Radiotherapy has always been an effective therapy for cancers. More than half of cancer patients are treated with radiotherapy alone or in combination with other treatments. It primarily exerts its role by direct physical damage to DNA or indirect damage from reactive oxygen species. Despite the appreciable advances in radiotherapy in recent years, most cancer patients remain poor prognosis. Tumor radioresistance has been considered as a powerful obstacle to impinge on radiotherapy efficacy, and EMT acts as one of the most important factors involving radioresistance (1, 2). Epithelial-to-mesenchymal transition (EMT) is a biological process that refers to the transition of epithelial cells to ones with mesenchymal phenotype. This process is generally accompanied by the alterations in cell morphology, cell-cell adhesion and cellular signaling factors. It is characterized by the loss of epithelial adhesion markers (E-cadherin) and the gain of mesenchymal markers such as N-cadherin, vimentin, fibronectin and alpha smooth muscle actin (a-SMA) (3).

In this review, we conduct a comprehensive review focusing on the recent advances in EMT-related mechanisms in regulating radioresistance and the influence of irradiation on EMT, and excavate the potential strategies to overcome radioresistance.

EMT is known to play a central role in inducing resistance of tumor cells to irradiation, and it is regulated by a variety of molecular mechanisms containing TGF-β, Wnt/β-catenin, PI3K/AKT, Notch, NF-κB, IL-6/STAT3, non-coding RNAs and so on. Also, cancer stem cells and tumor microenvironment participate in the acquisition of EMT program and tumor radioresistance. These mechanisms function mainly depending on the activation of EMT-related transcription factors (TFs) and markers, including Snail/Slug, ZEB1/2, Twist1/2, E-cadherin, N-cadherin, Vimentin (4–7). EMT-related mechanisms in regulating radioresistance are depicted in Table 1 .

There is an interactive network between EMT and irradiation. The processes of EMT induces radioresistance of tumors, and irradiation further enhances EMT, providing a positive feedback loop to increase the radioresistance. After several hour’s irradiation to cancer cells with X-rays, the migration and invasion capacities of these cells were increased, paralleled by a decrease of E-cadherin expression and an increase of Vimentin and Smad3 expression (126). Tsukamoto H et al. (127) also found that irradiation facilitated the migration and invasion of endometrial cancer cells by inducing the development of EMT in vitro.

In tumorigenesis and development, a variety of signaling pathways are significantly activated, such as MAPK/ERK, TGFβ, HIF-1, Wnt and Notch pathways, and irradiation will further activate these pathways to induce the tumor malignant phenotypes such as EMT, leading to the occurrence of radioresistance. Irradiation can increase the release of the reactive oxygen species (ROS), resulting in the activation of signaling pathways and alterations in the tumor microenvironment. ROS has been reported to be involved in irradiation-induced EMT by regulation of multiple EMT markers and transcriptional factors (128). Evaluated levels of ROS may contribute to activating NF-κB signaling, which increases IL-6 production by cancer cells and CAFs, thus facilitating the EMT program by regulating the expression of N-cadherin, E-cadherin, vimentin, Twist and Snail (129). In addition, irradiation could promote the upregulation of Snail via activating the ERK signaling and inhibiting glycogen synthase kinase 3β (GSK3β) (130, 131). Elimination of ROS using a ROS scavenger N-acetyl-L-cysteine (LNAC), suppressed the expression of vimentin, indicative of a vital role for ROS in irradiation-induced EMT (132).

P38 mitogen-activated protein kinase (p38MAPK) pathway was activated after irradiation, which contributes to the occurrence of EMT and invasion through increased expression of Snail (133). HIF-1 was also implicated in irradiation-induced EMT. Irradiation could increase the protein stability of HIF-1α under hypoxia, and then HIF-1α translocated to the nucleus, dimerized with HIF-1β, and regulated the gene expression of key EMT transcriptional factors, thereby inducing EMT, migration and invasion (134). Furthermore, irradiation could activate Wnt signaling pathway by mediating Wnt ligand expression, increasing β-catenin stabilization and transcriptional activities (70, 128, 135), which strengthens the expression of Snail to trigger EMT (71). Exposure to irradiation can promote the activation of the TGF-β signaling, leading to the EMT in many types of cancer cells (9, 136, 137). Disturbance of TGF-β pathway could increase the radiosensitivity via repressing EMT (10). Moreover, another study discovered that EMT induced by irradiation through TGF-β pathway was not limited to the radiation dose range, and both low- and high-level radiation could contribute to the induction of EMT (138). Likewise, Zhou Y C et al. (139) irradiated six cancer cells lines with 2Gy dose, and they found that the irradiated cancer cells presented enhanced invasion and metastatic abilities, which is associated with irradiated-induced EMT process by activating TGF-β signaling to regulate the expression of epithelial and mesenchymal markers. Li T et al. (140) demonstrated that irradiation strengthened the expression of TMPRSS4, and subsequently weakened the expression of E-cadherin, which triggers EMT to enhance the invasion and metastasis of cancer cells. Additionally, NF-κB pathway was activated by irradiation in A549 cells, involved in the induction of EMT (141). NF-κB could elicit STAT3 activation by transcriptional upregulation of HER2 expression after exposure to ionizing radiation, thus resulting in EMT-mediated radioresistance in breast cancer stem cells (142–144). Besides, evidence from another study of breast cancer uncovered that Notch signaling was highly activated in fractionally irradiated cancer cells, accompanied with the alteration of EMT phenotype. Afterward, it revealed that Notch provoked the EMT by regulating IL-6/JAK/STAT6 signaling pathway in response to fractionated-radiation (145).

