Current Pharmaceutical Biotechnology - Volume 10, Issue 2, 2009

Novel Targets in Cancer Therapy In this special issue six articles on current developments in the field of cancer research with special emphasis on novel approaches and or novel targets in cancer chemotherapy are presented. These include identification of novel targets in breast cancer such as the bone marrow microenvironment and the interaction with subsets of tumor cells including tumor stem cells (see article by Rameshwar), the notch signaling pathway (see article by Purow BJ), epigenetics (Humeniuk et al.), novel ideas regarding targeting the immune system for cancer immunotherapy (see Chaudhury et al.), targeting the tumor stromal interaction (article by Anton and Glod) and lastly targeting the tumor initiating cells or tumor stem cells (see article by Bansal and Banerjee). Rameshwar discusses a new way of thinking for identifying the breast cancer stem cells and other subsets in bone marrow. Following entry of breast cancer cells in the marrow, it is proposed that the first step involves formation of gap junctional intercellular communication between cancer cells and stromal cells close to the endosteum. An understanding of the mechanisms by which cancer cell subsets interact with other cells of the bone marrow could lead to an understanding of cancer behavior in bone marrow and may lead to identification of novel therapeutic targets. Chaudhury et al. propose that the immunosuppressive tumor environment can be altered to become immune activating, thus facilitating the infiltration of myeloid and lymphoid cells that can act in concert leading to tumor regression. In this regard, immunotherapeutic approaches such as DNA vaccines, dendritic cell based vaccines, HSP based vaccines and gene transfer technology, are being developed and further refined to overcome their inherent limitations. They argue that careful evaluation of the suppressive nature of the tumor microenvironment accompanied by qualitative and quantitative measurements of lymphocyte responses in patients may lead to development of more meaningful therapeutic strategies. Combined with development of advanced genetic technologies and continuous identification of novel tumor antigens, the field of cancer immunotherapy is poised to make major advances. The Notch signaling pathway has been found to play central roles in humans in stem cell maintenance, cell fate decisions, and in cancer as well. Notch family members are now being recognized as oncogenes in an ever-increasing number of cancers making them attractive therapeutic targets. Purow makes the case that although significant progress has been made in dissecting the complex workings of this signaling pathway, there are very limited options available for clinical use of Notch inhibitors. The review addresses current state-of-the-art, newer notch targeting agents in the pipeline, and potential strategies for use of future Notch inhibitors in the clinic as anticancer agents. Humeniuk et.al. propose that epigenetic modifications can be transmitted to the next generation and used to turn off and/or on certain genes or pathways that may confer survival benefit to a cancer cell. As the epigenetic changes are readily reversible, strong arguments can be made in favor of “epigenetic therapy”. They are quick to point out that a potential problem in this therapeutic approach is the lack of specificity as epigenetic modifications are used by both normal and cancer cells to regulate expression of various genes. They refer to ongoing studies to identify genes that are differentially expressed in cancer cells vs. normal cells are providing valuable information about molecular targets for epigenetic therapy. Humeniuk et al. summarize some of these studies and discus the differences between conventional and epigenetic therapy utilizing epigenetic drugs like DNA methyltransferase inhibitors or histone deacetylase inhibitors. Current thoughts on the future of epigenetic therapy are also discussed. It is now recognized that the stroma plays a role in processes such as nutritional support, the removal of waste products, and the creation of a barrier regulating the exchange of fluids, gases, and cells. The stroma provides support with growth factors and cytokines and promotes angiogenesis, tissue invasion, and metastasis. More recently, it has become evident that the stroma provides a chemoresistant capability to the tumor, preventing chemotherapeutics from reaching their target. Anton and Glod discuss the recent developments in targeting the various players involved in the tumor stroma interactions and argue that future development of more specific targets will depend upon further characterization of the cellular and molecular interactions in the tumor microenvironment.

