Immunohistochemical analysis of autophagy-related proteins.
1. Introduction
Autophagy is a cellular stress-adaptive process in which double-membrane structures called autophagosomes engage in protein degradation, cellular differentiation, apoptosis and antigen processing, and are recycled to sustain cellular metabolism [1-11]. It is a self-digesting mechanism responsible for removal of long-lived proteins and damaged organelles by lysosomes, and opposing roles in cell death and survival have been described for autophagy.
Autophagy is a multifaceted process, and alterations in autophagic signaling pathways are frequently observed in cancer. Cancer is a disease generated by mutation, selection and genome instability in the resulting tumor tissue, and is considered to be the second leading cause of death in western countries after heart disease [12, 13]. Autophagy can be activated by various stimuli including hypoxia during the tumor formation [14]. One hypothetical mechanism is that autophagy promotes tumor cell survival in response to diverse stresses [15]. Furthermore, autophagy spatially and temporally regulates tumor development by suppressing tumor growth through regulating cell proliferation in the early stages of tumorigenesis [16]. Conversely, when autophagy is reduced, it contributes to tumor formation and growth by the breakdown of tumor cells following autophagy-related cell death, leading to tumor cell survival [17]. There is a controversy about the roles of autophagy in cancer [1, 3, 18]. In this review, we outline the multiple roles of autophagy in cancer, including gene expression, gene mutation, and chemotherapy.
2. Autophagy-related genes in cancer
2.1. ATG genes
Most recently, molecular genetic analyses have focused on the function of autophagy-related gene (
Beclin-1 (encoded by
Frameshift mutations of genes with mononucleotide repeats are features of cancers with microsatellite instability (MSI). Mononucleotide repeat frameshift mutations in
2.2. UVRAG
As an
2.3. IRGM
In the autophagy pathway, the immunity-related guanosine triphosphatase (GTPase) family, M (IRGM), plays a central function and appears to have an important role in the activation of the pathway.
2.4. RASSF1
The RAS association domain family 1A (RASSF1A) is one of the most epigenetically silenced elements in human cancers. The tumor suppressor gene,
2.5. NOD2
The nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a member of the Nod-like receptor family and associates with the cell surface membrane. NOD2 activation controls the induction of autophagy, or apoptosis [68-70]. Four major
3. Analysis of autophagy by immunohistochemistry
Recently, the role of autophagy in cancer development and progression has been investigated using immunohistochemistry. Immunohistochemical methods have been developed that supplement the detection of autophagy via genetic analyses. Many antibodies for autophagy detection are routinely used for immunohistochemistry against proteins involved in autophagy pathways [83-86] (Table 1).
|
|
|
LC3 (rabbit polyclonal antibody) | [86] | |
|
Medical & Biological Laboratories, Japan | |
|
Pressure cooker (110C-120C) for 10 min; 10 mM citrate buffer, pH 6.0 | |
|
Formalin-fixed, paraffin-embedded specimens | |
|
Invariably granular cytoplasmic staining | |
LC3 | [100] | |
|
Novus Biologicals, USA; (1:400) | |
|
High temperature and pressure, citrate buffer | |
|
Formalin-fixed, paraffin-embedded specimens | |
|
Cytoplasmic staining | |
Beclin-1 (rabbit monoclonal antibody) | [95] | |
|
Abcam, UK; (1:100) | |
|
Microwave oven for 15 min, 10 mM citrate buffer, pH 6 | |
|
Formalin-fixed, paraffin-embedded specimens | |
|
Cytoplasmic staining | |
Beclin-1 (rabbit polyclonal antibody) | [97] | |
|
Abcam, UK; (1:100) | |
|
Microwave oven, 10 mM citrate buffer, pH 6 |
|
|
Formalin-fixed, paraffin-embedded specimens | |
|
Membrane-plasma, cytoplasm and nucleus in the cancer cells and no or modest staining in the adjacent noncancerous tissue | |
Beclin-1 (rabbit polyclonal antibody) | [25] | |
|
Novus Biologicals, USA | |
|
Pressure cooker inside a microwave oven at 700 W for 30 min, 10 mM citrate buffer, pH 6.0 | |
|
Microarray recipient block was constructed containing paraffin-embedded colorectal adenocarcinoma tissue samples from 103 archival patient specimens | |
|
Cytoplasmic staining | |
Beclin-1 | [100] | |
|
Cell Signaling, USA; (1:100) | |
|
High temperature and pressure, citrate buffer | |
|
Formalin-fixed, paraffin-embedded specimens | |
|
Cytoplasmic staining | |
BIF-1 (mouse monoclonal antibody) | [98] | |
|
Imgenex, USA; (1:2500) | |
|
standard cell conditioning (Ventana Medical Systems, USA) | |
|
Formalin-fixed, paraffin-embedded core sections on a tissue array | |
|
Cytoplasmic staining | |
ATG5 (rabbit polyclonal antibody) | [30] | |
|
Abcam, UK; (1:800) | |
|
Pressure cooker inside a microwave oven at 700 W for 30 min, 10 mM citrate buffer, pH 6.0 | |
|
Formalin-fixed, paraffin-embedded specimens | |
|
Cytoplasmic and/or nuclear |
3.1. Proteins involved in autophagy
3.1.1. LC3
Microtubule-associated protein 1 light chain 3 (LC3) is an autophagosomal orthologue of yeast ATG8, with approximately 30% amino acid homology [87, 88]. LC3 is a specific marker of autophagosome formation. LC3-I is localized to the cytoplasm, whereas LC3-II binds to autophagosomes [89].
