Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

Patient accrual was conducted according to Institutional Review Board of the Mater Domini/University Magna Graecia (Catanzaro, Italy). The study was approved by the Institutional Review Board of the Mater Domini/University Magna Graecia in the meeting of May 21st 2014. Tumor, normal mucosa and peripheral blood samples were obtained from patients referring to General Surgey Unit of University Hospital Magna Graecia of Catanzaro (Catanzaro, Italy), who underwent surgical resection for colon cancer since January 2013. Biopsies were immediately snap frozen and stored at − 80 °C. In all cases diagnosis was confirmed by reviewing hematoxylin/eosin-stained slides.

General demographic information, clinical findings, surgical treatment, histo-patological examination and follow-up data were collected for each patient. Detailed documentation of the histo-pathological findings allowed classification according to the current edition of UICC [26]. None of the patients received chemotherapy or radiation therapy prior to surgery. See Additional file 1 for complete characteristics of patients. Briefly, among the 37 patients, 13 were women and 24 were males. The mean age was 68 years old (range 47–84). Stage was known for 36 of the 37 patients: 7 patients had stage I disease, 13 patients had stage II disease, 12 patients had stage III disease and 4 patients had stage IV disease. Grade was known for 35 out of 37 patients: 26 patients had tumors that were graded G2 and 9 patients had tumors that were graded G3. Four patients presented distant metastasis.

Sequencing was performed with the Ion AmpliSeq™ Comprehensive Cancer Panel on the Ion Proton system (ThermoFisher Scientific, MA, USA) starting from 40 ng of DNA and following standard protocols. Primary bioinformatic analysis of data is described in a previous manuscript (Oliveira et al., under review). We set an average mean coverage of > 300, a coverage of > 40 for each variant and variant frequency > 5%. To further filter the number of non-pathogenic variants identified in the study the following criteria were used: i) variants with quality score ≤ 30 were excluded, ii) variants that were not annotated as pathogenic or likely pathogenic by SIFT and Polyphen2 algorithms were excluded, iii) variants detected in both normal and tumor samples were excluded. Finally, manual observation of the variants using the Integrated Genomic Viewer (IGV – Broad Institute) was performed to exclude inconclusive variants.

DNA obtained from matched normal and tumor samples was amplified using specific primers. The purified products were sequenced using BigDye terminator v3.1 (Applied Biosystems, Foster City, CA) with ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, CA).

Tumors obtained at surgery were snap frozen in liquid nitrogen and conserved at − 80 °C. RNA was prepared using Trizol (ThermoFisher Scientific, MA, USA) and purified using RNeasy mini-eluate clean-up kit (QIAGEN Inc. CA, USA). cDNA was retro-transcribed with random hexamers using SuperScript®III First-Strand Synthesis System (ThermoFisher Scientific, MA, USA).

Deparaffinized 4-μm sections were hydrated in decreasing ethanol gradient solutions and rinsed in wash solution (TBST, 0.05 mol/L Tris Buffered Saline with Tween20). Antigen retrieval was performed with citrate buffer pH 6 for antibodies to RET and phospho-RET Y905, and pH 9 for antibody to phospho-RET Y1062, respectively, for 20 min at 98 °C followed by washing in TBST. Sections were incubated with antibodies against RET (rabbit monoclonal antibody anti-RET, 1:250 dilution, #14556 Cell Signaling Technology, Danver, MA), phospho-RET Y905 (rabbit polyclonal antibody anti-pRET, 1:100 diluition, #3221 Cell Signaling Technology) or phospho-RET Y1062 (rabbit polyclonal antibody anti p-RET, 1:100 dilution) [27] for 60 min, followed by incubation with secondary Alexa Fluor 488F fragment of goat anti-rabbit IgG (H + L) antibody; 1:500 dilution (ThermoFisher Scientific, MA, USA #A11070) for 45 min. Cells were counterstained with DAPI,1:500 dilution, (ThermoFisher Scientific, MA, USA), mounted using anti-fade mounting medium #53023 (DAKO, Glostrup, Denmark) and observed at Fluorescence Microscopy (Leica Microsystems, Wetzlar Germany).

