The methylation-expression correlation of autophagy-related genes in colorectal cancer patients from southern Iran

Abstract

Colorectal cancer (CRC), which has high mortality and increasing morbidity is a major concern worldwide. The autophagy pathway plays a crucial role in carcinogenesis and drug resistance in this disease. Epigenetic modification is one of the main regulatory mechanisms for this pathway. This study aimed to investigate the impact of promoter methylation as one of the epigenetic modifications on the expression of autophagy-associated genes (ATGs) (ATG2B, ATG4D, ATG9A, and ATG9B) in 21 CRC patients from southern Iran. The tissue DNA and RNA were extracted by standard phenol-chloroform extraction method and A BIOZOL RNA isolation kit, respectively. The methylation status and transcript levels of desired genes were ascertained using the methylation-specific PCR and quantitative real-time PCR methods, respectively. In the majority of studied patients, the relative mRNA expressions of ATGs were significantly higher in CRC tissues compared to normal ones. There was no significant relationship between the methylation of the ATG genes and clinicopathological features of CRC patients. Interestingly, in most of the patients, the promoter hypermethylation of the ATG2B, ATG4D, ATG9A and ATG9B genes led to their high mRNA expression. Although promoter hypermethylation usually suppresses gene expression, the cancer type, stage, and compensatory mechanisms may reverse this association. This highlights the complexity of the epigenetic regulation of ATG2B, ATG4D, ATG9A and ATG9B genes in CRC. Further large-scale studies will contribute to discovering the exact influences of ATG methylation in CRC carcinogenesis and thereby may thereby provide novel targets and biomarkers for this lethal illness.

INTRODUCTION

As a usual digestive tract tumor, colorectal cancer (CRC) is the third main cause of cancer-associated deaths in the world [1]. Over the past decades, great advances have been made in the prevention, diagnosis, and treatment of cancer, such as personalized medicine, however, the survival rate of CRC patients is still insufficient [2]. Recently, the global burden of CRC will enhance by 60%, to over 1.1 million deaths and 2.2 million new cases by 2030 [3]. An understanding of pathogenesis, and study for new prognostic cancer biomarkers, may affect advancement in cancer prognoses and may also cause the identification of novel promising targets for anticancer therapies. A large body of evidence has revealed autophagy as a potential new biomarker in CRC development [4].

As a multistep catabolic pathway, autophagy is a critical process in cancer initiation and development [5]. Autophagy plays paradoxical and complicated roles in cancer via interactions with cell survival, cell metabolic reactions, and the turnover of proteins and organelles at multiple levels [6]. With both promoting and inhibitory effects on cancer cells, autophagy can prevent tumor formation in early stages while it can lead to tumor cell survival and malignant transformation in response to various stressful triggers in later stages of cancer [7, 8]. It has been proposed that multiple signaling cascades, such as epigenetic regulation, have crucial roles in autophagy deregulation. However, the effect of epigenetic regulation on autophagy is largely debatable [9].

CRC is a multifactorial disease that arises due to the cumulative accumulation of genetics as well as epigenetic alterations [10]. Epigenetic modifications, which change the expression of important genes related to physiological and pathological processes without influencing the DNA sequence, have been reported to play a critical role in the initiation and progression of various cancers, such as gastrointestinal cancers. Autophagy may be regulated by various epigenetic processes, including DNA methylation, microRNA-associated modulation, and histone modifications [9, 11, 12]. Current studies have associated autophagic flux with epigenetic regulation [13, 14]. The clinical and biological importance of epigenetic regulation of autophagy in cancer has increasingly received vast attention among researchers worldwide. Deviant epigenetic changes of autophagic regulators are considered key determinants of the cancer fate and are evident in multiple ways: promotion or prohibition of autophagy that can be protective or lethal, thereby contributing to tumor suppression, progression, metastasis, or development of chemo- or radio-resistance in established tumors [11].

