Formation of benign tumors by stem cell deregulation.

Within living organisms, stem cells respond to various cues, including to niche signals and growth factors. Niche signals originate from the stem cell's microenvironment and promote the undifferentiated state by preventing differentiation, allowing for stem cell self-renewal. On the other hand, growth factors promote stem cell growth and proliferation, while their sources comprise of a systemic input reflecting the animal's nutritional and metabolic status, and a localized, homeostatic feedback signal from the tissue that the stem cells serve. That homeostatic signal prevents unnecessary stem cell proliferation when the corresponding differentiated tissues already have optimal cell contents. Here, we recapitulate progresses made in our understanding of in vivo stem cell regulation, largely using simple models, and draw the conclusion that 2 types of stem cell deregulations can provoke the formation of benign tumors. Namely, constitutive niche signaling promotes the formation of undifferentiated "stem cell" tumors, while defective homeostatic signaling leads to the formation of differentiated tumors. Finally, we provide evidence that these general principles may be conserved in mammals and as such, may underlie benign tumor formation in humans, while benign tumors can evolve into cancer.

Non-autonomous regulation of germline stem cell proliferation by somatic MPK-1/MAPK activity in C. elegans.

Extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is a major positive regulator of cell proliferation, which is often upregulated in cancer. However, few studies have addressed ERK/MAPK regulation of proliferation within a complete organism. The Caenorhabditis elegans ERK/MAPK ortholog MPK-1 is best known for its control of somatic organogenesis and germline differentiation, but it also stimulates germline stem cell proliferation. Here, we show that the germline-specific MPK-1B isoform promotes germline differentiation but has no apparent role in germline stem cell proliferation. By contrast, the soma-specific MPK-1A isoform promotes germline stem cell proliferation non-autonomously. Indeed, MPK-1A functions in the intestine or somatic gonad to promote germline proliferation independent of its other known roles. We propose that a non-autonomous role of ERK/MAPK in stem cell proliferation may be conserved across species and various tissue types, with major clinical implications for cancer and other diseases.

The effect of age on stem cell function and utility for therapy.

During development, stem cells generate all of the differentiated cells that populate our tissues and organs. Stem cells are also responsible for tissue turnover and repair in adults, and as such, they hold tremendous promise for regenerative therapy. Aging, however, impairs the function of stem cells and is thus a significant roadblock to using stem cells for therapy. Paradoxically, the patients who would benefit the most from regenerative therapies are usually advanced in age. The use of stem cells from young donors or the rejuvenation of aged patient-derived stem cells may represent part of a solution. Nonetheless, the transplantation success of young or rejuvenated stem cells in aged patients is still problematic, since stem cell function is greatly influenced by extrinsic factors that become unsupportive with age. This article briefly reviews how aging impairs stem cell function, and how this has an impact on the use of stem cells for therapy.

DAF-18/PTEN signals through AAK-1/AMPK to inhibit MPK-1/MAPK in feedback control of germline stem cell proliferation.

Under replete growth conditions, abundant nutrient uptake leads to the systemic activation of insulin/IGF-1 signalling (IIS) and the promotion of stem cell growth/proliferation. Activated IIS can stimulate the ERK/MAPK pathway, the activation of which also supports optimal stem cell proliferation in various systems. Stem cell proliferation rates can further be locally refined to meet the resident tissue's need for differentiated progeny. We have recently shown that the accumulation of mature oocytes in the C. elegans germ line, through DAF-18/PTEN, inhibits adult germline stem cell (GSC) proliferation, despite high systemic IIS activation. We show here that this feedback occurs through a novel cryptic signalling pathway that requires PAR-4/LKB1, AAK-1/AMPK and PAR-5/14-3-3 to inhibit the activity of MPK-1/MAPK, antagonize IIS, and inhibit both GSC proliferation and the production of additional oocytes. Interestingly, our results imply that DAF-18/PTEN, through PAR-4/LKB1, can activate AAK-1/AMPK in the absence of apparent energy stress. As all components are conserved, similar signalling cascades may regulate stem cell activities in other organisms and be widely implicated in cancer.

DAF-18/PTEN locally antagonizes insulin signalling to couple germline stem cell proliferation to oocyte needs in C. elegans.

