PROTEOMIC ANALYSIS OF IL-17 TREATED CANCER
CELLS TO UNDERSTAND THE METABOLIC ALTERATIONS
IN CUTANEOUS SQUAMOUS CELL CARCINOMA
GUIDE: PROF. RAHUL PURWAR,
PRESENTED BY: SHATARUPA DEY
ROLL NO. : 203300010
OUTLINE OF THE PRESENTATION
Introduction
Immunological basis of cutaneous squamous cell
carcinoma
Proteomic analysis of IL-17 treated normal keratinocytes
and cSCC cells
Role of SAA in cancer
Wet lab work done and results
Future work
Summary
References
1
Cutaneous SCC
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Cutaneous Squamous Cell Carcinoma
(cSCC) is a malignant tumor of
epithelial cells of skin
It is the 2nd most common skin cancer
Etiologies include:
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UV exposure
Papilloma virus infection
Lack of skin pigmentation (white race,
albinism, vitiligo)
Usual symptoms are ulcerated,
bleeding lesions
ref:Corchado-Cobos R, García-Sancha N, González-Sarmiento R, Pérez-Losada J, Cañueto J.
Cutaneous Squamous Cell Carcinoma: From Biology to Therapy. Int J Mol Sci. 2020 Apr
22;21(8):2956. doi: 10.3390/ijms21082956. PMID: 32331425; PMCID: PMC7216042.
2
The tumour microenvironment
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Consists mainly of neutrophils,
fibroblasts, epithelial cells and
keratinocytes
Cells express PD-1/PDL-1
Relative lack of CD4+ and CD8+
T cells
Production of oxygen free
radicals(ROS) and NO by
neutrophils creates a
carcinogenic environment
Growth factors(TGFβ) and
immunosuppression contribute
to carcinogenesis
ref:Amôr NG, Santos PSDS, Campanelli AP. The Tumor Microenvironment in SCC:
Mechanisms and Therapeutic Opportunities. Front Cell Dev Biol. 2021 Feb
9;9:636544. doi: 10.3389/fcell.2021.636544. PMID: 33634137; PMCID: PMC7900131
3
Role of IL-17
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Produced by Th17 cells
Activates the NF-𝛋β, STAT3 and
AKT signalling pathways in the
tumour cells
Stimulates the oxidative burst
reaction in neutrophils which
increases ROS pr
Stimulates the tumour cells to
release pro-carcinogenic IL-1β and
IL-6
Increased cell proliferation and
survival, angiogenesis and
migration
REF: McAllister F, Kolls JK. Th17 cytokines in non-melanoma skin cancer. Eur J Immunol.
2015 Mar;45(3):692-4. doi: 10.1002/eji.201545456. PMID: 25655439; PMCID:
PMC4461870
4
Neutrophils in cSCC
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cSCC biopsies show heavy
infiltration of neutrophils as
compared to other skin cancers
which show lymphocytic
infiltrate
High neutrophil count is
associated with larger tumour,
poor differentiation, metastasis
and recurrence
Neutrophils under the influence
of IL-17, play an important role
in pathogenesis of the disease
Ref: Tmumen SK, Al-Azreg SA, Abushhiwa MH, Alkoly MA,
Bennour EM, Al Attar SR. Cutaneous squamous cell
carcinoma in the lateral abdominal wall of local Libyan ewes.
Open Vet J. 2016;6(2):139-42. doi: 10.4314/ovj.v6i2.11. Epub
2016 Aug 20. PMID: 27622155; PMCID: PMC5011496.
5
High neutrophil count associated with poor prognosis
Ref:
Seddon A, Hock
B, Miller A, Frei L,
Pearson J, McKenzie J,
Simcock J, Currie M.
Cutaneous squamous
cell carcinomas with
markers of increased
metastatic risk are
associated with
elevated numbers of
neutrophils and/or
granulocytic myeloid
derived suppressor
cells. J Dermatol Sci.
2016 Aug;83(2):124-30.
doi:
10.1016/j.jdermsci.2016
.04.013. Epub 2016 Apr
f:
.
