Cancer models in Caenorhabditis elegans

Abstract

Although now dogma, the idea that nonvertebrate organisms such as yeast, worms, and flies could inform, and in some cases even revolutionize, our understanding of oncogenesis in humans was not immediately obvious. Aided by the conservative nature of evolution and the persistence of a cohort of devoted researchers, the role of model organisms as a key tool in solving the cancer problem has, however, become widely accepted. In this review, we focus on the nematode Caenorhabditis elegans and its diverse and sometimes surprising contributions to our understanding of the tumorigenic process. Specifically, we discuss findings in the worm that address a well-defined set of processes known to be deregulated in cancer cells including cell cycle progression, growth factor signaling, terminal differentiation, apoptosis, the maintenance of genome stability, and developmental mechanisms relevant to invasion and metastasis.

Figure 1. Regulation of CKI-1

Model of factors controlling the expression and activity of CKI-1. LIN-1, LIN-14, LIN-29, LIN-31, and DAF-16 promote transcription of cki-1, whereas CDC-14 is thought to stabilize CKI-1 protein through removal of an inhibitory phosphate. The antagonist of the CDC-14 protein phosphatase, a presumed kinase, is unknown in C. elegans. Yellow shading indicates the existence of human orthologs. Dashed arrow indicates translation of cki-1 mRNA into protein.

Figure 2. Pocket protein functions are phylogenetically conserved

Comparison of the reported functions for mammalian pocket proteins in tumor suppression and the known functions of the sole C. elegans pocket protein ortholog, LIN-35/pRb. Identically colored lines correspond to related or analogous functions.

Figure 3. The EGFR-Ras-MAPK pathway in C. elegans vulval development

Activation of the EGFR-Ras-MAPK pathway in C. elegans vulval development is initiated when LIN-3/EGF ligand secreted by the gonadal anchor cell (AC) binds to the LET-23/EGFR receptor, causing it to dimerize and undergo autophosphorylation (Aroian et al., 1990; Hill and Sternberg, 1992). This event allows the SEM-5/Grb2 adaptor to bind phosphorylated LET-23 and recruit the SOS-1/Sos1 guanine nucleotide exchange factor (GEF) (Clark et al., 1992a; Chang et al., 2000). SOS-1 promotes the exchange of GDP for GTP on the LET-60/Ras GTPase, which in turn activates a protein kinase cascade that includes LIN-45/Raf, MEK-2/MEK, and MPK-1/MAPK/ERK (Han et al., 1993; Sternberg et al., 1993; Lackner et al., 1994; Wu and Han, 1994; Kornfeld et al., 1995a; Wu et al., 1995; Chong et al., 2003). Targets of MPK-1 phosphorylation include the complex of LIN-1/Ets and LIN-31/FoxB1, which dissociates after phosphorylation, thereby allowing activated LIN-31 to promote the acquisition of vulval cell fates (Tan et al., 1998). Additional positive regulators of vulval fates include SUR-2/Med23 and LIN-39/Hox-B5, a member of the homeobox family of proteins (Clark et al., 1993; Singh and Han, 1995). Known negative regulators include members of the SynMuv group of proteins and CBP-1/p300 (Eastburn and Han, 2005; Fay and Yochem, 2007). Pathway modulators include LIN-2/CASK, LIN-7/Lin-7C, and LIN-10/APBA2, which mediate the subcellular localization of LET-23 and UNC-101/AP-1μ-1 and APM-1/AP-1μ-2, which promote LET-23 endocytotic recycling (Lee et al., 1994; Sternberg et al., 1995; Kaech et al., 1998; Shim et al., 2000). SLI-1/c-Cbl negatively regulates the pathway by targeting activated LET-23 for internalization and degradation (Rubin et al., 2005; Swaminathan and Tsygankov, 2006). The roles of ARK-1/Ack and DEP-1/Dep1/Scc in inhibiting LET-23 signaling are not yet understood. RHO-1/RhoA and its GEF, ECT-2/Ect2, positively regulate the pathway downstream of SEM-5 (Canevascini et al., 2005). The role of the PTP-2/PTPN11 protein tyrosine phosphatase is not well understood, but it may act at several nodes upstream of LET-60 signaling (Gutch et al., 1998; Chang et al., 2000). LET-60 GTP hydrolysis is stimulated by GAP-1/Gap1 (Hajnal et al., 1997). The putative scaffold proteins KSR-1/Ksr1 and SUR-8/Sur8 are required for robust signaling downstream of LET-60 (Kornfeld et al., 1995b; Sundaram and Han, 1995; Sieburth et al., 1998; Stewart et al., 1999). SUR-5/Sur5/AACS, an aminoacyl CoA synthase, inhibits the pathway at the level of LET-60 through an unknown mechanism (Gu et al., 1998). MPK-1/MAPK/ERK is dephosphorylated by the MAPK phosphatase (MKP) family protein LIP-1/MKP, which renders it inactive (Berset et al., 2005). In addition, KSR-1 activity is regulated in part by zinc ion concentrations that are controlled through CDF-1/Znt1 and SUR-7/CDF, by the SUR-6/PP2A protein serine/threonine phosphotase, and by the PAR-1/MARK1 kinase (Jakubowski and Kornfeld, 1999; Sieburth et al., 1999; Bruinsma et al., 2002; Yoder et al., 2004). Yellow fill indicates proteins that were first implicated in EGFR-Ras-MAPK signaling through studies in C. elegans.

