Autophagy as a cytoprotective mechanism in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is amongst the most aggressive human malignancies, representing a significant health burden worldwide. Autophagy is an evolutionarily conserved catabolic process that degrades and recycles damaged organelles and misfolded proteins to maintain cellular homeostasis. Alterations in autophagy are associated with cancer pathogenesis, including ESCC; however, the functional role of autophagy in ESCC remains elusive. Here, we discuss the clinical relevance of autophagy effectors in ESCC and review current knowledge regarding the molecular mechanisms through which autophagy contributes to ESCC. We highlight the cytoprotective role of autophagy in ESCC and discuss autophagy inhibitors as novel experimental therapeutics to potentiate the effects of anti-cancer therapies and/or to overcome therapeutic resistance in ESCC.

Introduction

Esophageal squamous-cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer and amongst the most aggressive forms of human carcinoma, with a 5-year survival rate of <20% [1]. Factors contributing to ESCC patient mortality include late stage diagnosis, frequent metastasis, and limited therapeutic response. Despite extensive characterization of the genomic landscape of ESCC, which often features overexpression of epidermal growth factor receptor (EGFR) and Cyclin D1 as well as TP53 mutations [2–6], effective targeted therapies remain elusive. Thus, novel diagnostic, prognostic and therapeutic strategies are needed to improve ESCC patient outcomes.

Macroautophagy is an evolutionarily conserved catabolic process facilitating recycling of intracellular components, including damaged organelles and protein aggregates, and also serves as a homeostatic mechanism permitting adaptation to stressors including nutrient deprivation, oxidative stress and inflammation [7]. In response to autophagy-inducing stimuli, cytosolic cellular constituents are sequestered in double-membrane autophagic vesicles (AVs). AV biogenesis is induced by upstream signals, including mammalian target of rapamycin (mTOR) inactivation and AMP-activated protein kinase (AMPK) activation. The concerted activity of various proteins, including members of the autophagy-related (ATG) family and class III phosphoinositide 3-kinases (PI3Ks) [7], then directs AV nucleation and elongation, yielding mature AVs. Subsequent fusion of AVs with lysosomes is essential for acid hydrolase-mediated degradation of autophagic cargo, generating substrates for energy production and biosynthesis [7].

Alterations in autophagy have been linked to human pathologies, including neurodegeneration, inflammatory bowel disease, and cancer. Autophagy plays a complex role in carcinogenesis, initially limiting malignant transformation while later fostering growth and progression of established lesions. Autophagy-mediated tumor suppression is largely attributed to inhibition of oxidative stress and subsequent genomic instability via selective autophagic removal of dysfunctional mitochondria (termed mitophagy) [8]. In established lesions, however, tumor cells co-opt autophagy as a mechanism to survive under conditions of nutrient, microenvironmental and chemotherapeutic stress [9]. A therapeutic window for autophagy inhibition in cancer is supported by experimental evidence demonstrating that tumors are more sensitive to autophagy inhibition than normal tissues [10]. Currently, the only Food and Drug Administration-approved autophagy inhibitors are chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), anti-malarial agents that prevent lysosomal acidification, thereby blocking AV clearance [11]. Clinical trials of these agents alone or in combination with established therapies support the therapeutic potential of autophagy inhibition with partial response or stable disease reported in subsets of melanoma, colorectal cancer, myeloma and renal cell carcinoma patients [12–14]. Notably, autophagy inhibitors have yet to be tested in ESCC patients.

Here, we provide a comprehensive review of the current literature related to autophagy and its mediators in ESCC. Studies evaluating the expression of autophagy effectors in human specimens will be discussed, highlighting their clinical implications. We will review studies utilizing in vitro and in vivo models to examine the functional role of autophagy in ESCC carcinogenesis and therapy response and discuss the implications and limitations of the current literature, which supports autophagy as a cytoprotective mechanism in ESCC tumor cells that limits response to various therapeutic interventions. Finally, we will present future perspectives that may facilitate our understanding of the role of autophagy in ESCC with the ultimate goal of utilizing this knowledge to improve clinical practice and patient outcomes in this deadly disease.

