Numerous studies have reported that the expression levels of KRTs in many tumors were dysregulated16–20, and the roles of KRTs in occurrence, progression, and metastasis of multiple cancers has been partially confirmed36–39. However, systematic bioinformatics analyses about the function of KRTs in LUSC are limited and have yet to be performed. This is the first time to systematically study the expression of KRTs in LUSC and their prognostic values. We hope our findings will help to provide new therapeutic measures and improve the prognosis of patients with LUSC.
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KRTs family is a group of intermediate flament proteins which express in various types of epithelial cells40,41. Previous studies have confirmed that KRTs played significant roles in maintaining the structural stability of epithelial cells and were involved in numerous biological activities, including epithelial cell signal transduction, stress response, tumor cell apoptosis, and tumor cell proliferation42,43. Moreover, Nazarian et al.44 reported that the KRTs were dysregulated in a variety of tumor tissues, and played vital roles in invasion and metastasis of tumors. In summary, this kind of proteins has great potential in diagnosis, typing, and prognosis prediction of tumors. Therefore, the roles of KRTs as multifunctional regulators of epithelial tumorigenesis are worthy of further exploration. We used TCGA database to screen abnormally expressed KRTs between LUSC samples and adjacent normal lung samples, and found that 57 KRTs met our screening criteria. Subsequently, we used GEPIA database and HPA database to observe the transcriptional levels and protein levels of 57 KRTs. The results showed that the expression levels of KRT5, KRT6A, KRT6B, KRT6C, KRT13, KRT14, KRT15, KRT16, KRT17, KRT19, and KRT23 in LUSC tissues were significantly higher than in adjacent normal lung tissues. Survival analyses showed that the high expression levels of KRT5, KRT6A, KRT6B, KRT6C, KRT13, KRT14, KRT15, KRT16, KRT17, KRT19, and KRT23 were closely related to the poor OS and PFS of patients with LUSC. ROC curves indicated that the KRT5, KRT13, and KRT23 had potential diagnostic value in patients with LUSC.
KRT5 is usually specifically expressed in the basal layer of the epidermis, and its normal expression is essential for the protection of epithelial cells45. Moll et al.19 reported that KRT5 was specifically expressed in squamous cell carcinoma of multiple tissues. For lung cancer, the expression level of KRT5 in LUSC tissues was significantly higher than in adjacent normal lung tissues20,46,47, which was helpful for the differential diagnosis of LUSC and LUAD47. In our study, TCGA database revealed that the expression level of KRT5 was higher in LUSC tissues than in adjacent normal lung tissues. By analyzing the Kaplan-Meier plotter database and TCGA database, we determined the prognostic value of KRT5 in patients with LUSC, and the results indicated that the high KRT5 expression was significantly associated with poor OS and PFS in patients with LUSC followed up for 200 months. In addition, the AUC of KRT5 was 60.753, which had a certain predictive effect on the prognosis of LUSC.
KRT6 is a group of keratins associated with stratified epithelium, including KRT6A, KRT6B, and KRT6C. Studies have reported that KRT6A, KRT6B, and KRT6C were involved in the epidermization of squamous epithelium, so their expression in LUSC increases sharply46–49. The KRT6A is an important part of type II keratin family, and plays a considerable role in the epidermization of squamous epithelium50,51. The overexpression of KRT6A in LUSC and LUAD can be used as a prognostic marker of lung cancer47,52,53. In addition, studies have reported that KRT6A and KRT6B were related to the progression of renal carcinoma and breast cancer54,55. The expression levels of KRT6A, KRT6B, and KRT6C in squamous cell carcinoma-like (SCCL) subtype urothelial carcinoma were significantly increased, and were closely related to poor prognosis56. In our report, the expression levels of KRT6A, KRT6B, and KRT6C in LUSC tissues were higher than in normal lung tissues, which was consistent with the reported in the literature. However, there is no previous report on the relationship between KRT6 expression and tumor prognosis. Our research found that the high expression levels of KRT6A, KRT6B, and KRT6C were significantly associated with poor OS and PFS in patients with LUSC.
