Chapter IV : General discussion and perspectives

Chapter IV : General discussion and perspectives

Chapitre IV. General discussion and perspectives

The aim of this thesis was to study the transcriptome of thyroid carcinomas in order to give a better understanding of the molecular mechanisms that lead to the development and progression of these tumors.

Hybridization of 26 papillary thyroid carcinomas (PTCs) on Agilent microarray slides enabled us to generate tremendous amounts of data which were used to find signatures between different subtypes of PTCs. Interests of these signatures are multiple, and include correlation between the molecular and biological phenotypes, leading to a better understanding of the subtypes of these tumors, identification of potential therapeutic targets specific to a variant and discovery of new diagnostic or prognostic markers. The first question we asked was to know if it was possible to separate radio-induced and sporadic PTCs. We found that on a global scale, transcriptomes of sporadic and post- Chernobyl PTCs were similar, which confirmed a previous study of our laboratory based on a smaller number of genes and samples

. Nevertheless, although both types of tumors represent the same disease, we found that they could be accurately separated, with a more subtle analysis, using a multi-genes signature. Two other independent gene expression signatures were also able to separate post-Chernobyl and sporadic PTCs, one related to their presumed respective etiological agents (radiation vs. H

O

), the other related to the homologous recombination DNA repair mechanism. We interpreted these results as evidence for different and detectable cancer susceptibility factors between both types of tumors. Different additional experiments should be done to give weight to this hypothesis. Indeed, the susceptibility signature is weak, maybe due to the fact that each tumor is compared to its adjacent tissue, which could attenuate the signal. Therefore, direct comparison of post-Chernobyl and sporadic PTCs on Affymetrix slides could lead to a stronger signature. We have already performed these experiments and analyses are still ongoing. In addition, our study does not formally rule out potential confounders, such as age or ethnic factors which are clearly different between both types of tumors.

Thanks to our collaboration with the Chernobyl Tissue Bank, our laboratory has planned

to collect thyroid tumors from young Ukrainian patients born after the Chernobyl

explosion and to compare them with the radio-induced tumors. Finally, our potential radiation signature should normally also be found in healthy tissues. Therefore, our laboratory is trying to obtain blood from Ukrainian patients with radio-induced or sporadic PTC in order to see if the susceptibility signature is present in these cells as well.

In the long-term, a radiation susceptibility test could be set up.

Microarray analyses also enabled to identify a signature composed of 54 genes associated to the classical variant of PTCs. Analysis of this gene list by the DAVID software enabled us to relate, at least partially, this signature to the most important remodeling of the classical subtype compared to the other histological variants. This includes the uPA system (uPAR and uPA) which was overexpressed in the classical variant at the mRNA level. Confirmation of their regulation at protein level should allow to confirm the major role of this system in the remodeling of PTCs. A weakness of this study is the imbalance between the classical (n=17) and the other variants of PTC (n=9). Additional gene expression profiles of non-classical variants of PTCs could lead to a stronger signature, possibly with more genes.

RET/PTC rearrangements and activating BRAF mutations are the two most common

genetic alterations in PTCs. They both lead to the constitutive activation of the MAPK

ERK1/2 signaling pathway, considered as the primary event leading to papillary

carcinogenesis. Nevertheless, while BRAF only activates the ERK1/2 cascade, receptors

tyrosine kinase like RET are able to activate other cascades like the PI3K signaling

pathway. Therefore we would expect, at least partially, a different molecular profile

between tumors harboring the BRAF mutation and those harboring RET/PTC

rearrangements. Nevertheless, we did not succeed to find a signature between both types

of tumors with a supervised method (SAM) using Agilent slides. In accordance with our

data, Frattini et al showed that PTCs with RET/PTC or BRAF mutation were associated

with a similar gene expression profile

. Nevertheless, they showed subtle differences

existing between both types of tumors using supervised methods. These small differences

between their and our results could be explained by sample size, different DNA

microarray platforms and genetic or environmental differences in their Italian patients

compared to our French and post-Chernobyl patients. More surprisingly, Giordano et

harboring the different gene alteration (including RAS mutation) based on their global gene expression. This large discrepancy between Frattini and our results, compared to those of Giordano is difficult to explain. We postulated that these discordant results occured from the fact that Frattini et al and we used dual channel microarrays while Giordano et al used the one channel Affymetrix technology. Consequently, Giordano et al directly compared tumors without substraction of the adjacent tissue, which was not the case in the two other studies. Nevertheless, our last study of PTCs analyzed on Affymetrix slides did not support this hypothesis. Explanation of this large discrepancy still remains unknown.

