Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jan;134(1):133-140.
doi: 10.1038/jid.2013.293. Epub 2013 Jul 5.

A conditional zebrafish MITF mutation reveals MITF levels are critical for melanoma promotion vs. regression in vivo

James A Lister  1 , Amy Capper  2 , Zhiqiang Zeng  2 , Marie E Mathers  3 , Jennifer Richardson  2 , Karthika Paranthaman  2 , Ian J Jackson  4 , E Elizabeth Patton  5
Affiliations
Free PMC article

A conditional zebrafish MITF mutation reveals MITF levels are critical for melanoma promotion vs. regression in vivo

James A Lister et al. J Invest Dermatol. 2014 Jan.
Free PMC article

Abstract

The microphthalmia-associated transcription factor (MITF) is the "master melanocyte transcription factor" with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAF(V600E) to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAF(V600E)mitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The microphthalmia-associated transcription factor (MITF) is oncogenic with BRAFV600E in melanomagenesis. (a) Adult wild-type zebrafish or (b) adult mitfavc7 mutant zebrafish living in water at 28 °C or (c, d) <26 °C. At <26 °C some melanocytes are visible in the body (d: enlarged region, white arrows). (e) Adult transgenic line expressing human BRAFV600E in the melanocytes. (f–i) Genetic crosses of BRAFV600Emitf at the semirestrictive temperatures develop nevi (*) and melanoma (on the tail of the middle fish, and on the head of the bottom fish). (j) Melanoma incidence curves of BRAFV600Ep53 and BRAFV600Emitf (<26 °C) genetic crosses. (k) Real-time PCR (RT-PCR) analysis of the mitfa transcript in BRAFV600Ep53 and BRAFV600Emitf melanomas.
Figure 2
Figure 2
Comparative histopathology of BRAFV600E melanomas. (a) Cross-section of adult BRAFV600Emitf zebrafish with superficial spreading melanoma (dotted line). Infiltrating melanophages in the kidney are indicated (yellow arrows). i, intestine; k, kidney; l, liver; m, muscle; o, ovary; s, spinal column; sb, swimbladder. (Top and bottom panels) Hematoxylin and eosin (H&E) stain of BRAFV600Emitf melanoma, indicating large melanophages (red arrows), spindle or epithelioid cell shapes (yellow arrows), and pigmented melanoma cells (white arrow). Scale bars=20  μm. (b) Cross-section of adult BRAFV600Ep53 zebrafish with invasive melanoma (dotted line). (Top and bottom panels) H&E stain of BRAFV600Ep53 melanoma, indicating pigmented melanoma cells (white arrows) and nuclear pleomorphisms (yellow arrow). Scale bars=20 μm. (c–f) Immunohistochemistry staining for (c) phospho-extracellular signal–regulated kinase (ERK), (d) p53, (e) Melan-A, and (f) phospho-histone H3. Scale bars=50 μm. (g). Box plot of mean percentage phospho-histone H3–stained cells in BRAFV600Emitf and BRAFV600Ep53 tumors (n=11 melanomas of each genotype). Bars represent interquartile range; Student's t-test P=0.0078.
Figure 3
Figure 3
MITF target gene expression. (a–i) Box plots of quantitative real-time PCR (qRT-PCR) of MITF target genes and p53. The y-axis indicates the difference between the cycle threshold (Ct) value of the gene of interest and the Ct value of β-actin in each sample. Note that the y-axis is inverted for ease of interpretation. Bars represent interquartile range; P-values determined by Student's t-test. Also see Supplementary Table S1 online. MITF, microphtholmia associated transcription factor.
Figure 4
Figure 4
Abrogation of MITF activity causes melanoma regression. (a) Images of adult BRAFV600Emitf zebrafish at <26 °C, and transferred to water at 32 °C for 14 days (bottom images). Red dotted lines outline the tumors before and after the temperature shift. (b) Control BRAFV600Ep53 melanoma fish at <26 °C and at 32 °C. Areas of increased tumor growth are indicated with yellow asterisks. (c) Adult BRAFV600Emitf zebrafish at <26 °C and after 2 months at 32 °C. MITF, microphtholmia associated transcription factor.
Figure 5
Figure 5
Melanoma regression is associated with melanophage and apoptotic activity. (a) Hematoxylin and eosin (H&E) staining of a regressing BRAFV600Emitf melanoma showing almost total regression with prominent melanophages (scale bar=200 μm). Boxed region is enlarged in right panel (scale bar=100 μm). (b) A regressing tumor, showing subtotal regression with melanoma cells present (scale bar=100 μm). Boxed region is enlarged in right panel (scale bar=50 μm). (c) Images of nonregressing (<26 °C) and regressing (32 °C) BRAFV600Emitf melanomas (as shown in b), stained with an antibody to detect cleaved-Caspase-3 (scale bar=50 μm).
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bertolotto C, Lesueur F, Giuliano S, et al. A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma. Nature. 2011;480:94–98. - PubMed
    1. Beuret L, Flori E, Denoyelle C, et al. Up-regulation of MET expression by alpha-melanocyte-stimulating hormone and MITF allows hepatocyte growth factor to protect melanocytes and melanoma cells from apoptosis. J Biol Chem. 2007;282:14140–14147. - PubMed
    1. Ceol CJ, Houvras Y, Jane-Valbuena J, et al. The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature. 2011;471:513–517. - PMC - PubMed
    1. Cheli Y, Giuliano S, Fenouille N, et al. Hypoxia and MITF control metastatic behaviour in mouse and human melanoma cells. Oncogene. 2012;10:2461–2470. - PubMed
    1. Cheli Y, Giuliano S, Botton T, et al. Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny. Oncogene. 2011;30:2307–2318. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources