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Cartilage oligomeric matrix protein acts as a molecular biomarker in multiple cancer types

Bingjie Guo1 . Yajing Wang2 . Wenyu Liu3 . Sailong Zhang4(D

Received: 14 August 2022 / Accepted: 26 September 2022 / Published online: 18 October 2022 @ The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO) 2022

Abstract

Purpose The main function of cartilage oligomeric matrix protein (COMP) is to maintain the synthesis and stability of the extracellular matrix by interacting with collagen. At present, there are relatively few studies on the role of this protein in tumors. This study aimed to explore the relationship between COMP and pan-cancer, and analyzed its diagnostic and prognostic value.

Methods The Cancer Genome Atlas database, the Genotype-Tissue Expression database and the Cancer Cell Line Encyclo- pedia database was used for gene expression analysis. The receiver operating characteristic curve was used to assess the diag- nostic value of COMP in pan-cancer. Kaplan-Meier plots were used to assess the relationship between COMP expression and prognosis of cancers. R software v4.1.1 was used for statistical analysis, and the ggplot2 package was used for visualization. Results COMP was significantly overexpressed in 15 human cancers and showed significantly difference in 12 molecular subtypes and 16 immune subtypes. In addition, the expression of COMP is associated with tumor immune evasion. The ROC curve showed that the expression of COMP could predict the occurrence of 16 kinds of tumors with relative accuracy, including adrenocortical carcinoma (ACC) (AUC = 0.737), breast invasive carcinoma (BRCA) (AUC = 0.896), colon adeno- carcinoma (COAD) (AUC = 0.760), colon adenocarcinoma/rectum adenocarcinoma esophageal carcinoma (COADREAD) (AUC = 0.775), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC) (AUC = 0.875), kidney renal papillary cell carcinoma (KIRP) (AUC = 0.773), kidney chromophobe (KICH) (AUC = 0.809), ovarian serous cystadenocarcinoma (OV) (AUC = 0.906), prostate adenocarcinoma (PRAD) (AUC = 0.721), pancreatic adenocarcinoma (PAAD) (AUC = 0.944), rectum adenocarcinoma (READ) (AUC = 0.792), skin cutaneous melanoma (SKCM) (AUC = 0.746), stomach adenocar- cinoma (STAD) (AUC = 0.711), testicular germ cell tumors (TGCT) (AUC = 0.823), thymoma (THYM) (AUC = 0.777) and uterine carcinosarcoma (UCS) (AUC = 0.769). Furthermore, COMP expression was correlated with overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) in ACC (OS, HR = 4.95, DSS, HR = 5.55, PFI, HR = 2.79), BLCA (OS, HR = 1.59, DSS, HR = 1.72, PFI, HR = 1.36), KIRC (OS, HR = 1.36, DSS, HR = 1.94, PFI, HR = 1.57) and COADREAD (OS, HR = 1.46, DSS, HR = 1.98, PFI, HR = 1.43). We selected previously unreported blad- der urothelial carcinoma (BLCA) for further study and found that COMP could be an independent risk factor for OS, DSS and PFI. Moreover, we found differentially expressed genes of COMP in BLCA and obtained top 10 hub genes, including LGR4, LGR5, RSPO2, RSPO1, RSPO3, RNF43,ZNRF3, FYN, LYN and SYK. Finally, we verified the function of COMP at the cellular level by using J82 and T24 cells and found that knockdown of COMP could significantly inhibit migration and invasion. This finding supports that COMP could be a potential biomarker for pan-cancer diagnosis and prognosis encompassing tumor microenvironment, disease stage and prognosis.

Conclusion This finding supports that COMP could be a potential biomarker for pan-cancer diagnosis and prognosis encom- passing tumor microenvironment, disease stage and prognosis.

Keywords Cartilage oligomeric matrix protein (COMP) · Molecular biomarker · Pan-cancer · Multiple omics integrative analysis · Tumor microenvironment

Bingjie Guo and Yajing Wang have equally contributed to the article.

Introduction

Tumor tissue is a complex assembly of tumor cells and their surrounding tumor microenvironment (TME) [1]. TME is an ecological niche that stimulates the progression of can- cer, which contains various cells, such as pathochemical entities, extracellular matrices, normal stromal fibroblasts, and immune cells [2]. An increasing number of reports have shown that there are interactions between tumor cells, stromal cells, and immune cells in the TME, which could influence anti-tumor immunity and immunotherapy [3, 4]. Immunosuppressive cells in the TME can inhibit the func- tion of cytotoxic T cells, allowing tumor immune evasion and reducing therapeutic efficacy [5, 6]. Unfortunately, it is very complicated to simultaneously study various cellular constituents and their molecular mechanisms in the TME by the traditional molecular biology experiments, especially for the complex and diverse immune phase cells in the TME [7, 8]. However, the bioinformatics approach has proven to be one of the most effective strategies to address this challenge. In addition, bioinformatics can also exert its advantages in research fields, such as tumor pathogenesis, mutation cap- ture, and biomarker detection.

COMP, one of the thrombospondin family, consists of five matricellular calcium-binding proteins that regulate growth factors, cytokines, and responses to injury [9]. The protein is composed of five identical subunits, which are linked by disulfide bonds to form a protein with 524 kDa. COMP can bind to collagen with high affinity through its C-terminal domain [10, 11]. Elevated COMP expression is associated with various diseases, such as systemic sclerosis, vascular atherosclerosis, and rheumatoid arthritis [12-14]. A recent report suggested that COMP could be used as a novel biomarker to assess the risk of liver cirrhosis and hepatocel- lular carcinoma [15]. In addition, it has been reported that COMP can also be a potential biomarker in chronic hepatitis C and liver fibrosis process [16]. Studies have shown that COMP mRNA expression may be highly correlated with tumorigenesis, including the occurrence and progression of colon, breast, and lung cancer [17-20]. However, there is currently insufficient scientific evidence to indicate whether COMP can be a marker of cancer and its role in the immune microenvironment.

Therefore, in this study, we investigated the expression and prognostic significance of COMP in multiple cancer types and examined the potential role of COMP in OS, DSS, and PFI. In addition, we investigated the effect of COMP on TME in different tumors. Moreover, we further detected COMP-related co-expressed genes and differen- tially expressed genes in the BLCA.

Materials and methods

COMP expression analysis

The tumor tissue data for this study were obtained from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases, with a total of 15,776 samples for 33 tumor types. The tumor cell line data in this study were obtained from the Cancer Cell Line Encyclo- pedia (CCLE) database [21]. The above data are used for statistical analysis using R Software V4.1.1.

The relationship between COMP expression and tumor immunity

The relationship between the expression of COMP and the molecular subtypes and immune subtypes in different tumor types was analyzed using TISIDB database [22]. In addition, this database was used to analyze the correlation between COMP expression and immunomodulator.

This study explored the relationship between COMP expression and immune cell infiltration in different tumor cells using TIMER2 database. In addition, we used the TIMER2 database to explore the correlation between COMP expression and infiltration of 4 immunosuppressive cells (cancer-associated fibroblasts, M2 subtype of tumor-asso- ciated macrophages, myeloid-derived suppressor cells, and regulatory T) in different tumor types.

COMP mutation landscape in different tumors

We used the cBioPortal database to explore the COMP mutation landscape as well as the copy-number alteration [23, 24].

COMP-binding proteins’ interaction network

A total of 50 COMP-binding proteins were obtained using the STRING network, with the parameters’ set to minimum required interaction score [“medium confidence (0.400)”]. The protein-protein interaction (PPI) was visualized using Cytoscape (version 3.7.1).

Gene ontology and Kyoto encyclopedia of genes and genomes analyses

The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the enrichment of 50 COMP-binding proteins. “ClusterProfiler” package in R was used for analysis [25, 26].

Values of COMP expression in predicting tumorigenesis

We used receiver-operating characteristic (ROC) curves to evaluate the accuracy of COMP in predicting the occurrence of different tumor types. AUC (0.5-0.7) means low accu- racy, AUC (0.7-0.9) means a certain accuracy, and AUC (>0.9) means a high accuracy.

Survival analysis in different tumor types

The Kaplan-Meier plots were used to assess the relation- ship between COMP expression and survival prognosis (OS, DSS and PFI) in different tumor types by COX regression analysis. The visualization was performed using “surminer” in the R package.

