Cover/Title page
Supporting Information
Comparative Proteomic Profiling of Adrenocortical Neoplasia Using Mass Spectrometry
Jean Lucas Kremer1, Henrique Sanchez Ortega1, Talita Souza-Siqueira2, Claudia Blanes Angeli3, Leo Kei Iwai4, Claudimara Ferini Pacicco Lotfi1*
1. Institute of Biomedical Sciences, Department of Anatomy, University of São Paulo, São Paulo, Brazil, Av. Prof. Lineu Prestes, 2415, Butantan, São Paulo, SP, 05508-000.
2. School of Medicine, Department of Clinical Medicine, University of São Paulo, Brazil, Av. Dr. Arnaldo, 455, Cerqueira César, São Paulo, SP, 01246903.
3. Institute of Biomedical Sciences, Department of Parasitology, University of São Paulo, Brazil, Av. Prof. Lineu Prestes, 1374, Butantan, São Paulo, SP, 05508-000.
4. Butantan Institute, Laboratory of Applied Toxicology, Center of Toxins, Immune- response and Cell Signaling LETA/CeTICS Laboratory, Brazil, Av. Vital Brasil, 1500 - Butantã, São Paulo, SP, 05503-900.
*Corresponding author: clotfi@usp.br.
Table of Contents
Figure S1. The histopathological characteristics of A) normal human adrenal (NHA); B) adenoma adrenocortical (ACA); C) carcinoma adrenocortical; D) Primary macronodular hyperplasia adrenal (with mutation in the ARMC5 gene (PMAHw); E) Primary macronodular hyperplasia adrenal (without mutation in the ARMC5 gene (PMAHwt). Supplementary_File_Figure S1.pdf.
Figure S2. Correlation of proteomics-identified targets with clinical data in ACC using Gene Expression Profiling Interactive Analysis (GEPIA platform); Overall Survival (OS); disease-free survival (DFS). SRRM2 = serine/arginine repetitive matrix 2; NUP160 = nuclear pore complex protein; RBM3 = RNA-binding motif protein 3. Supplementary_File_Figure S2.pdf.
Figure S3. Differentially expressed protein of hyperplasia with and without ARMC5 mutation. A) The number of total proteins expressed in PMAHwt and PMAHw, the overlapping part of the circle indicates the number of proteins expressed in both groups; B) Heatmap of DEPs identified in PMAHw and PMAHwt; C) Volcano plots for significantly differentially expressed proteins PMAHwt vs. PMAHw. The -log10 (FDR) is plotted against log2 (Fold-Change). PMAHw = primary macronodular adrenal hyperplasia with ARMC5 mutation, PMAHwt = primary macronodular adrenal hyperplasia without ARMC5 mutation, DEPs = differentially expressed proteins, FDR = false discovery rate. Supplementary_File_Figure S3.pdf.
Figure S4. Biological processes identified by network enrichment analysis using Gene Ontology (GO) and STRING platforms of 64 adrenocortical carcinomas (ACC) upregulated proteins relative to adrenocortical adenomas (ACA). FDR = false discovery rate. Supplementary_File_Figure S4.pdf.
Figure S5. High expression of STMN1 and PHGDH is associated with aggressive behavior and poor patient outcomes in ACC, whereas NDRG4 expression is the opposite. Kaplan-Meier survival plot of overall survival (OS), disease-free survival (DFS), and expression across tumor stages of A) Stathmin 1 (STMN1); B) phosphoglycerate dehydrogenase (PHGDH); C) N-myc downregulated gene family 4 (NDRG4) expression from TCGA analysis. n = 38. Supplementary_File_Figure S5.pdf.
Table S1 - Total proteins identified in primary macronodular adrenocortical hyperplasia without ARMC5 mutation. Supplementary_File_Table S1.xlsx.
Table S2 - Total proteins quantified in all analyzed samples. Supplementary_File_Table S2.xlsx.
Table S3 - Proteins identified specifically in each group and Proteins exclusively identified comparing all groups. Supplementary_File_Table S3.xlsx.
Table S4 - Proteins identified as up- or downregulated between the two conditions. Supplementary_File_Table S4.xlsx.
Table S5 - Cross-validation of DEPs in ACC-SNUH identified in ACC-USP on comparison between the two conditions. Supplementary_File_Table S5.xlsx.
