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UNIVERSITY OF MEDICINE AND PHARMACY OF CRAIOVA

PhD Thesis

Abstract

Genetic markers involved in

inflammatory digestive pathology

PhD Tutor Prof. univ dr. Florica Popescu PhD Joint Tutor Prof. univ. dr. Francisc Mixich

PhD Student Elena-Raluca Nicoli

Craiova, 2013

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Investeşte în oameni !

FONDUL SOCIAL EUROPEAN

Programul Operaţional Sectorial Dezvoltarea Resurselor Umane 2007 – 2013

Axa prioritară 1

„Educaţia şi formarea profesională în sprijinul creşterii

economice şi dezvoltării societăţii bazate pe cunoaştere”

Domeniul major de intervenţie 1.5

„Programe doctorale şi postdoctorale în sprijinul cercetării”

Titlul proiectului

"Creşterea calităţii şi vizibilităţii rezultatelor cercetării ştiinţifice a doctoranzilor cu frecvenţă prin acordarea de burse doctorale"

Contract nr: POSDRU/107/1.5/S/82705

Beneficiar

Universitatea de Medicină şi Farmacie din Craiova

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INTRODUCTION

Inflammatory bowel disease (IBD) represents a heavy burden for the patients

and national health systems, due to their onset in adolescence or early adulthood

with potentially debilitating symptoms and chronic and unpredictable course

characterized by an increased risk of developing colorectal cancer (Høivik M. et al,

2012). With all the therapeutic improvements that have been made in the last years,

there are still a surprisingly large number of patients with IBD who have to undergo

surgery due to disease complications (Podolsky D.K., 2002). Thus, any molecular

marker that could be used for early diagnosis and to avoid complications like

colorectal cancer development is beneficial.

Key Words: TPC2, Inflammatory Bowel Diseases, Colorectal Cancer, Autophagy

PART I - STATE OF KNOWLEDGE

Chapter 1. General introduction

Main risk factors influence on IBD epidemiology

IBD represents an important public health problem, as it affects mostly young

adults and is marked by episodes of debilitating relapse and remission with negative

effects on socioeconomic status and quality of life. The epidemiology of IBD is

constantly changing, suggesting that environmental factors including infections, diet,

lifestyle factors, and medication play a major role in modifying disease progression

(Ng Siew C et al., 2013). Generally, incidence rates of ulcerative colitis (UC) and

Crohn’s Disease (CD) vary between 2.2 to 14.3 cases per 100,000 person/years for

UC and from 3.1 to 14.6 cases per 100,000 person/years for CD (Longo D.L., 2012).

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Autophagy – an important pathogenic pathway

Genome-wide association studies identified in their intent to discover the

genetic basis of IBD, beyond genes that modulate the innate immunity, a group of

genes related to autophagy that seem to be associated with increased disease

susceptibility, mainly CD (Nys K. et al., 2013; Van Limbergen J. et al., 2009). The

autophagy genes connected with an increased susceptibility to IBD (CD or UC or

both) identified through several genome-wide association studies are ATG16L1,

IRGM1 and LRRK2. It was demonstrated on several knockout animal models that

autophagy is modulated by LRRK2 activity: an increased activity of the gene tempers

the autophagic flux while reduced activity would amplify it (Gómez-Suaga P. et al.,

2012). Pathology involving LRRK2 defects depends on the cellular enviroment in

which the gene works (Lewis P. A. & Manzoni C., 2012). LRRK2 expression level

and defects influence the number of autophagosome in a calcium-dependent

manner: the calcium chelator BAPTA inhibits their number increase and, genetically

depletion of ER calcium stores blocks LRRK2 effects on autophagosome numbers,

increases the pH of a population of lysosomes and the number of lipid droplets

(Gómez-Suaga P. et al., 2012).

