Development Of Radioresistance Research Articles

TMT-Based Quantitative Proteomic Profiling of Human Esophageal Cancer Cells Reveals the Potential Mechanism and Potential Therapeutic Targets Associated With Radioresistance.

The recurrence of esophageal squamous cell carcinoma (ESCC) in radiation therapy treatment presents a complex challenge due to its resistance to radiation. However, the mechanism underlying the development of radioresistance in ESCC remains unclear. In this study, we aim to uncover the mechanisms underlying radioresistance in ESCC cells and identify potential targets for radiosensitization. We established two radio-resistant cell lines, TE-1R and KYSE-150R, from the parental ESCC cell lines TE-1 and KYSE-150 through fractionated irradiation. A TMT-based quantitative proteomic profiling approach was applied to identify changes in protein expression patterns. Cell Counting Kit-8, colony formation, γH2AX foci immunofluorescence and comet assays were utilized to validate our findings. The downstream effectors of the DNA repair pathway were confirmed using an HR/NHEJ reporter assay and Western blot analysis. Furthermore, we evaluated the expression of potential targets in ESCC tissues through immunohistochemistry combined with mass spectrometry. Over 2,000 proteins were quantitatively identified in the ESCC cell lysates. A comparison with radio-sensitive cells revealed 61 up-regulated and 14 down-regulated proteins in the radio-resistant cells. Additionally, radiation treatment induced 24 up-regulated and 12 down-regulated proteins in the radio-sensitive ESCC cells. Among the differentially expressed proteins, S100 calcium binding protein A6 (S100A6), glutamine gamma-glutamyltransferase 2 (TGM2), glycogen phosphorylase, brain form (PYGB), and Thymosin Beta 10 (TMSB10) were selected for further validation studies as they were found to be over-expressed in the accumulated radio-resistant ESCC cells and radio-resistant cells. Importantly, high S100A6 expression showed a positive correlation with cancer recurrence in ESCC patients. Our results suggest that several key proteins, including S100A6, TGM2, and PYGB, play a role in the development of radioresistance in ESCC. Our results revealed that several proteins including Protein S100-A6 (S100A6), Protein-glutamine gamma-glutamyltransferase 2 (TGM2), Glycogen phosphorylase, brain form (PYGB) were involved in radio-resistance development. These proteins could potentially serve as biomarkers for ESCC radio-resistance and as therapeutic targets to treat radio-resistant ESCC cells.

Blocking ACSL6 Compromises Autophagy via FLI1-Mediated Downregulation of COLs to Radiosensitize Lung Cancer.

Lung cancer (LC) is the leading cause of cancer-related mortality worldwide. Radiotherapy is the main component of LC treatment; however, its efficacy is often limited by radioresistance development, resulting in unsatisfactory clinical outcomes. Here, we foundthat LC radiosensitivity is up-regulated by decreased expression of long-chain acyl-CoA synthase 6 (ACSL6) after irradiation. Deletion of ACSL6 results in significant elevation of Friend leukemia integration 1 transcription factor (FLI1) and a marked decline of collagens (COLs). Blocking of ACSL6 impairs the tumor growth and upregulates FLI1, which reduces the levels of COLs and compromises irradiation-induced autophagy, leading to considerable therapeutic benefits during radiotherapy. Moreover, the direct interaction between ACSL6 and FLI1 and engagement between FLI1 and COLs indicates the involvement of the ACSL6-FLI1-COL axis. Finally, the potently adjusted autophagy flux reduces its otherwise contributive capability in surviving irradiation stress and leads to satisfactory radiosensitization for LC radiotherapy. These results demonstrate that enhanced ACSL6 expression promotes the aggressive performance of irradiated LC through increased FLI1-COL-mediated autophagy flux. Thus, the ACSL6-FLI1-Col-autophagy axis may be targeted to enhance the radiosensitivity of LC and improve the management of LC in radiotherapy.