Deriving a reference gene list from our study on Agilent microarray slides and two other studies

enabled us to identify many genes consistently regulated through PTCs compared to their adjacent tissue. This gene list was based on 50 tumors derived from patients of different ages presenting various histological variants and genetic alterations, and therefore constitutes the common molecular phenotype of the majority of PTCs.

Analysis of this regulated gene list with statistical tools led to several conclusions, including the identification of genes related to the lymphocytic infiltration of these tumors, a general uncompensated downregulation of immediate early genes and a correlation between the molecular phenotype and the probable collective migration mode of PTCs.

We also observed a regulation of many ECM proteins, proteases and proteases inhibitors consistent with the important remodeling of these tumors. Moreover, identification of a signature with a larger overexpression of proteases in the classical subtype compared to the other variants adds weight to the involvement of these genes in the remodeling.

We also investigated the regulation of the three major MAPK signaling pathways

ERK1/2, p38 and JNK. Contrary to the ERK1/2 and p38 cascades, the JNK was

statistically differentially activated by overexpression of their components. This result is

in accordance with a previously published immunohistochemistry study

. The JNK

cascade is commonly activated by cytokines and we could therefore imagine that the

general overexpression of cytokines as observed in our gene list leads to activation of this

pathway. Nevertheless, its role in tumor development remains controversial

. A role for

the JNK pathway in tumorigenesis is supported by the high levels of JNK activity found in several cancer cell lines

. Moreover, it has been shown that oncogenic Raf and JNK cooperate to induce massive hyperplasia in the eye of a Drosophila model

. In humans, a role for JNK in tumorigenesis has been reported in liver, where JNK was shown to promote chemically induced hepatocarcinogenesis

. This pro-oncogenic role of JNK may be related to its own ability to promote proliferation. In contrast, other studies have linked JNK to tumor suppression

. One mechanism of tumor suppression is mediated by a role for JNK in tumor surveillance by the immune system

. Tumor suppression may also be mediated by the pro-apoptotic effects of JNK activation

. Together, these data suggest that JNK may play a context-dependent role in tumorigenesis. In PTC, the role of the JNK pathway activation is not yet investigated and the study of this cascade in thyroid cell cultures stimulated by EGF/serum, a treatment that mimic papillary thyroid carcinogenesis

, could therefore lead to a better understanding of this signaling pathway in PTC.

Analysis of the regulated gene list also showed a statistically significant overexpression of the components involved in the EGF signaling pathway, among which several EGF ligands, which can supplement the genetic alterations commonly found in PTCs for the activation of the ERK1/2 cascade. Interestingly, while genetic alterations activate this cascade only in cells harboring these mutations, the EGF ligands can potentially also induce proliferation of neighbouring cells, by paracrine stimulations. This could explain the reported heterogeneity of some PTC, where only a small subset of cells harbor RET/PTC rearrangement

. Hypothesis of paracrine stimulations (EGF or other) from rearranged cells inducing proliferation of adjacent, non-rearranged thyrocytes, was therefore tested in vitro. Unfortunately, our results were negative, although many reasons can explain this, as discussed in §V of the results chapter. Nevertheless, activation of the EGF signaling cascade is also present in RET/PTC rearranged mice (Burniat et al, in preparation) and its role in papillary thyroid carcinogenesis should therefore be investigated in this model.

During the last year of the thesis, we decided to assess the gene expression profiles of the

very aggressive anaplastic thyroid carcinomas, and to compare them with the ones of

types of tumors. A muldidimensional scaling (MDS) analysis has shown that ATC displayed a gene expression profile intermediate between PTCs and thyroid cancer cell lines, suggesting a trend to dedifferentiation and higher proliferation rate from PTCs to cell lines. Because the MDS was performed on a global scale, this suggests that the major differences between ATCs and PTCs are related to dedifferentiation and proliferation.