Protein extracts were prepared with lysis buffer containing 50 mM HEPES pH 7.5, 5 mM EDTA, 250 mM NaCl, 1 mM dithiothreitol, 0.5% Nonidet P40, 1 mM Na₃VO₄, 1 mM NaF supplemented with 10 μg of aprotinin/ml, 10 μg of leupeptin/ml, 1 mM PMSF and a mix of protease inhibitors (SIGMAFAST protease inhibitor Tablets for general Use; Sigma-Aldrich St. Louis, MO, USA). Lysates were centrifuged at 13,000 rpm for 30 min at 4 °C and the supernatants were collected. Protein concentration was estimated with a modified Bradford assay (Bio-Rad Laboratories, Berkeley, CA, USA). Western blot analysis was carried out by standard methods.

To detect RET dimers, electrophoresis was carried out on a 6% SDS–PAGE in non-reducing conditions. In this case proteins extracts were prepared in the same lysis buffer as above devoid of dithiothreitol, and with sample loading buffer devoid of 2-mercaptoethanol. Samples were not heated upon loading.

Proteins were revealed by enhanced chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Berkeley, CA, USA). The antibodies used in this study were: anti-RET (E1N8X, #14556), anti-phospho-RET, (#3221), anti-phospho-ERK1/2 (#9101), anti-ERK1/2 (#9107) purchased from Cell Signaling Technology, Danver, MA, USA. The anti-phospho-RET Y1062 used in this study has been previously described [27].

RET mutants were obtained by site-specific mutagenesis of RET51-WT construct (pBabe vector encoding for the proto-RET gene long isoform). The RET51-C634R plasmid, used as positive control, is described elsewhere [28]. RET51-G533C and RET51-P1047S were obtained by site-directed mutagenesis of RET51-WT using an in vitro oligonucleotide mutagenesis system (Quik-Change XL site-directed mutagenesis; Agilent Technologies, CA, USA). Plasmid DNA was extracted using the QIAGEN Plasmid Maxi Kit (QIAGEN CA, USA) as suggested by the supplier. The presence of the specific mutations was verified by DNA direct sequencing. Mutant plasmids were sequenced entirely to exclude the presence of additional mutations.

Human HEK293T cells were maintained in Gibco™ DMEM with 10% FBS and 1% penicillin/streptomycin (ThermoFisher Scientific, MA, USA). Empty pBABE vector and/or plasmids encoding RET wild type or mutant alleles were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, MA, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were trypsinized and plated into 100-mm plates. Transfected cells were selected with 0.75 μg/ml puromycin for 1 week until all cells in the control plates were dead. Then cells were trypsinized, counted and 5000 cells were plated into 60-mm plates. Cells were kept in DMEM with 10% bovine serum. The culture medium was changed every 3–4 days. Approximately 10 days after transfection, cells were stained by crystal violet. The colony formation experiments were repeated three times.

Stably transfected cells expressing RET wild type and mutant alleles were seeded in 96 wells round bottom plates (1000 cells/well). After 24 h, 10 μl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; (Sigma-Aldrich St. Louis, MO, USA) was dissolved at the concentration of 5 mg/ml in warm assay medium was added to cells at the indicated time points (24 h, 48 h, 72 h and 96 h) to yield a final assay volume of 110 μl/well. Plates were incubated for 4 h at 37 °C and 5% CO₂. After incubation, supernatants were removed, and 100 μl of isopropanol was added. Plates were placed on an orbital shaker for 5 min, and the absorbance was recorded at 570 nm. Triplicates were made for each time point and two independent experiments were conducted. Statistical analysis was performed by two-way ANOVA and Dunnett’s multiple comparison test with GraphPad Software (La Jolla, CA, USA).

In a previous work, we have reported the analysis of 37 colon cancer patients using the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) (Oliveira et al., under review). In that work, patients who had undergone surgery for colon cancer at the General Surgery Unit of University Magna Graecia of Catanzaro in the years 2013–2015 were studied. Complete demographic and clinical information of patients are reported in Additional file 1. DNA was extracted from matched normal and pathological tissues and subjected to NGS analysis on the Ion Torrent platform using the CCP (ThermoFisher Scientific, MA, USA), that provides complete exon coverage of the 409 most important cancer-associated genes.

