Alterations in PGC1α expression levels are involved in colorectal cancer risk: a qualitative systematic review

Colorectal cancer (CRC) is a major global public health problem and the second leading cause of cancer-related death. It is the third most commonly diagnosed cancer in men and the second in women [1]. CRC is the seventh and fourth most common cause of death and loss of life expectancy in Western Europe, respectively, and is associated with an elevated consumption of resources [2, 3].

Mitochondrial dysfunction has long been suspected to be involved in this type of tumorigenesis, as supported by an accumulating body of research evidence. However, little is known about how mitochondrial alterations contribute to tumorigenesis [4‐7]. Mitochondrial biogenesis is a fundamental cellular process required to maintain functional mitochondria and as an adaptive mechanism in response to changing energy requirements [8]. Both endogenous and exogenous factors, as well as numerous signaling pathways and gene expression patterns, converge upon the mitochondrial biogenesis process to coordinate the energy needs of cells, tissues and the entire organism [4, 8, 9].

The master regulator of mitochondrial biogenesis is peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A or PGC1α), because it controls production of mitochondrial proteins [10]. This gene is a transcriptional coactivator of the PGC-1 (peroxisome proliferator-activated receptor gamma coactivator 1) gene family, which has three known members, PGC1α, PGC1β and PRC (PGC-1 related coactivator). PGC1α and PGC1β are expressed in tissues with high energy demand, while PRC is expressed ubiquitously [10, 11]. While all three members of this family are potent regulators of mitochondrial function and biogenesis, PGC1α is the most widely studied, and the other two are less well characterized [8, 11‐13]. However, [11, 14].

PGC1α acts as a master regulator of energy metabolism and mitochondrial biogenesis by integrating and coordinating the activity of other transcription factors, such as Nuclear respiratory factor 1, Nuclear factor 2, PPARα (peroxisome proliferator-activated receptors α) and Mitochondrial transcription factor A [15]. Various endogenous and exogenous factors also regulate mitochondrial biogenesis through this gene [4, 9]. In addition, expression levels of PGC1α appear to be directly related to mitochondrial biogenesis activity. As a multi-response factor, many agents and events regulate PGC1α expression via multiple intracellular mediators [16].

Several mutations in nuclear and mitochondrial genes encoding for mitochondrial components have been reported to be associated with increased cancer risk [17], and mitochondrial loss is known to precede the development of dysplasia [6]. We believe that there is a clear relationship between CRC and PGC1α; however, the role of mitochondria and PGC1α in CRC is poorly understood at present.

Several studies suggest that PGC1α and related genes can regulate different pathways, such as mitochondrial biogenesis, antioxidant systems, reactive oxygen species, de novo lipid synthesis, and glycolysis, there by playing a role in risk for and development of CRC (See discussion) [10, 11]. Although there is no widely accepted mechanism to explain how PGC1α is involved in human CRC, it is essential to understand this mechanism in order to reduce CRC risk, as well as the development of novel therapeutic tools to treat tumors and to support measures to reduce CRC risk.

In this paper, we report a systematic review to summarize current evidence on the role of PGC1α in the initiation and progression of CRC. Since there is a limited evidence on this specific question, we used a broad, inclusive search strategy, with the aim of providing a basis for more comprehensive investigation.

The central question of this review is clear: summarize current evidence on the role of PGC1α in the initiation and progression of CRC. We tested the effect of different PGC1α expression levels during the development of colorectal cancer. There is little prior evidence on this question, so only a small number of studies have been included in this review.

The studies included in this review aimed to evaluate whether an increase or decrease in PGC1α expression levels affects the development of CRC. Most studies (61.5%) were carried out using cell lines [10, 11, 20, 21, 23‐25]: in four studies, cells were injected subcutaneously into the flanks of mice [20, 21, 23, 24], and two studies evaluated expression levels in mice in vivo [14, 21]. Other studies were carried out in samples of patients with disease [6, 13, 26, 27], with similar results to the in vitro studies.

Each article proposes a possible molecular mechanism of action. First, we try to understand the normal mechanism of PGC1α action proposed by Ilenia D’Errico et al. [20], which is derived from work on cell cultures and two groups of transgenic mice: iPGC1α transgenic mice and iPGC1α Apc^Min/+ mice (more information in Table 2). The authors conclude that PGC1α is a metabolic regulator of intestinal cell fate. In addition, PGC1α seems to have different functions in tissues with high aerobic energy metabolism, such as at the bottom of the crypts, where PGC1α expression increases production of antioxidant enzymes that protect cells from reactive oxygen species. In contrast, at the top of the villi, where aerobic energy metabolism is low, PGC1α expression increases mitochondrial biogenesis, resulting in greater accumulation of reactive oxygen species than at the bottom of the crypts, which induces apoptosis [20]. In this way, there is a balance between cell proliferation and apoptosis under normal conditions.