Figure 4. Hermaphrodite wild-type and tumorous gonads

In wild-type gonads, mitosis is restricted to the distal portion of the germline. In this region, GLP-1/Notch is activated by LAG-2/DSL via the somatic distal tip cell (DTC). As divisions occur and mitotic cells move out of range of the LAG-2 signal, GLP-1 becomes inactive and germ cells exit mitosis and enter meiosis (transition zone). Moving further proximally, germ cells transit several stages of meiotic prophase, eventually forming male and female gametes that are used for fertilization. Tumorous gonads contain proliferating mitotic cells at ectopic locations. In the case of strong gain-of-function alleles such as glp-1 (oz112gf), meiosis is completely abolished and mitotic cells are detected throughout the gonad arm, resulting in a contiguous tumor. Loss-of-function mutations in other genes (e.g., pro-1 and gld-1) lead to more-limited tumor formation in the central-to-proximal gonad region.

Figure 5. The regulatory network that controls germ cell proliferation

Increased activity or ectopic expression of proteins in green boxes promotes mitotic proliferation. In the case of glp-1(gf) mutants, this results in germline tumor formation. Decreased activity of proteins in red boxes, either alone or in combination, also induces hyperproliferation and tumor formation. For simplicity, only a subset of known germline regulators is shown. In some cases genes depicted to act in parallel to GLP-1, such as TEG-4 and METT-10, may integrate their functions more directly within the GLP-1 pathway. Note the high degree of regulatory cross-talk between the core factors (gray lines). Dashed lines and question marks indicate predicted regulatory connections and missing components, respectively.

Figure 6. A delay in meiotic entry, in combination with somatic gonad signaling, promotes proximal tumor formation

Blue and red indicate mitotic and meiotic germ cell regions, respectively. The green crescent-shaped cell on the left represents the distal tip cell (DTC). Yellow-to-brown flattened oval cells indicate somatic gonad sheath cells; darker shading indicates more proximal sheath cells. The proximal sheath cells depicted in mid-L4 and adult gonads are the Sh3–5 pairs. Rounder proximal cells in early L3 and mid-L3 larvae have not yet completed divisions. Green arrows indicate DSL ligand signaling from the DTC and sheath cells. For simplicity, central portions of mid-L4 and adult gonads are not shown; the missing region is demarcated by parallel lines. Note the delay in the timing of initial meiosis in Pro-defective gonads at the mid-L3 stage. In the case of glp-1(gf) mutants (e.g., [glp-1(ar202)]), this is due to increased GLP-1 activity that prevents proximal germ cells from exiting mitosis even after moving a sufficient distance from the DTC. Because pro-1 mutants have reduced mitotic proliferation during early larval development, gonads fail to extend at the normal rate. This results in the prolonged exposure of proximal germ cells to the DTC pro-mitotic signal. Similarly, hlh-12 mutants, which are defective at DTC migration, also result in continuous ligand exposure. At later developmental stages, these non-differentiated proximal germ cells are susceptible to stimulation by DSL ligands expressed from the sheath, leading to sustained mitosis and the formation of tumors. This figure is modeled after Killian and Hubbard, 2005.

Figure 7. Schematic of AC invasion in C.elegans hermaphrodites

At the early L3 larval stage, a netrin signal from the ventral nerve cord (VNC) diffuses dorsally to polarize the anchor cell (AC). Polarization (red side of AC) involves the recruitment of UNC-40 by the integrin heterodimer INA-1–PAT-1 as well as redistribution of F-actin and other cytoskeletal modulatory proteins, leading to the creation of an invasive membrane domain. At the mid-L3 larval stage, an unidentified diffusible cue from the primary vulval cells (indicated by question mark), causes the anchor cell to send out protrusions to breach the basement membrane (BM) and penetrate the vulval cells. FOS-1A, which is produced by the AC, facilitates basement membrane removal by activating targets such as MMPs.

Figure 8. Schematic of DTC migration in C.elegans hermaphrodites

Starting at the L2 larval stage, two ventrally located distal tip cells (DTC; green) migrate in three distinct phases to attain U-shaped gonadal arms. Black arrows indicate the direction of migration during these phases. GON-1, which is secreted from both migrating DTCs and adjacent muscles (not shown), promotes DTC migration, possibly by degrading FBL-1/fibulin. Phase 2 of migration requires the functions of MIG-17 and UNC-6/netrin, the latter of which transmits a repulsive signal. MIG-17 and FBL-1 localize to the gonad basement membrane.