Relationship between COMP expression and clinical characteristics in BLCA

In BLCA, the relationship between COMP expression and different clinical characteristics was presented using box- plot. The downloaded fragments per kilobase per million data were converted into transcripts per million reads and analyzed after log2 conversion. The Wilcoxon rank sum test was used to detect two groups of data.

Univariate and multivariate cox regression analyses in BLCA

In BLCA, the expression of COMP and its clinical char- acteristics were analyzed by univariate and multivariate COX regression to further determine the prognostic value of COMP in OS, DSS, and PFI.

Searching for COMP co-expressed genes in BLCA

In BLCA, we explored 50 co-expressed genes that were posi- tively and negatively correlated with COMP expression. The volcano map was plotted in ggplot2 package with a threshold of |log2 fold-change|>1.0 and adjusted p value<0.05. In addition, we construct PPI network of different expression genes using a threshold of |log2 fold-change|>2.0. The MCC algorithm of “CytoHubba” in Cytoscape was used to analyze the central gene.

To elucidate the potential effects of selected genes in the BLCA, the high- and low-expression groups were analyzed by GSEA using the MSigDB collection (h.all.v7.2.symbols. gmt).

Cell culture

The human HCC cell lines J82, T24 were purchased from Cell bank of Chinese Academy of Sciences. The J82 and T24 cells were cultured in high DMEM (Hyclone, USA) and MEM (Hyclone, USA) respectively, which supplemented with 1% streptomycin and penicillin and 10% FBS (Gibco, USA). Cells were cultured in a humidified atmosphere of 5% CO2 at 37 °℃.

Wound-healing assay and Transwell assays

For wound-healing assay, J82 and T24 cells were digested into a suspension at the density of 1× 105 cells/well, and spread evenly on 12-well plates. The cells were draw a thin line at the bottom by using a 10 uL pipette tip gently. The wounds at 0 h and 24 h were recorded under the micro- scope (Leica, Germany). Relative wound closure (%)= [Area (24 h)-Area (0 h)] / Area (0 h).

The migration assay was performed by Transwell cham- bers (Corning, USA) on 24-well cell culture plates. The upper and lower culture chambers were separated by 8-um pore diameter. J82 and T24 cells in a density of 1× 104/ well were seeded in the upper chamber. For the invasion assays, the Matrigel (BD Biosciences, USA) was diluted with serum-free medium and added 100 µL/well to the upper chamber sand. J82 and T24 cells were stained with 0.1% crystal violet for 20 min, and were observed under the microscope (Leica, Germany).

Western blot analysis

Whole-cell lysates were prepared for the Western blot analy- sis of expression of COMP (Abcam, ab231977, UK) and B-actin (CST, 4970, USA). The concentration of protein was determined by BCA kit (Thermo Scientific, A53225, USA). Protein samples were isolated using 10% SDS-PAGE gel and then transferred to polyvinylidene difluoride mem- branes. The secondary antibodies (CST, 7074, USA) were incubated, and the membranes were washed and visualized using enhanced chemiluminescence kit (Thermo Scientific, USA) and Gel Imaging System (Syngene, USA).

Knockdown of COMP in cells

Small interfering RNA specific to COMP (si-COMP) (5’- AGAAACUUGAGCUGUUGAUGCC-3’, 5’-GGCUAU CAAGACAGCUCAAGUUUCU-3’) and the scramble siRNA (control) were purchased from Shanghai Bio-Link Company (Shanghai, China). J82 and T24 cells were spread on 6-well plates and then transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen, Eugene, OR, USA).

Results

COMP expression in different tumors

In this study, we first investigated the expression of COMP in normal tissues. COMP showed relatively high level in adipose tissue, blood vessel, breast, cervix uteri, lung, nerve, prostate, salivary, skin, testis, thyroid, and vagina. On the contrast, especially, the expression of COMP in blood vessels was the highest in all tissues. However, COMP showed relatively low level in adrenal gland, blood, bone marrow, brain, fallopian tube, liver, ovary, pituitary, and spleen (Fig. 1a). CCLE database results showed that COMP was expressed in all cell lines. As the figure shown, embryonal cell lines showed relative high expression of COMP and bile duct showed relative low expression of COMP (Fig. 1b). The TCGA database showed that COMP is significantly overexpressed in 10 cancer types, such as BLCA, colon adenocarcinoma (COAD), and kidney renal clear cell carcinoma (KIRC) (Fig. 1c). In addition, results from the GTEX database showed that COMP was sig- nificantly increased in 15 cancer types, such as BLCA, COAD, and KIRC (Fig. 1d).

Relationship between COMP expression and immune or molecular subtypes in different tumors

As the results from the TISIDB database shown, COMP expression was correlated with the immune subtypes of 16 kinds of tumors, including BLCA (Fig. 2a), breast invasive carcinoma (BRCA) (Fig. 2b), cervical squamous cell carci- noma and endocervical adenocarcinoma (CESC) (Fig. 2c), head and neck squamous cell carcinoma (HNSC) (Fig. 2d), kidney chromophobe (KICH) (Fig. 2e), kidney renal pap- illary cell carcinoma (KIRP) (Fig. 2f), liver hepatocel- lular carcinoma (LIHC) (Fig. 2g), lung adenocarcinoma (LUAD) (Fig. 2h), lung squamous cell carcinoma (LUSC) (Fig. 2i), mesothelioma (MESO) (Fig. 2j), ovarian serous cystadenocarcinoma (OV) (Fig. 2k), prostate adenocar- cinoma (PRAD) (Fig. 2l), sarcoma (SARC) (Fig. 2m), skin cutaneous melanoma (SKCM) (Fig. 2n), testicular germ cell tumors (TGCT) (Fig. 2o), and thyroid carcinoma (THCA) (Fig. 2p).

Moreover, we also explored the relationship between COMP expression and molecular subtypes, and found that COMP was correlated with 12 cancer types such as BRCA, COAD, and HNSC. For BRCA, COMP was identified to express less in basal (Fig. 3a). For COAD, COMP was expressed the highest in CIN (Fig. 3b). For HNSC, mesenchymal showed a higher COMP level than

other groups (Fig. 3c). COMP was expressed more in c2c-CIMP than any other groups in KIRP (Fig. 3d). For LIHC, COMP showed a higher expression in icluster1 than all the other groups (Fig. 3e). For LUSC, COMP was expressed more in secretory than anyone else (Fig. 3f). For OV, no groups expressed more COMP than mesen- chymal (Fig. 3g). No groups expressed COMP so much as kinase signaling in pheochromocytoma and paragan- glioma (PCPG) (Fig. 3h). For PRAD, COMP expressed ever so much in ETV4 (Fig. 3i). For stomach adenocar- cinoma (STAD), COMP was expressed the highest in GS (Fig. 3j). For SKCM, COMP was expressed the highest in hotspot mutants (Fig. 3k). For uterine corpus endometrial carcinoma (UCEC), COMP was expressed the highest in CN-HIGH (Fig. 3l).

Conspicuously, the COMP expression showed a signifi- cantly correlation with the immune stimulators (Supplemen- tary Fig. S1) and immune inhibitors (Supplementary Fig. S2) in the most tumors except for GBM and LGG. Further- more, we also revealed the association of chemokines with COMP, among which most chemokines could be regulated by COMP except CCL16, CCL24, and CCL25 (Supplemen- tary Fig. S3). Furthermore, COMP exerted a vital role in modulating most receptors in the most tumors except for GBM and LGG (Supplementary Fig. S4).

COMP mutation landscape in different tumors

A total of 10,967 samples from 32 studies in TCGA were used to explore COMP mutations using the cBioPortal data- base. The results showed that the COMP mutation frequency was the highest in OV and UCEC, both exceeding 5%. In SKCM, the mutation frequency of COMP was more than 4%, which is also at a relative high level (Fig. 4a). In terms of mutation counts, COMP levels were higher in SKCM, LUSC, and BLCA than other tumor types (Fig. 4b). In addi- tion, the mutation site of COMP showed that a total of 119 sites were mutated in COMP, which had a total of 757 amino acids. Among them, R672Q and R740S sites showed higher mutation frequency than any other sites (Fig. 4c).

