygenase 2 (Cox-2) expression in gastric cancer cells but GSK-3b inhibition stimulates only a modest Cox-2 expression [30], which might explain why Cox-2 inhibition can also result in GSK-3b increase with an anti-tumor effect. GSK-3b was shown to be essential in early pancreatitisinduced acinar-to-ductal metaplasia as GSK-3b ablation limited the acinar-to-ductal metaplasia [31]. Increased GSK-3b expression is associated with the lesion grade in cervical cancer and inversely correlated with Cyclin D1 [32]. Dextran sulfate sodium (DSS)induced intestinal fibrosis was improved via GSK-3b signaling and after the use of the peroxisome proliferator-activated receptor gamma (PPAR-g) modulator, GED-0507-34 Levo [33].
Carcinogenesis is often described as a process of cell growth gone awry with external factor(s) having damaged the genetic material of the cell, i.e. caused somatic mutations. However, in our view, consideration needs to be given to how a cell or tissue responds to an internal/external provocation. Thus, we hold that the tumor microenvironment plays an important role in carcinogenesis as shown by the identification of a multifactorial cytokine loop in bladder cancer in vitro: fibroblast-induced interleukin 8 (IL-8), hepatocyte growth factor (HGF), matrix metalloproteinase-2 (MMP-2), granulocyte macrophage colony-stimulating factor (gmCSF), and MCP-1 act together to facilitate metastasis [37].
Fibroblasts activation is known to trigger the deposition of ECM compounds such as vimentin, with aSMA resulting in the fibronectin matrix, linking both cells and collagen bundles [38]. We suggest that the imbalance of compounds such as fibronectin, decorin, and vimentin with ongoing fibrosis is of importance in carcinogenesis. Fibrogenesis, through aberrant accumulation and activation of myofibroblasts, triggers the deposition of ECM components and initiates tissue remodeling [38] with increased stiffness analogous to that seen in pulmonary or liver fibrosis that occurs prior to, for example, asbestos-induced mesotheolioma or smoking-associated lung cancer [39,40].
Matrix stiffening also stimulates the proliferation of fibroblasts with increased matrix synthesis [41]. This was shown in normal tissue with a "…stiffness-driven suppression of cyclooxygenase-2 (Cox-2) expression and prostaglandin E2 (PGE2) as a key link between matrix stiffening and fibroblast activation". Cytokines and TGF-b cause fibroblasts within the matrix to modulate ECM, including aSMA expression [42]. aSMA expression is induced by TGF-b [43] Syndecan-4 is also necessary for transduction of signals through protein kinase C alpha (PKCa) activation resulting in the assembly of the actin cytoskeleton, cell contractility, and metastasis [44]. A Bleomycin model of fibrosis resulted in a 6-fold increased tissue stiffness compared to normal lung tissue. In normal tissue, PGE2 results in fibroblast quiescence including a feedback pathway between matrix stiffening, Cox-2 suppression, and fibroblast activation in the development of fibrosis [41]. This cascade, when perpetuated, could explain how fibrogenesis can shift into a selfsustaining phase.
Fibroblasts are involved in creation of the PCN (Fig. 1). This process is supported by exosome releases (small vesicles) that activate the chemoresistance-inducing factor Snail in epithelial cells, promote proliferation, and induce drug resistance [45].
There is a renewed interest in assessing the contribution of inflammatory processes as prognostic variables in cancers [46]. The sequences (1) pathogenic stimulus, (2) chronic inflammation, (3) fibrosis and (6) normal to cancer cell transition are seen in viral hepatitis-associated liver cancer [47]. It also appears that ongoing pathogenic stimuli may result in epigenetic silencing and thereby increase virulence [48].

TGF
The TGF-b family of receptors specific for TGF-b include, TGF-bR1 (ALK5), TGF-bR2 and TGF-bR3 (b-glycan); TGF-bR1 and TGF-bR2 have a high affinity for TGF-b1 and low affinity for TGF-b2 while TGF-bR3 has a high affinity for both homodimeric TGF-b1 and TGF-b2. TGF-b1 ligand binds to either TGF-bRIII or TGF-bRII, which aggregates with TGF-bRI and phosphorylates TGF-bRI resulting in the transmembranous phosphorylation of Smad-2 and Smad3 by the serinethreonine kinase activity of TGF-bRI [49][50][51][52]. This builds up a complex with Smad4 allowing the complex to migrate into the nucleus [53]. Once inside the nucleus, (together with other factors), transcription is initiated. Increased stromal TGF-b gene expression has been associated with poor prognosis in CRC [54]. Investigating the mRNA expression of TGF-b1, Smad4, and apoptosis-inducing Smad7 in 97 samples of esophageal squamous cell carcinoma (ESCC) biopsies taken before neoadjuvant radiochemotherapy revealed that only Smad4 had predictive value in regard to patient survival [53].
