Abstract
Squamous cell carcinoma of the esophagus is associated with a dismal prognosis. Therefore, identification of new targets and implementation of new treatment modalities are issues of paramount importance. Based on a survey of the literature, we identified microRNAs conferring antitumoral activity in preclinical in vivo experiments. In the category of miRs targeting secreted factors and transmembrane receptors, four miRs were up-regulated and 10 were down-regulated compared with five out of nine in the category transcription factors, and six miRs were down-regulated in the category enzymes, including metabolic enzymes. The down-regulated miRs have targets which can be inhibited by small molecules or antibody-related entities, or re-expressed by reconstitution therapy. Up-regulated miRs have targets which can be reconstituted with small molecules or inhibited with antagomirs.
- miR-based target identification
- miR-related treatment modalities
- preclinical in vivo models
- squamous cell esophageal cancer
- review
More than 450,000 patients are affected by esophageal cancer worldwide (1). The highest incidence is found in eastern Asia, eastern and southern Africa, and southern Europe (1). In the USA, the disease claimed 16, 170 deaths in 2020 (2). Esophageal cancer is one of the most lethal types of cancer and surgical resection, chemotherapy and radiotherapy are primary treatments but have limited efficacy and severe side-effects (3). Two histological subtypes have been identified: esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC) as epidemiologically and biologically distinct types of cancer (4). ESCC is the major histological subtype and accounts for 80% of esophageal cancer worldwide (4). However, the incidence of esophageal adenocarcinoma and ESCC vary with geographic location (4). Barrett’s esophagus is a benign precursor lesion of esophageal adenocarcinoma (5).
Characterization of the genomic landscape of ESCC has revealed heterogeneity, and mutations in pathways of growth factor receptors, cell-cycle regulation, apoptosis, angiogenesis and DNA repair (6, 7). The US Food and Drug Administration recently approved pembrolizumab and nivolumab, which both target programmed death 1 for immunotherapeutic intervention for advanced ESCC. Pembrolizumab was approved for treatment of unresectable/metastatic tumors with high microsatellite instability, and nivolumab for unresectable, advanced or recurrent tumors (8, 9). Nevertheless, there is an urgent need to identify new targets and treatment modalities for ESCC. In this review, we focus on microRNAs (miRs) shown to be ESCC-related in preclinical in vivo systems for target identification and as new treatment modalities through inhibition or reconstitution of function for ESCC.
MicroRNAs and Cancer
miRs are small regulatory RNAs that derive from hairpin regions of precursor transcripts (10). Each miR associates with an Argonaute protein, forming a silencing complex which binds to complementary sequences of target transcripts and promotes destabilization or translational repression of the bound transcripts (10). miRs can interfere with several targets and act as oncogenes or tumor suppressors in a context-dependent manner (11, 12). They play a role in all aspects of cancer such as proliferation, apoptosis, invasion, metastasis and angiogenesis (13-16). In proof-of-concept experiments it has been shown that deletion of miR-15 and miR-16 induced B-cell lymphoma in mice by targeting BCL2 apoptosis regulator (BCL2) (17) and expression of miR-17 in the liver of transgenic mice caused hepatocellular carcinoma (18). The roles of miRs in ESCC have been summarized in several reviews (19-21). In this review, we focus on miRs in preclinical in vivo ESCC models in order to identify targets for therapeutic intervention and to explore new entities for therapeutic intervention by inhibition of deregulated miRs or replacement therapy with miR mimics.
miRs Targeting Secreted Factors and Transmembrane Receptors in In Vivo Models
Up-regulated miRs (Figure 1A).
Up-regulated (A) and down-regulated (B) miRs targeting secreted factors and transmembrane receptors in esophageal cancer cells in preclinical in vivo systems. ADAMTS1: A disintegin and metalloprotease with thrombospondin motifs 1; CD44, 47, 133: cluster of differentiation 44, 47, 133; EGFR: epidermal growth factor receptor; EI24: etoposide-induced 2.4 transcript; HER2: human epidermal growth factor receptor 2; IGF1R: insulin growth factor-like receptor 1; INTα6: integrin α6; INTβ6: integrin β6; INV: invasion; MET: metastasis; PROL: proliferation; RAGE: receptor for advanced glycation end-products; SARA: SMAD anchor for receptor activation; TG: tumor growth; TGFβR2: transforming growth factor receptor 2; TNFα: tumor necrosis factor α; VEGFA: vascular endothelial growth factor A.
miR-19a [targeting tumor necrosis factor α (TNFα)]. miR-19a induced proliferation of ESCC cell lines EC109, EC 9706 and KYSE150 (22). An antisense oligo directed against miR-19a induced apoptosis in EC9706 cells (22). Knockdown of miR-19a inhibited in vivo tumorigenicity of EC9706 cells in nude mice (22). TNFα was identified as a direct target of miR-19a (22). Pro- and anti-tumoral effects, such as stimulation of cancer progression and metastasis, direct cytotoxicity to tumor cells and stimulation of the immune response against cancer, have been reported (23-26). Therefore, the tumor-inhibitory function of TNFα in ESCC has to be resolved in more detail.