HMGB1/TREM2 positive feedback loop drives the development of radioresistance and immune escape of glioblastoma by regulating TLR4/Akt signaling

BackgroundRadioresistance and immune escape are crucial reasons for unsatisfactory therapeutic effects of glioblastoma (GBM). Although triggering receptor expressed on myeloid cells-2 (TREM2) involved in forming immunosuppressive microenvironment, but the underlying mechanism and its roles in mediating cancer radioresistance remain unclear, moreover, the efficient delivery of drugs targeting TREM2 to GBM encounters serious challenges. Hence, this study aimed to elucidate the effect and mechanisms of targeted TREM2 silencing on reversing the radioresistance and immune escape of GBM aided by a glutathione-responsive biomimetic nanoparticle (NP) platform.MethodsRadioresistant GBM cell lines and TREM2 stable knockdown GBM cell lines were firstly established. RNA sequencing, colony formation assay, western blot, enzyme-linked immunosorbent assay and co-immunoprecipitation assay were used to detect the molecular mechanisms of TREM2 in regulating the radioresistance and immune escape of GBM. The glutathione-responsive biomimetic NP, angiopep-2 (A2)- cell membrane (CM)-NP/siTREM2/spam1, was then constructed to triply and targeted inhibit TREM2 for in vivo study. Orthotopic GBM-bearing mouse models were established to evaluate the anti-GBM effect of TREM2 inhibition, multiplex immunofluorescence assay was conducted to detect the infiltration of immune cells.ResultsTREM2 was a regulator in accelerating the radioresistance and immune escape of GBM through participating in DNA damage repair and forming a positive feedback loop with high mobility group box 1 (HMGB1) to cascade the activation of Toll-like receptor 4 (TLR4)/protein kinase B (Akt) signaling. A2-CM-NP/siTREM2/spam1 was successfully synthesized with excellent passive targeting, active targeting and homologous targeting, and the in vivo results exhibited its remarkable anti-GBM therapeutic effect through promoting the infiltration of type 1 helper T cells and CD8+T cells, reducing the infiltration of type 2 helper T cells and regulatory T cells, repolarizing macrophages to M1-type, and decreasing the secretion of pro-tumor and immunosuppressive cytokines.ConclusionsTargeting TREM2 therapy is a promising avenue for optimizing radiotherapy and immunotherapy to improve the prognosis of GBM patients.

Studies on Human Cultured Fibroblasts and Cutaneous Squamous Cell Carcinomas Suggest That Overexpression of Histone Variant H2A.J Promotes Radioresistance and Oncogenic Transformation.

Background: Cellular senescence in response to ionizing radiation (IR) limits the replication of damaged cells by causing permanent cell cycle arrest. However, IR can induce pro-survival signaling pathways that reduce the extent of radiation-induced cytotoxicity and promote the development of radioresistance. The differential incorporation of histone variant H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of radiation-induced senescence. However, the precise epigenetic mechanism and function of H2A.J overexpression in response to IR exposure still needs to be elucidated. Methods: Primary (no target, NT) and genetically modified fibroblasts overexpressing H2A.J (H2A.J-OE) were exposed to 20 Gy and analyzed 2 weeks post-IR for radiation-induced senescence by immunohistochemistry and immunofluorescence microscopy. Transcriptome signatures were analyzed in (non-)irradiated NT and H2A.J-OE fibroblasts by RNA sequencing. Since H2A.J plays an important role in the epidermal homeostasis of human skin, the oncogenic potential of H2A.J was investigated in cutaneous squamous cell carcinoma (cSCC). The tissue microarrays of cSCC were analyzed for H2A.J protein expression pattern by automated image analysis. Results: In response to radiation-induced DNA damage, the overexpression of H2A.J impairs the formation of senescence-associated heterochromatin foci (SAHF), thereby inhibiting the SAHF-mediated silencing of proliferation-promoting genes. The dysregulated activation of cyclins and cyclin-dependent kinases disturbs cell cycle arrest in irradiated H2A.J-OE fibroblasts, thereby overcoming radiation-induced senescence. Comparative transcriptome analysis revealed significantly increased WNT16 signaling in H2A.J OE fibroblasts after IR exposure, promoting the fundamental mechanisms of tumor development and progression, including the activation of the epithelial-mesenchymal transition. The quantitative analysis of cSCCs revealed that undifferentiated tumors are associated with high nuclear H2A.J expression, related with greater oncogenic potential. Conclusion: H2A.J overexpression induces radioresistance and promotes oncogenic transformation through the activation of WNT16 signaling pathway functions. H2A.J-associated signatures may improve risk stratification by identifying patients with more aggressive cSCC who may require radiotherapy with increased doses.