It is known that proteasomic activity is higher in tumors [55]. The degradation of TGF-b1 receptors occurs by the ubiquitin E3 ubiquitin-protein ligase (Smurf2) and that post-transcriptional factors may alter the activity of genes [53]. A negative influence on this transcriptional complex is achieved by inhibitors such as the nuclear proto-oncogene 'Sloan Kettering Institute' (Ski), the Skinovel protein (SnoN) [56,57], the Smad transcriptional corepressor T cell growth inhibitory factor (TGIF) [58] and positive influences by the stimulation via Runx proteins [59].
As a result of transcription, the synthesis of inhibitors of the cell cycle, i.g.p15, p21 and the inhibition of the proliferation and apoptosis-induced protein, c-myc occurs [60]. A disruption of the complete TGF-b cascade takes place via phosphorylation of Smad3 by CDC2-kinasis and initiates the cell-cycle to proceed to the transition G1-/ S-phasis [61]. TGF-ß1-induced apoptosis ( Fig. 1) occurs by indirect activation of mitogen-activated protein kinases (MAP kinases) [62][63][64][65] and can also be induced by apoptosis of Smad7 [63,66,67]. Gluthatione-S-transferase is also relevant as it inhibits members of the MAP kinase family by building up a protein-protein interaction. This suggests that increasing the glutathione-S-transferases p level can lead to an inhibition of MAP kinases [68]. This model is supported by data from head and neck cancers [69].
The inhibition of heat-shock protein 27 (HSP27), highly expressed in fibrotic foci of idiopathic pulmonary fibrosis (IPF), results in Snail degradation followed by inhibition of TGF-b1 induced cell transition [75]. TGF-b1 stabilizes Snail and is associated with increased integrin b3 with consequent high Akt activity and elevated inhibitory phosphorylation of GSK-3b [76,77].
The nerve growth factor, IB (NGFIB, Nur77 or NR4A1), was shown to negatively regulate TGF-b1 suggesting the ability to limit pro-fibrotic TGF-b effects [78]. Also, Nur77 was shown to promote breast cancer invasion and metastasis by activating TGF-b signaling [79]. However, it was shown that the HCV core protein inhibits Nur77 and apoptosis [80] and that inhibition of apoptosis by Nur77 occurs through NF-kB [81]. These data demonstrate that the disruption of cellular homeostasis is of importance during carcinogenesis.
Snail stability and activity are activated by lysyl oxidase [82]. TGF-b1 induces lysyl oxidase expression ( Fig. 1), secretion, and proteolytic processing in normal as well as in mammary epithelial cells and lysyl oxidase downregulates the E-Cadherin suppressive effect [83] while upregulating vimentin [84,85]. Both the upregulation of vimentin and downregulation of E-Cadherin were observed at the mRNA level [86].
TGF-b1 increases accumulation of all forms of lysyl oxidase proteins which are usually decreased by PGE2 [86]. "Much of the PGE2 action on the expression of type I collagen alpha1, lysyl oxidase, and Cox-1 genes is mediated through the prostaglandin E2 (EP2) receptor and a subsequent increase in intracellular cAMP [87].
Blocking TGF-b on the surface of regulatory T cells (T Reg ) increases programmed cell death protein 1 (PD-1) on CD8+ T cells, which may suggest a role for T Reg in cancer therapy [88]. Even in rare small bowel carcinomas significant associations between mRNA levels for Cox-1, Cox-2, TGF-b1, prostaglandins of E class (PGEs), and pro-angiogenic factors, vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor C (VEGF-C) are likely to play a role in carcinogenesis [89]. Different vascular endothelial growth factors (VEGFs) seem to have different and specific effects in carcinogenesis and in disease progression depending on the histological subtype. VEGF-C plays a role during progression via lymphangiogenesis in ESCC, while this effect was not observed in esophageal adenocarcinomas [90].
