Abstract 3046: Lipocalin2, an iron transporter, functions as a novel oncogene and enhances the proliferation and survival of endometrial carcinoma cell.
Authors: Miyamoto Tsutomu , Asaka Ryoichi , Yamada Yasushi , Ishikawa Kaori , Kobara Hisanori , Suzuki Akihisa , Shiozawa Tanri 
Published: August 2014

Background: The mechanism of carcinogenesis and progression of endometrial carcinoma has not been fully understood. To elucidate the molecular machinery, we performed microarray analysis using mRNA extracted from normal endometria, endometrial hyperplasia and carcinoma and found lipocalin2 (LCN2), an iron transporter, to be markedly up-regulated along with disease progression. Furthermore, the over-expression of LCN2 and its receptor, SLC22A17, was associated with poor outcome of the patients with endometrial carcinoma. In the present study, we further analyzed the molecular functions of LCN2 using endometrial carcinoma cells.

Methods: An endometrial carcinoma cell line HHUA over-expressing both LCN2 and SLC22A17 was used. We established LCN2-silenced HHUA (HHUAsh) and control-HHUA (HHUAc) by transfection of LCN2-shRNA and scrambled shRNA producing vector, respectively. The growth and migration were examined by WST-1 assay and wound healing assay. Cell viability under stress conditions such as ultraviolet (UV) irradiation or cisplatin (CDDP) treatment, with or without iron-chelation, was measured by WST-1. The intensity of apoptosis was analyzed by TUNEL staining and Apostrand assay. The expression of phosphorylated Akt was examined by Western blotting. In vivo tumor formation was observed by the subcutaneous injection of those HHUA cells to nude mice. The colony formation of these HHUA cells in soft agar was evaluated. In addition, the LCN2 cDNA containing retrovirus vector (LCN2-RET) was transfected to NIH3T3 to observe the transformation ability.

Results: The cell migration of HHUAsh was decreased compared with HHUAc. After UV irradiation and CDDP treatment, the residual cell viability of HHUAsh (25.4% and 37.1%, respectively) was significantly lower than that of HHUAc (51.2% and 77.6%, p&lt;0.05). Under these stresses, the enhanced apoptosis and down-regulation of phosphorylated Akt was observed in HHUAsh. Interestingly, these differences were abolished by iron-chelation. The growth of HHUAsh showed no difference with that of HHUAc in vitro. However, LCN2-silencing significantly reduced the tumor growth in nude mice (84% reduction). Moreover, the down-regulation of phospho-Akt expression was observed in tumors of LCN2-silenced HHUA. The colony formation in soft agar of HHUAsh was significantly reduced (66.3% reduction) from that of HHUAc, and this difference was rescued to 12% by addition of recombinant LCN2. In addition, NIH3T3 transfected by LCN2-RET formed focuses.

Conclusions: The present study revealed that LCN2 is involved in the genesis and survival of endometrial carcinoma cells under stress conditions and in the tumor growth in vivo, via p-Akt signaling in an iron-dependent manner, suggesting LCN2 to be a novel oncogene.

Citation Format: Tsutomu Miyamoto, Ryoichi Asaka, Yasushi Yamada, Kaori Ishikawa, Hisanori Kobara, Akihisa Suzuki, Tanri Shiozawa. Lipocalin2, an iron transporter, functions as a novel oncogene and enhances the proliferation and survival of endometrial carcinoma cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2013-3046