Other mechanisms have been proposed on the basis of various studies of PGC1α overexpression under non-normal conditions, mainly in cell culture. One mechanism supports the idea that overexpression of PGC1α results in high levels of Sp1 (specificity protein 1), which is then thought to enhance expression of ACBP (Acyl-CoA-binding protein) via ACBP’s Sp1 binding site. ACBP upregulation increases cell proliferation and decreases sensitivity to H2O2-induced apoptosis [10, 14]. A second mechanism proposes that PGC1α is a key metabolic regulator of several aspects of glucose metabolism. PGC1α is thought to coordinate gene expression in metabolic pathways that convert glucose to fatty acids. In turn, fatty acid synthesis promotes tumor growth [21] and disrupts the balance between apoptosis and cell proliferation, promoting the development of colorectal cancer.

A third possible mechanism is based on overexpression of both PGC1α and PGC1β, which increases the electron transfer activity of the mitochondrial respiration chain and augments mitochondrial biogenesis [11, 14]. Both processes partly disrupt ΔΨm and cytosolic calcium [Ca2+] buffering ability, with two consequences: 1) Dysfunctional oxidative phosphorylation in mitochondria, and 2) stimulation of the Ca2+ signaling cascade, which in turn may activate genes involved in tumor invasion and metastasis. Both of these effects can upregulate PGC1α, generating a positive feedback loop [11]. In addition, the increase in mitochondrial biogenesis seems to give the cells the necessary energy for increased longevity and cellular division, promoting tumor growth [11, 14].

From these three molecular mechanism we can conclude that PGC1α and PGC1β allow cells to balance i) mitochondrial activity and cytotoxic protection in the production of reactive oxygen species, and ii) apoptosis and cell proliferation [10, 11, 14, 21]. In addition, we can observe that all studies in cell lines [10, 11, 21] and mice [14] support the idea that high expression of PGC1α carries increased CRC risk. Note that while none of these articles work with the same cell line, the cells used are similar since the majority of cell lines are epithelial (Table 2). Shin et al. [10] use human colon cancer cells (HT29 and SNU-C4) and mouse colon cancer cells (CT-26). Srivastava et al. [11] use VACO425 and VACO429, another type of human colon cancer cell. Bhalla et al. [21] use other human colon adenocarcinoma cells, Colo205, as well as HT29, like Shin et al. Despite this, all of these studies arrived at the same conclusions.

However, Ilenia D’Errico [24] show that Bax is necessary for the pro-apoptotic effect of PGC1α in colorectal cancer cells, which could be another molecular mechanism of action, although this is not consistent with other studies. In this study [24], PGC1α overexpression seems to induce Bax translocation to mitochondria, and Bax-protein mediates the pro-apoptotic effect of PGC1α [24]. In this case, the cell line used is HCT116 (human colon carcinoma, also epithelial cells) and they also use mice who were subcutaneously injected with these cells. Here there is some controversy: in absence of Bax, PGC1α overexpression was not able to oppose tumor growth.

In addition to this research on overexpression of PGC1a, other studies have focused on low expression, inhibition or silencing of PGC1α and PGC1β. Using the same culture cells as Ilenia D’Errico [24], as well as other cells and in inmunodeficient mice, Fisher et al. [23] showed that PGC1β is aberrantly expressed in human colon cell lines and tumors, and maintains ERRα levels, contributing to its tumorigenic properties. These authors also found that KSR1 (Kinase suppressor of Ras 1) and AMPK (AMP-activated protein kinase), which act upstream of the PGC1β promotor, were linked to the action of transcriptional regulators PGC1β/ERRα (estrogen-related receptor α). Reduced KSR1 and AMPK expression also downregulated expression of PGC1β and ERRα, which inhibits the survival of colorectal cancer cells [23].

Based on all of this evidence, it seems that PGC1α/PGC1β is overexpressed in human colon cell lines during tumor development, and this overexpression seems to be required for both cell proliferation and survival, and to reduce apoptosis [10, 11, 14, 21, 23]. Only one study obtained markedly different results despite using the same cell line as one of the above (HCT116), although this was based on Bax instead of PGC1α [24]. Note that while all of these studies were performed using different cell lines with genetic changes, most of these are human epithelial cells (Table 2). For this reason, different results could not be attributed to the use of different cell lines. We consider that we cannot explain the origin of this differences.

