Figure 4 - uploaded by Anne Marije van Harten
Content may be subject to copyright.
Effects of Wee1 inhibition on cell cycle regulation, DNA damage and mitosis. (a-d) Western blot analysis of premalignant cell lines VU-preSCC-M3 (a) and D19 (b), HNSCC cell line UM-SCC-22A (c) and ovarian cell line OVCAR3 (d) all showed decreased p-CDK1 Y15 expression upon treatment with 100 nM Adavosertib, already after 4 h of treatment (c). Increased levels of γH2Ax Ser139 indicated DNA damage induced by Wee1 inhibition. Additional cell lines are shown in Fig. S4a-c. (e,f) Precancerous cells VUpreSCC-M3 (e) and HNSCC UM-SCC-22A (f) were stained for α-tubulin (microtubules, in green) and DNA (DAPI, in blue). In untreated conditions, both the premalignant and tumor cell lines showed normal bipolar spindle formation with condensated chromosomes. Upon treatment with 100 nM Adavosertib for 48 h, (pro-) metaphase cells showed less dense spindles with problematic DNA alignment, as well as tripolar spindles and micronuclei formation in UM-SCC-22A. After treatment with 250 nM Adavosertib, chromosomal alignment and condensation was increasingly abnormal in both cell lines and giant cells with multiple nuclei or nuclei fragmentation were found, a result of mitotic failure. No monopolar spindles were found. (g,h) To investigate mitotic exhaustion due to SAC checkpoint activation, pole-to-pole length (µm) was measured of (pro-) metaphases in untreated and 100 nM Adavosertib treated cells. A significant increase of pole-to-pole length was found in both VU-preSCC-M3 (g, two-sided t-test, p = 0.007) and UM-SCC-22A (h, two-sided t-test, p = 0.0001) (see Fig, S4d for a representative picture).
Source publication
HPV-negative head and neck squamous cell carcinomas (HNSCCs) develop in precancerous changes in the mucosal lining of the upper-aerodigestive tract. These precancerous cells contain cancer-associated genomic changes and cause primary tumors and local relapses. Therapeutic strategies to eradicate these precancerous cells are very limited. Using func...
Contexts in source publication
Context 1
... inhibition initiates death in mitosis and micro-nucleation due to DNA damage induction and unsupervised entry of mitosis. Since Wee1 is a main inhibitor of CDK1, which controls mitosis, the molecular activation of a number of key regulators of mitosis was further investigated (Figs. 4a-d and S4a-c). Expression levels of CDK1 and its binding partner cyclin B1 (not shown) remained more or less stable upon Wee1 inhibition, although the large number of dying cells after 24 h of treatment might have hampered interpretation of the data. Upon treatment with 100 nM Adavosertib, the inhibitory phosphorylation of Y15 on CDK1 ...
Context 2
... we investigated abnormalities in mitotic cells in precancerous VU-preSCC-M3 and HNSCC UM-SCC-22A cells after 48 h of Wee1 inhibition. Microtubules and spindle formation were visualized using α-Tubulin (green) and DNA was stained with DAPI (blue) (Fig. 4e,f). In untreated conditions, both premalignant as well as HNSCC cells showed normal bipolar spindles and chromosomal condensation during mitosis. After Wee1 inhibition, the spindles showed abnormalities and the chromosomes failed to align in the metaphase plate. Both giant cells with fragmented DNA (arrows) and isolated DNA nuclei ...
Context 3
... and isolated DNA nuclei appeared frequently, indicating mitotic failure. Remarkably, upon treatment with 100 nM Adavosertib, we observed a significant increase in the spindle pole-to-pole distance in (pro-)metaphase cells, compared to untreated cells for both VU-preSCC-M3 (two-sided t-test, p = 0.007) and UM-SCC-22A (two-sided t-test, p < 0.0001) (Figs. 4g,h and S4d). This indicates mitotic arrest due to SAC-checkpoint activation, inward forces on the spindles due to DNA damage caused by Wee1 All graphs show the effect on cell viability upon knockdown compared to the negative transfection control used. Negative transfection control siCONTROL#2 was used for primary fibroblasts, UM-SCC-22A, and ...
