Methylation analysis as well as Xenograft experiments were performed by external co-laborators within the Colonet consortium. Methylation analysis was performed by Dr.
Sascha Tierling in the Institute of Genetics and Epigenetics in Saarbr¨ucken, Xenograft experiments were performed by Maria Rivera in the group of experimental pharmacology in the Max-Delbr¨uck Center Berlin-Buch.
2.12.1 Methylation analysis
The following protocols were written and operated by Dr. Sascha Tierling:
DNA preparation and bisulfite treatment DNA was prepared using standard protocols. Bisulfite treatment was performed on 500 ng genomic DNA. Briefly, DNA was treated with 2 M sodium bisulfite and 0.6 M NaOH, then denatured for 15 min at 99℃ and incubated for 30 min at 50 ℃. We introduced two thermo spikes of 99 ℃ for 5 min followed by two incubation steps of 1.5 h at 50 ℃. Purification was achieved by loading, desulfonation with 0.3 M NaOH and washing with 1 x TE on a microcon YM-30 column (Millipore, Schwalbach, Germany). Bisulfite DNA was eluted in 50 µl 1 x TE.
Promoter methylation analysis by 454 GS-FLX sequencing Amplicons were generated using region- specific primers with the recommended A and B adaptors (Roche, Mannheim, Germany) at their 5’ -end. PCR of bisulfite-treated DNA was performed in
a total volume of 30 µl containing 3 µl Hot Star-Taq reaction buffer (Qiagen, Hilde, Germany), 2.4 µl dNTPs, 2.5 mM each, 100 nmol of each primer, 1.25 U Hot Star-Taq (Qiagen, Hilden, Germany) and 2 µl of bisulfite DNA. After initial denaturation at 95
℃ for 15 min, 40 cycles were carried out (denaturation 95℃for 1 min, annealing 54 ℃ for 1 min, extension 72 ℃ for 1 min and final extension 72 ℃ for 5 min). PCR prod-ucts were visualized on 1.2 % agarose gels, purified using the Gel/PCR DNA Fragments extraction kit (AVEGENE, Taipei, Taiwan) and measured by intercalating fluorescence dye (Qubit HS-Kit, Invitrogen, Darmstadt, Germany) using Qubit Fluorometer (Invit-rogen, Darmstadt, Germany). After amplicon pooling, emulsion PCR was performed using Lib-A emPCR protocols. DNA-containing beads were recovered, enriched and sequenced from the A-adaptor on a XLR70 TitaniumPicoTiterPlate according to the manufacturers protocols (Roche, Mannheim, Germany).
MsSNuPE-experiments and HPLC separation Fifty ng of genomic DNA were used as a template in a 30µl reaction volume in the presence of 3 mM Tris-HCl, pH 8.8, 0.7 mM (NH4)2SO4, 50 mM KCl, 2.5 mM MgCl2, 0.06 mM of each dNTP, 3 U HotFire DNA polymerase (Solis BioDyne, Tartu, Estonia) and 1 µM primers. 5 µl of the PCR products were treated with 1 µl of ExoSAP (1:10 mixture of Exonuclease I and Shrimp Alkaline Phosphatase, USB) for 30 min at 37 ℃. To inactivate the ExoSAP enzymes the reaction was incubated for 15 min at 80 ℃. Afterwards, 14 µl primer extension mastermix (50 mM Tris-HCl, pH 9.5, 2.5 mM MgCl2, 0.05 mM of all four ddNTPs, 3.6 µM of each SNuPE primer, 2.5 U Termipol DNA polymerase (Solis BioDyne, Tartu, Estonia)) was added. Primer extension reactions were performed at 96 ℃ for 2 min followed by 50 cycles 96 ℃ / 30 sec, 50 ℃ / 30 sec, 60 ℃ / 2 min. Separation of products was conducted at 50 ℃ by continuously mixing buffer B (0.1 M TEAA, 25 % acetonitril) to buffer A (0.1 M TEAA), either over 15 min: 23-31 % (AREG A2), or 15 min: 22-35 % (EREG A2).
2.12.2 Xenograft experiments
Thein vivo experiments (generation and treatment of xenografts, as well as tissue collec-tion) were carried out by Maria Rivera in the group of experimental pharmacology in the Max-Delbr¨uck Center Berlin-Buch. 5x106 LIM1215 cells were injected subcutaneously into immunodeficient NMRI nu/nu mice. Per treatment five mice were used. Three inde-pendent experiments were performed. In the first experiment (MV10107) the influence of Valproat alone and in combination with Erlotinib on tumor growth was examined.