COMP is associated with immunoevasive TME via different mechanisms

For 39 tumor types in TCGA database, we investigated the association between COMP and 6 common types of immune cells infiltration (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells). The results showed that, except for ACC, DLBC, KICH, UCS, and UVM, the more expression of COMP, the more immune cells infiltrated in other TME. Furthermore, in HNSC, LIHC, LUSC, and PAAD, up-regulated COMP was positively associated with infiltration of all six types of immune cells.

Fig. 1 COMP expression analysis. a COMP expression in different normal tissues. b CCLE database of COMP in different tumor cell lines. c TCGA database of COMP in different tumors and normal tis- sues. d COMP expression in tumors and normal tissues with the data of the TCGA and GTEx database. * p<0.05, ** p<0.01, *** p<0.001

a

15

The expression of COMP Log2 (TPM+1)

0

10

o

00 D

IN GD

000

D

0

5

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300 000

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000 0

090

%

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A

o

4

D

-

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O

M

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0

®

Adipose Tissue

Adrenal Gland

Bladder

Blood

Blood Vessel

Bone Marrow

Brain

Breast

Cervix Uteri

Colon

Esophagus

Fallopian Tube

Heart

Kidney

Liver

Lung

Muscle

Nerve

Ovary

Pancreas

Pituitary

Prostate

Salivary Gland

Skin

Small Intestine

Spleen

Stomach

Testis

Thyroid

Uterus

Vagina

b

1.75

·

The expression of COMP

1.50

·

0

·

0

0

0

0

1.25

50

000 0

CEDIDO

00

o

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00

0

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0

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0

8

8

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1

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9

0.75

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8

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6

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0300

8

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0

0

0

0

0

0

0

0.50

Bile Duct

Bladder

Bone

Brain

Breast Cervical

o

Colon/Colorectal

Embryonal

Endometrial/Uterine

Engineered

Esophageal

Eye

Fibroblast

Gallbladder

Gastric

Head and Neck

Kidney

Leukemia

Liposarcoma

Liver

Lung

Lymphoma

Myeloma

Neuroblastoma

Non-ous

Ovarian

Pancreatic

Prostate

Rhabdoid

Sarcoma

Skin

Teratoma

Thyroid

c

10

ns

ns



Normal

The expression of COMP Log2 (TPM+1)


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8

Tumor


**

**


**

6


**

ns

ns

ns

4

2

0

T

BLCA

BRCA

CHOL

COAD

ESCA

HNSC

KICH

KIRC

KIRP

LIHC

T

T

LUAD

LUSC

PAAD

PRAD

READ

STAD

THCA

UCEC

0

12



ns



ÅR

ns


4%






*



ns










.

Normal

The expression of COMP Log2 (TPM+1)

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Tumor

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S

ACC

BLCA

BRCA

CESC

CHOL

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LAML

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC

SKCM

STAD

TGCT

THCA

THYM

UCEC

UCS

UVM

Fig. 2 Relationship between COMP expression and immune subtypes in different tumors. a BLCA. b BRCA. c CESC. d HNSC. e KICH. f KIRP. g LIHC. h LUAD. i LUSC. j MESO. k OV. l PRAD. m SARC. n SKCM. o TGCT. p THCA

BLCA :: COMP_exp

a

Pv=2.75e-08

b

BRCA :: COMP_exp Pv=1.06e-15

CESC :: COMP_exp Pv=3.17e-02

n=C1 369,C2 390,C3 191,C4 92,C6 40

c

HNSC : COMP_exp

n=C1 173,C2 164,C3 21,C4 36,C6 3

n=C1 77,C2 217,C4 6

d

Pv=4.6e-03

n=C1 128,C2 379,C3 2,C4 2,C6 3

Expression (log2CPM)

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Expression (log2CPM)

Expression (log2CPM)

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10

Expression (log2CPM)

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C1

C2

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C4

C6

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C2

C3

C4

C6

-10

C1

C2

C4

Subtype

C3

Subtype

Subtype

C1

C2

C4

C6

Subtype

KICH :: COMP_exp

e

Pv=4.9e-02 n=C1 2,C3 38,C4 12,C5 13

f

KIRP :: COMP_exp

Pv=6.02e-04

g

LIHC :: COMP_exp

Pv=3.91e-11

n=C1 22,C2 45,C3 135,C4 159,C6 1

h

LUAD : COMP_exp

n=C1 3,C2 4,C3 202,C4 66,C5 2,C6 2

Pv=6.8e-05

n=C1 83,C2 147,C3 179,C4 20,C6 28

Expression (log2CPM)

Expression (log2CPM)

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Expression (log2CPM)

10

Expression (log2CPM)

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10

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-5

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C1

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C5

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C2

C3

C4

C5

C6

C1

C2

C3

C4

C6

C1

C2

C3

C4

C6

Subtype

Subtype

Subtype

LUSC :: COMP_exp

Pv=1.47e-03

MESO :: COMP_exp Pv=5.09e-03

k

OV :: COMP_exp Pv=3.52e-03

PRAD :: COMP_exp

n=C1 275,C2 182,C3 8,C4 7,C6 14

n=C1 32,C2 21,C3 8,C4 11,C6 11

n=C1 46,C2 159,C3 3,C4 61

Pv=6.99e-05

n=C1 35,C2 18,C3 307,C4 45

Expression (log2CPM)

10

Expression (log2CPM)

10

Expression (log2CPM)

Expression (log2CPM)

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10

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8

5

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0

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C1

C2

C3

C4

C6

C1

C2

C3

C4

C6

-10

C1

C2

C3

C4

C1

C2

C3

C4

Subtype

Subtype

Subtype

Subtype

m

SARC :: COMP_exp Pv=4.6e-04 n=C1 64,C2 38,C3 42,C4 59,C6 20

n

SKCM :: COMP_exp Pv=1.48e-02 n=C1 41,C2 27,C3 14,C4 19,C6 2

o

TGCT :: COMP_exp Pv=3.19e-02 n=C1 42,C2 104,C3 2,C4 1

p

THCA: COMP_exp

Pv=7.17e-03

n=C1 2,C2 13,C3 459,C4 22,C6 3

Expression (log2CPM)

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Expression (log2CPM)

10

Expression (log2CPM)

10

Expression (log2CPM)

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10

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5

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C1

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C6

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C6

Subtype

Subtype

Subtype

Subtype

COMP showed an excellent correlation with immune cells infiltration in LUSC (r=0.23 ~ 0.48) and PAAD (r=0.17 ~ 0.43), and a good correlation in HNSC (r=0.14 ~ 0.35) and LIHC (r=0.25 ~ 0.37). Furthermore, we found the strong- est correlation between COMP expression and neutrophil infiltration in CHOL (r=0.55) (Fig. 5a).

In addition, we investigated the association between COMP expression and infiltration of common immuno- suppressive cells that promote T-cell exclusion (CAF,

Macrophage M2, MDSC, and Tregs). The results showed that the expression of COMP was positively correlated with the invasion of CAF, but negatively correlated with the invasion of macrophage M2 in most tumors except GBM, PCPG, and THYM. Notably, in KIRC, LIHC, MESO, and UCES, the expression of COMP expression had positive correlation with tumor infiltration of MSDC, while in BLCA, BRCA, COAD, HNSC, HNSC-HPV-, LUSC, PCPG showed the opposite. In addition, for Tregs,

Fig. 3 Relationship between COMP expression and molecular subtypes in different tumors. a BRCA. b COAD. c HNSC. d KIRP. e LIHC. f LUSC. g OV. h PCPG. i PRAD. j STAD. k SKCM. I UCEC

BRCA :: COMP_exp

COAD :: COMP_exp

a

Pv=2.91e-16 n=Basal 172,

b

Pv=3.24e-02 n=CIN 226, GS 49.

HNSC :: COMP_exp Pv=2.05e-14

KIRP :: COMP_exp Pv=2.46e-06 n=C1 95,

C

Her2 73.

n=Atypical 67, Basal 87, Classical 48, Mesenchymal 74

d

LumA 508,

HM-SNV 6.

C2a 35, C2b 22.

LumB 191.