Context 4
... cells were collected from excised uvulas of healthy adults as previously described 46 To investigate mitotic exhaustion due to SAC checkpoint activation, pole-to-pole length (µm) was measured of (pro-) metaphases in untreated and 100 nM Adavosertib treated cells. A significant increase of pole-to-pole length was found in both VU-preSCC-M3 (g, two-sided t-test, p = 0.007) and UM-SCC-22A (h, two-sided t-test, p = 0.0001) (see Fig, S4d for a representative picture). www.nature.com/scientificreports ...
Context 5
... inhibition initiates death in mitosis and micro-nucleation due to DNA damage induction and unsupervised entry of mitosis. Since Wee1 is a main inhibitor of CDK1, which controls mitosis, the molecular activation of a number of key regulators of mitosis was further investigated (Figs. 4a-d and S4a-c). Expression levels of CDK1 and its binding partner cyclin B1 (not shown) remained more or less stable upon Wee1 inhibition, although the large number of dying cells after 24 h of treatment might have hampered interpretation of the data. Upon treatment with 100 nM Adavosertib, the inhibitory phosphorylation of Y15 on CDK1 ...
Context 6
... we investigated abnormalities in mitotic cells in precancerous VU-preSCC-M3 and HNSCC UM-SCC-22A cells after 48 h of Wee1 inhibition. Microtubules and spindle formation were visualized using α-Tubulin (green) and DNA was stained with DAPI (blue) (Fig. 4e,f). In untreated conditions, both premalignant as well as HNSCC cells showed normal bipolar spindles and chromosomal condensation during mitosis. After Wee1 inhibition, the spindles showed abnormalities and the chromosomes failed to align in the metaphase plate. Both giant cells with fragmented DNA (arrows) and isolated DNA nuclei ...
Context 7
... and isolated DNA nuclei appeared frequently, indicating mitotic failure. Remarkably, upon treatment with 100 nM Adavosertib, we observed a significant increase in the spindle pole-to-pole distance in (pro-)metaphase cells, compared to untreated cells for both VU-preSCC-M3 (two-sided t-test, p = 0.007) and UM-SCC-22A (two-sided t-test, p < 0.0001) (Figs. 4g,h and S4d). This indicates mitotic arrest due to SAC-checkpoint activation, inward forces on the spindles due to DNA damage caused by Wee1 All graphs show the effect on cell viability upon knockdown compared to the negative transfection control used. Negative transfection control siCONTROL#2 was used for primary fibroblasts, UM-SCC-22A, and ...
Context 8
... cells were collected from excised uvulas of healthy adults as previously described 46 To investigate mitotic exhaustion due to SAC checkpoint activation, pole-to-pole length (µm) was measured of (pro-) metaphases in untreated and 100 nM Adavosertib treated cells. A significant increase of pole-to-pole length was found in both VU-preSCC-M3 (g, two-sided t-test, p = 0.007) and UM-SCC-22A (h, two-sided t-test, p = 0.0001) (see Fig, S4d for a representative picture). www.nature.com/scientificreports ...
Citations
... Cultures that proliferated well were inspected for cells with different morphology, and morphologically different cell clones were selected by differential trypsinization when possible. Cultures were genetically characterized as described below, immunohistochemical biomarkers as well as drug sensitivity were evaluated according to van Harten et al. [19]. The study was approved by the Institutional Review Board at Amsterdam UMC and patients were enrolled in the study after written informed consent. ...
