Journal of Clinical Medicine

MDPI

Article

A Systematic Pan-Cancer Analysis of MEIS1 in Human Tumors as Prognostic Biomarker and Immunotherapy Target

Han Li 1,+, Ying Tang 2,+, Lichun Hua 2,4, Zemin Wang 1, Guoping Du 3, Shuai Wang 1, Shifeng Lu 4,* and Wei Li 5,*

1 Key Laboratory of Environmental Medicine Engineering, Department of Epidemiology and Health Statistics, School of Public Health, Southeast University, Nanjing 210009, China

2 Department of Ultrasound Diagnostic, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China

3 Department of General Practice, Southeast University Hospital, Nanjing 210018, China

4 Department of Hematology and Oncology, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China

5 Department of Clinical Research, Children’s Hospital of Nanjing Medical University, Nanjing 210008, China

* Correspondence: maclsf@163.com (S.L.); weili126@126.com (W.L.)

+ These authors contributed equally to this work.

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Citation: Li, H .; Tang, Y .; Hua, L .; Wang, Z .; Du, G .; Wang, S .; Lu, S .; Li, W. A Systematic Pan-Cancer Analysis of MEIS1 in Human Tumors as Prognostic Biomarker and Immunotherapy Target. J. Clin. Med. 2023, 12, 1646. https://doi.org/ 10.3390/jcm12041646

Academic Editor: Emanuela Anastasi

Received: 20 January 2023

Accepted: 13 February 2023 Published: 18 February 2023

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Copyright: @ 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Abstract: Background: We intended to explore the potential immunological functions and prognostic value of Myeloid Ecotropic Viral Integration Site 1 (MEIS1) across 33 cancer types. Methods: The data were acquired from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Gene expression omnibus (GEO) datasets. Bioinformatics was used to excavate the potential mechanisms of MEIS1 across different cancers. Results: MEIS1 was downregulated in most tumors, and it was linked to the immune infiltration level of cancer patients. MEIS1 expression was different in various immune subtypes including C2 (IFN-gamma dominant), C5 (immunologically quiet), C3 (inflammatory), C4 (lymphocyte depleted), C6 (TGF-b dominant) and C1 (wound healing) in various cancers. MEIS1 expression was correlated with Macrophages_M2, CD8+T cells, Macrophages_M1, Macrophages_M0 and neutrophils in many cancers. MEIS1 expression was negatively related to tumor mutational burden (TMB), microsatellite instability (MSI) and neoantigen (NEO) in several cancers. Low MEIS1 expression predicts poor overall survival (OS) in adrenocortical carcinoma (ACC), head and neck squamous cell carcinoma (HNSC), and kidney renal clear cell carcinoma (KIRC) patients, while high MEIS1 expression predicts poor OS in colon adenocarcinoma (COAD) and low grade glioma (LGG) patients. Conclusion: Our findings revealed that MEIS1 is likely to be a potential new target for immuno-oncology.

Keywords: MEIS1; pan-cancer analysis; tumor; biomarkers; immunotherapy; prognosis

1. Introduction

Cancer is a disease that seriously affects human health, and it is also the most con- cerning disease by global research [1]. Globally, 18.1 million cases of cancer were newly diagnosed in 2020, and this number is expected to be 28.4 million in 2040; with the burden growing in almost every country, the prevention and treatment of cancer are significant public health challenges [2]. Currently, cancer treatment approaches primarily include chemotherapy, radiotherapy, surgery, targeted therapy, stem cell transplants, and im- munotherapy [3,4]. Recently, immunotherapy has shown positive outcomes in various cancer types previously limited due to lack of known intervention strategies [5]. Im- munotherapy is a form of cancer therapy that helps human to fight cancer by activating the immune system, which is assuredly one of the greatest innovation in cancer treatment in the last decade [6]. Biomarkers are important for predicting the outcomes in response to immunotherapy, numerous candidate biomarkers have been used; however, responses are only typically present in a small number of patients [7]. Novel immunotherapy targets can

be screened by conducting pan-cancer analysis of genes in human tumors [8], including those that characterize the tumor microenvironment and targeted signaling pathways [9].

MEIS proteins, which belong to the three amino acid loop extension (TALE) class of transcription factor family, whose members include MEIS1 (NC_000002.12, Figure S1), MEIS2, and MEIS3 [10]. MEIS1 was first described in a leukemia mouse model. According to the single cell type module of Human Protein Atlas (http://www.proteinatlas.org/, accessed on 12 January 2023), MEIS1 expression is specificity to oligodendrocytes, rod photoreceptor cells, and endometrial stromal cells (Figure S2a). Meanwhile, in different blood and immune cells, the expression of MEIS1 was higher in total peripheral blood mononuclear cells (PBMC), natural killer (NK)-cells, and plasmacytoid dendritic cells (DC) (Figure S2b).

The expression of MEIS1 was influenced by cell types, age, the environment humans stay in, their pathological state, and the metabolism features of cancer cells [10,11]. MEIS1 was engaged in numerous cellular processes including chromatin remodeling, cell cycle regulation, apoptosis, and transcription regulation of self-renewal genes [12]. Research has shown that MEIS1 was strongly related to HOX genes and their cofactors to exert its regulatory effects on numerous signaling pathways [13]. Moreover, MEIS1 was a directly repressed target of MYC, and via effects on HOXB13, links MYC activity to androgen receptor activity to mediate cancer development [14]. MEIS1 can promote leukemogenesis and supports leukemic cell homing and engraftment by inducing synaptotagmin-like 1 (SYTL1) [15]. The impaired expression of MEIS1 was highly correlated with the poor prognosis of colorectal cancer patients [16]. In addition, MEIS1 was identified to reduce major histocompatibility complex class II (MHCII)expression in acute myeloid leukemia (AML) cells [17]), MEIS1 overexpression improved survival in patients with AML [18].

As a transcription factor, MEIS1 drives cell growth [19], dysregulated MEIS1 expres- sion contributes to tumorigenesis in multiple tumor types [12,20-23]. Due to its role in cancer cell proliferation, MEIS1 can become a molecular biomarker for cancer diagnosis and even a target for cancer therapy [24]. However, the expression levels of MEIS1 in various tumors are different [10,24]. It is a negative regulator in non-small-cell lung cancer (NSCLC) [25], whereas it serves as a positive regulator in esophageal squamous cell car- cinoma (ESCC) [26], malignant peripheral nerve sheath tumor (MPNST) [19] and Ewing sarcoma [27]. Even different studies found inconsistent expression of MEIS1 in prostate cancer [28,29]. The function of MEIS1 in cancer needs reassessing, we speculated that the oncogenic role of MEIS1 was affected by multiple factors, and it is possible that the complex role of MEIS1 in proliferation may largely depend on the tumor microenvironment (TME) [30]; nevertheless, the function of MEIS1 in TME remains uncertain. Various online tools were used to analyze the data from TCGA, GTEx and GEO databases, which intended to explore the potential mechanisms of MEIS1 across different cancers.

