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Reduction of Derlin activity suppresses Notch-dependent tumours in the C. elegans germ line

['Ramya Singh', 'Department Of Biological Sciences', 'University Of Calgary', 'Calgary', 'Ryan B. Smit', 'Xin Wang', 'Chris Wang', 'Hilary Racher', 'Dave Hansen']

Date: 2021-11

Regulating the balance between self-renewal (proliferation) and differentiation is key to the long-term functioning of all stem cell pools. In the Caenorhabditis elegans germline, the primary signal controlling this balance is the conserved Notch signaling pathway. Gain-of-function mutations in the GLP-1/Notch receptor cause increased stem cell self-renewal, resulting in a tumour of proliferating germline stem cells. Notch gain-of-function mutations activate the receptor, even in the presence of little or no ligand, and have been associated with many human diseases, including cancers. We demonstrate that reduction in CUP-2 and DER-2 function, which are Derlin family proteins that function in endoplasmic reticulum-associated degradation (ERAD), suppresses the C. elegans germline over-proliferation phenotype associated with glp-1(gain-of-function) mutations. We further demonstrate that their reduction does not suppress other mutations that cause over-proliferation, suggesting that over-proliferation suppression due to loss of Derlin activity is specific to glp-1/Notch (gain-of-function) mutations. Reduction of CUP-2 Derlin activity reduces the expression of a read-out of GLP-1/Notch signaling, suggesting that the suppression of over-proliferation in Derlin loss-of-function mutants is due to a reduction in the activity of the mutated GLP-1/Notch(GF) receptor. Over-proliferation suppression in cup-2 mutants is only seen when the Unfolded Protein Response (UPR) is functioning properly, suggesting that the suppression, and reduction in GLP-1/Notch signaling levels, observed in Derlin mutants may be the result of activation of the UPR. Chemically inducing ER stress also suppress glp-1(gf) over-proliferation but not other mutations that cause over-proliferation. Therefore, ER stress and activation of the UPR may help correct for increased GLP-1/Notch signaling levels, and associated over-proliferation, in the C. elegans germline.

Notch signaling is a highly conserved signaling pathway that is utilized in many cell fate decisions in many organisms. In the C. elegans germline, Notch signaling is the primary signal that regulates the balance between stem cell proliferation and differentiation. Notch gain-of-function mutations cause the receptor to be active, even when a signal that is normally needed to activate the receptor is absent. In the germline of C. elegans, gain-of-function mutations in GLP-1, a Notch receptor, results in over-proliferation of the stem cells and tumour formation. Here we demonstrate that a reduction or loss of Derlin activity, which is a conserved family of proteins involved in endoplasmic reticulum-associated degradation (ERAD), suppresses over-proliferation due to GLP-1/Notch gain-of-function mutations. Furthermore, we demonstrate that a surveillance mechanism utilized in cells to monitor and react to proteins that are not folded properly (Unfolded Protein Response-UPR) must be functioning well in order for the loss of Derlin activity to supress over-proliferation caused by glp-1/Notch gain-of-function mutations. This suggests that activation of the UPR may be the mechanism at work for suppressing this type of over-proliferation, when Derlin activity is reduced. Therefore, decreasing Derlin activity may be a means of reducing the impact of phenotypes and diseases due to certain Notch gain-of-function mutations.

Funding: This work was supported by the Natural Sciences Research Council of Canada (06647-2015) and Canadian Institute of Health Research (PJT-155999) to DH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://www.nserc-crsng.gc.ca/index_eng.asp https://cihr-irsc.gc.ca/e/193.html .

Copyright: © 2021 Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here we describe the effect that ERAD and the UPR have on the Notch-dependent control of stem cell proliferation in the C. elegans germline. We show that germline tumour formation resulting from increased GLP-1/Notch signalling is suppressed by mutations in cup-2 and der-2, encoding Derlin proteins which are components of ERAD. We also show that ectopically induced ER stress suppresses germline stem cell over-proliferation caused by increased GLP-1/Notch signaling and that this suppression requires the UPR. Both loss of cup-2 and induced ER stress can only suppress GLP-1/Notch-dependent tumours, suggesting they act directly on the Notch pathway, in a context-specific manner that suppresses excess overproliferation. We propose that ER stress and the UPR have a protective role, correcting for aberrant over-proliferation caused by increased GLP-1/Notch signaling levels in the C. elegans germline and restoring proper balance between stem cell proliferation and differentiation. Therefore, this study contributes to our understanding of how affecting protein folding capacity by modulating ER stress, can regulate the balance between stem cell self-renewal and differentiation.

