Introduction
Lung cancer is the most lethal type of cancer accounting for nearly 1,600,000 deaths worldwide in 2012 (1). Non-small cell lung cancer (NSCLC) is the predominant histological type, accounting for 80–85% of all cases (2). Histological type and extension of disease at the time of diagnosis influence prognosis. Yet, even with the advent of new targeted therapies and improved methods for early diagnosis, patients with lung cancer have short-term survival (1). Highlighting biologic markers is essential to clarify malignant lung processes, to identify patient subtypes and thereby to improve the poor prognosis of lung cancer.
The apoptosis pathway has been extensively studied in cancer research since malignant cells have the ability to evade this pathway. The disruption in the balance between pro-survival and pro-death cascades could break the barrier to cancer (3). In this context, the inhibitors of apoptosis proteins comprise a range of molecules, which play a critical role in cell-acquired resistance to death. In many types of tumors, the levels of expression of certain molecules commonly appear upregulated, and one example is the X-linked inhibitor of apoptosis protein (XIAP) protein (4). XIAP-associated factor 1 (XAF1) is a pro-apoptotic protein and it was primarily identified as an antagonist of XIAP, counteracting XIAP anti-caspase activity (5). Data suggest a plethora of possible XAF1 functions favoring apoptosis either through survivin degradation (6), mitochondrial cytochrome c release (7,8), p53 stabilization (9,10), as a modulator of cell cycle G2/M phase (11), as an inductor of autophagy by upregulating Beclin 1 (12), inhibition of the AKT pathway (12) or also inhibition of VEGF (13).
Loss of XAF1 has been observed in many types of tumors and is associated with increased malignant potential (9,13–21). Exceptionally, testicular germ cell tumors show XAF1 overexpression, which could be one of the reasons behind their extraordinary sensitivity to chemotherapy (22). In vitro assays have shown that restoration of XAF1 expression sensitizes cancer cells to apoptotic stimuli (5,9).
Understanding the mechanism of XAF1 downregulation in tumors could be a promising strategy by which to introduce new agents capable of restoring XAF1 expression and improving drug response. There are a variety of processes under investigation. For example, epigenetic patterns altered within the promoter region of XAF1 genes have been identified in tumors. Hypermethylation of CpGs sites located in the 5′ proximal region was found to be associated with transcriptional silencing of XAF1 in gastric cancer (14,23), urogenital (24) and colon (25) cancer cell lines. However, epigenetic modifications are extremely complex and other XAF1 associations have been observed. Heat-shock factor 1 (HSF1) belongs to a family of transcriptional factors involved in cellular response under stress conditions. Once activated, HSF1 homotrimerizes, translocates to the nucleus and binds to heat shock response elements (HSEs) to regulate the transcription of target genes (26). Notably, a functional HSF1-binding element was recognized in the XAF1 promoter gene and was found to be associated with the repression of XAF1 transcription (27). Wang et al reported that gastrointestinal cancer (GI) overexpressed HSF1 compared to normal tissues and the XAF1 expression was inversely correlated with HSF1 in GI cancer cell lines (27).
The tumor microenvironment can also provide abnormal conditions for cells. Dai et al reported that HSF1 may promote oncogenesis by facilitating cellular adaptation to the malignant lifestyle (26). HSF1 was also reported as a coadjuvant at the p53 transcriptional machinery. In response to DNA damage, HSF1 can complex with p53 and regulate p53-responsive genes, but only in wild-type p53 cells (28).
Recently, a link between XAF1 and p53 was demonstrated. The p53 responsive element was found within the promoter of the XAF1 gene and p53 was found to suppress the transcription of XAF1 (29). Zou et al confirmed that there is a direct and functional interaction between p53 protein and the XAF1 promoter (10). This interaction was found in wild-type p53, but not mutant p53 cells. Furthermore, there are no data integrating XAF1 and HSF1 expression and the p53 status in patient cohorts. Since there are limited studies performed in lung cancer patients (21), we investigated the XAF1 expression in a prospective cohort of NSCLC patients, and also lung cancer cell lines. Our data may contribute to the understanding of the potential role of XAF1 as a tumor-suppressor gene and its suggestive regulations.
Discussion
The process of lung carcinogenesis is still not well understood and several studies have tried to investigate target molecules to improve therapy and survival. A particular molecule called XAF1, which belongs to the apoptosis pathway, was investigated by us. The putative tumor-suppressor gene XAF1 has been found underexpressed in various cancer tissues and cell lines and, in addition, data generated elsewhere showed that restoration of its expression could increase sensitivity to apoptotic stimuli (14–21,30).