As a highly conserved pathway, autophagy is severely orchestrated by key autophagy proteins, which are encoded by more than thirty autophagy-related genes (ATG) [4]. As the main components of the autophagy-mediated regulatory network, ATGs contribute to CRC occurrence and development [15]. ATGs regulate the autophagy pathway and are frequently controlled by epigenetic modifications, including DNA methylation, histone modification, and microRNA-mediated gene regulation [16]. Altered expression (either over- or under-expression) of autophagy genes may be considered a crucial factor in the initiation and progression of numerous cancers [17]. In humans, ATG genes are regulated at both transcriptional and post-translational levels, as well as by epigenetic modifications [18]. Based on previous studies, compared with other mentioned epigenetic mechanisms, DNA hypermethylation has a more distinguished role in the regulation of autophagy [19]. Epigenetic silencing of some key genes by DNA methylation is involved in the autophagy regulation in all stages of cancer [9].

In melanoma, ATG5 down-regulation by its promoter hypermethylation is correlated with cell proliferation and tumorigenesis [20]. ATG16L2 is frequently silenced by DNA methylation, and its inactivation is associated with poorer outcomes than imatinib treatment in hematological cancers [9]. In breast cancer, abnormal methylation of BECLIN-1, an autophagy-related gene that acts as a tumor suppressor, can be considered as a mechanism of autophagy prevention and induction of tumor [21-23]. Similarly, promoter hypermethylation of ATG4D, ATG2B, ATG9A, and ATG9B genes has been demonstrated in most invasive ductal carcinomas (IDC), and this enhanced methylation was associated with the cancer grade, reduced gene expression, and lymph node metastasis [24].

The activation and biological effects of cellular autophagy are controlled by various autophagy-related genes (ATG) and signaling pathways [25]. Among these pathways, epigenetic modifications greatly contribute to the process of cellular autophagy [11]. The epigenetic control of autophagy is very complicated and has a dual role in CRC. Therefore, it is still controversial whether autophagy activation or inhibition promotes cancer development [7]. Regarding the lack of knowledge on the effect of epigenetic modification of autophagy in CRC, the present study aimed to investigate the impacts of epigenetic alterations of the genes related to autophagy (ATG2B, ATG4D, ATG9A, and ATG9B) in CRC patients. It will contribute to detecting potential mechanisms that regulate autophagy in disease searching for novel targets for more efficient diagnosis and treatment of CRC.

MATERIALS AND METHODS

Patients and tumor samples:

The surgically resected tumor and normal adjacent tissues (the tissue surrounding the tumor that appears histologically normal but may have molecular alterations) were collected from 21 colorectal cancer patients at one university-related hospital in southern Iran, Shiraz, from 2021 to 2022. Informed consent was received from each patient or the patient’s supervisor. The institutional ethics committee (Ethical Approval ID: IR.SUMS. REC.1402.205) ethically confirmed this study. At once after surgical resection, the cancerous and normal adjacent tissues were snap-frozen and stored at -80ºC. Then, the histological diagnosis was done by an expert pathologist who ascertained the appropriate tissue sections for further extraction of DNA and RNA and molecular analyses. The clinicopathological features of patients were assessed from hospital records. Inclusion criteria were patients diagnosed with colorectal cancer, individuals aged 18 years or older at the time of diagnosis, patients with a pathologically confirmed adenocarcinoma of the colon or rectum, and those able to provide written informed consent for participation in studies. Exclusion criteria were patients diagnosed with other cancers, individuals with a strong family history of colorectal cancer or other relevant cancers, individuals younger than 18 at the time of diagnosis, patients without a pathologically confirmed adenocarcinoma of the colon or rectum, and those who are unable to provide written informed consent for participation in studies.

DNA extraction:

The canonical proteinase K digestion and phenol-chloroform technique were applied for the extraction of genomic DNA from cancerous and normal adjacent samples [26].

