C. elegans Germline as Three Distinct Tumor Models

1. Introduction

Tumorigenesis, the abnormal proliferation of cells leading to tumor formation, highlights various capabilities of cancer cells, such as growth signal self-sufficiency, insensitivity to anti-growth signals, apoptosis evasion, replicative potential, sustained angiogenesis, and tissue invasion [1]. Due to the complexity and overlapping genetic changes in humans, model organisms are crucial in studying tumorigenesis. The nematode Caenorhabditis elegans (C. elegans) is a preferred genetic model for in vitro tumorigenesis research. The transparency of C. elegans enables direct observation of cell growth and tumorigenesis in vivo [2]. Its highly conserved genes and pathways related to tumorigenesis make C. elegans an ideal model organism [2], especially for studying tumorigenesis in germline influenced by both germline and somatic signals [2].

1.1. C. elegans Germline Development

C. elegans exist as either hermaphrodites (XX) or males (XO). Hermaphrodites initially produce a limited number of sperm during the larval stage (L4) and then switch to producing oocytes in young adult stages; they are self-fertile during adulthood. However, males consistently produce sperm without switching to oogenesis. The germline is organized in a simple linear pattern in both sexes, progressing from germline stem cells (GSCs) at the distal region to maturing gametes at the proximal region (Figure 1A). Specifically, a mesenchymal somatic cell, known as the distal tip cell (DTC), functions as a GSC niche and plays a crucial role in GSC maintenance and the mitotic cell cycle in the distal germline [3]. Once a GSC moves away from the DTC niche, it enters the meiotic cell cycle and eventually differentiates into sperm or oocytes (Figure 1A). In addition, C. elegans is an attractive model organism due to the ease of generating mutant strains, genetic manipulation, phenotype analysis, microscopy, and imaging. Therefore, the C. elegans germline has been widely used as a model organism in various biomedical fields, including research on tumorigenesis.

1.2. Three Distinct Mechanisms of C. elegans Germline Tumorigenesis

Germline tumors observed in mutant C. elegans individuals can originate from different sources, depending on the mechanism of tumorigenesis. In the context of C. elegans, the term “tumorous” is used to define germlines that exhibit three distinctive features (Figure 1B): (1) a vast excess of mitotic cells through ectopic proliferation, (2) minimal or no germ cell differentiation due to meiotic entry failure, and (3) the generation of mitotic germ cells through dedifferentiation. This report outlines three distinct mechanisms of tumorigenesis.

2. GLP-1/Notch Activation-Mediated Tumorigenesis: Ectopic Proliferation

2.1. Notch Signaling

In most multicellular organisms, the Notch signaling pathway is highly conserved and controls various cellular processes, including proliferation, differentiation, cell fate specification, and other cellular responses (Artavanis-Tsakonas et al., 1999). Notch ligands (DSL: Delta/Serrate/LAG-2) are expressed on the membrane of donor cells adjacent to receiving cells expressing Notch receptors (Figure 2A). Upon interaction between the Notch receptor and a ligand, an ADAM-family metalloprotease cleaves the exterior of the Notch receptor, followed by g-secretase cleaving the inner portion of the Notch receptor within the cell membrane. This Notch intracellular domain (NICD) relocates to the nucleus, where it forms a tertiary complex with CSL (CBF1/Suppressor of Hairless/LAG-1) transcription factors and mastermind-like protein (MAML-1), leading to the activation of Notch target genes. Notably, aberrant Notch signaling can lead to uncontrolled cell growth, metastasis, and resistance to apoptosis [4]. Therefore, understanding the precise mechanism governing context-dependent outcomes of Notch signaling is crucial.

2.2. C. elegans Notch Signaling and Its Core Regulators

The Notch signaling pathway and its core regulators are highly conserved in C. elegans (Figure 2B). Two distinct Notch receptors exist in this organism: GLP-1 (GermLine Proliferation-1) and LIN-12 (cell LINeage-12). GLP-1 is primarily found in germ cells and plays a critical role in the maintenance and mitotic division of GSCs [3]. Conversely, LIN-12 is predominantly present in somatic cells and is essential for determining the fate of vulva cells during early larval development [5]. The activation of GLP-1 signaling requires two Notch ligands: LAG-2 and APX-1 [6,7]. LAG-2 is mainly found in the DTC, which acts as a niche for GSCs. When LAG-2 interacts with GLP-1, a cascade of cleavage events is initiated, leading to the release of the NICD [8]. Subsequently, GLP-1(NICD) forms a tertiary complex with LAG-1 and LAG-3, activating transcription for its target genes [8]. Two target genes, lateral signaling target (lst-1) and synthetic GLP (sygl-1), have been extensively studied [9]. These genes function redundantly in the maintenance and mitotic division of GSCs [9]. While a single mutation for either sygl-1 or lst-1 can sustain GSCs, double mutations show no GSC phenotype, similar to glp-1(null) mutants [9]. Notably, overexpression of either sygl-1 or lst-1 induces the formation of germline tumors, resembling the phenotype observed in glp-1 gain-of-function (gf) mutants [10]. These findings highlight the crucial role of two GLP-1 target genes in the maintenance and mitotic division of GSCs.

