Many different functions of G protein-coupled receptor kinase 6 have been reported in various cancers, such as tumor progression, angiogenesis, metastasis and cell proliferation etc. Chen et al suggested that GRK6 plays an important role in cell adhesion and migration of prostate cancer cell lines, breast cancer cell lines and cervical cancer cell lines . In addition, GRK6 provides a signal transduction scaffold that regulates cell adhesion and cytoskeletal organization through ArfGAP 1 interaction with G protein coupled receptor kinase and indirect transactivation of epidermal growth factor receptors. This in turn affects the migration and invasion of cancer cells . Due to secondary messengers (including cAMP and calmodulin), GRK6 can affect the migration and invasion of cancer cells . We conducted a bioinformatics analysis of public sequencing data to gain a more thorough understanding of GRK6's functions in ccRCC and its regulatory network, and it will better guide the future research of ccRCC.
RCC lacks particular symptoms at early stage and effective noninvasive methods for screening, leading to the present situation that the treatment of early RCC is hampered. Therefore, new RCC markers are needed to improve early diagnosis. Analysis of transcript sequencing data from more than 1,000 clinical samples in the GEO and TCGA databases including five different ccRCC study data sets [18–21] confirmed that GRK6 mRNA levels and CNV in ccRCC were significantly higher than normal kidney tissues. We find that GRK6 overexpression occurs in multiple cases of ccRCC, which is worthy of further clinical verification and has the potential to be a valuable marker for diagnosis and prognosis.
CNVs can have significant genomic implications, disrupting genes and altering genetic content, resulting in phenotypic differences . The increase in the copy number of GRK6 in ccRCC was found by our research, and that the major type of GRK6 alteration was amplification, which was associated with shorter survival period. We speculate that altered GRK6 expression and GRK6 dysfunction in ccRCC may be caused by alterations in chromosomal structure. Due to GRK6 plays many important physiological functions, its alteration may lead to changes in a wide variety of downstream signaling pathways. In fact, neighboring gene networks close to GRK6 usually show varying degrees of amplification in ccRCC. Related functional networks are involved in chemokine signaling pathway, cytokine-cytokine receptor interaction and neuroactive ligand-receptor interaction. Thus, the network of GRK6 alterations is involved in the core node of post-transcriptional regulation, which is closely related to regulating various enzymes stability and functions. Accumulating results show the nonnegligible roles of modification enzymes in tumor progression, such as the ubiquitinases , deubiquitinases , kinases , and phosphatases . GRKs were first reported to catalyze the phosphorylation and desensitization of G protein-coupled receptors (GPCRs). Nevertheless, recent studies identified its GPCR-independent roles in regulating cellular functions . For example, GRK2, GRK5, and GRK6 can mediate TNFa-induced NF-kB signaling via direct phosphorylation of IkBa . Moreover, GRKs were reported to be involved in tumor development and progression. GRK5 can functionally regulate well-known cancer-related proteins such as Wnt and tumor suppressor p53 . Another example is the upregulation of GRK5 in glioblastoma stem cells, indicating its significance in promoting tumor proliferation . The above research is consistent with the results of our functional network analysis.
The important network of target kinases, miRNAs and transcription factors can be revealed by the enrichment analysis of target gene sets by GSEA. Our results suggest that the functional network of GRK6 participates primarily in cytokine metabolic process, interferon-gamma production and adaptive immune response. These findings are consistent with the fact that GRK6 can mediate TNFa-induced NF-kB signaling via direct phosphorylation of IkBa . It is critical to understand how alteration in a protein important for ensuring normal transcription can lead to major dysfunction and even to cancer such as ccRCC.