During development, stem cell populations rapidly proliferate to populate the expanding tissues and organs. During this phase, nutrient status, by systemically affecting insulin/IGF-1 signalling, largely dictates stem cell proliferation rates. In adults, however, differentiated stem cell progeny requirements are generally reduced and vary according to the spatiotemporal needs of each tissue. We demonstrate here that differential regulation of germline stem cell proliferation rates in Caenorhabditis elegans adults is accomplished through localized neutralization of insulin/IGF-1 signalling, requiring DAF-18/PTEN, but not DAF-16/FOXO. Indeed, the specific accumulation of oocytes, the terminally differentiated stem cell progeny, triggers a feedback signal that locally antagonizes insulin/IGF-1 signalling outputs in the germ line, regardless of their systemic levels, to block germline stem cell proliferation. Thus, during adulthood, stem cells can differentially respond within tissues to otherwise equal insulin/IGF-1 signalling inputs, according to the needs for production of their immediate terminally differentiated progeny.

Reprogramming and development in nuclear transfer embryos and in interspecific systems.

Nuclear transfer (NT) remains the most effective method to reprogram somatic cells to totipotency. Somatic cell nuclear transfer (SCNT) efficiency however remains low, but recurrent problems occurring in partially reprogrammed cloned embryos have recently been identified and some remedied. In particular, the trophectoderm has been identified as a lineage whose reprogramming success has a large influence on SCNT embryo development. Several interspecific hybrid and cybrid reprogramming systems have been developed as they offer various technical advantages and potential applications, and together with SCNT, they have led to the identification of a series of reprogramming events and responsible reprogramming factors. Interspecific incompatibilities hinder full exploitation of cross-species reprogramming systems, yet recent findings suggest that these may not constitute insurmountable obstacles.

On the cellular and developmental lethality of a Xenopus nucleocytoplasmic hybrid.

Nucleocytoplasmic hybrid (cybrid) embryos result from the combination of the nucleus of one species, and the egg cytoplasm of another species. Cybrid embryos can be obtained either in the haploid state by the cross-fertilization or intra-cytoplasmic injection of an enucleated egg with sperm from another species, or in the diploid state by the technique of interspecies somatic cell nuclear transfer (iSCNT). Cybrids that originate from the combination of the nucleus and the cytoplasm of distantly related species commonly expire during early embryonic development, and the cause of this arrest is currently under investigation. Here we show that cells isolated from a Xenopus cybrid (Xenopus (Silurana) tropicalis haploid nucleus combined with Xenopus laevis egg cytoplasm) embryo are unable to proliferate and expand normally in vitro. We also provide evidence that the lack of nuclear donor species maternal poly(A)(+) RNA-dependent factors in the recipient species egg may contribute to the developmental dead-end of distantly-related cybrid embryos. Overall, the data are consistent with the view that the development promoted by one species' nucleus is dependent on the presence of maternally-derived, mRNA encoded, species-specific factors. These results also show that cybrid development can be improved without nuclear species mitochondria supplementation or replacement.

Amphibian interorder nuclear transfer embryos reveal conserved embryonic gene transcription, but deficient DNA replication or chromosome segregation.

Early interspecies nuclear transfer (iNT) experiments suggested that a foreign nucleus may become permanently damaged after a few rounds of cell division in the cytoplasm of another species. That is, in some distant species combinations, nucleocytoplasmic hybrid (cybrid) blastula nuclei can no longer support development, even if they are back-transferred into their own kind of egg cytoplasm. We monitored foreign DNA amplification and RNA production by quantitative PCR (qPCR) and RT-qPCR in interorder amphibian hybrids and cybrids formed by the transfer of newt (Pleurodeles waltl) embryonic nuclei into intact and enucleated frog (Xenopus laevis) eggs. We found a dramatic reduction in the expansion of foreign DNA and cell numbers in developing cybrid embryos that correlated with reduced gene transcription. Interestingly, expansion in cell numbers was rescued by the recipient species (Xenopus) maternal genome in iNT hybrids, but it did not improve P. waltl DNA expansion or gene transcription. Also, foreign gene transcripts, normalized to DNA copy numbers, were mostly normal in both iNT hybrids and cybrids. Thus, incomplete foreign DNA replication and/or chromosome segregation during cell division may be the major form of nuclear damage occurring as a result of nuclear replication in a foreign cytoplasmic environment. It also shows that the mechanisms of embryonic gene transcription are highly conserved across amphibians and may not be a major cause of cybrid lethality.

