Ross A. Kalman

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.

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 & AIMS The 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. METHODS Normal 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. RESULTS NOTCH1 (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. CONCLUSIONS Notch 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.

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.

Abstract 5194: Notch regulates squamous differentiation, cell plasticity and tumor heterogeneity in esophageal carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: The Notch receptor family regulates cell fates and may act as a tumor suppressor in the squamous epithelia. While squamous differentiation is maintained in esophageal squamous cell carcinoma (ESCC) and its precursor lesions, the roles of Notch in esophageal tumor biology remain elusive. Methods: Transformed human esophageal cells expressing EGFR, p53R175H and cyclin D1 (EPC2-T) and ESCC cell lines were stably transduced with ICN1, an active form of NOTCH1 (N1) in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or gamma-secretase inhibitors (GSI). 8xCSL-luciferase reporter was transiently transfected to assess Notch activity. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2. Squamous epithelium was reconstituted in organotypic (3D) culture, a form of tissue engineering. In addition, cell growth was assessed in soft agar and immunodeficient mice. Quantitative RT-PCR, Western blotting, immunohistochemistry (IHC) and flow cytometry were done to determine gene expression. Primary ESCC tissues (n=20) were analyzed by IHC. Results: In primary ESCC, N1 was localized to well-differentiated tumor cell nests expressing involucrin (IVL), a squamous differentiation marker, while downregulated in poorly-differentiated cells lacking IVL within a single tumor tissue, implying N1 in tumor heterogeneity. In cell lines, ICN1 activated Notch-dependent transcription and squamous differentiation in the absence of DNMAML1. In 3D culture, DNMAML1 and GSI not only blocked squamous differentiation but induced massive invasion and epithelial-to-mesenchymal transition (EMT) with Wnt activation as suggested by loss of E-cadherin and nuclear localization of β-catenin. In agreement, DNMAML1 upregulated LEF1, Zinc finger E-box binding proteins ZEB1 and ZEB2, transcription factors all essential in EMT and maintenance of cancer stem cells, facilitating transforming growth factor (TGF)-β1-induced EMT in EPC2-T cells. Moreover, DNMAML1 enriched a unique subset of CD44-bright and CD24-dim cells and augmented colony formation, tumorigenicity as well as chemotherapeutic drug resistance against Cisplatin. Importantly, Cre-mediated removal of chromosomally integrated floxed DNMAML1 reactivated Notch to allow terminal differentiation with IVL induction. Moreover, ZEB knockdown greatly impaired TGF-β-mediated EMT while restoring chemotherapeutic drug sensitivity. Conclusions: These data indicate that Notch activation promotes squamous differentiation whereas Notch inhibition enriches poorly differentiated tumor cells with cancer stem cell potential with drug resistance, which involves ZEB and Wnt activation, thus providing a novel mechanistic insight into how Notch signaling may contribute to tumor heterogeneity in ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2011-5194

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.