This review focuses on the properties of different breast cancer cell subsets, including cancer stem cells (CSCs) and cancer progenitors. The premise is that an understanding of self-renewal, the effects of aging microenvironment on the behavior of cancer cell subsets will map the path of development from CSCs to progenitors. The basic characterization of different cancer cell subsets will lead to their signatures and open the field to novel methods of prognosis and diagnosis. The identification of cancer progenitors would allow scientists to modify the cells genetically for dedifferention to CSCs. This will benefit the field to understand the method by which CSCs are developed. The review discusses a reductionist approach for identifying the CSCs and other subsets in bone marrow. An understanding of the mechanisms by which cancer cell subsets interact with other cells of the bone marrow could lead to an understanding of cancer behavior in bone marrow. A protective role of mesenchymal stem cells after the cancer cells enter the marrow is proposed as the first step in the cancer cells forming gap junctional intercellular communication with stromal cells close to the endosteum. It is possible that microRNAs could be shared between the cancer cells and stroma. The review recapitulates two stages of breast cancer: an early stage when the cancer cells enter bone marrow, perhaps prior to clinical detection and during the stage of heavy tumor burden when a subset of cancer cells survive and can resurge years after remission. This review argues for new approaches to identify breast cancer stem cells, and to understand how this population of cells interacts with the bone marrow microenvironment. In summary, the presented approach could lead to the development of new drug targets and approaches to treat breast cancer.

Notch was first recognized as an important developmental pathway in Drosophila in the first half of the 20th century. Many decades later, this pathway has been found to play central roles in humans in stem cell maintenance, cell fate decisions, and in cancer as well. Notch family members are being revealed as oncogenes in an ever-increasing number of cancers. Though significant progress has been made in dissecting the complex workings of this signaling pathway, there are very limited options available for Notch inhibitors. However, the pioneering class of Notch inhibitors is already in clinical trials for two cancer types. This review will address the current state-of-the-art, agents in the pipeline, and potential strategies for future Notch inhibitors. Successful development of Notch inhibitors in the clinic holds great promise as a new anti-cancer strategy.

Recent advances in cancer research showed that changes of the cell “epigenome” contribute significantly to the development and progression of cancer. Similar to genetic alterations, epigenetic modifications can be transmitted to the next generation and used to turn off and/or on certain genes or pathways that may confer survival benefit to a malignant cell. However, epigenetic changes are readily reversible raising the possibility of “epigenetic therapy”. A potential problem in this therapeutic approach is the lack of specificity, as epigenetic modifications are used by both normal and cancer cells to regulate expression of various genes. Ongoing studies to identify genes that are differentially expressed in cancer cells vs. normal cells are providing valuable information about molecular targets for epigenetic therapy. The present article will focus on summarizing some of these studies and will discus the differences between conventional chemotherapy and epigenetic therapy utilizing epigenetic drugs like DNA methyltransferase inhibitors or histone deacetylase inhibitors. Current perspectives on the future of epigenetic therapy are also discussed.

The concept of cancer immunotherapy provides a fresh perspective as it is not associated with many of the drawbacks of conventional therapies such as chemotherapy, radiotherapy and surgery. When fully activated the immune system has immense potential as is evident from mis-matched transplanted organs undergoing rapid immunological attack and rejection. However, the development of immune strategies for cancer therapy has been associated with challenges of their own. Early attempts at cancer vaccination were carried out in an empirical manner that did not always lead to reproducibility. This led to a search of tumor associated antigens with the belief that specific targeting of these antigens would lead to successful tumor elimination. Active vaccination with TAA peptides or passive vaccination with specific lymphocytes against these TAAs did not however demonstrate encouraging results in clinical trials. This was mainly because of the lack of an activating immune response which is required for continuous stimulation of lymphocytes and also because of the selection of tumor escape variants that did not express the particular TAA. On the positive side, attempts at characterizing TAAs illuminated the molecular changes that attribute a malignant phenotype to cancer cells. Attempts at cytokine therapy were also met with challenges of high systemic toxicity and a lack of specific lymphocyte activation. It was therefore realized that an ideal vaccinating agent should be able to combine the effects of both these therapeutic strategies, i.e., it should be able to induce an innate immune response which can be tailored to a tumor specific adaptive immune response. By this, the immunosuppressive tumor environment can be altered to become immune activating, thus facilitating the infiltration of myeloid and lymphoid cells that can act in concert leading to tumor regression. In this regard, immunotherapeutic approaches such as DNA vaccines, dendritic cell based vaccines, HSP based vaccines and gene transfer technology, are being developed and further refined to overcome their inherent limitations. Animal experiments with these therapeutic modalities have demonstrated exciting results, although their evaluation in clinical trials has not indicated exceptional tumor protection in a large percentage of the patients. These observations only further underscore the multivariate and dynamic nature of the immune system and the many ways in which tumor cells modulate themselves and their surroundings to escape immune surveillance. Assessment of successful therapeutic intervention will require periodic evaluations of the suppressive nature of the tumor microenvironment accompanied by qualitative and quantitative measurements of lymphocyte responses in patients. With the development of advanced genetic technologies and continuous identification of tumor antigens, the field of cancer immunotherapy is progressing at an exciting pace giving us hope for the advent of effective treatment modalities that will prolong tumor free survival and enhance the quality of life in patients with malignant disease.

