Abstract 2294: IGF-independent regulation of EMT by insulin-like growth factor binding protein-3 in transformed human esophageal cells
Authors: Natsuizaka Mitsuteru , Kalman Ross A. , Ohashi Shinya , Takaoka Munenori , Gimotty Phyllis , El-Deiry Wafik S. , Klein-Szanto Andres , Diehl J. Alan , Herlyn Meenhard , Nakagawa Hiroshi 
Published: February 2011

Abstract
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-RasV12 and SV40 T-antigen (T-Ag) or epidermal growth factor receptor (EGFR) and p53R175H 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-p53R175H 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.