Abstract 1148: Epithelial to mesenchymal transition might be induced via CD44 isoform switch in colorectal cancer
Authors: Mashita Naoki , Yamada Suguru , Iwata Naoki , Kanda Mitsuro , Kobayashi Daisuke , Tanaka Chie , Fujii Tsutomu , Nakayama Goro , Sugimoto Hiroyuki , Koike Masahiko , Nomoto Shuji , Fujiwara Michitaka , Kodera Yashuhiro 
Published: May 2015

Backgrounds: The epithelial to mesenchymal transition (EMT) is well known to play a crucial role in cancer invasion and metastasis and has been actively investigated in colorectal cancer (CRC). In recent years, the isoform switch of CD44, which is recognized to be a cancer stem cell marker, has been reported to associate with EMT, especially in breast cancer and hepatocellular carcinoma. However, the clinical impact of CD44 isoform switch to EMT in CRC is still unknown.

Methods: E-cadherin, vimentin, CD44 standard (CD44s) and CD44 variant9 (CD44v9) expression were measured in 14 CRC cell lines and 150 CRC patients by real-time PCR. EMT status and CD44 status was determined by calculating vimentin/E-cadherin and CD44s/CD44v9 expression ratio, respectively. The CRC cell lines and patients were divided into two groups based on the EMT and CD44 status. We examined the association between the EMT status and CD44 isoform switch, and analyzed the correlation with clinicopathological factors and prognosis. Finally, we evaluated the effect of CD44 knockdown by siRNA in CRC cell lines by measuring the proliferation, migration and invasion ability.

Results: 1. The CRC cell lines were classified into 8 epithelial, 4 mesenchymal and 2 intermediate type. Of these cell lines, CD44s was highly expressed in mesenchymal type cell lines, whereas, CD44v9 was highly expressed in epithelial type cell lines.

2. The 150 CRC patients were divided into 115 epithelial group and 35 mesenchymal group based on vimentin/E-cadherin expression ratio. Mesenchymal group showed significantly poorer survival than epithelial group (5-year survival rate; epithelial: 85.5% vs. mesenchymal: 62.1%, P = 0.009). EMT status was significantly correlated with invasion depth of tumor (P = 0.036), lymphatic vessel involvement (P = 0.034) and histological grade (P = 0.001).

3. High CD44 status group showed poorer prognosis than low CD44 status group (5- year survival rate; low group: 85.0% vs. high group: 62.1%, P = 0.025). There was a significant correlation between EMT status and CD44 status (r = 0.298, P &lt; 0.001). On multivariate analysis, pathological T4 stage (P = 0.011), liver metastasis positive (P = 0.001), high CA19-9 level (≥37) (P = 0.003) and CD44 status (P = 0.001) were independent prognostic factors for CRC patients.

4. In COL-3-JCK, which is identified to be mesenchymal type, CD44 knockdown by siRNA decreased the vimentin expression at protein level. When the functional analysis were performed, CD44 knockdown significantly reduced the abilities of cell proliferation (P &lt; 0.001), migration (P = 0.004) and invasion (P = 0.025).

Discussion: EMT was a critical prognostic factor and CD44 isoform switch might contribute to EMT induction in CRC. These results could support a better understanding of the relationship between EMT and cancer stem cell. Therefore, further investigation of the specific splice isoform of CD44 might lead to novel therapeutic intervention for CRC patients.

Citation Format: Naoki Mashita, Suguru Yamada, Naoki Iwata, Mitsuro Kanda, Daisuke Kobayashi, Chie Tanaka, Tsutomu Fujii, Goro Nakayama, Hiroyuki Sugimoto, Masahiko Koike, Shuji Nomoto, Michitaka Fujiwara, Yashuhiro Kodera. Epithelial to mesenchymal transition might be induced via CD44 isoform switch in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1148. doi:10.1158/1538-7445.AM2014-1148