Low expression of XAF1 was observed in squamous cell lung cancer (21). In the present study, we identified low expression of XAF1 in NSCLC cells lines compared to that noted in normal lung (Fig. 1). However, to the best of our knowledge, this is the first study to describe the same pattern in both lung adenocarcinoma cell lines and patient specimens.
Despite no significant evidence in prognosis (Fig. 6), our cohort showed an association between XAF1 and increased malignancy. An association between reduced XAF1 and a higher histopathological grade was observed in our tumor specimens (Fig. 2) corroborating the results of previous studies (15,21,31–33). This may reinforce the potential role of XAF1 in lung tumor progression. Restoration of XAF1 could be a target mechanism to suppress malignant progression in the lung, as described in the literature for other tissue types. To that end a better understanding of the mechanisms leading to its downregulation is warranted. The first evidence for a possible cause of downregulation has been focused in hypermethylated regions at the XAF1 promoter gene (14). Therefore, we explored hypermethylation of CpG sites near the 5′ proximal region (−2 to −246 nt) that appeared strongly correlated with transcriptional silencing. We frequently observed the same profile in both non-tumor and tumor tissues by the methylation-specific PCR technique (data not shown). In order to investigate specific CpG sites and quantify the methylated percentage, we performed quantitative real-time PCR. One primer covered proximal CpGs sites −3 and −4 and the second one covered −5 to −7 CpG sites. After evaluating the methylation pattern in all matched paired lung tissues, we observed that 13 (31%) tumor tissues were hypermethylated (data not shown). The percentage of methylation varied from 2 to 30% in the CpG site −3 and −4 and from 2 to 88% in CpG site −5 to −7. When CpG −3 and −4 site appeared hypermethylated, the CpG site −5 to −7 was usually highlighted too. Almost all cases of non-tumor adjacent tissues appeared unmethylated or <1% methylated (data not shown). Our intention was to explore methylated areas with possible useful techniques applicable to the clinic setting, such as PCR. Since the XAF1 promoter region does not contain defined CpG islands but instead it contains CpG sites too closely, we found it difficult to deeply explore methylated areas using these techniques.
Meanwhile, the silencing of XAF1 in tumors has been investigated by other groups. Wang et al observed a functional HSF1-binding element (HSE/XAF1) located at −862/-821 of the XAF1 gene. They showed that HSF1 could repress XAF1 transcription and this could be reversed by blocking HSF1 binding (23). Under oncogenic stimulus, HSF1 could affect cell transformation, proliferation, signal transduction and metabolic process in favor of survival. Therefore, HSF1 could respond to oncogenic stimuli and add on the global network to support tumorigenesis (26). HSF1 can also have a dual action under stressful conditions. In normal cells it was described as a molecule, which protects cells from pathophysiological conditions such as hypoxia, thermal injury and age-related neurodegeneration (34,35). Since normal physiology is altered in the tumor microenvironment, HSF1 could also enhance malignancy levels. Notably, breast cancer containing high HSF1 was associated with poor prognosis (36). Another study showed that HSF1−/− mice presented superior survival to HSF1+/+ mice after tumor induction, as well as enhanced resistance to tumor formation (26).
In the present study, we investigated HSF1 protein and we observed that higher levels of HSF1 protein were translocated to the nucleus (where HSF1 is active) in tumor tissues (Fig. 5). Moreover, absence of expression was detected in non-tumor paired tissues indicating that HSF1 may be activated during the lung carcinogenesis process. This is consistent with the findings in gastric and colon cancer, where Li et al observed higher expression of HSF1 in gastrointestinal cancer tissues than in normal tissues (37). They also showed that stress stimuli of gastrointestinal cancer cell lines could upregulate HSF1 expression, but inversely downregulate XAF1 expression (37). In our immunohistochemical results (Figs. 3 and 5), a reduction in XAF1 expression was observed in lung tumor samples (Fig. 3), whereas a strong nuclear staining of HSF1 was observed in the tumor tissues (Fig. 5). Since HSF1 was shown to downregulate XAF1 expression in tumors, it may be informative to develop a selective HSF1 inhibitor in order to observe whether the levels of XAF1 could be restored in cancer cells. One difficulty which must be overcome is the fact that HSF1 is highly versatile, involved in numerous stress and non-stress related cell processes (38). Furthermore, it is also an important link for the translational machinery, since it was shown that a block in translation in cancer cells resulted in inhibition of HSF1 binding to its target genes (39). Thus, another possible way to block HSF1 activity may be to interfere with the ribosomal function in the cancer cells.