Methylation-specific PCR (MSP) assay of the methylation of gene promoter:

The MSP method was performed for the determination of promoter methylation status of 4 ATGs (ATG9A, ATG9B, ATG4D, and ATG2B) in normal and tumor tissues [27]. Briefly, genomic DNA (1 µg) was exposed to sodium bisulfite, and then methylated and unmethylated specific primers were utilized for PCR amplification (Table S1). Finally, the obtained products were observed utilizing electrophoresis on 2% agarose gel and UV illumination.

Gene expression analysis using quantitative Real-Time PCR (qRT-PCR):

A BIOZOL RNA isolation kit (BiofluxBioer, China) was used for total RNA extraction from cancerous and normal adjacent tissues, based on the instructions of the manufacturer. To validate the RNA integrity, 1.5% denaturing agarose gel electrophoresis with 2% formaldehyde was used.

As described previously, the relative expression levels of four ATGs (ATG9A, ATG9B, ATG4D, and ATG2B) in cancerous and normal adjacent tissues were analyzed by real-time RT-PCR [28]. In brief, the complementary DNA (cDNA) was synthesized based on the manufac-turer’s instructions (Cinagene, Iran). For each studied gene, real-time PCR amplification was performed on 1 µl cDNA in 25 µl reaction mixture using SYBR Green master mix (Ampliqon, Danmark) and gene-specific primer pairs (Table S2) in a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, USA). The gene amplification was done in triplicate with precycling heat activation at 95°C for 10 min, accompanied by 40 cycles (95°C/15 s, 58°C/30 s, 72°C/30 s, and a final extension at 72°C/10 min). To normalize the levels of gene expression, the 2^-ΔΔCT formula was used, and he β-actin was also applied as a housekeeping gene.

Statistical analyses:

The SPSS version 18 (SPSS Inc., Chicago, IL) was applied for the statistical evaluation. The results are represented as mean±standard deviation (SD). The difference between the two groups was analyzed by an unpaired Student’s t-test. Descriptive frequencies and median statistics were applied to evaluate the association between promoter hypermethylation status and mRNA transcript levels for four studied genes. The p-value of < 0.05 was considered statistically significant.

RESULTS

The clinicopathological characteristics of the patients are indicated in Table 1. Twenty-one (21) patients were included in this study. Most patients were male (66.7%) and older than 60 years (57.1%). Most patients were diagnosed with distal CRC (85.7%, 18 cases) in stages II and III (81%, 17 cases). Most tumors were also well and moderately differentiated (90.5%, 19 cases). As described in the materials and methods section, hypermethylation of CpG islands in tumors was evaluated by the MSP method. The related results are shown in Figure 1.

Table 1.

Distributions of choosed features of the participants

Variables		n (%)
Age	< 60 years ≥ 60 years	9 (42.9) 12 (57.1)
Sex	Male Female	14 (66.7) 7 (33.3)
Stage	I II III	4 (19) 9 (42.9) 8 (38.1)
Site	Distal Proximal	18 (85.7) 3 (14.3)
Differentiation	Well Moderate Poor	13 (61.9) 6 (28.6) 2 (9.5)

Figure 1.

The results of MSP for promoter methylation of ATG9A, ATG9B, ATG4D, and ATG2B genes in colorectal cancer tumors. M: methylated genes; U: unmethylated genes; T: tumor samples, N: Normal adjacent tissues. Lane M demonstrates the 50 bp DNA marker. ATG2B: M=146 bp, U=144 bp; ATG4D: M=157 bp, U=154 bp; ATG9A: M=108 bp, U=108 bp; ATG9B: M=167 bp, U=169 bp.

The most common methylated locus was ATG9B (95.2%; 20 of 21), followed by ATG4D (76.2%; 16 of 21), ATG2B (57.1%; 12 of 21), and ATG9A (47.6%; 10 of 21). Simultaneous promoter hypermethylation of all evaluated genes was detected in six (28.6%) patients. Normal samples were unmethylated for all genes. There was no significant relationship between the ATG genes methylation and clinicopathological characteristics of CRC patients (Table 2).

