Autophagy refers to a series of biochemical processes in eukaryotic cells involving ‘self-digestion’ by degrading their own cytoplasm and organelles. Under nutritional deficiency, starvation or anoxia, autophagy is described as a pathway capable of promoting cell survival (1). Under these conditions, it is essential for autophagy to provide energy for cells. A previous study indicated that autophagy is associated with the effects of antitumor agents on tumor cells. Subsequently, tumor cells overcome nutritional deficiency and hypoxia by autophagy and are therefore protected from entering the apoptotic pathway (2).

One of the important factors for the poor prognosis of tumors is their tolerance to radiotherapy and chemotherapy. Recently, it was found that autophagy is one of the mechanisms involved in the resistance of tumors to chemotherapy (3). Autophagy inhibitor, chloroquine, improves the sensitivity of human lymphoma cells to apoptosis inducers, and thus it is considered to treat tumors by combining autophagy inhibitors and apoptosis inducers (4). Tumor cells grow in low-vascularized environments by autophagy and the treatment of tumors may be elucidated by inhibiting autophagic activities. Li et al(5) found that the inhibition of autophagy may improve the sensitivity of intestinal cancer cells to fluorouracil (5-FU). Therefore, it is of great importance to identify anti-tumor agents that target the molecular mechanisms of autophagy.

According to the concept of bioinformatics and network pharmacology, theoretically, if the inhibition of autophagic genes (or gene knockout) prevents autophagy, agents inhibiting the same genes may also suppress autophagy (6). At present, there are ~500 biological targets for therapeutic agents and it is estimated that human genome data may contain ~5,000–10,000 novel targets, which is 10- to 20-fold higher than currently known targets. It is not difficult to obtain a number of novel agents, which may be 10-fold higher than the currently available drugs if genomic data are sufficiently mined (7). The present study screened differentially expressed protein kinases during autophagy using bioinformatics and constructed the kinase-kinase inhibitor regulatory network. The aim therefore was to explore kinase inhibitors capable of arresting autophagy with potential research values.

A number of anti-tumor treatments may induce autophagy (12). It was found that in breast cancer cells, tamoxifen and other estrogen receptor antagonists induced autophagy (13). Chemotherapy at varied doses may result in autophagy at different degrees in breast cancer cells, colorectal cancer cells and prostate cancer cells (14). Autophagy of tumor cells may be induced during treatment on spongiocytoma using arsenic trioxide, temozolomide and rapamycin (15), treatments on ovarian cancer using resveratrol (16) and treatments on cervical cancer using histone deacetylase (17). Therefore, it has been estimated that the therapeutic alliance of autophagic inhibitors may improve therapeutic efficacy and prognosis (18). Mathew et al(19) demonstrated from animal testing that the inhibition of autophagy may aid anti-tumor therapy. They indicated that a large concentration of tumor cells died following cytotoxic treatment, while autophagy promoted resilience and hibernation of tumor cells, and autophagy inhibitors may directly eliminate these cells. When the autophagy inhibitor, chloroquine, was used in combination with alkylating agents, satisfactory therapeutic efficacy was obtained (20). The phenomenon of the induction of apoptosis by autophagy inhibitors was observed in previously mentioned studies (21); therefore, sensitization may be induced in anti-tumor treatments if autophagy inhibitors were used in combination to block the autophagic pathway. However, only a limited number of autophagy inhibitors are available for current clinical application, including bafilomycin and chloroquine (22).

Numerous molecules are involved in autophagy and the regulatory mechanisms are extremely complex. The majority of regulatory mechanisms remain unclear; however, a number of efforts have been made for exploration and many signaling molecules are shared in regulating autophagy and apoptosis. The determined signaling molecules involved in regulating autophagy include cytokine, MAPK and mitochondrial signaling for which most of the signals exert regulatory functions by mTOR kinase (23).

