ISSN: 2455-2283

Archives of Clinical Gastroenterology

Review Article       Open Access      Peer-Reviewed

Flies to Humans - Humans to Flies: A Virtuous Circle of Colorectal Cancer Prevention

Elena Kamilari, Yiorgos Apidianakis* and Myrofora Panagi*

Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus Dates: Received: 22 May, 2017; Accepted: 02 June, 2017; Published: 06 June, 2017 *Corresponding author: Yiorgos Apidianakis, 1 Panepistimiou Ave, Department of Biological Sciences, University of Cyprus, 2109, Nicosia, Cyprus, Tel. 357-22893767; E-mail: @ Myrofora Panagi, 1 Panepistimiou Ave, Department of Biological Sciences, University of Cyprus, 2109, Nicosia, Cyprus, Tel. 357-22893967 E-mail: @

Author and article information

*Corresponding author: Yiorgos Apidianakis, 1 Panepistimiou Ave, Department of Biological Sciences, University of Cyprus, 2109, Nicosia, Cyprus, Tel. 357-22893767; E-mail: [email protected]
doi : 10.17352/2455-2283.000038
Submitted: 22 May, 2017 | Accepted: 02 June, 2017 | Published: 06 June, 2017
Keywords: Biomarkers; Drosophila; Human; Model host; Colorectal cancer; Inflammation

Cite this as

Kamilari E, Apidianakis Y, Panagi M (2017) Flies to Humans - Humans to Flies: A Virtuous Circle of Colorectal Cancer Prevention. Arch Clin Gastroenterol. 2017; 3(3): 47-60. Available from: 10.17352/2455-2283.000038

Copyright License

© 2017 Kamilari E, 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.

Abstract

The two Nobel prizes in physiology or medicine of 1995 and 2011 establish Drosophila genetics as a signifi cant contributor of genes and signaling pathways relevant to human disease, including innate immunity and cancer. Other than providing clues on mammalian gene homologue function, relatively little attention has been paid on the translational aspect of Drosophila genes, microbes and environmental factors that infl uence homeostasis and disease. This is particularly important for colorectal cancer (CRC) prevention, for which molecular diagnostic tools are non-existent. While clinical studies provide a wealth of information on genes and microbes linked to infl ammatory bowel disease (IBD) and CRC, it is unknown if they can serve as biomarkers in terms of CRC prevention. We discuss the line of research showing that many biomarkers of intestinal infl ammation and CRC in humans may be modeled and mechanistically tested in fl ies. Vise versa, genes and processes, such as regenerative infl ammation and aging- associated DNA damage, found in fl ies to promote tumorigenesis may be tested as biomarkers of CRC risk in humans. Thus, modeling human intestinal infl ammation and cancer in fl ies can provide a means to assess causality of conserved genes and microbes that can colonize the fl y intestine. Moreover, successful modeling in fl ies enables the “treatability” of the pertinent biomarkers via dietary, probiotic and pharmacological interventions and paves the way for clinical trials of treatments that may alleviate intestinal infl ammation and the risk for CRC.

Indexing and Abstracting

Introduction

The genetic and histological suitability of flies for basic research on CRC

During the last two decades Drosophila has become a powerful model for exploring the links between inflammation and colorectal cancer (CRC). The fully sequenced genome, the high degree of human disease-related gene homology with flies (up to 75%), the reduced genetic redundancy, the great availability of genetic tools enabling spatial and temporal manipulation of cells, as well as, the evolutionary conservation of signaling pathways controlling vital biological processes and immunity, make Drosophila a suitable model host for the identification of candidate biomarkers implicated in tumor-promoting inflammation [1,2]. In addition, the small size, the low cost of maintenance and the easy delivery of orally administrated drugs facilitate whole-animal screening for molecular compounds affecting stem cell mediated carcinogenesis [3,4]. Remarkably, there are at least 60 chemical compounds originally known for their activity in human cells that demonstrably have the same molecular mechanism of action in flies [2].

The intestine is the most rapidly self-renewing tissue of the human body. Intestinal epithelium is continuously exposed to pathogens and chemicals of the lumen leading to enterocyte damage and concomitant regenerative inflammation that completely replenishes the damaged or lost cells by asymmetrically dividing intestinal stem cells (ISCs) [5-7]. ISCs reside at the bottom of the crypt of Lieberkühn along with the secretory Paneth cells. ISCs proliferate to give rise to transient cells that amplify and differentiate, while moving upwards the crypt. Differentiated cells are found at the rim of the crypts and the villi. These are absorptive enterocytes, but also secretory cells, namely, Paneth, enderoendocrine or goblet cells. Between the villi and the lumen there is a goblet cell derived-mucus layer that protects cells from direct bacterial contact. The tissue is supported by stromal cells of various types and surrounded by visceral muscle. Similarly, Drosophila intestine is maintained by ISCs that divide giving rise to new ISCs and transient enteroblasts, which normally differentiate without further divisions into either absorptive enterocytes or enteroendocrine cells [8,9]. Although the mammalian Paneth, goblet and stromal cells are absent in flies, many of their immunity and barrier physiology functions are fulfilled by highly endoreplicating enterocytes and the visceral muscle [1]. The fly intestine generally lacks crypts that support multiple progenitor cells and villi. These appear necessary for maximal nutrient absorption in mammals, but in flies a monolayer of epithelial cells surrounded by two layers of visceral muscle suffices for homeostasis. In addition to the mucus layer, the Drosophila gut lumen is surrounded by a chitin layer, the peritrophic matrix that confers extra protection against pathogens [10].

