Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status

Macroautophagy, herein referred to as autophagy, is a process that allows cells to deliver intracellular proteins, lipids and organelles to lysosomes where degradation can take place [1]. After autophagic degradation, the intracellular material is removed from the lysosomal compartment and recycling occurs in the cytoplasm [2]. The impact of autophagy on cellular physiology varies and depends upon the circumstances of the affected cell. The process of autophagy is also involved in tumorigenesis and can be both tumour promoting as well as tumour suppressing. Tumour-suppressive effects are achieved by degradation of oncogenic protein substrates, toxic proteins and defective organelles. However, autophagy-related intracellular recycling of substrates necessary for mitochondrial activity exerts a tumour-promoting effect in cancer cells. Autophagy can also function as a cancer cell survival pathway. Several studies have shown that autophagy is upregulated in RAS-driven cancers [3] and is especially induced in cancer areas with hypoxic conditions, where it supports tumour cell survival [4].

LC3, p62, Beclin-1 and uncoordinated (UNC) 51-like kinase 1 (ULK1) are central autophagy-related proteins involved in the autophagy flux. LC3 (microtubule-associated protein 1 light chain 3) is a well-established marker of autophagy activity in cancer cells [5]. LC3 is a mammalian homolog of the yeast ATG8 protein, a ubiquitin-like protein that becomes lipidated and tightly associated with autophagosomal membranes [6]. The hallmark of autophagosome formation is characterized by the insertion of LC3 II (isoform II of light chain) within the inner and outer layers of the vesicle. Measurement of LC3 expression by immunohistochemistry is a frequently used method to reliably quantify autophagosome formation [7].

p62 is an adaptor protein with several binding motifs and functions in assembling protein complexes [8]. p62 is involved in several important signalling pathways such as the NF-kB pathway [9]. In addition, p62 functions in the regulation of apoptosis via activation of polyubiquitinated caspase 8 [10] and influences the oxidative stress response, which is regulated by the Keap1-Nrf2 system. Under oxidative stress, p62 binds to Keap, which in turn blocks Nrf2 degradation, leading to increased expression of cytoprotective Nrf2 target genes.

Beclin-1 is an autophagy-specific protein that regulates autophagosome formation [11]. Until recently, the BECN1 gene that encodes the Beclin-1 protein was proposed to function as a tumour suppressor gene. However, current studies have revealed no evidence for BECN1 mutation or loss in cancers other than breast and ovarian cancers [12]. The allelic loss of BECN1 in breast and ovarian cancer seems to not be a driver mutation, but a passenger mutation along with BRCA1 mutation due to the proximity of BECN1 to BRCA1. Data from the Cancer Genome Atlas Research Network, accessible via the cBioPortal for Cancer Genomics, show that only 2.5 % of colorectal cancers (CRCs) show genetic alterations of the BECN1 gene [13, 14]. According to the cBIOPortal data, the p62 gene is altered in 1.5 % of CRC and the LC3 gene is altered in up to 14 %.

Beclin-1 interacts with several binding partners and can both induce and suppress the autophagy pathway. Interaction with PI3K can lead to upregulation of autophagy while interaction with Bcl-2 can result in inhibition of autophagy [15]. While Beclin-1 was thought to localize mainly in the cytosolic department, several studies demonstrated both nuclear and cytoplasmic localization of Beclin-1. Indeed, in a study of a cohort of CRCs, nearly half of the cancers exhibited a significant nuclear Beclin-1 staining pattern [16].

ULK1 is a serine/threonine kinase which is essential for autophagy and is involved in early autophagosome formation. Under nutrient deprivation, ULK1 is activated by the activated AMP-activated protein kinase (AMPK) and induces initiation of autophagy [17].

In vitro studies demonstrated that treatment of tumour cell lines with autophagy inhibitors such as chloroquine increases chemotherapy-induced cell death, thus establishing autophagy as a promising novel therapeutic target [18‐20]. Whether key autophagy proteins may predict chemoresistance in CRC is not known. Here we investigated the potential prognostic value of the autophagy-related proteins Beclin-1, p62, LC3 and ULK1 in a cohort of CRC specimens with a focus on patients with an unfavourable outcome due to UICC stage III/IV CRC treated with chemotherapy and/or KRAS-mutated CRCs.

Autophagy is a cellular pathway that regulates transportation of cytoplasmic macromolecules and organelles to lysosomes for degradation [22] and ensures protein and organelle homeostasis [1, 23]. Cancer cells have a high demand for energy equivalents and specific metabolites, and these resources can be provided by autophagy.