miR-25 (targeting E-cadherin). Overexpression of miR-25 in patients with ESCC was associated with lymph node metastasis and poor survival (27). miR-25 induced migration but not proliferation in KYSE150 ESCCs (27). Targeting miR-25 reversed epithelial–mesenchymal transition (EMT) transition in ESCCs in vitro (27). E-Cadherin was identified as a direct target of miR-25 (27). Up-regulation of miR-25 induced lung metastases after tail vein injection of EC-109 ESCC cells in immunocompromised mice (27). Down-regulation of E-cadherin was correlated with development and progression of cancer, high tumor grade and low patient survival (28, 29). It has been shown that modulation of expression of E-cadherin promotes migration of ESCC cells (30). However, due to specificity issues inherent to up-regulation of E-cadherin with small molecules, targeting E-cadherin is a difficult issue from a drug-discovery point of view.
miR-483-3p [targeting etoposide-induced 2.4 transcript (EI24)]. miR-483-3p was overexpressed in ESCC, promoted proliferation, G1/G2 transition and migration of ESCCs, and inhibited sensitivity of ESCC to chemotherapy (31). miR-483-3p promoted growth of ESCC xenografts in immunocompromised mice (31). EI24 has been identified as a direct target of miR-483-3p (31). EI24, a p53-response protein, contains six transmembrane domains and suppresses cell growth through caspase 9 and mitochondrial pathways including in ESCC (32, 33). Furthermore, EI24 has been shown to contribute to EMT (34).
miR-584k [targeting A disentegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1)]. miR-548k overexpression correlated with poor prognosis of patients with ESCC (35). miR-548k mediated proliferation and cell-cycle progression of KYSE30 and KYSE510 ESCCs and induced migration and tube formation by human dermal lymphatic endothelial cells (35). In nude mice implanted with KYSE30-derived ESCCs transfected with miR-548k, lymphangiogenesis, increased microlymphatic density and metastasis to lymph nodes were observed (35). Tail vein injection experiments indicated that miR-548k promotes lung metastasis (35). ADAMTS1 was identified as a target of miR-548 (35, 36). Overexpression of miR-548k promoted tyrosine phosphorylation in dermal lymphatic endothelial cells (35). Vascular endothelial growth factor receptor 3 (VEGFR3), a stimulator of lymphangiogenesis and promoter of lymphatic metastasis, is stimulated by ADAMTS1 (36-39). miR-548k also down-regulates Kruppel-like factor 10 (KLF10), a repressor of epidermal growth factor receptor (EGFR) (40).
Down-regulated miRs (Figure 1B).
miR-126 (targeting VEGFA). Lower expression of miR-126 in ESCC tissues and cell lines compared to corresponding normal tissues was observed (41). VEGFA was identified as a target of miR-126 (41). miR-126 inhibited proliferation of JH-Eso Ad1, OE19 and OE33 ESCC cells (41). In nude mice, tumor growth was inhibited in OE33 cells transfected with miR-126 (41). VEGFA can increase permeabilization of blood vessels and growth of new blood vessels (42, 43). Targeting of VEGFA with monoclonal antibodies has been pursued successfully in clinical trials, leading to approval for several tumor indications, but not ESCC (44).
miR-133b (targeting EGFR). miR-133b repressed proliferation, apoptosis, anchorage-independent growth and EMT of KYSE150 and ECA109 ESCCs and its expression was reduced in ESCC tissues (45). miR-133b inhibited tumor growth and lung metastases of KYSE150 and ECA109 ESCCs in nude mice (45). EGFR was identified as a target of miR-133b (45). miR-133b was shown to inhibit anchorage-independent growth, migration and invasion of ESCC via integrin β4 (INTB4)/focal kinase/growth-factor receptor bound protein 2, AKT serine/threonine kinase 1 and extracellular signal-regulated kinase signaling pathways (45). Gene amplification, aberrant activation and activation mutations of EGFR have been observed in several types of cancer (46, 47). EGFR has been identified as an important target for ESCC (48): 70-88% of patients with ESCC show high expression of EGFR and its high expression correlates with poor prognosis (49, 50).
miR-193a-5p [targeting human epidermal growth factor receptor 2 (HER2)]. miR-193a-5p inhibited HER2 expression and enhanced radiosensitivity of ESCCs in vitro and in vivo in mice (51). It reduced colony formation in KYSE70 and KYSE510 ESCCs and inhibited tumor growth by down-regulation of HER2 in vitro and in vivo (51). High expression of miR-193a-5p was correlated with successful chemoradiation treatment of ESCC (51). HER2 was also targeted in an orthotopic model of esophageal carcinoma (52). HER2 is up-regulated in 10-20% of ESCC and its up-regulation is correlated with worse prognosis (53-56).
