Potential Role of CTNNA3 And FRMPD4 In Vascular Thrombosis of Colon Adenocarcinoma

Background: Vascular tumor thrombus is an important pathological feature of malignant tumors, closely related to lymph node metastasis, and considered to be tumor micrometastasis. Two down-regulated genes, Catenin Alpha 3 (CTNNA3) and FERM And PDZ Domain Containing 4 (FRMPD4) were selected via analyzing the differential expression of vascular tumor thrombus in colon adenocarcinoma and paracancerous tissues. Furthermore, we observed the potential role of them in vascular thrombosis of colon adenocarcinoma. Method: Candidate genes for vascular thrombosis of colon adenocarcinoma were screened by GSE127069, and then pan-cancer veri�cation and immune in�ltration analysis were performed. The relationship between gene and vascular thrombosis was analyzed from the level of gene mutation by cBioPortal. Finally, the clinical samples collected were used for expression veri�cation. Results: CTNNA3 and FRMPD4 were low-expressed in vascular tumor thrombus of colon adenocarcinoma, which were positively correlated with microsatellite instability (MSI). They were also closely related to immune microenvironment and immune cell subtype in�ltration. According to the analysis of gene mutation, their gene deletion was related to the vascular invasion indicator. Finally, the protein and mRNA expression of CTNNA3 and FRMPD4 were down-regulated in the samples of vascular thrombosis of colon adenocarcinoma, compared with the normal glands which from paracancerous tissues. Conclusion: Our study demonstrates that CTNNA3 and FRMPD4 may be potential biomarkers of vascular thrombosis in colon adenocarcinoma. These results may provide a new strategy to identify the micrometastasis in colon adenocarcinoma.


Introduction
Colorectal cancer is the third most common cancer in men worldwide, after prostate cancer and lung cancer, and the second most common cancer in women, after breast cancer (1).In recent years, the incidence of colon cancer has been an increasing trend, and most of patients are diagnosed in the middle or advanced stages (2).Although postoperative overall survival of colon cancer has been prolonged in recent years, however, there are still 30%~50% of patients with postoperative recurrence, tumor metastasis, and eventually death (3).
The invasion and metastasis of cancer cells are important factors that determine the prognosis of patients with colon cancer, and the occurrence of tumor thrombus in blood vessels is a prerequisite for tumor invasion of vascular system and lymph node metastasis (4,5).Vascular tumor thrombus in ltration is an important prognostic factor for many malignant tumors, such as endometrial cancer (6), breast cancer (7), gastric cancer and colon cancer (8).Different studies have suggested that the incidence of tumor thrombus in the midvein of colon cancer ranges from 10-89.5% (9)(10)(11).Colon cancer thrombus mainly includes direct tumor invasion, blood re ux, tumor cell exfoliation, adhesion, in ltration, proliferation (12).The cancer cells can in ltrate into submucosa which is rich in vascular system, easily in ltrate and form vascular tumor thrombus.Genetic mutations are critical tovascular tumor thrombus in colon cancer (13).However, the relation between gene alteration and tumor thrombus is not clear.Therefore, it is of great signi cance to nd potential biomarkers of vascular thrombosis for treating colon cancer.
Vascular tumor thrombus leads tumor cells to better tolerate injury, promote tumor cell metastasis (14).
Study has shown that, compared with the negative group, the risk of death in the vascular tumor thrombus positive group increased by 1.748 times, and OS signi cantly decreased (15).Lymphatic tumor thrombus in ltration is an important risk factor for sentinel lymph node metastasis (16).Therefore, patients with II stage of colon cancer with positive vascular tumor thrombus in ltration should receive chemotherapy even if there is no lymph node metastasis after operation.it also has guiding value for preoperative neoadjuvant therapy.
Antivascular and antilymphatic therapy is of great signi cance for patients with vascular tumor thrombus, such as bevacizumab and Shunitinib can inhibit angiogenesis, and reduce lymph node metastasis (17,18).At present, the common method to detect vascular tumor thrombus is tissue section hematoxylin-eosin (HE) staining (19), but the detection rate of vascular tumor thrombus in II colorectal cancer varies from 5.2-30% (20).Unfortunately, the ability to distinguish lymphovascular invasion from blood vessel invasion is poor.
In this study, two signi cantly low-expressed genes Catenin Alpha 3 (CTNNA3) and FERM And PDZ Domain Containing 4 (FRMPD4) were obtained from the analysis of the difference between intratumoral tumor thrombus group and normal adjacent tissues of colon adenocarcinoma.After TCGA pan-cancer veri cation, it was found that these two genes may be associated with microsatellite instability (MSI) and immune in ltration.According to the analysis of gene mutations, we found two genes had different degrees of gene mutation, such as ampli cation, deep deletion and missense mutation, and were related to methylation.Immune cell in ltration analysis also shown that these two genes were associated with different immune cell in ltration.Finally, we found that these two genes were down-regulated in patients with colon adenocarcinoma thrombus at the mRNA and protein levels.In short, we found potential biomarkers in patients with colon adenocarcinoma thrombus, which has a certain guiding signi cance for the therapy of patients with cancer thrombus.