Expression of ATG genes and proteins is altered in ESCC

Studies evaluating the expression of autophagy-associated factors in ESCC patient cohorts have revealed clinically significant findings (Table 1). Consistent with a tumor suppressor role for autophagy, human ESCC lesions display decreased levels of ATG genes and proteins. Integrated genomic characterization of esophageal tumors identified deep deletions in ATG7, the gene product of which mediates AV elongation, in two molecular subtypes of ESCC [4]. Co-occurrence of ATG7 deletions with amplification in NFE2L2 [4], encoding the antioxidant mediator NRF2, support ATG7 as a critical mediator of oxidative stress [15–17]. Decreased and/or lost expression of BECLIN 1, an effector of AV nucleation, was also reported in ESCC tumors [18,19]. Complementary BECLIN 1 knockdown and overexpression experiments further revealed a respective increase and decrease in invasion of EC9706 ESCC cells, supporting a potential functional role for BECLIN 1 downregulation in ESCC progression [19]. Following post-translational cleavage and lipidation, microtubule-associated protein light chain 3 (LC3) is incorporated into autophagic membranes, serving as a molecular marker for AVs. Aberrant methylation of LC3Av1, transcriptional variant 1 of the LC3A isoform, was identified in a subset of human ESCC tumors concurrent with diminished protein expression [20]. Forty five percent of ESCC cell lines additionally displayed LC3Av1 gene silencing and ectopic LC3Av1 expression inhibited xenograft tumor growth of KYSE170 ESCC cells [20].

Table 1.

Clinical significance of autophagy-associated factors in ESCC

Target	Clinical Significance	Method(s)	Reference
ATG7	Deep deletions in ATG7 identified in 2 molecular subtypes of ESCC	DNA sequencing	4
BECLIN 1	BECLIN 1 expression is lower in ESCC than adjacent normal esophageal epithelium Tumors from early clinical stages (I/II) display higher BECLIN 1 mid-to-late stage (III/IV) tumors Low BECLIN 1 expression is associated with poor overall survival, poor tumor differentiation, advanced pathologic stage and LN metastasis	qRT-PCR, immunoblotting	18
BECLIN 1	BECLIN 1 expression is lost in ESCC (33%) Negative BECLIN 1 expression is associated with poor overall survival, increased depth of invasion, advanced clinical stage and LN metastasis	IHC	19
LC3Av1	LC3Av1 expression is reduced >50% in primary tumor compared to corresponding non-cancer tissue in 19/38 cases LC3Av1 methylation detected in 6/38 cases	qRT-PCR, COBRA	20
LC3	High LC3 expression in ESCC (53%) LC3 expression increased gradually in IEN and T1 carcinoma, but did not change in T2-T4 carcinoma LC3 expression does not correlate with survival	IHC	21
LC3	High LC3 expression in ESCC (62.5%) High LC3 expression is associated with decreased overall and disease-free survival	IHC	22
LC3	High cleaved LC3 is associated with decreased post-surgical survival	IHC	29
LC3	Low LC3 expression is associated with depth of invasion, advanced tumor stage, LN metastasis, lymphatic invasion and venous invasion	IHC	30
LC3	High LC3 expression detected in ESCC (53%), LC3 undetectable in non-cancerous tissue LC3 expression increased in IEN, ESCC compared to normal No significant difference between early (T1) and advanced ESCC (T2-T4)	IHC, immunoblotting	21
LC3, PINK1	High LC3 expression in ESCC (47.9%) is associated with poor response to CRT High PINK1 expression in ESCC (44.9%) is associated with reduced overall survival and poor response to CRT PINK1 expression is elevated in patients with pre-operative CRT compared to CRT naive	IHC	23
LC3B, BECLIN 1	High LC3B expression is associated with poor overall survival BECLIN 1 expression does not correlate with overall survival	IHC	27
LC3, BECLIN 1	LC3 positivity is associated with lower median survival time following CRT LC3 and BECLIN 1 positivity is associated with lower median survival time following CRT	IHC, immunoblotting	28
ATG5	ATG5 positivity detected in tumors, absent in adjacent normal tissue ATG5 SNPs (rs1322178, rs3804329, rs671116) is associated with mortality of early-stage ESCC patients ATG5 SNPs (rs1322178, rs3804329) is associated with early distant metastasis of early-stage ESCC patients	IHC, genotyping	24
ULK1	ULK1 expression detected in ESCC (70.2%), marginally detectable in normal tissue High ULK1 expression is associated with decreased 5-year survival time	IHC, immunoblotting	25
ULK1	Low ULK1 expression is associated with decreased median and 5-year survival time, increased LN metastasis	IHC	26

Abbreviations: ATG, Autophagy-related; COBRA, combined bisulfite restriction analysis; CRT, chemoradiotherapy; ESCC, esophageal squamous cell carcinoma; IEN, intraepithelial neoplasia; IHC, immunohistochemistry; LC3, microtubule-associated protein light chain 3; LC3Av1, LC3 isoform A variant 3; LN, lymph node; PINK, PTEN-induced putative kinase; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; SNP, single nucleotide polymorphism