2. Materials and Methods

2.1. The Analysis of MEIS1 Expression in Different Cancers

MEIS1 gene differential expression between tumor tissues and normal tissues in 33 tumor types was explored in Gene_DE module of Tumor Immune Estimation Resource 2.0 (TIMER 2.0) [31]. Concerning many tumors without control data in TIMER 2.0, the Gene Expression Profile module of Gene Expression Profiling Interactive Analysis version 2 (GEPIA2) was used to match TCGA with GTEx data [32]. The differential expression analysis method was ANOVA and log2 (TPM + 1) for log-scale was used (we set | Log2FC | Cutoff as 1 with q-value Cutoff of 0.01) for box plots. “Stage Plots” in GEPIA2 can present MEIS1 gene expression in different stages in 26 tumor types. Log2(TPM + 1) for log-scale was used for violin plots.

The University of Alabama at Birmingham Cancer (UALCAN) data analysis Portal was used to analyze MEIS1 protein expression, phosphoprotein level and promoter methy- lation [33]. Breast cancer, clear cell renal cell carcinoma (ccRCC), colon cancer, lung ade-

nocarcinoma (LUAD), ovarian cancer, and uterine corpus endometrial carcinoma (UCEC) were available for total-protein, phosphorylation and promoter methylation level analyses.

2.2. Association of MEIS1 with Survival in Different Tumors

In GEPIA2, the “Survival Analysis” module was used to reveal OS and disease- free survival (DFS) curves using the Kaplan-Meier method for the high and low MEIS1 expression groups in different cancer types. High and low expression groups were classified according to a 50% (median) cutoff of MEIS1 expression, and the hypothesis test method was the log-rank test.

2.3. Gene Alteration and Immune Infiltration Analysis of MEIS1

cBioPortal (https://www.cbioportal.org/, accessed on 28 September 2022) was equipped with the function to analyze gene alteration frequency, alteration types (mutation, ampli- fication, multiple alterations), and mutation sites. The expression of MEIS1 in 22 tumor- infiltrating immune cells (TIICs) was evaluated by CIBERSORT on the SangerBox website (http://sangerbox.com/Tool, accessed on 28 September 2022). Meanwhile, the relationship between the expression of MEIS1 and immune checkpoint genes (ICPGs), TME biomarkers including TMB, MSI, and NEO were also explored. Furthermore, the relevance between MEIS1 expression and immune subtypes of 30 tumor types and molecular subtypes of 17 tumor types were analyzed utilizing the TISIDB database (http://cis.hku.hk/TISIDB/ index.php, accessed on 28 September 2022).

2.4. Gene Enrichment Analysis of MEIS1

Firstly, the top-50 proteins binding with MEISI1 were obtained in STRING (https: / /string-db.org/, accessed on 7 October 2022) by setting “the minimum required interac- tion score”, “the meaning of network edges”, “active interaction sources” and “the max number of interactors to show” to “Low confidence”, “Evidence”, “Experiments” and “No more than 50 interactors”, respectively. The top-50 MEIS1-binding genes network was also presented.

The “Similar Gene Detection” module of GEPIA2 was used to detect 100 similar genes to MEIS1. In addition, the first five genes correlated with MEIS1 were selected according to Pearson’s correlation coefficients using the “Correlation Analysis” module of GEPIA2.

VENNY2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed on 7 October 2022) was used to compare interacted genes with similar genes. GO and KEGG analysis was performed on 147 genes in two sets. “org.Hs.eg.db” package was applied to ID transform and the “clusterProfiler” package was applied to enrichment analysis. Data visualization was achieved through the “ggplot2” packages. This analysis was realized by R software [R-4.1.0, 64-bit, Vienna, Austria].

2.5. Ethical Approval

Our study was conducted in accordance with the principles stated in the Declaration of Helsinki, the secondary analysis of existing data of public use data sets does not require informed consent and ethical approval.

3. Results

3.1. Differential Expression of MEIS1 in Cancers

The result of MEIS1 expression in different cancers by the TIMER2.0 webserver was shown in Figure 1a, MEIS1 was down-regulated (the gene expression in the tumor group was lower than that in the control group) in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), COAD, HNSC, kidney renal papillary cell carcinoma (KIRP), LUAD, lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), thyroid carcinoma (THCA) and uterine corpus endometrial car- cinoma (UCEC) (p < 0.001), pheochromocytoma and paraganglioma (PCPG) (p < 0.01), stomach adenocarcinoma (STAD), cervical squamous cell carcinoma and endocervical ade-

nocarcinoma (CESC) (p < 0.05). MEIS1 expression in tumor tissues of cholangiocarcinoma (CHOL), Liver hepatocellular carcinoma (LIHC) (p < 0.001) and glioblastoma multiforme (GBM) (p < 0.05) was higher than normal tissues.

Figure 1. MEIS1gene and protein expression in 33 tumors and pathological stages of tumors. (a) MEIS1 gene expression level in 33 tumor tissues and normal tissues, even specific subtypes of tumors through TIMER2 (Blue represents normal tissues, red represents tumor tissues and purple represents metastatic tumors). *** p < 0.001; ** p < 0.01; * p < 0.05. (b) MEIS1 gene expression in tumors without normal tissues in TCGA, including ACC, LAML, SKCM, TGCT, THYM and UCS. * p < 0.05. (c) Total protein expression level of MEIS1 in tumor tissues and normal tissues of Breast cancer, Colon cancer, ccRCC, UCEC and LUAD. *** p < 0.001. (d) MEIS1 expression level in different pathological stages of ACC, COAD, KIRC, KIRP and LIHC.

a

TCGA dataset

MEIS1 Expression Level (log2 TPM)


*


*




**



*

*



7.5

-

4

5.0

2.

HIN.