... Previously, we reported on a panel of 319 tumor-lethal siRNAs that emerged from genome-wide screens in two cancer cell lines. These were rescreened on a large panel of HNSCC cell lines including FA-HNSCC derived cell lines 15 . Here, we aimed to identify druggable gene targets that could be exploited for treatment of FA-HNSCC and we provide preclinical data on gemcitabine toxicity in FA-HNSCC cell lines, FA-de cient cells and FA-de cient mice. ...
... RRM1 and RRM2 are essential genes in FA-HNSCC As a starting point of this study, we analyzed the viability data of all cell lines from the previously executed 319 tumor-lethal siRNA re-screen 15 , focusing speci cally on the FA-HNSCC cell lines. A viability score of ≤ -0.5 relative to the positive and negative controls was considered as lethal 15 (Fig. 1a). ...
... RRM1 and RRM2 are essential genes in FA-HNSCC As a starting point of this study, we analyzed the viability data of all cell lines from the previously executed 319 tumor-lethal siRNA re-screen 15 , focusing speci cally on the FA-HNSCC cell lines. A viability score of ≤ -0.5 relative to the positive and negative controls was considered as lethal 15 (Fig. 1a). We noted that the knockdown of both RRM1 and RRM2 resulted in a highly signi cant reduction of viability, which was observed in all FA-derived and the majority of non-FA-derived cell lines tested. ...
Fanconi anemia (FA) is a rare hereditary disease characterized by an inactivating mutation in the FA/BRCA pathway, critical for the effective repair of DNA interstrand crosslinks (ICLs). The disease is characterized by progressing bone marrow failure, congenital abnormalities and an increased risk to develop malignancies early in life, in particular head and neck squamous cell carcinoma (HNSCC). While ICL-inducing cisplatin combined with radiotherapy are a mainstay of HNSCC treatment, cisplatin is contraindicated for FA-HNSCC patients. This dilemma necessitates the identification of novel treatment modalities tolerated by FA-HNSCC patients.
To identify druggable targets, an siRNA-based genetic screen was performed previously in HNSCC-derived cell lines from FA and non-FA tumor origin. Here we report that the Ribonucleotide Reductase (RNR) complex, consisting of the RRM1 and RRM2 subunits, was identified as a therapeutic target for both, FA and non-FA-HNSCC. While non-FA-HNSCC cells responded differentially to RNR depletion, FA-HNSCC cells were consistently found hypersensitive. This insight was confirmed pharmacologically using 2', 2'-difluoro 2'deoxycytidine (dFdC), also known as gemcitabine, a clinically used nucleotide analogue that is a potent inhibitor of the RNR complex. Importantly, while cisplatin exposure displayed a severe, long-lasting toxicity on the hematopoietic stem and progenitor compartments in Fancg-/- mice, gemcitabine was well tolerated and had only a mild, transient impact.
Taken together, our data implicate that gemcitabine-based chemoradiotherapy could serve as an alternative HNSCC treatment in Fanconi patients, and deserves clinical testing.
... The therapeutic landscape for treatment of head and neck squamous cell carcinoma still relies on surgery and radiotherapy in combination with chemotherapeutics and immunotherapy, particularly for advanced cases [49]. Several novel small molecule therapies are under clinical investigation with the goal of improving radiation treatment efficacy, targeting nonresponsive cancer types, and decreasing toxicities associated with current regimens [22,[50][51][52]. Combination therapy with agents that individually or synergistically induce cancer cell death represents an avenue for enhancing efficacy across a wide range of cancers with mutations affecting different cell death pathways, while limiting treatment-associated toxicity [53]. ...
... As such, protein ubiquitination and the DNA damage response represent promising targets for novel therapeutic combinations [54,55]. This study investigates a previously unreported combination of the cIAP and WEE1 inhibitor drug classes, which target pathways known to be dysregulated in and critical for cancer cell survival and progression [52,56]. Both drug classes have shown to induce cancer cell death in vitro and in vivo across a panel of HSNCC cell lines [30,48]. ...
