The Drosophila Accessory Gland as a Model for Prostate Cancer and Other Pathologies.

The human prostate is a gland of the male reproductive tract, which together with the seminal vesicles, is responsible for most seminal fluid production. It is a common site of cancer, and unlike other glands, it typically enlarges in aging men. In flies, the male accessory glands make many major seminal fluid components. Like their human equivalents, they secrete proteins from several conserved families, including proteases, lectins, and cysteine-rich secretory proteins, some of which interact with sperm and affect fertility. A key protein, sex peptide, is not conserved in vertebrates but plays a central role in mediating long-term effects on females after mating. Although postmitotic, one epithelial cell type in the accessory glands, the secondary cell, continues to grow in adults. It secretes microvesicles called exosomes from the endosomal multivesicular body, which, after mating, fuse with sperm. They also appear to affect female postmating behavior. Remarkably, the human prostate epithelium also secretes exosomes, which fuse to sperm in vitro to modulate their activity. Exosomes from prostate and other cancer cells are increasingly proposed to play fundamental roles in modulating the tumor microenvironment and in metastasis. Here we review a diverse accessory gland literature, which highlights functional analogies between the male reproductive glands of flies and humans, and a critical role for extracellular vesicles in allowing seminal fluid to promote male interests within the female. We postulate that secondary cells and prostate epithelial cells use common mechanisms to control growth, secretion, and signaling, which are relevant to prostate and other cancers, and can be genetically dissected in the uniquely tractable fly model.

PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.

The emerging role of mTORC1 signaling in placental nutrient-sensing.

Nutrient-sensing signaling pathways regulate cell metabolism and growth in response to altered nutrient levels and growth factor signaling. Because trophoblast cell metabolism and associated signaling influence fetal nutrient availability, trophoblast nutrient sensors may have a unique role in regulating fetal growth. We review data in support of a role for mammalian target of rapamycin complex 1 (mTORC1) in placental nutrient-sensing. Placental insulin/IGF-I signaling and fetal levels of oxygen, glucose and amino acids (AAs) are altered in pregnancy complications such as intrauterine growth restriction, and all these factors are well-established upstream regulators of mTORC1. Furthermore, mTORC1 is a positive regulator of placental AA transporters, suggesting that trophoblast mTORC1 modulates AA transfer across the placenta. In addition, placental mTORC1 signaling is also known to be modulated in pregnancy complications associated with altered fetal growth and in animal models in which maternal nutrient availability has been altered experimentally. Recently, significant progress has been made in identifying the molecular mechanisms by which mTORC1 senses AAs, a process requiring shuttling of mTOR to late endosomal and lysosomal compartments (LELs). We recently identified members of the proton-assisted amino acid transporter (PAT/SLC36) family as critical components of the AA-sensing system or 'nutrisome' that regulates mTORC1 on LEL membranes, placing AA transporters and their subcellular regulation both upstream and downstream of mTORC1-driven processes. We propose a model in which placental mTORC1 signaling constitutes a critical link between maternal nutrient availability and fetal growth, thereby influencing the long-term health of the fetus.

Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation.

The phosphoinositide3-kinase (PI3K)/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signalling cascades promote normal growth and are frequently hyperactivated in tumour cells. mTORC1 is also regulated by local nutrients, particularly amino acids, but the mechanisms involved are poorly understood. Unexpectedly, members of the proton-assisted amino-acid transporter (PAT or SLC36) family emerged from in vivo genetic screens in Drosophila as transporters with uniquely potent effects on mTORC1-mediated growth. In this study, we show the two human PATs that are widely expressed in normal tissues and cancer cell lines, namely PAT1 and PAT4, behave similarly to fly PATs when expressed in Drosophila. Small interfering RNA knockdown shows that these molecules are required for the activation of mTORC1 targets and for proliferation in human MCF-7 breast cancer and HEK-293 embryonic kidney cell lines. Furthermore, activation of mTORC1 in starved HEK-293 cells stimulated by amino acids requires PAT1 and PAT4, and is elevated in PAT1-overexpressing cells. Importantly, in HEK-293 cells, PAT1 is highly concentrated in intracellular compartments, including endosomes, wherein mTOR shuttles upon amino-acid stimulation. Therefore our data are consistent with a model in which PATs modulate the activity of mTORC1 not by transporting amino acids into the cell but by modulating the intracellular response to amino acids.