0.0

ACC.Tumor (n=79)

BLCA.Tumor (n=408)

BLCA.Normal (n=19)

BRCA. Tumor (n=1093)

BRCA.Normal (n=112)

BRCA-Basal. Tumor (n=190)

BRCA-Her2.Tumor (n=82)

BRCA-LumA. Tumor (n=564)

BRCA-LumB. Tumor (n=217)

CESC.Tumor (n=304)-

CESC.Normal (n=3)

CHOL. Tumor (n=36)

CHOL.Normal (n=9)

COAD.Tumor (n=457)

COAD.Normal (n=41)-

DLBC. Tumor (n=48)

ESCA.Tumor (n=184)

ESCA.Normal (n=11)

GBM.Tumor (n=153)

GBM.Normal (n=5)

HNSC.Tumor (n=520)

HNSC.Normal (n=44)

HNSC-HPV+.Tumor (n=97)

HNSC-HPV -. Tumor (n=421)

KICH. Tumor (n=66)

KICH.Normal (n=25)

KIRC.Tumor (n=533)

KIRC.Normal (n=72)

KIRP.Tumor (n=290)

KIRP.Normal (n=32)

LAML. Tumor (n=173)

LGG.Tumor (n=516)

LIHC.Tumor (n=371)

LIHC.Normal (n=50)

LUAD.Tumor (n=515)

LUAD.Normal (n=59)

LUSC.Tumor (n=501)

LUSC.Normal (n=51)

MESO.Tumor (n=87)

OV.Tumor (n=303)

PAAD.Tumor (n=178)

PAAD.Normal (n=4)

PCPG.Tumor (n=179)

PCPG.Normal (n=3)

PRAD.Tumor (n=497)

PRAD.Normal (n=52)

READ.Tumor (n=166)

READ.Normal (n=10)

SARC.Tumor (n=259)

SKCM.Tumor (n=103)

SKCM.Metastasis (n=368)

STAD.Tumor (n=415)

STAD.Normal (n=35)

TGCT.Tumor (n=150)

THCA.Tumor (n=501)

THCA.Normal (n=59)

THYM.Tumor (n=120)-

UCEC.Tumor (n=545)

UCEC.Normal (n=35)

UCS. Tumor (n=57)

UVM. Tumor (n=80)

b TCGA+GTEx dataset

00

MEIS1 Expression log2(TPM+1)

2

0

3

..:- 1.

«

E.

-

2

o

Tumor (N=77)

ACC

LAML

Normal (N=128)

Tumor (N=173)

SKCM Normal

TGCT Tumor (N=137)

THYM Tumor (N=118)

UCS

Normal

Tumor (N=461) (N=558)

Normal (N=165)

Normal (N=339)

Tumor (N=57)

Normal (N=78)

(N=70)

C CPTAC dataset

Protein Expression of MEIS1 (Zvalue)

Breast cancer ***

Colon cancer ***

Clear cell RCC ***

UCEC ***

LUAD ***

Normal N=18

Tumor

Normal N=100

Tumor

Normal N=84

Tumor N=110

Normal N=31

Tumor

Normal N=111

Tumor N=111

N=125

N=97

N=100

d

TCGA dataset

MEIS1Expression Jog2(TPM+1)

ACC

F value = 3.45 Pr[F] = 0.0221

COAD

F waive = 2.9 Pr(F) = 0.0056

KIRC

F value = 4.73 Pip-F) - 0.00291

KIRP

F value = 6.06

Pm(F) = 0.000526

LIHC

F value = 4.13

PmpF) = 0.0068

Stage

II

Ill

IV

Stage

MI

III

IV

Stage

I

Il

HI

IV

Stage

1

İl

ill

IV

Stage

1

Il

İlI

IV

For those tumors without control tissues in TIMER 2.0 datasets, GEPIA2 was used to match these tumor tissues with the control tissues in the GTEx database to obtain boxplots of expression differences. As shown in Figure 1b (p < 0.05), MEIS1 was differentially expressed in ACC, acute myeloid leukemia (LAML), skin cutaneous melanoma (SKCM), testicular germ cell tumors (TGCT), thymoma (THYM) and uterine carcinosarcoma (UCS), which was down-regulated in ACC, SKCM, TGCT and UCS, and up-regulated in LAML and THYM. Furthermore, the statistically significant results were verified through GEO database. These results were presented in Figure S3, which were consistent with the results of MEIS1 differential expression analysis using the TCGA and GTEx database. MEIS1 protein expression in six common tumors was executed utilizing the CPTAC dataset. Figure 1c showed the total MEIS1 protein expression in breast cancer, colon cancer, LUAD, UCEC and ccRCC was higher than that in normal tissues (p < 0.001). Figure 1d revealed the correlation between pathological stages and MEIS1 expression levels, including ACC, COAD, KIRC, KIRP, and LIHC (p < 0.05). The correlation between pathological stages and MEIS1 expression levels in other cancer types with no statistical significance were shown in Figure S4.

3.2. Prognostic Index of MEIS1 in Different Cancers

As shown in Figure 2a, GEPIA2 survival analysis showed that low MEIS1 expression predicts poor OS of ACC, HNSC and KIRC patients (p < 0.01), while high MEIS1 expression predicts poor OS of COAD patients (p < 0.05) and LGG patients (p <0.001). As shown in Figure 2b, high MEIS1 expression predicts poor DFS of COAD and KIRP patients (p < 0.01), and LGG patients (p < 0.001), while low MEIS1 expression predicts poor DFS of HNSC patients (p <0.05).

a

Figure 2. Correlation between MEIS1 expression and survival of patients of cancers in TCGA. The survival plots and Kaplan-Meier curves with positive outcomes were given. (a) Correlation between MEIS1 expression and OS of ACC, COAD, HNSC, KIRC, LGG and UVM patients. (b) Correlation between MEIS1 expression and DFS of COAD, HNSC, KIRP and LGG patients.

Overall Survival

log10(HR)

ENSG00000143995.19

0.25

(MEIS1)

0.00

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

-025

9

Low MEIS1 Group High MEIS1 Group

:

Low MEIST Group High MEIST Group Logrank p=0.021 HR(high)=1.8

9

Low MEIS1 Group

1

Low MEIST Group Non MEIST Group Logrank p=0.02 HR (high)a) 7

2

Low MEIST Group

9

High MEIS1 Group Logrank p=9.80-07 HR(high) 2 5

Low MEIS1 Group On MEIST Group

Logrank pu0.0087

HRInich| 0 33 ((199)=0.012

Hon MEIST Group Logrank p=0.002 HR(high)=0.66

8

8

n(high)=38 n(jow)=38

POUR)-0.022

0

P(HR)=0.0021 nghigh)=258

12

Percent survival

D(HR)-0.021 n(high)=258

0

n(high)=134

p(HR)-2.30-06 n(high)=257 n(ow)-257

S

HR(hg )=0.38

PIHR TO 041 ningh)39 n[low)=39

8

ngow)=135

8

n(low)=259

0

n(low)-258

8

8

5

2

0

2

3

0

8

8

2

2

2

8

ACC

COAD

HNSC

KIRC

LGG

UVM

8

0

8

0

2

8

0

50

100

150

0

50

100

150

0

50

100

150

200

0

50

Months

100

150

0

50

100

150

200

0

20

40

60

80

Months

Months

Months

Months

Months

b Disease Free Survival

log10(HR)