2.3. GLP-1 Mutant Alleles

The primary distinction between the two groups of mutants lies in their germline phenotype. Loss-of-function mutants significantly reduce GSCs, while gain-of-function mutations form germline tumors. These glp-1 mutants have served as valuable tools to identify genes associated with GLP-1 signaling. The representative mutant alleles are listed in Table 1.

2.4. Positive or Negative Regulators of GLP-1/Notch Signaling

While most glp-1(ar202) and glp-1(bn18) mutants are fertility at 15°C or 20°C, the majority of glp-1(ar202) gain-of-function mutants develop germline tumors upon shifting to 25°C [14], whereas most glp-1(bn18) loss-of-function mutants exhibit defects in germline proliferation at 25°C, resulting in no germ cells [11]. These intriguing phenotypes provide an opportunity to identify genes that positively or negatively regulate GLP-1/Notch signaling (Figure 2C).

• CYE-1 and CDK-2: Fox et al. found that germline CYE-1 and CDK-2 are required for GLP-1/Notch-mediated germ cell proliferation. RNAi-mediated depletion of CYE-1 or CDK-2 significantly suppressed germ cell proliferative fate in glp-1(bn18) mutants even at 20°C [15]. Additional genetic analysis suggests that CYE-1 and CDK-2 act independently of GLP-1/Notch signaling to promote proliferative fate [15].
• Subunits of the DNA polymerase alpha-primase complex: Yoon et al. found that DIV-1 (regulatory subunit) is indispensable for GLP-1/Notch-mediated germ cell proliferation during early larval development, whereas POLA-1 (catalytic subunit) and two primase subunits, PRI-1 and PRI-2, play a crucial role in GLP-1/Notch-mediated maintenance of proliferative cell fate during adulthood [16]. Robinson-Thiewes et al. also identified POLE-1 (the catalytic subunit of DNA polymerase e) as a regulator of germ cell proliferation [17].
• Chaperone HSP90: Lissemore et al. performed a genetic screening to identify genes that promote GLP-1/Notch signaling and found that HSP-90, a molecular chaperone, plays an essential role in stem cell maintenance [18]. It was a novel finding demonstrating the essential role of HSP90 in Notch signaling in development.
• Ribosomal Protein S6 kinase (S6K): Roy et al. identified RSKS-1/S6K as a positive regulator of GLP-1/Notch signaling-mediated germline proliferation [19]. Additional screening also found that Hedgehog-related ligands may share functional relationships with GLP-1/Notch and RSKS-1/S6K in maintaining GSCs [19].
• Bro1-domain protein: Liu and Maine identified the ego-2 (enhancer of glp-1) gene as a positive regulator of germline proliferation that interacts genetically with the GLP-1/Notch signaling pathway [20]. Notably, ego-2 also promotes LIN-12/Notch signaling in somatic tissues [20]. They found that the EGO-1 protein contains a Bro1 domain, which localizes to specific endosomal compartments in other systems. Thus, they suggest that EGO-2 may promote GLP-1/Notch signaling through endocytic process function [20].
• Derlin family proteins: Singh et al. demonstrated that reduced CUP-2 and DER-2 function suppresses GLP-1/Notch-mediated germline tumorigenesis [21]. CUP-2 and DER-2 are Derlin family proteins that function in endoplasmic reticulum-associated degradation (ERAD). Therefore, they suggest that Derlin activity may be associated with GLP-1/Notch-mediated tumorigenesis [21].
• U/T level: Chi et al. demonstrated that GLP-1 expression in the distal gonad region is repressed by low Uridine/Thymidine (U/T) levels [22]. C. elegans CDD-1/-2 Cytidine Deaminases are involved in uridine biosynthesis. Notably, worms lacking both CDD-1 and CDD-2 exhibited germline proliferation defects, whose phenotype was rescued by Uridine supplementation [22].
• TRIM-NHL protein: Brenner et al. identified nhl-2 as an inhibitor of glp-1(ar202)-mediated tumorigenesis [23]. NHL-2, a conserved TRIM-NHL protein family member, suppresses germ cell proliferation by inhibiting PUF-3 and PUF-11. They also found that CGH-1 RNA helicase and ALG-5 work with NHL-2 to inhibit glp-1(ar202)-mediated tumorigenesis.
• E3 Ubiquitin ligase: Gutnik et al. reported that the splicing factor PRP-19 (a candidate E3 ubiquitin ligase) inhibits the nuclear accumulation of the GLP-1/Notch intracellular domain [24].
• PUF RNA-binding protein: PUF-8 is a conserved PUF RNA-binding protein that inhibits the translation of target mRNAs. In C. elegans germline, PUF-8 is involved in decisions regarding proliferation/differentiation, differentiation/dedifferentiation, and sperm/oocyte fates, depending on the genetic context [25]. Racher and Hansen demonstrated that PUF-8 inhibits glp-1(ar202)-mediated tumorigenesis in the C. elegans germline [26]. Other PUF proteins (FBF-1/2 and PUF-3/11) also play a critical role in GSC maintenance [27].
• Syndecan: Gopal et al. identified SDN-1 (a syndecan transmembrane proteoglycan) as a positive regulator of GLP-1/Notch signaling. SDN-1 promotes GLP-1 expression and mitotic germ cell fate by controlling a somatic TRP calcium channel. The TRP enhances glp-1 expression by governing the calcium-dependent binding of an AP-2 transcription factor (APTF-2) [28].

