1. The Effects of Phosphorylation at T118
and S251-3 of SOX2 on its Function in
Relation to Lung Squamous Cell
Carcinoma.
Niralini. Thayaparan, School of Life Sciences, University of Nottingham, UK
2. Acknowledgements
• Supervisor: Paul Scotting
• PhD Student: Elisa Marelli
Colleagues:
• Lawrence Farrell
• Scotty Jennings
• Andrew Wilkinson
3. Outline
• SOX2 is commonly associated with Lung Squamous
Cell Carcinoma (LSCC).
• This current study aimed to investigate in vitro effects
of phosphorylation at T118 and S251-3 of SOX2 on its
role in relation to LSCC.
• Desired phosphorylation mutants of mouse SOX2
(mSOX2) were generated and derivative genes were
overexpressed in HeLa, however desired reporter
construct was not generated.
4. Background
• LSCC is a sub-type of lung cancer that
causes ½ million global
deaths/annually.
• There is still lack of targeted
treatments due to incomplete
elucidation of causative mechanisms
(Yang et al., 2015).
• SOX2 is a transcription factor that is
commonly associated with LSCC but
little is known about its post-
translational effects (Malak et al.,
2015).
Protein Kinase C1
SOX2
P
HHAT
SHH
T118
5. Aims and objectives
Aim: Investigate in vitro effects of phosphorylation of SOX2 on
its function in proliferation in relation to LSCC.
Hypothesis: Phosphorylation of SOX2 enhances its
proliferation activity in relation to LSCC.
Objectives:
1) Generate mSOX2 phosphorylation mutants of interest.
2) Overexpress genes encoding mSOX2 wild-type (WT) and
desired phosphorylation mutants in HeLa cell line.
3) Generate HHAT reporter construct.
4) Determine in vitro effects of mSOX2 phosphorylation
mutants on HHAT promoter.
6. Methods
1) Generation of site-specific mSOX2
phosphorylation mutants
Mutagenic primer design then site-directed
mutagenesis.
2) Overexpression of genes encoding mSOX2 WT
and desired phosphorylation mutants in HeLa:
Transfection and western blot analysis.
8. 1) Mutagenic primer design
and SDM
Sequencing analysis
showed sequences of
site-directed mutants
resembled mSOX2
coding sequence except
at sites of interest. Figure 3: Sequence traces produced by
FinchTV, Geospiza, UK.
mSOX2 T118 mSOX2 T118D mSOX2 T118A
SOX2
P
T118
SOX2
P
T118D
SOX2
P
T118A
9. 2) Western blot analyses
WB: Mouse anti-Myc and rabbit anti-HA
IP: LiCor green anti-mouse and red anti-
rabbit
SUMO2
49
Figure 4: WB analysis of HeLa cells overexpressed with
genes encoding mSOX2 WT and desired phosphorylation
mutants.
38
28
14
17
kD
mSOX2
6
• Genes
encoding
mSOX2 WT/
mutants were
all
overexpressed
in HeLa.
• mSOX2 was
more degraded
in presence
than absence of
SUMO2 and
unaffected by
phosphorylation
at desired sites.
11. 4) Luciferase analysis
Figure 5: Nanog promoter activity caused by mSOX2
mutants relative to that by mSOX2. Anomalous results
have been excluded from calculation of mean luciferase
activity. Error bars represent ±SD. * p < 0.05.
Nanog promoter
activity relative to
that caused by
pcDNA3-mSOX2
WT:
• ~8000-fold lower
by pGL3-Nanog
alone.
• ~two-fold higher
by pcDNA3-
mSOX2S251-3D.
• Insignificantly
different by
pcDNA3-
mSOX2T118 mutant
conditions.
0
0.5
1
1.5
2
2.5
3
3.5
4
NanogPromoterActivity
(RelativetomSOX2WT)
mSOX2 WT/mutant
*
*
12. Summary
• Aim of this study was to investigate in vitro effects of
phosphorylation of SOX2 on its function in proliferation
in relation to LSCC.
• Phosphorylation mutants of interest were generated and
corresponding genes were successfully overexpressed
in HeLa, however desired reporter construct was
unsuccessfully generated.