15

Normal 137

HM-indel 60

C2c-CIMP 9

Expression (log2CPM)

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Expression (log2CPM)

Expression (log2CPM)

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Expression (log2CPM)

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10

:

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5

5

:

:

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-5

-5

-5

-5

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-10

-15

Basal

Her2

LumA

LumB

Normal

Atypical

Basal

Classical

Mesenchymal

CIN

GS

HM-SNV

HM-indel

C1

C2a

C2b

C2c-CIMP

Subtype

Subtype

Subtype

Subtype

e

LIHC :: COMP_exp

Pv=4.79e-06

f

LUSC :: COMP_exp

Pv=1.53e-06

n=basal 42, classical 63, primitive 26, secretory 39

g

OV :: COMP_exp

Pv=1.02e-16

n=Differentiated 66, Immunoreactive 78, Mesenchymal 71. Proliferative 78

h

PCPG : COMP_exp

n=iCluster:1 64,

Pv=1.93e-08

iCluster:2 55,

iCluster:3 63

Expression (log2CPM)

10

Expression (log2CPM)

10

Expression (log2CPM)

n=Corticaladmixture 22, Kinasesignaling 68, Pseudohypoxia 61. Wnt-altered 22

15

Expression (log2CPM)

10

5

10

5

5

5

0

8

!

0

0

0

:

-5

-5

-5

-5

:

-10

-10

-10

iCluster:1

iCluster:2

iCluster:3

basal

classical

primitive

secretory

Differentiated

Immunoreactive

Mesenchymal

Proliferative

Corticaladmixture

Kinasesignaling

Pseudohypoxia

Wnt-altered

Subtype

Subtype

Subtype

Subtype

i

PRAD :: COMP_exp

STAD :: COMP_exp Pv=4.4e-03

k

UCEC :: COMP_exp

Pv=3.7e-03

SKCM :: COMP_exp Pv=8.02e-03

Pv=5.61e-04

n=1-ERG 152,

n=CIN 223, EBV 30, GS 50.

Expression (log2CPM)

n=BRAF_Hotspot_Mutants 150, NF1_Any_Mutants 27. RAS_Hotspot_Mutants 92. Triple_WT 46

n=CN_HIGH 160, CN_LOW 144, MSI 124, POLE 79

2-ETV1 28.

3-ETV4 14.

4-FLI1 4,

5-SPOP 37.

HM-SNV 7, HM-indel 73

6-FOXA1 9.

15

Expression (log2CPM)

15

7-IDH1 3,

Expression (log2CPM)

Expression (log2CPM)

8-other 86

10

10

5

10

10

5

0

:

-5

5

5

0

8

8

C

G

0

6

-10

0

_Hotspot_Mutants

NF1_Any_Mutants

RAS_Hotspot_Mutants

Triple_WT

0

-5

-5

-5

-10

-10

1-ERG

2-ETV1

3-ETV4

4-FLI1

5-SPOP

Sub. 6-FOXA1

7-IDH1

8-other

CIN

EBV

GS

HM-SNV

HM-indel

CN_HIGH

CN_LOW

MSI

POLE

Subtype

Subtype

Subtype

Subtype

COMP expression was positively correlated in PCPG and THCA, and negatively correlated in SKCM and SKCM- metastasis (Fig. 5b).

We next explored the impact of COMP on immune check- point blockade. Moreover, we also compared with other bio- markers to explore the accuracy of COMP in predicting OS. The results showed that in 25 cohort studies, COMP could function in 9 groups (AUC>0.5). The predictive ability of COMP is comparable to that of T. Clonality, but lower than that of TIDE, MSI. score, CD274 CD8, IFNG, and Merck18. This indicates that COMP is at a moderate level for OS pre- diction accuracy (Fig. 5c).

GO and KEGG analyses of COMP-binding proteins

We screened 50 COMP-related binding proteins using STRING, visualized them with Cytoscape, and performed GO and KEGG enrichment analysis. As the results shown, GO enrichment mainly contained extracellular structure organization, plasma membrane receptor complex, and extracellular matrix structural constituent (Fig. 6c). KEGG analysis showed that the pathway related to ECM-receptor interaction, PI3K-Akt signaling pathway, Human papillo- mavirus infection, and Focal adhesion (Fig. 6d).

Clinical and Translational Oncology (2023) 25:535-554

Mutation

Structural Variant

Amplification

Deep Deletion

☒ Multiple Alterations

TGCT (TCGA, PanCancer Atlas)

Amplification

PCPG (TCGA, PanCancer Atlas)

KIRP (TCGA, PanCancer Atlas)

Shallow Deletion

KIRC (TCGA, PanCancer Atlas)

KICH (TCGA, PanCancer Atlas)

THCA (TCGA, PanCancer Atlas) LAML (TCGA, PanCancer Atlas) ☐

☒ Gain

☐ Diploid

Deep Deletion

Structural Variant Splice (VUS)

☐ GBM (TCGA, PanCancer Atlas)

Truncating (VUS)

HNSC (TCGA, PanCancer Atlas)

Inframe (VUS)

PRAD (TCGA, PanCancer Atlas)

Missense (VUS)

THYM (TCGA, PanCancer Atlas)

PAAD (TCGA, PanCancer Atlas) LGG (TCGA, PanCancer Atlas)

Not mutated

☒ LUAD (TCGA, PanCancer Atlas)

UVM (TCGA, PanCancer Atlas) BRCA (TCGA, PanCancer Atlas) STAD (TCGA, PanCancer Atlas) LUSC (TCGA, PanCancer Atlas)

DLBC (TCGA, PanCancer Atlas)

BLCA (TCGA, PanCancer Atlas)

Melanoma

ACC (TCGA, PanCancer Atlas)

Non-Small Cell Lung Cancer Bladder Urothelial Carcinoma

6%

MESO (TCGA, PanCancer Atlas)

Esophagogastric Adenocarcinoma

542

5%

SARC (TCGA, PanCancer Atlas) COAD (TCGA, PanCancer Atlas)

Mature B-Cell Neoplasms

Colorectal Adenocarcinoma

a

Alteration Frequency

4%

CESC(TCGA, PanCancer Atlas)

000098880099000006

Head and Neck Squamous Cell Carcinoma

3%

ESCA (TCGA, PanCancer Atlas)

Esophageal Squamous Cell Carcinoma

Cervical Squamous Cell Carcinoma

757aa

Fig. 4 COMP mutation landscape in different tumors. a The mutation frequency of COMP in different TCGA studies. b The counts of mutations

2%

CHOL (TCGA, PanCancer Atlas)

LIHICI (TCGA, PanCancer Atlas)

Undifferentiated Stomach Adenocarcinoma

Hepatocellular Carcinoma

1%

UCS (TCGA, PanCancer Atlas)

Cervical Adenocarcinoma

Structural variant data

SKCM (TCGA, PanCancer Atlas)

Ovarian Epithelial Tumor

Mutation data CNA data

SV/Fusion (1)

UCEC (TCGA, PanCancer Atlas)

Endometrial Carcinoma

Ov (TCGA, PanCancer Atlas)

Renal Non-Clear Cell Carcinoma

TSP_C

Renal Clear Cell Carcinoma

Glioblastoma

Sarcoma Invasive Breast Carcinoma

Splice (8)

600

Cholangiocarcinoma

Pancreatic Adenocarcinoma

Pleural Mesothelioma

Diffuse Glioma

TSP_3

Adrenocortical Carcinoma

14

Prostate Adenocarcinoma

Inframe (1)

TSP_3

b

Mutation Count (log2(value + 1))

12

Fibrolamellar Carcinoma

Thymic Epithelial Tumor Leukemia

TSP_3

10

TS

Non-Seminomatous Germ Cell Tumor

400

8

Seminoma

TSP_3

Ocular Melanoma

6

Well-Differentiated Thyroid Cancer

Truncating

Pheochromocytoma

TSP_3

4

Miscellaneous Neuroepithelial Tumor

2

Encapsulated Glioma

0

Missense (102)

