Current Cancer Research in the MCB Department

Cancer arises by a multi-step, Darwinian process of variation and selection, involving the accumulation of activating mutations in proto-oncogenes and inactivating mutations in tumor suppressor genes. The process is accelerated by the genetic instability of cancer cells, which is believed to result from passage through “telomere crisis.” Thus, cancer cells may contain many – perhaps hundreds – of genetic changes. One of the challenges we now face is to be able to develop a complete description of the genetic changes that have taken place in each individual tumor, so that therapies can be targeted that are specific for the tumors of each individual patient.

One of the most promising developments in the field is the development of new “rational” or “targeted” treatments directed against the altered gene products found in cancer cells. Classical cancer treatments usually rely on chemo- or radiation therapies to induce apoptosis of tumor cells.  These treatments have many undesirable side effects because normal cells, although retaining many of the protective checkpoint mechanisms that tumor cells have lost, are nevertheless still sensitive to these agents. Drugs that target specific oncogene products or proteins involved in the regulation of proliferation, motility and apoptosis are therefore needed. The most spectacular example of the success of such an approach is the drug Gleevec, a chemical agent that inhibits the Abl protein kinase and has proven effective in controlling human chronic myelogenous leukemia (CML).  Similarly treatment with antibodies specific for CD20 is helpful in combating human non-Hodgkin’s lymphomas and antibodies specific for the neu (HER2) oncoprotein are helpful in controlling human breast cancer.

Thus the goals of basic laboratory research are to identify the genetic changes that occur in cancer, the define the roles of different oncoproteins and tumor suppressors in cancer, and to use our knowledge of protein structural biology and tumor immunology to develop rational targeted therapies.

1. Tumor Immunology

Immunotherapy may provide novel ways of attacking of tumor cells.  Treatment with antibodies specific for CD20 is effective in combating human non-Hodgkin’s lymphomas and antibodies specific for the neu (HER2) oncoprotein are used in the therapy of human breast cancer.   In addition to specific silencing of tumor causing proteins, an alternative novel cancer therapy that harnesses the power of the immune system has recently proven effective in the mouse system and shows promise in some early human clinical trials.   Cancer eradication is achieved by making the tumor cells "immunogenic", which can lead to tumor clearance by the immune system arming host T lymphocytes and natural killer cells so that they can attack and kill tumor cells.  Research activities in the Division of Immunology (Department of Molecular Cell Biology at Berkeley) focus on devising strategies for cancer immuno-therapy (David Raulet, William Sha) and on understanding the signal transduction pathways that control cell proliferation, apoptosis and differentiation. The goal of the latter studies is to identify new molecular targets for leukemia/lymphoma therapy and to understand how tumor cells arise (Astar Winoto, Mark Schlissel, Laurent Coscoy).

•The Winoto laboratory is interested in the molecular mechanism of tumorigenesis and the molecular differences between normal and tumor cells.  Using gene targeting techniques, Winoto's lab has studied the receptor for TRAIL, a protein related to tumor necrosis factor (TNF) that can kill tumor but not normal cells, and has found that elimination of the TRAIL/TRAIL-R interaction leads to enhancement of the immune system. Winoto's lab also found that FADD, a pro-cell death protein for the TRAIL and TNF receptor family members, and survivin, an inhibitory apoptosis protein and a "universal tumor marker" that is an active target of cancer drug therapy, are both important for cell cycle progression. In another line of research, Winoto's lab found that a protein kinase called ERK5 is crucial for angiogenesis and the regulation of VEGF, an angiogenesis inducer important for cancer cell growth. Finally, the Winoto lab is studying a mouse model of human cancer in which T cells deficient for PTEN, a tumor suppressor mutated in many human tumors, develop T cell lymphomas.