Recently, studies have highlighted an emerging link between Notch signalling, ER stress and the UPR. Disruptions to ER zinc homeostasis affect Notch trafficking and activity in human cancer cell lines and Drosophila imaginal wing discs [ 56 , 57 ]. Mutations in p97, a key ERAD component, also disrupt Notch signalling in Drosophila wing development [ 58 ]. Inducing ER stress in human cell culture induces expression of the Notch ligand DLL4 [ 59 ]. Whether ERAD plays a role in physiological Notch signalling, or whether Notch signalling is modulated only in response to ER stress is unclear from these latest studies.

As part of another study to identify potential mRNA targets of PUF-8 (to be published elsewhere), we found that loss of cup-2 activity strongly suppressed the overproliferation phenotype observed in puf-8(0); glp-1(gf) animals (see below). PUF-8 is a pumilio homolog that is known to play a role in the proliferation vs differentiation balance in the C. elegans germline and loss of puf-8 strongly enhances the stem cell overproliferation phenotype of glp-1(gf) mutants [ 53 – 55 ]. Although cup-2 does not appear to be a direct target of PUF-8 ( S2 Fig ), the loss of cup-2 suppressing overproliferation provides an inroad into studying the role of ERAD in affecting GLP-1/Notch signaling and the proliferation vs. differentiation decision. Previous studies have shown that worms mutant for the ERAD component CUP-2, have increased expression of HSP-4::GFP, a hallmark of ER stress, as well as activation of the Unfolded Protein Response (UPR) [ 43 , 44 ]. These studies have also shown that der-2, the other worm Derlin, is partially redundant with cup-2 in the activation of the UPR. Here we investigate the effect that these Derlin mutants and ER stress have on the balance between stem cell proliferation and differentiation in the C. elegans germ line.

CUP-2 (coelomocyte uptake defective) was first identified in C. elegans as being required for endocytosis by the scavenger-like cells, the coelomocytes [ 50 ]. Later, CUP-2 was found to bind SNX-1 (sorting nexin), a component of the retromer complex in early endosomes [ 51 , 52 ]. This interaction was also observed with human Derlins and Sorting Nexins [ 52 ]. Similar to its role in ERAD, in endocytosis CUP-2 is thought to aid in recognition of misfolded plasma membrane proteins and their transportation to the ER for degradation [ 52 ].

CUP-2 is a member of the Derlin (degradation in the ER) family of proteins, that function in ERAD [ 14 , 43 – 45 ]. Derlins were initially discovered in yeast with Der1 and Dfm1 [ 45 , 46 ]. Mammals have three Derlin family members, Derlin-1, Derlin-2 and Derlin-3 [ 47 ]. CUP-2 is most similar to human Derlin-1 and a second C. elegans Derlin, DER-2, is most similar to human Derlin-2 and Derlin-3 [ 43 , 47 , 48 ]. As expected for proteins functioning in ERAD, loss of cup-2 and der-2 result in activation of the UPR [ 43 , 44 ]. Further support for cup-2 and der-2’s role in ERAD is provided by the fact that as is the case with yeast der1, cup-2 is also synthetically lethal with ire-1, a sensor for the UPR and overexpression of der-2 in Δder1 Δire1 yeast strains partially suppresses the conditional lethality associated with the strain [ 43 , 46 , 49 ].