EGF_CA

200

EGF_CA

in different tumors. c The mutation sites of COMP

COMP

C

# COMP Mutations

5

0

0

Fig. 5 Correlations of COMP expression with immunoevasive TME via different mechanisms. a Infiltration by immune cell in different tumors. b Infiltration by immunosuppressive cell types in pan-cancer. c Comparing COMP with other standardized cancer immune evasion biomarkers. CAF, cancer-associated fibroblast; MDSC, myeloid- derived suppressor cells; Tregs, regulatory T cell

a

ACC

b

ACC

C

Random

BLCA

BLCA

COMP

BRCA

BRCA

BRCA-Basal

BRCA-Basal

BRCA-Her2

BRCA-Her2

BRCA-Luminal

BRCA-LumA

TIDE

CESC

BRCA-LumB

CHOL

CESC

Zhao2019_PD1_Glioblastoma_Pre

COAD

CHOL

Pos=8,Neg=7

MSI.Score

847

Zhao2019_PD1_Glioblastoma_Post

DLBC

COAD

Pos=6,Nog=3

VanAllen2015_CTLA4_Melanoma

ESCA

DLBC

Pos=19,Neg=23

Uppaluri2020_PD1_HNSC_Pre

GBM

ESCA

P value

Pos=8,Neg=15

P value

GBM

Uppaluri2020_PD1_HNSC_Post

HNSC

1.00

1.00

147

Pos=9,Neg=13

HNSC-HPVneg

HNSC

6.45

Ruppin2021_PD1_NSCLC

0.75

HNSC-HPV-

0.75

TMB

Pos=7,Neg=15

HNSC-HPVpos

Riaz2017_PD1_Melanoma_Ipi.Prog

KICH

HNSC-HPV+

0.50

0.50

Pos=4,Neg=22

KIRC

KICH

Riaz2017_PD1_Melanoma_Ipi.Naive

Pos=6,Neg=19

0.25

KIRC

0.25

CD274

Prat2017_PD1_NSCLC-HNSC-Melanoma Pos=21,Neg=12

KIRP

0.00

KIRP

0.00

Nathanson2017_CTLA4_Melanoma_Pre

LGG

LGG

Pos=4,Neg=5

LIHC

Correlation

LIHC

Correlation

Nathanson2017_CTLA4_Melanoma_Post

Pos=4,Nog=11

LUAD

1.0

1.0

Miao2018_JCB_Kidney_Clear

LUAD

Pos=20,Neg=13

LUSC

0.5

LUSC

0.5

CD8

McDermott2018_PDL 1_Kidney_Clear

Pos=20,Neg=61

MESO

0.0

MESO

0.0

Mariathasan2018_PDL1_Bladder_mUC

OV

Pos=68,Neg=230

-0.5

OV

-0.5

Liu2019_PD1_Melanoma_Ipi.Prog

PAAD

PAAD

Pos=16,Neg=31

-1.0

Liu2019_PD1_Melanoma_Ipi.Naive

PCPG

-1.0

PCPG

IFNG

.

Pos=33,Neg=41

PRAD

PRAD

Lauss2017_ACT_Melanoma

Pos=10,Neg=15

READ

READ

Kim2018_PD1_Gastric

Pos=12,Neg=33

SARC

SARC

T.Clonality

Hugo2016_PD1_Melanoma

SKCM

SKCM

Pos=14,Neg=12

Hee2020_PD1_NSCLC_Oncomine

SKCM-Metastasis

SKCM-Metastasis

Pos=9,Nog=12

Gide2019_PD1_Melanoma

SKCM-Primary

SKCM-Primary

Pos=19,Neg=22

Gide2019_PD1+CTLA4_Melanoma

STAD

STAD

B.Clonality

Pos=21,Neg=11

-

TGCT

TGCT

Chen2016_PD1_Melanoma_Nanostring

Pos=6,Neg=9

THCA

THCA

Chen2016_CTLA4_Melanoma_Nanostring

Pos=5,Neg=11

THYM

THYM

Braun2020_PD1_Kidney_Clear

UCEC

Pos=201,Neg=94

UCEC

UCS

UCS

UVM

UVM

Purity

B Cell

CD8+ T Cell

CD4+ T Cell

Macrophage

Neutrophil

Dendritic Cell

CAF

Macrophage M2

Merck18

MDSC

Tregs

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

AUC

Diagnostic value analysis of COMP in pan-cancer

The ROC curve was used to evaluate the predictive diag- nostic value of COMP. As the results shown, COMP had a certain accuracy (AUC>0.7) in predicting 16 cancer types, including ACC, BRCA, COAD, COADREAD, DLBC, KIRP, KICH, OV, PRAD, PAAD, READ, SKCM, STAD, TGCT, THYM, and UCS. Among them, COMP showed the highest accuracy (AUC>0.9) in predicting OV and PAAD (Fig. 7).

Prognostic performance of COMP in pan-cancer

Conspicuously, the expression level of COMP was mark- edly associated with the OS, DSS, and PFI of ACC, BLCA, KIRC, and COADREAD. Cox regression results showed

that poor prognosis was correlated with higher COMP expression in ACC, OS (HR 4.95) (Fig. 8a), DSS (HR 5.55) (Fig. 8b), and PFI (HR 2.79) (Fig. 8c). For BLCA, KIRC, and COADREAD, the same conclusion could be obtained that the upregulation of COMP predicted poor progno- sis. For BLCA, OS (HR 1.59) (Fig. 8d), DSS (HR 1.72) (Fig. 8e), and PFI (HR 1.36) (Fig. 8f). For KIRC, OS (HR 1.36) (Fig. 8g), DSS (HR 1.94) (Fig. 8h), and PFI (HR 1.57) (Fig. 8i). For COADREAD, OS (HR 1.46) (Fig. 8j), DSS (HR 1.98) (Fig. 8k), and PFI (HR 1.43) (Fig. 8l).

Furthermore, we concluded that elevated expression of COMP was correlated with a worse prognosis in most clini- cal subgroups of BCLA. For OS, age < = 70, gender (Male), N stage (N0), race (White), radiation therapy (No), smoker (Yes), lymphovascular invasion (Yes), primary therapy out- come (CR), and histologic grade (High Grade) (Fig. 9a); For

Fig. 6 PPI network, GO analysis, and KEGG analysis of 50 targeted binding proteins of COMP. a PPI network. b Visual network of GO and KEGG analyses. c GO analysis. d KEGG analysis

a

ITGA4

PTK2

DCN ACAN

ITGA9

b

extracellular structure organization extracellular matrix organization integrin-mediated signaling pathway

SOX9

MMP13

ITGB3

ITGA3

CD47

integrin complex

protein complex involved in cell adhesion

CILP

ITGA2

plasma membrane receptor complex

extracellular matrix structural constituent

ITGB4

COL9A1

FN1

ITGB5

collagen binding

ITGA2B

integrin binding

MMP3

ECM-receptor interaction

Focal adhesion

ITGB8

ITGAV

PI3K-Akt signaling pathway

Counts

BGN

COL9A2

COL9A3

CD36

13

FMOD

COMP

SDC1

28

ITGB7

ITGA5

44

ITGA1

COL2A1

COL1A1

VWF

SDC4

ANGPT1

ITGB6

ITGB1

ADAMTS14

MATN4

ITGA7

ITGA11

PRG4

MATN1

CHAD

ASPN

ITGA10

ADAMTS12

ITGA6

ADAMTS7

ITGA8

MATN3

C

d

extracellular structure organization

ECM-receptor interaction

extracellular matrix organization

of

PI3K-Akt signaling pathway

cell-substrate adhesion

p.adjust

Human papillomavirus infection

p.adjust

integrin-mediated signaling pathway

1.312720e-14

5e-09

9.845401e-15

Focal adhesion

4e-09

3e-09

plasma membrane receptor complex

6.563601e-15

2e-09

3.281800e-15

1e-09

protein complex involved in cell adhesion

Regulation of actin cytoskeleton

8

5.400890e-63

integrin complex

Counts

Dilated cardiomyopathy

Counts

10

9

focal adhesion

☐ 27

Hypertrophic cardiomyopathy

☐ 21

☐ 33

44

extracellular matrix structural constituent

Arrhythmogenic right ventricular cardiomyopathy

integrin binding

collagen binding

MA

Proteoglycans in cancer

Hematopoietic cell lineage

extracellular matrix binding

0.2

0.4

0.6

0.8

0.2

0.4

0.6

0.8

GeneRatio

GeneRatio

DSS, age< =70, gender (Male), N stage (N0), race (White), radiation therapy (No), smoker (Yes), lymphovascular inva- sion (Yes), histologic grade (High Grade), and BMI < = 25 (Fig. 9b); For PFI, age < = 70, gender (Male), race (White), radiation therapy (No), smoker (Yes), lymphovascular inva- sion (Yes), BMI 25, subtype (Papillary), and pathologic stage (Stage III) (Fig. 9c).