A similar mechanism of action was described in the case of nicotinic acid

adenine dinucleotide phosphate (NAADP), an agonist-generated second

messenger, able to trigger complex calcium signals, initiated from acidic stores and

subsequently amplified by ER calcium release channels (Churchill G. C. et al., 2002;

Patel S. & Docampo R., 2010; Morgan A. J. et al., 2011). It was showed that NAADP

main action is on the endolysosomal two-pore channels TPC1 and TPC2 (Calcraft

P.J. et al., 2009; Brailoiu E. et al., 2009; Patel S. et al., 2011), but it seems that the

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molecule is not directly attached to TPCs, an unidentified associated low-molecular

weight binding protein being responsible for this connection (Lin-Moshier Y. et al.,

2012). TPCs role is to release calcium under NAADP action, a molecule for which

TPC2 presents high and low affinity binding sites, suggesting that TPC2 may be one

of the components of an NAADP-dependent, lysosome-targeted Ca2+ release

channel that is neither activated, nor inhibited by Ca2+ (Zhu M.X. et al., 2009).

Thus, demonstration and definition of TPC2 potential role in the

pathophysiological mechanisms involved in autophagy defective diseases such as

Parkinson’s disease or IBD require more elaborate studies on knockout animal

models associated with the assessment of TPC2 gene expression levels in the

damaged tissues versus healthy ones .

PART II - PERSONAL CONTRIBUTIONS

Chapter 2. TPC2 Mouse Model – preliminary result

The Aim of this chapter was to obtain more information about TPC2 KO and

WT animals that could be used in further studies to show the role played by TPC2 in

pathophysiological mechanisms involved in autophagy defective diseases such as

neurodegenerative diseases, IBD or tumour processes.

Materials and Methods

Animals. All studied animals were TPC2 KO and strain-matched WT

littermates provided by the Parrington/Galione labs (Department of Pharmacology,

University of Oxford). Animals were housed under standard conditions, in solid-

bottomed polycarbonate cages that were individually ventilated, at constant

temperature (22°C), 45-65% relative humidity and a 12:12-h light–dark cycle and

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were provided with irradiated water and food ad libitum. All procedures were carried

out in accordance with the UK Animals (Scientific Procedures) Act, 1986 and

experiments were carried out with UK Home Office approval. This experiment was

conducted on a number of twelve animals from which five were TPC2 KO (two

females and three males) and six TPC2 WT (three males and three females)

animals.

Cell preparation. Single cell suspensions of spleen, lymph nodes (pooled

inguinal) and thymus were accomplished according to standard procedures, were

prepared using DPBS between two glass microscopic slides. FACS staining. The

multicolour antibody cocktails used for labelling the cells of interest were: FITC-

CD69, PE-CD25 (IL2Rα, p55), Alexa Flour-CD335 (NKp46), PerCP-Cy5.5-CD45,

APC-H7-CD19 and Pacific Blue-CD3e.

Flow cytometry. All samples were analysed on a FACSCanto II (BD

Biosciences) that was standardised using BD Cytometer Setup and Tracking beads

and software. Data acquisition was controlled with BD FACS Diva v6 software.

Results and Discussions

Comparison of the two animal model TPC2 (KO and WT) organs between

them was revealed:

in spleen, lymphocytes expression is significant different in cell population of:

T cells especially for CD69 positive, NK cells mostly CD25 positive and B

cells both for CD69 and CD25 positive;

in thymus was revealed significant differences for: T cells expression both for

CD69 and CD25 positive, and B cells expression especially for CD69 positive.

NK cells population presented no differences;

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in lymph nodes was remarked a significant difference in: T cells CD25

positive expression, NK and B cells expression both for CD69 positive and

CD25 positive;

In this study, we have used the previously characterised TPC2 KO and WT

mouse models to assess lymphocytes expression in the spleen, lymph nodes

(pooled inguinal) and thymus of TPC2 KO compared with TPC2 WT animals based

on that isolation of lymphocytes and other hematopoietic-derived cells from tissues

to highlight their phenotype that has become increasingly relevant to immunology

over the last decade (Sheridan, B. S et al, 2012).

