Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements

Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets. Due to the selective lethal effect on cancer cells harboring specific genetic alterations, it is expected that targeting synthetic lethal genes would provide wider therapeutic windows compared with cytotoxic chemotherapeutics. Here, we review the current status of the application of synthetic lethal screening in cancer research fields from biological and methodological viewpoints. Very recent studies seeking to identify synthetic lethal genes with K-RAS and p53, which are known to be the most frequently occurring oncogenes and TSGs, respectively, are introduced. Among the accumulating amount of research on synthetic lethal interactions, the synthetic lethality between BRCA1/2 and PARP1 inhibition has been clinically proven. Thus, both preclinical and clinical data showing a preferential anti-tumor effect on BRCA1/2 deficient tumors by a PARP1 inhibitor are the best examples of the synthetic lethal approach of cancer therapeutics. Finally, methodological progress regarding synthetic lethal screening, including barcode shRNA screening and in vivo synthetic lethal screening, is described. Given the fact that an increasing number of synthetic lethal genes for major cancerous genes have been validated in preclinical studies, this intriguing approach awaits clinical verification of preferential benefits for cancer patients with specific genetic alterations as a clear predictive factor for tumor response.     

Using Functional Genetics to Understand Breast Cancer Biology

Genetic screens were for long the prerogative of those that studied model organisms. The discovery in 2001 that gene silencing through RNA interference (RNAi) can also be brought about in mammalian cells paved the way for large scale loss-of-function genetic screens in higher organisms. In this article, we describe how functional genetic studies can help us understand the biology of breast cancer, how it can be used to identify novel targets for breast cancer therapy, and how it can help in the identification of those patients that are most likely to respond to a given therapy.Much remains to be learned regarding the function of mammalian genes. Only some quarter of all human genes have well-described functions. It is likely that quite a few of these currently unannotated genes will turn out to play key parts in cancer biology. For example, a 70-gene gene signature that can discriminate breast tumors of good and poor prognosis contained some 20 genes of currently unknown function (van ‘t Veer et al. 2002). The fact that these genes of unknown function foretell breast cancer prognosis hints at a role for at least some of these genes in breast cancer biology. The unbiased search for genes that contribute to breast cancer development is therefore likely to yield a rich harvest of new insights. RNA interference allows us to suppress genes systematically on a large scale and study the effects of gene suppression on specific cellular processes or signaling pathways. Consequently, RNA interference-based genetic screens have the potential to deepen our understanding of the molecular events that cause breast cancer, to find novel targets for therapy and to find biomarkers of drug responsiveness. In this article, we will describe the technologies available to perform both gain-of-function and loss-of-function genetic screens and will illustrate how such functional genetic screens have been used in the recent past to study a variety of outstanding questions in the biology of breast cancer.     

A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor

Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.Key words: homologous recombination, PARP inhibitor, BRCA1, BRCA2, PTEN, PALB2, EMSY, double strand break repair     

