Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia.

Cancer cachexia is a common deleterious paraneoplastic syndrome that represents an area of unmet clinical need, partly due to its poorly understood aetiology and complex multifactorial nature. We have interrogated multiple genetically defined larval Drosophila models of tumourigenesis against key features of human cancer cachexia. Our results indicate that cachectic tissue wasting is dependent on the genetic characteristics of the tumour and demonstrate that host malnutrition or tumour burden are not sufficient to drive wasting. We show that JAK/STAT and TNF-α/Egr signalling are elevated in cachectic muscle and promote tissue wasting. Furthermore, we introduce a dual driver system that allows independent genetic manipulation of tumour and host skeletal muscle. Overall, we present a novel Drosophila larval paradigm to study tumour/host tissue crosstalk in vivo, which may contribute to future research in cancer cachexia and impact the design of therapeutic approaches for this pathology.

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells.

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours.

Identification of genes for engineering the male germline of Aedes aegypti and Ceratitis capitata.

BACKGROUND: Synthetic biology approaches are promising new strategies for control of pest insects that transmit disease and cause agricultural damage. These strategies require characterised modular components that can direct appropriate expression of effector sequences, with components conserved across species being particularly useful. The goal of this study was to identify genes from which new potential components could be derived for manipulation of the male germline in two major pest species, the mosquito Aedes aegypti and the tephritid fruit fly Ceratitis capitata. RESULTS: Using RNA-seq data from staged testis samples, we identified several candidate genes with testis-specific expression and suitable expression timing for use of their regulatory regions in synthetic control constructs. We also developed a novel computational pipeline to identify candidate genes with testis-specific splicing from this data; use of alternative splicing is another method for restricting expression in synthetic systems. Some of the genes identified display testis-specific expression or splicing that is conserved across species; these are particularly promising candidates for construct development. CONCLUSIONS: In this study we have identified a set of genes with testis-specific expression or splicing. In addition to their interest from a basic biology perspective, these findings provide a basis from which to develop synthetic systems to control important pest insects via manipulation of the male germline.

The RNA export factor, Nxt1, is required for tissue specific transcriptional regulation.

The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex.

Histone variant Htz1 promotes histone H3 acetylation to enhance nucleotide excision repair in Htz1 nucleosomes.

Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. We show here that a histone H2A variant, Htz1 (H2A.Z), in nucleosomes has a positive function in promoting efficient NER in yeast. Htz1 inherently enhances the occupancy of the histone acetyltransferase Gcn5 on chromatin to promote histone H3 acetylation after UV irradiation. Consequently, this results in an increased binding of a NER protein, Rad14, to damaged DNA. Cells without Htz1 show increased UV sensitivity and defective removal of UV-induced DNA damage in the Htz1-bearing nucleosomes at the repressed MFA2 promoter, but not in the HMRa locus where Htz1 is normally absent. Thus, the effect of Htz1 on NER is specifically relevant to its presence in chromatin within a damaged region. The chromatin accessibility to micrococcal nuclease in the MFA2 promoter is unaffected by HTZ1 deletion. Acetylation on previously identified lysines of Htz1 plays little role in NER or cell survival after UV. In summary, we have identified a novel aspect of chromatin that regulates efficient NER, and we provide a model for how Htz1 influences NER in Htz1 nucleosomes.

Silenced yeast chromatin is maintained by Sir2 in preference to permitting histone acetylations for efficient NER.

Very little is currently known about how nucleotide excision repair (NER) functions at the ends of chromosomes. To examine this, we introduced the URA3 gene into either transcriptionally active or repressed subtelomeric regions of the yeast genome. This enabled us to examine the repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in identical sequences under both circumstances. We found that NER is significantly more efficient in the non-repressed subtelomere than the repressed one. At the non-repressed subtelomere, UV radiation stimulates both histones H3 and H4 acetylation in a similar fashion to that seen at other regions of the yeast genome. These modifications occur regardless of the presence of the Sir2 histone deacetylase. On the other hand, at the repressed subtelomere, where repair is much less efficient, UV radiation is unable to stimulate histone H4 or H3 acetylation in the presence of Sir2. In the absence of Sir2 both of these UV-induced modifications are detected, resulting in a significant increase in NER efficiency in the region. Our experiments reveal that there are instances in the yeast genome where the maintenance of the existing chromatin structures dominates over the action of chromatin modifications associated with efficient NER.