ENSG00000143995.19 (MEIS1)

02

CESC CHOL

0.0

ACC

BLCA

BRCA

COAD

DLBC

ESCA

GBM

HNSC

KICH

KIRC

KIRP

LGG

LIHC

LUAD

LUSC

MESO

OV

PAAD

PCPG

PRAD

READ

SARC

SKCM

STAD

TGCT

THCA

THYM

UCEC

UCS

UVM

-02

9

Low MEIST Group High MEIS1 Group Logrank p=0.0038

e

Low MEIST Group COMEIST Group Logrank p=0.049 HR(high)=0.72

0

Low MEIS! Group High MEIST Group Logrank paQ 0043

1

Low MEIST Group

High MEIS1

8

HR[high)=2

8

8

HR(high)=23

Logrank pm1.Ge-05

(HR)=0.0046

p(HR)=0.049 n(high).258

p(HR)=0.0055 n(high)=141 n(low)m141

0

HR(high)=2

Percent survival

n(high)=134 n(low)= 135

P(HR)=2:26-05 n(high)-257 n(ow)-257

de

4

8

now)-259

8

8

o

8

2

2

8

0

2

2

COAD

HNSC

KIRP

LGG

:

0

6

0

0

50

100

150

0

50

100

150

200

0

50

100

150

200

0

50

100

150

Months

Months

Months

Months

3.3. Alteration of MEIS1 Gene Analysis Data

According to Figure 3a, in NSCLC, the frequency of gene change was the highest (3.51%) and mutation is the main type of gene change. Mutation is the main type of genetic change in ESCC, melanoma, endometrial carcinoma, esophagogastric adenocarcinoma, colorectal cancer, cervical squamous cell carcinoma, adrenal cortical cancer and hepato- cellular carcinoma as well. Likewise, amplification became the primary type of MEIS1 gene alteration in ovarian epithelial tumor, bladder urothelial carcinoma, mature B-cell neoplasms and prostate adenocarcinoma. Furthermore, Figure 3b contains more details, such as mutation types, sites and the number of cases with “Missense” as the main type of mutation. R102Afs*20 mutation can be observed in 2 COAD patients while R102Pfs*18 can be detected in a UCEC patient. We did not find any hotspot mutation in the MEIS1 gene.

Figure 3. MEIS1 gene alteration frequency, types, sites and cases. (a) MEIS1 alteration types and frequency. (b) MEIS1 mutation types, sites and cases. "+" represents that data are available.

a

Mutation

Structural Variant

Amplification

Deep Deletion

Multiple Alterations

3%-

Alteration Frequency

2%

1%-

Structural variant data

Mutation data

CNA data

Non-Small Cell Lung Cancer

Ovarian Epithelial Tumor

Esophageal Squamous Cell Carcinoma

Melanoma

Colorectal Adenocarcinoma

Bladder Urothelial Carcinoma

Endometrial Carcinoma

Esophagogastric Adenocarcinoma

Mature B-Cell Neoplasms

Cervical Squamous Cell Carcinoma

Sarcoma

Head and Neck Squamous Cell Carcinoma

Adrenocortical Carcinoma

Hepatocellular Carcinoma

Prostate Adenocarcinoma

Invasive Breast Carcinoma

Renal Clear Cell Carcinoma

Thymic Epithelial Tumor

Undifferentiated Stomach Adenocarcinoma

Renal Non-Clear Cell Carcinoma

Well-Differentiated Thyroid Cancer

Pleural Mesothelioma

Pheochromocytoma

Miscellaneous Neuroepithelial Tumor

Cervical Adenocarcinoma

Diffuse Glioma

Ocular Melanoma

Leukemia

Glioblastoma

Cholangiocarcinoma

Pancreatic Adenocarcinoma

Seminoma

Non-Seminomatous Germ Cell Tumor

b

5

# MEIS1 Mutations

91

Missense

COAD (n=2)

10 Truncating

R102Afs*20/Pfs*18

0 Inframe

UCEC (n=1)

6 Splice

1 SV/Fusion

0

Homeobo ..

0

100

200

300

390aa

3.4. Phosphorylation and Promoter Methylation Expression of MEIS1 across Different Cancers

Changes in phosphorylation pathway are closely related to cancer. Based on the CPTAC dataset, breast cancer, ovarian cancer, colon cancer, ccRCC, UCEC, LUAD and pediatric brain cancer were included to explore phosphorylation level between their tumor tissues and normal tissues. Ultimately, the box plots of four types of cancer are available in Figure 4a. In ovarian cancer, MEIS1 phosphoprotein level (S194, S196 and T202) between tumor and normal tissues has no statistical differences, while the phosphorylation level of S196 in LUAD (p <0.001), ccRCC (p <0.001) and UCEC (p <0.001) were higher in normal tissues compared to tumor tissues. More experiments evidence is needed to identify the function of MEIS1 phosphorylation at the S196 site in tumorigenesis. In addition, it is found that promoter methylation level of MEIS1 in BLCA, HNSC, KIRC, KIRP, PRAD and UCEC were lower in primary tumors compared to normal tissues (p < 0.05) (Figure 4b).

Figure 4. Phosphorylation and promoter methylation analyses of MEIS1 protein in different tumors. (a) Phosphorylation level of MEIS1 between their tumor tissues and normal tissues. (b) Promoter methylation level of MEIS1 between their tumor tissues and normal tissues.

a

Ovarian cancer

3

Ovarian cancer

3-

Ovarian cancer

2

2

2.

Z value

P = 2.8 × 10-1

1

1-

P = 3.0 × 10-1

1 -

P = 2.0 × 10-1

0

0

0

-1

-1

-1

-2

-2

-2

S194

S196

T202

-3

Normal N=19

Tumor N=84

-3

Normal N=19

Tumor N=84

-3

Normal N=19

Tumor N=84

3

6

Lung adenocarcinoma

4

Clear cell RCC

UCEC

2

4

2

Z value

1

2

0

0

0

-1

P = 2.5 × 10-30

P = 2.4 × 10-31

P = 3.8 x 10-15

-2

-2

S196

S196

-2

S196

-4

Normal N=102

-4

Tumor N=111

Normal N=83

Tumor N=110

-3

Normal N=31

Tumor N=100

b

Promoter methylation level of MEIS1 in BLCA

Promoter methylation level of MEIS1 in HNSC

Promoter methylation level of MEIS1 in KIRC

0.1

0.1

0.08

Beta value

P = 9.4 × 10-4

0.09

P = 6.8 x 10-3

P = 6.0 × 10-5

0.08

0.07

0.08

0.07

0.06

0.06

0.06

0.05

0.04

0.05

0.04

0.04

0.02

0.03

Normal (n=21)

Primary tumor (n=418)