Oncogenes and Tumor Suppressor Genes

Breast cancer progression involves multiple genetic events, which can activate dominant-acting oncogenes and disrupt the function of specific tumor suppressor genes. This article describes several key oncogene and tumor suppressor signaling networks that have been implicated in breast cancer progression. Among the tumor suppressors, the article emphasizes BRCA1/2 and p53 tumor suppressors. In addition to these well characterized tumor suppressors, the article highlights the importance of PTEN tumor suppressor in counteracting PI3K signaling from activated oncogenes such as ErbB2. This article discusses the use of mouse models of human breast that recapitulate the key genetic events involved in the initiation and progression of breast cancer. Finally, the therapeutic potential of targeting these key tumor suppressor and oncogene signaling networks is discussed.Karyotypic and epidemiological analyses of mammary tumors at various stages suggest that breast carcinomas become increasingly aggressive through the stepwise accumulation of genetic changes. The majority of genetic changes found in human breast cancer fall into two categories: gain-of-function mutations in proto-oncogenes, which stimulate cell growth, division, and survival; and loss-of-function mutations in tumor suppressor genes that normally help prevent unrestrained cellular growth and promote DNA repair and cell cycle checkpoint activation. Epigenetic deregulation also contributes to the abnormal expression of these genes. For example, genes that encode enzymes involved in histone modification are mutated in primary renal cell carcinoma (Dalgliesh et al. 2010; van Haaften et al. 2009). In addition, the involvement of noncoding RNAs in tumorigenesis and tumor metastasis has been recently documented (Croce 2009; Shimono et al. 2009). These can act as oncogenes or tumor suppressor genes, depending on the context. Here, we discuss genes that are frequently altered in breast cancer, focusing on ErbB2, PI3K (phosphatidylinositol 3 kinase) pathways, TP53, BRCA1/2, and PTEN (phosphatase and tensin homolog deleted on chromosome 10). Genetically engineered mouse models are emphasized because these provide a wealth of biological information. We consider in detail genetic and biochemical studies that have shown that oncogenic proteins and tumor suppressors provide a critical balance in regulation of key pathways that control cell number and cell behavior.     

On Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis

Epigenetic programs have been extensively studied in embryonic stem cells. However, epigenetic controls in mammary gland development and in the differentiation of mammary epithelial stem cells have not been defined.The role of epigenetic programs, including DNA methylation, chromatin (histone) modification, and noncoding RNAs, in cellular differentiation and tumorigenesis is well established and, with recent technological improvements, increasingly well understood at the molecular level. Increasing evidence also implicates epigenetic alterations in mediating the long-term effects of environmental risk factors such as diet, exposure to allergens, and various chemicals in various human diseases, including cancer, asthma, and mental disorders (Heijmans et al. 2009; Feinberg 2010). DNA and histone modification patterns have been the most extensively studied in embryonic stem cells (ESCs) (Mikkelsen et al. 2007; Meissner et al. 2008; Lister et al. 2009), whereas the roles of epigenetic changes in mammary gland development and in the differentiation of mammary epithelial stem cells have not been analyzed in either humans or laboratory animals.Huang and Esteller (2011) provide an overview of epigenetic modifications and the technologies developed for their characterization and profiling studies performed in normal mammary epithelial cells and breast cancer. The role of epigenetic programs in regulating human mammary epithelial cell differentiation has not been defined, largely owing to difficulties and controversies associated with the purification and functional characterization of various progenitor and differentiated cells. As discussed by Borowsky (2011) and Visvader and Smith (2011), currently there is no consensus on the identity of bipotential human mammary epithelial stem cells and luminal and myoepithelial progenitors. Further hampering progress in this area are the lack of technologies suitable for the characterization of genome-wide DNA methylation and histone modification profiles of small numbers of cells that can be recovered from tissue samples. Advances in single-molecule sequencing platforms and their application to epigenetic studies will likely solve this problem as methods allowing genome-wide gene expression, DNA methylation, and histone methylation profiling of minute cell numbers have recently been described (Adli et al. 2010; Gu et al. 2010; Ozsolak et al. 2010). The lack of defined human mammary epithelial stem cell hierarchy also makes the interpretation of epigenetic alterations identified in breast cancer problematic, owing to uncertainties about what normal cell to use for comparison. This is especially problematic when using bulk tissue samples, which is the case in the majority of published studies. Numerous genes have been identified as being epigenetically altered in breast cancer and some of these are likely to reflect true malignancy-associated events, but many events may just reflect cell-type-specific differences between normal and cancer tissues. Although this issue does not influence the use of these markers for cancer diagnosis and prognostication, it complicates attempts to understand their potential role in tumorigenesis.One of the most exciting areas of investigation is the role of epigenetic alterations in the long-term effects of various life events on breast cancer risk. For example, in utero exposure to chemicals such as bisphenols (BPA) may increase breast cancer risk by inducing epigenetic alterations in mammary epithelial stem and progenitor cells. Similarly, the reduced risk of postmenopausal breast cancer associated with full-term pregnancy in young adulthood may also be explained by epigenetic alterations in stem cells. The development of new technologies and improved understanding of human mammary epithelial cell types will assure rapid progress in these areas.Finally, the most important question is how we can use the knowledge we have gained for the prevention and treatment of breast cancer. Drug discovery efforts aimed at the identification of inhibitors of specific DNA- (and histone) modifying enzymes will likely lead to the discovery of clinically useful agents. The number of studies published on these topics in the past few years and the number of pharmaceutical companies pursuing epigenetic targets guarantee that progress in these areas will be made soon.     