NEK2 contributes to radioresistance in esophageal squamous cell carcinoma by inducing protective autophagy via regulating TRIM21

BackgroundRadiotherapy (RT) has been identified as a vital treatment for esophageal squamous cell carcinoma (ESCC), while the development of radioresistance remains a major obstacle in ESCC management. The aim of this study was to investigate the effect of NIMA-related kinase 2 (NEK2) on radioresistance in ESCC cells and to reveal potential molecular mechanisms.MethodsHuman esophageal epithelial cells (HEEC) and human ESCC cell lines were obtained from the Research Center of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Cell Counting Kit-8 (CCK-8) and flow cytometry assays were applied to assess the proliferation ability, cell cycle, apoptosis rates, and ROS production of ESCC cells. The colony-forming assay was used to estimate the effect of NEK2 on radiosensitivity. Autophagy was investigated by western blotting analysis, GFP-mRFP-LC3 fluorescence assay, and transmission electron microscopy (TEM).ResultsIn the present study, our results showed that NEK2 was associated with radioresistance, cell cycle arrest, apoptosis, ROS production, and survival of ESCC. NEK2 knockdown could significantly inhibit growth while enhancing radiosensitivity and ROS production in ESCC cells. Interestingly, NEK2 knockdown inhibited ESCC cell autophagy and reduced autophagic flux, ultimately reversing NEK2-induced radioresistance. Mechanistically, NEK2 bound to and regulated the stability of tripartite motif-containing protein 21 (TRIM21). The accumulation of NEK2-induced light chain 3 beta 2 (LC3B II) can be reversed by the knockdown of TRIM21.ConclusionThese results demonstrated that NEK2 activated autophagy through TRIM21, which may provide a promising therapeutic strategy for elucidating NEK2-mediated radioresistance in ESCC.

Abstract 2887: Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain

Abstract This study aims to analyze irradiation-induced metabolic alterations in glioblastoma cells and astrocytes to enhance understanding of cellular responses and facilitate the development of more effective radiotherapeutic strategies. Metabolic reprogramming of tumor cells is considered one of the hallmarks of cancer. Studies have shown that abnormal activation of oncogenes and cancer-related signaling pathways, as well as the inactivation of tumor suppressor genes can induce metabolic reprogramming. These metabolic aberrations facilitate rapid proliferation, continuous growth, invasion, metastasis, and immune evasion. Following the application of radiotherapy, the activity of several metabolic pathways significantly changes, potentially leading to the development of radioresistance. However, a differential effect of ionizing radiation on tumor and normal cells is not well understood. Hence, quantifying irradiation-induced metabolic changes in glioblastoma cells and astrocytes is of high importance. We have developed a novel approach to measure dynamic metabolite changes upon irradiation, in vivo, in situ, and in real-time. This tool combines imaging of recently developed fluorescent biosensors via 2-photon microscopy and millimeter-scaled irradiation. Murine glioblastoma cells and astrocytes were generated with stable expression of biosensors for the quantification of intracellular concentrations of various metabolites, including lactate. Cytosolic concentrations of lactate depend on the balance between glycolytic production, mitochondrial consumption of pyruvate and lactate, and the exchange with the extracellular lactate pool via monocarboxylate transporters. Many cancer cells are characterized by an excessive conversion of glucose to lactate even under normoxic conditions. We quantified this so-called Warburg Effect in glioblastoma cells and astrocytes as the ratio between basal lactate production and the lactate increase after OXPHOS inhibition and determined irradiation-induced metabolic changes on the single-cell level. Complementarily, we used metabolomics and Seahorse analyses before and after irradiation. Our data indicate that irradiation increases oxygen consumption in glioblastoma cells but not in astrocytes. Also, we established an orthotopic glioblastoma mouse model via injection of sensor-expressing tumor cells for in vivo measurements of metabolite changes during and after irradiation. These data are expected to significantly advance the understanding of the cellular response to irradiation on a molecular level in situ and in real-time. This knowledge will help to develop combination treatments and increase the efficiency of radiotherapy. Citation Format: Marvin Kreuzer, Pascal Imseng, Bruno Weber, Martin Pruschy. Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2887.