Simple Summary
Both IAP and WEE1 inhibitors have demonstrated therapeutic efficacy in in vitro pre-clinical models of head and neck squamous cell carcinoma (HNSCC). Here, we demonstrate that dual treatment with IAP and WEE1 inhibitors sensitizes both HPV-negative and HPV-positive HNSCC cells to both TNFα-dependent and radiation-associated cell death, demonstrating a potential therapeutic combination to treat these cancers.
Abstract
Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK–NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC.
... In summary, field cancerization has been well characterized in genetic terms, the cells can be cultured and even used for therapeutic target screening [49,50]. A field should be defined as a group of cells with tumour-associated somatic genetic alterations. ...
Simple Summary
Oral cavity is the most common site of head and neck cancer which is ranked as the eighth most common cancer worldwide. Oral cancer treatment is often associated with significant morbidity and is sometimes ineffective. These cancers, mainly due to tobacco and alcohol consumption, can develop from oral potentially malignant disorders, the most common of which is oral leukoplakia. Some of these oral potentially malignant disorders disappear, while others will transform to oral cancer. Patients may also develop cancer in the field of cancerization. Unfortunately, except for the surgical excision of lesions with dysplasia, there is no effective intervention to effectively prevent transformation or cancer development in the field of cancerization. Moreover, no standardized biomarker has been clearly identified as sufficient to predict malignant transformation. In this article, several experts discuss the main challenges in oral cancer prevention, in particular the need (i) to define new a new classification system integrating cellular and molecular features aiming (ii) at better identifying patients at high risk of malignant transformation, and (iii) at developing treatment strategies to prevent their malignant transformation of oral potentially malignant disorders.
Abstract
Oral potentially malignant disorders (OPMD) may precede oral squamous cell carcinoma (OSCC). Reported rates of malignant transformation of OPMD range from 3 to 50%. While some clinical, histological, and molecular factors have been associated with a high-risk OPMD, they are, to date, insufficiently accurate for treatment decision-making. Moreover, this range highlights differences in the clinical definition of OPMD, variation in follow-up periods, and molecular and biological heterogeneity of OPMD. Finally, while treatment of OPMD may improve outcome, standard therapy has been shown to be ineffective to prevent OSCC development in patients with OPMD. In this perspective paper, several experts discuss the main challenges in oral cancer prevention, in particular the need to (i) to define an OPMD classification system by integrating new pathological and molecular characteristics, aiming (ii) to better identify OPMD at high risk of malignant transformation, and (iii) to develop treatment strategies to eradicate OPMD or prevent malignant transformation.
... Head and neck cancer (HNC) is generally defined as a span of malignant neoplasms that originate from the soft tissues include the oral and nasal cavities, sinuses, lips, salivary glands, throat, and larynx (Joshi et al., 2014). HNC is mostly caused by tobacco and alcohol consumption, and recently, the human papillomavirus (HPV) has been found as another important cause of HNC (van Harten et al., 2020). According to the GLOBOCAN 2018 statistics, the estimated number of new HNC diagnosed and deaths were about 705,781 and 358,144, respectively. ...
Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.
... However, exploiting rewired pathways such as those that regulate cell cycle control in cancer cells by targeting an additional cell cycle regulating protein also follows the concept of synthetic lethal interactions. Recent studies suggest that loss of cell cycle control by p53 and/or p16 Ink4A loss of functions sensitize (pre)HNSCC cells to Wee1, Chk1 and PLK1 inhibition [84,[109][110][111][112]. Normal cells may stay arrested in G1/G0, while cancer cells are forced to enter S-phase and progress through the cell cycle and have to rely on the rewired control mechanisms that act during DNA replication and G2-M. ...
... Subsequent interference with the paralogue gene may lead to decreased cell survival (Figure 2, Right panel). To identify redundant paralogue genes after loss of a passenger gene, integrated analysis is required to match genomic copy number losses with vulnerabilities to interference with known paralogues [110,[113][114][115]. ...