Morphogen Gradient Formation

How morphogen gradients are formed in target tissues is a key question for understanding the mechanisms of morphological patterning. Here, we review different mechanisms of morphogen gradient formation from theoretical and experimental points of view. First, a simple, comprehensive overview of the underlying biophysical principles of several mechanisms of gradient formation is provided. We then discuss the advantages and limitations of different experimental approaches to gradient formation analysis.How a multicellular organism develops from a single fertilized cell has fascinated people throughout history. By looking at chick embryos of different developmental stages, Aristotle first noted that development is characterized by growing complexity and organization of the embryo (Balme 2002). During the 19th century, two events were recognized as key in development: cell proliferation and differentiation. Driesch first noted that to form organisms with correct morphological pattern and size, these processes must be controlled at the level of the whole organism. When he separated two sea urchin blastomeres, they produced two half-sized blastula, showing that cells are potentially independent, but function together to form a whole organism (Driesch 1891, 1908). Morgan noted the polarity of organisms and that regeneration in worms occurs with different rates at different positions. This led him to postulate that regeneration phenomena are influenced by gradients of “formative substances” (Morgan 1901).The idea that organisms are patterned by gradients of form-providing substances was explored by Boveri and Hörstadius to explain the patterning of the sea urchin embryo (Boveri 1901; Hörstadius 1935). The discovery of the Spemann organizer, i.e., a group of dorsal cells that when grafted onto the opposite ventral pole of a host gastrula induce a secondary body axis (Spemann and Mangold 1924), suggested that morphogenesis results from the action of signals that are released from localized groups of cells (“organizing centers”) to induce the differentiation of the cells around them (De Robertis 2006). Child proposed that these patterning “signals” represent metabolic gradients (Child 1941), but the mechanisms of their formation, regulation, and translation into pattern remained elusive.In 1952, Turing showed that chemical substances, which he called morphogens (to convey the idea of “form producers”), could self-organize into spatial patterns, starting from homogenous distributions (Turing 1952). Turing’s reaction–diffusion model shows that two or more morphogens with slightly different diffusion properties that react by auto- and cross-catalyzing or inhibiting their production, can generate spatial patterns of morphogen concentration. The reaction–diffusion formalism was used to model regeneration in hydra (Turing 1952), pigmentation of fish (Kondo and Asai 1995; Kondo 2002), and snails (Meinhardt 2003).At the same time that Turing showed that pattern can self-organize from the production, diffusion, and reaction of morphogens in all cells, the idea that morphogens are released from localized sources (“organizers” à la Spemann) and form concentration gradients was still explored. This idea was formalized by Wolpert with the French flag model for generation of positional information (Wolpert 1969). According to this model, morphogen is secreted from a group of source cells and forms a gradient of concentration in the target tissue. Different target genes are expressed above distinct concentration thresholds, i.e., at different distances to the source, hence generating a spatial pattern of gene expression (Fig. 1C).Open in a separate windowFigure 1.Tissue geometry and simplifications. (A) Gradients in epithelia (left) and mesenchymal tissues (right). Because of symmetry considerations, one row of cells (red outline) is representative for the whole gradient. (B) Magnified view of the red row of cells shown in A. Cells with differently colored nuclei (brown, orange, and blue) express different target genes. (C) A continuum model in which individual cells are ignored and the concentration is a function of the positions x. The morphogen activates different target genes above different concentration thresholds (brown and orange).Experiments in the 1970s and later confirmed that tissues are patterned by morphogen gradients. Sander showed that a morphogen released from the posterior cytoplasm specifies anterioposterior position in the insect egg (Sander 1976). Chick wing bud development was explained by a morphogen gradient emanating from the zone of polarizing activity to specify digit positions (Saunders 1972; Tickle, et al. 1975; Tickle 1999). The most definitive example of a morphogen was provided with the identification of Bicoid function in the Drosophila embryo (Nüsslein-Volhard and Wieschaus 1980; Frohnhöfer and Nüsslein-Volhard 1986; Nüsslein-Volhard et al. 1987) and the visualization of its gradient by antibody staining (Driever and Nüsslein-Volhard 1988b, 1988a; reviewed in Ephrussi and St Johnston 2004). Since then, many examples of morphogen gradients acting in different organs and species have been found.In an attempt to understand pattern formation in more depth, quantitative models of gradient formation have been developed. An early model by Crick shows that freely diffusing morphogen produced in a source cell and destroyed in a “sink” cell at a distance would produce a linear gradient in developmentally relevant timescales (Crick 1970). Today, it is known that a localized “sink” is not necessary for gradient formation: Gradients can form if all cells act as sinks and degrade morphogen, or even if morphogen is not degraded at all. Here, we review different mechanisms of gradient formation, the properties of these gradients, and the implications for patterning. We discuss the theory behind these mechanisms and the supporting experimental data.     