Using flies for identifying genetic and microbial biomarkers of risk for CRC

Fly immunity shares evolutionary conserved mechanisms with human innate immunity. Drosophila midgut responds to uracil from intestinal pathogenic bacteria by inducing the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway [11,12], which in turn activates the conserved NAPDH oxidase, Duox, leading to the release of reactive oxygen species (ROS) [13-15]. The antimicrobial activity of ROS is complemented with the production of antimicrobial peptides (AMPs). Toll signaling pathway is responsible for the systemic AMP response mediated by the nuclear translocation of the NF-κB-like transcription factor(s) Dorsal and/or Dif. The second NF-κB-like pathway of Drosophila, named immune deficiency (IMD) pathway, is stimulated by peptidoglycan recognition proteins (PGRPs). IMD is induced by bacterial peptidoglycan leading to the nuclear translocation of Relish and AMPs expression both systemically and in the fly gut [16,17]. Intestinal damage and stress are also capable of stimulating particular AMP expression following secretion of the Upd3 inflammatory cytokine, an analog of the human interleukin (IL)-6, which activates the JAK/STAT signaling in both ISCs and the visceral muscle [18,19]. JAK/STAT pathway activation leads to the expression of epidermal growth factors and consequently induction of EGFR/Ras/MAPK cascade inducing ISC proliferation [20,21]. Drosophila stem cells are further modulated by the Target of Rapamycin (TOR), Hippo and wingless pathways [22-24]. This inflammation induced tissue regeneration process referred to as regenerative inflammation may contribute to tumor initiation and progression and is conserved between flies and mammals [6,7].

Inflammation is pivotal for host defense, but it can lead to pathogenesis when chronic and predispose for cancer. The inflammatory microenvironment facilitates tumor initiation and progression, although a direct causality has not been established for CRC. Germline mutations account as a driving force for the 10% of CRC incidence, whereas the vast majority of cases associate with somatic mutations and environmental factors, including chronic inflammation and bacterial infections [25,26]. For instance, the chronic inflammation of the gastrointestinal mucosa present in IBD patients is a key predisposing factor for developing CRC [27]. Nevertheless, so far only Helicobacter pylori infection has been established as a causative agent of gastrointestinal inflammation and cancer [28]. In mammals, areas with active inflammatory responses accompanied by a high rate of epithelial cell-turnover and sustained DNA damage are sufficient to drive carcinogenesis [29]. This setting of increased predisposition to tumorigenesis is also found in the aging midgut of Drosophila [30]. Moreover, pathogenic bacterial infection promotes intestinal tumorigenesis in genetically predisposed adult Drosophila [31].

Previous studies have used fruit flies as model hosts to induce intrinsic and extrinsic oxidative damage that resembles the aging associated changes in progenitor cells [32]. Collective evidence indicates accumulation of γH2AvD foci in ISCs, analogous to the mammalian γH2AX, a DNA damage marker. γH2AvD foci correlate with γ-ray-induced DNA damage in ISCs and age-related accumulation of 8-oxo-2′deoxyguanosine. Interestingly, age and oxidative stress related DNA damage in Drosophila can be alleviated by the chemotherapy drug Metformin via downregulation of the insulin-like growth factor-I receptor/insulin receptor (IGF1R/IR) and the AKT [33]. As ISCs age, they acquire persistent chromatin lesions bearing double strand breaks (DSB) and thereby, initiating a continuous secretion of inflammatory cytokines. In mammals, damaged ISCs that have entered senescence preserve their capacity to secrete different factors and interact with the surrounding microenvironment [34]. Similarly, age-related DNA damage and JNK-driven dysplasia correlate with barrier failure and excessive systemic inflammatory signaling attributed to the bacterial translocation across the Drosophila gut [35,36]. Somatic inactivation of Notch tumor suppressor during aging in flies causes spontaneous neoplasia driven by somatic recombination, genomic deletions and rearrangements [30].