miR-375 [targeting insulin-growth factor like 1 receptor (IGF1R)]. Down-regulation of miR-375 correlated with advanced stage, distant metastasis, poor overall survival and disease-free survival in patients with ESCC (57). Down-regulation of miR-375 in ESCC was due to promoter methylation (57). IGFR1 has been identified as a direct target of miR-375 (57). miR-375 suppressed proliferation and invasion of KYSE10 and KYSE30 ESCCs and their metastasis to the liver after tail vein injection into nude mice (57). In clinical specimens of ESCC, IGF1R expression was negatively correlated with miR-375 expression (57). Targeting of the IGF1R pathway has been pursued in many types of cancer (58). In ESCC, overexpression of IGF1R was noted in 60% of tumors in patients (59). IGF1R is a marker for prognosis and a potential therapeutic target for ESCC (60, 61). The IGF1R pathway has been identified as a key axis in progression of ESCC (62). Antitumoral effects have been observed with figitumumab, a monoclonal antibody directed against IGF1R in several preclinical in vitro and in vivo models of ESCC (63).
miR-17/20a [targeting transforming growth factor β receptor 2 (TGFRβ2) and SMAD anchor for receptor activation (SARA)]. Down-regulation of miR-17/20a correlated with ESCC lymph node metastasis and inhibited migration and invasion of 30D and 180-U ESCCs (64). miR-17/20a suppressed lung metastasis of 30D cells after tail vein injection into nude mice (64). miR-17/20a did not influence proliferation and apoptosis of ESCCs (64). TGFRβ2 and SARA were identified as bona fide targets of miR-17/20a (64). A heterotrimeric SMAD2, -3, -4 complex is translocated into the nucleus to regulate transcription of genes such as INTB6, which mediates migration and invasion (64, 65). The other target of miR-17/20a, SARA, recruits SMAD2/SMAD3 complexes to TGFRβ2 at intracellular membranes (66). However, TGFβ signaling can exert pro- as well as antitumoral effects in a context-dependent manner and therefore has to be investigated in further detail in ESCC (67, 68).
miR-92b (targeting INTαv). miR-92b inhibited lymph node metastasis and was associated with favorable prognosis in patients with ESCC (69). miR-92b inhibited migration and invasion of 30D ESCCs in vitro (69). In vivo, miR-92b inhibited invasion into the peri-esophageal muscle after orthotopic implantation of 30D cells into nude mice (69). In the tail vein injection model, miR-92b inhibited lung metastasis (69). INTαv has been identified as a bone fide target of miR-92b (69). Furthermore, miR-92b reduced phosphorylation of focal adhesion kinase and reduced activation of RAS-related C3 botulinum toxin substrate (RAC1), both essential mediators of cellular motility in ESCCs (69, 70). Integrins consist of 18α and 8β transmembrane receptors, inclusive 24 heterodimers, which can cross-link components of the extracellular matrix with the cytoskeleton and thus have an impact on invasion and metastasis of tumor cells (71, 72).
miR-34a [targeting cluster of differentiation 44 CD44)]. miR-34a was down-regulated in ESSC tissues and cell lines and inhibited invasion and migration of ECA109 and TE-13 ESCC cells (73). miR-34 inhibited tumor growth and metastasis in nude mice (73). CD44 was identified as a target of miR-34a (73). CD44 is a transmembrane protein with hyaluronic acid as the major ligand, resulting in activation of pathways involved in proliferation, survival, cytoskeletal changes, motility and metastasis (74, 75). CD44 standard form and several splice variants which contain additional peptide motifs that can interact with growth factors and cytokines at the cell surface may play a role in EMT and adaptive plasticity of cancer cells (76, 77). It has been shown that up-regulation of CD44 in E-cadherin-negative esophageal cancer cells results in activation of invasion (78). Tumor-initiating cells in ESCC express high levels of CD44 (79) and overexpression of CD44 is associated with poor prognosis in patients with ESCC (80).
miR-133a (targeting CD47). CD47 expression in ESCC was associated with lymph node metastasis (81). Expression of CD47 was significantly lower in ESCC samples in comparison to corresponding non-cancerous tissues (81). CD47 was identified as a target of miR-133a (81). Pre-miR 133a strongly inhibited the tumorigenic potential of TE-8 ESCC in vivo in nude mice (81). CD47 is a transmembrane molecule which inhibits phagocytosis and therefore is a target which has received much attention in translational oncology (82-84). It was shown that following blocking of CD47 with monoclonal antibodies, ESCCs were phagocytosed by M2 macrophages (85).