0.03

Normal (n=50)

Primary tumor (n=528)

Normal (n=160)

Primary tumor (n=324)

Promoter methylation level of MEIS1 in KIRP

Promoter methylation level of MEIS1 in PRAD

Promoter methylation level of MEIS1 in UCEC

0.08

0.175

0.09

0.15

P = 2.2 × 10-16

0.08

P = 1.2 × 10-2

Beta value

0.07

P = 7.4 × 10-4

0.07

0.125

0.06

0.06

0.1

0.05

0.05

0.075

0.04

0.04

0.05

0.03

0.03

0.025

0.02

Normal (n=45)

Primary tumor (n=275)

Normal (n=50)

Primary tumor (n=502)

Normal (n=46)

Primary tumor (n=438)

3.5. MEIS1 Expression in Immune and Molecular Subtypes across Different Cancers

As shown in Figure 5a, the expressions of MEIS1 were different in various immune subtypes including C1-C6 in ACC, BLCA, BRCA, CHOL, COAD, KIRC, LGG, LUAD, LUSC, pancreatic adenocarcinoma (PAAD), PRAD, sarcoma (SARC), STAD, TGCT (all p < 0.05). In addition, MEIS1 was diversely expressed in multiple molecular subtypes of ACC, BRCA, esophageal carcinoma (ESCA), GBM, HNSC, KIRP, LGG, LIHC, LUSC, ovarian serous cystadenocarcinoma (OV), PCPG, PRAD, STAD and UCEC (all p < 0.05) (Figure 5b).

Figure 5. MEIS1 expression in immune and molecular subtypes of cancers (a) The relationship between MEIS1 expression and immune subtypes of cancers. (b) The relationship between MEIS1 expression and molecular subtypes of cancers.

a

ACC MEIS1_exp Pv = 3.11 x 10 -*

BLCA MEIS1_exp Pv = 5.11 * 10 -*

BRCA MEIS1_exp Pv = 3.37 * 10 -*

CHOL MEIS1_exp Pv = 3.9 * 10-

COAD MEIS1_exp Pv = 2.72 * 10-2

n=C1 1,C2 1,C3 23,C4 49,C5 3,C6 1

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

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

n=C1 7,C2 2,C3 17,C4 8,C6 1

n=C1 332,C2 85,C3 9,C4 12,C6 3

Expression (log2CPM)

10.0

8

8

6

7.5

:

.

7.5

Y

6

5.0

H

H

!

0

4

9

U

B

4.

H

H

H

:

!

8

4

!

2.5-

H

8

I

5.0

8

1

2

0

H

0.0

2.5

0

2

2.5

4

C1

C2

C3

C4

C5

C6

2

C1

C2

C3

C4

C6

C1

C2

C3

C4

C6

C1

C2

C3

C4

C6

C1

C2

C3

C4

C6

KIRC MEIS1_exp Pv = 1.29 x 10-

LGG MEIS1_exp Pv = 3.49 x 10 -*

LUAD MEIS1_exp Pv = 5.42 x 10-

LUSC MEIS1_exp Pv = 2.16 x 10-

PAAD MEIS1_exp Pv = 9.68 x 10-

n=C1 7,C2 20,C3 445,C4 27,C5 3,C6 13

n=C3 10,C4 147,C5 356,C6 1

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

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

Expression (log2CPM)

8

8

n=C1 57,C2 32,C3 40,C4 1,C6 21

8

5.0

7.5

U

8

H

9

6

6

6

J

E

5.0-

I

I

1

A

H

1

i

8

2.5

4

H

!

:

8

4

:

I

H

2.5

8

8

4

0.0-

2

2

0.0

2

C1

C2

C3

C4

C5

C6

C3

C4

C5

SARC MEIS1_exp Pv = 4.21 x 10

C6

C1

C2

C3

C4

C6

0

C1

C2

C3

C4

C6

C1

C2

C3

C4

PRAD MEIS1_exp

Pv = 1.34 x 10-

STAD MEIS1_exp Pv = 3.11 x 10-1

C6

TGCT MEIS1_exp Pv = 2.42 * 10-

Subtype

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

n=C1 64,C2 38,C3 42,C4 59,C6 20

12

n=C1 129,C2 210,C3 36,C4 9,C6 7

n=C1 42,C2 104,C3 2,C4 1

Expression (log2CPM)

5.0

H

:

8

·

7.5

.

8

2.5

I

İ

!

5.0

4

!

!

I

1

8

Y

U

8

4

I

0.0

2.5

0

:

0

-2.5

0.0

C1

C2

C3

C4

-4

Subtype

C1

C2

C3

C4

C6

Subtype

C1

C2

C3

C4

C6

C1

C2

C3

C4

b

Subtype

Subtype

ACC MEIS1_exp Pv = 2.21× 10-

BRCA MEIS1_exp Pv = 1.19 * 10 -**

ESCA MEIS1_exp Pv = 1.62 x 10 -*

GBM MEIS1_exp

naBasal 172, SOR LumA 508. LumB 191. Normal 137

Pv = 3 x 10-1

naCIN 74. ESCC 90, GS 1. HM-SNV 2 HM-indel 2

naClassic-like 47. high2. G-CIMP-low 5, LGm6-GBM 12. Mesenchymal-like 53

HNSC MEIS1_exp Pv = 8.56 * 10-

CIMp.Internae 27

Expression (log2CPM)

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

CIMP-low 32

10.0

8

8

7.5

6

6

6

H

:

6

:

H

H

4

H

i

H

H

5.0

3

H

:

8

8

4

I

4

I

2.5

0

2.

Ş

2

2

A

CIMP-high

MP-intermediate

CIMP-low

3

0

O

Basal

Her2

LumA

LumB

Normal

CIN

ESCC

GS

HM-SNV

HM-indel

Classic-like

G-CIMP-high

G-CIMP-low

LGm6-GBM

Mesenchymal-like

Atypical

Basal

Classical

Mesenchymal

KIRP MEIS1_exp Pv = 5.8 x 10-+

LGG MEIS1_exp

Pv = 7.44 x 10-%

nClassic-like 23.

Expression (log2CPM)

G-CIMP-high 234, G-CIMP-low 12. Mesenchymal-like 45. PA-like 26

LIHC MEIS1_exp Pv = 2.95 x 10 -* n=Cluster:1 64. iCluster:2 55, iCluster:3 63

LUSC MEIS1_exp Pv = 2.6 x 10-+ n=basal 42. classical 63. primitive 26. secretory 39

OV MEIS1_exp Pv = 1 x 10

C2a 35.

C2b 22

Codel 171.

C2c-CIMP 9

n=Differentiated 66.

6

Immunoreactive 78. Mesenchymal 71. Proliferative 78

3

8

8

10.0

I

i

.