Immune assessment of animal models revealed that both lymphocytes and

hematopoietic-derived cells are characterized by significant heterogeneity in

morphology, surface antigen expression, and function (Croitoru K., et al, 1991).

Based on the above preliminary results and comparing with literature data, we

can say that the immune cells expression in TPC2 KO model tissues might be

different compared with TPC2 WT mouse, suggesting that TPC2 could be an

important player in immune response. More studies are needed in order to fully

characterize and understand the immune system of TPC2 KO animals.

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Chapter 3. The role of acidic-store calcium in susceptibility to colitis:

Insights gained from the TPC2 knockout mouse – preliminary results

The aim of this chapter was to assess the response of the TPC2 KO mouse

model to dextran sodium sulphate (DSS)-induced acute colitis compared to wild-type

(TPC2 WT) animals.

Material and methods

Animals. The studied animals were TPC2 KO and strain-matched WT

colonies provided by the Parrington/Galione labs (Department of Pharmacology,

University of Oxford). All animal studies were conducted using protocols approved by

the UK Home Office for the conduct of regulated procedures under licence (Animal

scientific Procedures Act, 1986) and experiments were carried out with UK Home

Office approval under PPL 30/2524. Twenty four animals were included in this study.

For induction of colitis, twelve animals (six TPC2 KO and six TPC2 WT, mixed

groups with equal numbers of males and females), at 6 weeks of age were

administered with 2% DSS in drinking water over the course of one week then

switched to normal drinking water for three days. Twelve animals (six TPC2 KO and

six TPC2 WT, mixed groups with equal numbers of males and females) were also

included in the study as control. Ten days following the first day of DSS

administration, mice were sacrificed by CO2 asphyxiation and tissues were collected

for histopathology and molecular biology.

Drug Administration. Dextran sodium sulphate (DSS, MP Biomedicals,

Catalog no.: 160110, 500g, M.W.=36,000-50,000) was dissolved in water in a

concentration of 2% and administered to animals in drinking water for 7 days starting

on day 0 (D0) ad libitum. All animals were provided the normal or 2% DSS drinking

water ad libitum.

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Assessment of the disease. Throughout the course of the ten days of colitis

induction, mice weight, physical condition, stool consistency, and the presence of

gross and occult blood in the faeces and/or from the anus were examined and

documented daily. Weigh loss of animals was assessed by daily monitoring of body

weight loss compared to D0.

Histopathology Analysis. Evaluation of histological changes and inflammatory

cell infiltration in colon was performed on colon sections fixed in 10% neutral-

buffered formalin. Samples were processed, paraffin-embedded, and thereafter

sectioned using a Leica RM2126 microtome. Tissue sections (4 µm) were stained

with hematoxylin and eosin (H&E). Five sections, 50 mm apart per colon sample

were evaluated and repeated in all mice per group in a blinded manner, and scored.

Sample preparation

Paraffin embedding protocol. All samples were kept in formalin for exactly 7

days before processing and were processed identically following the same protocol.

H&E staining protocol. The staining with hematoxylin (Harrison Hematoxylin,

Sigma UK) was done by incubating the sections directly in hematoxylin a couple of

dips and then rinsed in tap water followed by one or two dips to differentiate in acid

alcohol (1% HCL in 70% ethanol) and 30 seconds in Scott’s tap water. The next step

was the dipping in eosin (water eosin) and a brief rinse under tap water. The

sections were dehydrated though graded alcohol, cleared in Histoclear Solution and

mounted with a permanent mounting medium (DePeX).

Tissue collection. Animals were sacrificed by CO2 asphyxiation followed by

decapitation. Blood was removed by cardiac puncture. Peripheral tissues were

perfused with 0.9% saline at 20ml/minute for 1-2 minutes. Whole liver lobes were

dissected and part was kept in 10% formalin and part snap frozen on isopentane on

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dry ice (approximately -78°C). Intestines were removed for histopathology and

molecular biology and dissected into regions for qPCR.

