(A) Soft agar assay of SNU449 cells was performed for 27 days. (B) Cells were injected subcutaneously to mice (1 × 10⁷ cells/mouse). Tumor volume values and body weights were measured as described in Methods and plotted as mean ± SD. Values for SNU449T16m and SNU449T3m were zero, since they did not form tumors in mice. (C) Tumors from SNU449Cp or T16 cell–injected mice were stained for H&E or for p27^Kip1 or TM4SF5. Original magnification, ×40 (A); ×100 (C). (D) Extracts from control or TM4SF5-expressing cell–injected mice were immunoblotted for p27^Kip1, pS¹⁰p27^Kip1, and TM4SF5. (E) Working model of TM4SF5-mediated EMT and contact inhibition loss. Overexpression of TM4SF5 appears to enhance p27^Kip1 expression and cytosolic stabilization, being correlated with p27^Kip1 Ser10 phosphorylation, presumably by hKIS and/or PKB/Akt. TM4SF5 also affects FAK signaling to RhoA via RhoGAPs. These TM4SF5 effects indicate a linkage between cytosolic p27^Kip1 expression and stabilization and RhoA activity regulation, which may modulate cellular morphology. Morphological change in turn causes EMT, which involves E-cadherin suppression and α-SMA induction in TM4SF5-expressing cells. Due to cell-cell contact loss, one cell may not recognize adjacent cells and grow in an uncontrollable manner, leading to contact inhibition loss (i.e., piling up of cells). Solid-line connections with question mark indicate that it is currently unknown how TM4SF5 exerts its effects, and dotted lines indicate either a cross-talk between the 2 receptors not evidenced but prospective from another report (23) or a functional relationship between 2 parties with unknown mechanisms. pi, inorganic phosphate.