6

4

:

A

N

6

:

6

0

4

7.5

:

:

B

:

O

8

2

2

:

N

C

I

4

4

!

i

H

5.0

3

C2c-CIMP

Classic-like

Codel

G-CIMP-high

G-CIMP-low

Mesenchymal-like

PA-like

0

2

2.5

C1

C2a

C2b

O

classical

primitive

secretory

Differentiated

Immunoreactive

Mesenchymal

Proliferative

basal

PRAD MEIS1_exp Pv = 3.67 x 10-1

iCluster:1

iCluster:2

iCluster:3

Subtype

PCPG MEIS1_exp

2-ETV1 28.

Pv = 1.33 x 10 -*

3-ETV4 14,

STAD MEIS1_exp

n=Corticaladmixture 22.

4-FLI1 4,

Pv = 2 * 10*

UCEC MEIS1_exp

Kinasesignaling 68, Pseudohypoxia 61, Wnt-altered 22

5-SPOP 37.

naCIN 223, EBV 30. GS 50.

Pv = 1.36 x 10-7

6-FOXA1 9.

Expression (log2CPM)

CN LOW 144, MSI 124, POLE 79

7-IDH1 3,

HM-SNV 7. HM-indel 73

8-other 86

7

6

7.5

.

8

5

:

:

S

H

3

8

8

8

H

8

0

8

I

H

5.0

i

İ

i

9

N

!

0

N

!

4

3

2.5

Orticaladmixture

Kinasesignaling

Pseudohypoxia

Wnt-altered

3

1-ERG

2-ETV1

3-ETV4

4-FLI1

5-SPOP

6-FOXA1

7-IDH1

8-other

0.0

0

CIN

EBV

GS

HM-SNV

HM-indel

CN_HIGH

CN_LOW

MSI

POLE

Subtype

Subtype

Subtype

Subtype

Subsequently, the correlation between MEIS1 expression and 60 ICPGs expression was explored (Figure 6). In most cancers, such as lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), PRAD, READ, COAD, OV, KIRC and LIHC, MEIS1 expression was positively related to the expression of most ICPGs. It means that the patients with high expression of MEIS1 will show better immunotherapy effects by using immune checkpoint inhibitors (ICIs). However, in TCGT and SARC, MEIS1 expression was negatively correlated with most ICPGs expression. It showed that patients with high MEIS1 expression will have a poor prognosis when targeting these ICPGs. Therefore, it can be proved that MEIS1 is equipped with strong potential in immunotherapy.

Figure 6. The correlation matrix between MEIS1 expression and ICPG expressions. * p < 0.05.

Type

CD276

correlation coefficient

VEGFA

HAVCR2

-1.0-0.5

0.0

0.5

1.0

IL10

pValue

TGFB1

C10orf54

0.0

0.5

1.0

EDNRB

Type:

IL12A

Inhibitory

VEGFB

Stimulaotry

VTCNI

ARGI

IL13

IL4

KIR2DL1

KIR2DL3

ADORA2A

BTLA

IDO1

TIGIT

CTLA4

SLAMF7

CD274

LAG3

PDCDI

ICOSLG

TNFRSF14

ILIA

IL1B

TNFSF9

IFNA1

IFNA2

IFNG

PRF1

GZMA

CCL5

CD70

TNFRSF18

TNFRSF4

CD28

ITGB2

CD80

IL2RA

IL2

TNF

CD40LG

CD27

ICOS

CXCL10

CXCL9

CD40

ICAM1

TNFRSF9

HMGB1

SELP

ENTPD1

TNFSF4

CX3CL1

TLR4

BTN3A1

BTN3A2

3.7. The Correlation between MEIS1 Expression and Immune Cell Infiltration and TME in Different Cancers

According to results in Figure 7, MEIS1 expression was closely correlated with most immune cells in various cancers. The expression of MEIS1 was correlated with macrophages_M2 in 20 cancer types, CD8+T cells in 19 cancer types, Macrophages_M1 in 15 cancer types, macrophages_M0 in 13 cancer types and neutrophils in 10 cancer types. For evaluating anti-tumor immunity, as shown in Figure 8, the relationships between MEIS1 and TMB, MSI and NEO were explored in all cancers. MEIS1 expression was negatively associated with TMB in ACC, STAD, STES, SARC, BLCA and KIRC. As for MSI, MEIS1 expression was negatively correlated with MSI in UCS, UCEC, SARC, STAD, STES, GBM, LGG, KIPAN, PRAD and HNSC, while it was positively associated with MSI in TGCT. In addition, MEIS1 expression had a negative correlation with NEO in SARC, UCEC, KIRP, KIPAN, KIRC and LUAD.

correlation coefficient

0.5

0.0

0.5

p Value

0.0

0.5

1.0

1.5

2.0

Figure 7. The correlation matrix between MEIS1 expression and immune cell content. * p < 0.05.

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TCGA-TGCT(N=132)

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TCGA-HNSC(N-517)

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TARGET-ALL-R(N-99)

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TCGA-STAD(N-388)

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TCGA-CESC(N-291)

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TCGA-STES(N-569)

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TOGA-KIRP(N-285)

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TCGA-COAD(N=282)

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TCGA-LUSC(N=491)

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TCGA-BLCA(N=405)

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TCGA-KIRC(N=528)

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TCGA-LIHC(N-363)

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TCGA-DLBC(N-46)

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TCGA-UVM(N-79)

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TCGA-PAAD(N=177)

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TCGA-SKCM(N-452)

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TARGET-ALL(N-86)

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TCGA-OV(N=417)

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ICGA-SARC(N 258)

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TCGA-THCA(N-503)

TCGA-UCS(N-56)

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TARGET-NB(N=153)

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TCGA-ESCA(N-181)

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TARGET-I.AMI.(N=142)

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TCGA-SKCM-M(N=351)

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TCGA-GBM(N-152)

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B_cells_naive

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NK_cells_resting

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Monocytes

Macrophages_MO

Macrophages_MI

Macrophages_M2

Dendritic_cells_resting

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Mast_cells_resting

Mast_cells_activated

Eosinophils

Neutrophils

3.8. MEIS1-Related Genes Are Correlated to Cell Proliferation and Differentiation

Based on STRING, 50 MEIS1-binding proteins supported by experiments were ob- tained. The protein-protein interaction networks are presented in Figure 9a. After that, 100 similar genes were acquired from correlation analysis in GEPIA2, 50 interacted genes and 100 similar genes have 3 overlap genes in Figure 9b. Then, the 147 genes were integrated to perform GO and KEGG analysis presented in Figure 9c. KEGG analysis indicates that “Transcriptional misregulation in cancer” might play a vital role in the effect of MEIS1 on cancers. Furthermore, by GO analysis, at the biological process (BP) level, most of the genes

are involved in cell differentiation and cell fate determination. At the molecular function (MF) level, most of the genes took part in regulating DNA-binding transcription activator and repressor activity, RNA polymerase II-specific, enhancer sequence-specific and acti- vating transcription factor binding. At last, the cellular component (CC) also proved that these genes were related to transcription.