Given the putative bimodal role of autophagy in carcinogenesis, it is not surprising that ESCC lesions may also feature enhanced expression of ATG factors. Three independent studies demonstrated increased LC3 expression when comparing normal and ESCC tissues [21–23]. LC3 immunoreactivity gradually increased during progression from premalignant lesions to early ESCC, positively correlating with the proliferation marker Ki-67, while remaining unchanged in late stage tumors [21]. Early-stage ESCC tumors were found to exhibit higher expression of ATG5, a mediator of AV elongation, compared to adjacent normal tissue [24]. Unc-51-like kinase 1 (ULK1), the kinase that phosphorylates BECLIN 1 to induce autophagy, is upregulated when comparing ESCC tumors to adjacent non-cancerous tissues in vivo or ESCC cell lines to normal esophageal keratinocytes in vitro, potentially as a result of enhanced protein stability [25,26]. Further, ESCC lesions feature expression of PTEN-induced putative kinase (PINK)1, a molecular tag that accumulates on damaged mitochondria to mediate mitophagy, providing a potential link between mitophagy and ESCC [23].

ATG factors as prognostic biomarkers in ESCC

Beyond establishing that alterations in expression of autophagy-associated factors occur in the context of esophageal carcinogenesis, various studies have found that expression of ATG genes and proteins function as prognostic biomarkers in ESCC. In early-stage ESCC tumors, single nucleotide polymorphisms (SNPs) in the ATG5 gene locus correlated with prognosis and predicted early distant metastasis [24]. As increased ATG5 expression did not correlate with ATG5 genotype [24], further investigation is required to elucidate how ATG5 SNPs may affect ESCC prognosis. In two independent ESCC cohorts, low or negative expression of BECLIN 1 was associated with poor patient outcomes [19,27]. While BECLIN 1 expression alone failed to correlate with chemoradiotherapy response, increased post-treatment survival was observed in ESCC lesions with negative expression of both BECLIN 1 and LC3 [28]. We have reported that increased cleaved LC3A expression predicts poor prognosis in ESCC patients [29], a finding that was recapitulated upon evaluation of LC3 or LC3B in two additional studies [22,27]. By contrast, Sakurai and colleagues determined that low LC3 expression predicts poor prognosis as well as depth of invasion, lymphatic invasion and lymph node metastasis in ESCC patients [30]. An additional study failed to detect a significant correlation between LC3 and ESCC patient survival [21]. Discrepant results with regard to the use of ULK1 as a biomarker in ESCC have also been reported [25,26]. Demographic and clinicopathologic factors of distinct patient cohorts, the use of antibodies recognizing distinct antigens and variability in staining protocols could explain divergent findings. It is possible that co-analysis of LC3 along with other ESCC-relevant factors may prove to be an effective strategy toward improving the clinical utility of LC3 an ESCC biomarker as co-expression of LC3 and p53 has been shown to correlate poor prognosis [31]. With regard to post-therapeutic ESCC prognosis, high expression of LC3 has been shown to correlate with poor response to neoadjuvant chemotherapy [23]. Finally, a role for mitophagy in ESCC therapeutic response is suggested as upregulation of PINK1 was found in ESCC patients treated with preoperative chemotherapy as compared their treatment naïve counterparts and high PINK1 expression predicted poor chemotherapeutic response [23]. Given the limited scope of the described studies and the noted discrepancies, large-scale investigations evaluating the reliability and validity of autophagy mediators as prognostic biomarkers in ESCC will be required prior to their translation to clinical medicine.

Mechanisms through which autophagy contributes to ESCC carcinogenesis

There are currently few functional investigations into the role of autophagy in ESCC. Along with Wu et al, we have defined autophagy as regulator of ESCC cancer stem cell (CSCs), which have been identified by cell surface antigens, including CD44 and OV6 [29,32]. In transformed esophageal keratinocytes, autophagy regulated epithelial-mesenchymal transition (EMT)-mediated conversion of CD44^-/Low non-CSCs to CD44^High CSCs. Specifically, mitophagy facilitated EMT in response to transforming growth factor-β by clearing dysfunctional mitochondria and modulating oxidative stress [29]. In established ESCC xenografts, autophagy or mitophagy inhibition depleted CD44^High CSCs and induced oxidative stress, but failed to cause tumor regression [29]. Autophagy activation also promoted OV6+ CSC expansion by activating ATG7-dependent Wnt/β-catenin signaling in Eca109 and TE-1 ESCC cells [32]. Recently, autophagy was shown to be a downstream effector of metastasis-associated colon cancer 1 (MACC1), a protein positively associated with lymph node metastasis in ESCC patients [33]. MACC1 depletion reduced ESCC cell proliferation, migration and invasion concurrent with inhibition of autophagy [33]. Autophagy inhibition using 3-methlyadenine (3-MA), which blocks AV formation by inhibiting activity of class III PI3Ks, also reversed these phenotypes downstream of MACC1 [33]. In sum, these studies indicate that autophagy acts as tumor-promoting factor in ESCC cells, permitting the expansion and maintenance of CSCs with enhanced malignant potential as well as metastasis-associated phenotypes; however, further in vivo studies are necessary to directly link to autophagy to ESCC metastasis.

