The BAX gene as a candidate for negative autophagy-related genes regulator on mRNA levels in colorectal cancer

Carcinogenesis is a complex, multistep process during which acquired genomic alterations may lead to chromosomal, microsatellite and epigenetic instability and thus result in cancer progression [1]. Cancers are the second leading cause of morbidity and mortality worldwide [2]. Colorectal cancer (CRC) is one of the most common cancers in developed regions, such as Australia, Europe and North America, and the leading cause of cancer-related deaths [2]. Its incidence in these regions is high (about 55% of all cancer cases) and ranges from the second to third (depending on population ethnicity) most common type of cancer among both sexes [2]. Most CRCs are sporadic, and individual susceptibility to disease is determined by: (1) environmental factors, such as occupational exposure, dietary habits and lack of physical activity, as well as (2) genetic makeup, including polymorphic variants in genes responsible for cellular metabolism and DNA repair (low-risk variants) [3, 4]. Despite immense progress in knowledge of genetic and environmental factors in CRC etiology, along with new treatment approaches which have been recently introduced into clinical practice, this cancer is usually diagnosed at a late stage of disease and thus 5-year overall survival is not frequent [5]. Recently, macroautophagy (hereafter autophagy) has emerged as a promising independent prognostic molecular biomarker and a potential target in cancer therapy [6]. Autophagy is a catabolic process enabling the maintenance of normal cell homeostasis by degrading redundant molecules and organelles (“self-eating”), but is also responsible for intracellular recycling, e.g., reuse of amino acids from degraded proteins [7]. Briefly, a cargo designed for degradation is engulfed by a double-membraned vesicle, called an autophagosome, which fuses with lysosome and thus its content is decomposed by acidic enzymes [8]. Autophagy is a fundamental cellular process which is highly conserved from yeasts to humans, and many yeast genes involved in autophagy have human orthologs (AuTophaGy related; ATG). Autophagy, as a defense process, is usually upregulated in cells under conditions of stress, e.g., starvation [8]. Thus, the energy essential for maintaining basic cellular functions may be acquired by the process of degrading proteins or organelles which are less necessary for cell survival (pivotal structures remain intact) in the process of autophagy [8]. The induction of autophagy is regulated by a variety of genes, including ULK1, ATG13, UVRAG, Bif-1 and BECN1. The following steps of autophagy result in the elongation and maturation of autophagosomes [8]. Eventually, autophagosomes fuse with lysosomes, thus forming autophagolysosomes and their content may be degraded by hydrolases [8].

Recently, autophagy has been extensively studied in different types of tumors, e.g., breast, pulmonary, prostate, brain and colorectal [7]. Up to now, autophagy in carcinogenesis has been described as a double-edged sword because of its dual function. On the one hand, autophagy protects normal cells against neoplastic transformation by maintaining intracellular homeostasis, but, on the other hand, may result in cancer cells being more likely to survive than normal cells under adverse circumstances, such as hypoxia and starvation, as well as during anticancer therapy [7, 9]. To date, the results of many studies on autophagy in CRC are conflicting and inconclusive; thus, its function in CRC development and progression remains unclear. Recently, a complex interaction between autophagy and apoptosis was reported. However, studies have shown conflicting results [10].

Because of inconclusive research data, we have focused on the mRNA expression levels of five genes involved in the induction of autophagy: BECN1, UVRAG, ULK1, ATG13 and Bif-1 and two genes involved in apoptosis: the antiapoptotic BCL2 and the proapoptotic BAX. These expression levels were observed in both colorectal cancer cells and paired relatively normal, adjacent tissue.

Genetic mutations leading to the activation of protooncogenes and/or loss of functioning of tumor suppressor genes may lead to the deregulation of various cellular pathways, including autophagy, and thus to cancer formation [13]. Autophagy is an intracellular mechanism responsible for defense against cellular stress [14]. However, its role in cancer initiation, tumor growth, anticancer therapy and treatment still remains an unanswered question [14].