Effects of COMP in different clinical characteristics of BLCA

We investigated the relationship between the expression of COMP and the different clinical features of BLCA. The results showed that the expression of COMP was

significantly correlated with pathologic stage, T stage, N stage, radiation therapy, race, histologic grade, and sub- type of BLCA (Table 1). In addition, we found that COMP was up-regulated in patients with age > 70 (Fig. 10a), path- ologic stage III/IV (Fig. 10b), T stage III/IV (Fig. 10c), and N stage II/III (Fig. 10d), while it was down-regulated in radiation therapy (Yes) (Fig. 10e), primary therapy outcome (CR) (Fig. 10f), subtype (Papillary) (Fig. 10g), lymphovascular invasion (No) (Fig. 10h), and race (Asian) (Fig. 10i), respectively.

Fig. 7 ROC curve for COMP in pan-cancer. a ACC. b BRCA. c COAD. d COADREAD. e DLBC. f KIRP. g KICH. h OV. i PRAD. j PAAD. k READ. l SKCM. m STAD. n TGCT. o THYM. p UCS

a

ACC

b

BRCA

C

COAD

d

COADREAD

1.0

1.0

1.0

1.0

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

0.6

0.6

0.6

0.6

0.4

0.4

0.4

0.4

0.2

COMP

COMP

COMP

COMP

AUC: 0.737

0.2

AUC: 0.896

0.2

AUC: 0.760

0.2

AUC: 0.775

0.0

CI: 0.657-0.816

0.0

CI: 0.877-0.914

0.0

CI: 0.722-0.799

Cl: 0.741-0.809

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

e

DLBC

f

KIRP

g

KICH

h

OV

1.0

1.0

1.0

1.0

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

0.6

0.6

0.6

0.6

0.4

0.4

0.4

0.4

0.2

COMP

0.2

COMP

0.2

COMP

COMP

AUC: 0.875

AUC: 0.773

AUC: 0.809

0.2

AUC: 0.906

0.0

CI: 0.807-0.943

0.0

CI: 0.716-0.829

0.0

CI: 0.732-0.886

0.0

CI: 0.871-0.940

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

i

PRAD

j

PAAD

k

1.0

READ

I

SKCM

1.0

1.0

1.0

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

0.6

0.6

0.6

0.6

0.4

0.4

0.4

0.4

0.2

COMP

0.2

COMP

0.2

COMP

COMP

AUC: 0.721

AUC: 0.944

AUC: 0.792

0.2

AUC: 0.746

0.0

Cl: 0.677-0.764

0.0

CI: 0.914-0.974

0.0

Cl: 0.730-0.855

0.0

CI: 0.718-0.774

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

m

STAD

n

TGCT

o

1.0

THYM

p

UCS

1.0

1.0

1.0

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

Sensitivity (TPR)

0.8

0.6

0.6

0.6

0.6

0.4

0.4

0.4

0.4

0.2

COMP

AUC: 0.711

0.2

COMP

AUC: 0.823

0.2

COMP

AUC: 0.777

0.2

COMP

AUC: 0.769

0.0

Cl: 0.670-0.751

0.0

CI: 0.772-0.873

0.0

CI: 0.722-0.832

0.0

CI: 0.689-0.849

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

1-Specificity (FPR)

Univariate and multivariate cox regression analyses in BLCA

We used univariate and multivariate Cox regression analy- sis in BLCA to explore the correlation between COMP expression and clinical characteristics. As the results

shown, in OS, age, primary therapy outcome, pathologic stage, and COMP expression were prognostic risk fac- tors (Table 2), while in DSS, pathologic stage, primary, therapy outcome, and COMP were prognostic risk factors (Supplementary Table S1), and in PFI, pathologic stage,

Fig. 8 Correlations between COMP expression and the prognosis OS, DSS, and PFI of cancers. a-c ACC. d-f BLCA. g-i KIRC. j-I COADREAD

a

ACC

b

ACC

C

ACC

1.0

COMP

1.0

COMP

1.0

4

COMP

Survival probability

Low

Survival probability

Low

Low

0.8

High

0.8

High

Survival probability

0.8

High

0.6

0.6

0.6

#

0.4

0.4

0.4

0.2

Overall Survival

Disease Specific Survival

HR = 4.95 (2.00-12.23)

0.2

0.2

Progress Free Interval

HR = 5.55 (2.08-14.76)

HR = 2.79 (1.45-5.37)

0.0

P = 0.001

0.0

P = 0.001

0.0

P = 0.002

0

50

100

150

0

50

100

150

0

50

100

150

Time (months)

Time (months)

Time (months)

Low

39

16

2

Low

38

15

7

2

Low

39

13

6

2

High

40

12

0

High

39

12

0

High

40

6

0

0)

d

BLCA

e

BLCA

f

BLCA

1.0

COMP

1.0

-

COMP

1.0

-

COMP

Survival probability

Low

Survival probability

Low

0.8

High

Survival probability

Low

0.8

High

0.8

High

0.6

0.6

0.6

0.4

0.4

H

0.4

+

0.2

Overall Survival

++

0.2

Disease Specific Survival

HR = 1.59 (1.18-2.14)

HR = 1.72 (1.20-2.48)

0.2

Progress Free Interval

HR = 1.36 (1.01-1.83)

0.0

P = 0.002

0.0

P = 0.004

0.0

P = 0.04

0

40

80

120

160

0

40

80

120

160

0

40

80

120

160

Time (months)

Time (months)

Time (months)

Low

207

42

9

2

1

Low

199

42

9

2

1

Low

207

35

8

2

1

High

206

39

14

4

2

High

200

39

14

4

2

High

207

32

11

3

1

g

KIRC

h

KIRC

İ

KIRC

1.0

COMP

1.0

COMP

1.0

J

COMP

Survival probability

Low

Survival probability

Low

0.8

High

0.8

High

Survival probability

Low

0.8

High

0.6

0.6

0.6

0.4

0.4

0.4

H

0.2

Overall Survival HR = 1.36 (1.01-1.84)

0.2

Disease Specific Survival

HR = 1.94 (1.31-2.88)

0.2

Progress Free Interval HR = 1.57 (1.14-2.15)

0.0

P = 0.043

0.0

P = 0.001

0.0

P = 0.005

0

50

100

150

0

50

100

150

0

50

100

Time (months)

Time (months)

Time (months)

Low

269

106

18

1

Low

262

104

18

1

Low

268

90

13

0

High

270

101

22

0

High

266

99

22

0

High

269

83

15

0

COADREAD

k

COADREAD

I

COADREAD

1.0

COMP

1.0

COMP

1.0

COMP

Survival probability

Low

Survival probability

Low

Survival probability

Low

0.8

High

0.8

++ High

0.8

High

0.6

0.6

0.6

+

0.4

+

0.4

0.4

+

0.2

Overall Survival HR = 1.46 (1.03-2.08)

0.2

Disease Specific Survival HR = 1.98 (1.23-3.16)

0.2

Progress Free Interval

HR = 1.43 (1.05-1.94)

0.0

P = 0.033

0.0

P = 0.005

0.0

P = 0.024

0

50

100

150

0

50

100

150

0

50

100

150

Time (months)

Time (months)

Time (months)

Low

321

43

9

0

Low

306

38

9

0

Low

321

35

8

0

High

322

32

7

1

High

315

31

7

1

High

322

24

6

1

Springer

Fig. 9 Relationship of COMP expression with the a OS, b DSS, and c PFI in different clinical subgroups of BLCA

a

CharacteristicsHR (95% CI)OSP value
Age ( <= 70)1.73(1.13-2.67)0.013
Gender (Male)1.60(1.12-2.28)0.01
N stage (NO)2.35(1.20-4.58)0.012
Race (White)1.67(1.21-2.31)0.002
Radiation therapy (No)1.73(1.26-2.39)0.001
Smorker (Yes)2.05(1.44-2.92)<0.001
Lymphovascular invasion (Yes)1.78(1.14-2.79)0.011
Primary therapy outcome (CR)2.18(1.22-3.90)0.008
Histologic grade (High Grade)1.51(1.12-2.03)0.007