Tissue collection for histopathology. Weight loss and histology are the most

important ways to assess disease. The colon (starting from 0.5 cm above the anus

up to the cecum), small intestine (starting from above the cecum up to the stomach),

spleen, thymus, and part of the liver were carefully dissected, removed, and

weighted. For histology, sections of mid, distal, proximal colon, ileum, jejunum,

duodenum (~1 cm) as well as cecum and stomach were collected into 10% formalin

(in PBS) and then embedded in paraffin, sectioned (4 μm) and stained with H&E.

Tissue collection for RT-PCR. For RT-PCR; slices (~1 mm) of mid, distal,

proximal colon, cecum, ileum, jejunum, duodenum, stomach and liver, were snap-

frozen in liquid nitrogen and stored at -80°C until extraction of mRNA.

mRNA extraction from intestinal tissue. Intestine specimens (~10 mg of tissue

per sample) were homogenized using a pestle homogenizer in lysis buffer (Buffer

RLT, Qiagen) with 1% β-MCE (mercaptoethanol, Sigma, UK), and total RNA

extraction was performed according to the manufacturer instructions

(QiagenRNEasy Mini Kit).

cDNA synthesis from intestinal tissue. To determine the mRNA expression in

the mouse colonic tissue, quantitative real-time reverse-transcription polymerase

chain reaction (qRT-PCR) was performed using High Capacity cDNA Reverse

Transcription Kit from Applied Biosystems, UK.

qRT-PCR from intestinal tissue. Quantification of the different cytokines and

genes for mRNA expression in colon tissues was assessed by qRT-PCR with

specific primers for each gene (IL1β, IL6, TNFα, TLR4, TGFβ, Caveolin, ICAM,

VCAM, β-Actin). The expression level of each transcript within the different samples

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was quantified as the relative value against β-Actin. The primers used in qRT-PCR

reactions were purchased Roche, UK.

Results and Discussions

Daily measurements during the experiment. Daily body weight measurements

revealed that DSS-induced colitis was associated with a higher weight loss in the

group TPC2 KO animals compared with TPC2 WT group (***p<0.0001).

Measurements of organs parameters. Analysing the colon weight/length ratio,

we observed an extremely significant differences between the two groups: TPC2 KO

and WT, whilst in small intestine, this ratio was not significantly different between the

two groups. The weight/length ratio recorded in spleen showed also no significant

differences between the two genotypes.

Histopathological assessment of colonic sections:

At the end of the experiment, no histological differences were found between

WT and KO control groups (on water). However, when comparing the DSS groups

with their control groups, histological assessment revealed important differences in

the mid and distal colon of WT and KO groups as follows:

for TPC2 WT group receiving DSS, a moderate hyperplasia associated with

mild goblet cell depletion and low epithelial cells loss was noticed. Also, the

area of affected tissue was up to 25% coverage, showing a gentle infiltration

with leukocytes in lamina propria;

the TPC2 KO group receiving DSS has shown unrelenting depletion of the

goblet cell accompanied by a substantial hyperplasia and epithelial cells loss.

Also, several histological features of severe inflammation were observed

including loss of crypt structure, oedema, and crypt abscesses associated

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with stern submucosal inflammation. More than 50% of the colonic tissue was

affected, having almost no structural architecture.

Histolopathology scoring. Histopathology scores of proximal colon were not

significantly different between the two genotypes. Due to measurements of length

and weight before processing the sections for histology (embedding in paraffin

blocks and staining with H&E), a lot of the H&E sections have shown processing

artefacts and hence not all the sections from each animal have been scored.

Expression analysis of inflammatory gene markers mRNAs in TPC2 animals

colonic sections after induction of colitis:

the studied model of colitis induced by DSS in TPC2 animals is suggesting a

possible confirmation of the histopathology results, showing a relative

increased percentage fold changes of IL-6 and IL-1β mRNA expression.