The adult C. elegans germline harbours stem cells whose self-renewal is regulated by the GLP-1/Notch signalling pathway. The gonads of the C. elegans hermaphrodite comprise of two U-shaped tubes that meet at a common uterus [ 28 , 29 ]. Germ cells are born at the distal end of each arm and mature as they move proximally towards the vulva. The most distal population of germ cells are mitotically dividing stem cells [ 29 ]. As cells move proximally, they enter into meiosis and mature in an assembly line like manner to produce gametes. The pool of distal stem cells is maintained by GLP-1/Notch signalling and loss of GLP-1/Notch signalling results in a loss of the stem cell population, while increased GLP-1/Notch signalling leads to tumour formation [ 30 – 32 ] ( S1A and S1B Fig ). Two redundant pathways comprising of GLD-1 and GLD-2 function downstream of GLP-1/Notch signaling to promote differentiation. If the activities of both these pathways is reduced or eliminated a germline tumour results, similar to that due to increased GLP-1/Notch signaling [ 33 – 35 ] ( S1C Fig ). Many other regulatory controls ranging from factors controlling cell division such as CYE-1/CDK-2, subunits of the DNA polymerase alpha-primase complex, or proteasomal activity to signalling pathways such as MPK-1 ERK, Insulin, TGF- β, and TOR have been identified that modulate the balance between self-renewal vs differentiation to provide robust control [ 36 – 42 ]. They also add a layer of modulatory control necessary for the germline to adapt. Disruption of any one of these modulatory controls have weak effects on the balance between self-renewal and differentiation in an otherwise wildtype genetic background under ideal conditions; however, the redundancy of these pathways combine to create a robust system necessary for the balance between germline stem cell self-renewal and differentiation.

The housekeeping function of ERAD has important physiological implications for protein homeostasis. For example, as much as 75% of the wild type cystic fibrosis transmembrane conductance regulator (CFTR) protein is targeted for degradation through ERAD [ 16 – 20 ]. Single amino acid mutations in the 140 kDa, twelve transmembrane domain CFTR protein disrupt its proper folding such that all CFTR protein is degraded by ERAD leading to cystic fibrosis [ 17 ][ 21 ]. Dysregulation of ERAD can lead to the accumulation of misfolded proteins in the ER, which induces ER stress [ 14 ]. In response to ER stress, a series of protective cellular events are triggered to deal with the accumulation of misfolded proteins. Translation is attenuated to limit protein folding burden, the ER expands and becomes more elaborate to increase protein folding capacity, expression of chaperone proteins is increased and expression of ERAD components are increased to assist in protein folding. Collectively this response is called the Unfolded Protein Response (UPR)[ 14 , 22 ]. If the UPR fails, apoptosis can be triggered to eliminate the stressed cell in both animal models and human disease[ 14 , 23 ]. The increased levels of protein synthesis required for overproliferation in cancer cells is thought to increase basal levels of ER stress and the UPR [ 24 ]. This increase in ER stress is thought to either make cancer cells more resilient, or more susceptible to artificially inducing ER stress [ 25 , 26 ]. Understanding how cancer cells (and all stem cells) regulate and respond to ER stress is crucial in order to be able to understand how therapeutics act on them [ 27 ].

An indirect mechanism to modulate stem cell systems is through the regulation of protein folding and protein quality control. For example, recently it has been shown that increasing the genesis of misfolded proteins in hematopoietic stem cells (HSCs) impairs self-renewal of HSCs [ 3 ]. In the case of muscle stem cells, impairment of autophagy, the lysosomal degradation of long-lived proteins and damaged organelles, leads to senescence and stem cell exhaustion [ 4 ]. As another example, the transcription factor NRF3 is significantly mutated across twelve cancer cell lines and promotes cancer cell proliferation [ 5 , 6 ]. NRF3 is regulated by ER retention and endoplasmic-reticulum-associated degradation (ERAD), two cellular mechanisms responsible for surveillance of protein folding [ 7 , 8 ]. ERAD is a multi-step process in which misfolded proteins are recognized, retrotranslocated into the cytoplasm and targeted for degradation by the proteasome [ 8 – 11 ]. Recognition of misfolded proteins involves lectins and chaperone proteins in the ER [ 8 ]. Retrotranslocation occurs in protein complexes containing E3 ubiquitin ligases that also ubiquitinate the misfolded protein. In yeast, the Doa10/Ubc7 complex retrotranslocates and ubiquitinates proteins with misfolded cytosolic domains (ERAD-C pathway), while the Hrd1/Hrd3/Der1 complex acts on proteins with misfolded ER luminal domains (ERAD-L pathway) [ 12 , 13 ]. This distinction between different ERAD pathways is less clear in mammalian systems [ 9 , 14 ]. In both yeast and mammals, a p97 (Cdc48)/Npl4/Ufd1 complex extracts many of the targeted proteins in an ATPase-dependent manner allowing them to be degraded by the proteasome [ 12 , 15 ].

