The tumor progression process is characterized by rapid cell growth accompanied by alterations in the microenvironment of the tumor cells. Among these changes, is the appearance of multiple areas of hypoxia (low oxygen tension), a hallmark feature of human and experimental tumors [40, 41]. Hypoxic regions within tumors arise for several reasons. First of all, rapid tumor cell growth can outpace the distance that allows adequate delivery of O2 by surrounding blood vessels, causing hypoxic areas to form. Secondly, new blood vessels formed in tumors are usually disorganized and are prone to collapse, resulting in regions of inadequate perfusion and hypoxia. Additionally, diminished tumor oxygenation can be exarcerbated by reduced O2 transport caused by cancer-related or cancer treatment-induced anemia .
Hypoxia has recently emerged as a major factor that influences malignant progression. The effects of hypoxia are mainly mediated by a series of hypoxia-induced proteomic and genomic changes that enable tumor cells to survive or escape their oxygen deficient environment [43, 44]. But how does hypoxia contribute to tumor progression? One of the key factors in mediating tumor cell survival and tumor progression is the hypoxically regulated production of growth factors that induce new blood vessel formation (angiogenesis). In tumors, the ability to induce angiogenesis, in addition to provide O2 and nutrients to support cell survival and proliferation, is associated with the development of metastasis by increasing the opportunity of tumoral cells to access blood circulation and disseminate at distal sites [45, 46].
Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular response to hypoxia [47, 48]. This transcription factor is a heterodimer composed of two subunits: an oxygen sensitive alpha-subunit (HIF-1 a) and a constitutively expressed beta-subunit (ARNT/HIF-1^) [49, 50]. Under normoxic conditions, HIF-1 a is hydroxylated on several specific proline residues by one of three HIF-1 oxygen-dependant prolyl hydroxylases, PHD1-3, a reaction that depends on the presence of oxygen. Hydroxylation permits the binding of HIF-1 a to the von Hippel Lindau tumor suppressor (VHL), a component of an E3 ubiquitin ligase complex which targets HIF-1 a for proteasomal degradation, consequently maintaining HIF-1 a at low levels [51, 52]. Under hypoxic conditions, the prolyl hydroxylases are no longer active and the unmodified HIF-1 a no longer interacts with VHL and thereby accumulates. The stabilized HIF-1a then translocates to the nucleus where it heterodimerizes with the HIF-1 p subunit. The HIF-1 complex activates transcription by binding to a specific DNA sequence called Hypoxia Responsive Element (HRE) found within the promoter of target genes [53, 54].
Up to now, more than 70 genes are known to be transcriptionally activated by HIF-1 . Several of these genes convey the survival and proliferation of tumor cells by mediating angiogenesis, glucose uptake, invasion and metastasis, thereby promoting tumorigenesis. Nuclear accumulation of HIF-1 protein has been reported in human common cancers and cells lines, including head and neck, glioblastomas, breast, colon, pancreatic and prostate, which are pathological conditions where furin was also found to be overexpressed [2, 5, 56-59]. In accordance with this, furin was recently identified as a novel HIF-1 target .
Computer-assisted analysis of furin promoters P1, P1A and P1B uncovered the presence of several putative HREs similar to those found in many genes regulated by oxygen deprivation such as vascular endothelial growth factor, erythropoietin and glucose transporter-1 [53, 60, 61]. This suggested that furin expression may be regulated under hypoxic conditions to achieve adequate proteolytic maturation of angiogenic and/or tumorigenic substrates within tumors.
To test this hypothesis, HepG2 cells, derived from a human hepatoma, were cultured under hypoxic conditions (1% O2). A marked increase in furin mRNA levels was observed with a maximum increase of 18-fold after 24 hours of low oxygen exposure. As a comparison, the convertases PACE4 and PC7 mRNA levels were barely affected by hypoxia, indicating that hypoxia-induced expression is not extended to all of the human proprotein convertase family members. Interestingly, a similar increase in furin mRNA was observed in other cell lines, including RAW 264.7 mouse macrophages, primary rat synoviocytes, as well as mouse Hepa-1 hepatoma cell line, indicating that the regulation of fur gene expression by hypoxia is extended to various cells types and species .
