Cancer arises through an evolutionary process whereby normal cells acquire mutations that erode growth controls, leading to the inappropriate expansion of aberrantly proliferating cells. Such mutations can involve activation of oncogenes or inactivation of tumor-suppressor genes, each contributing one or more new capabilities to the developing cancer cell. However, cancer is not an inevitable consequence of oncogenic mutations; instead, cells acquiring such mutations can be eliminated or kept in check by innate tumor-suppressor programs that can be activated in these damaged cells.
Our laboratory studies the tumor-suppressor networks that control apoptosis (programmed cell death) and cellular senescence (permanent proliferation arrest) and how disruption of these processes influences malignant behavior. We have previously shown that apoptosis and cellular senescence are potent barriers to oncogene-driven tumorigenesis and that each contributes to the antitumor action of many chemotherapeutic drugs. Thus, not only do mutations that disrupt apoptosis and senescence promote tumor progression but, depending on the particular lesion, they can also reduce the efficacy of cancer therapy. Our current research integrates advanced genetic and genomic tools, such as mosaic mouse models, RNA interference (RNAi), and cancer genomics on human patient samples. We use these tools to identify new components of tumor-suppressor gene networks and to characterize their impact on tumorigenesis and treatment response.
We have recently developed new mouse cancer models based on the genetic manipulation of stem and progenitor cells ex vivo, followed by transplantation of the altered cells into the appropriate organ of syngeneic recipient mice. This approach allows us to rapidly study the impact of many genes and gene combinations on tumorigenesis in a mosaic setting in which tumor-initiating cells are embedded in normal tissues; this closely resembles the situation in human tumors.
We also have developed powerful methods for using RNAi to suppress gene function in vivo in either a stable or a reversible manner. Working with Gregory Hannon (HHMI, Cold Spring Harbor Laboratory), we have developed short hairpin RNA (shRNA) technologies for conditionally controlling expression of the p53 tumor-suppressor gene. Using these tools, we showed that reactivation of endogenous p53 in established tumors can lead to apoptosis or senescence, depending on tumor type and genetic context. In addition to demonstrating the potential for targeting the p53 pathway for therapy, our studies illustrate how transgenic mice harboring conditional RNAi expression systems might be used to spatially, temporally, and reversibly control the expression of any endogenous gene. We are developing methods to produce such mice in a high-throughput manner.
Much of our current work on apoptosis and survival signaling exploits the Eμ-myc transgenic mouse model of B cell lymphoma. We previously showed that disruption of the ARF-p53 tumor-suppressor network and overexpression of the prosurvival genes Bcl-2 or Akt cooperate to accelerate Myc-induced lymphomagenesis. More recently, we used this same system to demonstrate that the eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that acts downstream of Akt to promote tumorigenesis and resistance to certain cancer therapies. We also found that the oncogenic activity of eIF4E correlates with its ability to activate translation and become phosphorylated on serine 209. These results suggest that translational control of cell survival might be a therapeutic target. Indeed, our collaborator Jerry Pelletier (McGill University) used these models to test new inhibitors of translation initiation and found they have antitumor activity in the Eμ-myc system.
In parallel to our studies of survival signaling, we continue to explore the roles and regulation of cellular senescence. Our laboratory was the first to demonstrate that deregulated mitogenic oncogenes could drive cells into a senescent state, thereby preventing transformation, and that senescence could contribute to the outcome of chemotherapy in vivo. Based on the hypothesis that senescence is an important tumor-suppressive mechanism in vivo, we continue to characterize new biological processes in which senescence contributes and are taking a genome-wide approach toward studying its regulation.
We also are interested in the biology of senescence and its potential roles beyond cancer. Although senescent cells have been observed in some aged and damaged tissues, their functional contribution to noncancer pathologies had not been examined. Recently, we showed that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce much of the extracellular matrix deposited in the fibrotic scar. In mice lacking p53, a key senescence regulator, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells activate a genetic program that limits fibrosis by reducing extracellular matrix production and stimulating their clearance by the innate immune system.
These results suggest that the senescence program acts physiologically to limit the fibrogenic response to acute tissue damage and that it acts in other wound-healing responses as well. We continue to explore the interplay between senescence and the immune system in the immune surveillance of developing tumor cells and in normal tissues.
To identify mechanisms of drug resistance and develop new therapeutic targets, we are also focused on factors that influence the cellular response to conventional chemotherapy. These efforts involve a combination of experiments using RNA interference to characterize drug sensitivity and resistance genes, genomic approaches designed to identify genes that are linked to poor treatment responses in patients, and animal-modeling studies to test new drugs and drug combinations that might circumvent drug resistance.
We are, for example, studying factors that influence therapy responses in mouse models of acute myeloid leukemia (AML), an aggressive form of leukemia. Many AML patients display or eventually acquire drug resistance. We produced mosaic AML models that harbor common AML genetic lesions, established the standard-of-care chemotherapy protocol for mice, and integrated sensitive imaging methods to monitor leukemia response in vivo. We then used these tools to characterize genotype-response relationships in AML and to explore the underlying molecular basis for these effects.
We showed that a subtype of leukemia linked to poor treatment response in the clinic (harboring genetic translocations of the MLL [mixed-lineage leukemia] gene) respond poorly to chemotherapy in mice, and that this results, surprisingly, from an attenuated p53 response. Our studies provide insights into the differential response patterns of human leukemia, suggest strategies for improving the use of conventional chemotherapy, and produce tractable preclinical systems for testing new therapeutic strategies. Developing strategies to target chemoresistant leukemia is a major goal of our current research.
To accelerate the pace at which we can gain information about cancer genes and cancer biology, we have initiated a comprehensive program to integrate our animal-modeling and RNAi technologies with human oncogenomics to identify new components of tumor-suppressor gene networks. As one example, we identified the deleted in liver cancer-1 (DLC1) gene as a relevant tumor suppressor in hepatocellular carcinoma. DLC1 is located on human chromosome 8p, which is commonly deleted in many tumor types but had not been decisively implicated in tumor suppression. However, using RNAi, we showed that DLC1 knockdown cooperates with Myc to promote HCC in mice, and that deregulation of RhoA is both necessary and sufficient for this effect. Our results validate DLC1 as a potent tumor-suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.
In addition to probing tumor-suppressor genes in vivo in a one-by-one manner, we used pooled RNAi screens to target the mouse orthologs of genes recurrently deleted in a series of human HCCs, and tested their ability to promote tumorigenesis in a mosaic mouse model. In contrast to randomly selected RNAi pools, many deletion-specific pools accelerated hepatocarcinogenesis in mice. Through further analysis, we identified and validated 13 tumor-suppressor genes, 12 of which had not been linked to cancer previously. One gene, XPO4, encodes a nuclear export protein whose substrate, EIF5A2, is amplified in human tumors, is required for proliferation of XPO4-deficient tumor cells, and promotes HCC in mice. Our results establish the feasibility of in vivo RNAi screens and illustrate how combining cancer genomics, RNAi, and mosaic mouse models can facilitate the functional annotation of the cancer genome. Parallel approaches in other mouse models are ongoing and continue to identify new tumor-suppressor genes.