Stem cell populations provide the source material for future tissue generation and play an important role in the development and maintenance of many tissues. A defining feature of stem cells, their ability to both self-renew and differentiate, is key to their function. Stem cells must maintain a balance between self-renewal and differentiation as excessive self-renewal can lead to tumour formation while too much differentiation leads to a depleted stem cell pool. The decision to self-renew or differentiate is essential for the proper development of their tissues. Critical systems like this require many layers of redundancy in order to have a high level of robustness[ 1 , 2 ]. This way, if pressure is applied to one layer, other layers are able to ensure proper decision-making. These layers of redundancy can also allow external inputs to impinge on the system, giving it the ability to adapt. Understanding these layers of redundancy will aid in research in stem cell-related diseases and in using stem cells as therapeutic agents.

Results

Loss of the cup-2 paralog, der-2, also suppress glp-1(gf) mediated overproliferation cup-2 encodes a Derlin protein that has previously been shown to be involved in ERAD [43,44], functioning partially redundantly with DER-2, the other C. elegans Derlin protein (Schaheen et al. 2009). DER-2 is thought to be the functional ortholog of yeast Der1p since overexpression of C. elegans DER-2 in yeast Δder1 Δire1 strains partially restores degradation of an Der1p associated ERAD substrate and partially suppresses the conditional lethality phenotype of the double mutant [46]. If disruption of ERAD is responsible for cup-2(0)’s ability to suppress germline overproliferation, then we would expect loss of der-2 to likewise suppress overproliferation. We found that loss of der-2 does decrease the size of the distal proliferative zone in glp-1(ar202gf) animals from ~610 cells in glp-1(ar202gf) to ~405 in der-2(tm6098) glp-1(ar202gf) double mutants (Fig 2A and 2C and Table 2). Importantly, the size of the distal proliferative zone in der-2(tm6098) single mutants is similar to that in wild-type animals (~227 and ~221 respectively), suggesting that the suppression of glp-1(ar202gf) is not simply due to an overall reduced rate of proliferation. The suppression of glp-1(ar202gf) is most pronounced when the activities of both cup-2 and der-2 are removed (Fig 2A and 2C and Table 2), reducing the size of the proliferative zone from ~610 to ~122. This suggests that cup-2 and der-2 may have some redundant function. We also noticed that genotypes mutant for both cup-2 and der-2 tend to have slightly narrower and smaller gonads, overall (Fig 2A). Indeed, the size of the distal proliferative zone in the cup-2(tm2838); der-2(tm6098) double mutant (~142) is smaller than either single mutant (~195 for cup-2 and ~227 for der-2), or the wild-type proliferative zone (~221)(Fig 2C and Table 2). Moreover, while loss of cup-2 suppresses the complete tumour phenotype from 100% tumourous gonads in puf-8(q725); glp-1(ar202gf) double mutants to 92% tumourous animals in cup-2(tm2838); puf-8(q725); glp-1(ar202) triple mutants, also eliminating der-2 function in cup-2(tm2838); puf-8(q725); der-2(tm6098) glp-1(ar202) quadruple mutants significantly reduces the percentage of completely tumourous animals to 67% (Fig 1B and Table 1). Therefore, the suppression of glp-1(ar202gf) (proliferative zone counts), and the suppression of puf-8(0); glp-1(ar202gf) by loss of cup-2 and der-2 suggests that cup-2 and der-2 function redundantly in promoting robust germline proliferation.

Derlin loss-of-function mutants have smaller proliferative zone sizes In order to ascertain whether Derlin mutants affect cell proliferation, we first asked whether the size of the proliferative zone is altered in Derlin mutants, cup-2 and der-2. As noted above, we found that cup-2 mutant worms have a slightly smaller proliferative zone than wild-type, whereas der-2 single mutants do not significantly alter the proliferative zone size (Fig 2C and Table 2). Moreover, cup-2; der-2 double mutants have a statistically significantly smaller proliferative zone size than wild-type (p = 2.182 X 10−5, t-test independent samples with Bonferroni correction) and cup-2 single mutants (p = 2.014 X 10−4, t-test independent samples with Bonferroni correction) (Fig 2C and Table 2). This implies that cup-2 and der-2 may have overlapping but partially redundant roles in regulating cell proliferation. Of the two Derlins, cup-2’s interaction with glp-1 gain-of-function alleles is stronger; however, cup-2 and der-2 additively have the strongest effect. We conclude that Derlin’s interaction with the GLP-1/Notch signalling pathway could be proportional to the strength of excessive GLP-1/Notch signalling and is most pronounced with strong glp-1 gain-of-function alleles.