In our study, we have shown relative downregulation of all but one of the examined autophagy-related genes, along with antiapoptotic BCL2, whereas proapoptotic BAX was relatively upregulated. We have observed its higher expression in the early stages of CRC in comparison with normal tissue. However, BAX expression successively decreases as a cancer progresses and is the lowest in patients with distant metastasis. Our results are in agreement with the observations published by Jansson and Sun [15]. They examined the protein expression level of BAX in normal colorectal mucosa, as well as in primary colorectal adenocarcinomas from early to advanced stages, including cases with metastases to regional lymph nodes. They reported more intense expression in primary tumors in comparison with normal tissue, but in metastatic CRC samples, lower expression levels have been observed [15]. Similar results have been obtained by Cobanoglu et al., who examined expression levels of BAX and AIF (apoptosis-inducing factor). BAX staining levels were markedly higher in adenomas and carcinomas than in normal mucosa. Moreover, the BAX level was higher in carcinomas than in adenomas [16].

Therefore, we may conclude that during the early stages of CRC carcinogenesis apoptosis is more prone to occur than autophagy, while during tumor progression an accumulation of genetic alterations may disturb the process of apoptosis and thus contribute to tumor progression and promotion.

We have observed a statistically significant correlation between a high expression of BAX and a decrease in expression of UVRAG. UVRAG is a well-known protein involved in autophagy initiation, through interaction with BECN1, as well as in the maturation of autophagosomes [8]. Recently, UVRAG has been reported as a crucial factor in apoptosis. Yin et al. [17] found that UVRAG possesses both autophagic and antiapoptotic properties mediated by its direct interaction with BAX in the cytosol, as confirmed by coimmunoprecipitation studies. These researchers have formulated the hypothesis that UVRAG exerts its cytoprotective function by controlling the localization of the BAX protein through interaction with this protein and inhibits translocation of BAX to the mitochondria and therefore prevents apoptosis [17]. Increased expression of UVRAG has been observed in cells exposed to stress, such as chemotherapy and/or UV radiation. The influence of the underexpression of UVRAG on anticancer therapy has been studied in experiments in which UVRAG expression has been inhibited by specific short hairpin RNAs (sshRNAs) transfection [17]. A decreased index of autophagy and increased level of apoptosis were detected [17]. Therefore, the authors suggested that decreased UVRAG activity directly influences BAX-induced apoptosis in cancer cells. The authors also showed that the antiapoptotic activity of UVRAG does not affect BAX expression [17]. However, UVRAG does not influence apoptosis induced by other proapoptotic proteins, such as Bad or Bid. Moreover, its direct role in the regulation of apoptosis seems to be an independent event, besides its proautophagic function [17]. Thus, it was assumed that in tumor cells UVRAG plays a central role in the modulation of apoptosis in response to stressful conditions (UVRAG-BAX complex) as a negative regulator and autophagy (UVRAG-BECN1 complex) as a positive regulator [17]. In our study, an elevated level of mRNA in BAX was shown to be associated with downregulation of the mRNA levels of UVRAG. Hence, we hypothesized that the promotion of apoptosis may influence the expression of UVRAG and therefore counteracts the induction of autophagy in CRC cells. Consequently, we conclude that high BAX expression may be a negative regulator of UVRAG gene expression.

Among the analyzed genes, we found that Bif-1 (BAX-interacting factor 1) expression was the highest, both in normal and in cancer tissues. Bif-1 is also known as SH3GLB1 (SH3 domain GBR2-like endophilin B1) and belongs to the endophilin protein family [18]. Bif-1 was identified as a BAX-binding protein and a necessary factor in the promotion of apoptosis [19]. It has been proven that the loss of Bif-1 inhibits the following: (1) BAX/Bak conformational activation, (2) release of cytochrome c and (3) caspase activation in response to intrinsic signals of death [19]. Overexpression of Bif-1 stimulates BAX and thus stimulates apoptosis. It has been hypothesized that Bif-1 may be a new type of BAX activator controlling apoptosis in the mitochondrial pathway [20]. Moreover, Bif-1 is also involved in autophagy and its complex with BECN1 in conjunction with UVRAG is required for the induction of autophagosome formation [19]. Coppola et al. [20] found decreased levels of both Bif-1 mRNA and protein in CRC tissues. These results are in agreement with our results. The Bif-1 gene is located on the short arm of chromosome 1 (locus: 1p22). This region is frequently deleted in many human cancers, including CRC [21‐24]. Therefore, it has been proposed that Bif-1 is a tumor suppressor gene. Loss of Bif-1 functioning may suppress apoptosis, as well as autophagy [20].