As microbial intestinal load increases with age, it becomes challenging to maintain symbiosis between the host and its microbes. Alterations within the microbiome structure could elicit an acute inflammatory signaling through the increased production of ROS and AMPs and the release of inflammatory cytokines and growth factors that regenerate the intestinal epithelium. However, chronic inflammatory responses and the excessive exposure of cells to oxidative stress have adverse effects on homeostasis, leading to dysbiosis. Dysbiosis is correlated with IBD [37] and cancer [38]. Drosophila is characterized by a simple microbiome of less than 30 microbial species [35] compared to that of humans, which is composed with hundreds of different bacterial species [39]. Lactobacillus and occasionally Enterobacteriaceae are part of both the fly and human microbiome. While huge differences exist, symbiotic bacteria are critical for host physiology in both species and the number of human bacteria that can colonize flies is far more than those found naturally. They promote growth by modulating nutrient metabolism and absorption [40] and participate in the shaping of gastrointestinal immune landscape [41,42]. Therefore, pinpointing the mechanisms by which gut microbiota affects health and disease, may help to suggest new therapeutic approaches to alleviate microbiota-directed inflammation and CRC incidence. Bacterial mono-associations or poly-associations with germ-free flies would provide insights regarding the contribution of symbiotic bacteria at the species level in intestinal disease.

Mammalian genetic and microbial biomarkers of risk for CRC

Most CRC cases are sporadic, that is, with no known genetic component attributed to them (70%-80% of all cases) and usually appear at an old age [43,44]. Hereditary forms of CRC include familial adenomatous polyposis (~1%), non-polyposis hereditary CRC or Lynch syndrome (2%-5%) and MYH-gene associated polyposis (<1%) [45]. Interestingly, molecular and cellular alterations precede morphological changes of the intestinal mucosa, and may predispose for tumorigenesis [46,47]. This must be true also for intestinal microbes and their balance [48]. Such alterations may be blamed for the recurrence of adenomatous polyps after surgical excision [49]. Therefore, ongoing efforts turn towards finding specific genetic and microbial markers that will allow the early detection of CRC appearance in terms of personalized medicine and treatment.

A hallmark of transition from normal colonic epithelium to neoplastic is genomic instability (GI), which is divided to chromosomal instability (CIN), microsatellite instability (MSI) and epigenetic instability (EI) [44]. The most familiar form of GI is CIN, which is implicated in 80%–85% of colorectal tumors [50]. GI is characterized by a) the presence of aneuploidy, which involves changes in chromosome number, b) modifications in the gene structure, such as insertions, deletions or base substitutions, which are also caused by MSI, c) chromosome rearrangements and d) gene multiplying. The basic concept of GI involves the loss of function of tumor-suppressor genes, such as adenomatous polyposis coli (APC), p53, SMAD4, and tumor-suppressor genes on chromosome 18q the area deleted in colon cancer (DCC), or the activation of the ,K-Ras oncogene [51].

APC is mutated in up to 80% of sporadic CRC cases and is involved in the negative regulation of Wingless-Int (WNT) signaling pathway. WNT pathway regulates various biological processes, such as cell proliferation, differentiation, polarity, and movement, and maintains intestinal epithelial cell (IEC) homeostasis [52-54]. The canonical WNT pathway is highly conserved and initiates with the binding of Wnts to Frizzled (Fz) receptors [55-57]. Downstream of Fz, Dishevelled (Dsh) the glycogen synthase kinase 3 beta (GSK3β) and casein kinase I-γ (CK1γ) result in the docking of the scaffold protein Axin and APC and the stabilization of β-catenin. The complex with β-catenin is disrupted upon ligand signaling and β-catenin moves to the nucleus where it binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors activating specific Wnt target genes. In the absence of Wnt, the serine/threonine kinases, CK1 and GSK3α/β, phosphorylate β-catenin, which is guided by the F box/WD repeat protein β-TrCP, for degradation to the proteasome. In the absence of signaling, TCF/LEF is not activated by β-catenin and its targets are suppressed by Groucho [58]. The TCF binding sites are also similar between vertebrates and Drosophila [59]. The function of β-catenin as a TCF activator is strictly regulated by the multiprotein complex that involves APC, which when mutated β-catenin is activated to induce tumor-promoting genes, such as Myc [60].