cup-2 loss-of-function mutant reduces the expression of SYGL-1, a readout of GLP-1/Notch signalling, in glp-1(gf) gonads We have demonstrated that loss of Derlin activity suppresses overproliferation due to increased GLP-1/Notch signalling, but not overproliferation in GLD-1/GLD-2 pathway mutants. This raises the possibility that loss of Derlin activity reduces the amount of GLP-1/Notch signalling in glp-1(gf) mutants. sygl-1 is a downstream transcriptional target of GLP-1/Notch signalling in the germline, functioning redundantly with lst-1, and is expressed in the distal proliferating cells in the germline [67–71]. To determine the effect of loss of Derlin activity on GLP-1/Notch signalling, we analyzed SYGL-1 expression in the relevant mutants (Fig 4A). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Loss of cup-2 decreases SYGL-1 protein levels in glp-1(ar202) germlines. A. Representative images of SYGL-1 protein expression observed in sygl-1(am307); glp-1(ar202) and cup-2(tm2838) sygl-1(am307); glp-1(ar202) genetic backgrounds by α-FLAG immunostaining. The sygl-1(am307) allele represents a 3XFLAG tagged version of endogenous SYGL-1. Asterisk, distal tip. Scale bar = 10μm. B. Normalized, fitted average SYGL-1 intensities measured by α-FLAG immunostaining of the indicated genotypes, each harbouring the sygl-1(am307) allele. Shaded areas indicate unscaled fitted standard deviation of the intensity measurements for each genotype. Standard deviation for average sygl-1(am307) has not been shown for ease of visualization but can be seen in S4 Fig. Average normalized intensities and standard deviations were fit to a sixth order polynomial. Fifteen gonads were analyzed for intensity measurements. Arrowheads point to the average location of the transition zone measured in at least seven gonads of each genotype. Distances from distal end (DE) were measured in microns and converted to germ cell diameters (g.c.d) as a reference, by assuming 1 g.c.d. = 2.833 microns. WT intensity measurement shown is the average sygl-1(am307) intensity across the three experiments used for scaling. https://doi.org/10.1371/journal.pgen.1009687.g004 Consistent with previous reports of SYGL-1 expression levels in glp-1(gf) mutants, we found that the zone of SYGL-1 expression expanded proximally in glp-1(ar202gf) gonads as compared to wild-type (S4B Fig)[67,68]. In wild-type gonads, SYGL-1 expression peaks around five cell diameters from the distal end, then decreases gradually until plateauing around 20 cell diameters from the distal end (Fig 4B). In glp-1(ar202gf) gonads the peak is around seven cell diameters and the plateau is around 24 cell diameters (Fig 4B). In cup-2(tm2838); glp-1(ar202gf) double mutants the expansion of SYGL-1 expression is suppressed, with the pattern of SYGL-1 expression being very similar to wild-type (Figs 4B and S4C). Interestingly, the SYGL-1 pattern in cup-2 single mutants is also shifted ~two cell diameters distally as compared to wild-type (Figs 4B and S4A). Since the data shown in Fig 4B was generated by comparing genotypes imaged on different slides and normalizing against SYGL-1 intensity of a wild-type background as an internal control (see Materials and Methods) we wanted to more directly compare the effect of loss of cup-2 activity on SYGL-1 expression in glp-1(ar202gf) animals, without normalization. Therefore, we compared SYGL-1 expression in glp-1(ar202gf) animals with cup-2(tm2838); glp-1(ar202gf) animals on the same slide (S4D Fig). This experiment yielded similar results to those obtained with normalization; the loss of cup-2 activity supresses the expansion of SYGL-1 expression along the distal-proximal axis in glp-1(ar202gf) animals. The distal movement of the SYGL-1 expression pattern in cup-2(0) as compared to wild-type, and in cup-2(tm2838); glp-1(ar202gf) as compared to glp-1(ar202gf), suggests that reduction of Derlin activity results in a decrease in GLP-1/Notch signaling, and that this reduction in GLP-1/Notch signaling is the likely cause of the suppression of the glp-1(gf) overproliferation by the loss/reduction of Derlin activity.

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