3. GLD-1 Loss-Mediated Tumorigenesis: Meiotic Entry Failure

3.1. STAR Family of RNA-Binding Proteins

The signal transduction activator of RNA metabolism (STAR) family of KH domain RNA-binding proteins is a highly conserved group of proteins among eukaryotes [29]. The STAR protein family operates at the post-transcriptional level, being involved in the stability, alternative splicing, and translational efficiency of their mRNA targets, thus influencing downstream gene expression [30]. Although STAR proteins are highly conserved, they exhibit variability in specific regions and specificity to certain RNA sequences in each model organism [31]. Moreover, RNA-recognition mechanisms by STAR proteins are yet to be explicitly defined for many models. Many STAR proteins have been identified with diverse roles across animal models [32]. For example, the held-out wing (HOW) gene in Drosophila is involved in embryonic cardiac development, and the alternative splicing defective-2 (ASD-2) gene in C. elegans is involved in the developmental control of alternative splicing [33,34]. STAR proteins also play an essential role in the development of vertebrate models. Most notably, mutations and loss of function in the quaking gene (QK1) in mice models have been shown to cause deficiencies in adult mice astrocyte maturation [35]. Furthermore, STAR proteins have been implicated in human disease [35,36]. More research is needed to elucidate the full pleiotropic effects and mechanisms of STAR proteins and their role in developmental maturation.

3.2. C. elegans gld-1 and Its Partners

C. elegans STAR RNA-binding protein GLD-1 (GermLine development Defective) plays multiple critical roles in C. elegans germline development. One well-known function is inhibiting germ cell proliferation [37,38]. Germ cells lacking GLD-1 enter the meiotic cell cycle and revert to the mitotic cell cycle, leading to the formation of germline tumors (Figure 1B). GLD-1 is predominantly expressed in the cytoplasm of premeiotic and pachytene cells in the C. elegans germline [38]. GLD-1 exerts its regulatory functions by binding to conserved sequence motifs (AGAAGC, CUACUAAC, or GAACGA) in the 5’ and 3’ UTRs of its mRNA target [39,40] (Figure 3A). Thereby modulating their stability and/or translation [41]. Several GLD-1 target mRNAs have been identified through biochemical and functional analyses, including rme-2, gna-2 [42], lin-45, tra-2, glp-1 [43], pos-1, pal-1 [44], cye-1, cep-1 [45], mes-3 [46], and four puf genes (puf-5, 6, 7, 8 and 10) [40,47]. RNA-IP/Chip analysis has further identified putative GLD-1 targets, primarily involved in reproduction, embryogenesis, cell division, and cell cycle [41]. GLD-1 and its partner NOS-3 (a member of the Nanos family of zinc finger proteins) act as a translational repressor to promote meiotic prophase progression in the C. elegans germline [48,49]. Moreover, the GLD-1/NOS-3 complex works with the GLD-2/GLD-3 complex, which promotes germline differentiation [48,49]. Thus, the GLD-1/NOS-3 and GLD-2/GLD-3 complexes are essential for germline differentiation, particularly through meiotic entry (Figure 3B).

