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The statistical significance of the differences in survival distribution among the prognostic groups was evaluated by the log-rank test [ 31 ]. The Cox proportional hazards model was applied to the multivariate survival analysis [ 32 ]. The prognostic variables on overall survival included: SPSS software version In hypoxic conditions, the two cell lines showed similar behavior. Nuclear accumulation of the protein was evident after hours, reaching a peak at about 4 hours and then declining to basal level after 12 hours.
HIF-2 alpha did not seem significantly involved in the response to hypoxic stress given no variation in mRNA and only a slight nuclear protein up-regulation in MSTO was observed after hours stimulation. In both cell lines, HIF-3 alpha nuclear protein accumulation was detectable from 48 and still present at 96 hours of hypoxia.
A representative experiment for each gene is shown.
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- NF-kappaB and cancer: how intimate is this relationship.
Hypoxia dependent activation of NF-kB was confirmed by a clean-cut and precocious up-regulation of nuclear phospho P65 Serthe transcriptionally active form of P Therefore, in both cell lines NF-kB pathway resulted highly responsive to hypoxia, although MSTO cells showed a more marked and prolonged activation. Hypoxic regulation of inflammatory-related genes in MM cells The ability of MM cells to adapt to low oxygen concentrations through the modulation of their pro-inflammatory phenotype was investigated by real-time PCR, in time course experiments under normoxic and hypoxic conditions Figure 2.
Oxygen deprivation up-regulated the expression in a large part of the studied genes with a comparable kinetic in the two cell lines. Except for P2X7R in MSTO, which was induced at 8 hours of hypoxic treatment, transcription of the other molecules started increasing at shorter stimulation times.
Modulation of pro-inflammatory genes under hypoxic stimulation.
NF-kappaB and cancer: how intimate is this relationship.
A representative experiment is shown. Interestingly, this effect did not rebound on the protein level where parthenolide did not seem to affect nuclear HIF-1 alpha protein accumulation at 4 and 8 hours of oxygen deprivation Figures 3B bottom panel and S2A. HIF- 1 alpha did not appear to be involved in NF-kB nuclear increase in MSTO, since no differences in the basal and hypoxic level of both p65 and phospho p65 were observed between the non silenced and silenced clones at 30 min of hypoxia Figures 3C and S2B.
One representative experiment is shown. In Figure 5, the results obtained at the time points where hypoxia exerted its maximum increase in transcription, are shown.
P2X7R expression was not affected by HIF-1 alpha but strongly down-regulated by parthenolide both in normal and oxygen deprived environment.
Therefore, its expression level seems controlled mainly by NF-kB. Consequently, for this gene, HIF-1 alpha control seems to prevail.
HMOX gene expression underwent a dual and complex modulation. The table in Figure 6 presents summary results from immunohistochemical analysis of 29 MM specimens.
Histological tumor specimens contained 16 epithelioid, 6 sarcomatoid and 7 mixed MM. The histological type did not significantly correlate with immunohistochemical parameters and was not associated with outcome and overall survival data not shown. Chemotherapy and radiotherapy did not show any impact on overall survival in univariate analysis data not shown. Sections were stained with streptavidin-biotin complex, DAB and hematoxylin.
Original magnification x Discussion In this study, we attempted to further our understanding in adaptation to hypoxia of MM by analyzing the mechanisms underlying inflammation-related gene expression under oxygen deprivation in two cellular models, MSTO and MPP.
At the same time, we looked for potential prognostic markers and therapeutic targets among the molecules involved in the response to low oxygen concentration, in particular HIFs and inflammatory related factors, as has already been done for other tumors [ 33 - 35 ]. HIF-1 alpha, among the three HIF isoforms, was confirmed to be the main player in hypoxic adaptation in both MSTO and MPP cells, with precocious modifications in transcription and nuclear accumulation comparable to those observed in other tumor cell lines [ 36 ].
HIF-2 alpha did not seem to be involved in hypoxic response in MM cell models since; a slight accumulation of nuclear protein was detected only in MSTO, whereas mRNA levels remained unchanged in both cell lines. Differently, a delayed HIF-3 alpha mRNA and nuclear protein up-regulation was observed in both cell lines, revealing, for the first time, its role in the MM hypoxic response. HIF-3 alpha is the less known transcriptional factor responsive to hypoxia, it is cell specific and its function is still under debate [ 37 ].
Interestingly, the immunohistochemical analysis performed on MM human samples showed an increased expression of HIF-3 alpha in the tumor tissues compared to non tumor and a border-line correlation between HIF-3 alpha expression and survival. Knowledge on predictive factors for mesothelioma outcome is poor, thus, the identification of molecular targets is required.
