Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma
Sin-Ae Lee, … , Ki Hun Park, Jung Weon Lee
Sin-Ae Lee, … , Ki Hun Park, Jung Weon Lee
Published March 20, 2008
Citation Information: J Clin Invest. 2008;118(4):1354-1366. https://doi.org/10.1172/JCI33768.
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Research Article

Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma

Abstract

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Authors

Sin-Ae Lee, Sung-Yul Lee, Ik-Hyun Cho, Min-A Oh, Eun-Sil Kang, Yong-Bae Kim, Woo Duck Seo, Suyong Choi, Ju-Ock Nam, Mimi Tamamori-Adachi, Shigetaka Kitajima, Sang-Kyu Ye, Semi Kim, Yoon-Jin Hwang, In-San Kim, Ki Hun Park, Jung Weon Lee

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Figure 2

TM4SF5-mediated morphological elongation involves RhoA inactivation.

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TM4SF5-mediated morphological elongation involves RhoA inactivation. Who...
Whole-cell lysates were immunoblotted (A) or used for RhoA assay and immunoblots (C). Parental, SNU449Cp, or TM4SF5-expressing cells transfected with shRNA against GFP, scrambled sequence (Scr), or TM4SF5 (shTM4SF5) were harvested for immunoblots (B) or RhoA activity assay and immunoblots (D). (E) SNU449Tp cells were cotransfected with pEGFP and shTM4SF5. After 24 hours, cells were replated on 10% FBS/RPMI-1640–precoated coverslips. After an additional 24 hours, cells were stained for actin and DNA and analyzed by confocal microscopy. Scale bars: 20 μm. (F) Scheme showing the linkage from FAK to RhoA through RhoGAPs. FERM, FAK N-terminal band 4.1, ezrin, radixin, moesin homology domain; pxxp, proline-rich domain. (G) Increased association of RhoGAPs with FAK in SNU449Tp cells. Lysates from control (Cp) or SNU449Tp (Tp) cells were immunoprecipitated with anti-FAK antibody before immunoblot analysis. hc, heavy chain. (H) SNU449Tp cells were transiently transfected with (HA)3-FAK WT or Y577F mutant. After 48 hours, lysates were prepared and immunoprecipitated with anti-HA antibody, before immunoblot analysis. (I) SNU449Cp cells were transfected with pEGFP-FLAG-GRAF or p190RhoGAP. After 24 hours, cells were replated on coverslips, as above. After an additional 24 hours, cells were double-stained for actin and then for FLAG-tag or GFP. Scale bars: 10 μm. (J) Cell extracts from Huh7 cells stably transfected with scrambled shRNA or shTM4SF5 were used in immunoblots. Data shown represent 3 different experiments.