The KRAS oncogene is an activated form of the endogenous gene and present in up to 43% of human CRC tumors. It encodes the guanosine diphosphate (GDP) and guanosine triphosphate (GTP) binding proteins. Wild type KRAS is induced by the epidermal growth factor receptor (EGFR) pathway, but the KRAS oncogene is constitutively active independently of such stimulation [61]. Activation of EGFR promotes an excessive mitogenic signaling cascade through the activation of numerous pathways, including the RAS – RAF – mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3-kinase (PI3K) – Akt, and the phospholipase C pathway [62, 63]. BRAF V600E (involved in 10-15% of CRC tumors), which encodes a guanosine triphosphate (GTPase), is also involved in the EGFR pathway activation [64]. PIK3CA gene of PI3K pathway is mutated in ∼15% of CRCs [65], while some cases include mutations in PTEN, a tumor suppressor, which normally inhibits PI3K [66]. Additional mutations present in CRC tumors include TCF7L2, , NRAS, FAM123B, CTNNB1 and SMAD2 [67] (Table 1). Mutations in the MSI pathway are primarily due to the loss of function of DNA repair proteins, MLH1, MLH3, PMS1, PMS2, MSH2, MSH3, MSH6 and Exo1 [68]. MSI also affects cell mitosis (TGF-β, GRB1, TCF-4, WISP3, activin receptor-2, IGF-2 receptor, axin-2, and CDX), apoptosis (BAX, caspase-5, RIZ, BCL-10, PTEN, hG4-1, and FAS), and additional DNA repair genes (MBD-4, BLM, CHK1 and RAD50) [69, 70]. The most known, clinically evaluated MSI markers are mononucleotide (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) exhibiting high sensitivity and specificity [64] (Table 2). Two kinds of EI have been mostly described in CRC: CpG island methylator phenotype (CIMP), and global DNA hypomethylation. Both mechanisms cause silencing of gene expression. Known biomarkers for CIMP-positive tumors are CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 and methylation must occur in at least 3 of them [71]. Indicative lists of mouse and fly homologs of human genetic and epigenetic biomarkers of inflammation and CRC are provided in Tables 1, 2.

Table 1: Genomic Instability and Epigenetic Instability related genes and their homology in human, mice and flies.

PROCESS

HUMAN
SNPs/GENES
[136]

MOUSE
SNPs/GENES
[137]

FLY GENES

HOMOLOGY

Chromosomal
Instability

APC

APC

APC-like [138]

Human-mouse: 90%

TP53

Trp53

p53 [139]

Human-mouse: 77%

SMAD4

Smad4

Med [140]

Human-mouse: 98% Human-fly: 78%

KRAS

Kras

Ras85D [141]

Human-mouse: 96% Human-fly: 85%

MYC

Myc

dMyc [142]

Human-mouse: 87%

RAF1

Raf1

D-raf [143]

Human-mouse: 98%

PIK3CA

Pik3ca

PI3K [144]

Human-mouse: 99%

BRAF

Braf

Raf [145]

Human-mouse: 86% Human-fly: 45%

PTEN

Pten

Pten [146]

Human-mouse: 99% Human-fly: 47%

TCF7L2

Fbxw7

Archipelago [147]

Human-mouse: 95%

TCF7L2

Tcf7l2

dTCF [59]

Human-mouse: 97%

NRAS

Nras

NRas [148]

Human-mouse: 99%

AMER1

Amer1

 

Human-mouse: 77%

CTNNB1

Ctnnb1

arm [149]

Human-mouse: 99% Human-fly: 67%

SMAD2

Smad2

Smad2 [150]

Human-mouse: 99%

Microsatellite Instability

MLH1

Mlh1

Mlh1 [151]

Human-mouse: 88% Human-fly: 58%

 

MLH3

Mlh3

 

Human-mouse: 70%

PMS1

Pms1

 

Human-mouse: 75%

PMS2

Pms2

Pms2 [152]

Human-mouse: 77%

MSH2

Msh2

spel1 [153]

Human-mouse: 93% Human-fly: 45%

MSH3

Msh3

 

Human-mouse: 82%

MSH6

Msh6

Msh6 [152]

Human-mouse: 85% Human-fly: 44%

Exo1

Exo1

 

Human-mouse: 73%

TGFB1

Tgfb1

Dlk1/Pref-1 [154]

Human-mouse: 90%

PIK3R1

Pik3r1

Pi3K21B [155]

Human-mouse: 96%

TCF-4

Tcf4

dTCF [59]

Human-mouse: 96%

WISP3

Wisp3

Ccn [156]

Human-mouse: 79%

ACVR2A

Acvr2a

put [157]

Human-mouse: 99% Human-fly: 47%

IGF2R

Igf2r

DInR [158]

Human-mouse: 81%

AXIN2

Axin2

Daxin [159]

Human-mouse: 88%

CDX

Cdx1

 

Human-mouse: 84%

BAX

Bax

 

Human-mouse: 92%

PRDM2

Prdm2

 

Human-mouse: 81%

BCL10

Bcl10

 

Human-mouse: 91%

 

PA2G4

Pa2g4

CG10576 [160]

Human-mouse: 99% Human-fly: 56%

FAS

Fas

 

Human-mouse: 49%

MBD4

Mbd4

 

Human-mouse: 96%

BLM

Blm

 

Human-mouse: 75%

CHEK1

Chek1

grp [161]

Human-mouse: 93% Human-fly: 47%

RAD50

Rad50

rad50 [162]

Human-mouse: 92% Human-fly: 29%

Epigenomic
Instability

CACNA1G

Cacna1g

Ca-α1T [163]

Human-mouse: 94%

IGF2

Igf2

Igf [164]

Human-mouse: 82%

NEUROG1

Neurog1

Atonal [165]

Human-mouse: 77%

RUNX3

Runx3

Runt [166]

Human-mouse: 89%

SOCS1

Socs1

SOCS36E [167]

Human-mouse: 92%
Table 2: Processes and genes affecting susceptibility to intestinal infection, stress or inflammation in humans, mice and flies, per Vaiserman, 2015[89].