3.1.2. Beclin-1 (ATG6)
Beclin-1 is a mammalian homolog of the yeast ATG6 protein. The expression of Beclin-1 protein has been reported in tumor tissues such as breast, ovarian, prostate, lung, brain, stomach and colorectum [25, 90]. Beclin-1 was found to be deregulated in human cancers and may play a role in the tumorigenesis and/ or progression of human cancers [21, 91]. It is required for autophagic induction and is a haploinsufficient tumor suppressor.
3.1.3. ATG5
ATG5 is a key regulator of autophagic and apoptotic cell death, and is involved in the early stages of autophagosome formation [18, 28]; binding of ATG5 with ATG12 contributes to autophagosome formation, which sequesters cytoplasmic materials before lysosomal delivery [18]. It is suggested that ATG5 is involved in both apoptotic and autophagic cell death [92].
3.1.4. Bax-interacting factor -1
Bax-interacting factor-1 (Bif-1) protein is a member of the endophilin B family, which plays a critical role in cell death, including autophagy and apoptosis. Loss of Bif-1 suppresses programmed cell death and promotes tumorigenesis [93, 94].
3.1.5. GABARAP
Gamma-aminobutyric acid type A receptor-associated protein (GABARAP) is one of the mammalian homologue of yeast ATG8. It is involved in autophagosome formation during autophagy and was first identified in the brain, but is widely expressed in a variety of normal tissues. Recent reports have suggested that GABARAP is an essential component of autophagic vacuoles in addition to its role as an intracellular trafficking molecule [87,88].
3.2. Expression of autophagy-related proteins in gastrointestinal cancers
Resent reports have demonstrated the expression of autophagy-related proteins in gastrointestinal carcinomas. Chen et al. examined the expression levels of Beclin1 in gastric carcinomas and adjacent normal gastric mucosal tissues by immunohistochemistry. According to their results, high levels of Beclin-1 expression were observed in 90/155 (58.1%) of gastric carcinomas, in 24/60 (40.0%) of adjacent mucosal tissues and in 13/30 (43.3%) of normal gastric mucosa tissues (
In contrast, using a tissue microarray approach, Ahn et al. investigated Beclin-1 protein expression in 103 colorectal and 60 gastric carcinoma tissues by immunohistochemistry. The expression of Beclin-1 was detected in 50/60 (83%) of gastric carcinomas and 98/103 (95%) of colorectal carcinomas. Conversely, the normal mucosal cells of both the stomach and colon showed no or very weak expression of Beclin-1. There was no significant association of Beclin-1 expression with clinicopathological characteristics, including invasion, metastasis and stage. Their data indicate that Beclin-1 inactivation by loss of expression may not occur in colorectal and gastric cancers. Rather, increased expression of Beclin-1 in the malignant colorectal and gastric epithelial cells compared with their normal mucosal epithelial cells suggests that neo-expression of Beclin-1 may play a role in both colorectal and gastric tumorigenesis [25].
An et al. analyzed ATG5 protein expression by immunohistochemistry and
Colorectal carcinoma is one of the most common cancers in the world and the incidence rate is rising. Miao et al. performed experiments to investigate a possible correlation between GABARAP expression in colorectal carcinoma and clinicopathological parameters, including patient survival times. Their results showed that the expression of GABARAP protein was significantly higher in colorectal cancers (51.5%) than the adjacent matched non-tumor tissues (33.0%), and overexpression of GABARAP was significantly correlated with a low grade of differentiation and shortened overall survival. They described GABARAP protein expression as a new prognosis marker in colorectal carcinoma [96].
Li et al. analyzed the expression of Beclin-1 protein in stage IIIB colon carcinoma by immunohistochemistry and correlated it with survival. Their results showed Beclin-1 immunostaining was distributed in the plasma membrane, cytoplasm and nuclei of tumor cells in 98/115 cases (85.2%). Modest or no Beclin-1 expression was observed in adjacent non-cancerous tissues. Higher levels of Beclin-1 expression were strongly associated with longer survival. Both univariate analysis and multivariate analysis showed that Beclin-1 expression levels and invasive depth of primary mass (T stage) were independent prognostic factors. They suggested that Beclin-1 is a favorable prognostic biomarker in locally advanced colon carcinomas [97].