Patient Data acquisition
RNA sequencing (GSE118389) for colon adenocarcinoma tissues and adjacent tissues of third-stage colon cancer patients with blood vascular thrombus was downloaded from GEO (Gene Expression Omnibus) (https://www.ncbi.nlm.nih.gov/geo/).Twenty cases of colon adenocarcinoma tumor thrombus and adjacent normal tissues came from the Second People's Hospital of Deyang City for detection of gene expression.The patients with left colon cancer included 9 males and 11 females.The clinical stages were II stage (n = 15) and III stage (n = 5).The pathological types of the samples were all tubular adenocarcinoma.This study has been approved by the Second People's Hospital ethics committee and the patient's informed consent.All the methods were performed in accordance with the relevant guidelines and regulations.

Differentially Expressed genes
'Limma' package was used to perform the batch correction of the GSE118389 database.The R software (version 4.0.5) was used to perform differentially expressed genes in the normal groups (n=3) and colon cancer thrombus (n=3) groups.The cutoff criteria were FDR p-value < 0.05 and logFC > 1.
2.3 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis R software were used to perform GO and KEGG analysis.88 differentially expressed genes and logFC values were selected for functional enrichment analysis of GO and KEGG.

Protein-protein interaction
The protein-protein interaction (PPI) network was analyzed online through STRING website (https://stringdb.org/).The co-expression relationship of 88 differential genes was visualized by PPI network.

Association between gene expression and MSI in pancancer
The MSI score was obtained from TCGA.Spearman's method was employed to evaluate the correlation between cancer gene expression and MSI.The radar map was designed using the 'fmsb' package from R software to visualize the both indicators.

Correlation of gene expression with tumor immune microenvironment and immune cell in ltration
The ESTIMATE algorithm ("estimate" and "limma" packages in R software) was employed to calculate immune scores, for predicting the tumor purity and in ltrating immune cells in colon adenocarcinoma.The "ggplot2", "ggpubr" and "ggExtra" packages in R software were employed to evaluate the correlation between CTNNA3 or FRPMD4 mRNA expression and the tumor immune microenvironment.

Correlation analysis of immune cell in ltration
The Pearson' method was used to analysis the correlation between CTNNA3 or FRPMD4 mRNA expression and immune cell in ltration.The in ltration data of B cells, CD4 + T cells, CD8 + T cells, dendritic cells, macrophages, neutrophils, T cell regulatory (Tregs) can be downloaded from the TIMER 2.0 database (http://timer.cistrome.org/).

Immunohistochemistry (IHC).
Human colon adenocarcinoma samples, colon adenocarcinoma thrombus, and adjacent normal tissue were used for IHC staining.IHC was performed according to the manufacturer's instructions (cat.no.SP9001; ZSGB-BIO).The primary antibody (anti-FRMPD, ab113420, abcam), anti-CTNNA3 (ab184916, abcam), and anti-CD34 (ab81289, abcam)) were used for IHC.The speci c operation steps were performed as previously described (21).The protein expression level was determined according to the staining intensity and the percentage of immunoreactive cells.

Statistical analysis
GraphPad Prism 7.0 was used for statistical analysis.Student's t-tests were used to perform differential comparisons of two groups.P value <0.05 considered to be statistically signi cant.The integrated analysis of these data was performed using R software (version 4.0.3).