Interestingly, autophagy processes in cancer may show both tumour-promoting and tumour-suppressing effects depending on many factors, such as tissue type, tumour stage and the type of oncogenic mutations involved in the different tumour types [2, 22]. Thus, it is not surprising that immunohistochemical evaluation of autophagy-related markers has revealed different results according to prognosis. However, autophagy inhibitors may serve as a potential therapeutic intervention in some cancers, and thus autophagy may become a clinically relevant focus in cancer diagnosis and therapy.

In this study, we performed immunohistochemically analysis of the autophagy-related markers p62, LC3, Beclin-1 and ULK1 in a cohort of CRCs. We acknowledge that measurement of the autophagy flux is challenging, since autophagy flux is a dynamic, multiple-stage process, including autophagosome formation, maturation and fusion with lysosomes as well as breakdown and release of macromolecules back to the cytosol. Thus, the accumulation of autophagosomes as roughly measured by immunohistochemistry could indicate either autophagic activation or a blockage of downstream actions, such as inefficient fusion or decreased lysosomal degradation [1]. However, immunohistochemical analysis of autophagy-related markers such as LC3 offers the opportunity to generate a temporary picture of the amount of autophagosome formation in tumour tissue at a specific point of time in a certain clinical setting [7].

Autophagy-related markers have been a subject of recent immunohistochemical studies in gastrointestinal adenocarcinomas (cholangiocarcinoma, pancreatic adenocarcinoma, oesophageal adenocarcinoma, gastric adenocarcinoma). Table 4 summarizes previously published study results and gives an overview about the expression of autophagy markers in cancer tissue and their potential clinical relevance in gastrointestinal carcinomas other than CRC.

The prognostic relevance of LC3 expression in CRC has been addressed in only a few studies so far. One study demonstrated a prognostic impact of LC3 expression in CRC, while another failed to demonstrate prognostic relevance of LC3 expression [24, 25]. Our study showed a statistical trend demonstrating reduced OS in CRC patients with LC3-positive CRC.

Only few studies on p62 and colorectal cancer have been published thus far. In a recent study on 178 colon carcinomas treated with adjuvant 5-FU, Beclin-1 overexpression (but not p62) was significantly associated with worse OS [25]. Another study was performed on p62 expression in various gastrointestinal cancers; however, this study included only 45 colon cancers and thus the results may not be reliable [26]. Immunohistochemical analysis of p62 expression has been examined in various other human malignancies with differing results. High cytoplasmic p62 expression and LC3 overexpression were associated with an unfavourable prognosis in oral squamous cell carcinoma [27, 28]. High cytoplasmic p62 expression was associated with decreased OS in triple negative breast cancer [29] and lung adenocarcinoma [30]. In these studies, increased cytoplasmic p62 expression was accompanied by an unfavourable course of cancer disease. However, in the present study, cytoplasmic negativity was significantly associated with a reduced cancer-specific OS, but did not qualify as an independent prognostic marker in multivariate survival analysis. At first glance, our results seem surprising, since the previously published studies revealed increased and not decreased p62 expression as a negative prognostic marker. On the other hand, there are also diverging reports about the potential prognostic relevance of p62 in human cancer [27]. p62 is a protein, which is embedded in multiple pathways and can exert manifold functions. It is well known that p62 may act as an adaptor protein that transports ubiquitinated protein aggregates to autophagosomes via its association with LC3B. In addition, p62 influences the oxidative stress response, which is regulated by the Keap1-Nrf2 system. However, given the fact that negative (and not increased) p62 cytoplasmic expression is associated with worse OS in our study, there has to be another explanation for this rather surprising finding. Indeed, at least two p62-related mechanisms may explain the aggressive course of p62-negative colorectal cancer:

1) p62 may also act as a tumour suppressor since it is capable of inducing autophagic degradation of regulators of the Wnt signalling pathway [31]. It is tempting to speculate that deficiency of p62 might upregulate this oncogenic pathway resulting in increased biological aggressiveness.

2) Recent studies demonstrated that the lack of cdk1-mediated phosphorylation of p62 leads to enhanced cell proliferation and tumorigenesis in response to RAS-induced transformation [32]. A decrease in p62 protein thus may lead to a faster exit from mitosis, which translates into enhanced cell proliferation. These mechanisms might provide a rationale explanation for the worse prognosis of p62-negative CRC. In addition, this finding might also explain the significant prognostic impact in the KRAS-mutated subgroup due to increased RAS-signalling.

