What is MITF?
As noted by Carreira et al[1]:
It is widely held that cells with metastatic properties
such as invasiveness and expression of matrix metalloproteinases arise through
the stepwise accumulation of genetic lesions arising from genetic instability and
“clonal evolution.”
By contrast, we show here that in melanomas invasiveness
can be regulated epigenetically by the microphthalmia-associated transcription
factor, Mitf, via regulation of the DIAPH1 gene encoding the
diaphanous-related formin Dia1 that promotes actin polymerization and
coordinates the actin cytoskeleton and microtubule networks at the cell
periphery.
Low Mitf levels lead to down-regulation of Dia1, reorganization
of the actin cytoskeleton, and increased ROCK-dependent invasiveness, whereas
increased Mitf expression leads to decreased invasiveness.
Significantly the regulation of Dia1 by Mitf also
controls p27Kip1-degradation such
that reduced Mitf levels lead to a p27Kip1-dependent
G1 arrest. Thus Mitf, via regulation of Dia1, can both inhibit invasiveness and
promote proliferation.
The results imply variations in the repertoire of
environmental cues that determine Mitf activity will dictate the
differentiation, proliferative, and invasive/migratory potential of melanoma
cells through a dynamic epigenetic mechanism.
Note that the discussion above shows that overexpression of
MITF leads to less invasiveness. We show the details of the pathway dynamics
below.
They continue:
Although Mitf is clearly required for melanoma
proliferation, why it is necessary has not been previously established. To
understand how Mitf depletion led to a block in G1/S transition, we examined a
number of known markers of proliferation and the cell cycle. Western blotting
of cells transfected with control or Mitfspecific siRNA revealed that depletion of Mitf led, as
expected from our previous work, to decreased expression of p21Cip1, but intriguingly also induced expression
of the p27Kip1 cyclin-dependent kinase
inhibitor. Note that similar results were obtained using a second Mitf siRNA
directed against a different region of Mitf. We also observed reduced
expression of cyclin E, and PCNA, most likely as an indirect effect of the cell
cycle arrest, but no change in cyclin D1 or Cdk2 levels. Tubulin and lamin B
were used as loading controls.
Finally they note:
In summary, Mitf appears to lie right at the heart of the
melanocyte, coordinating survival, cell cycle entry and exit, cytoskeletal
organization, melanosome assembly and transport, differentiation and migration/metastasis.
As such, understanding Mitf regulation and function may well be the key to
achieving one of the major aims of cancer research, an effective melanoma
therapy.
Thus the MITF function, in their view, is critical.
MITF Targeting and Control
From a current Eureka posting, Zoufal writes of work
recently reported in Genes &
Development by Pogenberg[2] et
al[3]
focused upon targeting and thus controlling MITF:
The results, published in the scientific journal
"Genes & Development", throw new light on the workings of the
so-called Microphthalmia-associated Transcription Factor MITF, that is not only
connected to skin cancer, but also to a variety of hereditary diseases where
the production of the skin pigment melanin is disturbed, and to certain aspects
of ageing. "Our data could provide a rational basis for the development of
tailor-made drugs targeting MITF", explains first author Vivian Pogenberg
from the Hamburg branch of the European Molecular Biology Laboratory
But MITF also makes stem cells turn into melanocytes in
the first place and controls cell proliferation and death in these cells.
That's why MITF is called a master regulator. In fact, it also has functions in
other cell types like mast cells of the immune system and bone eating
osteoclasts...
Mutations in MITF not only play a role in the development
of skin cancer, but also cause severe genetic diseases like the Tietz and
Waardenburg syndromes that lead to deafness, skin and hair pigmentation
defects, abnormal eye anatomy and altered vision. The transcription factor also
plays a role in our hair turning grey with age and other age-related
pigmentation alterations…
Crystals scatter X-rays in characteristic ways and
produce diffraction patterns from which the structure of the crystal - and here
MITF - can be reconstructed. The analysis revealed unexpected molecular
insertions that give MITF a unique kink. MITF forms a dimer with a long
coiled-coil protein "zipper", and the kink in this zipper limits
MITF's ability to bind to other transcription factors.
This paper thus appears to provide a targeted means to
control MITF and then perhaps melanoma metastasis. Identification of binding
sites is essential to control.
More on MITF
We can provide a bit more history on MITF and its functions.
As NCBI states[4],
MITF, microphthalmia-associated transcription factor (3p14.2-p14.1):
This gene encodes a transcription factor that contains
both basic helix-loop-helix and leucine zipper structural features. It
regulates the differentiation and development of melanocytes retinal pigment
epithelium and is also responsible for pigment cell-specific transcription of the
melanogenesis enzyme genes. Heterozygous mutations in the this gene cause
auditory-pigmentary syndromes, such as Waardenburg syndrome type 2 and Tietz
syndrome
MITF has been known to be a key player in melanoma
metastasis. As Chin et al state in their review paper[5]:
MITF, a melanoma oncogene targeted by amplification
The promise of DNA-based structural alterations as the
entry point for gene discovery has been illustrated by the recent
identification of MITF as a melanoma oncogene. The discovery of MITF amplification
in melanoma derived from an integrated analysis of genomic copy gains and
losses, together with sample-matched mRNA expression data.
When clustering algorithms were applied to SNP
array-derived chromosomal copy number data generated for the NCI-60 cancer cell
line collection, some of these cell lines aggregated according to tissue of
origin, including several melanoma cell lines. The bidimensionality of the
hierarchical algorithm also enabled the identification of chromosomal alterations
driving these lineage-restricted clustering patterns, and suggested that
lineage-specific cancer genes might reside within the genomic regions
implicated.