Modulating autophagy to improve therapeutic efficacy in ESCC

Although ESCC therapy often comprises radiation therapy alone or in combination with chemotherapeutic agents, including cisplatin and 5-fluorouracil (5-FU), radio- and chemoresistance remain significant clinical challenges. Given that the aforementioned experimental data support autophagy as a tumor-promoting factor in ESCC, it is tempting to speculate the autophagy inhibition may improve therapeutic response in ESCC patients. Indeed, various studies using in vitro and in vivo ESCC models support this notion, demonstrating that autophagy inhibitors augment the efficacy of anti-cancer therapies. Ionizing radiation activates autophagy in ESCC cell lines and autophagy inhibition enhanced radiation-induced apoptosis and cell cycle arrest in vitro, supporting a protective role for autophagy [34]. Autophagy inhibition also sensitized ESCC xenograft tumors to radiation by potentiating tumor growth suppression and inhibiting angiogenesis [34,35]. Autophagy is activated in response to cisplatin in ESCC cell lines [36,37] and autophagy inhibition enhances ESCC cell sensitivity to cisplatin in vitro and in vivo [37–39]. Furthermore, enhanced autophagy is associated with cisplatin- or 5-FU-resistantance in ESCC cell lines [38–40].

In addition to chemo- and radiotherapy, autophagy enhances the efficacy of various experimental therapeutics in preclinical ESCC models. The anti-angiogenesis drug Endostar, which benefits ESCC patients when used in combination with chemo- or chemoradiotherapy [41–43], induced autophagy via inhibition of mTOR signaling in Eca109 and TE-1 ESCC cells [44]. Additionally, CQ markedly increased the anti-cancer effects of Endostar in vitro [44]. ESCC cell apoptosis mediated by the proteasome inhibitor MG-132, the Bcl2 homologous 3-mimetic GX15-070, the natural compound resveratrol features autophagy [40,45–47] and combination of these treatments with autophagy inhibition potentiates tumor cell death [45–47].

Taken together, the described studies support autophagy as a protective mechanism in ESCC cells that promotes cell survival in response to anti-tumor therapies, thereby contributing to therapeutic resistance. It is also worth noting, however, that autophagy activation by the anti-EGFR monoclonal antibody nimotuzumab was demonstrated to promote chemosensitivity to cisplatin and paclitaxel in vitro [48]. Moreover, autophagy activation by the mTOR inhibitor rapamycin enhanced cisplatin and paclitaxel cytotoxicity [48]. This study implicates autophagy as a mediator of chemotherapy-induced apoptosis. As EGFR overexpression is common in ESCC, further understanding the role of autophagy in EGFR-positive ESCC patients may have applications for precision medicine related to the use of EGFR-targeted therapies.

Summary & Future Perspectives

In sum, the current literature supports autophagy an ESCC tumor promoter that contributes to tumor cell proliferation, invasion, and migration as well as cancer stem cell maintenance while also promoting therapy resistance (Figure 1). As findings related to the functional role of autophagy in ESCC are presently limited in number to investigations performed in tumor cells, the role of autophagy in esophageal carcinogenesis, particularly at the early stages, requires further study. Indeed, clinical studies identifying alterations in autophagy mediators in premalignant intraepithelial neoplasia lesions and early stage ESCC lesions indicate that determination of the functional significance of such alterations may provide valuable insight into the pathophysiology of ESCC. With regard to patient outcomes, there is certainly potential for autophagy effectors to serve as prognostic ESCC biomarkers following appropriate validation studies as noted above. Given that only a limited portion of ESCC patients respond to current standard of care and preclinical studies support a cytoprotective role for autophagy in response to chemo- and radiotherapy, evaluation of ATG proteins as predictive biomarkers in the context of specific interventions may have implications for precision medicine in ESCC patients.

Figure 1. Autophagy is a tumor promoting factor in ESCC.