In fact, results of a very recent study on 116 oesophageal adenocarcinomas are in line with our results. The immunohistochemical study of Adams et al. established missing/low (and not increased) p62 immunostaining as a predictor of worse prognosis in this cohort [33].

To complicate the evaluation of autophagy-related proteins, both cytoplasmic and nuclear staining has been described in previous studies. Although autophagy-related markers are thought to exert their effects mainly in the cytoplasm, these markers may be localized both in the cytoplasmic compartment as well as in the nuclear compartment [34]. Indeed, in a previous study on CRCs, nearly half of the cancers exhibited a significant nuclear Beclin-1 staining pattern [16].

Autophagy can exert both tumour-suppressive and tumour-promoting effects in human malignancies [22]. Recent studies showed that activation of oncogenic RAS, which is capable of inducing tumour growth, is sufficient to upregulate basal autophagy [35]. RAS mutation is a frequent and early tumour-promoting event in CRC. We analysed p62 expression in KRAS wildtype and KRAS-mutated-type CRC in this study and investigated the prognostic power of p62 expression. p62 expression was not correlated to RAS mutational status, but the prognostic power of p62 was lost in the wildtype KRAS subgroup, whereas it was observed as a trend (p = 0.062) in the KRAS-mutated subgroup. However, these results must be interpreted with caution. Cytoplasmic and nuclear Beclin-1 expression failed to show prognostic power in the complete cohort and especially in the wildtype KRAS CRC subgroup. However, in the mutated KRAS CRC subgroup, increased nuclear Beclin-1 expression was significantly associated with decreased OS.

The altered prognostic effect of autophagy-related markers in KRAS-mutated CRC represents a novel finding of our study. Interestingly, similar to Beclin-1, LC3-positive CRC with mutated KRAS demonstrated a significantly reduced OS but not in the KRAS wildtype CRC group, strengthening this finding. Thus there seems to be an alteration of the autophagy flux in the setting of KRAS mutation. A larger sample size of this subgroup with RAS-mutated cancers should be examined to confirm these results.

A previous study on CRCs reported the presence of nuclear Beclin-1 staining; however, the nuclear staining pattern lacked any prognostic relevance [16]. Nevertheless, the results of the present study need to be reproduced in larger cohorts. It is particularly important to conduct additional studies of autophagy-related markers with a focus on the cellular localizations of the proteins. Taking into account the results of previous studies on the prognostic relevance of p62 expression in CRC, why both underexpression and overexpression of this autophagy-related protein were demonstrated to influence survival in CRC as well as in other malignancies remains unclear. This question cannot be answered by the present study, since this study remains descriptive and lacks functional investigations.

ULK1 expression has been addressed in only one study on CRC so far [36]. Zou et al. were able to demonstrate that increased expression levels of ULK1 predicted poor prognosis in CRC. However, in our study with a smaller number of patients, we were not able to confirm these results. Nevertheless, in our study, increased ULK1 expression was significantly associated with the presence of lymph node metastasis. It has to be critically emphasized that in the study by Zhou et al., level of significance was rather low.

Analysis of autophagy-related proteins in human malignancies represents a valuable area of research due to the potential future therapeutic relevance.

Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemotherapy and radiation therapy [37‐40].

Clinical studies with autophagy inhibition are already being conducted [41] with most of the studies using hydroxychloroquine as an autophagy inhibitor. However, autophagy inhibition in human malignancies remains a topic of clinical studies and is not yet part of established therapeutic approaches. Our data show that p62, LC3 and Beclin-1 represent promising novel therapeutic targets especially in the subgroup of KRAS-mutated colorectal cancer patients.

Our study showed that decreased cytoplasmic p62 expression was significantly associated with an unfavourable tumour-specific OS, especially in the subgroup of KRAS-mutated CRCs. Nuclear Beclin-1 and dot-like LC3 expression were also associated with decreased OS solely in the mutated KRAS CRC subgroup. ULK1 expression completely lacked prognostic relevance. Thus p62, Beclin-1 and LC3 represent promising markers of novel targeted therapies, especially in the clinically relevant group of advanced CRCs treated by chemotherapy and prognostic unfavourable KRAS-mutated CRCs.

AMPK, AMP-activated protein kinase; BRCA1, breast cancer-associated antigen; CRC, colorectal carcinoma; H&E, haematoxylin and eosin; LC3, light chain 3; KRAS, Kirsten RAS; PI3K, phosphoinositide-3 kinase; UICC, Union internationale contre le cancer; ULK1, uncoordinated (UNC) 51-like kinase 1; 5-FU, 5-fluorouracilk