Determination of whole-genome expression differences in larynx cancers

Authors

Abstract

AimMore than 200.000 new laryngeal cancer cases have been diagnosed worldwide, annually. The incidence and prevalence have increased during the past decades. It also has a high mortality rate. To have a better knowledge about this particular cancer type, we aimed to conduct a comparative analysis of whole genome expression differences between laryngeal squamous cell carcinoma and adjacent normal tissues
MethodsTissue samples were collected from specimens of laryngeal carcinoma and normal epithelium tissues adjacent to the carcinoma. RNAs isolated from these specimens were used for genome-wide gene expression analysis using microarrays. Genes that were expressed significantly differently in the tumor sample compared to normal tissues were identified. Pathway analysis of these genes was also performed.
ResultsIt was observed that 24 genes were significantly differentially expressed in cancer tissue. Expressions of MMP1, MMP12, S100A2, S100A3, CK14, CK16, SLC2A1, ITGA6, CEP55, KLK6, LAMC2, IL1F9, TP63, VSNL1, CXCL1, COL4A5, COL4A6, FSCN1, KRT6B, KRT17, WDR66 and ACOT7 genes were increased, while expressions of ITM2A and CFD genes were decreased.
ConclusionSome of the genes mentioned above are known to be involved in the etiology of laryngeal cancer, but to the best of our knowledge, 10 genes have been associated with laryngeal cancer for the first time in our study. These genes might be useful as biomarkers in the clinic, for early diagnosis, prognosis and personalized targeted therapy.

Keywords

Introduction

laryngeal carcinoma remains a considerable cause of morbidity and mortality with ~180,000 new cases and 99,000 deaths, accounting for 50% of mortality worldwide in 2020.¹ several genetic and environmental factors have been identified to be associated with the development and progression of laryngeal squamous cell carcinoma (lscc). among them, the major etiological agents are tobacco and alcohol consumption. in recent years, these agents have been shown to cause molecular changes and a sequence of events in cells, including oncogene activation, inhibition of tumor suppressor genes, and changes in gene expression profiling. clinical outcomes in laryngeal cancer patients are thought to be based on clinicopathological features; however, few studies have indicated that gene expression alterations, as well as genomic and epigenetic changes, can also be used to predict prognosis. due to this reason, molecular characterization of laryngeal cancers seems to be extremely important. certain success has been achieved in molecular studies on the etiology of larynx cancer, but previous studies on larynx cancers were uncertain and incomprehensive.^2,3
lately, gene expression profiling (gep) using microarrays has been identified as a hopeful method to identify variations occurring in the pathophysiology of complex diseases such as cancers. identification of gene expression changes associated with laryngeal cancer gives us information about the mechanism of the disease and the pathways associated with this disease. thus, new biological and prognostic markers can be identified.⁴
there are many studies in the literature aimed at clarifying the etiology of the disease, but the pathogenesis of this neoplasm and associated pathways are still not fully understood. the lack of significant improvement in survival rates over the past 25 years is proof of this.⁵ that is why we aimed to conduct a comparative analysis of whole genome expression differences between laryngeal squamous cell carcinoma and adjacent normal tissues. these efforts will ensure a better understanding of molecular mechanisms underlying laryngeal carcinoma, as well as enable identification of novel biomarkers and therapeutic targets.

Materials and Methods

Tissue SamplesFresh tumor and adjacent normal tissue biopsy specimens were taken from n = 12 patients diagnosed with larynx cancer between March 2015 and April 2016. Clinical characteristics of the patients are shown in Table 1. These tissues were placed in RNA Later solution and stocked at -80 C until use. Informed consent was obtained from patients or their families. This study was approved by the ethics committee of Necmettin Erbakan University (decision number 2015/146) and financial support was provided by the Scientific Research Projects of Necmettin Erbakan University (Project Number:151518008).
RNA IsolationRNA isolation was performed by Norgen Total RNA Purf. Kit (cat.no. 25700). RNA concentration was detected by Thermo Scientific Nonodrop 2000c. Then, RNA quality and quantity were identified by Agilent RNA 6000 Nano Kit (reorder-no 5067-1511). Thus, RNA was purified from other molecules with the same absorbance.
Synthesis of CDNA and In Vitro Transcription of Biotin-CRNAThe obtained RNA was converted to cDNA using the TargetAmp™-Nano Labeling Kit (Illumina® Expression BeadChip®) before the hybridization step. First-strand cDNA synthesis was catalyzed by Super Script III Reverse Transcriptase. The produced cDNA was converted to double-stranded cDNA for use in the subsequent in vitro transcription reaction. Then, in vitro transcription was performed to obtain biotinylated cRNA. The synthesized biotin-labeled RNA was cleaned up again (Norgen RNA Clean-Up and Conc. Micro Kit 23600,43200) and cRNA levels of all samples were equalized before being sent to the microarray platform.
Microarray AnalysisThe biotin-labeled RNA was processed through hybridization, washing, labeling with streptavidin, and drying before installation. The labeled RNA strand was hybridized to the bead on the BeadChip (HumanHT-12 v4.0 Exp. BeadChip Kit) containing the complementary gene-specific sequence. This kit presents a comprehensive analysis of genome-wide expression. Each channel on the BeadChip contains approximately 47,000 different bead types distributed throughout the genome. Contents of the kit were created with reference to the NCBI RefSeq database. Hybridization was performed at 58 C for 20 h. BeadChips were washed after overnight hybridization. Unbound RNAs were removed. BeadChips were labeled with streptavidin, centrifuged immediately after washing, and stored in a dark, ozone-free environment until scanning. Finally, BeadChips were read in a microarray laser reader (Illumina® Whole-Genome Gene Expression Direct Hybridization Assay system).
Bioinformatics AnalysisPreliminary analyses and quality controls of the obtained data were performed using the Genome Studio software of the Illumina iScan Microarray system. Fold change (FC) is a strong marker indicating statistical significance of genes observed in the gene expression profile. By FC analysis, changes larger than two-fold were accepted as increase or decrease for each probe. Heat-map analysis for differentially expressed genes was generated and GO (gene ontology) analysis was performed.
Ethical ApprovalEthics Committee approval for the study was obtained.

