Dual inhibition of cMET and EGFR by microRNA-338-5p suppresses metastasis of esophageal squamous cell carcinoma.

MicroRNAs, as a group of post-transcriptional regulators, regulate multiple pathological processes including metastasis during tumor development. Here, we demonstrated the metastasis-suppressive function of microRNA (miR)-338-5p in esophageal squamous cell carcinoma (ESCC). Overexpression of miR-338-5p had inhibitory effect on invasive ability of ESCC cells and extracellular matrix degradation, whereas silencing miR-338-5p had opposite effects. Mechanistically, miR-338-5p directly targeted the 3' untranslated regions of hepatocellular growth factor receptor cMet (cMET) and epidermal growth factor receptor (EGFR). As a result, miR-338-5p inhibited the downstream signaling cascades of cMET and EGFR and repressed cMET- and EGFR-mediated ESCC cell invasion. Re-expression of cMET or EGFR in miR-338-5p overexpressing ESCC cells was sufficient to derepress ESCC cell invasion both in vitro and in vivo. We further showed that such manipulation downregulated the expression and secretion of matrix metalloproteinases 2 and 9, which resulted in impaired extracellular matrix degradation and cell invasion. Most importantly, systemic delivery of miR-338-5p mimic significantly inhibited metastasis of ESCC cells in nude mice. Taken together, our results uncovered a previously unknown mechanism through which miR-338-5p suppresses ESCC invasion and metastasis by regulating cMET/EGFR-matrix metalloproteinase 2/9 axis and highlighted the potential significance of miR-338-5p-based therapy in treating patients with metastatic ESCC.

FBX4 mediates rapid cyclin D1 proteolysis upon DNA damage in immortalized esophageal epithelial cells.

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

MicroRNA-338-5p reverses chemoresistance and inhibits invasion of esophageal squamous cell carcinoma cells by targeting Id-1.

5-Fluorouracil (5-FU) is a chemotherapeutic agent commonly used to treat esophageal squamous cell carcinoma (ESCC), but acquisition of chemoresistance frequently occurs and the underlying mechanisms are not fully understood. We found that microRNA (miR)-338-5p was underexpressed in ESCC cells with acquired 5-FU chemoresistance. Forced expression of miR-338-5p in these cells resulted in downregulation of Id-1, and restoration of both in vitro and in vivo sensitivity to 5-FU treatment. The effects were abolished by reexpression of Id-1. In contrast, miR-338-5p knockdown induced 5-FU resistance in chemosensitive esophageal cell lines, and knockdown of both miR-338-5p and Id-1 resensitized the cells to 5-FU. In addition, miR-338-5p had suppressive effects on migration and invasion of ESCC cells. Luciferase reporter assay confirmed a direct interaction between miR-338-5p and the 3'-UTR of Id-1. We also found that miR-338-5p was significantly downregulated in tumor tissue and serum samples of patients with ESCC. Notably, low serum miR-338-5p expression level was associated with poorer survival and poor response to 5-FU/cisplatin-based neoadjuvant chemoradiotherapy. In summary, we found that miR-338-5p can modulate 5-FU chemoresistance and inhibit invasion-related functions in ESCC by negatively regulating Id-1, and that serum miR-338-5p could be a novel noninvasive prognostic and predictive biomarker in ESCC.

Overexpression of F-box only protein 31 predicts poor prognosis and deregulates p38α- and JNK-mediated apoptosis in esophageal squamous cell carcinoma.

F-box only protein 31 (FBXO31), a subunit of the Skp1-Cul1-F box ubiquitin ligase, plays a crucial role in DNA damage response and tumorigenesis. Yet its expression and function vary in different types of human cancer. The expression of FBXO31 in esophageal squamous cell carcinoma (ESCC) and its association with clinicopathological features is not well studied. The underlying mechanism by which deregulated FBXO31 contributes to ESCC tumorigenesis is largely unknown. By immunohistochemical analysis of a tissue microarray containing 85 cases of ESCC and matched adjacent noncancerous tissue and an additional 10 cases of ESCC tissue samples, we found that FBXO31 was overexpressed in ESCC, and that its expression was significantly correlated with histological grade (p = 0.04) and clinical stage (p = 0.022). Higher expression of FBXO31 was associated with poor prognosis in univariate (p = 0.013) and multivariate (p = 0.014) analyses. We found that FBXO31 functioned as an antiapoptotic molecule in ESCC cells exposed to different types of genotoxic stress. Knockdown of FBXO31 inhibited serum-starved cell viability and decreased tumorigenicity of ESCC cells. In addition, the antiapoptotic effects of FBXO31 were associated with deactivation of stress-induced MAPK p38α and JNK. Furthermore, in vitro and in vivo data showed that silencing of FBXO31-sensitized ESCC cells and tumors to cisplatin treatment. Taken together, in addition to revealing that FBXO31 is an independent prognostic marker for ESCC, our findings substantiate a novel regulatory role of FBXO31 in tumorigenesis and drug resistance of ESCC.

