Mitsuteru Natsuizaka

Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors.

Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.

A NOTCH3-Mediated Squamous Cell Differentiation Program Limits Expansion of EMT-Competent Cells That Express the ZEB Transcription Factors

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-β-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.

NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.

BACKGROUND & AIMSnThe Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation, and proliferation in the hematopoietic system, the intestine, the pancreas, and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology are unknown.nnnMETHODSnNormal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic 3-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like 1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation assays, quantitative reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, and immunohistochemistry.nnnRESULTSnNOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia.nnnCONCLUSIONSnNotch signaling is important for esophageal epithelial homeostasis. In particular, the cross talk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.

Hypoxia activates the cyclooxygenase-2–prostaglandin E synthase axis

Hypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1alpha target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1alpha by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1alpha. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E(2) (PGE(2)) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE(2) production in a HIF-1alpha-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1alpha and IGFBP3. Activation of the COX-2-PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1beta and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1alpha target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.

Insulin-like growth factor-binding protein-3 promotes transforming growth factor-β1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells

Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-beta1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2-hTERT-EGFR-p53(R175H). In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-beta target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-beta1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-beta1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3(I56G/L80G/L81G), a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-beta1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3(I56G/L80G/L81G) also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-beta1-mediated EMT and cell invasion.

Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4

Barretts esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

Abstract 70: Loss of cellular senescence checkpoint functions reveals the oncogene characteristics of Notch1 in squamous cell carcinomas

Introduction: Notch signaling regulates cell fates that are dependent upon the CSL transcription factor. Both tumor suppressor and oncogenic roles of Notch have been implicated in the pathogenesis of squamous cell carcinomas (SCCs). We investigated the functional consequences of Notch activation in head and neck as well as esophageal SCCs (HNSCC and ESCC). Methods: Primary tumor tissues annotated with known clinical outcomes were analyzed. Human esophageal cells immortalized with telomerase or human papilloma virus E6/E7 genes, their derivatives expressing mutant p53 and ESCC cell lines were stably transduced with ICN1, an active form of Notch1 in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or γ-secretase inhibitors (GSI). 8xCSL-luciferase reporter was used to assess Notch-mediated transcriptional activity. RNA interference was done targeting either CSL or p16INK4A. Senescence was determined by cell growth inhibition and senescence-associated β-galactosidase assays. Cell growth was tested in soft agar and immunodeficient mice. Gene expression was determined by quantitative RT-PCR, Western blotting and immunohistochemistry (IHC). Results: In primary tumors, the active form of Notch1 (ICN1Val1744) was detected as intense nuclear staining of tumor cells in the invasive fronts in 29% of HNSCC (n=17) and 56% of ESCC (n=171). Nuclear ICN1Val1744 was significantly associated with a poor 5-year survival in postsurgical ESCC patients (n=115). In culture, ICN1 induced cellular senescence through CSL-dependent p16INK4A induction, which was antagonized by DNMAML1 or knockdown of either CSL or p16INK4A. Moreover, ICN1 failed to induce senescence in immortalized cells expressing E6/E7 or ESCC cells with a deleted INK4 locus. However, p53 mutation did not prevent ICN1-induced senescence. In soft agar and xenograft transplantation, ICN1 stimulated colony formation and tumor growth of ESCC cells that negated senescence. Histology revealed an increased number of less-differentiated tumor cells upon ICN1 induction. Moreover, GSI treatment of mice with xenografted ESCC cells resulted in tumor necrosis. Conclusions: These data indicate that Notch activation contributes to disease progression in ESCC. While ICN1 induces senescence, loss of p16INK4A-mediated senescence checkpoint point functions may be required in ICN1-mediated malignant transformation, thus providing a novel mechanistic insight into how Notch signaling may contribute to the pathogenesis of SCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 70. doi:1538-7445.AM2012-70

