FGF4 dissociates anti-tumorigenic from differentiation signals of retinoic acid in human embryonal carcinomas

Abstract

A subset of male germ cell cancers presenting with advanced stage abundantly express the ®broblast growth factor-4 (FGF4). FGF4 expression is restricted in vitro to undi erentiated embryonal carcinomas (ECs). During induced di erentiation, FGF4 expression is repressed in maturation sensitive but not resistant human ECs, suggesting FGF4 plays an important role in malignant growth or di erentiation of ECs. To explore these FGF4 signals in male germ cell cancers, the multipotent human EC NTERA-2 clone D1 (NT2/D1) cell line was studied. All-trans-retinoic acid (RA)-treatment of these cells induces a neuronal phenotype and represses tumorigenicity and FGF4 expression. In contrast, RA-treatment of retinoid resistant lines derived from NT2/D1 cells failed to repress FGF4 expression. This implicated FGF4 directly in regulating human EC growth or di erentiation. To evaluate further this FGF4 role, FGF4 was constitutively over-expressed in NT2/D1 cells using a CMV-driven expression vector containing the neomycin resistance gene. Three stable transfectants expressing exogenous FGF4 were studied as was a control transfectant only expressing the neomycin resistance gene. RA-treatment repressed endogenous but not exogenous FGF4 expression. RA-treatment of these transfectants induced morphologic and immunophenotypic maturation, changes in RA-regulated genes, and a G1 cell cycle arrest in a manner similar to parental NT2/D1 cells. This indicated FGF4 over-expression did not block RA-mediated di erentiation. As expected, RA-treatment repressed tumorigenicity of the control transfectant after subcutaneous injection into athymic mice. Despite RAtreatment, this repressed tumorigenicity was overcome in all the transfectants over-expressing FGF4. The histopathology and neovascularization did not appreciably di er between xenograft tumors derived from FGF4 over-expressing versus control transfectants. FGF4 expression studies were extended to patient-derived germ cell tumors using total cellular RNA Northern analysis and an immunohistochemical assay developed to detect FGF4 protein expression. Germ cell tumors with EC components were signi®cantly more likely to express FGF4 mRNA (P40.0179) than other examined germ cell tumors without EC components. Immunohistochemical results from 43 germ cell tumors demonstrated increased FGF4 expression especially in non-seminomas having EC components. Thus, FGF4 promotes directly malignant growth of cultured ECs, overcomes the antitumorigenic actions of RA, and is selectively expressed in speci®c histopathologic subsets of germ cell tumors. Taken together, these ®ndings indicate how di erentiation and anti-tumorigenic retinoic acid signals can be dissociated in germ cell cancer.

The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells

“…For example, expression of the growth factors FGF4 and TGFa is downregulated and expression of Wnt-13 is induced following treatment with either RA or HMBA, consistent with the hypothesis that common extracellular signaling pathways are important for multiple NTERA-2 di erentiation programs (Miller et al, 1994;Wakeman et al, 1998). Down-regulation of the growth factors FGF4, TGFa, and TDGF-1 (CRIPTO) has been shown to contribute to the reduced proliferative and tumorigenic potential in di erentiated NTERA-2 cells (Baldassarre et al, 1996(Baldassarre et al, , 1997Baselga et al, 1993;Maerz et al, 1998). In contrast, addition of recombinant bone morphogenetic protein-7 has been shown to induce di erentiation of NTERA-2 cells along a pathway distinct from that induced by RA or HMBA (Andrews et al, 1994).…”

“…To our knowledge, this is the ®rst study examining the e ects of overexpressing a secreted protein that is up-regulated during NTERA-2 di erentiation. Interestingly, the phenotype observed when CREG is overexpressed in these cells is di erent from the e ects of constitutive expression of other proteins implicated in the control of NTERA-2 cell growth and di erentiation (Baselga et al, 1993;Maerz et al, 1998;Moasser et al, 1995;Spinella et al, 1998). We have found that CREG increases the number of cells that di erentiate both in the absence of an inducer and in response to RA.…”

Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones

“…In order to avoid massive cell death, observed when Sox2 levels were elevated at least 4-fold above the endogenous level (Mitsui et al, 2003;Kopp et al, 2008;Tonge and Andrews, 2010), we generated cell clones with SOX2 expression driven by cytomegalovirus immediate-early promoter (CMV-IE), which exhibited weak activity in ES and EC cells (Niwa et al, 1991;Liew et al, 2007). The use of CMV-driven constitutive overexpression in NT2/D1 cells has been reported for several genes, such as pluripotency-related fibroblast growth factor-4 (FGF-4) and p27, as well as for FLJ11259/DRAM (Maerz et al, 1998;Baldassarre et al, 2000;Kerley-Hamilton et al, 2007). Using this expression system we obtained 54 resistant colonies.…”

Retinoic acid activates p53 in human embryonal carcinoma through retinoid receptor-dependent stimulation of p53 transactivation function

“…This RA-and di erentiationsensitive enhancer element contains OCT3/4 and Sox2 sites, but does not contain an RARE. The decline of FGF4 mRNA and protein expression re¯ects e ective RA-induced di erentiation and growth suppression of murine teratocarcinomas and human embryonal carcinomas and is`downstream' of direct retinoid receptordriven transactivation in this di erentiation program (Moasser et al, 1994;Yuan et al, 1995;Maerz et al, 1998). E ects of wild-type p53 and DN-p53 overexpression on FGF4 transcription were studied using this reporter assay.…”