Assessment of IL-6 and IL-1β expression levels in both TPC2 models, WT

and KO, revealed a significant difference between TPC2 KO receiving DSS

compared with TPC2 KO on water and Wild-type on water or DSS;

the TPC2 group receiving DSS showed a significant increase in relative

percentage fold change of Caveolin and VCAM mRNA expression when

comparing between KO and WT animals;

relative percentage fold change of TLR4 mRNA expression in TPC2 KO group

treated with DSS was significantly higher compared to TPC2 KO and also

Wild-type groups receiving water (*p = 0.0189);

relative percentage fold change of TNF-α mRNA expression in the Wild-type

group treated with DSS was very significantly higher than both in the TPC2

KO receiving water and the Wild-type group on water.

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assessment of TGF-β and ICAM expression levels in both TPC2 groups (WT

and KO) revealed no significant difference.

We have assessed the response of the previously characterized TPC2 KO

and WT mouse models (Chapter 2) to DSS-induced acute colitis. We have also tried

to evaluate if their response to DSS is associated with altered gene expression

levels of some inflammatory gene markers such as IL-6, IL-1β, Caveolin, VCAM,

ICAM, TNF-α, TLR-4 or TGF-β that have previously shown to be implicated in IBD

pathogenesis (Carta, S. et al, 2013).

DSS is the most commonly polysaccharide used to induce colitis in animal

models in the study of chronic gastrointestinal inflammation pathogenesis and

preclinical studies (Neurath, M. et al, 2000; Wirtz, S., & Neurath, M. F. 2007; Wirtz,

S. et al, 2007). Mice are given cycles of DSS added to their drinking water resulting

in the development of an acute or chronic intestinal inflammation. While chronic DSS

colitis is especially driven by T lymphocytes, acute DSS colitis can occur in the

absence of T cells (Wirtz S. et al 2007). Several studies showed that genetic factors

modulate susceptibility and responsiveness to DSS-induced colitis in animal models,

like the interleukin-10 deficient mouse model or C3H mouse model (Wirtz S. et al

2007; Berg DJ, et al, 2002; Perše, M. et al 2012; Mähler, M. et al 1998). Several

studies carried out between1999–2005 tried to show the involvement of genetic

factors in regulations of inflammatory response in mouse models, which represents

an important direction for future research (Stevceva, L. et al, 1999; Kitajima, S. et al,

2001; Melgar, S. et al, 2005)

Based on the above preliminary results and comparing them with literature

data, we can conclude that the TPC2 KO mouse model is more susceptible to DSS

induced colitis compared with TPC2 WT.

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Chapter 4. Determination of TPCN Gene Expression in Human Colorectal

Cancer Samples

Main objectives of this chapter was to quantify the expression of total TPCN1

and TPCN2 mRNA expression level in a Romanian cohort of patients diagnosed with

colorectal cancer (CRC), for further analysis to see whether there might be a

correlation between the gene expression and clinicopathological features of the

tumours in Romanian population.

Material and Methods

Patients and samples. 25 patients with colorectal tumour who had undergone

surgery at the County Clinical Emergency Hospital of Craiova, Romania between

2008 and 2012 were included. The Ethics Committee of University of Medicine and

Pharmacy of Craiova, Romania approved this study and written informed consent

was obtained from all subjects. The age of the patients included in the study was

64.24 ± 10.81 years (mean+/- sem), with a sex ratio of 14:11 (m:f).Both tumour (T)

and peritumour (PT) corresponding to large intestine mucosa were harvested during

surgery for all the patients. The samples were collected in RNAlater solution

(Ambion) and stored at -80°C until RNA isolation. The representative steps are

presented in next figure (Fig. 4.1).