... We previously reported on indications that collateral lethality may occur in premalignant oral cells and HNSCC tumor cells in a customized screen in multiple cell lines models with 319 siRNAs, preselected from prior HNSCC lethality screens, [110]. Although we were able to identify collateral lethality for some of these hits, no druggable targets were found in this small RNA interference screen. ...
Simple Summary
Head and neck squamous cell carcinomas (HNSCCs) develop from mucosal cells in the oral cavity, pharynx and larynx after either prolonged exposure to tobacco and alcohol, or a transforming infection with high-risk human papillomavirus (HPV). HPV-negative HNSCCs develop in a zone of premalignant mucosal cells centimeters in diameter and characterized by tumor-associated genetic changes, also referred to as ‘fields’, which can present as white leukoplakia lesions but are mostly invisible. Patients with HPV-negative HNSCC have an overall 5-years survival rate of 50–60%, despite application of intense treatment protocols, and current treatment regimens seem to have reached their plateau. Recently, immunotherapy using immune checkpoint inhibitors has been introduced, but seems effective in only some patients. Targeted treatments have failed to find their way into the clinic while novel therapies are urgently awaited that could target the tumor as well as the precancerous cells. However, recent data suggest that we are at the dawn of a new era. This review focusses on the preclinical identification of druggable targets for therapy for HPV-negative HNSCC and their preceding precancerous changes.
Abstract
Head and neck squamous cell carcinomas (HNSCC) develop in the mucosal lining of the upper-aerodigestive tract. In carcinogen-induced HNSCC, tumors emerge from premalignant mucosal changes characterized by tumor-associated genetic alterations, also coined as ‘fields’ that are occasionally visible as leukoplakia or erythroplakia lesions but are mostly invisible. Consequently, HNSCC is generally diagnosed de novo at more advanced stages in about 70% of new diagnosis. Despite intense multimodality treatment protocols, the overall 5-years survival rate is 50–60% for patients with advanced stage of disease and seems to have reached a plateau. Of notable concern is the lack of further improvement in prognosis despite advances in treatment. This can be attributed to the late clinical presentation, failure of advanced HNSCC to respond to treatment, the deficit of effective targeted therapies to eradicate tumors and precancerous changes, and the lack of suitable markers for screening and personalized therapy. The molecular landscape of head and neck cancer has been elucidated in great detail, but the absence of oncogenic mutations hampers the identification of druggable targets for therapy to improve outcome of HNSCC. Currently, functional genomic approaches are being explored to identify potential therapeutic targets. Identification and validation of essential genes for both HNSCC and oral premalignancies, accompanied with biomarkers for therapy response, are being investigated. Attentive diagnosis and targeted therapy of the preceding oral premalignant (preHNSCC) changes may prevent the development of tumors. As classic oncogene addiction through activating mutations is not a realistic concept for treatment of HNSCC, synthetic lethality and collateral lethality need to be exploited, next to immune therapies. In recent studies it was shown that cell cycle regulation and DNA damage response pathways become significantly altered in HNSCC causing replication stress, which is an avenue that deserves further exploitation as an HNSCC vulnerability for treatment. The focus of this review is to summarize the current literature on the preclinical identification of potential druggable targets for therapy of (pre)HNSCC, emerging from the variety of gene knockdown and knockout strategies, and the testing of targeted inhibitors. We will conclude with a future perspective on targeted therapy of HNSCC and premalignant changes.
... It is clearly important to extend this work to OPMD where there may be greater therapeutic benefit for the prevention of cancer development. Interestingly, using an RNA interference screen, van Harten et al. [130] identified Wee1 as a potential target in the inhibition of a cell line derived from a high risk OPMD lesion; such a chemo-preventive strategy might be valuable in attenuating the progression of such lesions in the pre-cancerous field. ...