PITPNC1 Suppress CD8+ T cell immune function and promote radioresistance in rectal cancer by modulating FASN/CD155

BackgroundRadioresistance is a primary factor contributing to the failure of rectal cancer treatment. Immune suppression plays a significant role in the development of radioresistance. We have investigated the potential role of phosphatidylinositol transfer protein cytoplasmic 1 (PITPNC1) in regulating immune suppression associated with radioresistance.MethodsTo elucidate the mechanisms by which PITPNC1 influences radioresistance, we established HT29, SW480, and MC38 radioresistant cell lines. The relationship between radioresistance and changes in the proportion of immune cells was verified through subcutaneous tumor models and flow cytometry. Changes in the expression levels of PITPNC1, FASN, and CD155 were determined using immunohistochemistry and western blotting techniques. The interplay between these proteins was investigated using immunofluorescence co-localization and immunoprecipitation assays. Additionally, siRNA and lentivirus-mediated gene knockdown or overexpression, as well as co-culture of tumor cells with PBMCs or CD8+ T cells and establishment of stable transgenic cell lines in vivo, were employed to validate the impact of the PITPNC1/FASN/CD155 pathway on CD8+ T cell immune function.ResultsUnder irradiation, the apoptosis rate and expression of apoptosis-related proteins in radioresistant colorectal cancer cell lines were significantly decreased, while the cell proliferation rate increased. In radioresistant tumor-bearing mice, the proportion of CD8+ T cells and IFN-γ production within immune cells decreased. Immunohistochemical analysis of human and animal tissue specimens resistant to radiotherapy showed a significant increase in the expression levels of PITPNC1, FASN, and CD155. Gene knockdown and rescue experiments demonstrated that PITPNC1 can regulate the expression of CD155 on the surface of tumor cells through FASN. In addition, co-culture experiments and in vivo tumor-bearing experiments have shown that silencing PITPNC1 can inhibit FASN/CD155, enhance CD8+ T cell immune function, promote colorectal cancer cell death, and ultimately reduce radioresistance in tumor-bearing models.ConclusionsPITPNC1 regulates the expression of CD155 through FASN, inhibits CD8+ T cell immune function, and promotes radioresistance in rectal cancer.

FBW7/GSK3β mediated degradation of IGF2BP2 inhibits IGF2BP2-SLC7A5 positive feedback loop and radioresistance in lung cancer

BackgroundThe development of radioresistance seriously hinders the efficacy of radiotherapy in lung cancer. However, the underlying mechanisms by which radioresistance occurs are still incompletely understood. The N6-Methyladenosine (m6A) modification of RNA is involved in cancer progression, but its role in lung cancer radioresistance remains elusive. This study aimed to identify m6A regulators involved in lung cancer radiosensitivity and further explore the underlying mechanisms to identify therapeutic targets to overcome lung cancer radioresistance.MethodsBioinformatic mining was used to identify the m6A regulator IGF2BP2 involved in lung cancer radiosensitivity. Transcriptome sequencing was used to explore the downstream factors. Clonogenic survival assays, neutral comet assays, Rad51 foci formation assays, and Annexin V/propidium iodide assays were used to determine the significance of FBW7/IGF2BP2/SLC7A5 axis in lung cancer radioresistance. Chromatin immunoprecipitation (ChIP)-qPCR analyses, RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)-qPCR analyses, RNA pull-down analyses, co-immunoprecipitation analyses, and ubiquitination assays were used to determine the feedback loop between IGF2BP2 and SLC7A5 and the regulatory effect of FBW7/GSK3β on IGF2BP2. Mice models and tissue microarrays were used to verify the effects in vivo.ResultsWe identified IGF2BP2, an m6A “reader”, that is overexpressed in lung cancer and facilitates radioresistance. We showed that inhibition of IGF2BP2 impairs radioresistance in lung cancer both in vitro and in vivo. Furthermore, we found that IGF2BP2 enhances the stability and translation of SLC7A5 mRNA through m6A modification, resulting in enhanced SLC7A5-mediated transport of methionine to produce S-adenosylmethionine. This feeds back upon the IGF2BP2 promoter region by further increasing the trimethyl modification at lysine 4 of histone H3 (H3K4me3) level to upregulate IGF2BP2 expression. We demonstrated that this positive feedback loop between IGF2BP2 and SLC7A5 promotes lung cancer radioresistance through the AKT/mTOR pathway. Moreover, we found that the ubiquitin ligase FBW7 functions with GSK3β kinase to recognize and degrade IGF2BP2.ConclusionsCollectively, our study revealed that the m6A “reader” IGF2BP2 promotes lung cancer radioresistance by forming a positive feedback loop with SLC7A5, suggesting that IGF2BP2 may be a potential therapeutic target to control radioresistance in lung cancer.