Several experiments were conducted in order to delineate the molecular mechanisms responsible for hypoxia-induced expression of furin . The transient trans-fection of HepG2 cells with constructs encompassing the luciferase reporter gene under the control of each of the three furin promoters (P1, P1A and P1B) revealed that the fur P1 promoter is the most sensitive to hypoxia. Cotransfection of a HIF-1 a dominant negative form in HepG2 cells or tranfection of the P1 promoterreporter gene plasmid alone in HIF-1 deficient cells (Hepa-1c4 cells) indicated the requirement of HIF-1 for furin promoter activation by hypoxia. Further analysis of the hypoxia sensitive P1 promoter revealed that deletion of the -1221 to -413
region, containing two putative HREs, abolished the response to both hypoxia and to exogenous expression of HIF-1, suggesting that one or both of these HREs takes part in the furin transcriptional unit in response to hypoxia. Directed mutagenesis of each of these HREs permitted to define the critical HRE responsible for the HIF-1-dependent response of P1 promoter under hypoxia. This HRE, possessing enhancer capability, shares sequence similarities with those identified within the promoter of several tumor related genes, such as VEGF, MT1-MMP, and endoglin [44, 62, 63].
Several studies suggest that furin-induced expression within tumors supports tumoral development through the increased bioavailability of pro-tumorigenic mediators. Interestingly, the hypoxia/HIF-1-dependent increase in furin gene expression was found to be associated with increased maturation/activation of the angiogenic/tumorigenic mediators MT1-MMP and TGF^1 . Both mediators are well characterized furin substrates that have been found to profoundly affect many aspects of tumor progression [14, 17, 64]. MT1-MMP, through proteolytic events, regulates various cell functions, including extracellular matrix turnover, promotion of cell migration and invasion. MT1-MMP acts either through direct degradation of extracellular matrix components or indirectly by activating MMP-2 . In addition, these metalloproteinases are involved in the angiogenic process by mediating endothelial cell invasion as well as the release or the activation of growth factors [66, 67]. TGF^1, in turn, creates a favorable environment for tumor establishment by repressing immune surveillance, inducing the production of potent angiogenic factors such as vascular endothelial growth factor and basic fibroblast growth factor, and by increasing the production of extracellular matrix proteases, which promote tumor proliferation, invasion and metastasis [68, 69]. Moreover, in addition to these molecules, the multiplicity of other established furin substrates involved in cell growth and survival (insulin-like growth factor receptor-1, hepatocyte growth factor receptor c-Met, platelet-derived growth factor), cell invasion (E-cadherin and integrins), and angiogenesis (vascular endothelial growth factor-C) supports the contention that the regulation of furin activity within hypoxic/HIF-1 expressing zones of tumors could profoundly impact the course of tumor growth, invasion, and metastasis in a detrimental manner [70-75]. Thus, further studies are required in order to establish a link between increased maturation of furin substrates under hypoxic conditions and promotion of tumorigenesis. Preliminary experiments performed in our laboratory with the human fibrosarcoma cell line, HT1080, tend to confirm this link. Results obtained from fluorescent immuno-labelling of furin within mice xenografts, revealed a striking overexpression of furin within hypoxic areas of tumors, indicating that the proprotein convertase hypoxic regulation takes place in vivo (Grandmont S. et al., unpublished data).