This study reviews the molecular landscape of oral potentially malignant disorders (OPMD). We examine the impact of tumour heterogeneity, the spectrum of driver mutations (TP53, CDKN2A, TERT, NOTCH1, AJUBA, PIK3CA, CASP8) and gene transcription on tumour progression. We comment on how some of these mutations impact cellular senescence, field cancerization and cancer stem cells. We propose that OPMD can be monitored more closely and more dynamically through the use of liquid biopsies using an appropriate biomarker of transformation. We describe new gene interactions through the use of a systems biology approach and we highlight some of the first studies to identify functional genes using CRISPR-Cas9 technology. We believe that this information has translational implications for the use of re-purposed existing drugs and/or new drug development. Further, we argue that the use of digital technology encompassing clinical and laboratory-based data will create relevant datasets for machine learning/artificial intelligence. We believe that therapeutic intervention at an early molecular premalignant stage should be an important preventative strategy to inhibit the development of oral squamous cell carcinoma and that this approach is applicable to other aerodigestive tract cancers.
Precancerous cells in the oral cavity may appear as oral potentially malignant disorders, but they may also present as dysplasia without visual manifestation in tumor-adjacent tissue. As it is currently not possible to prevent the malignant transformation of these oral precancers, new treatments are urgently awaited. Here, we generated precancer culture models using a previously established method for the generation of oral keratinocyte cultures and incorporated CRISPR/Cas9 editing. The generated cell lines were used to investigate the efficacy of a set of small molecule inhibitors. Tumor-adjacent mucosa and oral leukoplakia biopsies were cultured and genetically characterized. Mutations were introduced in CDKN2A and TP53 using CRISPR/Cas9 and combined with the ectopic activation of telomerase to generate cell lines with prolonged proliferation. The method was tested in normal oral keratinocytes and tumor-adjacent biopsies and subsequently applied to a large set of oral leukoplakia biopsies. Finally, a subset of the immortalized cell lines was used to assess the efficacy of a set of small molecule inhibitors. Culturing and genomic engineering was highly efficient for normal and tumor-adjacent oral keratinocytes, but success rates in oral leukoplakia were remarkably low. Knock-out of CDKN2A in combination with either the activation of telomerase or knock-out of TP53 seemed a prerequisite for immortalization. Prolonged culturing was accompanied by additional genetic aberrations in these cultures. The generated cell lines were more sensitive than normal keratinocytes to small molecule inhibitors of previously identified targets. In conclusion, while very effective for normal keratinocytes and tumor-adjacent biopsies, the success rate of oral leukoplakia cell culturing methods was very low. Genomic engineering enabled the prolonged culturing of OL-derived keratinocytes but was associated with acquired genetic changes. Further studies are required to assess to what extent the immortalized cultures faithfully represent characteristics of the cells in vivo.
Cells experience both endogenous and exogenous DNA damage daily. To maintain genome integrity and suppress tumorigenesis, individuals have evolutionarily acquired a series of repair functions, termed DNA damage response (DDR), to repair DNA damage and ensure the accurate transmission of genetic information. Defects in DNA damage repair pathways may lead to various diseases, including tumors. Accumulating evidence suggests that alterations in DDR-related genes, such as somatic or germline mutations, single nucleotide polymorphisms (SNPs), and promoter methylation, are closely related to the occurrence, development, and treatment of head and neck squamous cell carcinoma (HNSCC). Despite recent advances in surgery combined with radiotherapy, chemotherapy, or immunotherapy, there has been no substantial improvement in the survival rate of patients with HNSCC. Therefore, targeting DNA repair pathways may be a promising treatment for HNSCC. In this review, we summarized the sources of DNA damage and DNA damage repair pathways. Further, the role of DNA damage repair pathways in the development of HNSCC and the application of small molecule inhibitors targeting these pathways in the treatment of HNSCC were focused.
Objective:
Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro.
Materials and methods:
WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined.
Results:
Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest.
Conclusion:
These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radio-sensitizer in OTSCC irrespective of p53 mutational status.
