Anaplastic thyroid cancers harbor novel oncogenic mutations of the ALK gene

Abstract

Thyroid cancer is the most common endocrine cancer, and targeted approaches to treat it pose considerable interest. In this study, we report the discovery of ALK gene mutations in thyroid cancer that may rationalize clinical evaluation of anaplastic lymphoma kinase (ALK) inhibitors in this setting. In undifferentiated anaplastic thyroid cancer (ATC), we identified two novel point mutations, C3592T and G3602A, in exon 23 of the ALK gene, with a prevalence of 11.11%, but found no mutations in the matched normal tissues or in well-differentiated thyroid cancers. These two mutations, resulting in L1198F and G1201E amino acid changes, respectively, both reside within the ALK tyrosine kinase domain where they dramatically increased tyrosine kinase activities. Similarly, these mutations heightened the ability of ALK to activate the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein (MAP) kinase pathways in established mouse cells. Further investigations showed that these two ALK mutants strongly promoted cell focus formation, anchorage-independent growth, and cell invasion. Similar oncogenic properties were observed in the neuroblastoma-associated ALK mutants K1062M and F1174L but not in wild-type ALK. Overall, our results reveal two novel gain-of-function mutations of ALK in certain ATCs, and they suggest efforts to clinically evaluate the use of ALK kinase inhibitors to treat patients who harbor ATCs with these mutations.

Figure 1. Identification of novel somatic ALK mutations in anaplastic thyroid cancer

A. Sequencing electropherogram of the ALK gene. Presented in the left portion of Fig 1A are the sequencing results of the matched normal tissues of the two ATC cases, showing the wild-type ALK gene. Presented in the middle and right portions of Fig 1A are the sequencing results of two ATC tumors: shown in the upper panel are the sequencing results of sense and antisense strands of a region of exon 23 of the ALK gene in an ATC showing the heterozygous C>T mutation at nucleotide position 3592 in codon 1198, resulting in the L1198F amino acid change of ALK; shown in the lower panel are the sequencing results of sense and antisense strands of a region of exon 23 of the ALK gene in another ATC showing the heterozygous G>A mutation at nucleotide position 3602 in codon 1201, resulting in the G1201E amino acid change. Arrows indicate the mutated nucleotides. Nucleotide numbers refer to the position within the coding sequence of the ALK gene, where position 1 corresponds to the first position of the translation initiation codon. All samples were sequenced in two repeated experiments with independent PCR by sense and antisense primers. B. Schematic diagram of the ALK. Shown are the relative positions of the novel somatic ALK mutations L1198F and G1201E and the previously characterized mutations K1062M and F1174L from neuroblastoma. L1198F and G1201E are located in the tyrosine kinase domain of the ALK. C. Amino-acid sequence alignment of the ALK proteins from 6 species. Shown are the L1198 and G1201 residues that are evolutionarily completely conserved among these different species. Numbers indicate amino acid or codon positions. Amino-acid sequences are numbered with the initiation codon (methionine) of each protein defined as number 1.

Figure 2. Increased tyrosine kinase activities of ALK mutants L1198F and G1201E and their activation of the PI3K/Akt and MAP kinase pathways

A. In vitro assay of tyrosine kinase activities of ALK mutants. NIH3T3 cells stably expressing Flag-tagged vector, wild-type ALK (wt), and each of ALK mutants as indicated were lyzed. The cell lysates were assayed for tyrosine kinase activity as described in the Materials and Methods. The enzymatic activities were expressed as measured O.D. value×20. Results represent mean ± S.D. of three independent experiments. B. Activation of the PI3K/Akt and MAP kinase pathways. This is reflected by increased phosphorylation of Akt (p-Akt) and phosphorylation of ERK (p-ERK), respectively. NIH3T3 cells stably transfected with the indicated vector constructs as described in Fig 2A cell lysate proteins were subjected to Western blotting analyses for the indicated proteins using appropriate antibodies as described in the Materials and Methods. Successful protein expression of Flag-tagged wild-type ALK and each of the ALK mutants is shown in the top row of Fig 2B. The key molecules of the two pathways are shown in the subsequent rows. Total Akt, ERK and β-actin were used for quality control of loading proteins.

Figure 3. Focus-formation and anchorage-independent growth of cells promoted by ALK mutants

A. Cell focus-forming activities of ALK mutants. Shown are images of adherent growth of NIH3T3 cells transfected with Flag-tagged vector, wild-type ALK, and each of the ALK mutants indicated. Cells were cultured in regular medium with 10% FCS under standard conditions. Images of cell foci were photographed with 10X magnification after appropriate culture of cells as described in the Materials and Methods. B. Number of cell foci formed with the indicated transfections. The number of transformed foci was counted 14 days after cell transfection. Results represent mean ± S.D. of three independent experiments. C. Anchorage-independent cell growth of ALK mutants on soft agar. NIH3T3 cells stably transfected with Flag-tagged vector, wild-type ALK, and each of the ALK mutants indicated were seeded in soft agar and colonies formed 4 weeks later were photographed with 40X magnification. D. Analyses of number of colonies. The number of cell colonies corresponding to Fig 3C that were > 0.1 mm in diameter was counted. Results represent mean ± S.D. of three independent experiments.

Figure 4. Cell invasion promoted by ALK mutants

A. In vitro invasion assay of NIH3T3 cells with various transfections. Cells transfected with Flag-tagged vector, wild-type ALK, and each construct of the indicated ALK mutants. Cell invasion assay was performed as described in the Materials and Methods. Shown are the cells that invaded on the matrigel matrix-coated polycarbonate filter membrane after removal of the non-invasive cells. B. Number of invasive cells with the indicated transfections. Results of each column represent the mean ± S.D. of the numbers of invasive cells from three independent experiments.