Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity.

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Delta) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Delta cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Delta cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Delta cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, beta-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Delta cells with a decrease in G(1) and increase in S and G(2) fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Delta cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor.

Cyclin-dependent kinases and S phase control in mammalian cells.

Mammalian DNA replication is an elegantly choreographed process in which multiple components are assembled at the origins to form the prereplication complex. Formation and activation of the prereplication complex requires coordinate actions of G1and S phase cyclin-dependent kinases. Cyclin E-CDK2 and cyclin A-CDK2, together with DBF4-CDC7, phosphorylate several components of the prereplication complex and replication machinery. In this review, we summarize the current understanding of the mechanism of initiation of DNA replication in mammalian cells. The roles of cyclin A/E-CDK2 complexes in driving replication, their relationship with other regulators of S phase, and their role in keeping replication to only once per cell cycle will be discussed. In addition, an important issue is the checks and balances that prevent inappropriate DNA replication, and how a breakdown in these checkpoints can lead to genomic instability and cancer. A critical mediator of these checkpoints, ATM, signals through a comprehensive network of proteins leading to CDK2 inhibition thus preventing DNA synthesis. This will be reviewed in addition to other mechanisms involved in the intra-S phase DNA damage checkpoint.

Inhibition of mesothelin as a novel strategy for targeting cancer cells.

Mesothelin, a differentiation antigen present in a series of malignancies such as mesothelioma, ovarian, lung and pancreatic cancer, has been studied as a marker for diagnosis and a target for immunotherapy. We, however, were interested in evaluating the effects of direct targeting of Mesothelin on the viability of cancer cells as the first step towards developing a novel therapeutic strategy. We report here that gene specific silencing for Mesothelin by distinct methods (siRNA and microRNA) decreased viability of cancer cells from different origins such as mesothelioma (H2373), ovarian cancer (Skov3 and Ovcar-5) and pancreatic cancer (Miapaca2 and Panc-1). Additionally, the invasiveness of cancer cells was also significantly decreased upon such treatment. We then investigated pro-oncogenic signaling characteristics of cells upon mesothelin-silencing which revealed a significant decrease in phospho-ERK1 and PI3K/AKT activity. The molecular mechanism of reduced invasiveness was connected to the reduced expression of ß-Catenin, an important marker of EMT (epithelial-mesenchymal transition). Ero1, a protein involved in clearing unfolded proteins and a member of the ER-Stress (endoplasmic reticulum-stress) pathway was also markedly reduced. Furthermore, Mesothelin silencing caused a significant increase in fraction of cancer cells in S-phase. In next step, treatment of ovarian cancer cells (OVca429) with a lentivirus expressing anti-mesothelin microRNA resulted in significant loss of viability, invasiveness, and morphological alterations. Therefore, we propose the inhibition of Mesothelin as a potential novel strategy for targeting human malignancies.

Ral overactivation in malignant peripheral nerve sheath tumors.

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.

Activated oncogenes promote and cooperate with chromosomal instability for neoplastic transformation.

Most cancer cells are aneuploid. The chromosomal instability hypothesis contends that aneuploidy is the catalyst for transformation, whereas the gene mutation hypothesis asserts that cancer is driven by mutations to proto-oncogenes and tumor-suppressor genes, with the aneuploidy a side effect of tumorigenesis. Because genotoxic stress induced by "culture shock" can obscure the transforming potential of exogenous genes, we cultured wild-type and p53(-/-) mouse embryo fibroblasts in a more physiological (serum-free) environment. Under these conditions, the cells were immortal and, more importantly, chromosomally stable. Expression of oncogenic H-RasV12 did not induce senescence, but sensitized these cells to p53-dependent apoptosis. In addition, H-RasV12 induced chromosomal instability, as well as accumulation and phosphorylation of p53. Significantly, whereas cells grown under standard conditions could be transformed by coexpression of H-RasV12 and E1A, the chromosomally stable cells were refractory to transformation, as measured by anchorage-independent growth and tumor formation in nude mice. These oncogenes required a third genetic alteration that abolished the p53 pathway to create a permissive environment that promotes rapid chromosomal instability and transformation. Oncogene-induced chromosomal instability and transformation was attenuated by antioxidants. These data indicate that chromosomal instability could be a catalyst for oncogenic transformation, and bring together aspects of the chromosomal instability hypothesis and the gene mutation hypothesis for tumorigenesis.