Increasing evidence shows that the interaction between neoplastic cells and the surrounding stroma is a critical factor in solid tumor growth. The tumor stroma is made up of diverse cellular populations including macrophages, lymphocytes, vascular cells, and carcinoma-associated fibroblasts. The complex interactions between the stroma and neoplastic cells are largely unexplored. Initial therapies aimed at disrupting angiogenesis within the tumor microenvironment have met with success in a number of tumor types. An improved understanding of stromal signaling pathways is likely to identify additional novel therapeutic targets.

Cancer Stem cells (CSC) are defined as a population of cells found within a tumor that have characteristics similar to normal stem cells. Like normal stem cells they have the potential to self renew and differentiate. The cellular origin of these cancer stem cells - whether they originate from stem cells that have lost the ability to regulate proliferation, or they arise from more differentiated population of progenitor cells that have acquired abilities to self-renew is still unclear. Investigators have reported isolation of cancer stem cells or tumor initiating cells using techniques developed for isolating hematopoietic stem cells and assays that identify a small subset of tumor initiating cells. The TICs are thought to play an important role in tumor development, progression as well response to therapy and relapse. Strategies that combine conventional therapies with newer approaches that target the TICs may be more effective in tumor cell kill are discussed.

After the publication of the draft human genome sequence, now complete or partial genome sequences of 25 mammalian species are available in public genome databases. Obviously, sequence comparisons among the species are crucial for identification of similarities and differences between humans and other mammals in genome evolution. In the post genomic era, however, attention has been paid to uncovering gene function and identifying gene products that might possess therapeutic value. Although, systems biology and in silico analysis will help to predict in vivo gene function in the future, genetic modification using model organisms are necessary to confirm the predictions experimentally in vivo. Because of experimental limitation in human, other mammalian models are essential for this purpose. The laboratory mouse (Mus musclus) has a long history as a human model in biomedical research since early days of genetics. For most of the 20th century, production, identification, and analysis of mouse mutant strains were carried out independently in small scale. During this period, the spontaneous or induced mutant strains were archived and maintained by individual institution or scientist. Through the development of transgenic and gene targeting techniques in mice, the situation changed dramatically. The number of scientists working with mice rapidly increased. Engineering of mouse genome attracted many molecular biologists, because it seemed the most efficient way to study functions of gene in vivo and to generate models for human diseases. During same period, several large integrated projects were undertaken. They include the sequencing of the mouse genome, the production of numerous new mutations using chemical mutagens or by gene trap, and systematic phenotyping of many inbred and mutant strains. Despite the large body size and technical inabilities, chemical mutagenesis projects in rats are in progress. The resources also contain increased number of genetically engineered mutants and gene targeted ES clones from the knockout mouse project, which is a high-throughput international effort to produce knockout ES cells for all mouse genes. These precious and valuable resources, however, are not connected to efficient systems to produce novel therapeutic strategies or drug development. In this special issue of Current Pharmaceutical Biotechnology, we aimed to enhance and increase interaction between the mammalian mutant resource projects and pharmaceutical science. For this purpose, we selected three categories for review articles: 1) introduction of mammalian resource projects; 2) technology aspects for developing the resources; 3) applications of the mammalian resources to human diseases. First, chemically induced mutant resources are described; Soewarto et al. and Mashimo & Serikawa reviewed recent progress of ethyl nitrosourea (ENU) mutagenesis projects in the mouse and the rat, respectively. Araki et al. introduced ES cell based gene-driven mutagenesis projects, the gene trap project, and the usage of web-based database to obtain the information on trapped clones. Matsushima introduces that genetic variation in wild mice derived strains is also a valuable source for mutations. In addition to these four reviews on mutant resources, two reviews were selected to expand on technological usage of mutant resources. Fuchs et al. described the German Mouse Clinic, a large phenotyping centre for mutant mice, and its output. The standardization of the phenotyping is becoming more and more important in order to share phenotype information in the future. Ohtsuka et al. focused on recent progress and improvement on techniques in DNA construction for genetic engineering. The developing techniques are critical for production of genetically engineered animals in a highthroughput manner. Finally, usages and application of the mutant resources for human disease are described. Abe & Yu reviewed a positional cloning strategy for mutant strains with inflammatory arthritis. Miyamoto et al. focused on analysis of genetic engineered mice for preclinical studies. Many examples show that mutants of experimental animals are used as in vivo confirmation for pathogenesis of human diseases. More frequently, aspects from analysis of various mammalian mutants could be applied to development of new treatments and drugs in next decades. Thus mammalian mutant resources hold vast potential to accelerate the novel therapeutic challenges and thereby contribute to human health. We hope that this issue helps to the bridging and development of the interaction among different fields for the aspiration.