p53-based Cancer Therapy

Inactivation of p53 functions is an almost universal feature of human cancer cells. This has spurred a tremendous effort to develop p53 based cancer therapies. Gene therapy using wild-type p53, delivered by adenovirus vectors, is now in widespread use in China. Other biologic approaches include the development of oncolytic viruses designed to replicate and kill only p53 defective cells and also the development of siRNA and antisense RNA''s that activate p53 by inhibiting the function of the negative regulators Mdm2, MdmX, and HPV E6. The altered processing of p53 that occurs in tumor cells can elicit T-cell and B-cell responses to p53 that could be effective in eliminating cancer cells and p53 based vaccines are now in clinical trial. A number of small molecules that directly or indirectly activate the p53 response have also reached the clinic, of which the most advanced are the p53 mdm2 interaction inhibitors. Increased understanding of the p53 response is also allowing the development of powerful drug combinations that may increase the selectivity and safety of chemotherapy, by selective protection of normal cells and tissues.Thirty years of research on p53 have produced a detailed understanding of its structure and function. The almost universal loss of p53 activity in tumors has spurred an enormous effort to develop new cancer treatments based on this fact. Sophisticated animal models have shown that activation of the p53 response in even advanced tumors can be curative (Martins et al. 2006; Ventura et al. 2007; Xue et al. 2007). The p53 gene therapy, Gendicine, is approved in China and its US counterpart, Advexin, has shown activity in number of clinical trials. The p53 protein level is raised in many tumors by virtue of an increase in the protein''s half life and this tumor specific alteration in p53 processing has attracted tumor immunologists, who are now testing a number of p53 based vaccines in cancer patients (Speetjens et al. 2009).In more conventional approaches a range of small druglike molecules targeting the p53 system have been developed and several are now in clinical trials. Of critical importance has been the development of small-molecule inhibitors of the p53–Mdm2 protein interaction such as the Nutlins (Vassilev et al. 2004), which have shown activity against human xenografts in preclinical models. Advanced structural approaches have provided compelling support for the idea that some mutant p53 proteins can be targets for small molecules that would cause them to regain wild-type function (Joerger et al. 2006). Cell based screening methods have identified small molecules that can activate both mutant and wild-type p53 proteins in tumor cells to induce apoptosis. These screens, and RNAi based approaches, have revealed many new targets for therapy in the p53 pathway. In an exciting new approach, that has been validated in other tumor suppressor pathways, the search is on for targets in pathways that will show synthetic lethal interactions with loss of p53 function. Finally drug combinations have been developed that can selectively kill cancer cells that lack p53 function while protecting normal cells (Sur et al. 2009). The next few years hold out the prospect of new p53 based therapies that will be of wide application in cancer and other diseases.     