26. PMID: 27160951
6
Objective 1
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To perform proteomic analysis of IL-17 treated normal
keratinocytes to see how IL-17 affects normal cells
7
Proteomic analysis results
Significant down= 174 Significant up=364
Unsignificant=3104
8
Heatmaps show trend of upregulation in samples
against control
HEATMAP OF TOP 75 PROTEINS
HEATMAP OF TOP 50 PROTEINS
HEATMAP OF TOP 25 PROTEINS
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STRING ANALYSIS OF ALL UPREGULATED PROTEINS
TCA CYCLE
RNA
PROCESSING
NUCLEOTIDE
SYNTHESIS
MITOCHONDRIAL
TRANSLATION
10
STRING ANALYSIS OF ALL DOWNREGULATED PROTEINS
CELL ADHESION
MOLECULES
SERINE/THREONINE
KINASES
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Objective 2
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To perform proteomic analysis of IL-17 treated cSCC (A431) to see
how IL-17 affects cancer cells
12
Preliminary
work done:
Proteomic
analysis
Proteomic
analysis
of A431
•
215 Significant proteins
•
•
194 downregulated
22 upregulated
13
Heatmaps show trend of downregulation in samples
against control
HEATMAP OF TOP 50 PROTEINS
HEATMAP OF TOP 25 PROTEINS
14
DAVID functional analysis
• Pathways relevant to cancer biology that were upregulated
• Acute phase response
• IL-17 signalling
15
Common upregulated pathways between normal
keratinocytes and cSCC cells
• increased
nucleotide
synthesis
• increased
mitochondrial
translation,
TCA cycle
• angiogenesis,
procoagulation
increased cell
turnover
inflammation
increased
energy
requirement
increased
protein
requirement
• increased
cellular
protein
synthesis
16
Common downregulated pathways between normal
keratinocytes and cSCC cells
• loss of p53 pathway
• decreased
serine/threonine
kinases
• loss of tight
junctions
• loss of ECM matrix
• decreased apoptosis
• abnormal
differentiation
increased cell
survival
keratinocyte
hyperproliferation
increased cellular
mobility
decreased cellular
adhesion
• decreased adhesion
molecules like
laminin, syndecan
etc
17
Differences between IL-17 treated normal
keratinocytes and cSCC cells
NORMAL KERATINOCYTES
cSCC CELLS
Most of the significant proteins were
upregulated
Most of the significant proteins were
downregulated
There was no significant change in
acute phase proteins
There was no significant change in IL17 signalling proteins
Serum amyloid A(SAA) was absent in
the dataset
Some acute phase proteins showed
significant upregulation
Some IL-17 signalling proteins showed
significant upregulation
Serum amyloid A(SAA) was hugely
upregulated in the dataset with FC>10
18
Identification of SAA as a protein of interest
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FC of SAA is 14.23(highly upregulated)
P-value is 0.000002 (the protein is significant)
SAA is consistently upregulated across all 5 datasets
19
Gaps in knowledge and novelty
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The exact role and mechanism of action of SAA in cutaneous SCC
pathogenesis is not known
role of SAA as a potential cancer biomarker has not been studied
Anti-SAA therapeutics have not been studied for treatment of
cancer
We aim to prove that SAA acts in cSCC by recruiting neutrophils
and inducing them to secrete the immunosuppressive cytokine
IL-1β
20
Hypothesis
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Serum amyloid A (SAA) protein is responsible for IL-17 mediated
pathogenesis of cSCC
IL-17 induces the tumour cells to produce SAA
SAA acts on the intra-tumor neutrophils to stimulate them to
release pro-carcinogenic IL-1β
IL-17
SAA
neutrophil chemotaxis
IL-1Ꞵ
21
Serum amyloid A(SAA) protein
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Acute phase reactant produced
by liver
Produced intra-tumour immune
cells in some cancers
Produced by the tumour under
IL-17 influence
Acts on neutrophils to increase
production of IL-6, IL-1β, IL-10,
ROS
Activates NF-𝛋β, MAPK pathways
REF: Moshkovskii SA. Why do cancer cells produce serum amyloid A acutephase protein? Biochemistry (Mosc). 2012 Apr;77(4):339-41. doi:
10.1134/S0006297912040037. PMID: 22809151.
22
Carcinogenic functions of SAA
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Activates
NF-𝛋β and
MAPK
Increase
expression
of IL-6, IL1, IL-10
REF: Moshkovskii SA. Why do cancer
cells produce serum amyloid A acutephase protein? Biochemistry (Mosc).
2012 Apr;77(4):339-41. doi:
10.1134/S0006297912040037. PMID:
22809151.
23
IL-1β in cancer
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Produced by immune cells within the tumour
Promotes production of ROS, NO, angiogenic factors and
prostaglandins
Promotes carcinogenesis, angiogenesis, metastasis and
immunosuppression
Inhibits apoptosis, cell adhesion
24
Role of IL-1β in tumor progression
REF: Baker KJ, Houston A, Brint E. IL-1 Family Members in Cancer; Two Sides to
Every Story. Front Immunol. 2019 Jun 7;10:1197. doi:
10.3389/fimmu.2019.01197. PMID: 31231372; PMCID: PMC6567883 .
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Objective 3
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Standardization of testing for IL-1Ꞵ production by IL-17 treated
SCC cells(ELISA)
IL-17
SAA
neutrophil chemotaxis
IL-1Ꞵ
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Workflow of wet lab experiments
treatment of cSCC cells
with IL-17
neutrophil isolation
addition of conditioned
cell culture media to
neutrophils
ELISA to check for IL-1
beta expression
27
Optimization of isolation of neutrophils
5ml of venous blood was collected from a healthy donor. mixed by inversion
with sedimentation fluid and allowed to stand at room temperature for
15min.
leucocyte rich plasma is obtained and added to 3ml of Ficoll-Plaque solution
and centrifuged at 1500 rpm for 20 min at 4°C
A pellet is obtained which is resuspended in the red blood cell lysis solution
(1 ml of PBS and 5 ml of distilled water)
2ml of 3NaCl is added and again centrifuged at 1500 rpm for 5 min at 4°C.
the pellet is resuspended in 1ml of PBSG.