As indicated in Oliveira et al., (under review), the sequencing performance achieved was: 13 × 10⁶ mean number reads/sample, 791 (range 102.5–2656) mean sequence coverage depth of targeted exonic regions of the 409 genes analysed, with median uniformity of sequenced genes 97% (range 0.7–0.99). Common germ-line variants were removed by filtering sequentially through a pool of peripheral blood samples available from some patients (n = 12), the dbSNP141 and the 1000 Genomes Project data sets. Variants were functionally annotated using the algorithms Sift and Polyphen2 to predict their pathogenicity.

In this manuscript, we have re-analysed the data generated in the manuscript from Oliveira et al., focusing on genes encoding receptor-type tyrosine kinase (RTK). The number of RTK genes contained in the Comprehensive Cancer Panel that were object of the present study is 31. Overall, upon analysis of the data reported in Oliveira et al. we have observed 101 different potentially damaging variants distributed across 31 RTK genes. See Additional file 2 for a general summary of the analysis. We have found that 28 patients out of 37 presented variants in at least one gene encoding RTK, with the remaining 9 patients showing no variant in any of the 31 RTK genes. The mean number of mutated RTK genes/patient was 3.4 (range 1–13). Of the 28 samples that presented mutations in RTK genes, 8 patients had only one mutated gene, 6 had two different mutated genes and 14 presented 3 or more mutated genes. See Additional file 3 for patient-by-patient analysis of the results. Among RTK genes that presented variants, we observed variants in FLT4 (G1131S, R104Q, E1052K, D1003N, D728N, P707L, R362L, R282*, Q213*, G180R, P138S, E36* and 3 frameshifts) in 10 patients, ROS1 (S277Y, P437L, S653F, T804 N, Q1127*, G1709C and QG1708HC) in 7 patients, EPHA7 (D839G, P805L and P278S) in 6 patients, RET (R77C, P270L, G533C, P1047S) in 4 patients and MET (A320V, R988C) in 2 patients. In addition, ERBB2 variants (S310F, W482C, Q533*, T631A, S819F, A1216D) were detected in 6 patients, EGFR (R669*, W731*, D807E, L933P) and ERBB4 (R983K, M977 V, R847H, T244I) variants were detected in 4 patients, and ERBB3 variants (T389I, V850 M) were detected in 2 patients. FGFR1 (D784E, A131V) and FGFR2 (G302 K, L104P) variants were detected in 2 patients, whereas FGFR3 (D320N, A352E, A571V, T653I) and FGFR4 variants (R130C, KE144RG, F859 L, T912I) were detected in 4 patients. Eight tumors presented variants in only one RTK gene (FLT4, EGFR, ERBB4, ROS1, and EPHA7), which suggests that these variants can represent driving mutations. Accordingly, the variants detected in the EGFR, ERBB4 and FLT4 were present in the COSMIC database. Conversely, co-occurrence of variants in different genes encoding RTKs was frequent, occurring in 20 tumors out of 37 analysed (54%). Co-occurrence of variants included 2 different genes (FGFR4/NTRK1, ERRB3/FLT3, ALK/ERBB4, EPHA3/EPHA7, EPHB4/ROS1, and FLT4/LTK) in 6 cases and three or more genes in the remaining 14 cases. Notably, some samples showed variants in numerous genes encoding RTK (CC27, 11 genes; CC34, 13 genes; CC12, 8 genes).

Among the RTKs showing variants identified in this study, we decided to biologically validate the variants identified in the gene encoding the RET receptor. The reasoning for this choice was that, at difference with what happens in thyroid and lung cancer, the involvement of RET proto-oncogene to the development of colon cancer is unclear. Variants in the RET gene were identified in four colon cancer patients (patients CC12, CC20, CC25 and CC33), as indicated in Additional file 2.