In renal tubular epithelial cells, TGF-b1 triggered nuclear translocation of b-catenin and transcriptional induction of Slug resulting in a decrease of E-cadherin transcription [93]. E-cadherin suppresses uncontrolled cancer cell growth [94] while upregulation of Twist and N-cadherin results in increased invasion in TGF-b1 stimulated CCC cells [95].
Lysyl oxidase activates PI3K [100] and both, lysyl oxidase and MMPs, are induced by TGF-b [101]. Furthermore, lysyl oxidase triggers PI3K/Akt signaling HIF-1a protein synthesis and HIF-1a increases lysyl oxidase in vitro and in vivo demonstrating that lysyl oxidase and HIF-1a synergistically trigger remodeling of the ECM [100] plausibly creating the PCN [102,103]. More importantly, lysyl oxidase-induced HIF-1a expression was shown not to be restricted to colon cancer cells only but also was observed in primary human fibroblasts [Supplementary Fig. S2C in 100].
In vitro silencing of FOXO3a by transfection increases IL-8 levels in TNF-treated HT29 cells [114]. This pro-inflammatory effect was observed in pAktnegative primary breast cancer in which one key modulator of the inflammatory NF-kB, IkB kinase b was increased and shown to be associated with decreased survival [115]. Decreased FOXO3a activity was also observed in patients with ulcerative colitis, an inflammatory condition [114] and gastric cancer with poor survival [116]. Paradoxically, increased FOXO3a in gastric cancer was associated with improved survival [117]. FOXO3a was more common in tumor samples than in adjacent normal tissues. FOX3a induces apoptosis via Bim signaling in bladder cancer cells T24 [118] and in breast cancer FOXO3a upregulates Bim-inducing apoptosis [119]. In breast cancer cells, the non-phosphorylated form of FOXO3a inhibits carcinogenesis and promotes apoptosis through upregulation of p27, Bim, and cleaved caspase3 proteins [120]. However, here too, there is a disruption of homeostasis in multiple pathways leading to carcinogenesis as ERK downregulates (via MDM2 mediation) FOXO3a, inhibiting p27Kip1 and Bim transcription [120].
Puma is required for apoptosis [121] and is involved, independently of p53, in the induction of mast-cell death following cytokine deprivation in both mucosal-like and connective tissue-like mast cells [122]. As FOXO3a increases Puma [123], decreased FOXFO3a can be expected to result in the opposite effect and, via ERKsignaling, increase the downregulation of Bim and Puma with decreased apoptosis (Fig. 1).

E2F4/5
The cytoplasmic complex of Smad3, retinoblastomalike protein 1 (P107, RBL1), E2F4/5 and D-prostanoid (DP1) moves to the nucleus aided by TGF-b and with Smad4 repressing c-myc [124]. TGF-b mediated c-myc response by the Smad-E2F4/5-p107 complex occurs in vitro about an hour after TGF-b addition and this is described as a "cell cycle-independent event contributing to, rather than deriving from, G1 arrest". TGF-b promotes binding of Smad2/3, Smad4, p107, and the transcription factor E2F4/5 to the c-myc promoter in vivo and E2F4 and E2F5 repress transcription in combination with p107 or p130. E2F4/5 and p107 interact as "transducers of TGFb receptor signals upstream of cdk" [124]. The mechanism of silenced, repressed, or mutated c-myc associated with increased proliferation and cancer is incompletely understood as it has been shown that systemic c-myc repression can result in normal reversible regenerating tissue repressing Ras-induced lung tumors in mice [125].
