1. The study investigates the relationship between the BRAF and ERK3 proteins in melanoma. Knocking down BRAF decreases ERK3 expression at both the protein and mRNA levels, indicating BRAF regulates ERK3. 2. Overexpression of the mutated BRAF V600E protein increases ERK3 protein levels more than wild type BRAF. 3. Knocking down ERK3 in melanoma cell lines increases their migration in functional assays, suggesting ERK3 normally suppresses migration.

1. Minyi Chen1, Weiwen Long Ph.D. 1, Michael Kent Ph.D.2,3, Michael Markey Ph.D.1
1Wright State University Department of Biochemistry and Molecular Biology, 2Wright State University
Department of Dermatology, 3Dermatopathology Laboratory of Central States
Aims
• Investigate the clinic significance
of ERK3 and BRAF/BRAF V600E in
human melanoma, in
collaboration with DLCS.
• Elucidate how BRAF/BRAF V600E
upregulates ERK3.
• Identify the functions of the
BRAF-ERK3 signaling axis in
melanoma cell growth and
invasiveness.
Introduction
Arising from melanocytes, the melanin
pigment producing cell, melanoma makes up
75% of skin cancer deaths even though it
accounts for less than 5% of skin cancer
cases. As with other types of cancers,
melanoma is caused by genetic alterations.
Notably, BRAF is frequently mutated in
melanoma and the substitution of glutamic
acid to valine at codon 600 (V600E)
contributes to approximately 90% of all
BRAF activating mutations. BRAF V600E is
known to promote melanoma initiation and
metastatic progression by activating
multiple downstream targets, including
ERK1/2, and ERK3.
ERK3, an atypical MAPK, is usually an
unstable protein. Unlike other well-studied
MAPKs such as ERK1 and ERK2, little is
known regarding the regulation of ERK3
signaling. ERK3 is shown to be dysregulated
in melanoma and many other cancers. In
addition, a previous study demonstrated that
ERK3 level was upregulated BRAF V600E in
A375 melanoma cells. The current project
aims to elucidate the molecular mechanisms
by which BRAF V600E upregulates ERK3 and
investigate the clinical relevance of the
BRAF/ERK3 signaling axis in melanoma.
Melanocytes
Results
a) Western blots showing the
efficient knockdown of ERK3
by either stably expressing
shERK3 in A375 or by
transiently expressing siERK3.
Cells were then used for b)
trans-well cell migration assay
and c) scratch healing assay.
Knock down of ERK3 by either
transient siRNA or stable
lentiviral shRNA system in
both melanoma cell lines
significantly increased cell
migration under both
conditions.
a) Preliminary study of ERK3 expression in melanoma by
immunohistochemical (IHC) staining. Upper image: Nevus tissue showing
low level of ERK3 staining (brown color) Lower image: invasive Melanoma
tissue sample with strong ERK3 staining.
Knock down of BRAF downregulates ERK3 at both the protein level and mRNA level.
a) Western blots of BRAF, ERK3 and β-actin in A375 and OCM3 cells transfected with a non-targeting control
siRNA (siCtrl) or siRNA against BRAF (siBRAF).
b & c) mRNA levels were measured with RT-qPCR.
In both cell line, further experiment will be performed to test if BRAF also impacts ERK3 protein stability.
1.00 1.00
0.46
0.06
ERK3 BRAF
Foldchangeofgeneexpression
(normalizedtoGAPDH)
OCM3 shCtrl
shBRAF
1.00 1.00
0.80
0.53
ERK3 BRAF
Foldchangeofgeneexpression
(normalizedtoGAPDH)
A375 siCtrl
siBRAF
Me
Methods• DNA cloning
• Cell Transfection
• siRNA knock down of BRAF or ERK3 in melanoma cell lines with BRAF V600E
mutation: A375 (skin) & OCM3 (ophthalmic)
• A375 stably expressing shERK3 by lentiviral transduction
• 293T cells overexpressing BRAF or BRAF V600E
• Gene expression assays
• Real-time quantitative PCR (RT-qPCR)
• Western blotting
• Immunohistochemistry in clinical tissue specimens (from DLCS)
• Functional Assays
• Trans-well cell migration and scratch healing
• Data analysis
• Student’s t-test Trans-well assay with the upper serum
free media and lower complete media
Figure Source: Sean Crosson's HHMI online biology
• Analyze ERK3 expression by IHC and BRAF mutation status by
PCR in clinical tissue specimens of benign nevi, melanomas
and other skin cancers.
• Gene microarray of clinical tissue specimens for identifying
possible genetic regulation of the ERK3 pathway by BRAF.
• Examine the roles of ERK3 in melanoma cell proliferation,
invasion, and colony formation.
• Elucidate the underlying mechanism(s) and signaling pathways
by which knockdown of ERK3 greatly increases cell migration
Future Directions
• BRAF V600E has higher expression levels than wild type BRAF.
• Overexpression of BRAF upregulates ERK3 expression.
Conversely, knock down of BRAF downregulates ERK3, at least
partly through decreasing ERK3 mRNA level.
• Knock down of ERK3 in melanoma cell lines A375 and OCM3
stimulates cell migration ability, which is opposite to its role in
promoting migration of lung cancer cells.
Conclusions
Wright State University
Michael Markey’s lab, Weiwen Long’s lab
Dermatopathology Laboratory of Central States
Michael Kent, Ph.D. and DLCS laboratory
Acknowledgements
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shGIPZ Ctrl shERK3
AVERAGEDISTANCE
CHANGES(MM)
A375 shERK3 SCRATCH HEALING
ASSAY
A375 OCM3
shCtrl shERK3 siCtrl siERK3
ERK3
BRAF
β-Actin 110
335
79
294
0
100
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300
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Ctrl ERK3 knock down
CELLNUMBERPERFIELD
A375 & OCM3 shERK3 OR siERK3
TRANS-WELL MIGRATION
A375
OCM3
a)
a)
a)
b) Overexpression of
either BRAF or BRAF
V600E increases ERK3
protein level. West blots
of ERK3, BRAF and β-
actin of 293T cells
transfected with BRAF,
BRAF V600E or pcDNA3
empty vector.
b)
b)
b)
c)
c)
Nevus
Melanoma
A375 shGIPZ Ctrl
OCM3 siCtrl OCM3 siERK3
A375 shERK3
A375 shGIPZ Ctrl A375 shERK3