•The Schlissel laboratory is studying the mechanism of pre-B cell transformation by the Abelson Murine Leukemia Virus (AMuLV), which causes acute lymphocytic leukemia in mice though expression of the viral oncogene v-Abl.  Its cellular homologue, c-Abl is involved in a chromosomal translocation associated with 90% of cases of human CML. The Schlissel laboratory has identified hundreds of downstream target genes affected by v-abl transformation, some of which could provide novel targets for cancer therapeutics in the future. and is using c-Abl deficient mice to define the role of normal c-Abl activity in hematopoiesis. The Schlissel lab is also studying the V(D)J recombination that is crucial for proper development of B and T lymphocytes but which may also be involved in  the genetic instability and chromosomal translocation that are often associated with lymphoid malignancies, and is studying how errors during V(D)J recombination can give rise to lymphoid malignancy.

•The Coscoy laboratory is interested in pathogenesis by Kaposi’s Sarcoma-associated herpesvirus (KSHV), which causes Kaposi’s sarcoma, a cancer which is endemic in Africa and which is the most common cancer in AIDS patients. Coscoy's laboratory found that KSHV subverts host immunity by expressing two E3-ubiquitin ligases, called MIR1 and MIR2, which induce degradation of key components of immune recognition such as Major Histocompatibility Complex type I (MHC-I) molecules.  Coscoy has identified cellular MIR homologs and cellular co-factors required for the MIR protein function. These may provide additional targets for cancer therapy, since in a mouse model altering MIR function dramatically attenuates viral pathogenesis.

•The Raulet laboratory is studying another arm of the immune system, NK cells or natural killer cells, which can also mount an effective response against tumor cells although most tumor cells are not "immunogenic". The Raulet laboratory identified the protein ligands that bind and activate NK cells through NKG2D, an important NK cell receptor, and found that these ligands are not expressed in normal cells but are up-regulated in many tumor cells. Raulet holds a patent for the use of NKG2D ligands for tumor vaccines.  Raulet's laboratory has also found that NKG2D ligands are regulated by the ATR/ATM DNA damage signaling pathway, suggesting that induction in tumor cells might be tied to cancer genomic instability.

•The Sha laboratory is using a recently described co-stimulatory molecule B7h to render tumor cells "visible" to T lymphocytes.  Sha demonstrated that exogenous introduction of B7h into tumor cells led to their rejection by killer T cells.   B7h is effective in mobilizing recall but not primary responses of killer T cells.  Thus, B7h might complement other B7 molecules as an effective anti tumor immunotherapy.  Sha's laboratory is continuing to explore the unique ability of B7h to elicit T cell memory response against tumor cells.

2. Tumor Cell Biology and Cell Signaling

The mutations that occur in cancer result in alterations in the expression or activity of the proteins that in normal cells regulate cell proliferation, motility, differentiation and survival.  These proteins interact with each other to form a complex signaling network that conveys signals from cell surface receptors to intracellular effector systems, such as the nucleus, the cytoskeleton, metabolic enzymes and protein trafficking machinery.  Components of this signaling network include receptor tyrosine kinases such as the EGF receptor; non-receptor tyrosine kinases such as Src and Abl; small GTPases such as Ras; lipid kinases such as PI-3-kinase; protein serine/threonine kinases such as Raf and Akt; and transcription factors such as Fos and Jun.  Through a variety of mechanisms, these signaling pathways control progression through the cell cycle, largely by regulating the activity of cyclins, cyclin-dependent kinases and Cdk inhibitors.  Not only are many of these proteins found to be mutated in human cancer, but their continued expression and activity is required to maintain the malignant state.  Thus may of these proteins are key targets for therapeutic agents.

•Steven Martin is working on the non-receptor tyrosine kinase Src.  This was the first oncoprotein to be identified, and its activity is elevated in breast and colon cancers, so that it is a target for a number of cancer therapeutics now in clinical trials.  Martin has identified many of the protein substrates that are phosphorylated by this kinase.  Most recently, he has been studying how the expression of activated Src induces the formation of invasive adhesions called podosone or invadopodia, structures that are responsible for the ability of the transformed cells to invade through the extracellular matrix.