A
B
C
ZR
ZF
ZG
C
0.1 mm
0,1 mm
0.1 mm
D
E
0.0 mm
0.1 mm
SRRM2
Disease Free Survival
1.0
Low SRRM2 TPM
High SRRM2 TPM
Logrank p=0.026
0.8
HR(high)-2.1
p(HR)-0.031
Percent survival
n[high)-38
0.6
n(low)-38
0.4
0.2
0.0
0
50
100
150
Months
Overall Survival
1.0
Low SRRM2 TPM
High SRRM2 TPM
Logrank p=0.31
0.8
HR(high)=1.5
p(HR)-0.33
Percent survival
n(high)-38
0.6
n(low)-38
0.4
0.2
0.0
0
50
100
150
Months
Expression across tumor stages
:
F value = 2.14
Pr(>F) = 0.103
9
→
-
Stage I
Stage II
Stage III
Stage IV
NUP160
Overall Survival
1.0
Low NUP160 TPM
High NUP160 TPM
Logrank p=0.085
0.8
HR(high)-2
p(HR)-0.09
Percent survival
n(high)=38
0.6
n(low)-38
0.4
0.2
0.0
0
50
100
150
Months
Disease Free Survival
0
Low NUP160 TPM
High NUP160 TPM
Logrank p=0.0039
0.8
HR(high)=2.7
p(HR)-0.0058
Percent survival
n(high)-38
0.6
n(low)-38
0.4
0.2
0.0
0
50
100
150
Months
Expression across tumor stages
F value = 1.39
Pr(>F) = 0.254
+
(
2
-
Stage 1
Stage II
Stage III
Stage IV
RBM3
Disease Free Survival
1.0
Overall Survival
Low RBM3 TPM
High RBM3 TPM
1.0
Low RBM3 TPM
Logrank p=0.32
High RBM3 TPM
0.8
HR(high)-1.4
Logrank p=0.75
p(HR)-0.32
0.8
HR(high)-1.1
Percent survival
n(high)-38
p[HR)-0.76
n(low)-38
n[high)-38
0.6
Percent survival
0.6
n(low)-38
0.4
0.4
0.2
0,2
0.0
0.0
0
50
100
150
Months
0
50
100
150
Months
Expression across tumor stages
=
F value = 0.656
Pr(>F) - 0.582
10
9
00
A
6
5
Stage I
Stage II
Stage III
Stage IV
A)
PMAHwt
PMAHw
717
4475
203
B)
PMAHW
PMAHW
PMAHW
PMAHW
PMAHW
PMAHwt
PMAHwt
PMAHwt
PMAHwt
PMAHwt
C)
PMAHwt_vs_PMAHw_DEPs.
NOSTRIN
PCP4
6
C4BPB
PSPH
AKAP7
SNU13
STAB1
POLR21
GPKOW
-log10(P-Value)
4
POLR2B
CES2
RNPS1
Regulation
RDH13
POTEJ
PNPT1
ACYP2
Downregulated
Not Significant
Upregulated
2
0
-1.0
-0.5
0.0
0.5
1.0
log2(Fold Change)
ACYP2
AKA7G
C4BPB
GPKOW NOSTN
PCP4
RPB2
RPB9
POTEJ
SERB
RNPS1 NH2L1
EST2
PNPT1
RDH13
STAB1
Biological Process (Gene Ontology) enrichment
FDR
Regulation of stem cell differentiation
3.4e-04
5.2e-04
Chromatin remodeling
7.7e-04
1.1e-03
Nu cleobase-containing compound metabolic process
1
1.7e-03
2.6e-03
Nucleic acid metabolic process
İ
Groups at similarity 0.8
Gene count
Cellular nitrogen compound metabolic
process
1
☒
5
☒
Macromolecule metabolic process
15
30
Nitrogen compound metabolic process
Organic substance metabolic process
40
Metabolic process
Primary metabolic process
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
Signal
Reactome Pathways enrichment
FDR
Positive epigenetic regulation of rRNA
2.0e-05
expression
1
5.8e-05
Processing of Capped Intron-Containing Pre-mRNA
1.6e-04
4.7e-04
Metabolism of RNA
1.3e-03
3.8e-03
mRNA Splicing - Major Pathway
☒
Groups at similarity 0.8
Gene count
Late Phase of HIV Life Cycle
1
☒
3
Regulation of TP53 Activity through Acetylation
☒ 6
Transport of the SLBP Dependant Mature mRNA
9
Transport of the SLBP independent Mature mRNA
12
Transport of Mature mRNA Derived from an Intronless Transcript
SUMOyla tion
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Signal
STMN1
Overall Survival
Disease Free Survival
Expression across tumor stages
1.0
Low STMN1 TPM
High STMN1 TPM
0
Logrank p=1.4e-06
HR(high)=5.6
0.8
p(HR)=1.4e-05
n(high)=38
CƠ
n(low)=38
0.6
CD
0.4
0.2
៛
0.0
2
0
50
100
150
Months
F value = 8.64
Pr(>F) = 5.82e-05
Stage I
Stage II
Stage III
Stage IV
B)
PHGDH
Disease Free Survival
1.0
Low PHGDH TPM
High PHGDH TPM
Logrank p=0.0051
HR(high)=2.6
0.8
p(HR)=0.007
n(high)=38
n(low)-38
0.6
0.4
0.2
0.0
0
50
100
150
Months
Expression across tumor stages
F value = 9.34
00
Pr(>F) = 2.78e-05
6
+
2
.
0
Stage I
Stage II
Stage III
Stage IV
C)
NDRG4
Disease Free Survival
Overall Survival
Expression across tumor stages
1.0
Low NDRG4 TPM
1.0
High NDRG4 TPM
Low NDRG4 TPM
High NDRG4 TPM
F value = 4.23 Pr(>F) = 0.00822
Logrank p=0.0012
HR(high)=0.33
Logrank p=5.6e-07
0.8
p(HR)=0.0019
0.8
HR(high)=0.097
0
n(high)=38 n(low)=38
-P(HR) :- 3.2e-05
Percent survival
Percent survival
n(high)=38 n(low)=38
-
0.6
0.6
6
0.4
0.4
៛
0.2
0.2
2
0.0
0.0
0
0
50
100
150
0
50
100
150
Stage I
Stage II
Stage III
Stage IV
Months
Months
1.0
Low STMN1 TPM
High STMN1 TPM
Logrank p=4.1e-06
0.8
HR(high)=7.3
p(HR)=7.2e-05
Percent survival
n(high)=38
0.6
n(low)=38
0.4
0.2
0.0
0
50
100
150
Months
Overall Survival
1.0
Low PHGDH TPM
High PHGDH TPM
Logrank p=0.0028
0.8
HR(high)=3.5
p(HR)=0.0047
Percent survival
n(high)=38
0.6
n(low)-38
0.4
0.2
0.0
0
50
100
150
Months
Percent survival
Percent survival