Andrei L. Gartel

Lost in Transcription: p21 Repression, Mechanisms, and Consequences

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a major player in cell cycle control and it is mainly regulated at the transcriptional level. Whereas induction of p21 predominantly leads to cell cycle arrest, repression of p21 may have a variety of outcomes depending on the context. In this review, we concentrate on transcriptional repression of p21 by cellular and viral factors, and delve in detail into its possible biological implications and its role in cancer. It seems that the major mode of p21 transcriptional repression by negative regulators is the interference with positive transcription factors without direct binding to the p21 promoter. Specifically, the negative factors may either inhibit binding of positive regulators to the promoter or hinder their transcriptional activity. The ability of p21 to inhibit proliferation may contribute to its tumor suppressor function. Because of this, it is not surprising that a number of oncogenes repress p21 to promote cell growth and tumorigenesis. However, p21 is also an inhibitor of apoptosis and p21 repression may also have an anticancer effect. For example, c-Myc and chemical p21 inhibitors, which repress p21, sensitize tumor cells to apoptosis by anticancer drugs. Further identification of factors that repress p21 is likely to contribute to the better understanding of its role in cancer.

p21—Negative Regulator of the Cell Cycle

Abstract Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (Cdks). The cyclin kinase inhibitor p21 (also known as WAF1, CIP1, SDI1, and MDA-6) can induce G1 arrest and block entry into S phase by inactivating Cdks or by inhibiting activity of proliferating cell nuclear antigen (PCNA). In normal cells, p21 exists in quaternary complexes with cyclin, Cdk, and PCNA. Transcription of the p21 gene is activated by p53-dependent and -independent mechanisms. Mice deficient in p21 exhibit no apparent phenotype, although p21 function has been demonstrated to be necessary for p53-mediated G1 arrest following irradiation of p21-deficient mouse embryonic fibroblasts. Thus, the function of p21 under normal circumstances appears to be redundant. p21 is expressed in terminally differentiating cells of a variety of tissues in a p53-independent manner. Overexpression of p21 results in G1 arrest and has been shown to suppress effectively tumor growth in vitro and in vivo. We review the recent literature describing the functional characterization of p21. This protein plays a key role in regulating the cell cycle and may have potential gene therapy applications.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G1 cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Activation of Akt/protein kinase B overcomes a G2/M cell cycle checkpoint induced by DNA damage

ABSTRACT Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G2/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G2/M to the G1 phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G2 arrest that is alleviated by activated Akt. Furthermore, the transition from the G2/M to the G1 phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G2/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G2/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.

Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes

Constitutive expression of the proto-oncogene c-myc results in oncogenic activation and contributes to progression of a wide range of human and animal tumors. Myc executes its multiple activities mostly through transcriptional regulation of the target genes. The special interest of this review is the mechanism of transcriptional repression of cell cycle inhibitors by Myc. Myc suppresses expression of cell cycle/growth arrest genes gas1, p15, p21, p27, and gadd34, -45, and -153. It appears that Myc represses growth arrest gene transcription by at least two distinct mechanisms. One mechanism is limited to the binding of Myc-Max heterodimers to the Inr element in their promoters and inhibition of Miz-1 or other transcriptional activators via the C-terminal domain of c-Myc. This mechanism requires DNA binding of the Myc-Max complex to Inr sequences. The other mechanism is dependent on c-Myc binding to the Sp1 transcription factor via the c-Myc central region and inhibition of Sp1 transcriptional activity. At this time it is not entirely clear which Sp1-containing promoters will be repressed by c-Myc and what other modes of c-Myc transcriptional repression may exist. The ability of c-Myc to repress transcription of growth arrest genes may contribute to its potential to promote proliferation and oncogenesis.