CTNNA3 and FRMPD4 are low expressed in colon adenocarcinoma tumor thrombus
The differential analysis of the gene expression pro les of the colon adenocarcinoma para-normal group (n=3) and the colon adenocarcinoma thrombus group (n=3) was performed in the GSE127069 chip.The volcano map (Fig. 1A) and the heat map (Figure 1B) were used to visualize differentially expressed genes.The results shown that CTNNA3 and FRMPD4 were speci cally down-regulated in colon adenocarcinoma tumor thrombus samples.The protein-protein interaction network also visualized the coexpression of differential genes (Fig. 2).According to the analysis of the number of PPI nodes, the results shown that the number of CTNNA3 and FRMPD4 nodes was the most (Fig. 1C).In addition, GO and KEGG enrichment analysis were performed on 88 differential expressed genes (Fig. 1D, E), GO analysis shown that the differential genes were related to second-messenger-mediated signaling, positive regulation of cytosolic calcium ion concentration, regulation of cytosolic calcium ion concentration, cellular calcium ion homeostasis, and calcium ion homeostasis.KEGG analysis shown that the differential genes are related to chemokine signaling pathway, cocaine addiction, amphetamine addiction, gastric acid secretion, and insulin secretion.
3.2 CTNNA3 and FRMPD4 are related to MSI and immune microenvironment.
In order to further verify the expression and function of CTNNA3 and FRMPD4 in colon adenocarcinoma, we performed pan-cancer analysis on the TCGA database.The results shown that the expression of CTNNA3 and FRMPD4 in colon adenocarcinoma was lower than that in normal tissues (Fig. 3A, C), but there was no signi cant difference in their expression between tumor stages (Fig. 3B, D).MSI is caused by a defect in the mismatch repair gene and is strongly correlated with the occurrence of tumors.Clinically, MSI has been used as an important molecular marker for the prognosis of colorectal cancer and the formulation of adjuvant treatment programs.We found that CTNNA3 and FRMPD4 were positively correlated with MSI in colon adenocarcinoma (Fig. 3E, F).Due to the tumor immune microenvironment, the heterogeneity between tumor cells is activated, thereby increasing multidrug resistance, causing tumor cell progression and metastasis.Therefore, it is reasonable to explore the correlation between tumor immune microenvironment and the expression of CTNNA3 or FRMPD4 in colon adenocarcinoma.The ESTIMATE algorithm was used to calculate immune cell scores for colon adenocarcinoma.The results shown that the expression of CTNNA3 or FRMPD4 were positively related to the immune scores in colon adenocarcinoma (Fig. 3G, H), indicating that the level of CTNNA3 and FRMPD4 expression was upgraded as immune cells increases.

Gene mutations of CTNNA3 and FRMPD4 are associated with vascular invasion indicator
In order to further explore the functions of CTNNA3 and FRMPD4 in colon adenocarcinoma thrombus, we used the cBioPortal online tool to analyze gene functions.As shown in Fig. 5A and 6A, the genetic changes of CTNNA3 or FRMPD4 and the relationship between vascular invasion indicator were shown respectively.The results indicated that missense mutations of CTNNA3 were related to vascular invasion indicator, and missense mutations and deep deletion of FRMPD4 were related to vascular invasion indicator.Next, we analyzed the genes related to genetic altered, and the results shown that the genetic altered of CTNNA3 were related to TNN, PIK3CA, MACF1, TENM3, and RIMS1 (Fig. 5B), while the genetic altered of FRMPD4 were related to TLR7, FAM9B, WWC3, MXRA5, and DMD related (Fig. 6B).The analysis of correlation between gene mRNA expression and copy number variation shown that the mRNA expression of CTNNA3 and FRMPD4 were all related to shallow deletion, diploid, gain, etc. (Fig. 5C, Fig. 6C).Interestingly, the mRNA expression of CTNNA3 was also related to gene methylation (HM450) (Fig. 5D, Spearman, R=0.34; Pearson, R=0.42.P<0.001).The mutation site analysis results shown that the mutation of CTNNA3 The site was located at Y414C (Fig. 5E), and the mutation site of FRMPD4 was located at E619A/D (Fig. 6D).

CTNNA3 and FRMPD4 were down-regulated in patients with colon adenocarcinoma thrombus
Finally, in order to validate the expression of CTNNA3 and FRMPD4 in clinical practice, we collected 20 patients with colon adenocarcinoma thrombus.HE staining and CD34 staining were used to determine the morphology of tumor tissue and tumor thrombus, respectively (Fig. 7A).The expression of CTNNA3 and FRMPD4 in tumor thrombus tissues was lower than that in normal tissues (Fig. 7A, D, E).We also performed gene mRNA expression veri cation, which shown that the expression of CTNNA3 and FRMPD4 mRNA in colon adenocarcinoma embolus was lower than that in normal adjacent tissues (Fig. 7B, C).