miR-377 (targeting CD133 and VEGF). Down-regulation of miR-377 correlated with poor chemotherapy response and poor survival in patients with ESCC (86). miR-377 inhibited tumor-initiating cell properties (86). CD133 and VEGF were identified as direct targets of miR-377 (86). In the tail vein injection model, miR-377 inhibited lung colonization of KYSE270 cells in mice (86). A systemic formulation of a miR-377 mimic inhibited tumor growth, angiogenesis and metastasis of ESCC cells in nude mice (86). CD133 is a marker of tumor-initiating cells in cancer, but its quantitation has been hampered by the inability of current antibodies to detect CD133 variants and deglycosylated epitopes (87, 88). CD133 is a regulator of metastasis through cancer stem cells (89). CD133 promotes stemness in ESCC cells and its higher expression is associated with lymph node metastasis, clinical stage and histopathological grade of ESCC (90, 91). As outlined previously, VEGFA is a validated target for several types of cancer (42).
miR-185 [targeting receptor for advanced glycation end-products (RAGE)]. Overexpression of miR-185 suppressed migration and invasion of TE-11 and ECA109 ESCCs in vitro (92). In the tail vein metastasis model, miR-185 suppressed metastasis of ECA109 cells (92). In patients with ESCC, the level of plasma miR-185 was found to be decreased (92). RAGE was identified as a target of miR-185 (92). RAGE, a 35-kDa transmembrane receptor of the immuno-globulin super-family, and its ligands, high motility group box 1 (HMGB1) and S100 group of proteins, release cytokines after their interaction. Transcription factors which are involved in TG and survival, EMT and metastasis are also activated. RAGE is being evaluated as a target forcancer therapy (93, 94). Expression levels of RAGE are related to poor prognosis in patients with ESCC (95).
miRs Targeting Transcription Factors
Up-regulated miRs (Figure 2A).
Up-regulated (A) and down-regulated (B) miRs targeting transcription factors in esophageal cancer cells in preclinical in vivo systems. AKT: Serine-threonine kinase AKT; ARID3A: AT-rich interactive domain-containing protein 3A; CXX5: CCX-type zinc finger protein 5; DDP res: cisplatin resistance; EMT: epithelial–mesenchymal transition; FOXK2: forkhead box protein K2; FOXO31: forkhead-box protein 31; FOXM1: forkhead box protein M1; GSK3β: glycogen synthase kinase 3β; INV: invasion; KLF4: Krüppel-like factor 4; MAPK: mitogen-activated protein kinase; MET: metastasis; MYC: transcription factor MYC; mTOR: mechanistic target of rapamycin; p38: mitogen-activated protein kinase p38; PITX2: paired-like homeodomain transcription factor 2; PPARγ: peroxisome proliferator-activated receptor γ; PROL: proliferation; SMAD4: signaling protein SMAD4; SNAI2: snail family transcriptional repressor 2; SOX4: sex determining region Y-box 4; STAT3: signal transducer and activator of transcription 3; TG: tumor growth; ZEB1: zinc finger E-box binding homeobox 2.
miR-7-5p [targeting Kruppel-like factor 4 (KLF4)]. miR-7-5p was up-regulated in ESCC tissues and inhibited proliferation of ECA109 and TE-9 ESCCs (96). A miR-7-5p antagonist induced apoptosis in ECA109 and TE-9 cells and suppressed migration and invasion of ECA109 cells (96). A miR-7-5p antagonist inhibited tumor growth of ECA109 cells in vivo (96). KLF4 was identified as a direct target of miR-7-5p (96). KLF4 is a member of the KLF family of zinc finger transcription factors and contains three C2H2 zinc fingers. KLF4 is a stem cell factor which, together with octamer-binding transcription factor 4, SOX2, homeobox protein NANOG and transcription factor MYC, is involved in the induction of pluripotent stem cells (97). Most studies suggest that KLF4 acts as a tumor suppressor, but pro-tumorigenic activity of KLF4 has also been reported (98). KLF4 promoted ESCC differentiation by up-regulation of keratin 13 expression (99). In ESCC, KLF4 was shown to act as a tumor suppressor and was correlated with good prognosis (100).
miR-10b [targeting peroxisome proliferator-activated receptor gamma (PPARγ)]. miR-10b was up-regulated in patients with ESCC and targeted PPARγ (101). Suppression of miR-10b in ESCC cell lines enhanced chemosensitivity to cisplatin in vitro and in vivo (101). PPARγ enhanced resistance to cisplatin by activating AKT/mechanistic target of rapamycin (mTOR)/ribosomal protein70 S6 kinase (p70S6K) signaling (101). PPARγ is a member of the nuclear receptor superfamily and was shown to act as a ligand-inducible transcription factor which can exert tumor-suppressive and pro-tumorigenic effects (102, 103). It has been shown that PPARγ ligands suppress proliferation and induce apoptosis of ESCCs by inhibiting toll-like receptor 4-dependent mitogen-activated protein kinase signaling (104). Reduced PPARγ expression is correlated with poor prognosis in patients with ESCC (105).