KRT13 encodes an intermediate cytoskeletal protein, which is usually expressed in the basal compartments of stratified epithelium57. Previous studies have reported that KRT13 was expressed at different levels in different tumors, low KRT13 expression was found in oral squamous carcinomas58, esophageal squamous cell carcinoma59, bladder cancer60, lymph node-positive uterine cervix cancer61, and head and neck squamous cell carcinoma cell lines62, by contrast, high KRT13 expression was detected in LUSC47, colorectal cancer63, gastric cancer64, and tongue squamous cell carcinoma65. The overexpression of KRT13 was associated with the squamous cell metaplasia of the upper airway tract epithelium66,67, and was prominent among the gene expression markers for LUSC46. Yang et al.68 found that the HOXC6 promotes lung cancer progression by upregulating KRT13 expression. In addition, KRT13 was implicated in urothelial and stem cell differentiation69. In our report, we demonstrated that the expression level of KRT13 in LUSC tissues was higher than in normal lung tissues, the high KRT13 expression was correlated with poor prognosis in patients with LUSC, and the sensitivity of KRT13 was highest, which seemed consistent with the role of KRT13 as an oncogene and a diagnosis factor of LUSC.
KRT14 is an intermediate filament protein, which is usually expressed by basal epithelial progenitor cells located in epithelial niches of healthy adult tissues. Previous study indicated that KRT14 was not detected in normal tissues, was significantly increased in many tumor tissues, and was expressed in squamous cell carcinoma of different origins and degrees of differentiation70. According to the IHC analysis of surgical specimens, the expression level of KRT14 in lung cancer tissues was significantly increased71, especially in LUSC tissues46,47, which indicated that KRT14 played an important role in the occurrence and development of LUSC. Chen et al.47 found that the high KRT14 expression could be helpful in the differential diagnosis of LUAD and LUSC. In addition, Huang et al.72 reported that KRT14 was not expressed in normal cervical tissue, but with the increase of the grade of cervical epithelial neoplasia, the expression level of KRT14 gradually increased, which indicated that KRT14 could be used as one of the indicators for the diagnosis of early cervical cancer. In our study, we demonstrated that the expression level of KRT14 in LUSC tissues was higher than in cancer-free lung tissues, and high KRT14 expression was related to poor OS and PFS in patients with LUSC.
KRT15 is a type I keratin, which is mainly expressed in basal keratinocytes of stratified epithelium and plays a significant role in maintaining cytoplasmic stability73,74. Previous studies conformed that the overexpression of KRT15 was involved in tumor formation and progression, including NSCLC75,76, breast cancer77,78, oral squamous neoplasms79, urothelial cell carcinomas80, and hepatocellular carcinoma81. Sanchez-Palencia et al.82 reported that KRT15 expression level was higher in LUSC tissues than in cancer-free lung tissues, and may be a marker of LUSC. In addition, the high expression KRT15 was associated with poor prognosis of colorectal cancer83 and gastric cancer84. In the present study, KRT15 was significantly overexpressed in LUSC tissues, and its high expression level was related to the poor prognosis in patients with LUSC.
KRT16 is an important part of type I cytoskeleton. KRT16 was reported to play a role in LUAD tumorigenicity via EMT, and increased KRT16 expression was associated with poor outcomes of tumors, including LUAD85, oral squamous cell carcinoma36, and metastatic breast cancer86, which indicated KRT16 had an oncogenic role in tumors. In our report, we demonstrated that the expression level of KRT16 was higher in LUSC tissues, which was significantly related to poor OS and PFS in patients with LUSC.
KRT17 is a 48 kDa type I keratin20. Extensive tissue screening results showed that KRT17 was low expressed in mature epithelial tissues, but regenerated and highly expressed in cancer tissues87, and high expression of KRT17 indicated poor prognosis in NSCLC88, which indicated that KRT17 can be a biomarker in predicting progression and poor prognosis in patients with LUSC87,88. Chen et al.47 reported that KRT17 was highly expressed in LUSC, and in LUSC was significantly higher than in LUAD, suggesting that KRT17 may be a tumor marker for differentiating LUSC and LUAD subtypes of lung cancer. In addition, KRT17 was also highly expressed in breast cancer89,90, cutaneous squamous cell carcinoma91, cervical carcinoma92, gastric carcinoma93, and oral sprays94, which was associated with poor prognosis of these tumors89–94. In this report, we found that KRT17 was highly expression in LUSC tissues, which was correlated with poor OS and PFS of the patients with LUSC.