Autophagy activation has been documented in esophageal squamous cell carcinoma (ESCC) cells in response to both endogenous and exogenous stimuli. Metastasis-associated colon cancer 1 (MACC1)-mediated induction of autophagy promotes ESSC cell proliferation, migration and invasion. Transforming growth factor (TGF)-β in the tumor microenvironment induces autophagy to promote expansion of CD44^High cancer stem cells (CSCs). Autophagy also promotes expansion of OV6+ CSCs via Wnt/β-catenin signaling; however, the mechanisms regulating autophagy activation in this context remain to be determined. Activation of autophagy by chemo- and radiotherapy as well as various experimental ESCC therapeutics induce autophagy that has largely been shown to act in a cytoprotective fashion to limit therapy-induced cell death and promote therapy resistance.

ESCC standard of care includes surgical intervention often in combination with chemoradiotherapy. Therapeutic resistance is a common feature of ESCC tumors as evidenced by clinical studies failing to detect significant improvement in overall survival when comparing chemoradiotherapy-treated patients to those treated with surgery alone [1]. As preclinical studies support autophagy as tumor-promoting factor in ESCC that limits therapeutic response, evaluating the effects of pharmacological autophagy inhibition in ESCC patients is of great interest. Autophagy supports expansion and maintenance of CSCs displaying resistance to chemotherapeutic reagents, including 5-FU and cisplatin [29,32]. Thus, it is tempting to speculate that autophagy inhibition may selectively target CSCs to improve therapy response and patient outcomes in ESCC. Although clinical trials utilize CQ and HCQ to inhibit autophagy, these drugs are only moderately potent and induce off-target effects, spurring efforts to develop novel pharmacological autophagy inhibitors (Figure 2). For example, the bisaminoquinoline Lys05 triggers a greater deacidification of the lysosome compared to HCQ and has been shown to more effectively block autophagy in preclinical models [49]. In addition to improving the ability of lysomotropic agents to inhibit autophagy, drugs aimed at preventing AV formation are currently under development. Promising preclinical studies have been executed using inhibitors of ULK1, or the class II PI3K VPS34, to limit AV nucleation, as well as antagonists of ATG4B, a mediator of LC3 processing, to suppress AV elongation [50–57]. Finally, autophagic flux occurs in normal esophageal keratinocytes and serves as a cytoprotective mechanism in gastroesophageal reflux disease flux and eosinophilic esophagitis [58], a comprehensive understanding of the functional and mechanistic role of autophagy in esophageal biology is needed to improve the development of experimental autophagy-targeted therapies that aim to improve esophageal diseases patient outcomes, including ESCC.

Figure 2. Strategies for pharmacological autophagy inhibition.

Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) act at the lysosome to prevent autophagic vesicle (AV) clearance. Clinical trials using CQ or HCQ have been executed for various cancer types with varying results in terms of both patient outcomes and effects on autophagy in tumor tissues. Lys05, a dimeric analog of CQ, exhibits increased potency with regard to single-agent antitumor activity and autophagy inhibition as compared to HCQ in preclinical models; however, the clinical utility of this agent has yet to be established. Inhibitors of more proximal steps in autophagy have been utilized in preclinical studies, including those targeting unc-51-like kinase 1 (ULK1) and the class III phosphoinositide 3-kinase VPS34, two positive effectors of AV nucleation, and the cysteine protease ATG4B, a mediator of AV elongation. As these agents are further developed, it will be of great interest to determine their effects on human tumors either alone or in combination with established therapeutic modalities. This is particularly true of esophageal squamous cell carcinoma in which current data supports a tumor promoting role for autophagy and therapeutic resistance represents as substantial clinical challenge.

Highlights.

• Clinical studies support autophagy effectors as prognostic biomarkers in ESCC

• Autophagy promotes invasion and cancer stem cell expansion/maintenance in ESCC

• Autophagy inhibition improves therapeutic efficacy in preclinical ESCC models

• The mechanistic contribution of autophagy to ESCC biology and therapy response remain elusive

Abbreviations

3-MA

3-Methyladenine

5-FU

5-fluorouracil

AMPK

AMP-activated protein kinase

ATG

autophagy-related

AV

autophagic vesicle

CQ

chloroquine

CSCs

cancer stem cells

EGFR

epidermal growth factor receptor

EMT

epithelial-mesenchymal-transition

ESCC

esophageal squamous cell carcinoma

HCQ

hydroxychloroquine

LC3

microtubule-associated protein light chain 3

MACC1

metastasis-associated colon cancer 1

mTOR

mammalian target of rapamycin

PI3K

phosphoinositide 3-kinase

PINK

PTEN-induced putative kinase

SNPs

single nucleotide polymorphisms

ULK1

unc-51 like autophagy activating kinase 1