Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 16  |  Issue : 7  |  Page : 1596--1602

Incomplete ablation of colon cancer cells may induce activation of dormant cells: Evidence from bioinformatics analysis


Wenli Lin1, Jie Liu1, Wei Lv2, Changling Liu1, Yuping Sun1, Taiyang Zuo1,  
1 Department of Oncology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China
2 Department of Cardiology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China

Correspondence Address:
Taiyang Zuo
Department of Oncology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, 105 Jiefang Road, Jinan, Shandong Province 250013
China
Yuping Sun
Department of Oncology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, 105 Jiefang Road, Jinan, Shandong Province 250013
China

Abstract

Purpose: It is not yet verified whether incomplete radiofrequency ablation (iRFA) induces tumor progression and hypoxia related to tumor dormancy. This study showed the relationship between iRFA and tumor dormancy. Materials and Methods: To identify the candidate genes in the control and iRFA-treated colon cancer cells, microarray datasets GSE138224 were downloaded from Gene Expression Omnibus database. Using NetworkAnalyst, the differentially expressed genes (DEGs) were identified, function enrichment analyses were performed, and the protein–protein interaction (PPI) network and key PPI network were constructed. Results: A total of 656 DEGs were identified, comprising 637 downregulated and 19 upregulated genes. The enriched functions and pathways of the upregulated DEGs include an immune effector process, regulation of tyrosine phosphorylation of signal transducer and activator of transcription (STAT) protein, tyrosine phosphorylation of STAT protein, JAK-STAT cascade, and regulating JAK-STAT cascade, and CCL5 gene participated in regulating the JAK-STAT signaling pathway. The downregulated DEGs were mainly enriched in extracellular matrix–receptor interaction, PI3K-Akt signaling, Wnt signaling, transforming growth factor-beta signaling, and mitogen-activated protein kinase signaling pathways. There are three key PPI networks of DEGs (degree ≥10 and hub genes >3). The dormancy-related genes Bmp4 and Ccl5 were regarded as hub genes in the PPI network with Bmp4 as a downregulated gene and CCL5 as an upregulated gene. Conclusion: The identified DEGs and function enrichment analyses in this study aid the understanding of molecular mechanisms underlying the relationship between iRFA and tumor dormancy.



How to cite this article:
Lin W, Liu J, Lv W, Liu C, Sun Y, Zuo T. Incomplete ablation of colon cancer cells may induce activation of dormant cells: Evidence from bioinformatics analysis.J Can Res Ther 2020;16:1596-1602


How to cite this URL:
Lin W, Liu J, Lv W, Liu C, Sun Y, Zuo T. Incomplete ablation of colon cancer cells may induce activation of dormant cells: Evidence from bioinformatics analysis. J Can Res Ther [serial online] 2020 [cited 2021 Apr 13 ];16:1596-1602
Available from: https://www.cancerjournal.net/text.asp?2020/16/7/1596/301575


Full Text



 Introduction



Tumor dormancy is observed in local recurrence or metastasis in the clinic. It is usually referred to the time after treatment when a patient is free from clinical symptoms, causing relapse and metastasis, and refractive to common chemotherapy.[1] The dormancy tumors are characterized by long periods of quiescence marked by cell cycle arrest in the G0/G1 phase.[2] In breast and prostate cancer, the proliferation of dormant cells can induce cancer recurrence after several years of surgical resection.[3] Epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer treated with EGFR and tyrosine kinase inhibitor (TKI) could induce tumor dormancy, and dormant tumor cells are drug resistant.[4] Multiple mechanisms are involved in tumor dormancy, such as hypoxic and the presence of microscopic tumors. While most tumor cells die under tumor hypoxic conditions, some of them can adapt and survive in a dormant state for many days or months.[5] Therefore, tumor dormancy may be in a natural state during tumor evolution.[6] Dormant tumor cells may develop nonspecific resistance mechanisms to cell death, such as deregulation of JAK/signal transducer and activator of transcription (STAT) pathway,[7] growth arrest-specific protein 6 (GAS6), bone morphogenetic protein 4 (BMP4), BMP7, transforming growth factor-β2 (TGF-β2).[8],[9],[10] The presence of dormant tumor cells is one of the reasons for poor patient prognosis.