Our systematic search returned two studies that analyzed the effects of chemotherapy on PGC1α expression in cultured cells and colonosphere cultures (Table 2) [25, 26]. These studies suggest three other molecular mechanism of action of PGC1α. 1) Chemotherapy of colorectal tumor cells induces a SIRT1/PGC1a-dependent increase in oxidative phosphorylation that promotes tumor survival during treatment because chemotherapy induces a shift in tumor energy metabolism that protects tumor cells from cytotoxic damage [26]. Mechanistically, chemotherapy-induced DNA damage results in increased expression of SIRT1, which deacetylates and thereby activates PGC1α as a transcriptional coactivator. PGC1a acts in concert with several transcription factors to stimulate the expression of genes involved in mitochondrial biogenesis and respiration, resulting in increased oxidative phosphorylation and helping them survive treatment. In this case, the authors worked with human colonosphere cultures [26]. 2) PGC1α-induced activation of Uridine phosphorylase (UPase) expression, which is mediated by an estrogen related receptor (ERR) binding site. Overexpression of PGC1α via this mechanism sensitizes colon cancer cells to growth inhibition by 5-deoxy-5-fluorouridine, presumably by inducing apoptosis in tumor cells [25]. In this way, 3) ERRα is primarily thought to regulate energy homeostasis by interacting with PGC1α or PGC1β. ERRα overexpression reduces sensitivity to chemotherapy, while inhibition of these genes reduces reactive oxygen species and ΔΨm, which increases sensitivity to chemotherapy [22, 23]. Here, Fisher et al. worked with HCT116 and other cell lines [23], but studied the effect of the chemotherapy on HepG2 [22].

These mechanisms support the conclusion mentioned above, that PGC1α and PGC1β allow cells to balance i) mitochondrial activity and cytotoxic protection in the production of reactive oxygen species, and ii) apoptosis and cell proliferation. In addition, they suggest that any therapy that reduces PGC1α expression will increase cancer cells’ sensitivity to chemotherapy. Thus, more and more authors support the idea of using PGC1α as a potential target in cancer therapy [21‐23, 25, 26]. In fact, Do et al. [28] reported a strategy to reduce PGC1α levels to improve cancer therapy, though not in colon cancer cells. They showed for the first time that metformin, an insulin-lowering agent [29], induces miR-34a which reduces Sirt1 in wild-type p53 cancer cells, but does not occur in altered p53 cell lines. This fact was shown in HCT116 and MCF-7 cell lines among others. However, they only could show in an MCF-7 cell line (Breast cancer cells) that the reduction of Sirt1 involved reduced PGC1α and, subsequently, reduced NRF2 and enhanced susceptibility to oxidative stress [28]. DeCensi et al. 2010 [29] showed using a meta-analysis that metformin is associated with decreased risk of cancer, including colon cancer, in diabetic patients compared with other treatments [29].

Our systematic search returned three articles based on human samples. First, Ussakli et al. [6] studied 9 non-dysplastic colon biopsies from Ulcerative Colitis (UC) patients with high-grade dysplasia or cancer and 9 dysplasia-free UC patients, and concluded that while the development of dysplasia is preceded by mitochondrial loss, mitochondria are restored in cancer cells, which suggests that they are needed for further proliferation. This bimodal pattern may be driven by transcriptional regulation of mitochondrial biogenesis by PGC1α [6], which is consistent with the results of the cell culture studies described above [11, 14].

Two other papers propose the same idea, that overexpression of PGC1α results in higher CRC risk, although these studies are not comparable with the one described above [6]. First, Gaustadnes et al. [27] screened a selection of SNPs in pooled DNA, and found that the rs96516 SNP in the PPARGC-1A (*604517) 3’UTR was significantly associated with sporadic CRC risk, although this result was not reproducible in the same study [27]. Second, Feilchenfeldt et al. [13] studied expression levels of all isoforms of PPAR-ϒ and transcriptional partners such as PGC1α in patients with different stages of colon cancer, and found that expression levels of PPAR-ϒ vary between isoforms and cancer stages, while those of PGC1 were reduced in all cancer samples, with respect to normal samples [13].

Summarizing, while D’Errico et al., 2011 [20] showed that PGC1α^−/− mice are susceptible to intestinal tumorigenesis, several papers addressing the role of PGC-1 in tumor cell lines showed a role of PGC-1 in tumor progression. Thus, the function of PGC1α in colorectal cancer risk is not entirely clear, although it seems likely that it has a role in this disease.

Colorectal cancer is a major global public health problem and the second leading cause of cancer-related death. Our systematic review indicates that altered expression of PGC1α modifies CRC risk. Most studies showed that overexpression of this gene increases CRC risk [10, 11, 14, 21, 23, 25, 26], while some studies indicated that lower than normal expression levels could increase CRC risk [6, 13]. Thus, various authors suggest that PGC1α is a good candidate as a molecular target for cancer therapy. Reducing expression of this gene could help to reduce risk or progression of CRC [22, 25, 26], at least in different cell lines and transgenic or nude mice. However, in our opinion, there are not enough data on the role of PGC1α using human tumor samples to conclude a role of this gene in CRC. This question should be investigated further.