Study on mechanical behavior of Cu-bearing antibacterial titanium alloy implant.

Cu-bearing titanium alloy has been proved to have excellent antibacterial properties, which can effectively reduce the incidence of infection caused by implantation. On the other hand, the addition of Cu in the titanium alloy can also significantly improve the strength of the alloy due to the dispersed precipitation of Ti2Cu compounds in matrix, which will enhance the biomechanical safety of Cu-bearing antibacterial titanium alloys. In this study, a Ti6Al4V5Cu antibacterial titanium alloy and the ordinary Ti6Al4V titanium alloy were used to make a variety of orthopedic trauma repair implants, including bone pin, bone screw and bone plate. Through the internationally accepted test methods, the mechanical properties such as tension, torsion and bending of the implants were studied, and the difference between the implants made of two materials was analyzed. The results showed that the tensile strength of Ti6Al4V5Cu pin was 25% higher than that of Ti6Al4V pin, the torsion strength of the screw was increased up to 89%, the static bending load of the plate was increased by 67-89%, and the maximum loading force of the plate after 1 million cycles of four point dynamic bending was increased by 41-91%. The above improvements should be attributed to the strengthening effect of Cu in the antibacterial titanium alloy, indicating that the Cu-bearing antibacterial titanium alloy implant possesses both advantages of reducing the bacterial infection and improving the biomechanical safety, and a broad clinical application prospects.

Dynamic adult tracheal plasticity drives stem cell adaptation to changes in intestinal homeostasis in Drosophila.

Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. Here we uncover a previously unrecognised crosstalk between adult intestinal stem cells in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species activate tracheal HIF-1α and bidirectional FGF/FGFR signalling, leading to reversible remodelling of gut-associated terminal tracheal cells and intestinal stem cell proliferation following damage. Unexpectedly, reactive oxygen species-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 messenger RNA-binding protein (IGF2BP2/Imp). Our results reveal an intestine-vasculature inter-organ communication programme that is essential to adapt the stem cell response to the proliferative demands of the intestinal epithelium.

RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis.

RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

Tilting at windmills? The nucleotide excision repair of chromosomal DNA.

A typical view of how DNA repair functions in chromatin usually depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin on the repair process. It may be that in this current interpretation the repair mechanisms are 'tilting at windmills', fighting an imaginary foe. An emerging picture suggests that we should not consider chromatin as an inhibitory force to be overcome like some quixotic giant by the DNA repair processes. Instead we should now recognize that DNA repair and chromatin metabolism are inextricably and mechanistically linked. Here we discuss the latest findings which are beginning to reveal how changes in chromatin dynamics integrate with the DNA repair process in response to UV induced DNA damage, with an emphasis on events in the yeast Saccharomyces cerevisiae.

The antimicrobial peptide defensin cooperates with tumour necrosis factor to drive tumour cell death in Drosophila.

Antimicrobial peptides (AMPs) are small cationic molecules best known as mediators of the innate defence against microbial infection. While in vitro and ex vivo evidence suggest AMPs' capacity to kill cancer cells, in vivo demonstration of an anti-tumour role of endogenous AMPs is lacking. Using a Drosophila model of tumourigenesis, we demonstrate a role for the AMP Defensin in the control of tumour progression. Our results reveal that Tumour Necrosis Factor mediates exposure of phosphatidylserine (PS), which makes tumour cells selectively sensitive to the action of Defensin remotely secreted from tracheal and fat tissues. Defensin binds tumour cells in PS-enriched areas, provoking cell death and tumour regression. Altogether, our results provide the first in vivo demonstration for a role of an endogenous AMP as an anti-cancer agent, as well as a mechanism that explains tumour cell sensitivity to the action of AMPs.

A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila.

The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. Here, we show that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon α, which signals via its neuronal receptor DLgr2. Bursicon α/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon α/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, our work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis.

RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes.

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

Histone acetylation, chromatin remodelling, transcription and nucleotide excision repair in S. cerevisiae: studies with two model genes.