When we analyzed the impact of hypoxia on the pro-inflammatory phenotype of MM cells, we highlighted that oxygen deprivation significantly induced the expression of a selected set of inflammationrelated genes. In particular, CXCR4, a gene over-expressed and associated with increased proliferation, invasion and migration in several tumors [ 26 ], showed a remarkable hypoxic up-regulation in both MM cell lines, as already described by Li et al.
Li et al showed that non tumor mesothelial cell line LP9 do not express CXCR4 while 5 mesothelioma cell lines out of 6 expressed CXCR4 at different levels and one was completely negative for this protein. In addition, our results deriving from the immunohistochemical analysis on MM human samples pointed out, for the first time, a highly significant correlation between CXCR4 expression level and survival. The positive statistically significant correlation recorded between CXCR4 and HIF-3 alpha expression level data not shown supports the assumption that these two molecules in association could represent a nucleus of prognostic markers for MM.
As well as HIF-1 alpha, also NF-kB was activated early following hypoxic stress in both MM cell lines, thus confirming what observed in other tumor cells.
HIF-1 alpha and NF-kB pathways are intimately associated and a significant cross-talk between them exists on different levels. In fact, a number of stimuli up-regulates the expression of HIF- 1 alpha through NF-kB-dependent mechanisms [ 1516 ]. In different tumors, genes involved in the inflammatory pathway are up-regulated and, in some cases, related to prognosis [ 39 ].
Some of them, including inducible NO synthase and cyclooxygenase-2 COX-2contain functional response elements for both NF-kB and HIF-1 alpha in their promoters, even though the relative contribution of these transcriptional regulators remains controversial [ 40 - 43 ].
Conversely, HIF-1 alpha silencing did not induce variation in the nuclear translocation of p65 and in the level of phosphorylated Ser P65, both in normoxia and hypoxia, strongly suggesting that NF-kB pathway is not dependent on HIF-1 alpha activity in this cell line.
Therefore, the tuning of the pro-inflammatory phenotype in MSTO, relies on a complex balance between the action of these transcription factors that in turn is strongly influenced by the environmental changes. The transcriptional modulation of the antiinflammatory gene HMOX1 appears to be even more complex. These results confirm the dual control of this molecule we observed in prostate cells [ 44 ], where NFkB acts as a repressor under normoxia and hypoxia and HIF-1 alpha as an inducer under hypoxia.
Conclusion We can summarize our findings as follows: For the first time, a cross talk between hypoxia adaptation and pro-inflammatory pathway was revealed in MM which was accomplished through the interaction of the transcription factors HIF- 1 alpha and NF-kB.
The involvement of HIF-3 alpha in MM response to hypoxic stimulation was demonstrated and a possible correlation between its expression in MM tissues and prognosis was suggested. TWIST protein activity is also tightly regulated by post-translational modifications, offering an alternative to quickly adapt its activity to the cellular context.
TWIST proteins also form labile homodimers that modulate the expression of distinct downstream target genes, providing them with specific functions Firulli et al. The balance between the two complex types is highly regulated during development and is also likely to evolve during tumor progression. In particular, the reported modulation of Id expression in response to cell environment and mechanical constrains might be determinant in regulating the balance between these two complexes Firulli and Conway, ; Castanon et al.
Epigenetic events might also contribute to alter TWIST expression as their promoter sequences are subjected to methylation, although this still remains a matter of discussion Martin et al. A growing body of evidence now supports this conclusion, highlighting a preponderant role of TWIST proteins in promoting invasion and metastatic dissemination in multiple cancer types including breast Mironchik et al.
TWIST pro-metastatic potential relies on their ability to induce an epithelial to mesenchymal transition EMTa process that converts joined and polarized epithelial cells into isolated and motile mesenchymal ones, able to bypass the basement membrane and infiltrate into the surrounding extracellular matrix Thiery and Sleeman, ; Yang and Weinberg, ; Nakaya and Sheng, This programmed embryonic transdifferentiation process involves the loss of cellular junctions tight and adherent junctions as well as desmosomes and a profound reorganization of the cytoskeleton.
It is determinant in promoting multiple morphogenetic movements during the embryonic development, including the mesoderm formation during gastrulation, the neural crest delamination, the palate fusion and the formation of the heart cushions Thiery et al.
EMT contributes to several human pathologies, including renal, hepatic and lung fibrosis, by turning epithelial cells into collagen-producing mesenchymal ones Iwano et al. More recently, it turns out to be a driving force of cancer cell dissemination during tumor development Thiery et al. As epithelial cells that have undergone an EMT are phenotypically indistinguishable from fibroblasts, a role of EMT in cancers was controversial.
