Cancer progression/recurrence and metastasis are substantial problems in oral cancer patients with adjuvant RT. Experimental and clinical observations indicate that radiation can facilitate tumor metastasis in some cancer cells [14, 15], the leading cause of disease progression and mortality following treatment with RT. Therefore, alternative strategies to improve RT efficiency to prevent metastatic dissemination are urgently needed. Olaparib is the first PARP inhibitor approved for patients, demonstrating good safety and significantly prolonged survival for cancer patients. Additionally, evidence has suggested that combining RT with PARP inhibitor could be a synergistic antimetastatic efficacy in certain cancer cell lines, including melanoma [16] and cervical cancer [17], by inhibiting migration [18] .
Although Olaparib has been shown to inhibit proliferation and invasion abilities in oral cancer [19], there may be a synergistic effect that inhibits oral cancer metastasis when combined with radiation is less clear. In the present study, we first used parental OML1 cells and radioresistant OML1-R cells to assess the radiosensitizing effect of Olaparib. We showed that Olaparib, combined with IR, substantially decreased the growth rate when compared with IR-alone control cells. This result indicated that Olaparib enhanced the radiosensitivity of oral cancer cells. In addition, it is necessary to evaluate Olaparib's combining effects with irradiation further and test whether it is an effective treatment for cancer metastasis. We showed that treatment with Olaparib had noticeably an inhibiting effect on the migration and invasion of irradiated cells, confirming the experimentally in OML1 and OML1-R cells.
Since PARP is involved in regulating inflammatory processes, the inhibition of PARP has also been demonstrated as an immunomodulator to treat various types of inflammatory diseases [20, 21]. Besides, the immunomodulatory properties of PARP inhibitors have also been exploited to enhance antitumor immunity [22]. To clarify the molecular mechanism of an immune-related protein involved in the radiosensitization of Olaparib, we applied the cytokine profile array approach. We observed a substantial change in IL-17A in Olaparib-treated cells. IL-17A is a pro-inflammatory cytokine that has multifaceted roles in tumor formation. It is now known that IL-17A may play pro-tumor [23] or antitumor properties [24] in different tumor contexts. It has been reported to be highly expressed in tongue squamous cell carcinoma (TSCC) and positively correlated with tumor metastasis and poor outcome [25]. Several studies have shown that PARP inhibition exhibit markedly reduced IL-17A, which correlates with improved inflammatory disease symptoms. For example, in adjuvant-induced arthritis (AIA) mice experiments, PARP inhibition 5-aminoisoquinoline (5-AIQ) treatment reduces IL-17A, NF-κB- p65 release levels and attenuates the severity of AIA [26].
Similarly, the PARP inhibitor 3-aminobenzamide (3-AB) is reported to ameliorate the outcome of ischemic stroke and may be critically involved in decreasing IL-17 [27]. However, Olaparib-mediated inhibition of IL-17A enhances potential antimetastatic and radiosensitizing activities in oral cancer has not been studied yet. In the present study, we subsequently confirmed that Olaparib could decrease IL-17A in OML1 and OML1-R cells, especially in combination with RT.
PARP inhibitor treatment suppresses cancer cell migration and invasion [19]. Our data showed that combining Olaparib with IR significantly inhibited migration/invasion ability by OML1 and OML1-R cells. IL-17A has been shown to promote metastatic progression [28]. To investigate the functional effects of Olaparib on the migration of oral cancer cells could be counteracted by blocking IL-17A mediated signal transduction. We observed that in both cells, adding IL-17A abrogated Olaparib-reduced migration effectively. Besides, evidence indicates that NF-κB and MAPK pathways are involved in the induction of IL-17A expression [12, 23]. PARP inhibitors exert anti-inflammatory properties and anticancer effects by inhibiting the NF-κB and MAPK signaling [29, 30]. The present study found that Olaparib treatment and irradiation effectively decreased the IL-17A level, which enhanced the radiosensitivity and inhibited migration and invasion of oral cancer cells by downregulating NF-κB and p38 activation. We designed in vivo subcutaneous tumor model utilizing murine oral cancer MOC2 cells to determine the synergistic antitumor effect of Olaparib and RT when compared with single-agent treatments. Our studies revealed that combining Olaparib with RT resulted in a more considerable reduction in tumor volume than single-agent treatments. This founding was like to be linked to the decreased expression of IL-17A.
Metastasis is chiefly responsible for poor OSCC outcomes of treatment. Our metastatic mouse model demonstrated that Olaparib enhances RT's effect on preventing metastasis. We observed a reduction of lung metastasis after Olaparib treatment, especially in combining RT. Currently, PARP inhibitors are being established as monotherapies and within combination therapies for cancer patients with advanced or metastatic [30, 31]. The results revealed that Olaparib enhances RT's therapeutic efficiency and reduces metastasis.
We evaluated the clinical significance of IL-17A expression in 122 pathological stage I-IV oral squamous cell carcinoma (OSCC) patients. IL-17A expression was significantly higher in lymph node metastasis. Additionally, Kaplan-Meier survival analysis showed that high expression of IL-17A was associated with statistically significantly worse overall survival and cancer-specific survival. In multivariate analysis, IL-17A has been determined as an independent predictor of overall survival in OSCC. It has been reported that IL-17A activation is linked to carcinogenesis and progression of OSCC [25] and is regarded as a poor prognostic factor in other cancers [32]. Herein, we confirmed that increased IL-17A is strongly associated with shorter survival, indicating that IL-17A plays a vital role in OSCC pathogenesis and progression. Because activation of IL-17A could activate NF-κB signal transduction, several previous studies have demonstrated that IL-17A regulates NF-κB and cancer invasion and metastasis [23, 32]. Thus, one possible mechanism by which IL-17A is positively correlated with the degree of activation of NF-κB signal transduction is to promote OSCC development.
Besides, our data showed that perineural invasion (PNI) was also independently prognostic of overall survival in OSCC patients. PNI has been recognized as a key pathological feature of poor survival in many malignancies [33]. In previous studies, it has also been considered an indicator of a poor prognosis in OSCC [34, 35]. Nerves, as components of the tumor microenvironment (TME), also crosstalks with the immune system, which could contribute to tumor progression via inflammation [36]. M2-like tumor-associated macrophages (TAMs) have been demonstrated to secrete various cytokines with metabolic functions, such as IL6, CCL5, and CCL18 in the hypoxic TME to enhance PNI [37]. Notably, IL-17 can be produced by Th17 cells, Tc17 cells, macrophages, and neutrophil[9, 38, 39]. The in the TME are considered one of cancers' tumorigenesis progression and metastasis characteristics. Increased tumor-infiltrating Th17 cells and IL-17 were correlated with poor survival in patients with gastric and colorectal tumors [40, 41]. It has been suggested that the macrophage in breast tumor tissues were stained with anti-IL-17 antibodies. Therefore, infiltrating macrophage in breast tumor tissues demonstrated that it produced IL-17 and proposed a direct association between macrophage, IL-17, and breast cancer invasion [42]. However, the detailed biological and clinical role of PNI on survival should be further investigated in OSCC patients.
Our results provide a novel molecular rationale for combining Olaparib and radiotherapy and demonstrate that Olaparib promotes suppression of the IL-17A signal, enhancing radiosensitivity and decreasing tumor growth and metastasis in OSCC. However, it is not clear whether IL-17A-expressing immune cell population infiltrating interacts with the invasive features during OSCC development. Moreover, in the future, it is worth investigating how combining Olaparib and radiation can modulate the antitumor immune response by infiltrating immune cells to develop a strategy for inhibiting both progression and metastasis of OSCC.