Endoreplication

Developmentally programmed polyploidy occurs by at least four different mechanisms, two of which (endoreduplication and endomitosis) involve switching from mitotic cell cycles to endocycles by the selective loss of mitotic cyclin-dependent kinase (CDK) activity and bypassing many of the processes of mitosis. Here we review the mechanisms of endoreplication, focusing on recent results from Drosophila and mice.Eukaryotic cells proliferate by undergoing a sequence of events termed the “mitotic cell cycle” in which the genome is duplicated once and only once between cell divisions. The result is a population of cells with two copies of each chromosome (diploid, or 2C). Agents that interfere with the mechanisms that govern genome duplication frequently induce reinitiation of nuclear DNA replication during S phase. This phenomenon, termed “DNA rereplication,” is an aberrant event that produces a population of cells with a heterogeneous DNA content that reflects incomplete chromosome duplication, stalled replication forks, and DNA damage. In most cells, these events can lead to inducing the cell’s DNA damage response and can lead to apoptosis (Lee et al. 2010).Remarkably, some cells are developmentally programmed to exit their mitotic cell cycle in response either to environmental signals or to injury or stress, and then differentiate into nonproliferating, viable, polyploid cells. This phenomenon, termed “developmentally programmed polyploidy,” is a normal part of animal and plant development that occurs frequently in ferns, flowering plants, mollusks, arthropods, amphibians, and fish, although rarely in mammals. In contrast to DNA rereplication, developmentally programmed polyploidy produces cells with a DNA content of >4C, but in integral multiples of 4C (e.g., 8C, 16C, 32C, etc.), consistent with multiple S phases in the absence of cytokinesis. These cells typically stop proliferating but remain viable in a terminally differentiated state that may serve to regulate tissue size or organization, to trigger cell differentiation or morphogenesis, to increase the number of genes dedicated to tissue-specific functions without increasing the number of cells, or to adapt to environmental conditions. Mitotic divisions of polyploid cells are common for plant species, but they are rarely found in animals. Although known for decades, polyploid mitosis in insects remained mostly unstudied until it was recently shown that the cells of the rectal papilla in Drosophila undergo mitosis after executing two or more endocycles (Fox et al. 2010). Thus, polyploidy is not an irreversible process, although the benefit of this cell cycle variant remains to be elucidated.Developmentally programmed polyploidy occurs by at least four different mechanisms (Ullah et al. 2009). Proliferating cells in the syncytial blastoderm of Drosophila embryos and some hepatocytes in the postnatal liver of mammals become multinucleated and therefore polyploid by failing to undergo cytokinesis after mitosis (“acytokinetic mitosis”). Differentiation of skeletal muscle myoblasts into myotubes, monocytes into osteoclasts, and formation of placental syncytiotrophoblasts involves “cell fusion” to produce multinucleated, terminally differentiated cells that are similarly polyploid. Alternatively, cells may exit their mitotic cell cycle by arresting mitosis during anaphase and failing to undergo cytokinesis. This phenomenon, termed “endomitosis,” produces cells with a single giant nucleus that may subsequently fragment into a multinuclear appearance. Endomitosis occurs in mammals when megakaryoblasts differentiate into megakaryocytes (Bluteau et al. 2009), and in some plant cells (Weingartner et al. 2004). However, the primary mechanism for developmentally programmed polyploidy in arthropods (Smith and Orr-Weaver 1991; Edgar and Orr-Weaver 2001), plants (de la Paz Sanchez et al. 2012), and possibly mammals (Ullah et al. 2009) is “endoreplication” (also referred to as “endoreduplication”). Endoreplication occurs when a cell exits the mitotic cell cycle in G₂ phase and undergoes multiple S phases without entering mitosis and undergoing cytokinesis. The result is a giant cell with a single, enlarged, polyploid nucleus.     