0

1

2

3

4

b

CharacteristicsHR (95% CI)DSSP value
Age ( <= 70)1.84(1.13-3.00)0.014
Gender (Male)1.85(1.19-2.87)0.006
N stage (NO)2.59(1.28-5.25)0.008
Race (White)1.77(1.19-2.64)0.005
Radiation therapy (No)2.05(1.39-3.03)<0.001
Smorker (Yes)2.19(1.42-3.35)<0.001
Lymphovascular invasion (Yes)2.00(1.16-3.45)0.013
Histologic grade (High Grade)1.61(1.12-2.31)0.011
BMI ( <= 25)2.29(1.14-4.61)0.02

0

1

2

3

4

5

C

CharacteristicsHR (95% CI)PFIP value
Age( <= 70)1.61(1.09-2.36)0.016
Gender (Male)1.69(1.19-2.41)0.003
Race (White)1.49(1.07-2.06)0.018
Radiation therapy (No)1.52(1.11-2.06)0.008
Smorker (Yes)1.60(1.13-2.26)0.008
Lymphovascular invasion (Yes)1.91(1.19-3.05)0.007
BMI ( <= 25)1.98(1.11-3.52)0.02
Subtype (Papillary)2.86(1.54-5.32)0.001
Pathologic stage (Stage III)0.47(0.25-0.91)0.024

0

1

2

3

4

5

6

Table 1 Clinical characteristics of BLCA patients
CharacteristicLow expres- sion of COMPHigh expres- p sion of COMP
n207207
T stage, n (%)<0.001
T15 (1.3%)0 (0%)
T277 (20.3%)42 (11.1%)
T373 (19.2%)123 (32.4%)
T424 (6.3%)36 (9.5%)
N stage, n (%)0.004
N0129 (34.9%)110 (29.7%)
N120 (5.4%)26 (7%)
N224 (6.5%)53 (14.3%)
N34 (1.1%)4 (1.1%)
Pathologic stage, n (%)<0.001
Stage I4 (1%)0 (0%)
Stage II91 (22.1%)39 (9.5%)
Stage III59 (14.3%)83 (20.1%)
Stage IV51 (12.4%)85 (20.6%)
Radiation therapy, n (%)0.028
No180 (46.4%)187 (48.2%)
Yes16 (4.1%)5 (1.3%)
Race, n (%)<0.001
Asian34 (8.6%)10 (2.5%)
Black or African American6 (1.5%)17 (4.3%)
White153 (38.5%)177 (44.6%)
Histologic grade, n (%)<0.001
High grade186 (45.3%)204 (49.6%)
Low grade19 (4.6%)2 (0.5%)
Subtype, n (%)<0.001
Non-papillary112 (27.4%)163 (39.9%)
Papillary94 (23%)40 (9.8%)

primary therapy outcome, and COMP were prognostic risk factors (Supplementary Table S2).

DEGs between different COMP expression groups and GSEA analysis in BLCA

We performed differential gene analysis in COMP high- and low-expression groups, and there were 3013 DEGs in total, including 2257 up-regulated genes and 756 down- regulated genes (Fig. 11a). Among them, we obtained 491 DEGs, including 440 up-regulated genes and 51 down-reg- ulated genes with the threshold values of |log2 fold-change (FC)|>2.0 and adjusted p value <0.05.

To clarify the potential impact of the expression levels of the selected genes in BLCA, GSEA analysis was performed with the high expression and low-expression group using the MSigDB collection (h.all.v7.2.symbols.gmt). There are 22

data sets satisfying FDR (qvalue) <0.25 and p.adjust <0.05. GSEA revealed that several pathways, such as those related to epithelial mesenchymal transition, allograft rejection, myogenesis, and inflammatory response, were enriched in the high expression group (Fig. 11b-e). These findings sug- gest potential roles for COMP-related genes in the progres- sion, tumor microenvironment, and immune responses of BLCA.

Furthermore, among 491 DEGs, we obtained top 10 hub genes, including LGR4, LGR5, RSPO2, RSPO1, RSPO3, RNF43, ZNRF3, FYN, LYN, and SYK (Fig. 11f). Among them, LGR-related genes, RSPO-related genes, RNF43, and ZNRF3 were all involved in the regulation of WNT pathway, indicating that COMP might affect tumor microenvironment through WNT, which needs to be verified by subsequent experiments.

COMP knockdown inhibited migration and invasion in BLCA cell lines

We used 2 BLCA cell lines, J28 and T24, to further ver- ify the inhibitory effect of COMP. As shown in results, in both J82 and T24 cell lines, the protein level of COMP was significantly decreased after RNA interference (Fig. 12a). Furthermore, COMP knockdown was able to significantly inhibit the migration and invasion of J82 and T24 (Fig. 12b). Moreover, Transwell assays showed that COMP knockdown could significantly reduce the wound healing of J82 and T24 (Fig. 12c).

Discussion

COMP is a glycoprotein expressed in various tissues, and is involved in the assembly and stabilization of the extracel- lular matrix [27]. Currently, high expression of COMP can be seen in a variety of diseases, such as multiple epiphyseal dysplasia and arthritis [28-30]. It has been reported that in patients with arthritis, COMP can be used as a diagnos- tic marker and correlate with disease severity [31]. COMP was also found to be up-regulated in tumors, and correlated with tumor volume increase, metastasis, and rate of cancer recurrence. These tumors include esophageal adenocarci- noma [32], gastric cancer [33], breast cancer [34], papillary thyroid carcinoma [35], colon cancer [17], lung adenocarci- noma [19], and hepatocellular cancer [36]. Taken together, COMP may be a promising candidate target or novel bio- marker for tumor.

Although an association between COMP and cancer has been reported, no studies have shown whether COMP can be a specific biomarker for the diagnosis of cancer. Based on this, we analyzed the relationship between COMP and pan-cancer through CCLE database, TCGA database, and

Fig. 10 Associations between COMP expression and different clinical characteristics in BLCA. a Age. b Pathologic stage. c T stage. d N stage. e Radiation therapy. f Primary therapy outcome. g Subtype. h Lymphovascular invasion. i Race. ns, p≥0.05. * p<0.05. ** p<0.01. *** p<0.001

a

b

15

ns

C

15

ns

12



The expression of COMP Log2 (TPM+1)

10

The expression of COMP Log2 (TPM+1)

The expression of COMP Log2 (TPM+1)

8

10

10

6

4

5

5

2

0

0

0

70

>70

Stage I&Stage IIStage III

Stage IV

T1&T2

T3

T4

Age

Pathologic stage

T stage

d

15

ns

e

**

f

10

ns

12

The expression of COMP Log2 (TPM+1)

The expression of COMP Log2 (TPM+1)

10

The expression of COMP Log2 (TPM+1)

8

10

8

6

6

5

4

4

2

2

0

0

0

NO

N1

N2&N3

No

Yes

CR

PD&SD&PR

N stage

Radiation therapy

Primary therapy outcome

g

İ


h

12

**

15

ns

12

**

The expression of COMP Log2 (TPM+1)

10

The expression of COMP Log2 (TPM+1)

10

The expression of COMP Log2 (TPM+1)

8

8

10

6

6

4

4

5

2

2

0

0

0

Non-Papillary

Papillary

No

Yes

Asian

White

Subtype

Lymphovascular invasion

Black or African American Race

GTEx database, and found that COMP was overexpressed in various human cancers. Further analysis revealed that COMP may play an important role in the development and prognosis of various tumors, and may serve as a spe- cific biomarker for cancer. In addition, prognostic analysis showed a significant correlation between the expression of COMP and different molecular subtypes of 12 cancers and different immune subtypes of 16 cancers. Recent studies have shown that COMP could affect the prognosis of liver

fibrosis and hepatocellular carcinoma, and COMP may be a promising therapeutic target and may be a promise thera- peutic target [36]. Moreover, it was reported that the serum levels of COMP may be a potential novel biomarker for the evaluation of the prognosis in breast cancer [34]. Fur- thermore, COMP could lead to liver fibrosis via regulating collagen-I deposition [37]. Our study not only confirms the previous studies, but also expands the function of COMP