Amino acid transporters and nutrient-sensing mechanisms: new targets for treating insulin-linked disorders?

The IIS (insulin/IGF (insulin-like growth factor) signalling) cascade has an important role in regulating normal development and physiology, as evidenced by its effects in a host of major human diseases including cancer, Type 2 diabetes and neurodegeneration. Recently, it has become clear that multiple types of local nutrient-sensing mechanisms have an impact on cellular insulin-sensitivity through the downstream kinase TOR (target of rapamycin). In vivo analysis in flies has surprisingly highlighted PATs (proton-assisted amino acid transporters) as having a uniquely potent role in regulating IIS/TOR activity and growth, potentially via a novel signalling mechanism. Other molecules such as the heterodimeric amino acid transporter, CD98, which provides the principal route for cellular uptake of leucine, an amino acid implicated in regulating TOR, also appear to have important effects. As our understanding of how nutrient sensing has an impact on IIS/TOR increases, novel targets to modulate aberrant IIS in disease are likely to emerge, which could complement current strategies designed to block kinases in this pathway.

Extracellular and subcellular regulation of the PI3K/Akt cassette: new mechanisms for controlling insulin and growth factor signalling.

The PI3K (phosphoinositide 3-kinase)/Akt (also called protein kinase B) signalling cassette plays a central role in the response to growth factors, particularly insulin-like molecules, and its misregulation is a characteristic feature of diabetes and many forms of human cancer. Recent molecular genetic studies initiated in the fruitfly, Drosophila melanogaster, have highlighted two new cell-type-specific mechanisms regulating PI3K/Akt signalling and its downstream effects. First, the cellular response to this cassette is modulated by several classes of cell-surface transporters and sensors, suggesting an important role for extracellular nutrients in insulin-sensitivity. Secondly, various cell types show a markedly different subcellular distribution of the activated kinase Akt, influencing the cellular functions of this molecule. These findings reveal new mechanisms by which processes such as growth, lipogenesis and insulin resistance can be differentially regulated and may suggest novel strategies for treating insulin-linked diseases.

Drosophila SnoN modulates growth and patterning by antagonizing TGF-beta signalling.

Signalling by TGF-beta ligands through the Smad family of transcription factors is critical for developmental patterning and growth. Disruption of this pathway has been observed in various cancers. In vertebrates, members of the Ski/Sno protein family can act as negative regulators of TGF-beta signalling, interfering with the Smad machinery to inhibit the transcriptional output of this pathway. In some contexts ski/sno genes function as tumour suppressors, but they were originally identified as oncogenes, whose expression is up-regulated in many tumours. These growth regulatory effects and the normal physiological functions of Ski/Sno proteins have been proposed to result from changes in TGF-beta signalling. However, this model is controversial and may be over-simplified, because recent findings indicate that Ski/Sno proteins can affect other signalling pathways. To address this issue in an in vivo context, we have analyzed the function of the Drosophila Ski/Sno orthologue, SnoN. We found that SnoN inhibits growth when overexpressed, indicating a tumour suppressor role in flies. It can act in multiple tissues to selectively and cell autonomously antagonise signalling by TGF-beta ligands from both the BMP and Activin sub-families. By contrast, analysis of a snoN mutant indicates that the gene does not play a global role in TGF-beta-mediated functions, but specifically inhibits TGF-beta-induced wing vein formation. We propose that SnoN normally functions redundantly with other TGF-beta pathway antagonists to finely adjust signalling levels, but that it can behave as an extremely potent inhibitor of TGF-beta signalling when highly expressed, highlighting the significance of its deregulation in cancer cells.

The Drosophila cell shape regulator c-Jun N-terminal kinase also functions as a stress-activated protein kinase.