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) promotes chromosomal maintenance 1 (CRM1, Exportin1)-dependent nuclear export of E2F4/5 inhibiting p16INK4a-RB signaling resulting in lower cell cycle arrest [126]. P107 ( Fig. 1) TGF-b triggers fast binding of Smad2/3, Smad4, E2F4/5, and p107 to the c-myc promoter in vivo [124]. The retinoblastoma protein family contains RB, p107 and RB2/p130, named 'pocket proteins' for their molecular pocket for binding onto other proteins. RB binds to E2F keeping E2 promoter-binding-protein-dimerization partner (E2F-DP) inactivated and thus acts as a growth inhibitor preventing replication from the G1 to S-phase and also by reducing transcription of the S-phase by binding to a histone deacetylase (HDAC) protein. CDKand cyclin-dependent phosphorylated Rb (pRb) À which is found in the S, G2 and M-phases À results in the dissociation of E2 promoter-binding-protein-dimerization partner (E2F-DP) from Rb while entering the S-phase with consequent activation. p107 (RBL1) [127] is ubiquitously expressed though with varying tissue distribution and levels of expression in different organs, primarily in breast and prostate epithelium and in B-and T-lymphocytes [128]. Proliferating human fibroblasts show increased levels of the tumor suppressor p107 [127] and E2F4-p107 complexes with decreased p130 levels [129]. E2F-4 binds selectively to hypo-phosphorylated tumor suppressor p107 [130] and p107 interacts with E2F controlling the F-box receptor Skp2 with consequent inhibition of cell proliferation and p27 stabilization [131].
The matrix protein, laminin A/C, is required for normal levels of pRB and p107 in mouse fibroblasts [132]. p107 represses SP1-mediated activation of fibroblast growth factor receptor 1 (FGFR1) and FGFR1 promoter activity in proliferating myoblasts. p107 interacts with the transcription factor SP-1 in myoblasts whereas p130 not; SP-1 directly interacts with the transcriptional complex consisting of E2F4 and p107 at the FGFR1 E2F4 binding site [133]. p107 was reported to be overexpressed in the early stages of CRC while it was lower in patients with metastasis [134] and higher nuclear p107 expression was shown in salivary gland tumors [135]. Otherwise, pRb/p107-deficient mice frequently developed head and neck cancers as HPV-16 E7 transgenic mice, both with comparable phenotypes [136]. On the other hand, RB2/ p130 was involved in the development and progression of oral carcinomas [137,138], breast and endometrial cancer [139], lung cancer [140], nasopharyngeal carcinoma [141], lymphomas [142], and is a strong predictor of clinical outcome in endometrial carcinomas [143], and in HCC [144 reviewed in 145].
MMPs degrade the ECM as well as its non-ECM proteins and are not cell-specific [147]. Their functionality has been extensively reviewed [151]. As MMPs regulate tumor suppression there is an opportunity for using anti-MMP therapy to treat cancer [152][153][154]. MMPs are activated by MMPs themselves (e.g., metalloproteinase 3, MMP3) as well as by plasmin, heparin, and intra-and extracellular protein degradation triggered by oxidants [147].
MMPs are inhibited by tissue inhibitors of metalloproteinase (TIMP) and a2-macroglobulins. In healthy tissues there is a balance between MMPs and TIMPs and disruption of this homeostasis results in abnormal tissue degradation. TIMP-1 inhibits all MMPs except metalloproteinase 14 (MMP-14) and has a greater affinity for metalloproteinase 9 (MMP-9) over MMP-2, while TIMP-2 exhibits a higher affinity for MMP-2 than MMP-9 [154, reviewed in 147, 151].
Neutrophils express TIMP-1, metalloproteinase 8 (MMP-8), MMP-9, and TIMP-1, and are predominantly localized to a distinct granules or vesicles within the neutrophil [155 reviewed in 147]. The interplay of gelatinases (matrix metalloproteinases MMP-2 and MMP-9) is complex, although simplified for illustration in Figure 1. Despite its role in remodeling the ECM, MMP-9 is activated by MMP-2 and metalloproteinase 3 (MMP-3). MMP-9 might be involved in tumor invasion, while MMP-2 appears to be involved in metastasis [156]. The disruption of homeostasis was seen with TIMP-1 within the stroma where increased TIMP-1 promoted liver metastasis by triggering HGF along with activating HGF and HGF-activating proteases [157].
Signaling by mast cells with T-cell interaction [158] induces neutrophils granulocytes (npGC) and macrophages with consequent induced cytokines and signaling pathways [159][160][161]. However, not all neutrophils trigger chronic inflammation as they undergo reverse transmigration, meaning that in situations where such cells do not die or are phagocytosed, they migrate via the lungs to up-regulate the C-X-C motif of chemokine receptor 4 (CXCR4) and end up in the bone marrow where they undergo apoptosis [162].
