One of the difficulties of modeling cancer is its complexity. However, a small number of solid tumors are dependent on mutations in single loci, including tuberous sclerosis, neurofibromatosis, and Ret-based tumors such as multiple endocrine neoplasia type 2 (MEN2). Tuberous Sclerosis is an autosomal dominant disorder characterized by benign tumors in multiple organs. Most patients have inactivating mutations in the tsc1 or tsc2 genes, which encode a complex that mediates phosphatidylinositol-3 kinase (PI3K)/mammalian target of rapamycin (mTor) signaling (Sparagana and Roach, 2000). Since the discovery of the Drosophila orthologs in 1999, a large body of work has shown that Tsc-containing complexes integrate inputs from insulin signaling, nutrient sensing, as well as stress response pathways; together, these pathways are relevant for disease progression (Potter et al., 2001; Tapon et al., 2001; Pan et al., 2004; Table 1).
Table 1. List of Genes and Pathways in Drosophila That Have Been Implicated in Human Tumors
Neurofibromatosis 1 (NF1) is typically a childhood cancer syndrome characterized by benign brain tumors and tumors of the peripheral nervous system. Even though only a small fraction of these cancers develop into fatal metastatic disease, most children/adolescents suffer from debilitating skeletal defects and learning disabilities. Most patients show mutations in the NF1 gene, which encodes a large protein called neurofibromin (Ferner, 2007). Experiments performed in Drosophila provided some of the first evidence that NF1 regulates Ras pathway signaling (Williams et al., 2001; Table 1). Later mouse models showed that reduced NF1 activity can cooperate with other pathways including p19ARF and p53 to drive disease progression. This has led to efforts to treat neurofibromatosis patients with Ras pathway inhibitors (Barkan et al., 2006).
Perhaps the most focused Drosophila studies for single mutation tumor syndromes are the Ret-based cancers that primarily give rise to tumors of the thyroid. Activating mutations in the Ret (rearranged during transformation) receptor tyrosine kinase gene result in hyperproliferation of calcitonin producing C cells of the thyroid and a condition known as medullary thyroid carcinoma (MTC). Other tumors can emerge with lower penetrance including pheochromocytomas, parathyroid adenomas, and mucosal neuromas, and collectively this condition is referred to as MEN2 (Hazard et al., 1959; Donis-Keller et al., 1993; Carlson et al., 1994; Eng et al., 1996). MTCs can be treated by surgery if detected early but often—especially in patients with spontaneous disease—they are diagnosed late, when tumors have already metastasized to distant tissues including the brain, lung and liver (Jimenez et al., 2008; Bobinski et al., 2009). Little was known regarding the mechanisms underlying Ret transformation, nor had a useful chemotherapeutic been identified. Attempts to establish a useful mouse transgenic had met with mixed success (Smith-Hicks et al., 2000; Cranston and Ponder, 2003) and have not contributed to the search for a useful therapeutic.
The monogenic nature of MTC made it attractive to model in Drosophila. Targeting Drosophila Ret oncogenic isoforms (dRetMEN2) to the developing fly eye led to a rough eye phenotype characterized by excess proliferation, cell death, and developmental abnormalities. A genetic modifier screen identified 140 different loci that mediated oncogenic dRetMEN2's activity. These represented mediators of a broad array of signaling pathways including Ras, Src, Jnk, and Hedgehog (Read et al., 2005; Table 1). The broad spectrum of pathways was perhaps surprising, and pointed to the potential complexity of even a monogenic tumor.
A closer examination of the strongest genetic modifiers of dRetMEN2 has highlighted Ret's ability to direct many aspects of cancer. For example, our data indicated that dRetMEN2 signals through Src and Jnk to trigger the multiple steps required for metastasis-like behavior of cells, matrix metalloprotease production, and cell invasion (Read et al., 2005; Vidal et al., 2006). Ras pathway signaling, by contrast, appears to act primarily to promote proliferation (Read et al., 2005). Similar to other RTKs, Ret has a large number of docking sites, and presumably these account for its ability to activate many pathways (Cakir and Grossman, 2009). But these data pose a central question of which we have very little understanding: Ret is expressed broadly throughout our bodies, yet only C cells consistently respond by transforming in the presence of oncogenic Ret.
Monogenic models like dRetMEN2 hold considerable promise for the development of next generation therapeutics. After delineating the pathways relevant to tumor progression, rational strategies to inhibit important nodes of these pathways can be developed and tested in the whole animal. This approach has been successful for MEN2 therapeutics, a point that we discuss below.
Other Translational Models of Cancer
Efforts at developing cancer models with slightly more complexity have been successful, and recently a Drosophila model of human glioma, glioblastoma multiforme (GBM), was developed (Read et al., 2009). Human gliomas are deadly locally invading tumors that arise from glia and their precursor cells. Treatment options for gliomas are limited due to the nature of their location: chemotherapy, radiation, and if possible surgical resection are options. But most often the prognosis is poor with a mere 25% survival rate within 2 years of diagnosis (Kleihues et al., 1995; Maher et al., 2001; Furnari et al., 2007).
Gliomas commonly display constitutive activation of the epidermal growth factor receptor (EGFR) and PI3K pathways (Maher et al., 2001; Furnari et al., 2007). Activation of these two pathways in Drosophila embryonic glial cells resulted in massive over-proliferation of glial cells and a significantly larger larval brain. These brain-derived tumors grew and invaded into local tissues when transplanted into the abdomen of adult flies. It also led to activation of a network of genes linked to oncogenesis including Myc, Rb, Cyclins, and dTor (Read et al., 2009). Thus this model successfully showed that cooperative interaction between two pathways can induce an “oncogenic signature” that is distinct from the signatures that are observed by activation of either pathway alone, i.e., an emergent property (Table 1). Of interest, EGFR/PI3K pathway co-activation slightly earlier in the neuroblasts did not elicit glial hyper-proliferation. This suggested that the Drosophila larval glial cells have a “state” conducive to transformation that is distinct from the neuroblasts. Future studies will be required to identify differences between larval neuroblasts and committed glial precursors and establish how combinatorial signaling through PI3K and EGFR is kept under check in neuroblasts. This may prove helpful for identifying mechanisms that counter combinatorial activation of these pathways as a step toward targeted therapeutics.
A wing-based Drosophila genetic screen helped resolve a long-standing issue in another tumor type, colorectal cancer. The retinoblastoma gene (Rb) is a tumor suppressor that is mutated in a variety of cancers, yet Rb/E2F1 mutations are absent in colorectal tumors (Nevins, 2001). Most colorectal tumors require loss-of-function mutations in the adenomatous polyposis coli (Apc) gene; these mutations lead to stabilization of β-catenin and aberrant activation of the downstream effector cellular-myelocytomatosis (c-Myc; Kinzler and Vogelstein, 1996; Clevers, 2006). The Drosophila studies established E2F1 as a functional antagonist of β-catenin signaling. Using mouse studies and patient tumor analysis it was subsequently established that, in colon cancer, Rb represses E2F1 activity to allow aberrant β-catenin and c-Myc activity (Morris et al., 2008). Therefore, while Rb acts as a tumor suppressor in many tumors it acts as an oncogene in colorectal cancer. This study demonstrates the insights that Drosophila can provide into mechanisms driving human tumors (Table 1).