We describe the technology and two model systems in yeast designed to study nucleotide excision repair (NER) in relation to transcription and chromatin modifications. We employed the MFA2 and MET16 genes as models. How transcription-coupled (TCR) and global genome repair (GGR) operate at the transcriptionally active and/or repressed S. cerevisiae MFA2 locus, and how this relates to nucleosome positioning are considered. We discuss the role of the Gcn5p histone acetyltransferase, also associated with MFA2's transcriptional activation, in facilitating efficient NER at the transcriptionally active and inactive genes. The effect of Gcn5p's absence in reducing NER was local and UV stimulates Gcn5p-mediated histone acetylation at the repressed MFA2 promoter. After UV irradiation Swi2p is partly responsible for facilitating access to restriction of DNA in the cores of the nucleosomes at the MFA2 promoter. The data suggest similarities between chromatin remodelling for NER and transcription, yet differences must exist to ensure this gene remains repressed in alpha cells during NER. For MET16, we consider experiments examining chromatin structure, transcription and repair in wild type and cbf1Delta cells under repressing or derepressing conditions. Cbf1p is a sequence specific DNA binding protein required for MET16 chromatin remodelling and transcription.

The Saccharomyces cerevisiae histone acetyltransferase Gcn5 has a role in the photoreactivation and nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers in the MFA2 gene.

How DNA repair enzymes or complexes gain access to chromatin is still not understood. Here, we have studied the role of the S. cerevisiae histone acetyltransferase Gcn5 in photoreactivation (PR) and nucleotide excision repair (NER) at the level of the genome, the MFA2 and RPB2 genes, and at specific nucleotides within MFA2. The deletion of GCN5 markedly reduced the PR and NER of UV-induced cyclobutane pyrimidine dimers in MFA2 but much less so in RPB2, whereas no detectable defect was seen for repair of the genome overall. In Delta(gcn5), the MFA2 mRNA level is reduced by fourfold, while transcription from RPB2 is reduced only to 80 %. These changes in transcription correlate with the changes in NER and PR found in the Delta(gcn5) mutant. However, changes in MFA2 transcription cannot account for the decrease in NER in the non-transcribed strand and the control region of MFA2 where global genome repair (GGR) operates. We conclude that the histone acetyltransferase Gcn5 influences PR and NER at MFA2 in both its transcribed and non-transcribed DNA, yet it has little effect on these processes for most of the yeast genome. As a result, we speculate that histone acetylation allows efficient access of the repair machinery to chromosomal DNA damages either indirectly via influencing transcription or directly via modifying chromatin structure irrespective of transcription.

Transformed epithelia trigger non-tissue-autonomous tumor suppressor response by adipocytes via activation of Toll and Eiger/TNF signaling.

High tumor burden is associated with increased levels of circulating inflammatory cytokines that influence the pathophysiology of the tumor and its environment. The cellular and molecular events mediating the organismal response to a growing tumor are poorly understood. Here, we report a bidirectional crosstalk between epithelial tumors and the fat body-a peripheral immune tissue-in Drosophila. Tumors trigger a systemic immune response through activation of Eiger/TNF signaling, which leads to Toll pathway upregulation in adipocytes. Reciprocally, Toll elicits a non-tissue-autonomous program in adipocytes, which drives tumor cell death. Hemocytes play a critical role in this system by producing the ligands Spätzle and Eiger, which are required for Toll activation in the fat body and tumor cell death. Altogether, our results provide a paradigm for a long-range tumor suppression function of adipocytes in Drosophila, which may represent an evolutionarily conserved mechanism in the organismal response to solid tumors.

Chromatin modifications and nucleotide excision repair.

We have developed an innovative approach to examine the incidence and frequency of repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution in yeast sequences of choice and have then adapted it for the footprinting of nucleosomes and regulatory proteins that bind to DNA. Using the mating-type-specific gene MFA2 as a model, we have determined DNA repair rates for individual DNA lesions throughout the sequence. Positioned nucleosomes occur when the gene is repressed and we have begun to unravel how they are modified after UV. This radiation triggers histone acetylation, primarily at H3, and is mediated by the Gcn5 histone acetyltransferase; its absence reduces repair substantially. UV also triggers chromatin remodelling as measured by increased accessibility of restriction sites at the cores of the two nucleosomes in the gene's upstream control region; this is partly mediated by Swi2, a yeast SWI/SNF factor. Surprisingly neither of these events require functional NER, but NER is needed to return the chromatin to its pre-UV state.