Nevertheless, the physiological relevance of EMT in cancers in vivo has now unquestionably been experimentally confirmed Spaderna et al. TWIST proteins promote EMT by turning-down the expression of epithelial specific proteins, such as the E-cadherin and by upregulating the expression of mesenchymal markers such as the N-cadherin, the vimentin and the smooth-muscle actin.
Particular attention has been devoted on E-cadherin regulation as it behaves as the guardian of the epithelial phenotype, its knock-down being sufficient to promote an EMT Onder et al. TWIST proteins display oncogenic properties Beyond their prometastatic properties, TWIST proteins were found by our own group to display oncogenic properties by preventing senescence and apoptosis induction in response to oncogenic insults Ansieau et al.
These two oncosuppressive mechanisms are known to function in premalignant lesions Gorgoulis et al. This observation was next supported by the detection of apoptotic osteoblasts in Saethre-Chotzen syndrome patients, a human syndrome of cranial malformation due to reduced Twist1 function Yousfi et al.
TWIST proteins were identified in a functional screen as proteins able to counteract c-MYC induced apoptosis, thus depicting them as potential survival factors promoting cell transformation Maestro et al. In line with this observation, we have shown that TWIST1 overexpression is a prevalent p53 inactivation mechanism in neuroblastomas, a tumor where the p53 gene is usually not mutated, promoting tumor growth by alleviating N-MYC induced apoptosis Valsesia-Wittmann et al.
Recently, we further demonstrated that TWIST proteins were able to prevent oncogene-induced senescence, by neutralizing both Rb- and p dependent pathways, and to cooperate with oncogenes such as RASV12 or ERBB2 in murine primary fibroblast transformation. By overriding failsafe programs, TWIST proteins are therefore likely to promote malignant conversion, playing a de facto role at the early steps of tumor progression. Since this discovery, TWIST1 expression was shown to be induced during the malignant conversion of bladder Zhang et al.
By inhibiting Rb and p53 tumor suppressors, TWIST proteins are suggested to provide cells with proliferative and survival advantages at the primary tumor sites.
Breast Cancer Chemoprevention: Old and New Approaches
Intimate crosstalk between failsafe program escape and cancer cell dissemination Growing evidence suggest that proteins such as TWIST, instead of being constitutive EMT inducers, behave as sensors, promoting EMT in response to appropriate micro-environmental signals. Although these signals might cooperate with EMT inducers through the induction of independent and complementary signaling pathways, they are also believed to directly activate EMT-inducer activity. Importantly, this cooperative effect raises the possibility that failsafe program escape, when associated with the reactivation of embryonic genes such as TWIST, might promote malignant conversion, and concomitantly be determinant in fostering cancer cell dissemination Figure 1.
The initiation of the metastatic process thus would not be restricted to late stages of tumor progression, as commonly claimed. Instead, at least in some cancers, dissemination may be initiated much earlier, giving rise to disseminated single cells evolving independently of the primary tumor.
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Consistent with this idea, in a murine mammary tumor progression model, precancerous cell dissemination was found to initiate from atypical ductal hyperplasia, an early stage in tumorigenesis, in association with TWIST1 reactivation Husemann et al. Some mechanisms associated with failsafe program escape might therefore lead to the pre-selection of cells particularly prone to disseminate in permissive conditions, giving a rationale to the established metastatic predictive signatures.
In the presence of a abnormal mitogenic activation and in response to a permissive environment, tumor cells concomitantly acquire a mesenchymal and motile phenotype through EMT favoring invasion and dissemination, acquisition of cancer stem cell properties, a widespread chemoresistance to therapeutics and a global genomic instability.
Furthermore, it also induces angiogenesis in hypoxic conditions, thus setting the table for the primary tumor growth as well as the formation of distant metastasis. Recently, we and the Weinberg laboratory demonstrated in vitro that EMT also provides mammary epithelial cells with stem-like properties including a CD44 high CD24low antigenic phenotype, the ability to form mammospheres in low adherent conditions and a high tumorigenic potential Mani et al.
This observation raises the possibility that "cancer-stem" cells, or "cancer-initiating" cells, may arise not only from the alteration of normal stem or progenitor cells but also from the dedifferentiation of differentiated cells. Knowing that EMT is highly sensitive to the microenvironment and is a reversible process, the balance between cancer-stem cells and differentiated cells in tumors is presumed to be highly dynamic Gupta et al. Growing evidence strengthens the relevance of this observation in vivo.
Additionally, the majority of disseminated cells arising from metastatic breast cancers, display mesenchymal and stem cell-like features Aktas et al.