Radiofrequency ablation (RFA) is the most used thermal technique. The thermal ablation of tumors is used locally at extreme temperatures to induce cell coagulative necrosis, thereby curing tumor or reducing the tumor load,[11],[12],[13] but many studies have reported incomplete RFA (iRFA)-induced tumor progression due to minimal residual lesion, and it is well known that iRFA could cause tumor hypoxia. It is not yet verified whether iRFA-induced tumor progression and hypoxia relate to tumor dormancy. This study showed the relationship between iRFA and tumor dormancy and also obtained essential genes using data analysis. Microarray technology enables us to explore the genetic alterations in iRFA-treated cells and has been proven to be a useful approach to identify new biomarkers in other diseases. Thermal ablation is used to induce tumor cell necrosis, and most gene expressions are inhibited. Therefore, it is required to study upregulated genes after thermal ablation.

 Materials and Methods



Microarray data

Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo)[14] is a public functional genomics data repository of high-throughput gene expression, chips, and microarrays. The gene expression dataset GSE138224[15] was downloaded from GEO (Illumina GPL17021 Platforms, Illumina HiSeq 2500, Mus musculus). The probes were converted to the corresponding gene symbol according to the annotation information in the platform. GSE138224 dataset contained 3 tumor control and 3 iRFA-treated tumor samples, and we detected the tumor mRNA expression profiles of CT26 colon cancer in Balb/c mice using high-throughput sequencing. The samples were untreated and iRFA-treated CT26 tumors on day 3 after iRFA.

Identification of differentially expressed genes and functional enrichment analysis

NetworkAnalyst (https://www.networkanalyst.ca/NetworkAnalyst/home.xhtml) is an interactive web tool used for comprehensive gene expression analysis, meta-analysis, and network biology.[16] In this study, NetworkAnalyst was used to conduct pathway and process enrichment analysis of differentially expressed genes (DEGs).

Identification of differentially expressed genes

The DEGs between CT26 and iRFA-treated CT26 colon cancer samples were screened using NetworkAnalyst. Use edgeR for non-repetitive differential expression analysis, probe with more than one sets were removed or averaged, respectively. Fold change (logFC) > 1 and adjusted P < 0.05 were considered statistically significant difference.

Kyoto Encyclopedia of Genes and Genomes and gene ontology enrichment analysis of differentially expressed genes

Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding high-level functions and biological systems from large-scale molecular datasets generated by high-throughput experimental technologies.[17],[18] Gene Ontology (GO) is a major bioinformatics tool to annotate genes and analyze the biological process of these genes.[19] To analyze the function of DEGs, the GO terms and KEGG pathways were enriched based on the NetworkAnalyst online tool. P < 0.05 was considered a statistically significant difference.

Protein–protein interaction network construction and key protein–protein interaction network selected

Protein–protein interaction (PPI) enrichment analysis was performed using the following database: STRING interactome.[20] Analyzing the functional interactions between proteins may provide insights into the mechanisms of generation or development of diseases. In this study, PPI network of DEGs was constructed with NetworkAnalyst online STRING database tool, and confidence score cutoff >900 was considered a statistically significant difference. The key PPI network was selected with degrees ≥10 and hub genes >3, and the network of the genes and their coexpression genes was also analyzed using NetworkAnalyst web tool.

 Results



Identification of differentially expressed genes in CT26

After edgeR, matched gene number was 16161, the differentially expressed genes number was 656, and downregulated genes number was 637, upregulated genes number was 19 [Figure 1]a and [Figure 1]c. It is interesting to study upregulated genes after thermal ablation; therefore, we showed the upregulated genes by [Table 1] and its expression by heat map [Figure 1]b. Thermal ablation is used to induce tumor cell necrosis, and gene expressions are suppressed; most genes are downregulated after ablation; therefore, the upregulated genes after ablation are more clinically significant.{Figure 1}{Table 1}

Kyoto Encyclopedia of Genes and Genomes and gene ontology enrichment analyses of differentially expressed genes