In recent years, many studies have found that the tumor microenvironment is closely related to tumor invasion, growth, and metastasis. The interaction between chemokines and chemokine receptors recruits different immune cell subgroups into the tumor microenvironment, and these groups have an important impact on the occurrence and development of tumors. We found that GRK6 in ccRCC is associated with a network of kinases including IKBKB, PAK1 and LCK. These kinases regulate chemokine, cytokines and the adaptive immune response [41, 42]. In fact, IKBKB can enhance expression of the cytokines activated, and alter most of the NF-κB activity discovered in solid tumors. As a fundamental regulator of NF-κB activity, not surprisingly, the activity of IKBKB is closely related to tumor development and progression, such as breast cancer , skin cancer , and gastric carcer . In ccRCC, GRK6 may regulate chemokine, cytokines and tumor microenvironment via IKBKB kinase.
In the promoters and enhancers of many genes, there are functional NF-κB binding sites, and activated NF-κB can bind to specific sequences on the DNA chain to initiate and regulate immune responses, mediate cell adhesion, differentiation, proliferation, apoptosis and inflammation, and play an important role in the occurrence and development of various immune inflammatory diseases and tumors [46, 47]. When the body is stimulated by stimulating factors such as viruses, radiation, oxygen free radicals, bacterial lipopolysaccharides, TNFa etc, the NFkB-IkBs complex is activated, and NF-κB is dissociated and transferred to the nucleus, and specifically binds to the corresponding site to promote the corresponding gene transcription, so that the biological reaction occurs. At the same time, most of the activators in NF-κB and its signaling pathways are considered to play an important role in the occurrence and development of tumors . NF-κB activation induces DNA damage, oncogenic mutations and genomic instability leading to tumor initiation. Chronic inflammation and NF-κB can also cause chromosomal instability and aneuploidy. NF-κB enhances the proliferation of initiated tumor cells by promoting the production of various cytokines, growth factors and cell cycle proteins. Further studies should test this hypothesis.
Our study identified some miRNAs that were associated with GRK6. Actually these short noncoding RNAs, normally involved in post-transcriptional regulation of gene expression, can contribute to human carcinogenesis . The particular miRNAs in our study have been linked to tumor proliferation, apoptosis, invasion, metastasis, cytokine metabolic. In fact, miR-190-5p is regulated by NFκB1/p50, thereby restoring the apoptotic response of cells. MiR-192, miR-194 and miR-215 participate in EMT progression, and suppress tumor migration and invasion . MiR194 is a marker for good prognosis in clear cell renal cell carcinoma . MiR-374 can promote the proliferation and migration of cancer cells . MiR-204/211 increases cancer cell proliferation by down-regulating tumor suppressor genes . The dysregulation of these miRNAs is closely related to the occurrence and development of tumors. We analyze the relationship between GRK6 and these miRNAs through bioinformatics to guide further research.
Our study strongly demonstrates the importance of GRK6 in carcinogenesis and its potential as a ccRCC marker, and the results show that GRK6 overexpression in ccRCC has a profound impact on genome stability, multiple steps of gene expression (DNA replication, RNA splicing and protein translation) and cytokine metabolism. GRK6 is related to several tumor-associated kinases (such as IKBKB), miRNAs (such as miRNA-190), and transcription factors (such as NF-κB). Our research is based on the most prevalent bioinformatics theory and uses online tools to analyze target genes from tumor data from public databases. Meanwhile, this method has the advantages of low cost, large sample size and simplicity compared with traditional chip screening . This enables large-scale ccRCC genomics research and subsequent functional studies.
However, the TCGA database has its own limitations. One is that the TCGA KIRC samples contain three ethnic groups. The genetic background and etiology of KIRC can differ significantly across ethnic groups. Another limitation is that ccRCC patients are not easy to be found in the early stage of the disease. Under normal circumstances, the disease has progressed to the middle and late stages when obvious symptoms appear. But the KIRC samples contain relatively few patients in stage 4. Therefore, we should increase the number of patients of different races and KIRC stages to enrich our clinical samples and make the results more accurate. The third limitation is that transcriptome sequencing can detect only static mutations; it cannot directly provide information on protein activity or expression level. These questions should be addressed in follow-up experiments.