Deficient induction response in a Xenopus nucleocytoplasmic hybrid.

Incompatibilities between the nucleus and the cytoplasm of sufficiently distant species result in developmental arrest of hybrid and nucleocytoplasmic hybrid (cybrid) embryos. Several hypotheses have been proposed to explain their lethality, including problems in embryonic genome activation (EGA) and/or nucleo-mitochondrial interactions. However, conclusive identification of the causes underlying developmental defects of cybrid embryos is still lacking. We show here that while over 80% of both Xenopus laevis and Xenopus (Silurana) tropicalis same-species androgenetic haploids develop to the swimming tadpole stage, the androgenetic cybrids formed by the combination of X. laevis egg cytoplasm and X. tropicalis sperm nucleus invariably fail to gastrulate properly and never reach the swimming tadpole stage. In spite of this arrest, these cybrids show quantitatively normal EGA and energy levels at the stage where their initial gastrulation defects are manifested. The nucleocytoplasmic incompatibility between these two species instead results from a combination of factors, including a reduced emission of induction signal from the vegetal half, a decreased sensitivity of animal cells to induction signals, and differences in a key embryonic protein (Xbra) concentration between the two species, together leading to inefficient induction and defective convergence-extension during gastrulation. Indeed, increased exposure to induction signals and/or Xbra signalling partially rescues the induction response in animal explants and whole cybrid embryos. Altogether, our study demonstrates that the egg cytoplasm of one species may not support the development promoted by the nucleus of another species, even if this nucleus does not interfere with the cytoplasmic/maternal functions of the egg, while the egg cytoplasm is also capable of activating the genome of that nucleus. Instead, our results provide evidence that inefficient signalling and differences in the concentrations of key proteins between species lead to developmental defects in cybrids. Finally, they show that the incompatibilities of cybrids can be corrected by appropriate treatments.

Differential requirements for STRAD in LKB1-dependent functions in C. elegans.

The protein kinase LKB1 is a crucial regulator of cell growth/proliferation and cell polarity and is the causative gene in the cancer-predisposing disease Peutz-Jeghers syndrome (PJS). The activity of LKB1 is greatly enhanced following its association with the Ste20-like adapter protein STRAD. Unlike LKB1 however, mutations in STRAD have not been identified in PJS patients and thus, the key tumour suppressive role(s) of LKB1 might be STRAD independent. Here, we report that Caenorhabditis elegans strd-1/STRAD mutants recapitulate many phenotypes typical of par-4/LKB1 loss of function, showing defects during early embryonic and dauer development. Interestingly, although the growth/proliferation defects in severe par-4 and strd-1 mutant dauers are comparable, strd-1 mutant embryos do not share the polarity defects of par-4 embryos. We demonstrate that most of par-4-dependent regulation of germline stem cell (GSC) quiescence occurs through AMPK, whereby PAR-4 requires STRD-1 to phosphorylate and activate AMPK. Consistent with this, even though AMPK plays a major role in the regulation of cell proliferation, like strd-1 it does not affect embryonic polarity. Instead, we found that the PAR-4-mediated phosphorylation of polarity regulators such as PAR-1 and MEX-5 in the early embryo occurs in the absence of STRD-1. Thus, PAR-4 requires STRD-1 to phosphorylate AMPK to regulate cell growth/proliferation under reduced insulin signalling conditions, whereas PAR-4 can promote phosphorylation of key proteins, including PAR-1 and MEX-5, to specify early embryonic polarity independently of STRD-1. Our results therefore identify a key strd-1/STRAD-independent function of par-4/LKB1 in polarity establishment that is likely to be important for tumour suppression in humans.