Table 2.

Gene promoter methylation association with clinicopathological features of colorectal cancerpatients

Variables	ATG2B, n				ATG4D, n				ATG9A, n				ATG9B, n
	M	U	p		M	U	p		M	U	p		M	U	p
Total	12	9	value		16	5	value		10	11	value		20	1	value
Age <60 (9) ≥60 (12)	6 6	3 6	0.66		7 9	2 3	1		3 7	6 5	0.387		8 12	1 0	0.429
Sex Male (14) Female (7)	7 5	7 2	0.642		10 6	4 1	0.624		6 4	8 3	0.659		14 6	0 1	0.074
Site Proximal (3) Distal (18)	0 12	3 6	0.063		2 14	1 4	1		0 10	3 8	0.214		3 17	0 1	1
Tumor Stage I (4) II (9) III (8)	1 6 5	3 3 3	0.448		4 6 6	0 3 2	0.670		1 3 6	3 6 2	0.190		4 8 8	0 1 0	1
Differentiation Well (13) Moderate (6) Poor (2)	7 3 2	6 3 0	0.650		10 4 2	3 2 0	1		5 4 1	8 2 1	0.659		12 6 2	1 0 0	1

As shown in Figure 2, the expression levels of the ATG9A, ATG9B, ATG4D, and ATG2B genes were analyzed using qRT-PCR. The relative mRNA expressions of ATG9A, ATG9B, ATG2B, and ATG4D were significantly greater in CRC tissues compared to normal ones. The upregulation of ATG2B and ATG4D was just not significant (p>0.05) in one evaluated patient (P21 for ATG4B and P20 for ATG4D). Another exception was observed in P14, which showed a decreased transcript level of ATG4D in tumors compared to normal tissues (p<0.01).

Figure 2.

The qRT-PCR analysis of relative expressions of ATG2B (A), ATG4D (B), ATG9A (C), and ATG9B (D) in the colorectal cancer tissues (n=10), comparing to the normal counterparts. The transcript levels were normalized using β-actin. The data are indicated as means ± SD of two independent experiments performed in triplicate. (*p<0.05, **p<0.01, and ***p<0.001).

To determine the promoter methylation of ATG2B, ATG4D, ATG9A, and ATG9B genes and its impact on their gene expression activity, the transcript level fold changes were compared in methylated and unmethylated CRCs from 10 patients. Our results demonstrated that only in 14.3, 9.5, 19, and 4.7% of cases the promoter hypermethylation of ATG2B, ATG4D, ATG9A, and ATG9B accompanied by decreased transcript levels of these genes, respectively. However, in other patients, the promoter hypermethylation of studied genes was followed by increased gene expression.

DISCUSSION

This study shed light on the association between DNA promoter methylation and expression of four ATG genes (ATG9A, ATG9B, ATG4D, and ATG2B) in CRC patients. Our findings challenge the available understanding of promoter hypermethylation as a mechanism of gene silencing, accentuating the complexity of epigenetic regulation in CRC.

ATG genes play key roles in CRC pathogenesis and therapeutic resistance. ATG2 homologs, ATG2A and ATG2B, are peripheral membrane proteins that contribute to cellular nucleation and the early steps of autophagosome generation. Considerably, the simultaneous silencing of ATG2A and ATG2B leads to autophagy dysfunction and aggregation of autophagic constructs comprising the most of ATG proteins [24, 29].