PROCESS

HUMAN
SNPs/GENES
[136]

MOUSE
SNPs/GENES
[137]

FLY GENES

HOMOLOGY

Autophagy

ATG16L1

Atg16l1

CG31033 [168]

Human-mouse: 94% Human-fly: 38%

IRGM1

IRGMI

 

Human-mouse: 57%

ULK1

ATG16L1

Atg1 [169]

Human-mouse: 89%

MTMR3

Mtmr3

CG3632
(Myotubularin-like) [170]

Human-mouse: 84%

VAMP3

Vamp3

n-Syb, Syb/dVAMP [171]

Human-mouse: 94%

DAP

Dap

 

Human-mouse: 96%

LRRK2

Lrrk2

dLRRK [172]

Human-mouse: 87%

CUL2

Cul2

Cul-2 [173]

Human-mouse: 97% Human-fly: 51%

PARK7

Park7

dj-1beta [174]

Human-mouse: 92% Human-fly: 56%

Innate Immunity

NOD2

Nod2

PGRPs [6]

Human-mouse: 79%

RIPK3

RIPK3

Imd homolog of RIPK2 [6]

Human-mouse: 59%

Nf-KB

Nf-KB

NF-κΒ-like transcription factor(s)
Dorsal and/or Dif [6]

Human-mouse: 86%

TNFa

Tnfa

Eiger [6]

Human-mouse: 79%

COX-2

Ptgs2

COX-like [6]

Human-mouse: 87%

TLR1-10

Tlr1-11

toll [53]

Human-mouse: 74%

TLR-4

Tlr4

toll [53]

Human-mouse: 67%

 

IL18RAP

Il18rap

 

Human-mouse: 67%

Β-defensins

Β-defensins

defensins [175]

 

JAK 2,3

Jak 2,3

hop [176]

Human-mouse: 94%
Human-mouse: 83%

TYK2

Tyk2

Tyk2 [177]

Human-mouse: 78%

IL-1

Il1

 

Human-mouse: 98%
Human-mouse: 68%

SLC11A1

Slc11a1

Nramp [178]

Human-mouse: 89%

FCGR2A

Fcgr3

 

Human-mouse: 60%

FCGR2B

Fcgr2b

 

Human-mouse: 56%

REL

Rel

Rel [179]

Human-mouse: 70%

CARD9

Card9

 

Human-mouse: 86%

MIF

Mif

 

Human-mouse: 90%

FOXO3

Foxo3

dFOXO [180]

Human-mouse: 94%

Adaptive Immunity

PRDM1

Prdm1

 

Human-mouse: 87%

LSP1

Lsp1

 

Human-mouse: 68%

SMAD3

Smad3

 

Smox [181]

Human-mouse: 100% Human-fly: 81%

 

 

 

 

 

SMAD7

Smad7

MAD [182]

Human-mouse: 98%

TGF-β

Tgfb1

 

Human-mouse: 90%

TNFRSF6

Fas

 

Human-mouse: 49%

 

TNFRSF9

Tnfrsf9

 

Human-mouse: 57%

TNFRSF14

Tnfrsf14

 

Human-mouse: 46%

IL4

Il4

 

Human-mouse: 41%

IL6

Il6

Upd [6]

Human-mouse: 40%

IL10

Il10

 

Human-mouse: 73%

IL12A

Il12a

 

Human-mouse: 59%

IL12B

Il12b

 

Human-mouse: 66%

IL13

Il13

 

Human-mouse: 58%

IL17A

Il17a

 

Human-mouse: 62%

IL18

Il18

 

Human-mouse: 65%

IL22

Iltifb

 

Human-mouse: 78%

IL1R

Il1r

 

Human-mouse: 69%

IL7R

Il7r

 

Human-mouse: 63%

IL8R

Cxcr2

 

Human-mouse: 71%

IL17AR

Il17ar

 

Human-mouse: 70%

IL23R

Il23r

 

Human-mouse: 67%

IFNG

Ifng

 

Human-mouse: 41%

VNN1

Vnn1

 

Human-mouse: 76%

 

TNFSF8

Tnfsf8

 

Human-mouse: 70%

TNFSF11

Tnfsf11

 