Nevertheless, the genes differentially expressed between both types of cancers have to be further analyzed with statistical tools to have more insight into the differences in their physiopathology. The different processes and signaling pathways that have been identified as regulated in PTCs compared to adjacent tissues should be analyzed in ATCs.

Because no other large scale investigations have been realized so far for ATCs, this study would be the starting point for a better understanding of this very aggressive thyroid tumor. This work is currently ongoing in the laboratory.

To conclude, this thesis can be considered as an initiating work trying to understand the

molecular mechanisms that regulate thyroid carcinogenesis, and more specifically the

physiopathology of PTCs. The strength of our study is that our analyses are based on in

vivo data, which generated many hypotheses about the initiation, the progression, the

aggressiveness and the invasion of PTCs. Nevertheless, in vitro studies are now required

to validate these hypotheses and to go further in the understanding of the mechanisms

involved.

Chapter V : Material and methods

Chapter V. Material and methods

Note: In this section, we describe the material and methods related to results from sections II, V, VI, and VII of the results chapter. The remaining material and methods are described in the 2 published papers, in section III and IV.

I. Material

I.1 Cell lines

- KAT 10: human thyroid cell line derived from a papillary thyroid carcinoma - FTC 13342: human thyroid cell line derived from a follicular thyroid carcinoma - BCPAP: human thyroid cell line derived from a papillary thyroid carcinoma and

containing an activating mutation of BRAF

- TPC1: human thyroid cancer cell line derived from a papillary thyroid carcinoma and containing the RET/PTC1 rearrangement

- PCCL3: differentiated rat thyroid cell line

I.2 Culture mediums

3H medium:

Coon’s modified Ham’s F12 1µg/ml insulin

5µg/ml transferrin

1mU/ml TSH

1% fungizone

2% penicilin/streptomycin 5% FBS

2H medium: 3H medium without TSH Quiescence medium:

Coon’s modified Ham’s F12 5µg/ml transferrin

1% fungizone

2% penicilin/streptomycin 0,05% BSA

LB agar dishes:

LB agar

Ampicillin (100 µg/ml)

I.3 Solutions

I.3.1 Protein extraction and quantification Lysis buffer:

Tris-HCl 0,06M pH 6,8 SDS 2%

DTT 100 mM Glycerol 10%

Phosphatases inhibitors: sodium vanadate 100 µM and NaF 20 mM

Proteases inhibitors: Pefabloc 100 µg/ml and leupeptine 5µg/ml

Staining solution:

0,5% coomassie blue G 7% acetic acid 100%

Destaining solution:

7% acetic acid 100%

Extraction solution:

methanol 66%

NH

OH 1%

I.3.2 Western blotting Separating gel:

poly-acrylamide 6,5%

Tris-HCl 375 mM pH 8,8 SDS 0,1%

Ammonium persulfate (APS) 0,05%

Temed 0,05%

Stacking gel:

poly-acrylamide 4%

Tris-HCl 125 mM pH 6,8 SDS 0,1%

Ammonium persulfate (APS) 0,05%

Temed 0,2%

Laëmmli buffer 1×:

Tris-HCl 0,06 M pH 6,2 SDS 2%

DTT 100 mM Glycerol 10%

bromophenol blue 0,1%

Electrophoresis buffer 1×:

Tris 25 mM Glycine 192 mM SDS 0,1%

Transfer buffer:

Tris 20 mM Glycine 154 mM Methanol 20%

PBS 1×:

NaCl 150 mM KH

PO

2mM Na

HPO

8mM KCl 3 mM pH 7,4 TBST:

Tris 1%

NaCl 150 mM

Tween 0,05%

Antibodies used:

Primary antibody:

- FAK

(BioSource, Paisley, UK): dilution 1:1000 in TBST 1×

- FAK

(BioSource, Paisley, UK): dilution 1:1000 in TBST 1×

- α-tubulin (NeoMarkers, Fremont, CA) : dilution 1:500 in TBST 1×

- β-actin (Sigma, St Louis, MO) : dilution 1:1000 in TBST 1×

Secondary antibody:

- mouse IgG antibody (Amersham, Buckinghamshire, UK): dilution 1:10000 in TBST 1×