Aim of this review is to demonstrate the relevance of animal models created by ENU mutagenesis for the pharmaceutical community to understand diseases and the modulation of disease status by pharmaceutical compounds. We give an overview of what ENU mutagenesis in mice implies and introduce the main research centers running ENU mutagenesis projects. The different strategies of ENU mutagenesis screens are explained as well as the latest advances in mapping and mutation detection strategies, which until recently have been the main limiting step in forward genetics/ phenotype- driven approaches. ENU mutagenesis in mice has shown its power by providing animal models for human monogenic diseases. Moreover, the development of modifier and sensitized screens extended this resource to models for multigenic diseases and thereby opened the perspective to understand the modulation of disease states. Finally, we provide information about the accessibility and availability of these models for academic research.

The laboratory rat is obviously an important model for physiology, pathology, pharmacology, toxicology, and transplantation experiments. The value for pharmacological research is immense since virtually every drug approved for human treatment passes through the body of laboratory rats. Hundreds of unique rat models have been developed to mimic pathological and physiological human clinical conditions, especially in the case of complex diseases. Many of the model rats are deposited into rat resource centers, from which researchers can use and share animals and rat related resources in biomedical research. Recent progressing technologies for genetically engineered rats, such as traditional transgenesis, chemical ENU mutagenesis, and transposon insertional mutagenesis, wil l provide thousands of useful rat models for functional genomics and human diseases. Globally acting rat resource centers are prerequisites for successful and sustainable research in the biomedical field where the rats are used as model species.

While the human genome project has been completed, analysis of functions of each gene is still underway. Knockout and knock down of gene products offer useful tools to understand functions of a single gene in vivo. Production of knockout mice using homologous recombination in embryonic stem (ES) cells is a powerful and established strategy. However, it is laborious, time-consuming and expensive if expanding large scale. In mice, the gene trap is an alternative strategy to disrupt gene functions by random disruption of gene. The functions of a gene in vivo can be analyzed by production of mice from trapped ES clones. Large-scale gene trap projects have been started in some research centers of the world, and the International Gene Trap Consortium (IGTC) was established to strengthen interactions among centers involved. Moreover, the website of the IGTC has been constructed to integrate information of trap clones from each gene trap project. The database of the IGTC is expanding rapidly because of accumulation of information about gene trap clones from ongoing gene trap projects; approximately 135,000 trapped ES lines are registered in June, 2008. These clones are freely available to academic community. At moment, the IGTC cell lines have covered approximately 10,000 genes in the mouse genome database. Therefore, it is recommended to check the IGTC database before starting knockout experiment, even when annotations of genes are not available. In this review, we introduce principle and short history of gene trap, and then use of the IGTC database is described to obtain trapped ES clones for the experiments.