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Sample collection in RNALater

Store at -80°C

Histopathology

Total RNA isolation

Evaluation of RNA concentration, purity, and quality

Reverse transcription of total RNA in cDNA

qRT-PCR

Analysis of results using 2-ΔΔC

Statistics: GraphPad Prism 5, GraphPad InStat, GenexPro 4.4.2.308©

Fig. 4.1 Study diagram

Results and Discussions

The expression of total TPCN1 and TPCN2 was assessed in 25 paired

samples of CRC tumours and adjacent tissue.

Comparative TPCN expression in paired tumour and non-malignant

peritumour of colorectal mucosa. The cycle threshold (Cts) for the target genes was

normalised to the GAPDH housekeeping gene Cts and the results are presented as

relative mRNA expression.

The fold difference between paired samples, calculated using the 2-ΔΔCt

method was considered biologically significant when the mRNA fold change was

over 1.8 (>1.8 fold change in expression in tumour, <0.55 fold change in expression

in peritumour mucosa, 0.55-1.8 - irrelevant difference between tumour and adjacent

mucosa).

Total TPCN1 showed increased expression in non-invaded peritumour

samples compared with paired tumour tissue in 17 cases (68%); in 3 (12%) cases

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the expression was higher in tumour tissue and in the remaining 5 cases (20%) the

difference between paired samples was biologically irrelevant.

The total TPCN2 expression was increased in non-invaded peritumour

samples compared with paired tumour tissue in 15 cases (60%); in 3 (12%) cases

the expression was higher in tumour tissue and in the remaining 7 cases (28%) the

difference between paired samples was biologically irrelevant.

The expression of total TPCN1 and total TPCN2 were significantly

(statistically) higher in non-invaded peritumour tissue compared with tumour tissue

(p=0.005 and p=0.001 respectively, Wilcoxon matched-pairs signed rank test).

The expression for the TPCN genes (TPCN1 and TPCN2) was extremely

significant in each individual sample (tumour or peritumour tissue) (p<0.0001,

Wilcoxon Signed Rank Test).

Gene expression analysis in relation to tumour pathological features.

The analysis of TPCN1 and TPCN2 expression according to tumour location

revealed that mRNA levels tend to be lower in rectosigmoid colon compared with

ascending and descending colon reaching a statistically significant different level

(p=0.0210, p=0.0323 respectively, Kruskal-Wallis test).

When assessed according to tumour Dukes stage (A, B, C, D), total TPCN1

and TPCN2 showed a similar expression pattern. Even though there’s a tendency

towards a higher mRNA levels in the first two stages (stage A+B), however, this

difference was not statistically significant (NS), (p=0.26, p=0.47 respectively,

Kruskal-Wallis test). Regarding the assessment according to tumour stage (I, II, III,

IV), total TPCN1 and TPCN2 showed a similar expression pattern. The mRNA levels

were similar in all four tumour stages, the difference was not statistically significant

(NS, Kruskal-Wallis test).

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In the majority of the cases, both TPCN1 and TPCN2 levels were similar

between well (G1), moderate (G2) and poor (G3) tumours (p=0.74, p=0.55

respectively, Kruskal-Wallis test). In two G3, two G2 and one G1 tumours TPCN1

and TPCN2 levels were biologically higher compared with the rest of the tumours but

this was not representative for the entire group.

Correlations between genes in tumour and peritumour mucosa. A very strong

correlation between TPCN1 and TPCN2 expression in both tumour (r=0.94,

p<0.0001) and peritumour tissue (r=0.97, p<0.0001) was identified. In both tumour

and peritumour tissue, Spearman coefficient also highlights an aggregation of

TPCN1 and TPCN2 mRNA levels (r=0.60, p<0.004 for tumor and r=0.64, p<0.002 for

peritumour tissue).

The role of TPCs to discrete acidic calcium stores as in the case of TPC2 for

lysosomes and of TPC1 and TPC3 for endosomes has been confirmed (Zhu, M.X. et

al 2010).

