Eiko Ozono

Identification of novel target genes specifically activated by deregulated E2F in human normal fibroblasts.

The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth‐related genes but also in tumor suppression by activating pro‐apoptotic and growth‐suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27Kip1 and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth‐related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F‐responsive promoter elements and to contribute to E2F1‐mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function.

Tumor suppressor TAp73 gene specifically responds to deregulated E2F activity in human normal fibroblasts

Discrimination of oncogenic growth signals from normal growth signals is crucial for tumor suppression. The transcription factor E2F, the main target of pRB, plays central role in cell proliferation by activating growth‐promoting genes. E2F also plays an important role in tumor suppression by activating growth‐suppressive genes such as pro‐apoptotic genes. The regulatory mechanism of the latter genes is not known in detail, especially in response to normal and oncogenic growth signals. E2F is physiologically activated by growth stimulation through phosphorylation of pRB. In contrast, upon dysfunction of pRB, a major oncogenic change, E2F is activated out of control by pRB, generating deregulated E2F activity. We show here that the tumor suppressor TAp73 gene, which can induce apoptosis independently of p53, responds to deregulated E2F activity, but not to physiological E2F activity induced by growth stimulation in human normal fibroblasts. We identified E2F‐responsive elements (ERE73s) in TAp73 promoter that can specifically sense deregulated E2F activity. Moreover, RB1‐deficient cancer cell lines harbored deregulated E2F activity that activated ERE73s and the TAp73 gene, which were suppressed by re‐introduction of pRB. These results underscore the important role of deregulated E2F in activation of the TAp73 gene, a component of major intrinsic tumor suppressor pathways.

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.

Ectopic expression of the CDK inhibitor p21(Cip1) enhances deregulated E2F activity and increases cancer cell-specific cytotoxic gene expression mediated by the ARF tumor suppressor promoter.

In cancer treatment, specifically targeting cancer cells is important for optimal therapeutic efficacy. One strategy is to utilize a cancer specific promoter to express a cytotoxic gene or a viral gene required for replication. In this approach, the therapeutic window is dependent on the relative promoter activity in cancer cells versus normal cells. Therefore, a promoter with optimal cancer cell-specificity should be used. The tumor suppressor ARF promoter, which specifically responds to deregulated E2F activity, is a potent candidate. Defects in the RB pathway resulting in deregulated E2F activity are observed in almost all cancers. Furthermore, the ARF promoter exhibits greater cancer cell specificity than the E2F1 promoter and consequently, adenovirus expressing HSV-TK under the control of the ARF promoter (Ad-ARF-TK) has more selective cytotoxicity in cancer cells than the analogous E2F1 construct. Ideally, cancer specific gene expression driven by the ARF promoter could be enhanced for optimal therapeutic efficacy, with minimal side effects. We show here that ectopic expression of the CDK inhibitor p21Cip1 enhanced deregulated E2F activity and pro-apoptotic E2F target gene expression in cancer cells. Moreover, ectopic expression of p21Cip1 augmented cancer specific cytotoxicity of Ad-ARF-TK, and apoptosis induced by p21Cip1 was dependent on deregulated E2F activity. These results suggest that p21Cip1 specifically enhances deregulated E2F activity and that a combination of the CDK inhibitor with Ad-ARF-TK could be effectively employed for cancer therapy.

Differential requirement for dimerization partner DP between E2F-dependent activation of tumor suppressor and growth-related genes

The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.

The phosphatidyl inositol 3 kinase pathway does not suppress activation of the ARF and BIM genes by deregulated E2F1 activity

The transcription factor E2F plays crucial roles in tumor suppression by activating pro-apoptotic genes such as the tumor suppressor ARF. The regulation of the ARF gene is distinct from that of growth-related E2F targets, in that it is specifically activated by deregulated E2F activity, induced by over-expression of E2F or forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. The phosphatidyl inositol 3 kinase (PI3K) pathway was reported to suppress expression of some atypical pro-apoptotic genes by over-expressed E2F1. However, the effects of the PI3K pathway on the distinct regulation of typical pro-apoptotic E2F targets have not been elucidated. We examined whether the PI3K pathway suppressed activation of the typical pro-apoptotic E2F targets ARF and BIM. Activation of the PI3K pathway by growth stimulation or introduction of a constitutively active Akt/PKB did not reduce induction of ARF or BIM gene expression or activation of their promoters by over-expressed E2F1. These results suggest that the PI3K pathway does not suppress induction of typical pro-apoptotic genes that are selectively activated by deregulated E2F1.

To Grow, Stop or Die? – Novel Tumor-Suppressive Mechanism Regulated by the Transcription Factor E2F

Proliferation of mammalian cells is strictly regulated by growth stimulation. Cell prolifera‐ tion is stimulated not only by normal growth stimulation but also by abnormal growth stim‐ ulation originated from oncogenic changes. Such abnormal growth stimulation leads to tumorigenesis, if not properly guarded by appropriate cellular response. Cells are endowed with intrinsic tumor suppressor pathways to protect cells from tumorigenesis upon such on‐ cogenic threat [1]. The tumor suppressor pathways halt cell proliferation either by restrain‐ ing cell cycle progression or by inducing apoptosis (programmed cell death) in case of being unable to stop aberrant cell cycle progression. Consequently, the cell-fate, whether to grow, stop growing or die, is dependent on the balance between growth-promoting effects origi‐ nated from oncogenic changes and growth-suppressive effects mediated by the tumor sup‐ pressor pathways upon oncogenic changes (Figure 1). When the tumor suppressor pathways are disabled by further oncogenic changes, the balance of cell-fate determination shifts from growth suppression to proliferation, and cells start deregulated proliferation, leading to tumorigenesis. Among the intrinsic tumor suppressor pathways, two major path‐ ways are the RB pathway and the p53 pathway. Both pathways are important for induction of cell cycle arrest or apoptosis [2]. In addition, accumulating evidence indicates that the tu‐ mor suppressor TAp73, a member of the p53 family, also plays crucial roles in tumor sup‐ pression by inducing apoptosis independent of p53 [3, 4].