miR-32 (targeting CXXC5-type zinc finger protein 5). Increased expression of miR-32 has been noted in ESCC tissues and cells (106). Down-regulation of miR-32 inhibited migration and invasion of EC9706 and KYSE450 ESCCs (106). In vivo, miR-32 inhibitors reduce tumor weight and number of metastatic nodules (106). CXXC5-type zinc finger protein 5 (CXXC5) was identified as a target of miR-32 (85). miR-32 inhibitors reduced migration, invasion, EMT and TGFβ signaling (106). CXXC5 inhibited WNT/βcatenin signaling, induced apoptosis by stimulating TGFβ/SMAD signaling and activated DNA repair via the serine-threonine kinase ataxic telangiectasia mutated/p53 pathway (107-109). In addition, CXXC5 promoted TGFβ induced cell-cycle arrest (110). The role of CXXC5 in ESCC remains to be resolved in further detail.
miR-99b/let7a/miR-125a [targeting AT-rich interaction domain 3A (ARID3A)]. The miR-99b/let7a/miR-125a cluster promoted ESCC migration and invasion in vitro and induced experimental metastases in vivo (111). Transcription factor zinc finger E-box binding homeobox factor 1 (ZEB1) bound to the promoter of this cluster and regulated its transcription (111). ARID3A was identified as a direct target of miR-99b/let7a/miR-125a (111). ARID3A is a transcription factor which regulates chromatin accessibility, proliferation, differentiation and is critical for B-cell development (112). ARID3A expression was found to be reduced in ESCC compared to normal corresponding tissues (113). ARID3A has been shown to be involved in carcinogenesis and development of ESCC (114).
miR-602 [targeting forkhead box protein K2 (FOXK2)]. miR-602 was increased in ESSC tissues and its expression level significantly correlated with poor survival (115). DNA hypomethylation was involved in increased expression of miR-602 in ESCC (115). miR-602 promoted proliferation, colony formation, migration and invasion by KYSE 150 and KYSE 450 ESCCs (115). In KYSE 450 cells transfected with miR-602, tumor growth was significantly accelerated (115). miR-602 antagomir directly injected into the tumor led to slower tumor growth (115). In the tail vein assay, miR-602 induced increased metastasis to the lungs, to liver, bones and adrenal glands (115). FOXK2 was identified as a target of miR-602 (116). FOXK2 binds to purine-rich motifs on DNA and is involved in proliferation, survival, DNA damage, metabolism, invasion, migration and metastasis. However, the function of FOXK2 is context-dependent and depends on the tumor type (117). Down-regulation of FOXK2 was associated with poor prognosis in patients with gastric cancer (118).
Down-regulated miRs (Figure 2B).
miR-124 [targeting signal transducer and activator of transcription 3 (STAT3)]. miR-124 was suppressed in ESCC tissues and cell lines, inhibited proliferation and migration and induced apoptosis in ECA190 and TE-1 ESCCs (119). In ECA109 engrafted tumors, miR-124 inhibited growth (119). STAT3 was identified as a direct target of miR-124 (119). Activation of STAT3 by phosphorylation leads to its dimer formation, translocation into the nucleus, recognition of STAT3 binding elements and transcriptional activation of target genes. STAT3 regulates expression of genes related to the cell cycle, survival and the immune response, and is associated with progression and malignancy in several types of cancer (120, 121). STAT3 is being evaluated as a potential target for treatment of cancer (122). It has been shown that STAT3 is constitutively activated in ESCC tissues (123), inhibits apoptosis (124) and mediates proliferation and migration of ESCCs (125).
miR-130-3p (targeting SMAD4). miR-130a-3p directly targeted SMAD4 in EC-1 ESCCs (126). miR-130a-3p inhibited tumor axillary lymph node metastasis in nude mice (126). miR-130a-3p inhibited EMT, invasion, and migration induced by TGFβ in EC-1 cells (126). Restoration of SMAD4 expression rescued miR-130a-3p-suppressed EMT, invasion and migration (126). SMAD4 forms heterotrimeric complexes with SMAD2 and SMAD3 which are translocated into the nucleus and regulate expression of selected genes (127). SMAD4 is required for TGFβ-induced EMT (128). In healthy and early-stage cancer cells, TGFβ signaling has tumor-suppressive functions; in late-stage cancer, it can promote tumor progression, metastasis and chemotherapy resistance (129, 130).
miR-133a (targeting SOX4). miR-133a was down-regulated in ESCC tissues and cell lines KYSE150, KYSE510, EC9706 and TE13 in comparison to SHEE non-transformed esophageal cell line (131). miR-133a inhibited proliferation, migration and invasion of ESCCs as mentioned above (131). In vivo, miR-133a inhibited tumorigenicity of TE13 cells in a mouse xenograft model (131). SOX4 was identified as a direct target of miR-133a (131). SOX4 levels inversely correlated with miR-133a in ESCC tissues (131). SOX4 is up-regulated in many types of cancer and contributes to cellular transformation, cell survival and metastasis (132, 133). SOX4 was found to induce WNT signaling and EMT (134, 135). The role of SOX4 in ESCC remains to be explored in further detail.
