FHL2 exhibits anti-proliferative and anti-apoptotic activities in liver cancer cells
Introduction
FHL2 is a LIM-only protein that belongs to the Four-and-a-Half LIM-only protein family. It consists of four LIM domains and one N-terminal half LIM domain. It is strongly expressed in cardiac and skeletal muscle cells but a much lower level was observed in other tissues and cell types [1], [2], [3], [4]. The LIM domains in this protein can function as an adapter or modifier in protein interactions [5]. The protein interactions mediated by LIM domains allow FHL2 to have diverse functions in the regulation of many cellular processes such as cell survival, proliferation, differentiation, adhesion, motility, regulation of gene expression and signal transduction.
The role of FHL2 in cancer is particularly intriguing since FHL2 binds to different proteins and can function in a cell-type dependent fashion as transcriptional co-activators of several transcription factors, including androgen receptor, AP-1, CREB, BRCA1, WT-1 and NF-кB in various transformed cell types [6], [7], [8], [9], [10], [11], or as transcriptional co-repressors of ERK2, PLZF, SRF and FOXO1 [12], [13], [14], [15]. FHL2 was first identified as being downregulated in human rhabdomyosarcoma cells, suggesting a role in tumor development [16]. Many recent studies have reported the differential expression of FHL2 in tumor tissues. Interestingly, FHL2 is overexpressed in breast cancer [17], prostate cancer [18], ovarian cancer [4], gastrointestinal cancer [19], glioma [20] but down-regulated in liver cancer [21], making the role of FHL2 in cancer development elusive. In breast cancer, there are reports showing that FHL2 is downregulated in many breast cancer cell lines but overexpressed in mammary carcinoma tissues [9], [17]. It is also notable that FHL2 triggers apoptosis in human RD, monkey kidney COS-1 and normal mouse fibroblast NIH 3T3 cell lines [22] but plays an anti-apoptotic role in glioblastoma cells [20]. Most recently, FHL2 was found to act as a potent EMT inducer by stimulating vimentin and MMP-9 expressions and causing a loss of E-cadherin in colon DLD1 cells [23] but increases the expression of E-cadherin in colon HT-29 cells but reduced activity of the transcription factor NF-kappaB [24]. The intriguing aspects of FHL2 being as oncoprotein or tumor suppressor may be related to its interaction with different partner proteins in different cell types. So far, the precise FHL2 function in liver cancer cells is still unclear.
In the present study, we aim at studying the effect of FHL2 on cell proliferation, apoptosis, motility and invasiveness in liver cancer Hep3B cell line. We found that FHL2 expression reduces the cell proliferation through the control of G1/S transition by cyclin D1, p21 and p27. It can also inhibit cell migration and invasion and these effects are probably mediated by the control of the epithelial–mesenchymal transition. Surprisingly, FHL2 also presents an anti-apoptotic role as shown by its effect on Bcl-2 and Bcl-xL, and the influence on doxorubicin-induced apoptosis signaling. Taken together, our results indicate FHL2 could exert anti-apoptotic effect independent of tumor growth suppression.
Section snippets
Cell culture
All culture reagents were purchased from Invitrogen. All the hepatoma cell lines were purchased from American Type Culture Collection. WRL68 and HuH7 were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin while Hep3B and HepG2 were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium with the same supplements. Cells were maintained at 37 °C in a 5% CO2 humidified incubator and
Expression of FHL2 in HCC cell lines and tumor tissues
Initially, we intended to evaluate the expression status of FHL2 in HCC cell lines and tumorous tissues. The mRNA and protein levels of FHL2 in four liver cell lines were determined by real-time PCR and Western blot analysis, respectively. Real-time PCR analysis showed that the FHL2 mRNA levels were much lower in the three HCC cell lines (Huh7, HepG2 and Hep3B) compared with the normal hepatocyte line WRL68 (Fig. 1A). Western blot showed that FHL2 protein was expressed in WRL68, but
Discussion
Identification of the differences in the genetics of cancer cells and normal cells and exploration of cancer-associated genes are important for the development of targeted therapies in cancer treatment. In this study, we examined the potential role of FHL2 in liver carcinogenesis. Our examination of the effects of overexpression of FHL2 in Hep3B cells has revealed an interesting finding of the dual functions of FHL2 in liver cancinoma cells. On one hand, it exerts anti-proliferative and
Conflict of interest
The authors declare that they have no conflict of interest.
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