- rabbit IgG antibody (Amersham, Buckinghamshire, UK): dilution 1:10000 in TBST 1×

I.3.3 Silver nitrate staining Fixator 1:

Methanol 50% (v/v) Acetic Acid 25% (v/v) Fixator 2:

Glutaraldehyde 10% (v/v) Washing solution:

Ethanol 10% (v/v) Acetic Acid 5% (v/v) Oxydation solution:

K

Cr

O

0.1% (w/v)

Nitric Acid 0.015% (v/v)

Silver Nitrate staining AgNO

0.2% (w/v) Developing solution

Na

CO

3% (w/v)

Formaldehyde 0.02% (v/v) Stopping solution

Acetic Acid 3% (v/v) Glycerol 2% (v/v)

II. Methods

II.1 Proteins manipulations

II.1.1 Extraction and quantification of proteins

Thyroid tissues were pulverized in powder with teflon glass in 200 µl of lysis buffer.

After homogenization, the solution was incubated at 100°C during 5 minutes and stored until use.

To quantify proteins, we used the spectrophotometric technique to assess our proteins concentration compared to a standard curve. 4 µl of proteins were deposited on squares of 1,5× 1,5 cm

on Wattman paper in parallel with 4 µl of blank and 8 standard solutions of BSA (respectively 1, 2, 3, 4, 5, 6, 8 and 12 µg of proteins). The Wattman paper was dried, and then successively incubated in methanol during 30 seconds to eliminate all substances interfering with the assay, in the staining solution during 30 minutes at RT, then finally in the destaining solution 3 times during 30 minutes. The Wattman paper was dried, squares were cut and placed in eppendorf tubes in 1 ml of extraction solution.

Tubes were vortexed twice at 5 minutes intervals. 300 µl of each tube were taken and

used to measure the optical density at 620 nm. The optical densities of the standards were

plotted on a graphic in function of their respective concentration and the concentration of the proteins of our samples was deduced.

II.1.2 Western blotting

10 µg of proteins dissolved in Laëmmli 1× buffer were loaded in a 4% poly-acrylamide stacking gel in parallel with 5 µl of ladder (Precision Plus Protein

Dual Color Standards, Bio-Rad, Hercules, CA). A 25 mA current was applied during 1 hour. First, samples migrated in the stacking gel to concentrate them. Then, they entered in the 6,5%

separating gel, enabling the separation of proteins according to their size. Proteins were transferred on a nitrocellulose membrane by application of a current with a constant voltage (100 V) during 1h30.

The blocking of the nitrocellulose membrane was realized with incubation in TBST 1×

containing 5% of milk powder during 1h at RT. Incubation with the primary antibody was then performed at 4°C overnight. The membrane was washed 3 times in TBST 1×

during 10 minutes before incubation with the secondary antibody during 45 minutes.

After 3 washes in TBST 1× during 10 minutes, an ECL solution (Enhanced Chemiluminescence, Perkin Elmer, Waltham, MA) was applied on the membrane during one minute. The membrane was afterwards exposed to a film (hyperfilm, Amersham, Buckinghamshire, UK) during an adequate time to obtain an interpretable signal (1 to 20 minutes).

II.1.3 Silver Nitrate staining

This staining was done after the transfer of proteins on a nitrocellulose membrane.

Consequently, only a few proteins remained on the separating gel but this was sufficient to visualize them.

All the incubations were done under agitation. After transfer of the main proteins on the

nitrocellulose membrane, the separating gel was incubated overnight and a second time

during 30 minutes with 200 ml of the fixator 1 solution. This solution was then replaced

by 200 ml of the fixator 2 solution during 30 minutes. Two washes were realized during

15 minutes before immersing the separating gel in 200 ml of the oxidant solution during 12 minutes. The staining was then performed in the presence of 200 ml of the Silver nitrate solution during 18 minutes. After a fast wash with water (30 seconds), 200 ml of the developing solution were used to detect the proteins. When the proteins clearly appeared, the stopping solution (200 ml) was added for 5 minutes. The gel was finally dried and stored.

