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. 2011 Mar;178(3):1395-402.
doi: 10.1016/j.ajpath.2010.12.011.

Mutant IDH1 confers an in vivo growth in a melanoma cell line with BRAF mutation

Tatsuhiro Shibata  1 , Akiko Kokubu, Masashi Miyamoto, Yuko Sasajima, Naoya Yamazaki
Affiliations

Mutant IDH1 confers an in vivo growth in a melanoma cell line with BRAF mutation

Tatsuhiro Shibata et al. Am J Pathol. 2011 Mar.

Abstract

Melanoma is the most deadly tumor of the skin, and systemic therapies for the advanced stage are still limited. Recent genetic analyses have revealed the molecular diversity of melanoma and potential therapeutic targets. By screening a cohort of 142 primary nonepithelial tumors, we discovered that about 10% of melanoma cases (4/39) harbored an IDH1 or IDH2 mutation. These mutations were found to coexist with BRAF or KIT mutation, and all IDH1 mutations were detected in metastatic lesions. BRAF-mutated melanoma cells, additionally expressing the cancer-related IDH1 mutant, acquired increased colony-forming and in vivo growth activities and showed enhanced activation of the MAPK and STAT3 pathways. Genome-wide gene expression profiling demonstrated that mutant IDH1 affected the expression of a set of genes. Especially, it caused the induction of growth-related transcriptional regulators (Jun, N-myc, Atf3) and the reduction of Rassf1 and two dehydrogenase genes (Dhrs1 and Adh5), which may be involved in the carcinogenesis of IDH1-mutated tumors. Our analyses demonstrate that IDH1 mutation works with other oncogenic mutations and could contribute to the metastasis in melanoma.

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Figures

Supplementary Figure S1
Supplementary Figure S1
GAK cells were obtained from Japanese Collection of Research Bioresources (Sennan-shi, Japan) and maintained in DMEM supplemented with 10% fetal bovine serum. The FLAG-tagged wild or mutant (R132H) IDH1 genes were introduced in BRAF-wild GAK cells. A: Immunoblot analysis of mock, wild-type IDH1 (WT1 and 2), and mutant IDH1 (MUT1 and 2). The cell lysates were electrophoresed and immunoblotted with anti-flag antibody. Arrow indicates expected IDH1 protein. Asterisk indicates nonspecific signal. B: In vitro cell proliferation of mock, wild-type (WT1 and 2), and mutant (MUT1 and 2) IDHI-expressing clones. The culture condition is DMEM supplemented with 10% serum. C: Colony-forming activity of mock, wild-type IDH1 (WT1 and 2), and mutant IDH1 (MUT1 and 2). Representative plate of each clone is shown at the bottom.
Figure 1
Figure 1
IDH1 and IDH2 mutations in melanoma. Amino acid alignments of IDH1 and IDH2 proteins among human, mouse, rat, and chicken homologues are shown in the top panels. Sequence chromatographies of the IDH1 and IDH2 genes in primary melanomas (T) and corresponding normal (N) tissues are shown in the bottom panels. Arrows indicate the heterozygous mutation in tumor samples. Note that mutated amino acids residues are well conserved among species.
Figure 2
Figure 2
Growth advantage by mutant IDH1 in BRAF-mutated melanoma cells. A: Immunoblot analysis of mock, wild-type IDH1 (WT1 and 2), and mutant IDH1 (MUT1 and 2). The FLAG-tagged wild or mutant (R132H) IDH1 genes were introduced in BRAF-mutated G361 cells. The cell lysates were electrophoresed and immunoblotted with anti-flag antibody. Asterisk indicates nonspecific signal. B: In vitro cell proliferation of mock, wild-type (WT1 and 2), and mutant (MUT1 and 2) IDH1-expressing clones under two culture conditions (DMEM supplemented with 10% or with 1% serum). C: Colony-forming activity of mock, wild-type IDH1 (WT1 and 2), and mutant IDH1 (MUT1 and 2). Representative plate of each clone is shown at the bottom. D: Migration activity of mock, wild-type IDH1 (WT1 and 2) and mutant IDH1 (MUT1 and 2). Representative picture of migrated cells in each clone is shown at the bottom. E: The weight of in vivo tumors produced by mock, wild-type (WT1), and mutant (MUT1) IDH1-expressing clones (Mock: n = 5, WT1: n = 5, MUT: n = 8) (left). Histological appearance (hematoxylin-eosin staining) of tumors formed by mock and mutant IDH1-expressing clones (right). Scale bar = 100 μm.
Figure 3
Figure 3
No significant accumulation of reactive oxygen species by mutant IDH1 in melanoma cells. A: Measurement of reactive oxygen species (ROS) in mock and IDH1-expressing clones by fluorescence-activated cell sorting analysis. Representative data of ROS accumulation (right peak) and control signal (left peak) are indicated. Positive cell fraction is shown by arrows. B: Frequency of fluorescence positive cells in mock, wild-type IDH1-, and mutant IDH1-expressing clones (n = 3).
Figure 4
Figure 4
Downstream signal pathways of mutant IDH1 in melanoma cells. A: Detection of phosphorylated and non-phosphorylated forms of MAPK, AKT, STAT3, and S6K in mock, wild-type IDH1-, and mutant IDH1-expressing clones. β-actin expression was a loading control. B and D: Quantitative RT-PCR analysis of the candidate genes regulated by mutant IDH1.C: In vitro cell proliferation of mock, wild-type (WT1 and 2), and mutant (MUT1 and 2) IDH1-expressing clones under hypoxic condition (1% O2).
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References

    1. Tsao H., Atkins M.B., Sober A.J. Management of cutaneous melanoma. N Engl J Med. 2004;351:998–1012. - PubMed
    1. Verma S., Quirt I., McCready D., Bak K., Charette M., Iscoe N. Systematic review of systemic adjuvant therapy for patients at high risk for recurrent melanoma. Cancer. 2006;106:1431–1442. - PubMed
    1. Kuphal S., Bosserhoff A. Recent progress in understanding the pathology of malignant melanoma. J Pathol. 2009;219:400–409. - PubMed
    1. Curtin J.A., Fridlyand J., Kageshita T., Patel H.N., Busam K.J., Kutzner H., Cho K.H., Aiba S., Bröcker E.B., LeBoit P.E., Pinkel D., Bastian B.C. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353:2135–2147. - PubMed
    1. Davies H., Bignell G.R., Cox C., Stephens P., Edkins S., Clegg S., Teague J., Woffendin H., Garnett M.J., Bottomley W., Davis N., Dicks E., Ewing R., Floyd Y., Gray K., Hall S., Hawes R., Hughes J., Kosmidou V., Menzies A., Mould C., Parker A., Stevens C., Watt S., Hooper S., Wilson R., Jayatilake H., Gusterson B.A., Cooper C., Shipley J., Hargrave D., Pritchard-Jones K., Maitland N., Chenevix-Trench G., Riggins G.J., Bigner D.D., Palmieri G., Cossu A., Flanagan A., Nicholson A., Ho J.W., Leung S.Y., Yuen S.T., Weber B.L., Seigler H.F., Darrow T.L., Paterson H., Marais R., Marshall C.J., Wooster R., Stratton M.R., Futreal P.A. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–954. - PubMed

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