Calcium is important for maintaining normal trafficking, vesicular fusion events

and recycling within the endolysosomal system (Lloyd-Evans and Platt, 2011). Via

two-pore channel family (TPC) lysosomal calcium can be released by intracellular

calcium-releasing second messenger nicotinic acid adenine dinucleotide phosphate

(NAADP) (Churchill et al., 2002).

Our results on human colorectal cancer subjects show a higher expression of

total TPCN1 and TPCN2 in non-invaded peritumour tissue compared with tumour

tissue. Multivariate analyses showed that the studied TPCs gene expression level in

mucosa compared with tumour, could be a prognostic parameter independent of the

clinicopathological factors with regard to a possible risk to colorectal cancer. These

findings would require a further research to answer some other key questions that

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need to be answered about the involvement of TPCs in carcinogenesis. More of

these systematic research will probably reveal new and exciting targets for cancer

therapy.

Chapter 5. Determination of an autophagy polymorphism in the Human

Colorectal Cancer Samples

The purpose of this chapter was to collect and genotype a Romanian cohort

of patients diagnosed with CRC for ATG16L1 polymorphism. Once this was

accomplished, the final goal was assessing whether ATG16L1 T300A (+898A>G)

influences the risk of CRC in Romanian population. This polymorphism was selected

on the basis of functional data relating to its potential role in autophagy and

carcinogenesis, particularly within the gastrointestinal tract (Deretic, V. et al, 2013;

Gillen, C. D., et al, 1994; Bolan, C. R. et al, 2010)

Material and Methods

Patients and samples. A total of 351 Romanian subjects (109 patients

diagnosed with sporadic CRC and 242 healthy controls) were included in this study.

Matched control (healthy) subjects of the same ethnic and geographical origins were

recruited among unrelated volunteers admitted at the same time to the same

hospital, with no previous history of tumour, inflammatory or infectious disease.

Blood samples were obtained from both groups. The Ethics Committee of University

of Medicine and Pharmacy of Craiova, Romania approved this study and written

informed consent was obtained from all subjects.

DNA extraction and genotyping. Genomic DNA was extracted from the

peripheral blood leukocytes using Wizard® Genomic DNA Purification Kit (Promega,

Madison, WI, USA), following the manufacturer’s protocol. Polymorphism genotyping

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was performed with a predesigned TaqMan assay from Applied Biosystems (Foster

City, CA, USA): ATG16L1 T300A (+898A>G, rs2241880, assay C_9095577_20).

Results and Discussions

Subjects characteristics. Genotyping was performed in 109 CRC patients and

242 controls. There were no differences in median age and gender between cases

and controls.

Genotype frequency and the risk of CRC in Romanian population. A

significant association was observed for male subjects carrying GG genotype that

were at a higher risk for CRC (OR 2.76, 95% CI: 1.12-6.79, p = 0.019) when

compared with the more frequent AA genotype. Furthermore, there was no

correlation between CRC cases and controls for female subjects carrying GG

genotype (OR 1.19, 95% CI: 0.445-3.2, p =0.722) or AG genotype (OR 1.26, 95% CI:

0.51-3.15, p =0.607).

Association of ATG16L1 T300A (+898A>G) polymorphism with Dukes' stage

and histological subtype was examined separately (A, B, C, D). In a stratified

analysis, the only association between CRC and autophagy polymorphism was

found for carriers of GG allele and was restricted to moderately and poorly

differentiated cases (OR 5.15, 95% CI: 1.47-18.06, p=0.005). No significant

differences were observed between tumour stage (A + B), localisation or histological

grading and controls in stratified analysis.

Autophagy is a catabolic intracellular process in which cytoplasmic

components are sequestered within vesicles and delivered to the lysosomes. It is the

pathway used by cells during nutrient starvation to break down non-vital components

and use them as nutrients (Naser, S. A et al, 2012). Autophagy is involved in

infectious processes by clearing the cells of foreign antigens released from the

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breakdown of the pathogen (Naser, S. A et al, 2012). This pathway can also be

implemented as a repair mechanism to degrade damaged organelles and proteins.

