Abstract. Four and a half LIM domain protein 2 (FHL2) can interact with many proteins and regulates different cellular processes, including proliferation and differentiation. FHL2 expression is often deregulated in cancer and may act as both tumor-promoter or tumor-suppressor depending on the type of cancer. Thus, a previous study found that increased FHL2 expression in colon cancer and suppression of FHL2 in a colon cancer cell line with endogenously high FHL2 expression inhibited tumor growth. We applied the opposite strategy, an FHL2 expression plasmid was stably transfected into HT-29 cells, a colon carcinoma cell line which exhibits very low basal levels of FHL2. Stable expression of FHL2 in HT-29 cells induced a G2/M arrest and inhibited anchorage-dependent and -independent growth in vitro. Further, FHL2 expressing HT-29 cell clones revealed significantly higher expression of the differentiation marker E-cadherin but reduced activity of the transcription factor NF-κB, which is known to promote colon cancer progression. These findings further underscore the complex role of FHL2 in tumorigenicity, with even different effects on cellular functions of cancer cell lines derived from the same type of tumor and distinctly suggest caution regarding therapeutic strategies targeting FHL2 to treat (colon) cancer. IntroductionColorectal cancer (CRC) is one of the most frequent human tumors and the third leading cause of cancer death (1). The prognosis of CRC, especially advanced cancer, has not substantially improved although the molecular mechanisms of CRC have become better understood in the past two decades (2).Four and a half LIM protein 2 (FHL2), also known as down-regulated in rhabdomyosarcoma LIM (DRAL) domain protein, is a member of a small family of five proteins with four and a half LIM domains (3). The abbreviation LIM represents the names of three transcription factors (Lin-11, Isl-1 and Mec-3) in which such a domain was first identified (4). The LIM domains are double zinc finger motifs that play multiple roles in protein-protein interaction (5). FHL2 can interact with >50 different proteins with diverse functions such as receptors, enzymes, transcription factors, cofactors or splicing factors (6). Thus, FHL2 is involved in the regulation of various cellular processes including proliferation, differentiation, migration and contraction (7-10). FHL2 can function as repressor or activator of transcriptional activity, and noteworthy, depending on the cell type even the expression of the same gene or distinguished cellular functions, respectively, may be regulated in different ways. Thus, FHL2 promotes expression of ·-smooth-muscle actin (·-SMA) and contractility in myofibroblasts of the skin (9), but inhibits ·-SMA expression and contractility of cardiac muscle cells (11).Also the role of FHL2 in tumorigenesis is complex, since it may act as an oncoprotein or as a tumor suppressor depending on the type of cancer (6,12). FHL2 was first identified in human rhabdomyosarcoma cells as a downregulated gene indicati...