Human-mouse: 84%

TNFSF15

Tnfsf15

 

Human-mouse: 65%

CCR3

Ccr3

 

Human-mouse: 70%

CCR9

Ccr9

 

Human-mouse: 86%

CXCR3

Cxcr3

 

Human-mouse: 86%

CXCR4

Cxcr4

 

Human-mouse: 89%

CXCL1

Cxcl1

 

Human-mouse: 73%

IL5

Il5

 

Human-mouse: 72%

GATA3

Gata3

Pannier [183]

Human-mouse: 96%

DENND1B

Dennd1b

 

Human-mouse: 83%

LNPEP

Lnpep

 

Human-mouse: 88%

Regeneration

Wnt

Wnt

 

Wnt [184]

 

Notch

Notch

Notch [185]

Human-mouse: 90% Human-fly: 49%

APC

APC

APC-like [138]

Human-mouse: 90%

 

 

 

 

 

STAT3

Stat3

Jak/Stat [19]

Human-mouse: 99%

STAT4

Stat4

Jak/Stat [19]

Human-mouse: 95%

STAT5

Stat5

Jak/Stat [19]

Human-mouse: 96%

 

JAK2

JAK2

Jak/Stat [19]

Human-mouse: 94%

KRAS

Kras

Ras85D [141]

Human-mouse: 96% Human-fly: 85%

PIK3CA

Pik3ca

PI3K [144]

Human-mouse: 99%

Oxidative stress

NOS2

Nos2

dNOS [186]

Human-mouse: 81%

DUOX2

Duox2

Duox [187]

Human-mouse: 84% Human-fly: 38%

ADO

Ado

CG7550 [188]

Human-mouse: 86% Human-fly: 31%

SLC22A4

Slc22a4

Orct [189]

Human-mouse: 85%

GPX1/4

Gpx1/4

GTPx-1 [190]

Human-mouse: 93%

UTS2

Uts2

 

Human-mouse: 50%

PEX13

Pex13

Pex13 [188]

Human-mouse: 91% Human-fly: 36%

PARK7

Park7

DJ-1alpha [191]

Human-mouse: 92% Human-fly: 53%

DLD

Dld

Dl [192]

Human-mouse: 88% Human-fly: 49%

BACH2

Bach2

 

Human-mouse: 89%

LRRK2

Lrrk2

 

Human-mouse: 87%

PRDX5

Prdx5

Prx5 [188]

Human-mouse: 93% Human-fly: 57%

CARD9

Card9

 

Human-mouse: 86%

SLC23A1

Slc23a1

 

Human-mouse: 89%

NOX1

Nox1

Nox [193]

Human-mouse: 84%

Epithelial Barrier

MUC1

MUC1

 

Mucin-like [194]

 

MUC3

A630081J09Rik

Mucin-like [194]

Human-mouse: 64%

MUC4

MUC4

Mucin-like [194]

 

MUC5B

Muc5b

Mucin-like [194]

Human-mouse: 76%

HNF4A

Hnf4a

HNF4a [195]

Human-mouse: 96%

CDH1

Cdh1

Cdh1 [196]

Human-mouse: 81%

CDH2

Cdh2

CadN2 [197]

Human-mouse: 97%

CDH3

Cdh3

 

Human-mouse: 82%

LAMB1

Lamb1

LanB1 [198]

Human-mouse: 93%

 

 

 

 

 

GNA12

Gna12

Cta [199]

Human-mouse: 94%

CDH11

Cdh11

Cadherin domain CG11059 [200]

Human-mouse: 97%

ERRFI1

Errfi1

 

Human-mouse: 82%

ITLN1

Itln1

 

Human-mouse: 81%

MMP1

Mmp1

dm1- and dm2-MMPs
[201]

Human-mouse: 59%

MMP3

Mmp3

dm1- and dm2-MMPs
[201]

Human-mouse: 77%

MMP7

Mmp7

dm1- and dm2-MMPs
[201]

Human-mouse: 70%

MMP9

Mmp9

dm1- and dm2-MMPs
[201]

Human-mouse: 81%

MMP10

Mmp10

dm1- and dm2-MMPs
[201]

Human-mouse: 76%

 

MMP12

Mmp12

dm1- and dm2-MMPs
[201]

Human-mouse: 62%

MMP13

Mmp13

dm1- and dm2-MMPs
[201]

Human-mouse: 86%

MMP14

Mmp14

Mmp1 [202]

Human-mouse: 97% Human-fly: 39%

TIMP1

Timp1

 

Human-mouse: 74%

TIMP2

Timp2

 

Human-mouse: 98%

TIMP3

Timp3

dN-TIMP [201]

Human-mouse: 96%

DLG5

Dlg5

Dlg5 [203]

Human-mouse: 92%

MLCK

Mylk3

Strn-Mlck [204]