Identification of a Chemical Inhibitor of the Oncogenic Transcription Factor Forkhead Box M1

The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in a number of different carcinomas, whereas its expression is turned off in terminally differentiated cells. For this reason, FoxM1 is an attractive target for therapeutic intervention in cancer treatment. As a first step toward realizing this goal, in this study, using a high-throughput, cell-based assay system, we screened for and isolated the antibiotic thiazole compound Siomycin A as an inhibitor of FoxM1. Interestingly, we observed that Siomycin A was able to down-regulate the transcriptional activity as well as the protein and mRNA abundance of FoxM1. Consequently, we found that the downstream target genes of FoxM1, such as Cdc25B, Survivin, and CENPB, were repressed. Also, we observed that consistent with earlier reports of FoxM1 inhibition, Siomycin A was able to reduce anchorage-independent growth of cells in soft agar. Furthermore, we found that Siomycin A was able to induce apoptosis selectively in transformed but not normal cells of the same origin. Taken together, our data suggest that FoxM1 inhibitor Siomycin A could represent a useful starting point for the development of anticancer therapeutics.

Thiazole antibiotics target FoxM1 and induce apoptosis in human cancer cells

Forkhead box M1 (FoxM1) oncogenic transcription factor represents an attractive therapeutic target in the fight against cancer, because it is overexpressed in a majority of human tumors. Recently, using a cell-based assay system we identified thiazole antibiotic Siomycin A as an inhibitor of FoxM1 transcriptional activity. Here, we report that structurally similar thiazole antibiotic, thiostrepton also inhibits the transcriptional activity of FoxM1. Furthermore, we found that these thiopeptides did not inhibit the transcriptional activity of other members of the Forkhead family or some non-related transcription factors. Further experiments revealed that thiazole antibiotics also inhibit FoxM1 expression, but not the expression of other members of the Forkhead box family. In addition, we found that the thiazole antibiotics efficiently inhibited the growth and induced potent apoptosis in human cancer cell lines of different origin. Thiopeptide-induced apoptosis correlated with the suppression of FoxM1 expression, while overexpression of FoxM1 partially protected cancer cells from the thiazole antibiotic-mediated cell death. These data suggest that Siomycin A and thiostrepton may specifically target FoxM1 to induce apoptosis in cancer cells and FoxM1 inhibitors/thiazole antibiotics could be potentially developed as novel anticancer drugs against human neoplasia.

FoxM1 Is a General Target for Proteasome Inhibitors

Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

miRNAs: Little known mediators of oncogenesis

Cancer progression is mediated by overexpression of oncogenes and downregulation or loss of tumor suppressors. Proteins, which were traditionally categorized into these groups, have been recently joined by a species of RNA molecules known as microRNAs (miRNAs). miRNAs belong to a class of approximately 22-nt-long non-coding RNAs found in eukaryotes that hinder gene expression by inducing degradation or inhibiting translation of select mRNAs. A growing number of miRNAs have been implicated in promoting or suppressing tumorigenesis in a variety of tissues. The supporting evidence ranges from suggestive expression profiling data to direct functional validation using methods of forward and reverse genetics. We discuss the nature of published results, as well as the merits and pitfalls of various approaches aimed at identification of cancer-related miRNAs and their mRNA targets.

FOX(M1) News—It Is Cancer

FOXM1 is an oncogenic transcription factor of the Forkhead family and it has a well-defined role in cell proliferation and cell-cycle progression. Expression of FOXM1 is excluded in quiescent or differentiated cells, but its level is highly elevated in proliferating and malignant cells. Overexpression of FOXM1 has been reported in more than 20 types of human cancer. In recent years, FOXM1 has been implicated in diverse cellular processes and also a growing body of experimental data has underlined the relevance of FOXM1 in tumorigenesis. Although FOXM1 is under the control of three major tumor suppressors (RB, p53, and p19ARF), it is still active in the majority of human cancers. The oncogenic potential of FOXM1 is mainly based on its ability to transcriptionally activate genes that are involved in different facets of cancer development. In this review, the contribution of FOXM1 to each of the hallmarks of cancer will be summarized and discussed. Mol Cancer Ther; 12(3); 245–54. ©2012 AACR.