Gene mutations and aneuploidy: the instability that causes cancer.

Cancer cells have been characterized with activated mutant oncogenes and inactivated or deleted tumor suppressor genes. Cancer cells are also aneuploid, displaying a jumble of chromosomal anomalies including gain or loss of whole chromosomes or transposed chromosomal fragments. Whether mutation of specific genes or aneuploidy is more critical for tumorigenesis is very much a contentious issue. We recently showed that activated oncogenes induce oxidative damage that is exacerbated by conventional cell culture conditions. This "culture shock" or a loss of p53 function creates a precarious environment that permits oncogenes to induce rapid chromosomal instability and transformation. We found that mutant genes and aneuploidy were prerequisites and collaborators for neoplastic transformation.

DNA-dependent protein kinase and checkpoint kinase 2 synergistically activate a latent population of p53 upon DNA damage.

The role of the checkpoint kinase 2 (Chk2) as an upstream activator of p53 following DNA damage has been controversial. We have recently shown that Chk2 and the DNA-dependent protein kinase (DNA-PK) are both involved in DNA damage-induced apoptosis but not G(1) arrest in mouse embryo fibroblasts. Here we demonstrate that Chk2 is required to activate p53 in vitro as measured by its ability to bind its consensus DNA target sequence following DNA damage and is in fact the previously unidentified factor working synergistically with DNA-PK to activate p53. The gene mutated in ataxia telangiectasia is not involved in this p53 activation. Using wortmannin, serine 15 mutants of p53, DNA-PK null cells and Chk2 null cells, we demonstrate that DNA-PK and Chk2 act independently and sequentially on p53. Furthermore, the p53 target of these two kinases represents a latent (preexisting) population of p53. Taken together, the results from these studies are consistent with a model in which DNA damage causes an immediate and sequential modification of latent p53 by DNA-PK and Chk2, which under appropriate conditions can lead to apoptosis.

DNA damage-induced apoptosis requires the DNA-dependent protein kinase, and is mediated by the latent population of p53.

Mouse embryo fibroblasts (MEFs) expressing the adenovirus E1A protein undergo apoptosis upon exposure to ionizing radiation. We show here that immediately following gamma-irradiation, latent p53 formed a complex with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK(CS)). The complex formation was DNase sensitive, suggesting that the proteins came together on the DNA, conceivably at strand breaks. This association was accompanied by phosphorylation of pre-existing, latent p53 at Ser18 (corresponding to Ser15 in human p53), which was not found in DNA-PK(CS)(-/-) cells. Most significantly, DNA damage-induced apoptosis was abolished in both DNA-PK(CS)(-/-) and p53(-/-) cells. In addition, blocking synthesis of inducible p53 by cycloheximide did not abrogate apoptosis, suggesting that the latent population of p53 is sufficient for executing the apoptotic program. Finally, E1A-expressing MEFs from a p53 "knock-in" mouse where Ser18 was mutated to an alanine had an attenuated apoptotic response, indicating that phosphorylation of this site by DNA-PK is a contributing factor for apoptosis.

Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent p53-mediated apoptotic response.

In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. We show here that Chk2-/- mouse embryo fibroblasts (MEFs), unlike Atm-/- or p53-/- MEFs, behaved like normal MEFs in manifesting p21 induction and G(1) arrest upon exposure to gamma-irradiation. Therefore, Chk2 is not involved in p53-mediated G(1) arrest. To examine the role of Chk2 in p53-dependent apoptotic response, we used adenovirus E1A-expressing MEFs. We show that Chk2-/- cells, like p53-/- cells, did not undergo DNA damage-induced apoptosis, whereas Atm-/- cells behaved like normal cells in invoking an apoptotic response. Furthermore, this apoptosis could occur in the absence of protein synthesis, suggesting that it is preexisting, or "latent," p53 that mediates this response. We conclude that Chk2 is not involved in Atm- and p53-dependent G(1) arrest, but is involved in the activation of latent p53, independently of Atm, in triggering DNA damage-induced apoptosis.