Results

In this study, we identified that 14,294 genes were differentially expressed between laryngeal squamous cancer tissues and non-neoplastic tissues. When the FC value is considered to be 2 for the significance of the expression differences, increased expression of 22 genes (MMP1, MMP12, S100A2, S100A3, CK14, CK16, SLC2A1, ITGA6, CEP55, KLK6, KRT17, LAMC2, IL1F9, TP63, VSNL1, CXCL1, COL4A5, COL4A6, FSCN1, KRT6B, WDR66 and ACOT7) and decreased expression of two genes (ITM2A and CFD) were observed. Fourteen of 24 genes were known to be involved in the etiology of laryngeal tumors in the literature, but as far as we know, the effect on laryngeal cancer of the remaining 10 genes (VSNL1, CXCL1, COL4A5, COL4A6, FSCN1, KRT6B, WDR66, ACOT7, ITM2A, CFD) was identified for the first time in our study. Differentially expressed genes between LSCC and adjacent normal tissues are shown in Table 2. The hierarchical clustering of the expression of these genes is shown in Figure 1.
Also, to determine the biological functions of differentially expressed genes, WikiPathway functional enrichment analysis was carried out using the Gene Spring gx (Agilent) program. WikiPathways was established to contribute to the information about the pathway and to facilitate the access by the biology community. WikiPathways is a new database including and complementing databases such as KEGG, Reactome and Pathway Commons.
The pathways such as cell cycle, G1 to S cell cycle control, cell cycle checkpoints, regulation of DNA replication, DNA replication, degradation of the extracellular matrix were upregulated; however, the pathways such as electron transport chain, oxidative phosphorylation, integrin mediated cell adhesion, the citric acid cycle and respiratory electron transport, adipogenesis, mitochondrial translation, fatty acid triacylglycerol and ketone body metabolism were downregulated. The WikiPathway analysis of the most affected pathways is shown in Table 3.