Taken together, multiple signaling pathways such as Wnt, TGF-β, NF-κB, Notch and HIF-1 pathways play a crucial role in the occurrence of radiotherapy-induced EMT. Meanwhile, the alterations in TME induced by radiotherapy such as increase of ROS may acts as a stimulator to involve in the activation of signaling pathways.

From the primary cancer to adjacent and distant organs, EMT confers the potential of tumor invasion and metastasis, as well as the capacity of inducing therapeutic resistance. Therefore, targeting EMT by attenuating the mesenchymal/CSC phenotypes, inhibiting EMT-related signaling pathways, and perturbing tumorigenic TME, will be considered as promising therapeutic strategies for tumor treatment. The studies of these therapeutic strategies are summarized in Table 2 .

Previous studies have verified that CSC traits are associated with mesenchymal phenotype, metastatic capacity and therapy resistance. Targeting the CSC phenotype was conducive to the acquisition of considerable therapeutic benefit (165). The ionophore salinomycin is confirmed to be 100-fold more active against CSCs than traditional chemotherapy agent paclitaxel in breast cancer (146). Notably, the treatment of tumors with salinomycin significantly reduced the tumor size compared with the control group (147).

Given the important role of signaling pathways in mediating EMT, it is promising to acquire radiosensitization by combining the inhibitors targeted against these pathways with radiotherapy. Oncomodulin M (OSM) is a member of the IL6 cytokine family, which has been identified as an important driver of mesenchymal and CSC phenotypes. Inhibition of JAK1/2 by ruxolitinib blocked the STAT3-mediated transcription of OSM receptors Zeb1 and Snai1 and disturbed the emergence of CSC phenotype in pancreatic ductal adenocarcinoma cells (148). Inhibition of the PI3K/Akt pathway has been proven to be an effective way to increase the sensitivity of cancer cells to radiotherapy. Chang et al. reported that application of PI3K/Akt/mTOR pathway inhibitor (BEZ235) in combined with RT could significantly weaken radioresistance by decreasing the expression of EMT/CSC markers (17). Simvastatin, a conventional drug that is utilized for cardiovascular diseases, has been reported to promote the radiosensitivity of esophageal cancer cells by suppressing PTEN-PI3K/Akt pathway (149). In preclinical models, a dual PI3K/mTOR inhibitor PF-05212384 effectively inhibited PI3K and mTOR, leading to increased sensitivity of HNSCC to radiation (150). Another Akt/mTOR inhibitor Everolimus (also known as Rad001) are currently undergoing clinical trials in combination with radiotherapy in a variety of tumors, and may become a useful method of radiosensitization (151–153). Additionally, Nimotuzumab, an anti-EGFR monoclonal antibody, exerts effective roles in improving the radioresistance of esophageal cancer KYSE-150R cells by inhibiting EGFR signaling pathway and DNA repair (154). Also, Cetuximab, another anti-EGFR pathway antibody, displays potential antitumor effect in combination with radiotherapy, and obtained a survival benefit of 10% in patients with head and neck squamous cell carcinoma (HNSCC) in a phase 3 clinical trial (155). Several types of Notch pathway inhibitors are in clinical development, especially γ−secretase inhibitors (GSIs), which disturb the proteolytic cleavage of Notch receptors and block the release of the active intracellular domain (NICD). In multiple preclinical models, these inhibitors have displayed significant anti-tumor activity. Noteworthily, GSI (RO4929097) in combination with RT has entered clinical trials in patients with gliomas (NTC01119599) or brain metastases (NCT01217411) (156). In gastric cancer (GC), tangeretin that acts as a Notch-1 inhibitor could enhance the radiosensitivity and counteract irradiation-induced EMT both in vitro and in vivo (157). Blockage of Notch signaling by rhamnetin and cirsiliol could result in the reversal of EMT phenotype and improvement of radiosensitivity in NSCLC (24). These findings implied that inhibiting EMT by blockage of the Notch pathway may be an effective means to reduce EMT-mediated radioresistance. Wnt pathway plays a protective role for cancer cells in response to IR, therefore, targeting the Wnt signaling may also be important for enhancing radiosensitization. FH535, a β-Catenin/Tcf inhibitor that can inhibit the activation of Wnt/β-catenin pathway, is known as a potential radiosensitizer. It could effectively suppress EMT to reverse the radioresistance of KYSE-150R cells by increasing E-cadherin expression (13). Other small molecule inhibitors have been used in preclinical trials or clinical trials and shown good efficacy in blocking the Wnt signaling pathway (156, 166–168). Currently, several HIF-1-targeting agents have been applied in clinical practice, such as sunitinib (158), paclitaxel (159), solafeinib (160) and bortezomib (161). These agents, as antineoplastic chemotherapy or targeted treatment, can be combined with radiotherapy to play the role of radiosensitization. Furthermore, with the rapid development of nanotechnology, the method generating oxygen in hypoxic tumor regions has been accomplished by intratumoral injection of albumin–MnO2 nanoparticles, which will conduce to the improvement of hypoxic environment and strengthen the radiosensitivity (162). Acriflavine has been revealed that could suppress HIF1 dimerization and transcriptional regulation via directly binding to the HIF1α subunit (163). The application of YC−1, a HIF1 inhibitor, also displayed the treatment benefits when administered after irradiation (164).