In the present study, we have identified a C/T transition that led to the replacement of P1047 with S (P1047S) in the cytoplasmic tail of RET in patient CC12, a C/T transition that led to the replacement of P270 with L (P270L) in the RET extracellular domain in patient CC33, an A/T transversion that led to the replacement of G533 with C (G533C) in the cysteine-rich domain of RET protein in patient CC20 and a G/A transition that led to the replacement of R77 with C (R77C) in the RET extracellular domain in patient CC25.

As to the technical aspects of NGS, patient CC12 showed total mean coverage of 1990, coverage of the variant of 45 and frequency of the variant 49%, patient CC33 showed total mean coverage of 789, coverage of the variant of 349 and frequency of the variant 5.9%, patient CC20 showed total coverage of 728, coverage of the variant of 442 and frequency of the variant 20.4% and patient CC25 showed total mean coverage of 761, coverage of the variant of 89 and frequency of the variant 30.5%. Raw data showing the detection of the different RET mutations by NGS are contained in Additional file 4.

Interestingly, mutation G533C is located in the RET extracellular domain and consists in the replacement of a glycine amino acid with a cysteine as often observed in families with familial medullary thyroid carcinoma (FMTC) or Multiple Endocrine Neoplasia (MEN) 2A. Notably, the G533C substitution was identified in patients with FMTC [29], suggesting that it represents a gain-of-function mutation. For the complete list of mutated genes in the patient harboring the G533C substitution see Additional file 5. First we confirmed the results obtained through NGS by traditional Sanger sequencing. We performed DNA sequencing of RET exons on matched normal and cancer samples to confirm the presence of the specific mutations identified by NGS (See Fig. 1). Among the 4 different RET mutants identified in colon cancer patients we excluded the P270L variant because it represented less than 10% of the cell population and the R77C since it has been observed in a case of Hirschsprung’s disease [30]. Conversely, variants G533C and P1047S were included in the subsequent analysis. We found that in the two patients, mutations were cancer-specific and were not found in the corresponding normal tissue. These findings led us to exclude the presence of germ-line mutations associated with inherited diseases such as FMTC, MEN2 type 2A, MEN2 type 2B and/or Hirschsprung’s disease. Conversely mutations were somatically acquired and were apparently selected for within the bulk of tumor cells since they represented 20–49% of RET alleles, respectively. We analysed RET expression in the tumors and in the corresponding normal mucosa from patients CC12 and CC20 where RET mutations were identified.

The expression of RET in the samples harbouring RET mutations was evaluated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) (Fig. 2) and by in situ immunofluorescence analysis (Fig. 3a). RT-PCR analysis revealed the presence of a RET transcript in normal mucosa and the corresponding cancer in all patients analysed. The detection of RET transcript by RT-PCR mRNA was corroborated by analysis of the RET protein in normal and cancerous tissues by immunofluorescence. Expression of RET protein and the levels of its phosphorylation at residues Y905 and Y1062 were assessed in colon cancer and the corresponding normal mucosa of patients CC12 and CC20 using immunofluorescence.

Tissues were scored as positive when showing uniform, intense cytoplasmic labelling with marked accentuation of membranes, and as negative or weakly positive when labelling was a faint cytoplasmic staining with few positive cells interspersed among the majority of negative cells. We found that normal colon mucosa of the two samples analysed were moderately positive for RET expression (see Fig. 3a).

Similarly, we found that the corresponding cancerous tissues of patients CC12 and CC20 retained expression of RET at levels that were comparable with normal mucosa. Moreover, tumor samples harbouring RET mutations showed positivity for phospho-Y905 and phospho-Y1062 residues while the normal mucosa samples were either negative or presented weak positivity (see Fig. 3b). These results suggest that, at least in the case of some RET mutants, the expression of the receptor is not impaired by selective pressure for loss of RET expression.

We investigated whether the RET variants identified in colon cancer patients are able to stimulate anchorage-dependent proliferation in HEK293 cells. This cellular system has already been successfully used to investigate the oncogenic characteristics of multiple oncoproteins [31]. To this aim, the G533C and P1047S RET variants were generated by site-specific mutagenesis of pBABE-puro plasmid carrying the cDNA encoding human wild type RET (RET-G533C, RET-P1047S and RET-WT, respectively).