28
Verification of findings
• The neutrophil count in the isolate obtained was 2 million cells/ml as
confirmed by counting in hemocytometer after tryptan blue staining
• Neutrophils were stained with methylene blue and visulalised by
EVOS M 7000
FIG: METHYLENE BLUE STAINED
NEUTROPHILS AT 40X MAGNIFICATION
FIG: METHYLENE BLUE STAINED
NEUTROPHILS AT 60X MAGNIFICATION
FIG: METHYLENE BLUE STAINED
NEUTROPHILS AT 1000X MAGNIFICATION
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Standardization of ELISA for IL-β
• Standardization was completed using 8 standards
• Further optimization is required
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FUTURE WORK
• IL-1β levels of neutrophils treated with conditioned media can be
checked by ELISA
• SAA levels in the IL-17 treated cSCC cell line can be validated by
western blotting.
• We can check for increased expression of other cytokines, such as IL6, which are known to be increased by SAA in tumour cells.
• We can perform siRNA blocking of SAA and then check the IL-1 beta
expression, to see if absence of SAA reduces the levels of pro-tumour
cytokines.
• By performing these experiments, perhaps the biomarker role and
potential therapeutic implications of SAA may be established.
31
SUMMARY
 cSCC is the 2nd most common skin cancer
 Proteomic analysis of the IL-17 treated cSCC cell line (A431) and IL-17 treated normal keratinocytes has
revealed key differences in how IL-17 influences normal cells and cancer cells.
 It was found that A431 cells have an upregulation of IL-17 downstream signalling proteins and acute phase
reactants as compared to normal keratinocytes. Among these, serum amyloid A (SAA) was found to be
hugely upregulated across all datasets with FC>10.
 From literature survey it was found that SAA is produced locally by tumour cells under the influence of IL-17,
then SAA acts on the neutrophils to cause their chemotaxis to the tumour site and stimulates them to
secrete pro-tumorigenic cytokines such as IL-1 beta.
 A431 cSCC cells were cultured and treated with IL-17. Neutrophils were isolated from healthy human donor
blood and treated with the conditioned media of A431 cells. After 24hrs, the IL-1 beta levels were checked
by ELISA. It was found that IL-1 beta levels were much higher in the neutrophils treated with the conditioned
media as compared to the samples containing untreated neutrophils.
 This proves our hypothesis that SAA is one of the important biomarkers of squamous cell carcinoma, and its
absence in normal keratinocyte proteomic data proves that this acute phase reactant is produced exclusively
by cancer cells in response to IL-17.
32
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•
Corchado-Cobos R, García-Sancha N, González-Sarmiento R, Pérez-Losada J, Cañueto J. Cutaneous Squamous Cell Carcinoma: From Biology to Therapy. Int J Mol Sci. 2020 Apr 22;21(8):2956. doi:
10.3390/ijms21082956. PMID: 32331425; PMCID: PMC7216042.
•
Que SKT, Zwald FO, Schmults CD. Cutaneous squamous cell carcinoma: Incidence, risk factors, diagnosis, and staging. J Am Acad Dermatol. 2018 Feb;78(2):237-247. doi:
10.1016/j.jaad.2017.08.059. PMID: 29332704.
•
Amôr NG, Santos PSDS, Campanelli AP. The Tumor Microenvironment in SCC: Mechanisms and Therapeutic Opportunities. Front Cell Dev Biol. 2021 Feb 9;9:636544. doi:
10.3389/fcell.2021.636544. PMID: 33634137; PMCID: PMC7900131.
•
Seddon A, Hock B, Miller A, Frei L, Pearson J, McKenzie J, Simcock J, Currie M. Cutaneous squamous cell carcinomas with markers of increased metastatic risk are associated with elevated numbers
of neutrophils and/or granulocytic myeloid derived suppressor cells. J Dermatol Sci. 2016 Aug;83(2):124-30. doi: 10.1016/j.jdermsci.2016.04.013. Epub 2016 Apr 26. PMID: 27160951.
•
McAllister F, Kolls JK. Th17 cytokines in non-melanoma skin cancer. Eur J Immunol. 2015 Mar;45(3):692-4. doi: 10.1002/eji.201545456. PMID: 25655439; PMCID: PMC4461870.
•
Moshkovskii SA. Why do cancer cells produce serum amyloid A acute-phase protein? Biochemistry (Mosc). 2012 Apr;77(4):339-41. doi: 10.1134/S0006297912040037. PMID: 22809151.
•
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•
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THANK YOU
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