C. elegans STRADalpha and SAD cooperatively regulate neuronal polarity and synaptic organization.

Neurons are polarized cells with morphologically and functionally distinct axons and dendrites. The SAD kinases are crucial for establishing the axon-dendrite identity across species. Previous studies suggest that a tumour suppressor kinase, LKB1, in the presence of a pseudokinase, STRADalpha, initiates axonal differentiation and growth through activating the SAD kinases in vertebrate neurons. STRADalpha was implicated in the localization, stabilization and activation of LKB1 in various cell culture studies. Its in vivo functions, however, have not been examined. In our present study, we analyzed the neuronal phenotypes of the first loss-of-function mutants for STRADalpha and examined their genetic interactions with LKB1 and SAD in C. elegans. Unexpectedly, only the C. elegans STRADalpha, STRD-1, functions exclusively through the SAD kinase, SAD-1, to regulate neuronal polarity and synaptic organization. Moreover, STRD-1 tightly associates with SAD-1 to coordinate its synaptic localizations. By contrast, the C. elegans LKB1, PAR-4, also functions in an additional genetic pathway independently of SAD-1 and STRD-1 to regulate neuronal polarity. We propose that STRD-1 establishes neuronal polarity and organizes synaptic proteins in a complex with the SAD-1 kinase. Our findings suggest that instead of a single, linear genetic pathway, STRADalpha and LKB1 regulate neuronal development through multiple effectors that are shared in some cellular contexts but distinct in others.

Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survival.

Many organisms can enter a dormant state or diapause to survive harsh environmental conditions for extended durations. When Caenorhabditis elegans larvae enter dauer they arrest feeding but remain active and motile, yet become stress-resistant, extremely long-lived and non-ageing. Entry into dauer is associated with a reduction in insulin-like signalling, the accumulation of nutritive resources and a concomitant global change in metabolism, yet the precise molecular and physiological processes that enable long-term survival in the absence of caloric intake remain largely unknown. We show here that C. elegans larvae that lack LKB1/AMPK (AMP-activated protein kinase) signalling enter dauer normally, but then rapidly consume their stored energy and prematurely expire following vital organ failure. We found that this signalling pathway acts in adipose-like tissues to downregulate triglyceride hydrolysis so that these lipid reserves are rationed to last the entire duration of the arrest. Indeed, the downregulation of adipose triglyceride lipase (ATGL-1) activity suppresses both the rapid depletion of stored lipids and reduced life span of AMPK mutant dauers, while AMPK directly phosphorylates ATGL-1. Finally, we show that the slow release of energy during dauer is critical for appropriate long-term osmoregulation, which fails as triglyceride resources become depleted. These mechanisms may be essential for survival through diapause, hibernation, or long-term fasting in diverse organisms and may also underlie AMPK-dependent life span extension.

Genes that affect both cell growth and polarity mediate stem cell quiescence.

Stem cells possess the capacity to expand and self-renew and do so by dividing in either a symmetrical or an asymmetrical manner. Under particular circumstances, some stem cell populations can undergo prolonged cell cycle arrest or quiescence, until they are triggered to divide by a given stimulus. In cancer treatment, these populations represent a significant roadblock to efficient therapies as their non-dividing state renders them refractory to most commonly used cytotoxic interventions. In certain organisms, germline stem cells undergo quiescence if animals experience inappropriate growth conditions, and recent studies have determined that the level of insulin signaling is key in the regulation of their proliferation rate, and that it functions through at least two tumor suppressor genes, PTEN and LKB1. These gene products regulate both growth and polarity in diverse cellular contexts, while it remains unclear how they can modulate cell division and prevent tumorigenesis through each of these functions, and whether indeed these functions are separable. We hope that understanding how these tumor suppressor genes impinge on quiescent stem cell populations could provide us with a means of designing more effective therapies to reduce the frequency of stem cell-derived tumor growth that occurs following treatment.

Regulation of germline stem cell proliferation downstream of nutrient sensing.

Stem cells have recently attracted significant attention largely due to their potential therapeutic properties, but also because of their role in tumorigenesis and their resemblance, in many aspects, to cancerous cells. Understanding how stem cells are regulated, namely with respect to the control of their proliferation and differentiation within a functional organism, is thus primordial to safely profit from their therapeutic benefits. Here, we review recent advances in the understanding of germline stem cell proliferation control by factors that respond to the nutritional status and/or insulin signaling, through studies performed in C. elegans and Drosophila. Together, these data uncover some shared fundamental features that underlie the central control of cellular proliferation within a target stem cell population in an organism. These features may indeed be conserved in higher organisms and may apply to various other stem cell populations.

Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling.

In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. We show here that the C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like ;dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-beta activity also triggers developmental quiescence independent of the insulin-like pathway. Our data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutations in TGF-beta family members, PTEN or LKB1 result in related tumour-predisposing syndromes. Our findings establish a developmental relationship that may underscore their shared, characteristic aetiology.