3.3. gld-1 Mutant Alleles

The gld-1 gene plays a crucial role in meiotic progression and oocyte development. The formation of germline tumors in gld-1(q485) loss-of-function mutants is dependent on the sexual fate of the germline [50]. In gld-1(q485) loss-of-function mutant germline, germ cells exit the meiotic prophase but return to the mitotic cell cycle in the oogenic germline, while these germ cells can differentiate into sperm in the spermatogenic germline [50]. The representative mutant alleles are listed in Table 2.

3.4. Positive or Negative Regulators of GLD-1

The Regulators of GLD-1 Are Depicted in Figure 3B.

• GLD-2 poly(A) polymerase (PAP): GLD-2 is a cytoplasmic poly(A) polymerase [52]. It plays a critical role in meiotic entry and progression [53,54]. Thus, no functional gametes are produced in the absence of GLD-2 [53]. Notably, the gld-1 mRNA is a direct target of GLD-2 [55]. GLD-2 poly(A) polymerase promotes meiotic entry, at least in part, by activating GLD-1 expression. Consequently, GLD-2 loss enhances the formation of germline tumors in gld-1 loss-of-function mutant worms [53].
• FBF/PUF RNA-binding protein: C. elegans FBF/PUF proteins play a crucial role in maintaining GSCs by regulating the expression of various target mRNAs, including the gld-1 mRNA [56]. Since GLD-1 is essential for inhibiting proliferation and maintaining the differentiation state of germ cells, FBF/PUF repression of gld-1 mRNAs is critical for GSC maintenance. In addition, C. elegans PUF-8 proteins negatively inhibit the expression of gld-1 mRNAs by inhibiting gld-2 mRNAs indirectly [57].
• CYE-1/CDK2: CYE-1/CDK2 influences the mitosis/meiosis balance through GLD-1 [15,58]. Specifically, GLD-1 has CDK2 phosphorylation sites and appears to be a direct substrate of CYE-1/CDK2. Functional analysis showed that CYE-1/CDK2 works with FBF-1 to maintain GSCs by inhibiting GLD-1. Notably, cye-1 mRNA is also a repressing target of GLD-1 [59]. Therefore, GLD-1 and CYE-1/CDK2 inhibit each other for the mitosis/meiosis balance.
• Pre-mRNA Splicing Factor (PRP-17): Kerins et al. reported that PRP-17 and other C. elegans splicing factor orthologs function to promote meiotic entry by positively regulating the splicing of mRNAs of genes in the GLD-1 pathway [60].

5. Conclusions

Tumorigenesis can occur through various mechanisms, such as genetic mutations, epigenetic alterations, chromosomal abnormalities, immune system dysfunction, changes in the tumor microenvironments, and metabolic changes. In this report, we describe three specific tumor models: tumorigenesis mediated by (1) aberrant GLP-1/Notch activation, (2) gld-1 loss-of-function-mediated differentiation failure, and (3) puf-8 loss-of-function-mediated dedifferentiation (Figure 1B). Notably, each model requires the mutation of distinct genes (glp-1, gld-1, and puf-8) and operates through different underlying mechanisms. However, regulators within each tumor model exhibit shared features. For example, a puf-8(q725) mutation initially triggers dedifferentiation-mediated tumorigenesis, further enhanced by the additional mutation of lip-1. Interestingly, the same puf-8(q725) mutation also induces glp-1(ar202)-mediated tumorigenesis through a different mechanism. Similarly, a gld-1(q485) mutation initially leads to the formation of germline tumors due to differentiation failure, but this mutation also enhances puf-8(q725) dedifferentiation-mediated tumorigenesis. We also found that GLP-1/Notch signaling is involved in puf-8(q725) dedifferentiation-mediated tumorigenesis (Jones et al., unpublished results). These observations hypothesize that there may be shared internal regulatory networks that maintain the original tumorigenesis pathway. This idea also suggests specific therapies targeting both the origin of tumorigenesis and the internal regulatory networks. Consequently, the three distinct tumor models in C. elegans serve as valuable tools for identifying genes and developing unique therapeutics targeted to specific tumor pathways. This conceptual framework also provides insights into more complex tumor models in other organisms, including humans.