Human-mouse: 68%

Additional
Pro-tumorigenic

IL11

Il11

 

 

Human-mouse: 88%

IL23

Il23

 

Human-mouse: 74%

IL33

Il33

 

Human-mouse: 52%

VEGF

Vegf

Pvf1/3 - VEGF-related factor 1/3 [66]

Human-mouse: 88%

Genetic biomarkers linked to inflammation (IBD) may also be linked to CRC. Chronic inflammation and tissue damage induce cell proliferation and aberrant differentiation of macroscopically normal-appearing colonic mucosa, which may lead to crypt enlargement and potentially to cancer initiation and progression [72-74]. The most used marker for cell proliferation during inflammation and cancer is KI67 [75-80]. Additional markers used to estimate colorectal tumor cell mitosis are MCM7 and its negative regulator Geminin, which are involved in the DNA replication [81-83], as well as, Aurora kinase A (AURKA), which plays a critical role in cell cycle regulation [84], and proliferating cell nuclear antigen (PCNA), which is necessary for DNA synthesis during replication [85,86]. Inflammation responses involve the recruitment of tissue-resident macrophages and mast cells, which produce a variety of inflammatory mediators, including cytokines, chemokines, proteases, matrix metalloproteinases, TNF-α, interleukins (IL), interferons (IFN), and enzymes such as cyclooxygenase-2 (COX-2), 5-lipoxygenase (5LOX), and phospholipase A2 (PLA2) responsible for eicosanoid formation [87]. Many other cytokines may be pro-tumorigenic, including IL-4, IL-6, IL-8, IL-11, IL-17A, IL-22, IL-23, IL-33, TNF, TGF-β, and VEGF [88]. These mediators could serve as prognostic biomarkers for CRC appearance. Additional processes and genes affecting susceptibility to intestinal infection, stress or inflammation in human, mice and flies are described in table 2 [89].

Bacteria have been linked both positively and negatively to inflammation (IBD) and CRC. Major disruption of the healthy microbiota caused by extensive and prolonged antibiotic use, especially in neonates and children, can result in life-threatening necrotizing enterocolitis. In this case intestinal dysbiosis results in excessive inflammation, mucosal injury and cell death without regeneration [90]. Shifting the balance of intestinal microbiota from a pathogenic to a protective complement of bacteria can protect the gut from inflammation and subsequent injury [91]. Accordingly, broad-spectrum antibiotics destroy the flora leading to intestinal inflammation and damage that can be prevented with oral administration of microbiota-derived molecular patterns, such as lipopolysaccharide (LPS) that induce a steady state of inflammatory cytokines and prime the epithelium again pathogens [92]. From the immunological perspective evidence reveals various properties of intestinal bacteria that distinguish them as commensals vs. pathogens [93]. Nevertheless, for the most part microbiota is just linked to the healthy or the diseased state with only a few clear cases of bacterial pathogens presented as causal for the disease. Reduced abundance of potentially beneficial bacteria has been reported for patients with IBD and CRC (Table 3). Some of these belong to the family Lachnospiraceae, which produce short chain fatty acids, such as the anti-inflammatory butyric acids [94] and include the genera Lachnospira [95-97], Blautia [95,98-102], Anaerostipes [95] and Roseburia [103-106], even though some reports indicate increase abundance in CRC [107,108]. The genus Lactobacillus was negatively correlated with CRC [109-115]. Lactobacillus strains have been well characterized for their probiotic properties including production of butyrate, which has an antiinflammatory function [94], as well as, the antibacterial lactic acid and bacteriocins. In addition, Lactobacilli reduce the secretion of virulence factors from enterovirulent pathogens alleviating their deleterious effects on the host [116]. Similarly, Clostridia members of clusters XIVa, IV and XVIII, have been reported to reduce inflammation [117] and have decreased abundance in IBD [117-122]. Moreover, some reports have found reduced amounts of Bifidobacteria in CRC patients [95], while others the opposite [123].

Table 3: Genera over/under represented in IBD and CRC and their oxygen sensitivity.

GENERA

OXYGEN SENSITIVITY

REPRESENTATION IN IBD AND/OR CRC

CULTIVATED

Bacteroides

obligately anaerobic

Overrepresented

[133]

Escherichia

facultatively anaerobic

Overrepresented

Enterococcus

facultatively anaerobic

Overrepresented

Enterobacter

facultatively anaerobic

Overrepresented

Prevotella

strictly anaerobic

Overrepresented

Ruminococcus

strictly anaerobic

Overrepresented in IBD/ Underrepresented in CRC

Porphyromonas

strictly anaerobic

Overrepresented

Streptococcus

facultatively anaerobic

Overrepresented

Peptostreptococcus

facultatively anaerobic

Overrepresented

Fusobacterium

strictly anaerobic

Overrepresented

Erysipelothrix

facultatively anaerobic

Overrepresented/ Underrepresented

[205]