Increased levels of phosphoglycerate kinase 1 (PGK1), CXCR4, C-X-C motif chemokine 12 (CXCL12) and b-catenin are all associated with peritoneal carcinomatosis in gastric cancer patients [163]. PGK1 was shown to regulate CXCR4 and b-catenin at the mRNA and protein levels with a negative feedback loop for CXCR4 [164]. This was observed in HCC [165]. Increasing evidence about the role of elevated CXCR4 levels in inflammation and cancer have been published [166][167][168][169] such that its use in identifying cancer therapy targets might be helpful [170] as CXRC4 also affects CXCR2, mitogen-activated protein kinase kinase (MEK, MAPK2, MAPKK), and PI3K signaling [171].
Expression profiles of MMPs have been correlated with poor clinical prognosis for several human tumor types [172]. The pro-invasive and metastasis-promoting activity of MMPs likely works through ECM remodeling [152]. Tumor cell invasion can be reduced [173,174] by endogenous MMPs together with disintegrin and metalloproteinase 10 (MMP-10, ADAM-10) [175] and TIMP-1 [176].
MMP-7 was discovered by Woessner [177] and facilitates the degradation of casein, fibronectin, and collagen type I, II, IV and V and, thereby, the breakdown of the ECM [178]. H. pylori cytotoxin-associated gene (+) selectively increases MMP-7 in vitro and in vivo in gastric pre-and cancerous tissue [179]. Knocking down MMP-7 in mice increased H. pylori-induced gastric inflammation with elevated M1 macrophage markers, which also were reproducible in hyperplasia and dysplasia.

Extracellular matrix and lysyl oxidase
The macromolecular components of the non-cellular ECM consist of collagens, fibronectin, elastin, laminins, hyaluronan and proteoglycans [183]. Collagen is composed of three a-chains which are (at least partially) folded to a triple-helix and there are 28 collagen types subdivided into nine families [184]. Lysyl oxidase converts lysine residues through oxidative deamination of peptidyl lysine in elastin and peptidyl lysine and hydroxylysine in collagen resulting in cross-links [185]. Fibrillar collagen consists of three a-chains bund into a triple helix and each a-chain has a N-terminal and C-terminal propeptide next to the N-and C-terminal telopeptide; this area is the one were modification of lysine into helical lysyl hydroxylase (Hyl) occurs by (telopeptidase) lysyl hydroxylase compared to proline modification which occurs in the triplehelix only À not at the N-and C-terminal telopeptides links and the conversion of lysine into 5-hydroxylysine (Hyl) in the a-chains of procollagen are catalyzed by lysyl hydroxylases.
Lysyl oxidase is synthesized as a 46-kDa pro-enzyme by fibroblasts, undergoes N-glycosylation which is then secreted as a 50-kDa N-glycosylated proenzyme [202] into the ECM and once there, is proteolytically transformed to a mature enzyme of 31 ± 1 kDa [203 reviewed in 204]. Li et al. provided "immunological, catalytic, and chemical evidence that this catalyst occurs and appears to function within the nuclei of fibrogenic cells" [204]. Lysyl oxidase activity is dependent on its co-factors, copper and lysyl tyrosyl quinone [205], and catalyzes aldehyde formation from lysine residues to a-aminoadipidic-d-semialdehydes (allysines) in collagen and elastin precursors [206].
Lysyl oxidase increases myeloid lineage cells and is relevant for the recruitment of CD11b+ cells followed by co-localization with fibronectin [207]. Lysyl oxidase is upregulated in fibrosis and in scleroderma [208], and its isoenzymes are expressed differently in different tissues [199]. This may be crucial to understanding different tumor biologies as well as the dynamics of different cancers.
Remodeling by lysyl oxidase occurs in cardiac hypertrophy aggravating angiotensin-II induced hypertrophy [209] and angiotensin-TGF-b1 crosstalk was reported serving as an autocrine path in human cardiac hypertrophy [27,210].
Lysyl oxidase was shown to act on the ECM in both the cytoplasm and nucleus [205 reviewed in 83]. Furthermore, lysyl oxidase imbues type I collagen with increased rigidity in the ECM-promoted proliferation of mammary epithelial cells in vitro [83] and stimulates collagen cross-linking and synthesis of fibronectin [207]. The crosslinking of collagen to elastin results in stromal rigidity and increased mechano-transduction [211,212]. Lysyl oxidase attenuated collagen IV at the basement membrane with consequent increase of adherent CD11b+ cells and increased MMP-2 expression resulting in the degradation of collagen IV and increased CD11b+ cell invasion [207].