HEK293 cells were transfected with empty vector (HEK293-C) or transfected with plasmids carrying wild type or mutant RET (HEK293-RET-WT, HEK293-RET-G533C and HEK293-RET-P1047S, respectively). Multiple clones of HEK293-C, HEK293-RET-WT, HEK293-RET-G533C and HEK293-RET-P1047S cells from transfection experiments were expanded and/or frozen for further studies. HEK293-RET-C634R cells were used as positive control.

Analysis of cell proliferation by MTT demonstrated that, similar to HEK293-RET-C634R cells, HEK293-RET-G533C duplicated with an increased rate in monolayer compared with HEK293-C (p < 0.01). Conversely, HEK293-RET-WT and HEK293-RET-P1047S cells showed a more limited increase, being intermediate between negative (HEK293-C) and positive controls (HEK293-RET-C634R), see Fig. 4a.

Similar results were obtained with colony assay experiments. Also in this type of assay, HEK293-RET-G533C cells showed greater proliferative potential in clonogenic assays than HEK293-C cells, generating more colonies than control cells. Similarly, HEK293-RET-WT and HEK293-RET-P1047S cells showed an intermediate behaviour (Fig. 4b). These results indicate that RET mutant G533C, but not wild type or P1047S RET mutants expressed at similar level, is able to stimulate anchorage-dependent proliferation of human epithelial cells in culture.

Subsequently, immunoblot analysis was performed to check for RET expression and for the activation status of MAPK pathway in transfected HEK293 cells (Fig. 5). Lysates from transfected HEK293 cells and derivatives were analysed by immunoblot with anti-phosphoY1062 or anti-RET antibody. In agreement with the data from MTT and colony assay experiments, the G533C RET mutant showed increased RET phosphorylation than wild type RET or P1047S mutant (Fig. 5a). Subsequently, we assessed the activation status of critical signalling pathways that function downstream RET [32], the MAPK pathway. The activity of the MAPK pathway was assessed with anti phospho-Y202/T204 ERK1/2 antibody (Fig. 5a). Anti-total ERK1/2 antibody was used for normalization. We found that the adoptive expression of RET mutant G533C, at difference with wild type or P1047S RET, induced a marked increase in the activation of MAPK pathway. Finally, since the mutation carried by the active mutant involved a cysteine residue in the extracellular domain, we investigated whether this amino acidic change facilitated the formation of RET dimers. To detect RET dimer formation we separated cell lysates from transfected HEK293 cells by SDS-PAGE under either non-reducing or reducing condition. As shown in Fig. 5b, we found that RET mutants G533C was particularly efficient in generating RET dimers in transfected HEK293 cells comparable to the positive control (PC), suggesting that the oncogenic mechanism whereby RET mutant G533C contribute to colon cancer development is through an increase in the amount of covalently linked dimers induced by the presence of the unpaired cysteine induced by the mutation.

We also found that RET-G533C mutant stimulated cell migration in vitro (Fig. 6). We investigated the effects of the RET-G533C mutant on the migration capability of HEK293 cells by using wound healing assay (Fig. 6a). RET-WT and RET-C634R were used as controls. The results of a triplicate set of independent experiments demonstrated that RET-G533C mutant enhanced migration of HEK-293 cells of about 70% at 48 h at difference with RET-WT, which was much less efficient in promoting migration (Fig. 6b).

Finally, we determined whether the RET inhibitor vandetanib was able to abolish the effects exerted by RET-533C mutant in transfected HEK293 cells. Treatment of HEK393 cells expressing RET mutants with vandetanib induced a dose-dependent reduction (almost 50% using 500 nM Vandetanib) in viable cell number of HEK293-RET-G533C (and HEK293-RET-C634R) cells, demonstrating growth inhibitory activity in vitro (Fig. 7a). Similarly, subsequent immunoblot analysis demonstrated that vandetanib markedly reduced phosphorylation of RET Y1062 and of ERK1/2 Y202/T204 in HEK293-RET-G533C and HEK293-RET-C634R cells (Fig. 7b).