Autophagy is a complex pathway, modulated by several molecular mechanisms that

remain elusive. There have been approximately 30 autophagy-related (Atg) genes

identified, with 2 proteins having ubiquitin-like characteristics, Atg12 and Atg8 (Read,

S. et al, 2000; Mehrpour, M. et al, 2012). The ATG16L1 protein is expressed in the

colon, small intestine, intestinal epithelial cells, leukocytes, and spleen (Fujita, N. et

al 2008). In 2010, a study has shown that an ATG16L mutation: ATG16L1 -

Thr300Ala (rs2241880), located on chromosome 2, is associated with the onset of

ileal CD, and is therefore a key molecule in elucidating the genetic aspects of this

disease (Sventoraityte, J. et al, 2010).

A second report from the North American CD genome-wide study also shows

an association with ATG16L using a case-control analysis in 988 CD patients and

1007 controls (Rioux, J. D. et al, 2007). Taking into account that patients with IBD,

especially CD, have a six-fold higher risk of developing colorectal cancer, several

studies investigating the link between autophagy genes, and susceptibility to this

type of cancer, were conducted (Brest P. et. al., 2010). Thus, a study conducted by

Kang M.R. and colleagues in 2009, indicates that frameshift mutations in ATG genes

with mononucleotide repeats appear in gastric and colorectal carcinomas suggesting

that these mutations may contribute to cancer development by deregulating the

autophagy process (Kang M.R. et al., 2009). In our hospital-based case-control

study, we assessed whether an autophagy polymorphism influenced sporadic CRC

development and progression in Romanian population.

It is necessary to study also if there is a link between ATG16L1 gene

expression, CD course and colorectal cancer predisposition. It is clear that variants

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of this gene have been proven to be directly associated with CD because autophagy

plays a critical role in disease pathogenesis. Further research needs to focus on

understanding how ATG16L1 variants contribute to disease susceptibility in IBD

patients, and their possible therapeutic implications, and, allows the link between the

ATG16L1 variants in IBD and colorectal cancer.

In this study, it was found that ATG16L1 T300A (+898A>G) polymorphism

influences the risk of CRC in Romanian population. The male carriers of GG

genotype are at a higher risk for CRC. While this study indicates a strong association

in the moderately and poorly differentiated CRC cases, the small subgroups size

ousts drawing conclusions. Further research is required in different ethnic

populations to improve the level of knowledge of autophagy in colon cancer

tumorigenesis.

Final conclusions

Our results showed that between TPC2 KO and WT model there are

significant differences regarding expression of immune surface antigens in

lymphocyte population from spleen, thymus and lymph nodes.

Also, we identified that TPC2 KO mouse model might be more susceptible to

DSS-induced colitis and develops more severe lesions after DSS

administration than the WT model.

Several genes involved in inflammation seem to have a higher expression

level in TPC2 KO mice compared with TPC2 WT model after DSS ingestion.

These results are important for sustaining future experimental studies,

especially on TPC2 KO model, in order to improve management and therapy

22

of chronic inflammatory diseases, such as Crohn’s disease, ulcerative colitis

or colorectal cancer.

TPCN1 and TPCN2 gene expression level in non-invaded peritumour tissue is

higher compared with tumour tissue indicating that TPCN1 and TPCN2 may

serve as a useful new marker for the study of colorectal cancer.

ATG16L1 T300A (+898A>G) polymorphism influences the risk of CRC in

Romanian population; the male carriers of GG genotype being at a higher risk

for CRC.

Further research is required in different ethnic populations to improve the level

of knowledge of autophagy in colon cancer tumorigenesis and to show if

exists a strong link between TPCN, autophagy and development of chronic

inflammatory lesion like IBD or CRC.

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