Logan et al supports the idea of HSF1 implicated in cell conjecture via p53-regulated transcription. It is well known that p53 can predominantly act in favor of cell cycle arrest and apoptosis under DNA damage. However it can also act as a pro-cell survival factor depending on the extent of damage or stress duration. Under genotoxic stress, evidence shows that p53 transcriptional activity is dependent upon HSF1 interaction and that HSF1 also facilitates phosphorylation of p53 serine residue 6 and 15 via the Chk1/ATR complex (28). Although these findings indicate pro-apoptotic effects, the author advocates that in response to oncogenic activation, HSF1 may redirect its function via p53 to promote cell survival and tumorigenesis (28).
XAF1 has been reported as a target gene for p53 since it carries a p53 responsive element (−86 to −95 nt) within the XAF1 promoter region (29). In cancer cells, the presence of wild-type p53 could lead to transcriptional repression of XAF1 while in mutated p53 cells this did not occur (29). Byun et al reported for the first time the inverse correlation between loss of XAF1 and p53 mutations in gastric cells and tumors (14), but to date there are scarce studies investigating other cohorts. In the present study, p53-deleted cells had higher XAF1 expression than wild-type ones indicating a possible feedback loop mechanism (Fig. 1). In lung tumor specimens, we observed a reduction in XAF1 expression compared to the paired non-tumoral adjacent tissue indicating that XAF1 expression could be influenced by the level of tumorigenesis (Fig. 2B). In our wild-type 53 patients, we observed a reduction in XAF1 protein levels in tumor samples when compared to adjacent non-tumor tissue (Table II). In contrast, we did not observe the same pattern in mutant p53 specimens (Table II). Although this could be further confirmed by increasing the cohort number, one possible reason for this result could be that XAF1 may be upregulated in order to compensate for mutant p53, indicating a possible feedback-loop mechanism. It was recently observed that XAF1 forms a positive feedback loop with p53 by acting as a molecular switch in p53-decision making and that different isoforms of XAF1 have different functions (10,40).
We also observed that all never-smoker patients did not lose XAF1 expression and among them, two exhibited a higher level of XAF1 in the tumor tissue than that in the adjacent tissue (Table III). Conversely, XAF1 expression was reduced in smokers, showing that XAF1 expression could be associated with smoking (Table III; p=0.05). Moreover, cells harboring EGFR mutation (an alteration more commonly observed in never-smokers) also had higher XAF1 expression when compared to EGFR wild-type cells. Altogether, these findings are congruent with the current distinction between tumors from smokers and never-smokers. In the mid 2000′s, non-smoking-related lung cancer became an independent disease from the well-established lung cancer in smokers (41). Different studies indicated a heavier mutation burden and a greater perturbation of gene expression levels in smoking-related lung cancer than in non-smoking, and different driver genes were identified in non-smoker vs. smoker tumors (42,43). In addition, distinct characteristics were observed in non-smoker patients, which could vary according to sex, histological type and ethnic origin. For example, most occurrences were in woman, Asian people and the predominant histological type was adenocarcinoma (41). Our findings should be further validated in a larger cohort of never-smoker patients.
In conclusion, our results indicated that XAF1 expression is altered in NSCLC tumor samples when compared to normal tissue and that the XAF1 status was influenced by different clinicopathological characteristics, such as smoking, EGFR and TP53 mutation status. However, no significant associations were observed for both tumor-recurrence or tumor-related death. The limited sample size could have influenced these results, since patients in the group of with lack of XAF1 tended to have poorly differentiated histological grade. It may be interesting to validate our findings in a larger cohort, including stage IV patients. In addition, XAF1 not only plays a role as a tumor suppressor but it is also important in the entire cell stress mechanism where the p53 status is crucial. It remains to be determined which molecules belonging to different signaling cascades may mediate XAF1 activation or downregulation in lung cancer. Moreover, further understanding of the molecular mechanisms underlying HSF1 must to be increased, and it may be valuable to determine whether HSF1 may be a therapeutic target by which to mediate XAF1 levels in lung cancer cells.