Discussion
In this study, we analyzed the differential expression pro les of genes from GSE127069 and selected biomarkers for vascular tumor thrombus of colon adenocarcinoma.Then the gene function was further explored in TCGA and cBioPortal databases.Finally, the gene expression was veri ed with the collected clinical samples.These results suggested that CTNNA3 and FRMPD4 might exist as biomarkers of vascular tumor thrombus in colon adenocarcinoma.Vascular tumor thrombus is one of the characteristics of tumor cells invading adjacent blood vessels and lymphatic vessels, which can be regarded as the early manifestation of lymph node metastasis (23).Both domestic and foreign guidelines regarded vascular tumor thrombus positive as one of the items to evaluate the risk factors of colorectal cancer (24,25).Vascular tumor thrombus is not only an indicator of poor prognosis, but also a manifestation of micrometastasis (11).Related studies have con rmed that vascular invasion is related to the depth of tumor invasion and lymph node metastasis (26).We found that CTNNA3 and FRMPD4 were downregulated in vascular thrombosis of colon cancer and were related to calcium metabolism.Calcium metabolism is closely related to thrombosis (27), which may be one of the reasons why CTNNA3 and FRMPD4 become potential biomarkers of vascular thrombosis in colon adenocarcinoma.MSI, a class of short tandem repeat DNA sequences in the genome, is generally composed of 1-6 nucleotides and is arranged in tandem repeats.Due to the difference in the number of core repeat units, MSI have population polymorphism.Popat S, et al (28) published meta-analysis of 7642 patients with II colorectal cancer in 32 studies shown that compared with MSS patients, the risk of death in MSI-H patients was 0.65 (95% CI, 0.59-0.71),a reduction of up to 35%.At present, there is a great deal of evidence that MSI-H is a good marker for the prognosis of patients with stage II colorectal cancer (29).It has also been found that tumors with MSI-H typing have high immune cell in ltration against new antigens, but are offset by immune checkpoint suppressor ligands such as PD-L1, which binding to PD-1 prevents T cell activation (30).Recently, it has been reported that immune checkpoint inhibitor (pembrolizumab) was effective in MSI-H patients with tumor thrombosis.This re ects the relationship between tumor thrombus and MSI and tumor immune microenvironment (31).Consistent with the previously studies, we found that CTNNA3 and FRMPD4 were positively correlated with MSI, as well as with immune microenvironment and immune cell subtype in ltration.
However, due to the difference in the detection technology of vascular cancer thrombus, the detection rate of vascular cancer thrombus is quite different, resulting in many false negative, which may weaken the value of vascular cancer thrombus, resulting in a lack of understanding of vascular cancer thrombus.The study of 75 cases of colorectal cancer found that the lymphatic in ltration rate of HE staining was 19%, while the detection rate of lymphatic invasion of monoclonal antibody D2-40 was 40% (P < 0.001) (32).Morgantetti G,et al (33) also believes that CD34 antibody has strong stability, sensitivity and repeatability in the detection of capillaries and small vascular endothelial cells in tumors, so CD34 antibody is the preferred vascular endothelial cell marker for the evaluation of vascular cancer thrombus.We also used CD34 as a marker of vascular thrombus when we veri ed the protein expression by immunohistochemistry, which further veri ed the expression of candidate genes in vascular tumor thrombus.
We further found that both CTNNA3 and FRMPD4 have a high proportion of gene deep deletion in gene mutations in colon adenocarcinoma.That also indicated that downregulation of CTNNA3 and FRMPD4 in vascular tumor thrombus of colon adenocarcinoma is understandable.CTNNA3 is a tumor suppressor, which often mutates in laryngeal carcinoma and is downregulation in bladder urothelial carcinoma (34).CTNNA3 is a tumor suppressor of hepatocellular carcinoma, which inhibits the proliferation, migration and invasion of hepatocellular carcinoma.CTNNA3 inhibits AKT signal, which in turn reduces PCNA and MMP-9 in tumor cells (35).There are few studies on FRMPD4 in tumors.FRMPD4 plays an important role in normal cognitive development and function in humans and mice, and FRMPD4 mutation may lead to X-linked mental retardation syndrome by destroying the morphology of dendritic spines in glutamatergic neurons (36).The above studies and results shown that CTNNA3 and FRMPD4 have the characteristics of biomarkers in vascular tumor thrombus of colon adenocarcinoma.
The advantage of this study is that multiple databases are used to analyze the selected candidate genes, and clinical samples are used to verify the results.Then, this study also has many limitations.For example, there are few clinical samples for veri cation, and the candidate genes have not been veri ed from the biological function level.In the future research, we will reasonably design the experimental scheme to further study the candidate genes.

Conclusion
In our study, we performed integrated analysis of the GEO, TCGA and cBioPortal databases and found that CTNNA3 and FRMPD4 are involved in cancer biological functions such as MSI, immune subtype in ltration, and methylation in colon adenocarcinoma, and their mutations are also associated with vascular invasion indicator.Viewed in total, CTNNA3 and FRMPD4 may be regarded as potential biomarkers in patients with colon adenocarcinoma thrombus.

Declarations
No funding information.

Consent for publication
The patient consented all the individual person's data to publish.The PPI network of differentially expressed genes.

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