•Kunxin Luo has been working on the role of two oncoproteins, Ski and SnoN, in malignant transformation and in signaling by the cytokine TGF-b.   She has found that these two oncoproteins function by antagonizing the transcriptional activity of the Smad proteins, which are transcription factors that mediate anti-mitogenic signaling by TGF-b and related cytokines.  This antagonism occurs both through the recruitment of transcriptional repressors such as N-Co-R and by disrupting the formation of the active Smad heteromeric complex. .

•Kathy Collins has been studying the enzyme telomerase, the activity of which is essential for the ability of cancer cells to escape replicative senescence and proliferate without limit.  She has characterized the RNA protein interactions that are required for the assembly of this multi-subunit ribonucleoprotein complex.  She has also discovered that malignant transformation and DNA damage have opposing effects on the intra-nuclear location of active telomerase that affect the enzyme’s access to its telomeric substrates.

• Gary Firestone is working on the regulation of epithelial cell proliferation by extracellular signals, such as steroid hormones, growth hormones and dietary components.  He has identified a novel serum and glucocorticoid-inducible Ser/Thr kinase, Sgk, that represents a unique convergence point for proliferative and stress-induced signaling pathways.  He has also shown that steroid regulation of tight junction formation involves a multi-step pathway that regulates Ras and RhoA signaling.  In addition, in collaboration with Len Bjeldanes, he has examined the mechanism by which indole-3-carbinol, an anti-cancer compound produced by Brassica plants such as broccoli, suppresses the proliferation of human breast and prostate cancer cells, and has defined the pathways by which this component inhibits the repression and activity of cyclin-dependent kinases.

• Stuart Linn works on DNA damage and repair. His lab studied proteins from human cells that recognize DNA damage.  These proteins directly or indirectly mediate the cell's responses to DNA repair, transcription response, cell cycle arrest and apoptosis. Studies are in progress to examine carcinogenesis in mice lacking several of the DNA damage proteins.

• Lin He's research aims are to identify and characterize novel non-coding RNAs (ncRNAs) that play essential roles during tumorigenesis and tumor maintenance. Particular efforts are focused on microRNAs (miRNAs), a novel class of small, ncRNAs that mediate post-transcriptional gene silencing. Using mouse tumor models and cell culture studies, she will elucidate the molecular basis of the miRNA functions in the oncogenic and tumor suppressor network, and explore the potential of miRNAs as diagnostic tools and/or
therapeutical targets.

3. Structural Biology and Cancer Drug Discovery

As mentioned earlier, the discovery and design of small molecule inhibitors that specifically block the activity of specific signaling proteins provides a window for therapeutic intervention, with the successful cancer drug Gleevec, which blocks the activity of the Abl tyrosine kinase, as a prime example. Other new cancer drugs that are being developed along these lines include, for example, molecules that inhibit the epidermal growth factor receptor, as well as signaling proteins that control the growth of blood vessels that feed cancerous tissue. In each of these cases knowledge of the three dimensional structure of the drug targets (the oncoproteins) as well detailed pictures of the drug bound to the protein provides information that is invaluable for understanding how the drug achieves specificity and affinity for its target. Such knowledge is a critical adjunct to the process of designing the next generation of drugs and dealing with issues of resistance, which invariably develops in patients who are treated over the long term. Fundamental research in structural biology at Berkeley provides such information for a range of targets in cancer biology.

•Nearly half of all chemotherapeutic regimens rely one or more agents that disrupt the function of an essential group of enzymes known as topoisomerases. Topoisomerases are essential for cell viability, in part because of their ability to disentangle newly replicated chromosomes prior the onset of cell division. Research in James Berger’s laboratory is concerned with the structure and function of topoisomerases, including the crystallographic analyses of these enzymes bound to various anti-topoisomerase drugs that are currently in clinical use for the treatment of cancer. Berger has recently determined structures of each of the key catalytic components of yeast topoisomerase II bound to dexrazoxane (ICRF-187), a drug that is provided as an adjuvant to patients undergoing chemotherapeutic treatments, and is also in phased clinical trials for the direct treatment of lymphomas and breast cancer. Berger’s work has revealed that the drug acts not by directly blocking the active site of the enzyme, as is usually the case, but rather by stabilizing the association of these enzyme molecules with one another and preventing enzyme turnover. The emphasis here is on uncovering fundamental principles that can help guide the search for new drugs.