Figure 8. The relationship between MEIS1 expression and anti-immunity indicators (a) The relation- ship between MEIS1 expression and TMB (b) The relationship between MEIS1 expression and MSI. (c) The relationship between MEIS1 expression and NEO.

a

TMB

CHOL(N=36)

SampleSize

ACC(N=77)

·

STAD(N=409)

STES(N=589)

200

SARC(N=234)

ESCA(N=180)

400

LAML(N=126)

BLCA(N=407)

600

KIRP(N=279)

GBM(N=149)

800

CESC(N-286)

SKCM(N=102)

PRAD(N=492)

KIRC(N=334)

LUAD(N=509)

pValue

DLBC(N=37)

0.0

LUSC(N=486)

PCPG(N=177)

-0.2

THYM(N=118)

UCEC(N-175)

-0.4

UVM(N=79)

HNSC(N=498)

-0.6

OV(N=303)

THCA(N=489)

-0.8

TGCT(N=143)

-1.0

KIPAN(N=679)

BRCA(N=981)

GBMLGG(N=650)

MESO(N=82)

READ(N=90)

UCS(N=57)

LGG(N=501)

PAAD(N=171)

COADREAD(N=372)

LIHC(N-357)

COAD(N=282)

KICH(N-66)

-0.2

-0.1

0.0

0.1

0.2

0.3

Correlation coefficient(pearson)

b

MSI

UCS(N=57)

SampleSize

UCEC(N-180)

.

DLBC(N=47)

SARC(N=252)

200

STAD(N=412)

STES(N-592)

400

GBMLGG(N-657)

ACC(N-77)

600

KIPAN(N=688)

PAAD(N=176)

800

PRAD(N=495)

ESCA(N=180)

= 1,000

HNSC(N=500)

SKCM(N=102)

UVM(N-79)

pValue

CESC(N=302)

0,0

PCPG(N-177)

THYM(N=118)

-0.2

KIRP(N-285)

COADREAD(N=374)

-0.4

READ(N 89)

COAD(N=285)

-0.6

BRCA(N=1039)

KIRČ(N=337)

-0.8

BLCA(N=407)

1.0

LGG(N=506)

THCA(N=493)

LIHC(N-367)

LUAD(N=511)

LUSC(N-490)

GBM(N=151)

MESO(N=83)

OV(N=303)

CHOL(N-36)

KICH(N=66)

LAML(N=129)

TGCT(N=148)

-0.4

-0.2

0.0

0.2

0.4

Correlation coefficient(pearson)

C

NEO

THYM(N=64)

SampleSize

PCPG(N-60)

·

UCS(N=50)

100

SARC(N-177)

200

UVM(N=38)

UCEC(N-166)

300

CHOL(N-30)

400

KICH(N-39)

500

KIRP(N-260)

KIPAN(N-636)

L

600

ACC(N=57)

KIRC(N-337)

LUAD(N=462)

pValue

BLCA(N-375)

0.0

CESC(N=244)

-0.2

PRAD(N=341)

BRCA(N=857)

-0.4

LUSC(N=447)

READ(N=81)

-0.6

COADREAD(N=336)

0.8

COAD(N-255)

HNSC(N=446)

-1.0

GBM(N=117)

LIHC(N=337)

GBMLGG(N=520)

PAAD(N=113)

MESO(N=65)

LGG(N=403)

THCA(N=175)

SKCM(N=83)

DLBC(N=33)

TGCT(N=94)

-0.2

-0.1

0.0

0.1

0.2

Correlation coefficient(pearson)

Figure 9. MEIS1-related genes' enrichment analysis. (a) 50 MEIS1-binding proteins' interaction net- work derived from STRING. (b) Venn diagram showing the intersection between MEIS1-interacted genes and MEISI-correlated genes. (c) Bubble diagram for GO and KEGG analysis of MEIS1- related genes.

a

HOXD11

A

HOXC13

C

HOXBB

2

Pattern specification process

GO-BP

p.adjust

6 x 10-12

Regionalization

4 = 10

19

HOXA11

HOXA2

HIOXB13

1 * 10-12

SUMO3

VY

HOXA10

A

HOXA13

Anterior/posterior pattern

A

specification

Courts

DLX3

Embryonic organ development

14

22

HOXD13

HOXD9

30

MX1B

FAM222A

Embryonic skeletal system development

EN2

ENI

PBX4

PS

N

PBX3

0.15

0.20

0.25

0.30

CRTC1

TLX1

PBX2

GeneRatio

PBX1

MAG21L1

SRF

CREB1

MEIS1

MEIS2

PAX6

HORAS

MAB21L2

Transcription factor complex

GO-CC

p.adjust

TLX3

0.06

Y

CREBBP

HOXB4

HOXE9

Nuclear chromatin

0.04

PKNOX

0.02

ETS1

HOXD4

HOX87

HOXB

CRTC2

NUB1

Nuclear transcription.

Counts

0 3

factor complex

9

15

YAP1

HOXA7

HOXA9

HOXD12

TSHZ2

Aggresome

MTX2

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

2

GeneRatio

OVOLf

KMT2A

DNA-binding transcription activator activity, RNA polymerase II-specific

GO-MF

p.adjust

PSIPT

MEN1

0.00 800

DNA-binding transcription repressor activity.

0.00075

RNA polymerase II-specific

0.00050

0.00025

Enhancer binding

Counts

b

Enhancer sequence-specific DNA binding

»

16

MEIS1

20

interacted genes

similar genes

RNA polymerase Il distal enhancer

sequence-specific DNA binding

0.10

0.15

0.20

025

0.30

GeneRatio

47

3

97

Transcriptional misregulation in cancer

KEGG

paqjust

0.03

Human T-cell leukemia virus 1 infection

0.02

0.01

Cushing syndrome

Counts

6

Lysine degradation

10

HOXD4

0.10

0.15

0.20

0.25

0.30

HOXB4

GeneRatio

PBX2

4. Discussion

Cancer is a leading cause of death in each country, and the number of newly confirmed patients continues to increase [2]. Although current cancer therapy options exhibit some clinical success, a large number of cancer patients still have poor prognoses because of drug resistance, adverse effects, and other issues [34]. Consequently, there is a need to identify new therapeutic targets and sensitive biomarkers for cancer diagnosis and treatment. The pan-cancer analysis can deliver deep perception for the design of cancer prevention and precision treatment strategies by revealing the similarities and differences between different cancers [34]. In our study, we comprehensively performed MEIS1 expression and its correlation with prognostic and immunotherapy value in pan-cancer via different databases. The 33 cancer types were studied to identify MEIS1 expression in this study and it finally suggests that MEIS1 expression is different between tumor tissues and normal tissues in 23 cancer types. Among these 23 cancer types, MEIS1 expression is down- regulated in 18 cancer types, on the contrary, the other 5 types were up-regulated.