ATG4 encodes a member of the ATG4 mammalian family (a group of four cysteine proteases, ATG4A‒D) with endopeptidase function, important for later steps of maturation of autophagosome and its fusion with lysosomes [30]. ATG4D not only exerts an evident role in autophagy regulation but also acts in the interaction between autophagy and apoptosis [31, 32]. Abnormal DNA methylation of ATG4D profoundly contributes to various human diseases, including cancers [33, 34]. ATG9 family, with two functional members, ATG9A and ATG9B, is also a multi-transmembrane protein that acts as a membrane transporter in the initial stage of the autophagy flux [35, 36]. ATG9 actively interacts with phagophores but does not convert a stable component of the autophagosome membrane [37]. Loss of ATG9A and ATG9B genes or disruption of the autophagy pathway is associated with a vast variety of cancers [38]. While promoter hypermethylation generally suppresses gene expression by blocking transcription factor binding or RNA polymerase recruitment, in the current study, we unexpectedly observed an association between promoter hypermethylation of ATG9A, ATG9B, ATG2B, and ATG4D genes and their increased expression in the majority of CRC cases.

On the contrary to our findings, in a previous study on breast tumors, promoter hypermethylation of ATG9A, ATG9B, ATG2B, and ATG4D genes was linked to reduced gene expression [24].

In parallel with our findings, it has been reported that the hypermethylation of the ZAR1 gene was also accompanied by its upregulation in neuroblastoma [39]. Another study on a PCa mouse model also revealed that the elevated expression level of the GSC (Goosecoid) was related to DNA methylation [40]. A study by Niknam et al. also found that hTERT promoter methylation is directly associated with gene expression that could be explained by the lack of methylation near the transcription start site of hTERT [41].

This paradoxical relationship highlights that epigenetic regulation has a context-dependent nature and may depend on factors such as tumor type, microenvironment, and compensatory pathways [42]. For instance, it has been found that specified genes possessing unmethylated CpG islands in their promoters are unable to generate efficient transcripts as a result of the lack of RNA Pol II recruitment [43]. In addition, local methylation of distinctive residues has been revealed to be critical for the regulation of gene expression and is thus able to counteract the methylation status of the genomic region as a whole [44, 45]. Furthermore, varieties of transcription factors favor binding methylated CpGs rather than unmethylated ones [46-49]. Both sparse and dense methylation of the promoter can hinder the binding of the transcriptional machinery and thereby repress transcription of the related genes. Although the presence of enhancers cannot reactivate the gene expression in densely methylated promoters, enhancers such as SV40 can overcome the sparse methylation and reactivate the gene expression [44, 50, 51]. Nonetheless, it is crucial to notice that there is little data concerning possible mechanisms of gene activation via hypermethylation of DNA and what type of proteins are recruited in these mechanisms.

The present study confirms that a significant fraction of the DNA methylation phenomenon in CRC can be associated with alterations in gene expression levels positively. This highlights the importance of epigenetic evaluations accompanied by analysis of gene expression modifications to define the real impact of promoter methylation on gene transcription activation.

There are some limitations and future directions of this study. The small sample size of 21 patients limited the generalizability of the findings. More multi center studies with larger sample sizes are required to validate these results. Furthermore, it is needed to elucidate the mechanisms by which promoter methylation influences gene expression activation in CRC. In this regard, identifying the transcription factors and enhancers involved in this process could lead to deeper insight into the regulation of ATG genes in CRC and thereby may introduce novel targets and biomarkers for this cancer.

In conclusion, this study highlights the complex relationship between promoter methylation and expression of ATG genes in CRC. In contrast to the conventional insight, that promoter hypermethylation leads to gene silencing, the current findings indicated expression enhance-ment of ATG genes in CRC following promoter hypermethylation in a context-dependent manner. This underscores the complexity of epigenetic regulation in cancer and shows the need for further investigations to fully understand its role in CRC pathogenesis. These insights pave the way for the development of novel biomarkers, therapeutic strategies, and ultimately CRC outcome improvement.

Conflict of Interest:

The authors declare that they have no competing interests.

Authors’ Contribution:

MN: Methodology, Data curation, Formal analysis, Writing-original draft. FN: Conceptualization, Writing-review and editing. SVH: Methodology, Writing-review and editing. MZ: Conceptualization, Methodology, Writing-review and editing. PM: Conceptualization, Writing-review and editing, Funding acquisition