F-box only protein 31 (FBXO31) negatively regulates p38 mitogen-activated protein kinase (MAPK) signaling by mediating lysine 48-linked ubiquitination and degradation of mitogen-activated protein kinase kinase 6 (MKK6).

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1 · Cul1 · F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.

Epstein-Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage.

Epstein-Barr virus (EBV) infection is closely associated with several human malignancies including nasopharyngeal carcinoma (NPC). The EBV immediate-early protein BZLF1 is the key mediator that switches EBV infection from latent to lytic forms. The lytic form of EBV infection has been implicated in human carcinogenesis but its molecular mechanisms remain unclear. BZLF1 has been shown to be a binding partner of several DNA damage response (DDR) proteins. Its functions in host DDR remain unknown. Thus, we explore the effects of BZLF1 on cellular response to DNA damage in NPC cells. We found that expression of BZLF1 impaired the binding between RNF8 and MDC1 (mediator of DNA damage checkpoint 1), which in turn interfered with the localization of RNF8 and 53BP1 to the DNA damage sites. The RNF8-53BP1 pathway is important for repair of DNA double-strand breaks and DNA damage-induced G2/M checkpoint activation. Our results showed that, by impairing DNA damage repair as well as abrogating G2/M checkpoint, BZLF1 induced genomic instability and rendered cells more sensitive to ionizing radiation. Moreover, the blockage of 53BP1 and RNF8 foci formation was recapitulated in EBV-infected cells. Taken together, our study raises the possibility that, by causing mis-localization of important DDR proteins, BZLF1 may function as a link between lytic EBV infection and impaired DNA damage repair, thus contributing to the carcinogenesis of EBV-associated human epithelial malignancies.

p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells.

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21.

Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer.

Rationale: Little is known about the roles of proteoglycans in esophageal cancer. This study aims to investigate the roles and mechanisms of serglycin (SRGN) proteoglycan in promoting metastasis of esophageal squamous cell carcinoma (ESCC). Methods: Reverse phase protein array analysis was used to identify activated signaling pathways in SRGN-overexpressing cells. Chemokine array was used to identify differentially secreted factors from SRGN-overexpressing cells. Binding between SRGN and potential interacting partners was evaluated using proximity ligation assay and co-immunoprecipitation. The glycosaminoglycan (GAG) chains of SRGN were characterized using fluorophore-assisted carbohydrate electrophoresis. Tissue microarray and serum samples were used to determine the correlation of SRGN expression with clinicopathological parameters and patient survival. Results: In vitro and in vivo experiments showed that SRGN promoted invasion and metastasis in ESCC via activating ERK pathway, stabilizing c-Myc and upregulating the secretion of matrix metalloproteinases. SRGN-knockdown suppressed tumorigenic hallmarks. These SRGN-elicited functions were carried out in an autocrine manner by inducing the secretion of midkine (MDK), which was further identified as a novel binding partner of SRGN for the formation of a SRGN/MDK/CD44 complex. In addition, SRGN interacted with MDK and matrix metalloproteinase 2 in ESCC via its GAG chains, which were mainly decorated with chondroitin sulfate comprising of ∆di-4S and ∆di-6S CS. Clinically, high expression of serum SRGN in serum of patients with ESCC was an independent prognostic marker for poor survival. Conclusions: This study provides the first evidence that elevated serum SRGN has prognostic significance in patients with ESCC, and sheds light on the molecular mechanism by which elevated circulating SRGN in cancer patients might promote cancer progression.

Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

Id-1 (inhibitor of differentiation or DNA binding) is a helix-loop-helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id-1 expression activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id-1 and phospho-AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id-1 in esophageal cancer progression, ESCC cells with stable ectopic Id-1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki-67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id-1-overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id-1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id-1 expression altered the expression levels of markers associated with epithelial-mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id-1-overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT-dependent modulation of matrix metalloproteinase-9 expression. In conclusion, our results provide the first evidence that Id-1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

MAD2 expression and its significance in mitotic checkpoint control in testicular germ cell tumour.

Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p<0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p=0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.

Telomere dysfunction, genome instability and cancer.

Telomeres are highly specialized structures at the ends of chromosomes that are made up of tandem 5'-TTAGGG-3' repeats and a number of telomere associated proteins. By forming loop structures, the very end of a telomere is concealed and distinguished from a DNA break, thus protecting chromosomes from end-to-end fusions, misrepair and degradation. Telomere length is maintained by an enzyme called telomerase which is very weak or undetectable in most normal human somatic cells. In telomerase-negative cells, telomeric DNA is progressively lost with cell divisions until the cells undergo replicative senescence, which serves as an intrinsic mechanism to prevent normal somatic cells from replicating indefinitely. In checkpoint defective cells, telomere dysfunction resulting from excessive telomere attrition or disruption of telomere structure may initiate chromosomal instability through end-to-end fusion of unprotected chromosomes. Through propagation of breakage-fusion-bridge (BFB) cycles, genetic aberrations characteristic of cancers, including aneuploidy, loss of heterozygosity, gene amplification and gene loss can be generated. In vitro, cells with extensive chromosomal instability succumb to crisis which is characterized by wide-spread cell death. It has been reported that cells surviving crisis either have activated telomerase, or use an alternative telomere lengthening (ALT) mechanism to stabilize the existing telomeres and alleviate chromosome instability. The immortalized post-crisis cells have the potential to acquire additional genetic alterations for malignant transformation. In this review, we summarize our knowledge on the association between telomere dysfunction, genomic instability and cancer development.