Selective radiosensitization of p53 mutant pancreatic cancer cells by combined inhibition of Chk1 and PARP1

We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells, and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double-strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used two isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild-type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G₂ checkpoint abrogation, inhibition of HRR and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 “deficient-like” phenotype in p53 mutant tumor cells, while sparing normal tissue.Key words: pancreatic cancer, Chk1, PARP1, radiosensitization, p53     

The Role of the Apoptotic Machinery in Tumor Suppression

Multicellular organisms have evolved processes to prevent abnormal proliferation or inappropriate tissue infiltration of cells, and these tumor suppressive mechanisms serve to prevent tissue hyperplasia, tumor development, and metastatic spread of tumors. These include potentially reversible processes such as cell cycle arrest and cellular senescence, as well as apoptotic cell death, which in contrast eliminates dangerous cells that may initiate tumor development. Tumor suppressive processes are organized as complex, extensive signaling networks, controlled by central “nodes.” These “nodes” are prominent tumor suppressors, such as P53 or PTEN, whose loss is responsible for the development of the majority of human cancers. In this review we discuss the processes by which some of these prominent tumor suppressors trigger apoptotic cell death and how this process protects us from cancer development.A malignant tumor is characterized by the ability to expand in an uncontrolled manner, destroy normal tissue architecture, and ultimately undergo metastatic spread (Hanahan and Weinberg 2000). Although the number of mutations required for neoplastic transformation may vary, all tumors are reliant on two critical mechanisms for their development; the activation of oncogenes that promote proliferation and survival of cancer cells, as well as the inactivation of tumor suppressor genes that normally repress development and growth of tumors (Hanahan and Weinberg 2000).Oncogenes can be activated via multiple mechanisms, including chromosomal translocations, deletions or insertions, as well as point mutations. One such example is the translocation between chromosomes 9 and 22 that is present in most cases of chronic myeloid leukemia. The juxtaposition of the BCR and c-ABL genes results in the production of an abnormal BCR-ABL fusion protein with constitutive kinase activity (Deininger et al. 2005). However, in other cancer-causing chromosomal translocations, such as the t[8;14] translocation in Burkitt’s lymphoma, the coding sequence of the oncogene, c-MYC, is unchanged; rather its activation results from deregulated expression in B lymphoid cells as a consequence of its proximity to the IGH gene enhancer (Cory et al. 1987). Tumorigenesis promoted by deregulated kinase activity frequently results from the acquisition of point mutations. In this context, a single amino acid substitution can dramatically enhance kinase activity by preventing binding of negative regulators or “locking” the catalytic domain in the active conformation. This is exemplified by the BRAF(V600E) mutation frequently observed in melanoma or colon carcinoma (Poulikakos and Rosen 2011) and the activating mutations in EGF-R observed in lung adenocarcinoma (Sharma et al. 2007).Analogous to the activation of oncogenes, tumor suppressor genes can be inactivated through multiple mechanisms, including large-scale chromosomal alterations or point mutations. However, in most cases both alleles of the gene must be compromised to abolish gene function, unless the mutated protein can act in a dominant-negative fashion to block the activity of its wild-type counterpart.Multicellular organisms have evolved a plethora of mechanisms to restrain the growth or even eliminate aberrant cells—these processes can all function as tumor suppressors. Notably, of the attributes that cells must acquire to become cancerous (“hallmarks of cancer”) discussed by Hanahan and Weinberg (2000), several relate to escape from regulatory processes that would normally suppress tumor growth. They include cell cycle arrest, cellular senescence, and cell death; of these only cell death is irreversible, all others can (at least potentially) be reversed. In this review, we describe the mechanisms by which tumor suppressors that are disabled in a broad range and large fraction of cancers trigger cell death, and how components of the apoptotic machinery can themselves act as tumor suppressors.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.     

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells

Background

Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.

Result

Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.

Conclusion

Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.