Novel radiation and targeted therapy combinations for improving rectal cancer outcomes.

Neoadjuvant radiotherapy (RT) is commonly used as standard treatment for rectal cancer. However, response rates are variable and survival outcomes remain poor, highlighting the need to develop new therapeutic strategies. Research is focused on identifying novel methods for sensitising rectal tumours to RT to enhance responses and improve patient outcomes. This can be achieved through harnessing tumour promoting effects of radiation or preventing development of radio-resistance in cancer cells. Many of the approaches being investigated involve targeting the recently published new dimensions of cancer hallmarks. This review article will discuss key radiation and targeted therapy combination strategies being investigated in the rectal cancer setting, with a focus on exploitation of mechanisms which target the hallmarks of cancer.

Upregulation of CoQ shifts ferroptosis dependence from GPX4 to FSP1 in acquired radioresistance

Acquired radioresistance is the primary contributor to treatment failure of radiotherapy, with ferroptosis is identified as a significant mechanism underlying cell death during radiotherapy. Although resistance to ferroptosis has been observed in both clinical samples of radioresistant cells and cell models, its mechanism remains unidentified. Herein, our investigation revealed that radioresistant cells exhibited greater tolerance to Glutathione Peroxidase 4 (GPX4) inhibitors and, conversely, increased sensitivity to ferroptosis suppressor protein 1 (FSP1) inhibitors compared to their sensitive counterparts. This observation suggested that FSP1 might play a dominant role in the development of radioresistance. Notably, the knockout of FSP1 demonstrated considerably superior efficacy in resensitizing cells to radiotherapy compared to the knockout of GPX4. To elucidate the driving force behind this functional shift, we conducted a metabolomic assay, which revealed an upregulation of Coenzyme Q (CoQ) synthesis and a downregulation of glutathione synthesis in the acquired radioresistance cells. Mechanistically, CoQ synthesis was found to be supported by aarF domain containing kinase 3-mediated phosphorylation of CoQ synthases, while the downregulation of Solute carrier family 7 member 11 led to decreased glutathione synthesis. Remarkably, our retrospective analysis of clinical response data further validated that the additional administration of statin during radiotherapy, which could impede CoQ production, effectively resensitized radioresistant cells to radiation. In summary, our findings demonstrate a dependency shift from GPX4 to FSP1 driven by altered metabolite synthesis during the acquisition of radioresistance. Moreover, we provide a promising therapeutic strategy for reversing radioresistance by inhibiting the FSP1-CoQ pathway.

Polymerase iota (POLI) confers radioresistance of esophageal squamous cell carcinoma by regulating RAD51 stability and facilitating homologous recombination

Radiotherapy resistance is an important and urgent challenge in the clinical management of esophageal squamous carcinoma (ESCC). However, the factors mediating the ESCC resistance to radiotherapy and its underlying molecular mechanisms are not fully clarified. Our previous studies have demonstrated the critical role of DNA polymerase iota (POLI) in ESCC development and progression, here, we aimed to investigate the involvement of POLI in ESCC radiotherapy resistance and elucidate the underlying molecular mechanism. We found that highly expressed POLI was correlated with shorter overall survival of ESCC patients received radiotherapy. Down-regulation of POLI sensitized ESCC to IR, prolonged γH2AX foci in nuclei and comet tails after IR. HR but not NHEJ repair is inhibited in POLI-deficient ESCC cells. POLI stabilizes RAD51 protein via competitively binding with and blocking the interaction between RAD51 and E3 ligase XIAP and XIAP-mediated ubiquitination. Furthermore, loss of POLI leads to the activation of GAS signaling. Our findings provide novel insight into the role of POLI in the development of radioresistance mediated by stabilizing RAD51 protein in ESCC.

The multifaceted role of extracellular vesicles in prostate cancer-a review.