Lysyl oxidase "increased matrix elastin synthesis by 40-80% to that in control cultures in a dose-dependent manner" [213]. Early stage lung adenocarcinoma tissue with high lysyl oxidase expression exhibited poor survival rates [214].
An ongoing pathogenic stimulus with unresolved chronic inflammation and TGF-b1 activation results in increased lysyl oxidase activity and increased ECM rigidity. HIF-1a mediated lysyl oxidase expression is increased by hypoxia [207] which also promotes integrinmediated focal adhesion formation and PI3K signaling [215]. By contrast, integrin a5 expression in ESCC "was associated with the survival of patients with lymph node metastasis but did not influence the survival of patients without lymph node metastasis" [216].
Paradoxical findings [208,217] may be explained by the fact that various pathways can activate lysyl oxidase and its isoforms [218]. Activation of lysyl oxidase can occur through both tumor growth factor beta (TGF-b1) with Smad and non-Smad JNK/AP-1 signaling can regulate lysyl oxidase through vascular cell adhesion molecule (VCAM-1), intracellular adhesion molecule (ICAM-1), and monocyte chemotactic protein (MCP-1) which may explain paradoxical findings based on the particular isoform.
The lysyl oxidase family is involved in various cellular processes such as cell motility, migration, signaling, regulation of transcription, altering chromatin condensation, and carcinogenesis [208 reviewed in 219]. Furthermore, lysyl oxidase generates chemotactic sensitivity to cells e.g. through oxidizing the cell surface protein betatype platelet-derived growth factor receptor (PDGFR-b) [220,221]. For example, LOXL1 and LOXL4 are reported to be epigenetically silenced by promoter methylation in bladder cancer cells and antagonizing both isoenzymes resulted in the decrease of colony formation ability and activation of Ras [222].
Lysyl oxidase modulates the ECM, cell migration and growth [223] and its activity is greater in human breast cancer than in normal tissues [224].
Precancerous niche (PCN) (Fig. 1) Endometriosis is epidemiologically a precancerous state of ovarian cancer [225] and contains LOX-like protein 4 [226,227] with increased TGF-b1 and phosphorylated Smad3, decreased e-cadherin and increased staining of collagen I and lysyl oxidase [228]. Another important finding supporting the PCN as the key point for transition of a normal cell to cancer cell occurs in the cancer-resistant species, the blind mole rat or Spalax [229]. Application of chemical carcinogens to this animal resulted in healing without malignancy.
Increased lysyl oxidase expression was found at the cancer invasion front, and was observed in (so-called) reactive per-cancerous fibrosis of breast cancer [230]. We contend that such changes constitute the necessity for the PCN and thus for the transition of a normal cell to a cancer cell. In cancer-resistant species, precancerous lesions heal. The naked mole rat (Heterocephalus glaber) [231], as well as the subterranean blind mole rat (Spalax), show low activity of hyaluronan synthase 2 (HAS2) [229]. HAS2 is an important enzyme in the ECM, as it interacts with the leukocyte receptor CD44, and overexpression or increased extracellular hyaluronan was associated with decreased survival, and mediated tumor metastasis in vivo and in vitro in CRC [232,233], stomach [234], esophagus [235], breast [236][237][238][239], head and neck [240] and ovarian cancer [241].
Hyaluronan, together with CD44, plays a significant role in cell and cancer adhesion as well as providing a less dense matrix and stimulates cell motility and protection against pathogens [242][243][244][245].
Inhibiting HAS2 in an aggressive MDA-MB-231 breast cancer cell line inhibited initiation as well as progression of primary and secondary cancers [246].
Protein arginine methyltransferase 1 (PRMT1) is necessary for "TGF-b-induced SMAD3 activation through a mechanism similar to that of TGF-b-related bone morphogenetic protein (BMP) induced SMAD6 methylation, and thus promotes the TGF-b-induced EMT and epithelial stem-cell generation" [247]. SMAD7 inhibits the TGF-b-induced SMAD3 activation but PRMT1 induced SMAD 7 methlyation enables and facilitates TGF-b signaling.