To analyze the biological classification of DEGs, functional and pathway enrichment analyses were performed using NetworkAnalyst. KEGG analysis results revealed that upregulated DEG changes in the toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, natural killer cell-mediated cytotoxicity, and others [Figure 2]b. GO analysis results showed that upregulated DEG changes in biological processes (BPs) were significantly enriched in immune the effector process, the regulation of tyrosine phosphorylation of STAT protein, tyrosine phosphorylation of STAT protein, JAK-STAT cascade, regulating JAK-STAT cascade, and others [Figure 2]a. Notably, CCL5 gene participated in regulating JAK-STAT signaling pathway [Table 2]. The upregulated DEG changes in molecular function (MF) were mainly enriched in enzyme activator activity, chemokine activity, kinase activator activity, and others [Figure 2]c. Changes in cell component (CC) of upregulated DEGs were not statistically significant.{Figure 2}{Table 2}

Observably, the downregulated DEGs were mainly enriched in extracellular matrix (ECM)–receptor interaction, PI3K-Akt signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and others [Figure 3]a. GO analysis results showed that the downregulated DEG changes in BP terms were mainly enriched in the enzyme-linked receptor protein signaling pathway, tissue morphogenesis, anatomical structure morphogenesis, and others [Figure 3]b. The downregulated DEG changes in MF terms were enriched in receptor binding, heparin binding, actin binding, and others [Figure 3]c. Changes in CC of downregulated DEGs were enriched in the extracellular space, extracellular region part, proteinaceous ECM, and others [Figure 3]d.{Figure 3}

Protein–protein interaction network construction and key protein–protein interaction network selected

The PPI network of DEGs was constructed [Figure 4]. Upregulated gene was marked in red dot, and downregulated gene was marked in green. The dot size represents the degree of genes; the larger the dot, the higher the degree.{Figure 4}

The key PPI network of DEGs was selected with degree ≥10 and hub genes >3 [Figure 5]. The results showed three networks. Interestingly, the dormancy-related genes Bmp4 and CCL5 were regarded as hub genes in the PPI network with Bmp4 as a downregulated gene and CCL5 as an upregulated gene.{Figure 5}

 Discussion



Dormant tumor cells are characterized by cell cycle arrest in the G0/G1 phase with low metabolism, when conditions improve, dormant cells will proliferate again, and the presence of dormant tumor cells could gradual growth of drug-resistant tumor. Most times, in the clinic, patients may enter complete remission in which dormant cells represent the minimal residual disease following treatment.[21] EGFR-mutant non-small cell lung cancer treated with the EGFR-TKI treatment could induce tumor dormancy by activating the YAP/TEAD pathway. YAP/TEAD engages the epithelial–mesenchymal transition transcription factor SLUG to directly repress proapoptotic BMF, limiting drug-induced apoptosis, coinhibition YAP and TEAD could deplete dormant cells.[4] Dormant tumor cells may develop nonspecific resistance mechanisms to cell death, such as deregulation of JAK/STAT and mTORC2/AKT pathways,[7] and may also overexpress B7H1 and B7.[21],[22] Dormant tumor cells are one of the quiescent tumor stem cells, and there are many factors that induce residual disease dormancy, for example, GAS6, BMP4, BMP7, TGF-β2,[8],[9],[10] and others. Studies have revealed that breast cancer cells with low CoCo (BMP4 inhibitor protein) expression remained dormant,[23] and leucine-rich repeats and immunoglobulin-like domains protein 1[24],[25],[26] could regulate tumor stem cell quiescence and dormancy. The dormant tumor cell is characterized by the persistence of residual tumor cell for a long period, and it could induce the tumor recurrence and drug resistant, which is one of the reasons for poor patient prognosis; it is well known that iRFA-induced tumor progression in the presence of minimal residual lesion, it is unclear whether the activation of dormant cells is involved in iRFA-induced tumor progression. In this study, we have explained this problem based on gene expression differences.