Prostate cancer is the second most prominent form of cancer in men and confers the highest mortality after lung cancer. The term "extracellular vesicles" refers to minute endosomal-derived membrane microvesicles and it was demonstrated that extracellular vesicles affect the environment in which tumors originate. Extracellular vesicles' involvement is also established in the development of drug resistance, angiogenesis, stemness, and radioresistance in various cancers including prostate cancer. Extracellular vesicles influence the general environment, processes, and growth of prostate cancer and can be a potential area that offers a significant lead in prostate cancer therapy. In this review, we have elaborated on the multifaceted role of extracellular vesicles in various processes involved in the development of prostate cancer, and their multitude of applications in the diagnosis and treatment of prostate cancer through the encapsulation of various bioactives.

Cyclin D1 promotes radioresistance through regulation of RAD51 in melanoma.

Melanoma is a notoriously radioresistant type of skin cancer. Elucidation of the specific mechanisms underlying radioresistance is necessary to improve the clinical efficacy of radiation therapy. To identify the key factors contributing to radioresistance, five melanoma cell lines were selected for study and genes that were upregulated in relatively radioresistant melanomas compared with radiosensitive melanoma cells determined via RNA sequencing technology. In particular, we focused on cyclin D1 (CCND1), a well known cell cycle regulatory molecule. In radiosensitive melanoma, overexpression of cyclin D1 reduced apoptosis. In radioresistant melanoma cell lines, suppression of cyclin D1 with a specific inhibitor or siRNA increased apoptosis and decreased cell proliferation in 2D and 3D spheroid cultures. In addition, we observed increased expression of γ-H2AX, a molecular marker of DNA damage, even at a later time after γ-irradiation, under conditions of inhibition of cyclin D1, with a response pattern similar to that of radiosensitive SK-Mel5. In the same context, expression and nuclear foci formation of RAD51, a key enzyme for homologous recombination (HR), were reduced upon inhibition of cyclin D1. Downregulation of RAD51 also reduced cell survival to irradiation. Overall, suppression of cyclin D1 expression or function led to reduced radiation-induced DNA damage response (DDR) and triggered cell death. Our collective findings indicate that the presence of increased cyclin D1 potentially contributes to the development of radioresistance through effects on RAD51 in melanoma and could therefore serve as a therapeutic target for improving the efficacy of radiation therapy.

Transcriptional pausing induced by ionizing radiation enables the acquisition of radioresistance in nasopharyngeal carcinoma.

Lesions on the DNA template can impact transcription via distinct regulatory pathways. Ionizing radiation (IR) as the mainstay modality for many malignancies elicits most of the cytotoxicity by inducing a variety of DNA damages in the genome. How the IR treatment alters the transcription cycle and whether it contributes to the development of radioresistance remain poorly understood. Here, we report an increase in the paused RNA polymerase II (RNAPII), as indicated by the phosphorylation at serine 5 residue of its C-terminal domain, in recurrent nasopharyngeal carcinoma (NPC) patient samples after IR treatment and cultured NPC cells developing IR resistance. Reducing the pool of paused RNAPII by either inhibiting TFIIH-associated CDK7 or stimulating the positive transcription elongation factor b, a CDK9-CycT1 heterodimer, attenuates IR resistance of NPC cells. Interestingly, the poly(ADP-ribosyl)ation of CycT1, which disrupts its phase separation, is elevated in the IR-resistant cells. Mutation of the major poly(ADP-ribosyl)ation sites of CycT1 decreases RNAPII pausing and restores IR sensitivity. Genome-wide chromatin immunoprecipitation followed by sequencing analyses reveal that several genes involved in radiation response and cell cycle control are subject to the regulation imposed by the paused RNAPII. Particularly, we identify the NIMA-related kinase NEK7 under such regulation as a new radioresistance factor, whose downregulation results in the increased chromosome instability, enabling the development of IR resistance. Overall, our results highlight a novel link between the alteration in the transcription cycle and the acquisition of IR resistance, opening up new opportunities to increase the efficacy of radiotherapy and thwart radioresistance in NPC.