KRT19 is a member of the keratin family, which is functionally related to maintaining the structural integrity of epithelial cells, including bronchial epithelial cells95. Yuan et al.96 discovered that compared with LUAD, KRT19 was upregulated in LUSC, and the high expression level of KRT19 was closely related to poor prognosis of LUSC. In addition, KRT19 has been identified as a tumor-associated biomarker for a variety of tumors, including esophageal squamous cell carcinoma97, cervical carcinoma98, breast cancer99, colorectal cancer100, and hepatocellular carcinoma101. Moreover, the overexpression of KRT19 was associated with poor prognosis of hepatocellular carcinoma102 and breast cancer103. In the present study, KRT19 was significantly overexpressed in LUSC tissues, which was related to poor OS and PFS in LUSC patients.
KRT23 is a improtant member of the KRT gene family, and locates on human chromosome 17q21.2. Previous studies have shown that KRT23 is abnormally expressed in a variety of tumor tissues, including pancreatic carcinoma104, colorectal carcinoma105, and hepatocellular carcinoma106. In our report, we demonstrated that the expression level of KRT23 was higher in LUSC tissues than in normal lung tissues, and the high KRT23 expression was significantly correlated with poor prognosis in patients with LUSC. ROC curve analysis found that the specificity of KRT23 was 86.31, which had a certain predictive effect on the prognosis of LUSC.
In our study, the analysis of co-expression of KRTs found that KRTs were co-expressed in different degrees in LUSC, which was consistent with the finding of Travis, who found that KRT5, KRT6, KRT13, KRT14, KRT16, KRT17, and KRT19 were the gene expression markers for LUSC46.
Subsequently, we used cbioportal to detect the possible genetic alterations of KRTs in LUSC. The result indicated that mutation was the commonest gene alteration in KRTs. Previous studies have shown that the mutations of transcription factors were related to the occurrence and development of cancer, the abnormal expression of oncogenic transcription factors and chromatin regulatory factors were the internal reason for the tumorigenesis of cancer cells107. The abnormal function of these regulatory factors provided some clues for the mechanisms of abnormal gene expression108,109.
GO function and KEGG pathway analyses found that the KRTs and their neighbor genes were mainly enriched in epidermis development, keratinization, keratinocyte differentiation, and Rap1 signaling pathway. This was consistent with the knowledge that cytokeratin was abundant and stable in epithelial cells, and it had high specificity in tissues and cells, so it could be used as an ideal biological indicator to identify the origin of tumor and predict prognosis110. Some studies have reported that keratinization of tumors was closely related to the poor prognosis of tumor patients111,112, suggesting the importance of keratinization in neoplastic prognosis111. Studies on Rap1 signaling pathway have reported that abnormality of Rap1 signaling pathway was highly associated with the occurrence and development of some tumors, including pancreatic cancer, melanoma, and human chronic myelogenous leukemia113–115.
Finally, we analyzed the relationship between the expression level of KRTs and the clinical characteristics of patients with LUSC. It turned out that the expression levels of KRT13, KRT16, KRT17, and KRT19 were positively correlated with nodal metastasis, the expression levels of KRT15 and KRT17 were positively related to primary tumor, and the expression level of KRT15 was positively correlated with TNM stage. There were some studies on the relationship between KRTs and clinical features of tumors. Liu et al.85 reported that KRT16 was upregulated in LUAD and the highly KRT16 expression was related to nodal metastasis. Huang et al.36 suggested that high expression of KRT16 indicates the poorer pathological differentiation, advanced stages, and increased nodal metastasis of OSCC patients. In addition, the expression level of KRT16 was significantly increased in the process of nodal metastasis in primary breast cancer17. Wang et al.88 found that the highly expressed KRT17 was associated with poor differentiation and nodal metastasis in patients with NSCLC. Hu et al.116 found that the expression level of KRT17 was significantly associated with the tumor size, depth of invasion, nodal metastasis, and TNM stage in patients with gastric cancer. Nordgard et al.117 confirmed that KRT19 can be used as a molecular biomarker of disseminated tumor cells in regional lymph nodes from NSCLC patients.
In this study, we comprehensively analyzed the effects of KRTs on LUSC, and provided a thorough understanding of the heterogeneity and complexity of the molecular mechanisms of LUSC. Our results indicated that the increased expression of KRT5, KRT6A, KRT6B, KRT6C, KRT13, KRT14, KRT15, KRT16, KRT17, KRT19, and KRT23 in LUSC tissues might play an important role in LUSC oncogenesis and progression, and could also serve as molecular markers to identify high-risk group of LUSC patients. In addition, our findings suggested that KRT5, KRT6A, KRT6B, KRT6C, KRT13, KRT14, KRT15, KRT16, KRT17, KRT19, and KRT23 were potential therapeutic targets for LUSC, and were potential prognostic markers for patients with LUSC.