In addition to the agents mentioned above, there are several old drugs that also have potential radiosensitizing effects. The anti-diabetic drug metformin can suppress the EMT program and stemness by regulating transcriptional reprogramming and inhibiting ZEB1, TWIST1 and SLUG (169, 170), and strengthens the sensitivity of cancer cells to irradiation (171). Berberine could reduce the expression of vimentin and evaluate the level of E-cadherin, thereby perturbing the TGF-β-induced EMT and sensitizing nasopharyngeal carcinoma cells to radiation (10).

Although many agents exibited effective roles in radiosensitization, they are rarely applied in clinical practice as radiosensitizers. We consider the possible reasons for blocking the transformation of these agents as follows. First, the efficacy of many agents is verified in vitro experiments or in preclinical animal experiments, however, no good therapeutic effects is observed in clinical trials. Second, the limitation of transformation may be partly attributed to safety of the agents. Combined application of the agents with radiotherapy may amplify adverse reactions, even leading to some serious complications, which is an important reason for restricting the clinical application of many agents. Third, the molecular mechanisms involved in EMT are extremely complex, and these factors do not operate isolation, but functions as a coordinated signaling network, ultimately leading to the output of EMT program. Therefore, a comprehensive understanding aiming to the key nodes in this network is needed for the development of EMT-targeting radiosensitizers, which increases the difficulty of drug transformation. Last, drug delivery into cancer cells is also an important factor that needs to be solved urgently. Drug transport into malignant tumors through blood relies on a variety of factors, and is affected by tumor vascular system and tissue characteristics. Moreover, irradiation has a significant impact on the revascularization of tumor tissues, so how to increase the drug concentration at the tumor site may also be a factor that limits the radiosensitivity. Currently, the application of microcarriers based on nanoparticles or liposomes may provide a potential method to improve the tumor-killing effect and increase the sensitivity of radiotherapy.

In summary, radioresistance is still regarded as a major challenge in current tumor treatment, and EMT as an important inducer of radioresistance has attracted large amount of attention. In this review, we hold the opinion that EMT is not only the cause to induce radioresistance, but the result of irradiation acting on cancer cells and TMEs, thus forming an infinitely amplified positive feedback network to further increase the resistance of tumor cells to radiotherapy ( Figure 3 ). We must comprehensively recognize that EMT plays a central role in eliciting resistance of tumor cells to radiotherapy, involved in multiple molecular mechanisms including signaling pathways, non-coding RNAs, CSCs and tumor microenvironment. The fact that there is a vicious circle between EMT and irradiation needs to be better understood. At the present, there are an array of agents in preclinical or clinical trials that work on EMT or its related signal pathways. Based on continuous progression on molecular medicine and novel biotechnology, it holds promise in overcoming radioresistance and improving the prognosis of cancer patients.

JZ and LQ conceived the framework of manuscript. LQ and YC were responsible for writing the manuscript. NL, YL, JX and XW conducted the figure and tables. FL, GD, and FC performed the revision of the manuscript. All authors have read and approved the final manuscript.

This project was funded by the National Natural Science Foundation of China (grant no. 81672974) and the Natural Science Foundation of Shandong Province (grant no. ZR2021QH356 and no. ZR2021LSW023). The funders are the corresponding author and first author of this paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.