Abstract 2293: ZEB1 and ZEB2 promote EMT and invasion in esophageal squamous cell carcinoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnIntroduction: Epithelial-mesenchymal transition (EMT) promotes tumor invasion and metastasis. Amongst transcription factors essential in EMT, zinc finger E-box binding proteins ZEB1 and ZEB2 (ZEB) are upregulated in several tumor types due to loss of the miR-200 family. However, the role of ZEB in esophageal squamous cell carcinoma (ESCC) remains to be elucidated. Methods: ESCC cell lines were grown. Immortalized human esophageal cells EPC2-hTERT were retrovirally co-transduced with EGFR and mutant p53 (p53R175H), early genetic lesions common in ESCC, leading to transformation as described previously (Genes Dev. 2007; 21:2788). Short hairpin RNA was expressed by lentivirus to knockdown ZEB1 and ZEB2. Epithelial cells were grown in organotypic 3-D culture, a form of tissue engineering. Laser capture microdissection (LCM) was performed to isolate RNA for gene expression profiling in the invasive cells in 3-D culture as well as microRNA determination in archived paraffin embedded primary ESCC tissue samples. Real-time RT-PCR, Western blotting and immunohistochemistry were done to determine gene expression. Senescence-associated β-galactosidase activity was determined. Results: In primary ESCC, ZEB expression was found upregulated in tumor cells at the invasive front with increased Vimentin and decreased E-cadherin expressions. Moreover, LCM documented a significant downregulation of the miR-200 family in the invasive tumor cells consistent with EMT. ZEB upregulation and concomitant loss of the miR-200 family were also found in two out of 11 ESCC cell lines, exhibiting EMT corroborated by spindle-shaped cell morphology as well as an E-cadherin to N-cadherin class switch. EGFR overexpression and p53 mutation in EPC2-hTERT cells resulted in ZEB upregulation and a significantly augmented susceptibility to EMT, where robust induction of ZEB and suppression of the miR-200 family were observed upon TGF-β treatment. In 3-D culture, the cells co-transduced with EGFR and mutant p53 markedly invaded into the underlying stroma compartment. The invasive cells had a gene signature compatible with EMT along with ZEB upregulation. Finally, knockdown of either ZEB1 or ZEB2 not only prevented TGF-β from inducing EMT, but also TGF-β-mediated massive senescence in EPC2-hTERT cells transformed by EGFR and mutant p53. Conclusions: Our data indicate that ZEB1 and ZEB2 are negatively regulated by the miR-200 family and induced upon invasion in ESCC. ZEB may regulate senescence and EMT in concert with TGF-β signaling, thus contributing to esophageal carcinogenesis and tumor progression.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2293.

Abstract 2294: IGF-independent regulation of EMT by insulin-like growth factor binding protein-3 in transformed human esophageal cells

Introduction: Insulin-like growth factor (IGF) binding protein-3 (IGFBP3), a key regulator of IGF bioactivity, exerts either proapoptotic or growth stimulatory effects in a cellular context dependent manner. IGFBP3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP3 in esophageal tumor biology remains elusive. Methods: Human esophageal epithelial cells transformed by combinations of either Ha-Ras V12 and SV40 T-antigen (T-Ag) or epidermal growth factor receptor (EGFR) and p53 R175H were stably transduced with wild-type (WT) or I56G/L80G/L81G (GGG) mutant IGFBP3, the latter incapable of binding IGFs. Short hairpin RNAs (shRNAs) were used to knockdown IGFBP3. Gene expression was determined by real-time RT-PCR, Western blotting and immunofluorescence. Organotypic 3-D culture, a form of tissue engineering was done. Xenograft transplantation was also performed. Results: TGF-beta induced IGFBP3 robustly upon epithelial to mesenchymal transition (EMT) in EGFR-p53 R175H transformed cells where a cadherin-class switch, induction of other mesenchymal markers and a cellular morphological change were documented. Interestingly, IGFBP3 knockdown prevented TGF-beta from inducing EMT while ectopically expressed WT or GGG-mutant IGFBP3 promoted EMT. In organotypic 3-D culture, IGFBP3 knockdown greatly impaired the invasive growth of the transformed cells. By contrast, WT or GGG-mutant IGFBP3 stimulated invasion. Upon xenograft transplantation, WT-IGFBP3 induced massive apoptosis and completely abrogated tumor formation by the Ras-transformed cells. By contrast, both empty vector control and GGG-IGFBP3 allowed tumor formation at a comparable rate. Interestingly, however, immunofluorescence revealed two major cell populations within the GGG-mutant IGFBP3 expressing tumors; SV40 T-Ag positive, pancytokeratin bright, and fibroblast specific protein 1 (FSP1) negative epithelioid tumor cells and T-Ag positive, pancytokeratin dim, and FSP1 positive spindle shaped tumor cells. The latter, representing a predominant subset of tumor cells, implied EMT and grew more aggressively. Such EMT-compatible tumor cells were present minimally in the empty vector control transduced tumors, suggesting that IGFBP3 may also facilitate EMT in vivo through an IGF-independent mechanism. Conclusions: Our innovative approach reveals that xenografted tumors may require IGF signaling for adaptation to the microenvironment in the host tissue where WT-IGFBP3 may antagonize a prosurvival effect of IGFs in vivo. However, once tumors are adapted to the local microenvironment, IGFBP3 may promote EMT in an IGF-independent fashion, a novel mechanistic finding. This has implications upon EMT in other cancers and may offer platforms for cancer therapy. Grants: NCI P01-CA098101 and NIH R01-DK077005 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2294.