In this study, GSE138224 datasets were analyzed to obtain DEGs between colon cancer cells and iRFA-treated colon cancer cells. A total of 656 DEGs were identified, comprising 637 downregulated and 19 upregulated genes. GO and KEGG enrichment analyses were performed to explore the interactions among DEGs. The unregulated genes were mainly enriched in the immune effector process, regulation of tyrosine phosphorylation of STAT protein, tyrosine phosphorylation of STAT protein, JAK-STAT cascade, and regulating JAK-STAT cascade, while the downregulated genes were mainly enriched in ECM–receptor interaction, PI3K-Akt signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, and MAPK signaling pathway. Studies have reported that JAK/STAT pathways play critical roles in the dormant tumor cells.[7],[21] Tumor cells that persist for long periods may deregulate their JAK/STAT pathways in the dormant tumor cells resisted apoptosis model,[7] and JAK/STAT pathways may be a possible escape mechanism of dormant tumor cells selected drug-resistant cells. A similar mechanism has reported that the overexpression of granulocyte-macrophage colony-stimulating factor by TKI-resistant cells protects sensitive cells through the JAK2/STAT5 pathway.[27] In our data analysis, we found that JAK/STAT pathway genes were upregulated after iRFA, suggesting that JAK/STAT pathways were activated, suggesting that dormant tumor cells may be activated by JAK/STAT pathways. Thus, a possible mechanism of iRFA-induced tumor progression is inducing dormant tumor cells activated by JAK/STAT pathways.

Notably, CCL5 gene participated in regulating JAK-STAT signaling pathways. CCL5 was one of the DEG upregulated genes, and after GO and KEGG enrichment, CCL5 participated in the JAK/STAT pathways activation, PPI analysis further confirmed that CCL5 played a critical role in the PPI network of DEGs and in the key PPI network of DEGs. CCL5, C-C chemokine ligand 5, belongs to the C-C chemokine family, known as regulated by activated T-cells.[28] Studies have also shown that chemokines promote tumor cell proliferation by activating tyrosine kinase receptors, the JAK-STAT or MAPK/MER signaling pathways.[29] CCL5 can induce migration by upregulating the activities of MMP-9 through STAT3.[30] In breast tumor cells, increased positivity and expression levels of CCL5 are significantly associated with disease progression,[31] relapse,[32] metastasis,[32],[33] and drug resistance.[34] Our study showed that CCL5 was overexpressed in DEGs after iRFA and participated in the activated JAK/STAT pathways, so CCL5 may be involved in tumor progression after iRFA by JAK/STAT pathways. As mentioned above, dormant tumor cells may be activated by JAK/STAT pathways after iRFA. It is speculated that CCL5 may participate in the activation of dormant tumor cells after iRFA through the JAK/STAT signaling pathway. This speculation provides a theoretical basis for ablation combined with CCL5 inhibitors to treat tumors.

BMP4 is another notable gene in our study. BMP4 was one of the downregulated genes of DEGs, PPI analysis confirmed that BMP4 also played a central role in the PPI network of DEGs and in the key PPI network of DEGs. Studies have reported that BMP4 induces residual disease dormancy in different types of cancer,[8],[10],[35] in order to escape dormancy and become proliferative dormant disseminated tumor cells can inhibit BMP signaling by expressing inhibitors.[36] The inhibition of BMP4 induces the proliferation of dormant mouse mammary 4T07 in the lungs.[9] Briefly, BMP4 is a crucial factor in regulating tumor cell dormancy. Our results showed BMP4 suppression, which further explained that tumor dormancy was inhibited after iRFA. After GO and KEGG enrichment of downregulated DEGs, the results also showed that PI3K-Akt signaling pathway was one of the enriched pathways of downregulated DEGs. In addition, studies have shown that reduced PI3K-Akt signaling is linked to dormancy-like phenotypes.[36],[37],[38] In the presence of nutritional stress, cancer cells secrete factors that inhibit the PI3K pathway resulting in quiescence and autophagy induction.[36] Therefore, suppression of PI3K-Akt signaling should initiate cell dormancy, which is contrary to our previous analysis. The following reasons can explain this phenomenon. First, the purpose of thermal ablation is to kill tumor cells, and most gene expression is suppressed. Second, there is an activation and suppression mechanism for tumor dormancy after iRFA, which requires further experimental results to verify the specific existing mechanisms.

 Conclusion



This study was designed to identify DEGs that may be involved in tumor dormancy of colon cancer after iRFA. CCL5 and BMP4 genes and JAK-STAT and PI3K-Akt signaling pathways were identified and may be regarded as diagnostic biomarkers for tumor dormancy of colon cancer after iRFA. However, further studies are required to elucidate the biological function of these genes in colon cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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