In the second experiment (MV10532) the treatment mode was changed to evaluate the effect of Valproat treatment prior to Erlotinib treatment on tumor growth. And in the third experiment, the influence on tumor growth of 5-Azacytidine alone and in combi-nation with Erlotinib was tested. After cell injection, tumor growth was measured two times a week. When the tumors reached a volume of approximately 0.1 to 0.2 cm3 the
treatment started, which was for MV10107 on day 19, for MV10532 on day 12 and for MV10533 on day 13 post injection. The treatment modes and the concentrations of the used inhibitors are shown in table 20. The concentrations correspond to the highly tol-erable doses in human treatment. After finishing the experiments, the mice were killed and the tumors were shock frozen in liquid nitrogen. Afterwards, the tumors were stored at -80 ℃. Parts of the tumors were used for RNA isolation and AREG/EREG mRNA expression analysis (see section 2.2.7) as well as immunohistochemistry experiments.
Table 20: Design of xenograft experiments to compare in vitro results in an in vivo system:
i.p.: intraperitoneal injection, p.o.: oral treatment
MV10107 Application Days Dose (mg/kg/inj.)
Solvent (PBS)
Valproat i.p. Mon-Fri, 2x/day 200
Erlotinib p.o. Mon -Fri 50
Cetuximab i.p. every 7th day 2x (q7dx2) 50
Valproat + i.p. Mon-Fri, 2x/day 200
Erlotinib p.o. Mon -Fri 50
MV10532 Application Days Dose (mg/kg/inj.)
Solvent (NaCl)
Solvent (NaCl) i.p. day 1 to 6
Erlotinib p.o. day 7 to 11 50
Erlotinib + p.o. day 1 to 6 50
Valproat i.p. day 7 to 11 200
Valproat + i.p. day 1 to 6 200
Erlotinib p.o. day 7 to 11 50
MV10533 Application Days Dose (mg/kg/inj.)
Solvent (NaCl)
Erlotinib p.o. days 1-5, 7, 13-17, 22, 23 50 5-Azacytidine i.p. days 1-5, 7, 13-17, 22, 23 5 5-Azacytidine + i.p. days 1-5, 7, 13-17, 22, 23 5 Erlotinib p.o. days 1-5, 7, 13-17, 22, 23 50
3 Results
3.1 Amphiregulin and Epiregulin are differentially expressed in colorectal cancer cell lines
Figure 3:
Figure 3: AREG and EREG expression in different colorectal cancer cells: Seven colorectal cancer cell lines were tested for theirAREG andEREGmRNA expression and protein levels at 4 different timepoints. 1st row: The ∆Ct values were calculated by subtraction of the AREG orEREG Ct-value from the control Ct-value. 2nd row: The AREG or EREG protein amounts in the supernatant were normalized to the corresponding RNA concentration. 3rd row: The AREG and EREG protein amounts are shown per 10µg total cell lysates (see section 2.5.2).
The mRNA expression of Amphiregulin and Epiregulin was tested in several co-lorectal cancer cells at four timepoints by real-time PCR. The amounts of the cor-responding proteins in the supernatant or the cell lysates were also tested by ELISA experiments (see figure 3). EREG protein was neither detected in the cell lysates nor in the supernatants by the ELISA assay used. Negative values in the ELISA-barplots
indicate that the ELISA raw data of these samples was equal or lower than the raw data of the negative controls. Therefore, although comparison with the standard curve led to negative values, these values should be considered as zero. In contrast to EREG, there was a good correlation between AREG mRNA expression and protein amounts.
LowAREG mRNA expression correlated with a lack of detectable AREG protein in the supernatant or in the cell lysate, whereas highAREG mRNA expression correlated with high AREG protein amounts. Although there was a discrepancy between the AREG pro-tein amount in HCT116 cells between the supernatant and the cell lysate, the data could still be used to divide the cells in three groups of AREG mRNA and protein expressing cells and two groups of EREG mRNA expressing cells. The AREG groups consisted of cells with low AREG mRNA expression and no or only very low detectable amounts of AREG protein (SW480, RKO and Colo678), cells with medium AREG mRNA ex-pression and low detectable amounts of AREG protein (LIM1215 and HT29) and cells with high AREG mRNA expression and high amounts of AREG protein (HCT116 and CaCO2). The EREG groups consisted of cells with no or very low EREG mRNA ex-pression (SW480, RKO, Colo678 and LIM1215) and cells with medium to high EREG mRNA expression (HCT116, CaCO2).
The EREG mRNA expression correlated with the AREG mRNA expression, too. The cells comprising the low AREG expressing group appeared in the group of no or very low EREG mRNA expressing cells (SW480, RKO, Colo678). In a similar way, the cells comprising the high AREG expressing group appeared in the group of medium to high EREG mRNA expressing cells (HCT116, CaCO2). However, this only holds true for the EREG mRNA expression, since EREG proteins were not detected.