• Papilloma viruses (PV) cause cervical cancer in humans and are the etiological agents of a family of diseases that cause more cancer deaths in women world wide than any other form of cancer. Research in Michael Botchan’s laboratory has resulted in several discoveries that present important targets for future drug intervention in preventing malignant tumors. The PV's hijack the cellular DNA replication machinery through a stepwise assembly process that has been dissected by the lab, leading to the purification and characterization of the viral proteins that are critical for this function.  Structural models of the protein: protein interactions as derived from X- ray diffraction have been helpful in understanding the molecular details of these multi- step pathways. Several lead compounds that prevent these interactions have been uncovered and they have been shown to prevent plasmid replication in cells. Botchan seeks to understand through crystallographic methods the nature of these binding interactions. Such structures could help chemists synthesize better inhibitors that may be useful in preventing cervical cancer.

4. Gene Discovery

We still do not know all the oncogenic mutations that occur during tumor evolution. One strategy that has been successful in identifying novel genes that are altered in human cancers is to first conduct genetic screens in model organisms that are more suited to such manipulations. These include yeast, worms and flies. Screens can be readily designed for mutations that increase cell proliferation, decrease cell death or alter other characteristics of cells such as motility and invasiveness – each a feature of human cancer cells. In these organisms, a variety of powerful genetic techniques facilitate the rapid identification of the mutated gene. Its human ortholog can then be sequenced from a large collection of human cancer lines representing cancers derived from a variety of tissues. If mutations are found in the human ortholog, this indicates that the gene is likely to have an important role in the pathogenesis of human cancer.

The research environment at Berkeley presents an ideal situation to use such a strategy to discover genes that are importance in human cancer. There are a number of investigators in the Department of Molecular and Cell Biology who are using a variety of screens in model organisms to identify genes that function in biological processes that are perturbed in human cancers. Researchers at the Lawrence Berkeley National Laboratory have the expertise to utilize a high throughput strategy to sequence the human orthologs of these genes from a large panel of cell lines.

•Iswar Hariharan’s work illustrates the power of this kind of approach. The archipelago gene in Drosophila was identified in a genetic screen for mutations that result in increased cell proliferation. Archipelago was found to facilitate the destruction of two key proteins that promote cell proliferation – Myc and Cyclin E in Drosophila. The human ortholog of Archipelago (also known as hcdc4 or FBW7) was examined in a panel of cancer cells. Mutations were found in four cell lines suggesting that Archipelago was likely to be a tumor-suppressor gene in humans. More recent studies have shown that Archipelago is mutated in 16% of endometrial cancers, 12% of colorectal cancers and a small number of ovarian cancers.

•David Bilder’s research also illustrates the power of Drosophila genetics in uncovering genes that regulate cell proliferation. Bilder’s laboratory has carried out several screens to identify mutations that disrupt apicobasal polarity of embryonic, imaginal, and adult epithelia. These screens have led us to the study of the ‘neoplastic tumor suppressor genes’ (nTSGs): discs-large, lethal giant larvae, and scribble. The nTSGs encode proteins that act to distinguish the basolateral domain of epithelia by antagonizing the activity of the apical Baz/Par-3 complex. Bilder is studying the mechanisms by which the nTSGs polarize tissue, the signaling pathways that instruct cells as well as entire tissues to cease proliferation, and why these signals require polarized cells for proper transduction.

•Other investigators using invertebrate genetics to uncover genes that could be candidate for human tumor genes include faculty working on Drosophila (Botchan, Karpen), C. elegans (Meyer, Garriga, Dernburg) and yeast (Thorner, Rine, Drubin).

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