Discussion

Cancers occur as a consequence of accumulation of genetic aberrations. Almost every neoplasm has its own unique molecular changes. Characterization of these changes is extremely important in matters of clinical care and the development of personalized treatment strategies.
Laryngeal carcinomas have been identified as aggressive tumors because of their high recurrence and metastasis rate. The first step for regional recurrence and distant metastasis is the deterioration of the extracellular matrix components. Matrix metalloproteinases (MMP) are a family of enzymes that play a key role in this process. Overexpression or dysregulation of these enzymes is known to be related to various types of cancer.⁶ Krecicki et al. demonstrated strong immunoreactivity of MMP1 in 36 (72%) of 50 laryngeal squamous cell carcinoma cases using immunohistochemical methods.⁷ Liu et al. reported a difference in MMP12 expression between tumor and normal tissues, also metastatic and non-metastatic tumors.⁸ Kallikrein-related peptidase 6 (KLK6), a member of the serine protease family, is involved in cellular processes such as degradation of the extracellular matrix in a similar way to MMP. In a study of n = 162 patients with head and neck tumors, KLK6 overexpression was detected in 42.6% of the cases.^9,10 In addition, overexpression of the ITGA6 gene, which is involved in cell-cell and cell-matrix interactions, has been shown to be effective in tumor invasion and metastasis in many cancers, including LSCC.^11,12 Similarly, laminin encoded by the LAMC2 gene, which plays a role in cell differentiation, migration, and metastasis, was found to be overexpressed in squamous laryngeal cancer cases by microarray technique.¹³ We also detected upregulated expression in MMP1, MMP12, KLK6, LAMC2, and ITGA6 genes in all of our cases.
S100 proteins are components of the Epidermal Differentiation Complex and are known to be related with skin diseases and human cancers, including squamous cell laryngeal cancer, lung, ovarian, renal, colorectal, skin, and gastric carcinomas. In the study by Tyszkiewicz et al., while S100A1 and S100A4 genes were down-regulated, S100A2, S100A3, S100A4, and S100A11 genes were significantly up-regulated in n = 93 cases of head and neck cancer. In our study, S100A2 and S100A3 genes were upregulated in all cases.¹⁴
CK14, CK16, KRT17, and KRT6 genes, encoding members of the keratin family, are responsible for the structural integrity of epithelial cells. Lauriola et al. reported that CK14 expression difference was detected in 54 of 62 laryngeal carcinoma cases by immunocytochemistry, and this expression was strictly associated with S100A2.¹⁵ Elazezy showed that keratin 16 overexpression might be related to more aggressive breast cancer.¹⁶ Khanom R. et al. demonstrated that KRT6 and KRT17 were overexpressed in oral squamous cell carcinoma (OSCC) cell lines.¹⁷
CEP55 (centrosomal protein 55kDa) has been identified as a tumor-associated antigen functioning as a regulator in the PI3K/AKT pathway. Its upregulation has been associated with poor prognosis in various neoplasms.¹⁸ In laryngeal squamous cell carcinoma cases, CEP55 expression was increased in early and advanced stages.¹⁹ Our results also support the oncogenic role of CEP55 in LSCC. IL1F9 (interleukin 1 family, member 9) activates NF-kappa B and PI3K pathways, increasing cell growth, differentiation, and survival while suppressing apoptosis.²⁰ PI3K pathway is upregulated in over 90% of head and neck carcinoma cases.²¹
In recent years, Glut-1 protein encoded by the SLC2A1 gene has become popular in cancer research. Starska et al. observed SLC2A1 gene expression upregulated in 83% of 106 laryngeal carcinoma cases, associated with poor prognosis.²² Another study showed increased SLC2A1 expression in 30 of 38 head and neck carcinoma patients.²³ In our study, SLC2A1 expression was significantly increased, consistent with the literature. TP63, P53 tumor suppressor gene homolog, is highly expressed in various cancers, including LSCC.²⁴
Although genes such as VSNL1, CXCL1, COL4A5, COL4A6, FSCN1, KRT6B, KRT17, WDR66, ACOT7, ITM2A, and CFD are known to be altered in other cancers, they have not been described before in LSCC. Type IV collagen protein plays an important role in migration and adhesion. Degradation of type IV collagen is related to cancer progression, invasion, and metastasis. In colorectal cancer patients, loss of COL4A5 and COL4A6 expression was observed due to promoter hypermethylation.²⁵ In esophageal squamous cell carcinoma specimens, increased COL4A5 and COL4A6 expression was found.²⁶ FSCN1 encodes a protein involved in cell migration, motility, adhesion, and interactions. Overexpression of FSCN1 increases metastasis via enhanced motility.²⁷ Increased FSCN1 expression was detected in n = 129 cases of oral and oropharyngeal carcinoma.²⁸
VSNL1 has been identified as a tumor suppressor in esophageal cancer, but as an oncogene in neuroblastoma. It can also be used as a marker for Alzheimer’s disease and other neurodegenerative diseases.^29,30 CXCL1 gene encodes a protein involved in inflammation and neutrophil chemoattraction. Abnormal expression is associated with melanomas.³¹ ACOT7 gene participates in several neoplasms including melanomas, and its upregulation is linked to poor prognosis in acute myeloid leukemia.^32,34
WDR66 is a large family of proteins involved in signal transduction, apoptosis, cell cycle control, transcription regulation, and autophagy.³⁵ Overexpression has been found in gastric, thyroid, lung, and esophageal carcinomas, suggested as tumor markers.^36,38 ITM2A’s role in tumorigenesis is unknown, but decreased expression was observed in ovarian cancer tissues, suggesting a tumor suppressor role.³⁹ CFD has anti-inflammatory effects via the complement system. Ye H et al. detected decreased CFD expression in tongue squamous cell carcinoma cases.⁴⁰
In this study, analysis of expression-altering genes and pathway analysis were performed. The cell cycle and extracellular matrix degradation pathways were over-represented, while the electron transport chain pathway was under-represented. All affected pathways are shown in Table 3.

Conclusion

We have identified several differentially expressed genes and described their functions in the cell and the affected pathways in laryngeal squamous cell carcinoma. These genes may be used as new biomarkers for diagnosis, prognosis and therapy of LSCC. It is also believed that these genes will shed light on the studies that we wish to do in the future.

Declarations

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About This Article

Received:

January 20, 2023

Accepted:

February 25, 2023

Published Online:

March 11, 2023

Printed:

March 25, 2023