Species like the Heterocephalus glaber and Spalax seem to have found an effective anticancer strategy by inhibiting the PCN. The interplay of disruption of homeostasis can be seen as the blind mole rat, Spalax galili, which reveals higher proteasome activity with increased levels of markers for autophagy [248].
The inflammatory microenvironment promotes carcinogenesis [249]. Ongoing remodeling of ECM leads to the development of the PCN [102,103]. Inhibition of PCN formation with the anti-inflammatory and anti-fibrotic atrial natriuretic peptide (ANP) [250] as well as observations in viral hepatitis-triggered liver cancer, indicate that fibrosis and the PCN are necessary for the proposed sixstep sequence that describes carcinogenesis without invoking the need for somatic mutations [102,103]. This "network of macromolecules with distinctive physical, biochemical, and biomechanical properties" is the major component [251] as reported earlier [102]. Loss of E-cadherin is also associated with ongoing remodeling of the ECM [252] in creating the PCN.
Stimulus of the ECM with chronic release of P2Y2R and activation by nucleotides leads to ongoing THP-1 monocyte recruitment, and HIF-1a overexpression with consistent release of lysyl oxidase crosslinking collagen, facilitates changes to the tumor microenvironment [253,254], which then cause the changes that result in the formation of the PCN.
Lysyl oxidase-like-2 (LOXL2) overexpression in AsPC-1 and BxPC-3 cells enhanced cell transition and increased both migratory and invasive activities. This biochemical micromileu promotes and creates the PCN. Higher LOXL2 expression is associated with elevated Snail1 and cytokines [255] and with invasiveness of pancreatic cancer cells and low survival rates of pancreatic cancer patients [256]. Increase of LOXL2 was shown in biopsies of human tumors, fibrotic liver and lung tissues compared to healthy tissues [257].
LOXL2 activity results in fibrosis in non-alcoholic fatty liver disease (NAFLD) and is enhanced by insulin resistance [258]. Monoclonal anti-LOXL2 antibody AB0023 [259] were used [257] and decreased activated fibroblasts, desmoplasia and growth factors including cytokines and TGF-b signaling.
Although increases of lysyl oxidase were shown in murine schistosomiasis some 25 years ago [260], an antilysyl oxidase approach was not investigated. Lysyl oxidase was expressed in Escherichia coli (E. coli) [261]. Lysyl oxidase homolog 2 (LOXL2) [262] was found in human lung fibrosis, bronchiolo-alveolar carcinomas, and in situ ductal breast tumors [263]. In S.mansoni-infected mice and in early stages of liver granuloma, the lysyl oxidase gene and LOX-like gene (LOXL) were upregulated. LOXL2 promotes Snail and decreases E-cadherin, both important for carcinogenesis and metastasis. Investigations of the ECM by atomic force microscopy showed that LOXL2 does not affect ECM properties [264]. Incubating oral epithelial cells with the natural di-thiol a-Lipoic acid was shown "to modulate periodontal bacterial induced NF-kB activation, pro-inflammatory gene expression and cytokine production" [265].
Pathologists recognize the phenomena of desmoplastic stroma in histopathologies of cancerous tissues, which is concordant with rigid fibrous and remodeled ECM and develops from unresolved chronic inflammation. That the PCN is an important step in carcinogenesis [102,103] was recently addressed by Willumson et al. applying this microenvironment condition for potential use of liquid biopsy biomarkers investigating the PCN [266]. Other future diagnostic tools might include electrochemical nanobiosensors and microfluidic devices [267].
Creating a prognostic normogram of inflammatory fibrotic stroma was demonstrated in CCC by combining histopathological charcteristics together with CD3, CD4 and a-smooth muscle actin (a-SMA) staining of tissue microarrays [268]. Tumor cells aggregate to fibroblasts which further drive the cascade of chronic inflammation, fibrosis, and remodeling [269].
The epithelial to mesenchymal cell transition (EMT) initiates the Golgi scaffolding protein, PAQR11-mediated complex promoting cell migration and metastasis [270]. Thus, cancer cell development as well as tissue invasion and metastasis are triggered by an inflammatory fibrotic stroma [271]. This is further supported by evidence that the combined inhibition of LOXL2 and TGF-b1 receptor (TbR) activities by trihydrophenolics attenuated lung and cancer fibrosis [272].