II.2 Cell lines manipulations

II.2.1 Trypsinisation

Cells were trypsinized when reaching 80-90% confluence. Dishes were washed with 5 ml of BME medium and incubated during 5 minutes at 37°C with 5 ml of trypsine-EDTA 0,25%. The solution was transferred in a Falcon tube with a medium containing 5% of serum to neutralize the trypsine. The tube was centrifuged 5 minutes at 1200 rpm and the pellet of cells was resuspended in culture medium. The cells were counted with a hemocytometer before seeding.

II.2.2 Bromodeoxyuridine staining and indirect immunofluorescence

Bromodeoxyuridine (BrdU) is an analog of thymidine which substitutes for thymidine during DNA replication. BrdU can then be detected by immunofluorescence to calculate the number of cells entered in the S phase of the cell cycle.

10 µl of BrdU (Sigma, St Louis, MO) and 5 µl of fluorodeoxyuridine (FdU) (Sigma, St Louis, MO) were added to 6 cm

dishes containing PCCL3 cells (final concentrations: 10

4

M for BrdU and 2×10

M for FdU). FdU inhibits the endogene thymidine synthesis.

Note that the washes described below were done at room temperature (RT) during 5

minutes with PBS 1×/ BSA 0,05%. The antibodies, the sheep serum, the streptavidin and

the propidium iodide were diluted in PBS 1×/ BSA 0,05%.

After an incubation of 24h with BrdU and FdU, the medium was removed and cells were fixed by adding 2 ml of methanol during 10 minutes at -20°C. The next incubations were done at RT under agitation. In order to enable the entrance of molecules required for the staining (antibodies, streptavidine coupled with a fluorophore), cells were incubated with 2 ml of triton 0,1% / PBS 1× during 10 minutes. The nuclear proteins were destroyed by incubation with 2 ml of HCl 2N during 10 minutes. HCl was then neutralized with 2 ml of borax (0,1 M Na

B

O

.10H

O pH 8,5) and cells were washed 3 times. 120 µl of sheep serum (dilution 1:20; Dako, Glostrup, Denmark) were deposited on the cells during 30 minutes. Cells were then incubated during 2h with a mouse antibody directed against BrdU (dilution 1:25; Becton Dickinson, Franklin lakes, NJ). Then, they were successively incubated with a biotinylated anti-mouse immunoglobulin antibody (dilution 1:200;

Amersham, Buckinghamshire, UK) during 1h, washed 3 times during 10 minutes, incubated with fluorescein-conjugated streptavidin (dilution 1:50; Amersham, Buckinghamshire, UK) during 1h and washed again 3 times. Finally, cells were incubated 5 minutes in the dark with propidium iodide (dilution 1:100) to color the nuclei before being washed with water and dried in the dark overnight. A fluorescence microscope was used to calculate the proportion of cells having incorporated BrdU.

II.3 Microarray analyses

II.3.1 Experiments on Agilent cDNA Microarray slides

RNA purification, amplification, cDNA synthesis and labeling were performed as

described in the supplementary information of the paper described in §III of the results

chapter. All the tumor/non-tumor tissue pairs (n=26) were hybridized according to the

manufacturer’s protocol on Human 1 cDNA microarray (Agilent Technologies, Palo Alto,

CA) containing 12000 cDNAs, covering 8000 genes. The microarrays were scanned with

a Genepix 4000B scanner. All the details of microarray data pre-processing,

normalization and detection of differentially expressed genes are described in the

supplementary information of the paper described in §III of the results chapter.