The fact that the Bif-1 gene had the highest level of expression among all the genes tested in our study may be explained by the fact that this protein is involved in two independent intracellular pathways connected with cell death, namely apoptosis and autophagy. We found decreased Bif-1 mRNA levels in CRC samples, and therefore, we hypothesize that this decrease may result in the suppression of autophagy. However, as we also observed increased BAX gene expression in CRC samples, it can be argued that upregulation of apoptosis in CRC cells may be a driving force which downregulates autophagy and Bif-1 downregulation leads to its inhibition.

One of the most important proteins engaged in the initiation of autophagy is BECN1 (beclin 1) encoded by the BECN1 gene located on the long arm of chromosome 17 (locus 17q21.31). It has been hypothesized that BECN1 acts as tumor suppressor gene, because of its frequent deletion in a variety of tumors such as breast, ovarian and prostate [25‐27]. We found its expression level to be average in both tumor and normal tissue, with the level of expression being lower in tumor samples than in healthy tissue. Interestingly, the results of other authors are conflicting, as some studies found an increased BECN1 protein level in CRC samples [28‐30], while some found a decreased level [31, 32]. Hence, its role in CRC pathogenesis remains unclear and needs to be elucidated by further analysis. We would like to emphasize that the most common methods used for the evaluation of BECN1 expression are immunohistochemistry (IHC) and Western blot. Both methods are semiquantitative, and it is difficult to found direct relationships between protein and mRNA levels, because of complex post-transcriptional and post-translational modifications [33].

Intriguingly, in our research we found that mRNA levels of BECN1 and UVRAG genes are positively correlated with age, as older people exhibited higher expressions of both of them in normal tissue. Higher levels of the expression of autophagy regulators responsible for the induction of autophagy in the normal tissue of older people may be explained by the age-related failure of lysosomal hydrolases and the ineffectiveness of autolysosomes (accumulation of autophagic vacuoles), which cause autophagic activity to decline [34]. Therefore, in older people’s cells the accumulation of redundant molecules may stimulate higher levels of expression of genes responsible for the induction of autophagy (positive feedback).

We also observed medium expression levels of the BCL2 gene and lower expression levels in CRC samples. The BCL2 gene negatively regulates two cell death pathways: apoptosis and autophagy [35]. As we found an elevated level of BAX and decreased level of BCL2, we suggest that in the early stages of CRC tumorigenesis, apoptosis is more prone to occur than autophagy. However, in metastatic samples a decrease in the expression level of BAX is surprisingly not accompanied by an increase in the expression of autophagy-related genes. This phenomenon should be studied more thoroughly.

We have found a complex correlation between two pathways connected with cell death. Autophagy, along with apoptosis, is responsible for normal cell development during morphogenesis and for maintaining intracellular homeostasis, as well as cell death in mature organisms [36]. The interaction between both pathways is critical for the cell life cycle. However, to date the studies published on this interaction have shown conflicting results. Some proteins, such as BECN1, UVRAG, ULK1, BCL2 and BAX, have revealed a dual role and may regulate both autophagy and apoptosis [37, 38]. In this research, we have found that the genes engaged in the induction of autophagy ULK1 and UVRAG have the lowest expression levels in both cancer and normal tissue. Medium to high expression of mRNA was found in BCL2, BECN1, ATG13 and BAX. The highest expression was found in Bif-1. Further functional analysis is needed to elucidate how these two pathways (autophagy and apoptosis) are interdependent.