IGF2 induces CD133 expression in esophageal cancer cells to promote cancer stemness.

Failure to eradicate cancer stem cells (CSC) during primary therapy may lead to cancer recurrence. We recently reported that CD133 is a functional biomarker for CSCs in esophageal squamous cell carcinoma (ESCC) but the molecular pathways critical for maintenance of CD133-positive CSCs are largely unknown. Here, we revealed that knockdown of IGF2 or treatment with PI3K/AKT inhibitors markedly inhibited the abilities of CD133-positive ESCC cells to self-renew, resist chemotherapeutic drugs, and form tumors. Further functional analysis identified miR-377 as a downstream regulator of PI3K/AKT signaling, and a mediator of the effects of IGF2 on CD133 expression and CSC properties. We found that the expression levels of IGF2 and CD133 were positively correlated with each other in primary ESCC, and that concurrent elevation of IGF2 and CD133 expression was significantly associated with poor patient survival. Furthermore, in vivo experiments demonstrated that IGF2-neutralizing antibody enhanced the sensitivity of tumor xenografts in nude mice to 5-fluorouracil treatment. This study underpins the importance of the IGF2-PI3K/AKT-miR-377-CD133 signaling axis in the maintenance of cancer stemness and in the development of novel therapeutic strategy for treatment of esophageal cancer.

Id-1 activation of PI3K/Akt/NFkappaB signaling pathway and its significance in promoting survival of esophageal cancer cells.

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

Significance of PI3K/AKT signaling pathway in metastasis of esophageal squamous cell carcinoma and its potential as a target for anti-metastasis therapy.

Metastasis is the most lethal hallmark of esophageal squamous cell carcinoma (ESCC). The aim of the study is to identify key signaling pathways that control metastasis in ESCC. Highly invasive ESCC sublines (designated I3 cells) were established through three rounds of selection of cancer cells invading through matrigel-coated chambers. Gene expression profile of one of the I3 sublines was compared with that of its parental cell line using cDNA microarray analysis. Gene ontology and pathway analyses of the differentially expressed genes (both upregulated and downregulated) indicated that genes associated with cellular movement and the AKT pathway were associated with increased cancer cell invasiveness. Western blot analysis confirmed increased phosphorylated AKT (p-AKT), N-cadherin and decreased E-cadherin expression in the I3 cells. Immunohistochemistry was used to evaluate the clinical significance of p-AKT expression in ESCC, and the results showed higher p-AKT nuclear expression in lymph node metastases when compared with primary carcinoma. Inactivation of the PI3K/AKT pathway with specific inhibitors, or with PTEN overexpression, resulted in reversed cadherin switching and inhibited cancer cell motility. Inhibition of the pathway by treatment with wortmannin markedly suppressed experimental metastasis in nude mice. Our data demonstrated the importance of the PI3K/AKT signaling pathway in ESCC metastasis and support PI3K/AKT as a valid therapeutic target in treatment of metastatic ESCC.

Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression.

Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression.

Significance of MAD2 expression to mitotic checkpoint control in ovarian cancer cells.

Chromosome instability is a commonly observed feature in ovarian carcinoma. Mitotic checkpoint controls are thought to be essential for accurate chromosomal segregation, and MAD2 is a key component of this checkpoint. In this study, we investigated the competence of the mitotic checkpoint and its relationship to the expression of MAD2 protein in seven ovarian cancer cell lines. We found that a significant number (43%, three of seven cell lines) of the tested ovarian cancer cells failed to arrest in the G(2)-M phase of the cell cycle in response to microtubule disruption. This loss of mitotic checkpoint control was associated with reduced expression of the MAD2 protein. To additionally understand the significance of the MAD2 to mitotic checkpoint control, we established an inducible expression system in which MAD2 was induced by the addition of ponasterone A. Notably, the induced expression of MAD2 in two checkpoint-defective ovarian cancer cell lines led to the restoration of mitotic checkpoint response to spindle-disrupting agents. Taken together, our findings suggest that the steady-state amount of MAD2 inside cells may represent a molecular switch for mitotic checkpoint control. This provides a novel insight into the molecular basis of CIN in ovarian carcinoma and has implications for effective use of checkpoint-targeting drugs.

Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression.

Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer.

Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance.

PURPOSE: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. EXPERIMENTAL DESIGN: In vitro and in vivo functional assays were performed to study the effects of Id1-E2F1-IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. RESULTS: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. CONCLUSIONS: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1-E2F1-IGF2 regulatory axis has important implications for cancer prognosis and treatment.