Table 2 Univariate and multivariate Cox regression analyses of clinical characteristics associated with OS of BLCA
CharacteristicsTotal (N)Univariate analysisMultivariate analysis
Hazard ratio (95% CI)P valueHazard ratio (95% CI)P value
Age ( <= 70 vs.> 70)4131.421 (1.063-1.901)0.0181.670 (1.121-2.489)0.012
Primary therapy outcome (CR vs. PD&SD&PR)3575.224 (3.710-7.354)<0.0014.304 (2.739-6.764)<0.001
Pathologic stage (Stage I & Stage II vs. Stage IV)2693.036 (2.050-4.498)<0.0011.968 (1.266-3.060)0.003
Fig. 11 DEGs and GSEA analysis in BLCA. a The volcano map of DEGs red: upregulation. blue: downregulation. b-e GSEA analysis in BLCA. f Hub genes of PPI network identified in the gene lists

a

b HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION

C

HALLMARK_ALLOGRAFT_REJECTION

0.8

NES = 2.584

NES = 2.326

0.6

p.adj = 0.004

0.6

p.adj = 0.004

FDR = 0.002

FDR = 0.002

100-

0.4

0.4

-Log,0(P.adj)

Enrichment Score

Enrichment Score

0.2

0.2

0.0

0.0

50

Ranked list metric

Ranked list metric

6

6

NONA9

4

0

-2

-2

-4

-Log2(Fold Change)

0

4

10000

20000

30000

10000

20000

30000

Rank in Ordered Dataset

Rank in Ordered Dataset

d

HALLMARK_MYOGENESIS

e

HALLMARK_INFLAMMATORY_RESPONSE

f

NES =2.309

0.6

NES = 2.034

LGRS

RSPO3

Enrichment Score

p.adj = 0.004

Enrichment Score

0.6

FDR = 0.002

p.adj = 0.004

0.4

0.4

FDR = 0.002

RSPO2

ZNRF3

0.2

0.2

FYN

SYK

0.0

0.0

Ranked list metric

6

Ranked list metric

LGR4

LYN

RNF43

A

6

20NAO

0

-2

-2

RSPO1

10000

20000

30000

Rank in Ordered Dataset

10000

20000

30000

Rank in Ordered Dataset

in other tumor and provides ideas for the future work for searching tumor biomarkers.

Another important finding of this study is that the expres- sion of COMP is highly correlated with TME. COMP expression was positively correlated with the degree of immune cell infiltration in most tumors, especially in HNSC, LIHC, LUSC, and PAAD. Furthermore, we found that upregulation of COMP was positively correlated with CAF in most tumors. CAF, one of the abundant cell species in TME, could reshape the TME through collagen deposi- tion and matrix metalloproteinase secretion [38, 39]. The

available evidence suggested that CAF could interact with infiltrating immune cells to form immunoevasive TME, which in turn promotes tumorigenesis and development [40, 41]. In addition, among the hub genes we found, LGR- related genes and RSPO-related genes encoded receptors for R-spondins and were involved in the canonical WNT signaling pathway. Meanwhile, RNF43 and ZNRF3 played anti-regulatory roles in WNT, which functioned in tumor stem cells and tumor microenvironment [42]. Furthermore, SYK was involved in coupling activated immunoreceptors to downstream signaling events [43]. Therefore, we supposed

Fig. 12 COMP knockdown inhibited migration and invasion in BLCA cell lines. a Expression of COMP in J28 and T24 cells after RNA interference. b Transwell assays after COMP knockdown in J28 and T24 cells. c Wound-healing assay after COMP knockdown in J28 and T24 cells. * p<0.05

a

Relative expression (COMP)

1.5

Relative expression (COMP)

1.5

Control

si-COMP

Control

si-COMP

COMP

1.0

COMP

1.0

J82

0.5

T24

0.5

ß-Actin

ß-Actin

0.0

Control si-COMP

0.0

Control si-COMP

b

J82

800

T24

1000

Migration cell number

Migration cell number

Control

si-COMP

600

Control

si-COMP

800

600

Migration

400

400

200

200

0

Control si-COMP

0

Control si-COMP

800

1000

Control

si-COMP

Invasion cell number

Control

si-COMP

600

Invasion cell number

800

600

Invasion

400

400

200

200

0

Control si-COMP

0

Control si-COMP

C

J82

T24

Control

si-COMP

Control

si-COMP

Relative wound closure (%) (ratio to 0 H)

Relative wound closure (%) (ratio to 0 H)

0 H

1.5

1.5

1.0

1.0

0.5

0.5

24 H

0.0

0.0

Control si-COMP

Control si-COMP

that COMP might mediate similar immune functions in cancer. It might be an immune co-stimulatory molecule, which can further promote tumor proliferation, metastasis, or drug resistance by mediating immunosuppression to form immunoevasive TME. Further work is needed to determine whether COMP performs these functions and its potential molecular mechanisms.

At present, there are many reports on the effect of COMP on tumors, but few on its mechanism. For instance, COMP interacted with TAGLN in colorectal cancer to promote malignant progression [44]. COMP contributed to the severity of the breast cancer by increasing invasiveness and switching metabolism [18]. COMP regulated the interac- tion between Notch3 and Jagged1 in breast cancer [45]. COMP collaborated with CD36 and subsequently played

an essential role in MEK/ERK and PI3K/AKT-mediated HCC progression [17, 46]. To provide a basis for future research on the mechanism of COMP, we conducted GSEA analysis in BLCA. The results showed that there may be the following mechanisms: mesenchymal transition, allograft rejection, myogenesis, and inflammatory response. Among them, mesenchymal transition had also been reported to be a mechanism in COMP, which also corroborates our analy- sis [47]. There were also some reports on the promoting effect of EMT on BLCA [48, 49]. Whether COMP specifi- cally affected BLCA through EMT need to be verified by further research, including the mechanisms of other related pathways.

In this study, we evaluated the diagnostic and prognostic value of COMP using ROC curve and KM survival curve.

The results showed that COMP had certain value for the prognosis of 16 kinds of cancer. In addition, COMP was significantly associated with OS, DSS, and PFI in ACC, BLCA, KIRC, and COADREAD. The results suggested that COMP may be a biomarker or therapeutic target for tumor diagnosis and treatment. To further investigate the possible mechanism of COMP, we performed the GO and KEGG pathway enrichment of 50 COMP-binding pro- teins. The results suggested that COMP may play a role in tumorigenesis through extracellular matrix interaction and PI3K-AKT pathway. It should be emphasized that COMP is critical not only for extracellular matrix, but also for signal transduction both inside and outside the cell.

Since no relevant reports on the effect of COMP on BLCA have been reported so far, we chose BLCA for further research and found that COMP had important implications for BLCA. High expression of COMP was significantly associated with worse OS, DSS, and PFI in various clinical subgroups of BLCA. Univariate and multivariate Cox regression analyses identified pathologi- cal stage, primary therapy outcome, and COMP expres- sion as independent risk factors for OS, DSS, and PFI in BLCA. In addition, GSEA analysis of the co-expressed genes of COMP showed that it functioned in the progres- sion, tumor microenvironment, and immune response of BLCA. Finally, we further verified the inhibitory effect of COMP in BLCA cell lines. The results showed that COMP knockdown could inhibit migration and invasion in BLCA cell lines, which is consistent with our previous analysis results.

Conclusions

COMP may serve as an important target in pan-cancer diagnosis and prognosis as well as provide a theoretical basis for a comprehensive understanding of tumorigenesis and development, especially in the aspect of immune eva- sion of TME.

Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s12094-022-02968-8.

Author contributions Bingjie Guo and Yajing Wang contributed equally to the article. Bingjie Guo and Yajing Wang performed the statistical analysis and drew the pictures. Sailong Zhang and Wenyu Liu contributed to the design and writing of the study. All authors approved the final version of the manuscript.

Funding This study was supported by grants from the National Natural Science Foundation of China (81803541) and Shanghai Science and Technology Development Foundation (22140901900).

Data availability All data reported are included and represented in the manuscript.

Declarations

Conflict of interest The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent For this type of study formal consent is not required.

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Authors and Affiliations

Bingjie Guo1 . Yajing Wang2 . Wenyu Liu3 . Sailong Zhang4(D

☒ Wenyu Liu lwywinner@hotmail.com

☒ Sailong Zhang sailongzhang@126.com

1 Department of Gastroenterology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China

2 Department of Traditional Chinese Medicine, Second Military Medical University/Naval Medical University, Shanghai, China

3 Department of Hepatobiliary and Pancreatic Surgery, Changhai Hospital Affiliated to Naval Medical University, 168 Chang Hai Road, Shanghai 200433, China

4 Department of Pharmacology, Second Military Medical University/Naval Medical University, 325 Guo He Road, Shanghai 200433, China