Molecular switches accurately control proteins during cascade reactions for a series of signaling transduction processes in cells by activation or deactivation mechanisms. Among the cascade reactions for a series of signaling transduction processes, positive and negative feedback mechanisms complementing each other are essential for accurate control, thus the function of molecule switches is influential (24). The proteins involved in signaling transduction processes include protein kinases and GTP-binding proteins. Protein kinases control the activities of 30% of the proteins in cells, which are associated with almost all of the cell functions (25). Protein kinases are particularly important for intracellular signaling transduction and implementation of complex cell functions, such as cell division and death. The core function of kinases in controlling cell behaviors subsequently caused them to be considered as targets for treating multiple diseases and thus they have been extensively investigated. In addition, in contrast to gene transfection and RNA interference, a number of small molecular compounds are capable of effectively regulating kinase activities (26). They have been commercialized and it is of practical significance to introduce them to switch autophagy, which may realize the transformation of laboratory achievements to clinical application, and meets with the concepts of translational medicine. Therefore, the present study examined the factors regulating autophagy. In total, 544 differentially expressed genes were screened using the genome-wide expression profile microarray in 2 groups of autophagy models induced by starvation, among which 11 upregulated protein kinases were used as the targets for intervention by kinase inhibitors. Among the upregulated kinases, BRAF and TRIB3 were found to have corresponding specific inhibitors in the kinase-kinase inhibitor regulatory network. Moreover, numerous inhibitors were available for BRAF (27) and thus BRAF was analyzed.

BRAF is one of the three isoforms of RAF and it is an important transduction factor in the MAPK pathway (28). The MAPK signaling pathway is one of the most important signaling pathways in cells, it exists in most cells and transmits extracellular stimuli into cells or even nuclei. It has important functions in inducing proliferation, differentiation, transformation, apoptosis and other biological reactions (29). Extracellular signals stimulate ERK and RAS, and RAS further activates RAF and phosphorylates MAPK kinase (MEK1/MEK2). ERKs are translocated into the nucleus, phosphorylated cytosolic proteins and certain nuclear transcription factors, such as c-FOS, c-JUN, ELK-1, c-MYC and ATF2; therefore, they are involved in the regulation of cell proliferation and differentiation. ERKl/2 is activated by the MAPK pathway by survival signals and promotes autophagy by PI3K via mTOR (30). Shimada et al(31) indicated that endoplasmic reticulum stress may induce autophagy by activating the MAPK signaling pathway. To determine the functions of BRAF, the present study constructed a correlation network between BRAF and the differentially expressed genes regulated by BRAF. GO-BP enrichment analysis on these differentially expressed genes showed that their major functions were associated with cell death and apoptosis; therefore, regulation of BRAF may result in significant effects on the mechanisms of cell death. Results of the present study have shown that RIOK3 was closely associated with BRAF by cluster analysis. RIOK3 was very important for maintaining normal physiological function of cells, and RIOK3 depletion may result in cell death or significant abnormality in physiological function. Shan et al(32) reported that RIOK3 may be involved in the regulation of apoptosis by coordinating caspase-10, caspase-2 and NF-κB activation, indicating that RIOK3 may be closely associated with cell death.

The present study has demonstrated by constructing the kinase-kinase inhibitor regulatory network, that certain kinase inhibitors had multiple targets, and may inhibit downregulated genes (AKT4 and CDK2) and simultaneously inhibit upregulated genes (BRAF), thus off-target side-effects may occur and should be excluded. Of the 16 included specific kinase inhibitors functioning on BRAF, sorafenib was previously confirmed to be capable of regulating autophagy, while regorafenib had also been reported. However, no report was available on the regulation of autophagy by afatinib, selumetinib, PD318088, axitinib, TAK-733, GDC-0980, GSK2126458, PLX-4720, AS703026, trametinib, GDC-0941 and PF-04217903. Thus, these kinase inhibitors are potential targets for investigating the regulation of autophagy in the future.