Bif-1 protein plays a critical role in cell death, including autophagy and apoptosis. Coppola et al. examined Bif-1 expression level in colorectal carcinoma using semiquantitative immunohistochemistry and microarray analysis of archival specimens. Bif-1 expression was negative in 23/102 (22.5%) of colorectal carcinomas. Moderate to strong Bif-1 staining was identified in 37/102 (36.3%) of the tumors, and weak staining was noted in 42/102 (41.2%). Moderate to strong Bif-1 immunoreactivity was shown in 26/38 (68.4%) normal colorectal mucosa, and none were negative. In 12/38 (31.6%) cases, the normal colorectal mucosa demonstrated weak Bif-1 stain. The mean staining scores (intensity and percentage of positively stained cells) for colorectal carcinomas and normal colorectal mucosa differed significantly (
LC3 is a one of the most useful markers of autophagy. Yoshioka et al. evaluated LC3 expression in gastrointestinal cancers by immunohistochemistry to elucidate the role of autophagy in human cancer development. LC3 expression was compared with Ki-67 staining and expression of carbonic anhydrase IX, a hypoxic marker. LC3 was expressed in the cytoplasm of cancer cells, but not in non-cancerous epithelial cells. Furthermore, high expression of LC3 was observed in 56/106 (53%) of esophageal, 22/38 (58%) of gastric and 12/19 (63%) of colorectal cancers. The immunoreactive score (intensity and percentage of positively stained cells) of LC3 gradually increased during the early stages of esophageal carcinogenesis in low- and high-grade intraepithelial neoplasia and T1 carcinoma, but did not change in later cancer progression (T2–T4 carcinomas). In early esophageal carcinogenesis, LC3 expression correlated with the Ki-67 labeling index (
Ahn et al. reported that Beclin-1 expression was detected in 95% of colorectal carcinomas examined. In contrast, normal mucosal cells of colon showed no or very weak expression of Beclin-1. There was no significant association of Beclin-1 expression with clinicopathological characteristics, including invasion, metastasis and stage [25].
Guo et al. performed experiments to investigate the utility of Beclin-1 and LC3, in predicting the efficiency of cetuximab in the treatment of advanced colorectal cancer. Their results showed that Beclin-1 and LC3 expression was significantly correlated (r=0.44,
4. Autophagy in cancer chemotherapy
One of the standard modalities for treatment of patients with cancer is chemotherapy. Cytotoxic drug treatment often triggers autophagy, particularly in apoptosis-defective cells, and this excessive cellular damage combined with attempts to remediate that damage through progressive autophagy can promote autophagic cell death [101]. Platinum-containing cisplatin is one of the most extensively used chemotherapeutic agents, and remains the first-line treatment in various types of cancer [102]. Cisplatin-based chemotherapy frequently resulted in acquired resistance of cancer cells. Sirichanchuen et al. indicated that the levels of LC3-related autophagy were significantly lower in cisplatin resistant cells, and autophagosome formation was dramatically reduced in the resistant cells [103]. Patients with low LC3 expression had a higher objective response rate amongst advanced colorectal cancer patients treated with cetuximab-containing chemotherapy [100]. Expression of
5. Conclusion
Autophagy is involved in metabolism, cell-death, stress response and carcinogenesis. Several key autophagic mediators containing ATG-related proteins, LC3, Bif-1, GABARAP, UVRAG, IRGM, RASSF1, or NOD2, play pivotal roles in autophagic signaling networks in cancer. By these tumor-suppressive mechanisms in early-stage carcinogenesis, autophagy promotes genomic stability in carcinomas, and possibly contributes to cancer development.
Furthermore, immunohistochemical methods have been developed that supplement the detection of autophagy via genetic analyses. These are especially important since diagnosis of autophagic vacuoles using the classical method of electron microscopy is time-consuming, labor-intensive and costly. Many antibodies for autophagy detection are routinely used for immunohistochemistry. These autophagosomes then fuse with lysosomes to generate autolysosomes. Therefore, LC3 is an efficient and reliable marker for the detection of autophagosome formation.
Autophagy or ‘self-eating’ is frequently activated in tumor cells treated with chemotherapy. In cancer therapy, adaptive autophagy in cancer cells sustains tumor growth and survival in the face of the toxicity of cancer therapy. However, in certain circumstances, autophagy mediates the therapeutic effects of some anticancer agents. During tumor development and in cancer therapy, autophagy has been reported to have paradoxical roles in promoting both cell survival and cell death.
Autophagy may play a variety of physiological roles in cancer progression at each stage in various cancers. Further investigations are required to clarify the biological role of autophagy-related proteins so as to estimate their potential value in the diagnosis and treatment of cancer.
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