Moreover, hypoxia was shown to be associated with an increase in cell invasive phenotype, in part through the up regulation of proteases, some of which are furin substrates [76, 77]. Interestingly, overexpression of the furin inhibitor a1-PDX in HT1080 cells blunted hypoxia-induced invasion to levels found in control cells
Figure 3. In vitro invasion was assessed in Boyden chambers using filters coated with type IV collagen, a major component of blood vessel basement membranes. Eighty thousand parental HT1080 cells or stable transfected cells with either the furin inhibitor «1-PDX (PDX-1, PDX-2, PDX-3) or the empty pcDNA3 vector (CTL-1 and CTL-2) were allowed to migrate under normoxic (21% O2) or hypoxic (1% O2) conditions for 8 hours. Cells that have reached the lower surface of filters were visually counted using a light microscope at 400-fold magnification. Northern blot analysis was performed to confirm the expression of furin inhibitor «1-PDX. Results are expressed as the mean ± SEM. A representative experiment out of three is shown
Figure 3. In vitro invasion was assessed in Boyden chambers using filters coated with type IV collagen, a major component of blood vessel basement membranes. Eighty thousand parental HT1080 cells or stable transfected cells with either the furin inhibitor «1-PDX (PDX-1, PDX-2, PDX-3) or the empty pcDNA3 vector (CTL-1 and CTL-2) were allowed to migrate under normoxic (21% O2) or hypoxic (1% O2) conditions for 8 hours. Cells that have reached the lower surface of filters were visually counted using a light microscope at 400-fold magnification. Northern blot analysis was performed to confirm the expression of furin inhibitor «1-PDX. Results are expressed as the mean ± SEM. A representative experiment out of three is shown cultured in normoxia, suggesting that furin may significantly contribute to this process (Figure 3).
Since the discovery of HIF-1, the scientific community has demonstrated a strong interest for the mechanisms that regulate the functions of this transcription factor. One of the most significant finding is the discovery that physiological stimuli other that hypoxia can regulate HIF-1 expression and activity. Several nonhypoxic stimuli, such as growth factors, hormones and cytokines were demonstrated to induce HIF-1 a accumulation and activity in normal oxygen conditions, indicating that the tumor microenvironment encompasses, in addition to hypoxia, multiple checkpoints capable of regulating HIF-1 levels [78-82]. As opposed to hypoxia, stabilization does not seem to play a predominant role in the accumulation of HIF-1 a in response to non-hypoxic stimuli. In fact, the HIF-1a degradation process is not altered in response to a variety of growth factors and cytokines [83-86]. The prime mechanisms underlying this induction involves the increase in HIF-1 a gene transcription and/or mRNA translation, which seem sufficient to reverse the balance between synthesis and degradation, favoring HIF-1a accumulation in normoxia [83, 84, 87]. Although, not much is known about the mechanisms implicated in the transcrip-tional regulation of HIF-1a, several studies identified the PI3 kinase pathway and its downstream effectors as the mediators of increased HIF-1 a translation in response to growth factors [88, 89].
Recent scientific developments indicate that furin can be regarded as a regulator of both HIF-1 expression and activity through its capacity to process/activate growth factors and receptors (Figure 4).
Among others, TGF|1 was shown to induce the expression of HIF-1 a under normoxic conditions leading to HIF-1 complex formation and induction of VEGF,
Figure 4. Schematic representation of furin impacts on HIF-1 expression and activity. Several furin substrates were shown to induce HIF-1 a accumulation and activity in normoxic conditions. As opposed to hypoxia, that induces HIF-1 a stabilization by repressing PHD activity, these non-hypoxic stimuli act by modifying HIF-1 a transcription or translation levels through parallel or cooperative interactions between the Smads, PI3K and ERK 1/2 pathways. This will result in increased expression of the fur gene, a known HIF-1 target, creating a regulatory loop of potential importance in the induction as well as in the activation of numerous factors implicated in the pathology of cancer
Figure 4. Schematic representation of furin impacts on HIF-1 expression and activity. Several furin substrates were shown to induce HIF-1 a accumulation and activity in normoxic conditions. As opposed to hypoxia, that induces HIF-1 a stabilization by repressing PHD activity, these non-hypoxic stimuli act by modifying HIF-1 a transcription or translation levels through parallel or cooperative interactions between the Smads, PI3K and ERK 1/2 pathways. This will result in increased expression of the fur gene, a known HIF-1 target, creating a regulatory loop of potential importance in the induction as well as in the activation of numerous factors implicated in the pathology of cancer a key target of HIF-1, indicating that the transcription factor is active [79, 90]. In that study, however, the molecular mechanism involved in this increase of HIF-1 a expression was not explored. Since previous studies have indicated that increased transcription plays a significant role in HIF-1 a protein induction in response to non-hypoxic stimuli, we evaluated whether a similar mechanism is involved in the induction of HIF-1 a by TGF^1. Northern blot analysis revealed a marked induction of HIF-1 a mRNA levels in HepG2 cells after a 6h incubation in the presence of TGF^1, indicating that, as seen for other growth factors, TGF^ impacts HIF-1a mRNA accumulation (McMahon et al., unpublished observation). Since this growth factor is actively secreted by several cancer cells, furin expression in tumors could be directly regulated by TGF^1 through the action of Smads and/or HIF-1 . This will result in an increased processing of the TGF^1 precursor into its bioactive form creating a regulatory cycle of potential importance in the induction, as well as in the activation of numerous factors implicated in the pathology of cancer.