miR-145 (targeting MYC). miR-145 was down-regulated in ESCC tissues and suppressed proliferation, invasion and tumor growth of ESCC xenografts by targeting MYC (136). MYC is a transcription factor which increases proliferation, cell-growth and inactivates cell-cycle inhibitors (137-139). MYC can be amplified in ESCC and its high expression is significantly correlated with poor prognosis in patients with ESCC (140, 141). Compounds identified by several MYC inhibition approaches are close to clinical trials (142).
miR-150 (targeting ZEB1). Low expression of miR-150 contributed to malignant potential, lymph node metastasis, lymphatic and venous invasion, clinical staging and poor prognosis of patients with ESCC (143). miR-150 inhibited proliferation, migration and regulated morphology of TE-8 ESCCs (143). In a TE-8 xenograft model, miR-150 inhibited tumor growth (143). ZEB1 was identified as a target of miR-150 (143). ZEB1 contains seven zinc-fingers and one homeodomain and is a transcription factor that controls EMT (144). Aberrant expression of ZEB1 fosters invasion, migration and metastasis (145). It has been shown that ZEB1 promotes invasiveness of ESCC and confers an unfavorable prognosis in patients with ESCC (146).
miR-204 (targeting FOXM1). miR-204 was down-regulated in ESCC tissues in comparison to corresponding normal tissues (147). miR-204 inversely regulated EMT in EC109 and TE10 ESCCs (147). Overexpression of miR-204 suppressed growth of EC109 cells in vivo (147). FOXM1 was identified as a direct target of miR-204 (147). FOXM1 is a member of the family of FOXO transcription factors which are deregulated in many diseases, including cancer (148). FOXM1 is required for proliferation of many normal cells and as a master regulator is implicated in all hallmarks of cancer (149). FOXM1 is overexpressed in many types of tumors. Its oncogenic role is mediated by interaction with β-catenin and SMADs to induce WNT and TGFβ signaling pathways (150). Silencing of FOXM1 inhibited proliferation and migration of ESCCs (151). In ESCC, FOXM1 can activate phosphoinosite-3 kinase (PI3K)/AKT signaling and its expression was correlated with poor prognosis (151).
miR-507 [targeting nuclear factor erythroid 2-related factor 2 (NRF2)]. Administration of miR-507 alone or in combination with cisplatin inhibited tumor growth in vivo (152). NRF2, a basic leucine zipper protein transcription factor, was identified as a target of miR-507 (153). Under high oxidative stress levels, the NRF2 pathway is activated. Normally, NRF2 is degraded by Kelch-like-ECH-associated protein (KEAP1) and Cullin3 through ubiquitinylation. Non-ubiquitinylated NRF2 is translocated into the nucleus, combines with musculoaponeurotic fibrosarcoma transcription factors and binds to anti-oxidative response elements in the promoter regions of anti-oxidative stress genes (153). Tumor-suppressive as well as oncogenic functions have been assigned to NRF2 (154-156). In NRF2-addicted cancer, NRF2 is constitutively activated due to somatic mutations in KEAP1 or NRF2 and other mechanisms that disrupt binding of KEAP1 to NRF2 (157). In ESCC, NRF2 has been shown to induce proliferation and to be associated with radio- and chemotherapy resistance (158, 159).
miR-630 [targeting snail family transcriptional repressor 2 (SNAI2)]. Reduced miR-630 expression was associated with poor survival in patients with ESCC (160, 161). Transcription factor SNAI2, also known as SLUG, has been identified as a direct target of miR-630 (160). Ectopic expression of miR-630 inhibited proliferation, invasion, EMT and metastasis of ESCC cell lines in vitro and in vivo. SNAI2 plays a role in EMT and mediates anti-apoptotic activity (162, 163). Independently it has been shown that its down-regulation by RNA interference inhibited invasion and growth of ESCC (164).
miR-664a [targeting paired-like homeodomain transcription factor 2 (PITX2)]. Low expression of miR-664a correlated with tumor recurrence or metastasis in patient samples (165). miR-664a overexpression in KYSE-140 and -109 ESCCs reduced cell growth, colony formation, migration and invasion in vitro (165). Tumor growth in vivo was inhibited by miR-664 in KYSE-140 and ECA109 cells in immunocompromised mice (165). The number and size of lung metastases of these cell lines in mice were dramatically reduced by miR-664 (165). PITX2 was identified as a direct target of miR-664a (165). miR-664a was down-regulated by promoter hypermethylation and inhibited the WNT/β catenin pathway by targeting PITX2 in ESCCs (165). PITX2 acts as a transcription factor and participates in muscle formation (166, 167). Up-regulation of miR-664a reduced ESCC cell stem-like traits (168).