• Robust conclusions could not be made regarding effects
of phosphorylation of SOX2 on its function in
proliferation in relation to LSCC.
13. References
• JUSTILIEN, V., WALSH, M. P., ALI, S. A., THOMPSON, E. A., MURRAY, N. R. & FIELDS, A.
P. 2014. The PRKCI and SOX2 oncogenes are coamplified and cooperate to activate
Hedgehog signaling in lung squamous cell carcinoma. Cancer Cell, 25, 139-51.
• YANG, L. L., ZHANG, X. C., CHUAI, S. K., CHEN, Z. H., XIE, Z., GUO, W. B., CHEN, S. L.,
LEI, Y. Y., GUO, L. H., GOU, L., SUN, H. W., ZHANG, Q., YANG, J. J., TU, H. Y., SU, J. & WU,
Y. L. 2015. Integrative Analyses of Lung Squamous Cell Carcinoma in Ten Chinese
Patients with Transcriptome Sequencing. J Genet Genomics, 42, 579-87.
Editor's Notes
Use background data
Explain luciferase system
Lung Squamous Cell carcinoma
Limited knowledge is available regarding phosphorylation effects of SOX2 on its proliferative role in LSCC.
Phosphorylation at T118 of SOX2 is suggested in vitro to be crucial for its recruitment to Hedgehog Acyltransferase gene (HHAT) promoter, which activates sonic hedgehog (SHH) proliferative pathway (Justilien et al., 2014).
Non-small cell lung cancer
DiagrAMS
tITLE
Simplify
Lung cancer causes 1.6 million deaths; more than 85% is explained by NSCLC.
Chromosomal region 3q 26 is the most commonly amplified, SOX2 is found between 3q26.3-27
Few binding pockets for inhibitors to bind
Could target the post-translational modifications of SOX2 which regulate SOX2 activity
Sumoylation at K247 was suggested to inhibit SOX2 from binding the fgf enhancer and from activating fgf transcription therefore inhibiting proliferation – luciferase assays.
Phosphorylation dependent SUMOylation - Western blot – assayed SUMOylation status of phosphorylation mutants
Phosphorylation of T118 is needed for the recruitment of SOX2 to HHAT promoter which processes and makes Shh active therefore driving Shh pathway
BMP4 has been identified as a downstream target of SOX2 by IHC assays
BMP4 promotes p21 which inhibits CDK2/cyclin E therefore BMP4 represses proliferation
Protein kinase mediated proliferation
Consecutive sites
Constitutive phosphorylation
Phosphorylation at S251-3 sites of mouse SOX2 (mSOX2) is suggested in vitro to promote its SUMOylation at K247 that inhibits its proliferative activity (Tsuruzoe et al., 2006, Van Hoof et al., 2009).
T118A and D – phosphorylation mimicking and eliminating mutants
Level of SUMO2 reduced in lanes 4-13 compared to that in lane 3
Non-specific bands in SUMO
Has been difficult to see SUMOylated SOX2 but also reporter SOX2 is utilised
Auto-regulation- thus undetectable SUMOylated SOX2 – could inhibit SOX2 from binding targets
HHAT protein produces hedgehog ligand. LSCC cells need protein kinase C1-SOX2-hedgehog signaling for proliferation.
Decrease in binding of Sox2 to HHAT in CHIP experiments in the presence of PKCi RNAI.
Differences in cell numbers- renilla but then leakage
Usage of LSCC cell line instead of HeLa – to mimic LSCC niche – different protein interactome
Generation of individual phosphorylation mutants based on S251-3 sites of mSOX2 – to see whether it is the additive or individual effect of mutating S251-3 sites that suggest that phosphorylation at these sites could promote proliferation.
HHAT reporter construct was not generated; if it had been, the importance of phosphorylation at T118 of SOX2 and thus appropriateness of usage of PKC1 inhibitors could have been verified.
Phosphorylation mutants of interest were generated; valuable resource for functional studies of SOX2.
Phosphorylation at S251-3 of mSOX2 was suggested to promote Nanog activation that is implicated in tumor development and embyrogenesis – further research needed.