Bifidobacterium

obligately anaerobic

Overrepresented/ Underrepresented

[133]

Lachnospira

strictly anaerobic

Underrepresented

Blautia

strictly anaerobic

Underrepresented

Anaerostipes

strictly anaerobic

Underrepresented

Roseburia

obligately anaerobic

Overrepresented/ Underrepresented

Lactobacillus

facultatively anaerobic

Underrepresented

 

Clostridia members

 

 

of clusters XIVa, IV and XVIII

obligately anaerobes

Underrepresented

 

Translational studies for identifying “treatable” biomarkers of risk for CRC using Drosophila

Deciphering the right inflammatory status in the intestine is necessary for designing clinical trials against IBD and maybe CRC. Mucosal healing in IBD patients has shown promise as it correlates with remission of ulcers in Crohn’s disease and erosions and ulcers in ulcerative colitis [7,124]. Transcriptomic studies in humans have led to the identification of genetic and microbial associations with IBD and CRC. Here, we emphasize the use of Drosophila as a whole-animal model to validate the effectiveness, causality and toxicity of identified “treatable” biomarkers in intestinal disease. Examples include the “humanized” Drosophila strains, which are genetically engineered to express human orthologs [2,125]. Notably, tens of chemicals originally selected to target human proteins, such as Rapamycin, BEZ235, SP600125 and DAPT, have been shown to have the same mechanism of action in Drosophila i.e. inhibition of PI3K/mTOR, JNK and γ-secretase/Notch signaling, respectively [125-130]. Thus, accumulating evidence suggests that Drosophila could fill the gap between in vitro and mammalian model host testing (Figure 1).

Figure 1: A virtuous circle of research on CRC prevention. Work on the facilitating hallmarks of cancer, tumor-promoting inflammation and genomic instability, can in addition to unbiased approaches identify genetic and microbial biomarkers linked to risk for CRC. Biomarkers can be further tested and manipulated therapeutically by diet, drug and probiotic administration in flies to assess causality and facilitate clinical trials in humans.

Moreover, flies could be used to examine the association between identified intestinal disease-related microbiota and host. For instance, the commensal microbe of Drosophila Acetobacter pomorum was found to modulate insulin pathway via acetic acid production and subsequently promote ISC proliferation and overall animal growth [131]. Also, a positive correlation between Enterococcus spp. and IBD patients has been also reported. Enterococcus strains that form better biofilms, adhere strongly on intestinal cells and possess antioxidant defense mechanisms are mostly found in IBD patients versus healthy people [132]. Therefore, dissecting host-microbiome interactions of overrepresented in IBD and CRC bacterial strains, such as Bacteroides, Escherichia, Enterococcus and Enterobacter in gnotobiotic flies could give insights regarding bacterial pathogenicity. This is more feasible nowadays due to the increasing number of human microbiota species that we are able to culture and thus test in model organisms, such as flies [133]. Similarly, Drosophila could be used to determine the beneficial impact of bacteria underrepresented in CRC like Clostridia and Lactobacillus.

Accumulated evidence in Drosophila highlights the role of diet in intestinal disease. Nutrient deprivation and reduced insulin pathway correlate with reduced ISC proliferation and number, a phenotype that is reversible upon feeding [134]. Dietary L-glutamate also stimulates intestinal cell proliferation and growth via regulation of Ca2+ signaling [135]. This plasticity of ISC to nutrient availability could be used to target the aberrant proliferation of dysplastic lesions. Given that CRC is a multifactorial disease, a sophisticated combination of probiotic, chemical and dietary interventions might be required to efficiently prevent the disease. In this regard, tumor-initiating inflammation may be successfully targeted by sequestration of regenerative chemokines/cytokines and selective inhibition of signaling molecules that promote tumor survival and growth [25].

Limitations

The use of animal models provides the ability to study the effects of biomarkers of fundamental signaling pathways, microbes and environmental factors and suggest therapeutic interventions against intestinal inflammation and tumorigenesis. A practical limitation of using Drosophila in translational studies on IBD and CRC is the inability to assess the disease promoting properties of human anaerobes that are highly sensitive to the presence of oxygen. An additional limitation of the fly model is the lack of adaptive immunity and the absence of lamina propria in which immune cells reside and infiltrate. Thus, alternative animal hosts such as mouse models should be used to validate and complement the assessment of biomarkers, especially those related to adaptive immunity and highly sensitive to oxygen microbes. Regardless, advantages such as the short lifespan of the fly facilitate assessments of drug-diet-microbial interventions against sporadic intestinal cancer during ageing that is impractical to perform in mice. Thus, Drosophila can be an attractive model host for studying well-conserved genetic, microbial, and environmental components of intestinal homeostasis and disease, the analogous features of which might play a pivotal role in human health.

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