miRs Targeting Metabolic Enzymes (Figure 3)
Down-regulated miRs targeting enzymes in esophageal cancer cells mediating efficacy in preclinical in vivo systems DNA polβ: DNA polymerase β; LDHB: lactate dehydrogenase B; MET: metastasis; MTDH1: metadherin 1; OGT: O-linked-N-acetylglucosaminacetylation transferase; PROL: proliferation; PSAT1: phosphoserine aminotransferase 1; INV: invasion; TG: tumor growth; TIGAR: p53-inducible glycolysis and apoptosis regulator; USP26: ubiquitin-specific protease 26.
miR-144 [targeting p53-inducible glycolysis and apoptosis regulator (TIGAR)]. miR-144 was down-regulated in patients with ESCC and correlated with poor prognosis (169). Proliferation of EC9706 and ECA109 ESCCs was inhibited by miR-144, which was associated with a pro-apoptotic effect (169). In vivo, miR-144 inhibited tumor growth of xenografts of these cell lines (169). TIGAR was identified as a direct target of miR-144 (169). TIGAR acts as a fructose-2,6 biphosphatase and as a regulator of glucose breakdown (170). In some types of cancer, TIGAR is aberrantly up-regulated and promotes carcinoma growth by metabolic intermediates derived from the pentose phosphate pathway (171). TIGAR also reprograms glucose metabolism from glycolysis to the glutamine pathway through AMP-activated kinase (171). In TIGAR-overexpressing xenografts and patient-derived xenografts, efficacy was significantly enhanced when a glutaminase inhibitor was combined with chemotherapy agents (172).
miR-203a-5p [targeting ubiquitin-specific peptidase 26 (USP26)]. miR-203-5p was significantly down-regulated in esophageal tumor tissue (173). Overexpression of miR-203a-5p inhibited invasion and migration of KYSE150 and TE-1 ESCCs (173). Nude mice injected with a miR-203-5p mimic showed reduced lung metastasis of KYSE150 cells after tail vein injection (173). miR-203a-5p targets USP26 which stabilizes EMT-related transcription factor SNAI1 (173). In normal tissues, USP26 is exclusively expressed in testis (174). USPs are critical for cancer progression and several approaches to develop inhibitors of USPs for treatment of cancer are being pursued (175-177). It was shown that USP26 promotes ESCC by stabilizing SNAI1 (178).
miR-340 and miR-365 [targeting phosphoserine aminotransferase 1 (PSAT1)]. Both these miRs were down-regulated in ESCC cell lines EC1, EC109, EC9706 and in ESCC tissues (179, 180). Both inhibited invasion of EC9706 and EC109 ESCCs (179, 180). miR-340 as well as miR-365 transfectants reduced growth of EC9706 xenografts in nude mice (179, 180). miR-340 and miR-365 inhibited EMT by up-regulation of E-cadherin, down-regulation of SNAI1 and vimentin, and reduction of phosphorylated glycogen synthase kinase 3β (179, 180). PSAT1, involved in serine biosynthesis, is amplified in a significant subset of tumors and RNAi directed against PSAT1 reduced cancer cell survival and growth; therefore, discovery of PSAT1 inhibitors as anticancer agents is being pursued (181). PSAT1 plays a crucial role in development of ESCC and its expression predicts poor survival (182).
miR-375 [targeting lactate dehydrogenase B and metadherin (MTDH)]. miR-375 was down-regulated in ESCC clinical specimens in comparison to neighboring normal tissue sections, and a low level of miR-375 was associated with poor prognosis (183). miR-375 inhibited proliferation and migration of TE2 and T.Tn ESCCs (183). Lactate dehydrogenase B (LDHB) and MTDH were identified as direct targets of miR-375 (183). A miR-375/atelocollagen complex administered subcutaneously suppressed growth of TE2 and T.Tn xenografts in nude mice (184). miR-375 target LDHB converts lactose to pyruvate, which is further oxidized and is critical for mTOR-mediated tumorigenesis (185). LDHB also controls apoptosis and autophagy in tumor cells (186). The other miR-375 target, MTDH, functions as a regulator of EMT in carcinomas (187). It plays a role in tumor progression, invasion, resistance to chemotherapy in many carcinomas including ESCC. MTDH is required for proliferation, migration and invasion of ESCC (188, 189).
miR-485-5p [targeting O-linked N-acetylglucosamine transferase (OGT)]. miR-485-5p was expressed at low levels in ESCC cell lines and inhibited cell proliferation, migration and invasion in TE-1 and ECA109 ESCCs (190). Tumor growth in nude mice was inhibited in TE-1 cells overexpressing miR-485-5p (190). OGT was identified as a direct target of miR-485-5p (190). OGT catalyzes addition of N-acetylglucosamine through O-glycosidic linkage to serine or threonine and an S-glycosidic linkage to cysteine (191, 192). OGT is aberrantly expressed in cancer and modifies signaling proteins, transcription factors, metabolic enzymes, histones and chromatin regulators (193).