Thioacetamide (TAA)-induced fibrosis in mice was inhibited and advanced parenchymal biliary and non-biliary fibrosis was reversed by the use of anti-LOXL2 monoclonal antibody, AB0023, which also promoted hepatic progenitor cell differentiation towards hepatocytes and attenuated ductular reaction [277]. Therapeutic approaches to attenuating fibrosis and antiinflammatory approaches, e.g., potential corneal wound healing could be helpful as the TGF-b/Smad pathway of fibrosis in human keratocytes was inhibited effectively in vitro [286].
Inhibition of the PCN can be achieved in multiple ways as recently reported. Signaling of regulatory T-cells and its TGF-b pathway was inhibited with neutralizing antibodies [88]. Stroma and remodeled ECM (i.e., the PCN), rather than the cancer cell itself, triggers cancer development as myeloid cell expression of EGFR, increased activation of STAT3, and expression of survivin in intestinal epithelial cells and expression of IL-6 in colon tissues. EGFR deletion from myeloid cells but not from intestinal epithelial cells protected mice from colitisinduced cancer [287]. Therefore, PCN rather than the cancerous cell itself triggers carcinogenesis. Lysyl oxidase functions as a tumor promoter in advanced high-grade serous ovarian cancer and in facilitating the cascade of events leading to metastasis [288]. Increased lysyl oxidase expression levels in laryngeal cancer are associated with lymph node and distant metastasis and poor patient outcome [289] and inhibition of lysyl oxidase decreases or prevents fibrosis, metastasis, cell migration and motility [273][274][275][276][277][278][279][280][281][282][283][284][285].
The role of chemical carcinogens in the tumor microenvironment and carcinogenesis had been reviewed in more detail within the following manuscript of this special issue "Chronic inflammation evoked by pathogenic stimulus during carcinogenesis" [290]. This paragraph as well as the other manuscripts of the Special Issue summarize the available evidence in carcinogenesis and explains that the onset of cancer involves chronic inflammation, fibrosis with its remodeling leading to the formation of the PCN induce chronic cell stress and a disruption of homeostasis such that cancer can result over decades in a subset of the exposed population.
The PCN is not just to be the starting point for how a normal cell to cancer cell transition occurs, and the process can be interdicted, but also explains why the completeness of radical cancer surgery as macroscopic and microscopic complete resections (R0-resection) needs to be changed in accordance to a local (paratumoral) or more distant precancerous niche (PCN). This even may provide a future need or basis for why (1) a different therapeutic approach may be of benefit for the patient's survival, progression free survival À with the goal of quality of life remaining being foremost [291] À and even (2) why this would be of help in R0 resected patients who progress due to primary locally advanced cancer stages.

Summary
Ongoing pathogenic stimuli induces chronic inflammation [290,292], promotes fibrosis with its remodeled version, the PCN. Thus, the PCN explains why it is not just external stimuli that brings about carcinogenesis but the response of the host tissue [293] to such stimuli and the ability or inability of the host to interdict the pro-inflammatory pathways, signaling cascades [290,292], and fibrotic changes that describe the dysregulation of the cellular and tissue equilibrium (Fig. 1) which are, in toto, critical to carcinogenesis and, perhaps, equally important to the onset of metastasis. Significant in this context are the influence by the microbiome and obesity [294] as well as findings investigating a key enzyme, lysyl oxidase, its isoforms and receptors, as well as the study of cancer-resistant species. The remodeled ECM within the "Disruption of signaling homeostasis induced crosstalk in the carcinogenesis paradigm Epistemology of the origin of cancer" resulting in the PCN does not need a mutation to occur. Therefore, the proposed 6-step sequence is important for carcinogenesis and metastases.  ). We express our gratitude to the discussions on the web group of the Theodor-Billroth-Academy ® (TBA ® ) on LinkedIn, the exchange with scientists at Researchgate. com, as well as personal exchanges with distinguished colleagues who stimulated our thinking all named individually earlier in publications À we thank each one. We further gratefully acknowledge the support of Marjan S. Rupnik, PhD, Professor of Physiology, Institute of Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria, for pre-submission review of the literature and our fruitful discussions.