Personalized medicine offers a custom-made treatment for each patient directed by information of individual's genetic variation. Despite plenty of information about human single nucleotide polymorphisms (SNPs) and gene expression profile, predicting functions of genetic variations in humans is still a difficult task. Genetic analysis using experimental animals is possible to provide information about the functions of genetic polymorphisms over experimental invention in humans. In particular, inbred strains established from wild mice are valuable resources for analyzing functions of genetic polymorphisms. In this article, first I describe history of inbred strains derived from Japanese wild mice, Mus musuclus molossinus. Next, I discuss a mouse model for hyperlipidemia, which was isolated from a colony of Japanese wild mice. Interestingly, the hyperlipimic phenotypes are varied in congenic strains on other genetic backgrounds, reflecting phenotype variation of hyperlipidemia in human populations. Thus, further genetic analysis of Japanese wild mice can contribute to functional analysis of human genetic variation leading to personalized medicine.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line - a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, hostpathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Genetically modified animals have been used as models in broad range of studies including pharmaceutical biology. Designing and construction of transgene constructs are the first indispensable task in generating model animals. In addition to the classical restriction enzyme-based method, still holds some advantages in generating precise constructs, site-specific recombinase-based and homologous recombination-based DNA engineering strategies (e.g. Gateway and Red/ET recombineering, respectively) have been developed and widely used for vector construction or BAC modification. In this review, the three construction methods are described and their applications are discussed such as tandem assemblies of multiple components and modification of large DNA molecules. Combinational use of these E. coli-based recombinant DNA technologies enables the generation of precisely designed vectors useful for desired genome modification for future analyses.

One of the upcoming next quests in the field of genetics might be molecular dissection of the genetic and environmental components of human complex diseases. In humans, however, there are certain experimental limitations for identification of a single component of the complex interactions by genetic analyses. Experimental animals offer simplified models for genetic and environmental interactions in human complex diseases. In particular, mice are the best mammalian models because of a long history and ample experience for genetic analyses. Forward genetics, which includes genetic screen and subsequent positional cloning of the causative genes, is a powerful strategy to dissect a complex phenomenon without preliminarily molecular knowledge of the process. In this review, first, we describe a general scheme of positional cloning in mice. Next, recent accomplishments on the patho-mechanisms of inflammatory arthritis by forward genetics approaches are introduced; Positional cloning effort for skg, Ali5, Ali18, cmo, and lupo mutants are provided as examples for the application to human complex diseases. As seen in the examples, the identification of genetic factors by positional cloning in the mouse have potential in solving molecular complexity of gene-environment interactions in human complex diseases.

Using gene knockout mice of particular genes is one of the most effective methods in conducting successful study on the mode of action of target gene products in targeted organs. So called the knockout technology is now a powerful tool that can lead us to find clear understanding on difficult questions such as the effects of full antagonist against target molecules. Cacna1b ( 1B) gene knockout mouse was generated to study mechanisms of N-type calcium (Ca2+) channel. The model was able to overcome physiological obstacles in studies of N-type Ca2+ channel selective blockers, such as unspecific binding to structurally similar molecules, and failed distribution to targeted organs. In the case of N-type Ca2+ channel studies, knockout technology was successfully applied to various cardiovascular, sympathetic, nociceptive, sleepawake cycles, metabolic and neurodegenerative experiments using homozygous mutants of the 1B gene that turned out to be viable. These studies were able to confirm not only the predicted phenotypes, but were able to present completely unexpected phenotypes that are great interest for future study. Thus the outputs from the knockout mouse studies lead to gain the proof of concept as a drug for specific inhibitors of the gene products and enabled us to make further prediction of side-effects of these inhibitors in the drug discovery and development process.