Mitf-Mdel, a novel melanocyte/melanoma-specific isoform of microphthalmia-associated transcription factor-M, as a candidate biomarker for melanoma

Abstract

Background: Melanoma incidence is on the rise and advanced melanoma carries an extremely poor prognosis. Treatment options, including chemotherapy and immunotherapy, are limited and offer low response rates and transient efficacy. Thus, identification of new melanocyte/melanoma antigens that serve as potential novel candidate biomarkers in melanoma is an important area for investigation.

Methods: Full length MITF-M and its splice variant cDNA were cloned from human melanoma cell line 624 mel by reverse transcription polymerase chain reaction (RT-PCR). Expression was investigated using regular and quantitative RT-PCR in three normal melanocytes (NHEM), 31 melanoma cell lines, 21 frozen melanoma tissue samples, 18 blood samples (peripheral blood mononuclear cell; PBMC) from healthy donors and 12 non-melanoma cancer cell lines, including three breast, five glioma, one sarcoma, two kidney and one ovarian cancer cell lines.

Results: A novel splice variant of MITF-M, which we named MITF-Mdel, was identified. The predicted MITF-Mdel protein contains two in frame deletions, 56- and 6- amino acid deletions in exon 2 (from V32 to E87) and exon 6 (from A187 to T192), respectively. MITF-Mdel was widely expressed in melanocytes, melanoma cell lines and tissues, but almost undetectable in non-melanoma cell lines or PBMC from healthy donors. Both isoforms were expressed significantly higher in melanoma tissues than in cell lines. Two of 31 melanoma cell lines expressed only one isoform or the other.

Conclusion: MITF-Mdel, a novel melanocyte/melanoma-specific isoform of MITF-M, may serve as a potential candidate biomarker for diagnostic and follow-up purposes in melanoma.

Figure 1

Mutation analysis. (A-E) Sequence traces showing the splicing site mutation of human microphthalmia-associated transcription factor (MITF)-M. (A&B) Sequence of exon 2 of wild-type MITF-M using the forward primer. (C) Sequence of exon 6 of wild-type MITF-M using the reverse primer. (D) Sequence of exon 2 of the MITF-Mdel using the forward primer. (E) Sequence of exon 6 of the MITF-Mdel using the reverse primer. The nucleotides between the underlined nucleotides indicate the deletion regions. (F) Schematic representation of the structures of MITF-M and MITF-Mdel. The MITF-Mdel isoform has a 168-bp in-frame deletion in exon 2 and an 18-bp in-frame deletion in exon 6, but still has the transcriptional activation domain, the b-HLH-LZ domain and the serine-rich region (S).

Figure 2

cDNA and amino-acid sequences of human microphthalmia-associated transcription factor (MITF)-M and MITF-Mdel. (A) Alignment of mRNA sequences of MITF-M and MITF-Mdel. MITF-Mdel contains 168-bp and 18-bp in-frame deletions at mRNA level. (B) Alignment of predicted protein sequences of MITF-M and MITF-Mdel. MITF-Mdel is predicted to encompass 357 amino acids. The underlined portion marks the basic-helix-loop-helix domain. Compared with MITF-M, MITF-Mdel has deletions of 56-amino acid (from V32 to E87) in exon 2 and 6-amino acid (from A187 to T192) in exon 6. Dash lines represent deletions. Red highlighting indicates alternate exons. Green highlighting indicates amino acids encoded across a splice junction.

Figure 3

Detection of microphthalmia-associated transcription factor (MITF)-Mdel and MITF-M in melanoma cell lines and other carcinoma cell lines by reverse transcriptase polymerase chain reaction (representative results). MITF-M and MITF-Mdel have similar expression profiles. Similar to MITF-M, MITF-Mdel was found to be expressed only in normal human melanocytes, melanoma cell lines, and fresh melanoma tissues. Both isoforms were also expressed in MDA-MB-435, a cell line originally thought to be a breast cancer cell line, but has been identified as a melanoma cell line. Most melanoma expressed both MITF-M isoforms while two melanoma cell lines expressed only one or the other. Melanoma cell lines (n = 31; only 16 shown): from 501 to 2351, and MDA-MB-435. Breast carcinoma cell lines (n = 3; only two shown): MDA-MB-231 and SKBR3. Colon cancer cell lines (n = 2): T84 and Colo320. Ovarian cancer cell line (n = 1): ES2. Glioma cell lines (n = 5; only two shown): U138 and U87. PBMC No.7 and No.8 from healthy donors. Normal human melanocytes (NHEM) 7251 and NHEM2486: normal human melanocytes.

Figure 4

Relative expression of microphthalmia-associated transcription factor (MITF)-M (A) and MITF-Mdel (B) in 31 melanoma cell lines, 21 frozen melanoma tissue samples, 18 PBMCs from healthy donors, and 12 non-melanoma cell lines, including three breast, five glioma, one sarcoma, two kidney and one ovarian cancer cell lines. Data was calculated using ΔΔCt method. Melanoma cell line 624 mel was used as normalization control and its MITF-M and MITF-Mdel relative expression levels were set as 100%. Bars indicated median of expression levels of MITF-M or MITF-Mdel mRNA in each group.