In fact, tumors express molecules, other than TGF^1, that are known to induce the expression of HIF-1a in normoxic conditions. Several are furin substrates such as the growth factors IGF-1, HGF, and PDGF as well as the receptors IGFR1 and c-Met, which transmit intracellular signals in response to IGF and HGF, respectively [71-73, 92, 93]. The inflammatory cytokines TNFa, which is liberated from cell membranes through the action of another furin substrate, TACE, also induces HIF-1 activity in normoxic conditions . Thus, the activation of these molecules by furin will likely confer to the convertase the ability to regulate HIF-1 expression, which, in turn, could participate in the overexpression of furin, as well as other HIF-1 targeted genes to exacerbate tumor progression. Supporting this, preliminary results obtained from our laboratory indicate that furin inhibition is associated with decreased basal levels of HIF-1 in cells, which is reverted by the addition of exogenous growth factors (McMahon S. et al., unpublished data).
As mentioned, Smad proteins are known to regulate TGF^-dependent gene expression by interacting with a variety of co-activators, including FAST, AP-1, SP1, as well as CBP/p300 [22, 32, 34, 35, 94, 95]. Several studies indicated that both TGF^1 and hypoxia signalling pathways can synergize, at the transcriptional level, to induce the expression of erythropoietin, endoglin and VEGF genes [63, 96, 97]. This cooperative effect was attributed to the direct interaction between Smad3 and HIF-1, as demonstrated by co-immunoprecipitation assays. Further experiments also revealed the formation of a tripartite complex composed of SP1/HIF-1/Smad3 upon cell exposition to both hypoxia and TGF^ . Since TGF^ treatment or Smad3 transfection consistently resulted in increased HIF-1a/SP1 co-mmunoprecipitation, Smad3 was regarded not only as a coactivator factor, but also as an adaptor molecule that reinforce HIF-1/SP1 complex formation . Because of its ability to bind to general transcription factors as well as elements of the basal transcription machinery, the presence of Sp1 in this multicomplex is though to be an additional way used by
HIF-1 to connect with theses molecules for more efficient transcriptional activity. In these studies, HIF-1/Smad cooperation was shown to be transmitted by the direct interaction of each transcription factor to respective and adjacent consensus sites [63, 96, 97]. Interestingly, the presence of putative Smad binding elements were uncovered surrounding the functional P1-HRE, indicating that in the context of the furin promoter, Smad may also serve as an adaptor to optimize furin expression.
In addition to hydroxylation, that controls HIF-1 a stability, other post-translational modifications are necessary to confer maximal transcriptional activity to HIF-1. As an example, the c-terminal region of the HIF-1 a subunit is directly phosphorylated by the MAPK p42/44 in vitro and in vivo, resulting in increased HIF-1 transcriptional activity [98, 99]. As opposed to hypoxia, several growth factors, including TGFp1, are known to activate this signalling pathway following binding to their specific receptors . Thus, furin can, through the bioactivation of TGFp1, as well as other growth factors and receptors, enhance HIF-1 transcriptional activity in hypoxic and normoxic condition.
Together, these observations suggest that in addition to be regulated by HIF-1, furin can impact the expression and activity of this transcription factor. This sheds light on novel mechanisms by which the tumor microenvironment can modulate furin expression, but also, reveals a novel pro-tumorigenic action of furin, through the induction of HIF-1 expression and activity. This, in turn, can directly influence the expression of the convertase but also of several other HIF-1 regulated genes that strongly influence the tumorigenesis process.
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