SLC16A7 AGTTCTTCTTGGCCCTCCTC CGCTTGCTGCTACCACAATA 101 bp

FABP5 ATGGCCAAGCCAGATTGTAT TGAACCAATGCACCATCTGT 176 bp

NELL2 AATTTGGATGGCGGATATGA AAGCCTTGGGTCACATTCAG 228 bp

ANXA1 TCAAGCCATGAAAGGTGTTG CACAAAGAGCCACCAGGATT 182 bp

EPHA4 CGCTGCACCAAATCAAGTAG AACGTTTTTCAACCCACCAG 99 bp

TRIM22 ACCAGATCTGAGTGGGATGC TGCAGGTGCGTACAGTTTTC 149 bp

MAP3K1 GGATGCACTCTTGCCATTTT TGGGCATGGTGATCTACAAA 130 bp

DAPK2 CCAAGAGGCTCTCAGACACC CGATGTCCTCCTCAGTGTCA 239 bp

ARMCX2 TGTCTTCCTTTTGGGCTCTG AAGGTTAAGGGATGCCTGCT 145 bp

SNX1 CAAGCCACACTGCAGAAGAA CGGACCACTGTTGAAATCCT 155 bp

NRAS CCAAGACCAGACAGGGTGTT GTCAGGACCAGGGTGTCAGT 201 bp

MCM10 CGTCAGTGAGCAGCATGAAT TCCCGTTCCCATTTGTAGAG 141 bp

MCM4 ACCCTCAGGACGAAGCCTAT CGAGGGTATGCAGAAACCAT 241 bp

CTSZ GGGAGAAGATGATGGCAGAA ATAGGTGCTGGTCACGATCC 245 bp

RXRG AGAGCGAGCTGAGAGTGAGG CCTCCAAGGTGAGGTCAGAG 239 bp

DUSP6 CCAAATCATGGGCTCACTTT TGCATTTGAGGTGACACTCC 110 bp

OCLN TGGCAAAGTGAATGACAAGC GCAGGTGCTCTTTTTGAAGG 165 bp

NRCAM CCAATTGGATTACCACCACCTATAA ATTGGAAAAATAAAGGTCCCCATT 111 bp

NEDD8 TGACCGGAAAGGAGATTGAGAT CCTCCACACGCTCCTTGATT 72 bp

TTC1 CGGAGAAGCTGTGAGGTTCTTTA TCCTCTGGAACCCCACAGTT 85 bp

Table 8. Nucleotide sequence of the primers used for RT-PCR

II.3.2 Experiments on Affymetrix slides

Because RNA from our samples were already amplified by the method described in §III of the results chapter, we had to adapt the protocol of Affymetrix to our samples. In deed, in the Affymetrix protocol with one cycle of amplification, cRNA were labeled with biotin, which was not the case in our protocol. Consequently, we used the Affymetrix protocol with two cycles of amplification and we started just after the first round of amplification, considering that both protocols generated non biotinylated-cRNA after the first round of amplification. So briefly, the cRNA generated by our protocol from all our tissues (9 ATCs, 20 PTCs, 20 adjacent tissues to PTCs) were purified according to the Affymetrix protocol. The first and second strands of cDNA were synthesized and purified.

Biotynilated cRNA was generated, purified, quantified and fragmented. The quality of the fragmented cRNA was assessed using the Agilent 2100 Bioanalyzer. 15 µg of cRNA were finally hybridized on Affymetrix slides (HGU133 plus 2.0) which were afterwards washed and analyzed according the manufacturer’s protocol. Affymetrix experiments were performed at the microarray facility of the Institut J.Bordet (headed by Dr.

C.Sotiriou).

II.4 Real-time RT-PCR experiments

RT-PCR experiments related to the correlation between the gene expression profile and the biological phenotype of PTCs are described in §IV of the results chapter. For the

“ATC” project (§VII of the results chapter), confirmation of the reliability of the

regulated gene list was performed on 9 ATCs, 20 PTCs and 20 adjacent tissues to PTCs

for 16 selected genes. The primers were designed with the Primer3 software

(http://frodo.wi.mit.edu/) and are listed in table 8. We used the facilities of the University

of Swansea (Wales, UK, collaboration with Dr. Gerry Thomas) to perform our qRT-PCR

experiments. 1 µg of cRNA was incubated with DNase I (Ambion, Foster city, CA) and

reverse-transcribed in a 100µl reaction volume with random primers and Superscript II

(Invitrogen, Paisley, UK). Approximately 20ng of reverse-transcribed RNA (based on the

RNA concentration evaluated after the DNase treatment) were used in duplicate as a

template for the PCR reaction. We used the kit of Roche (Basel, Switzerland) and followed the manufacturer’s instructions. PCRs were performed on a LightCycler® 2.0 System using the cycling profile recommended by the manufacturer. All PCR efficiencies, obtained with 4 serial dilutions points (ranging from 20ng to 200pg), were above 90%

and real-time RT-PCR experiments were performed in duplicate for each gene.

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