miR-499 (targeting DNA polymerase β). DNA polymerase β has been identified as the target of miR-499 in EC9706 and KYSE30 ESCCs (194). miR-499 inhibited proliferation, induced apoptosis and reduced the DNA-repair capacity of EC9706 and KYSE30 ESCCs by targeting DNA polymerase β (194). miR-499 also inhibited tumor growth of xenografts derived from these cell lines in nude mice (194). DNA polymerase β is essential for short-patch base excision repair. DNA polymerase β overexpression resulted in aneuploidy and tumorigenesis in nude immunodeficient mice (195, 196). Targeting DNA polymerase β is considered as an option for cancer therapy (197).
Additional Targets
In addition miR-34a and miR-133b should be mentioned. They target phospholipase epsilon 1 (PLCE1) (198) and squalene epoxidase (SQE) (199), respectively. SQE is a rate-limiting enzyme in cholesterol biosynthesis (199). In vivo activity in ESCC-related preclinical xenografts was demonstrated with the corresponding targets, not with miR-34a and miR-133b.
Concluding Remarks
Up-regulated miRs indicate targets which have to be reconstituted with low-molecular-weight compounds or inhibited by miR antagonists such as locked nucleic acids and antagomirs (200). All antagonists are oligonucleotides with sequences complementary to endogenous miRs (200-203). Down-regulated miRs identify targets which can be inhibited by small molecules or antibody-related moieties, or which can be re-expressed by replacement therapy with miR mimetics or vector-based expression (200-203). miR mimetics are double-stranded RNAs which reconstitute the function of the corresponding miRs.
When deregulated miRs were grouped into categories as secreted factors and transmembrane receptors, transcription factors and metabolic enzymes, for secreted factors and transmembrane receptors, four miRs were up-regulated and 10 were down-regulated in ESCC tissues in comparison to matching normal tissues (Figure 1). VEGF (miR-126), EGFR (miR-133b), HER2 (miR-193a), IGF1R (miR-375) and CD47 (133a) seem to be the most promising targets for inhibition with small molecules and biological agents or reconstitution of the corresponding miRs.
In the category of transcription factors, we identified five miRs up-regulated and nine down-regulated with efficacy in preclinical in vitro and in vivo systems (Figure 2). Drugs targeting transcription factors such as estrogen receptor and androgen receptor are among the most impactful drugs in oncology (5). STAT3 (miR-124), SMAD4 (miR-130-3p), SOX4 (miR-122a), MYC (miR-145), ZEB1 (miR-150), FOXM1 (miR-204), NRF2 (miR-507), SLUG (miR-630) and PITX2 (miR-664) have emerged as potential targets for inhibition at the protein level or reconstitution of the corresponding miRs. A subset of ESCCs is addicted to NRF2 this aspect is worth further detailed investigation (156). In general, targeting transcription factors is associated with technical issues of tractability with respect to inhibition and specificity, as well as toxicity issues. Inhibitory efforts may target protein–protein interactions with co-factors, inhibition of transcription factor DNA binding, inhibition of expression of regulators of transcription factors, altering levels of ubiquitinylation and subsequent proteolysis (204-208). Proteolysis targeting chimeras are under active investigation, as well as cysteine reactive inhibitors, which target intrinsically disordered domains of transcription factors (204-208).
Six miRs targeting enzymes were down-regulated (Figure 3). TIGAR (miR-144), PSAT1 (miR-340 and miR-365) and LDHB (miR-375) are metabolic enzymes. These targets and their corresponding miRs deserve further validation for treatment of ESCC. Transformed cells adopt metabolism and support tumor initiation and progression and therefore become addicted to these changes (209-211). Drugs targeting metabolic enzymes such as the dihydrofolate reductase inhibitor methotrexate and thymidylate inhibitor 5-fluorouracil are well established in cancer therapy.
Inhibition of miRs and replacement therapy face several technical problems which are not discussed in this review. Among these issues are: delivery of the agents to tumor tissues, optimization of their escape after internalization, inappropriate biodistribution and optimization of their pharmaco-kinetic and pharmaco-dynamic properties (211-213). Further critical issues are off-site effects and cytokine-release syndrome (213). Recently the field has witnessed several drawbacks (214). However, cobomarsen, an oligonucleotide-based inhibitor of miR-155, was shown to slow growth of diffuse large cell B-cell lymphoma xenografts without any toxic effects and is currently being evaluated in several clinical studies in patients with hematological malignancies and seems to be well tolerated (215, 216). Nevertheless, clinical proof-of-concept of miR-based therapies is still pending.
Footnotes
Authors’ Contributions
AN and UHW jointly designed and prepared the article.
Conflicts of Interest
AN is and UHW was an employee of Roche.
- Received November 23, 2021.
- Revision received January 17, 2022.
- Accepted January 19, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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