Mammalian c-Jun N-terminal kinases (JNKs) are members of a group of stress-activated intracellular signalling molecules within the MAP kinase family. Molecular genetic analysis of a highly evolutionarily conserved Drosophila JNK homologue, DJNK, has demonstrated that this molecule plays an essential developmental role in cell shape regulation. However, it remains to be determined whether DJNK also responds to the broad range of cellular stresses and other stimuli that affect its mammalian counterpart. Here we demonstrate that c-Jun, a substrate for mammalian JNKs, is a specific substrate for DJNK and that an antiserum that cross-reacts with activated mammalian JNK at the conserved threonyl-prolyl-tyrosyl (TPY) motif within the activation loop also specifically recognises the activated form of DJNK. Using these two assays, we show that DJNK activity is stimulated in cultured cells by several treatments that activate mammalian JNKs, including addition of arsenite, vanadate and ceramide derivatives. It is therefore concluded that in addition to its essential developmental functions, DJNK plays an important role in stress responses that mirrors its mammalian counterpart.

Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway.

The human tumor suppressor gene PTEN encodes a putative cytoskeleton-associated molecule with both protein phosphatase and phosphatidylinositol 3,4,5-trisphosphate (PIP3) 3-phosphatase activities. In cell culture, the lipid phosphatase activity of this protein is involved in regulating cell proliferation and survival, but the mechanism by which PTEN inhibits tumorigenesis in vivo is not fully established. Here we show that the highly evolutionarily conserved Drosophila PTEN homolog, DPTEN, suppresses hyperplastic growth in flies by reducing cell size and number. We demonstrate that DPTEN modulates tissue mass by acting antagonistically to the Drosophila Class I phosphatidylinositol 3-kinase, Dp110, and its upstream activator Chico, an insulin receptor substrate homolog. Surprisingly, although DPTEN does not generally affect cell fate determination, it does appear to regulate the subcellular organization of the actin cytoskeleton in multiple cell types. From these data, we propose that DPTEN has a complex role in regulating tissue and body size. It acts in opposition to Dp110 to control cell number and growth, while coordinately influencing events at the cell periphery via its effects on the actin cytoskeleton.

JNK, cytoskeletal regulator and stress response kinase? A Drosophila perspective.

c-Jun N-terminal kinases (JNKs) are intracellular stress-activated signalling molecules, which are controlled by a highly evolutionarily conserved signalling cascade. In mammalian cells, JNKs are regulated by a wide variety of cellular stresses and growth factors and have been implicated in the regulation of remarkably diverse biological processes, such as cell shape changes, immune responses and apoptosis. How can such different stimuli activate the JNK pathway and what roles does JNK play in vivo? Molecular genetic analysis of the Drosophila JNK gene has started to provide answers to these questions, confirming the role of this molecule in development and stress responses and suggesting a conserved function for JNK signalling in processes such as wound healing. Here, we review this work and discuss how future experiments in Drosophila should reveal the cell type-specific mechanisms by which JNKs perform their diverse functions.

A JNK signal transduction pathway that mediates morphogenesis and an immune response in Drosophila.

The Drosophila MAP kinase DJNK is a homolog of the mammalian c-Jun amino-terminal kinase (JNK). Mutations in the DJNK gene correspond to the complementation group basket. DJNK is phosphorylated and activated by the Drosophila MAP kinase kinase HEP. Substrates of DJNK include the transcription factor DJun. DJNK participates in multiple physiological processes. Exposure to endotoxic lipopolysaccharide initiates an insect immune response and leads to DJNK activation. In addition, embryos lacking DJNK are defective in dorsal closure, a process in which the lateral epithelial cells migrate over the embryo and join at the dorsal midline. These data demonstrate that the DJNK signal transduction pathway mediates an immune response and morphogenesis in vivo.

Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases.

We have identified a Drosophila gene, Dror, which encodes a putative receptor tyrosine kinase (RTK) and maps to cytological location 31B/C on the second chromosome. In embryos, this gene is expressed specifically in the developing nervous system. The Dror protein appears to be a homolog of two human RTKs, Ror1 and Ror2. Dror and Ror1 proteins share 36% amino acid identity in their extracellular domains and 61% identity in their catalytic tyrosine kinase (TK) domains. Ror1 and Ror2 were originally identified on the basis of the similarity of their TK domains to the TK domains of members of the Trk family of neurotrophin receptors. The Dror protein shows even greater similarity to the Trk proteins within this region than do the human Ror proteins. In light of its similarity to trk and its neural-specific expression pattern, we suggest that Dror may encode a neurotrophic receptor that functions during early stages of neural development in Drosophila.