Saccharomyces cerevisiae Rad16 mediates ultraviolet-dependent histone H3 acetylation required for efficient global genome nucleotide-excision repair.

In yeast, global genome nucleotide-excision repair (GG-NER) requires a protein complex containing Rad7 and Rad16. Rad16 is a member of the switch/sucrose nonfermentable superfamily, and it is presumed that chromatin remodelling is its primary function during repair. We show that RAD16 is required for ultraviolet-dependent hyperacetylation of histone H3 (Lys 9 and Lys 14) at the MFA2 promoter and throughout the genome. The yeast repressor complex Ssn6-Tup1 represses many genes including MFA2. TUP1 deletion results in constitutive hyperacetylation of histone H3, nucleosome disruption and derepression of gene transcription in Tup1-regulated genes. GG-NER in the MFA2 promoter proceeds more rapidly in tup1Delta alpha-cells compared with wild type, even when transcription is inhibited. We show that elevated histone H3 acetylation levels in the MFA2 promoter in tup1Delta alpha-cells result in Rad7- and Rad16-independent GG-NER, and that Rad16 mediates the ultraviolet-induced acetylation of histone H3, necessary for efficient GG-NER.

Histone acetylation, chromatin remodelling and nucleotide excision repair: hint from the study on MFA2 in Saccharomyces cerevisiae.

Nucleotide excision repair (NER) is a sophisticated repair pathway that the cell utilizes to remove a broad range of DNA damage to help maintain the functional integrity of the genome. In the context of DNA packaged into chromosomes it is clear that the NER machinery does not repair all regions with equal efficiency. Recently, we found after UV that histone acetylation and chromatin remodelling were activated. UV irradiation triggers genome-wide histone hyperacetylation at both histone H3 and H4. However, in nucleosomes at the repressed MFA2 promoter only histone H3, but not histone H4, is hyperacetylated following UV. This Gcn5p-mediated histone H3 hyperacetylation enables efficient NER at MFA2. Chromatin in this promoter also becomes more accessible after UV. This is not dependent on Gcn5p, yet it is partially dependent on Swi2p. In later repair times both events gradually return to the pre-UV state. The post-UV histone modifications and chromatin remodelling at the repressed MFA2 promoter do not activate MFA2 transcription, nor do they require damage recognition by Rad4p or Rad14p. These experiments indicate early events are triggered in chromatin in response to UV treatment, and they are likely needed for efficient NER.

UV irradiation stimulates histone acetylation and chromatin remodeling at a repressed yeast locus.

Chromatin immunoprecipitation with anti-acetyl histone H3 (K9 and K14) and anti-acetyl histone H4 (K5, K8, K12, and K16) antibodies shows that Lys-9 and/or Lys-14 of histone H3, but not the relevant sites of histone H4 in nucleosomes at the repressed MFA2 promoter, are hyperacetylated after UV irradiation. This level of histone hyperacetylation diminishes gradually as repair proceeds. Accompanying this, chromatin in the promoter becomes more accessible to restriction enzymes after UV irradiation and returns to the pre-UV state gradually. UV-related histone hyperacetylation and chromatin remodeling in the MFA2 promoter depend on Gcn5p and partially on Swi2p, respectively. Deletion of GCN5, but not of SWI2, impairs repair of DNA damage in the MFA2 promoter. The post-UV histone modifications and chromatin remodeling at the repressed MFA2 promoter do not activate MFA2 transcriptionally, nor do they require damage recognition by Rad4p or Rad14p. Furthermore, we show that UV irradiation triggers genome-wide histone hyperacetylation at both histone H3 and H4. These experiments indicate that chromatin at a yeast repressed locus undergoes active change after UV radiation treatment and that failure to achieve histone H3 hyperacetylation impairs the repair of DNA damage.