Another major finding is that MEIS1 expression correlates with survival in cancer patients, which indicated that the abnormal expression of MEIS1 can be responsible for the poor prognosis of tumors. For the value of MEIS1 on the prognosis of cancer patients, it could be determined by the activity of the MEIS1 protein which depends on the en- vironment in which the cell exists. A similar finding was also demonstrated by Schulte et al. and Meng et al. by performing studies on MEIS1 [30,35]. MEIS1 is a hub gene in the differentially expressed gene interaction network for lung adenocarcinoma (LUAD) and participated in the occurrence and prognosis of LUAD [23]. MEIS1 overexpression was inversely correlated with relapse and overall survival in children with acute leukemias [36].

Down-regulated expression of MEIS1 was detected in colorectal cancer and predicted poor survival of colorectal cancer patients [16]. It is believed that abnormal DNA methylation plays a critical role in cancer development and progression [37]. Tumors and benign tis- sues exhibit different methylation patterns [38,39]. Our study found that the promoter methylation levels of MEIS1 in BLCA, HNSC, KIRC, KIRP, PRAD, and UCEC were lower in primary tumors compared to normal tissues. In conclusion, MEIS1 may be able to regulate some tumors at an epigenetic level, but more studies are needed to clarify the deeper mechanisms.

Immune infiltration is highly associated with the prognosis of tumor patients [40]. Six types of immune infiltration had been explored in cancer patients, maybe promoting or inhibiting tumor cell growth [41]. Among them, C5 is positively correlated to MEIS1. A high level of C5 can impair the curative effect of immune checkpoint inhibitors, as reported by Lehrer and his colleagues [42]. Our findings showed that MEIS1 expression was strongly related to most immune cells in various types of cancer. Moreover, MEIS1 expression is related to some immune subtypes and molecular subtypes in different types of cancer. We indicated that MEIS1 can play a role in the growth and progression of cancer and have a close correlation with immune regulation. The TMB, MSI, and NEO which characterize anti-tumor immunity were negatively related to MEIS1 expression in some cancers. Therefore, the immunotherapeutic effect of cancer patients can be predicted according to MEIS1 expression. It can contribute to guide immunotherapy more accurately. The MEIS1 expression was negatively related to TMB, MSI, and NEO in SARC. Thus, there is an inference that patients suffering from SARC with low MEIS1 expression might predicted better survival after immunotherapy. In most cancers, MEIS1 expression was positively related to most ICPGs expression. There is bold speculation that using immune checkpoint inhibitors along with interfering in MEIS expression will have an effective impact on the patient [30].

In gene enrichment analysis, HOXD4, HOXB4, and PBX2 were co-expressed with MEIS1. HOX were transcription factors that serve as an essential regulator for cell fate determination, stem cell functions, and gastrointestinal development. And they need TALE family proteins such as MEIS and PBX to improve their transcriptional efficiencies [43]. MEIS and PBX were correlated with cell growth, differentiation and apoptosis [44,45]. MEIS1 accompanied by HOX protein can form a complex to recruit transcriptional core- pressors or coactivators [13]. Meanwhile, the activation of MEIS1 always couples with the activation of HOXA7 or HOXA9 [46]. Previous papers have summarized the impact of HOX genes on tumors and concluded that HOX genes take part in the development of different tumors [47]. There was research which found that MEIS1 and PBX2 overexpressed in nephroblastomas [48]. So, there may be interference with HOX genes and PBX genes during the role of MEIS1 in cancers. A further mechanism of MEIS1 for different tumors is needed, to explore in the future.

According to KEGG analysis in this study, “Transcriptional misregulation in cancer” might play a vital role in the effect of MEIS1 on cancers. As an important transcription factor, if MEIS1 is over-expressed or low-expressed, transcriptional misregulation will emerge and then play a role in cancer development [49]. Based on GO analysis, MEIS1 may take part in tumor development by regulating cell differentiation and “DNA-binding transcription activator and repressor activity”, “RNA polymerase II-specific”, “enhancer sequence-specific” and “activating transcription factor binding”. Several researches have shown that MEIS1 serves as a tumor suppressor in ccRCC [50], prostate [29], NSCLC [25], gastric [51], and colorectal cancers [52] by promoting cell differentiation and inhibiting epithelial cell proliferation.

5. Conclusions

To sum up, our findings offer a comprehensive understanding of the functions of MEIS1 in prognostic and immunotherapy in different types of cancer. MEIS1 has the potential as a cancer immunotherapy target and is worthy of more attention. Moreover, our

results may provide valuable theoretical guidance for further research on the mechanism of MEIS1 in vivo and in vitro.

6. Limitations

This research had some restrictions. Initially, we only performed a series of bioin- formatics analysis of MEIS1 in multiple databases, the mechanisms of MEIS1 in different cancer types were not verified. Secondly, the small samples for some tumor types may cause inaccurate results or bias.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jcm12041646/s1, Figure S1: The Genomic context of MEIS1, Figure S2: The specificity expression of MEIS1 in single cell. (a) The single cell type specificity expression of MEIS1. (b) The specificity expression of MEIS1 in blood and immune cells. Figure S3: MEIS1 gene expression level in 16 tumor tissues and normal tissues in GEO database. **** p < 0.0001; ** p < 0.01; * p < 0.05. Figure S4: MEIS1 expression level in different pathological stages of THCA, BRCA, CESC, UCEC, DLBC, ESCA, HNSC, kidney chromophobe (KICH), LUAD, LUSC, UCS, PAAD, READ, SKCM, STAD, TGCT, BLCA, CHOL, and OV (p > 0.05).

Author Contributions: W.L .: conceptualization, writing-original draft preparation. H.L .: methodol- ogy, formal analysis. Y.T .: formal analysis. Z.W. and G.D .: software, validation. S.W .: data curation. L.H .: writing-reviewing and editing. S.L .: conceptualization, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding: The study will be financed by China Postdoctoral Science Foundation (2022M721682) and the Medical Scientific Research Project of Jiangsu Provincial Health Commission (ZD2022053).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The datasets presented in this study can be found in online repositories. The names of the repository /repositories and accession number(s) can be found in the article.

Conflicts of Interest: The authors declare no conflict of interest.

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