For the melanoma cell lines, the common genomic
alteration was a region of copy gain at chromosome 3p14-3p13. To facilitate the
identification of an oncogene targeted by this amplification event, the NCI- 60
collection was partitioned based on the presence or absence of copy gain at the
relevant chromosome 3p locus. This partitioning served as a two-class
distinction that drove a supervised analysis of sample-matched gene expression
data. Although the gene expression signature that emerged was dominated by
melanocyte lineage genes (as expected given that only melanoma cell lines comprised
the 3p-amplified class), MITF was the only gene
showing significantly increased expression in association with the 3p-amplified
melanoma cell lines that also mapped to the common region of 3p copy gain.
MITF amplification was
subsequently detected in 10% of primary cutaneous and 15%–20% of metastatic
melanomas.
Although the majority of amplifications were low level
(e.g., four to six copies per cell), high-level amplicons were also observed,
including one sample that exhibited >100 copies per diploid genome. A
Kaplan- Meier analysis performed on metastatic melanomas suggested that MITF
amplification in this setting correlated with adverse 5-yr patient
survival.
Finally, ectopic MITF overexpression complemented BRAFV600E in conferring soft agar colony growth to
immortalized melanocytes engineered to express TERT, and to lack the pRB and
p53 pathways. These functional studies thereby suggested a genetic context that
might characterize a subset of human melanomas whose survival is dependent on
MITF.
MITF also exemplifies a
newly recognized “lineage survival” oncogenic mechanism, wherein tumor genetic alterations
may target survival functions also operant in the relevant cellular lineages
during development and differentiation.
Thus, while the discovery of MITF
amplification began as a systematic genomics-based survey of many human
cancer types, it provides a striking convergence of melanoma oncogene discovery
and melanocyte development.
Next, as Genovese et al state[6]:
Histidine triad nucleotide-binding protein 1 (HINT1) is a
haploinsufficient tumor suppressor gene that inhibits the Wnt/β-catenin pathway
in colon cancer cells and Microphthalmia-associated transcription factor (MITF)
activity in human mast cells. MITF and β-catenin play a central role in
melanocyte and melanoma cell survival, and this study aimed to investigate the
effects of HINT1 on the MITF and β-catenin pathways in malignant melanoma
cells.
We found that HINT1 inhibits MITF and β-catenin
transcriptional activity, and both proteins can be co-immunoprecipitated with
an anti-HINT1-specific antibody in melanoma cell lines. Stable, constitutive
overexpression of the HINT1 protein in human melanoma cells significantly
impaired cell proliferation in vitro and tumorigenesis in vivo.
These effects were associated with a decreased expression
of cyclin D1 and BCL2, well known MITF and β-catenin transcription targets,
respectively. We also demonstrated that BCL2 and cyclin D1 can partially rescue
the HINT1-driven phenotype. Moreover, we found in ChIP assays that HINT1 binds the
chromatin at MITF and β-catenin sites in BCL2 and cyclin D1 promoters,
respectively, and that mSIN3a and HDAC1, well known transcriptional repressors,
can be co-immunoprecipitated with an anti-HINT1-specific antibody. These
findings support the tumor suppressor activity of HINT1 gene in melanoma cells
by promoting the formation of non-functional complexes with oncogenic
transcription factors like MITF and β-catenin.
Finally as Larribere et al state[7]:
Microphthalmia-associated transcription factor (MITF) M-form
is a melanocyte-specific transcription factor that plays a key role in
melanocyte development, survival, and differentiation. Here, we identified MITF
as a new substrate of caspases and we characterized the cleavage site after Asp
345 in the C-terminal domain.
We show that expression of a non-cleavable form of MITF
renders melanoma cells resistant to apoptotic stimuli, and we found that the
C-terminal fragment generated upon caspase cleavage is endowed with a
proapoptotic activity that sensitizes melanoma cells to death signals. The
proapoptotic function gained by MITF following its processing by caspases provides
a tissue-restricted means to modulate death in melanocyte and melanoma cells.
Their observation does show the impact of MITF in the
control of melanoma.
Observations
The recent work demonstrates that we can through an
understanding of the pathways then target specific pathway control proteins by
understanding their structure. We can already control B RAF in certain
circumstances by targeting its specificity and that controlling the path but
allowing MITF control in a broad sense may actually be much more powerful if
the results hold for clinical applications.
The ability to find, characterize, and design binding site
specific blocking agents is an essential step in a broader control of multiple
cancers.
[1]
Carreira, S., et al, Mitf regulation of Dia1 controls melanoma proliferation
and invasiveness, Genes Dev. 2006 20: 3426-3439.
[2]
See Pogenberg in http://genesdev.cshlp.org/
[3]
Zoufal, T., X-ray
analysis deciphers master regulator important for skin cancer, http://www.eurekalert.org/pub_releases/2012-12/ded-xad113012.php
[5]
Chin et al, Malignant melanoma: genetics and
therapeutics in the genomic era, Genes Dev. 2006 20: 2149-2182
[6]
Genovese G, et al, The tumor suppressor HINT1
regulates MITF and β-catenin transcriptional activity in melanoma cells, Cell
Cycle. 2012 Jun 1; 11(11):2206-15.
[7]
Larribere, et al, The cleavage of microphthalmia-associated transcription
factor, MITF, by caspases plays an essential role in melanocyte and melanoma
cell apoptosis, Genes Dev. 2005 19: 1980-1985.
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