Biological and therapeutic viewpoints towards role of miR-218 in human cancers: Revisiting molecular interactions and future clinical translations

Understanding the exact pathogenesis of cancer is difficult due to heterogenous nature of tumor cells and multiple factors that cause its initiation and development. Treatment of cancer is mainly based on surgical resection, chemotherapy, radiotherapy and their combination, while gene therapy has been emerged as a new kind of therapy for cancer. Post-transcriptional regulation of genes has been of interest in recent years and among various types of epigenetic factors that can modulate gene expression, short non-coding RNAs known as microRNAs (miRNAs) have obtained much attention. The stability of mRNA decreases by miRNAs to repress gene expression. miRNAs can regulate tumor malignancy and biological behavior of cancer cells and understanding their function in tumorigenesis can pave the way towards developing new therapeutics in future. One of the new emerging miRNAs in cancer therapy is miR-218 that increasing evidence highlights its anti-cancer activity, while a few studies demonstrate its oncogenic function. The miR-218 transfection is promising in reducing progression of tumor cells. miR-218 shows interactions with molecular mechanisms including apoptosis, autophagy, glycolysis and EMT, and the interaction is different. miR-218 induces apoptosis, while it suppresses glycolysis, cytoprotective autophagy and EMT. Low expression of miR-218 can result in development of chemoresistance and radio-resistance in tumor cells and direct targeting of miR-218 as a key player is promising in cancer therapy. LncRNAs and circRNAs are nonprotein coding transcripts that can regulate miR-218 expression in human cancers. Moreover, low expression level of miR-218 can be observed in human cancers such as brain, gastrointestinal and urological cancers that mediate poor prognosis and low survival rate.

Radiotherapy and radio-sensitization in H3K27M -mutated diffuse midline gliomas.

H3K27M mutated diffuse midline gliomas (DMGs) are extremely aggressive and the leading cause of cancer-related deaths in pediatric brain tumors with 5-year survival <1%. Radiotherapy is the only established adjuvant treatment of H3K27M DMGs; however, the radio-resistance is commonly observed. We summarized current understandings of the molecular responses of H3K27M DMGs to radiotherapy and provide crucial insights into current advances in radiosensitivity enhancement. Ionizing radiation (IR) can mainly inhibit tumor cell growth by inducing DNA damage regulated by the cell cycle checkpoints and DNA damage repair (DDR) system. In H3K27M DMGs, the aberrant genetic and epigenetic changes, stemness genotype, and epithelial-mesenchymal transition (EMT) disrupt the cell cycle checkpoints and DDR system by altering the associated regulatory signaling pathways, which leads to the development of radio-resistance. The advances in mechanisms of radio-resistance in H3K27M DMGs promote the potential targets to enhance the sensitivity to radiotherapy.

Circ_0085616 contributes to the radio-resistance and progression of cervical cancer by targeting miR-541-3p/ARL2 signaling.

Circular RNAs (circRNAs) play crucial regulatory roles in cancer progression and the development of radio-resistance. Here, we intended to explore the role of circ_0085616 in cervical cancer progression and its associated mechanism. Colony formation assay was employed to analyze the radio-resistance and proliferation of cervical cancer cells. Cell proliferation ability was also assessed by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell apoptosis was analyzed by flow cytometry. Tube formation assay was performed to analyze cell angiogenesis ability. Transwell assays were conducted to measure cell migration and invasion abilities. Dual-luciferase reporter assay was utilized to verify the target relationships. Xenograft mice model was used to analyze the role of circ_0085616 in tumor growth in vivo. Circ_0085616 expression was elevated in cervical cancer tissues and cell lines. Circ_0085616 interference suppressed the radio-resistance, proliferation, tube formation, migration, and invasion and elevated the apoptosis rate of cervical cancer cells. Circ_0085616 acted as a sponge for microRNA-541-3p (miR-541-3p), and miR-541-3p was negatively regulated by circ_0085616 in cervical cancer cells. Circ_0085616 absence-induced changes in the behaviors of cervical cancer cells were largely overturned by anti-miR-541-3p. miR-541-3p negatively regulated ADP ribosylation factor like GTPase 2 (ARL2) expression by binding to its 3' untranslated region (3'UTR). miR-541-3p mimic-induced effects were largely reversed by pcDNA-ARL2 in cervical cancer cells. Circ_0085616 positively regulated ARL2 expression by sequestering miR-541-3p. Circ_0085616 absence significantly inhibited the tumor growth in vivo. Circ_0085616 contributed to the radio-resistance and progression of cervical cancer partly through mediating the miR-541-3p/ARL2 axis.

Role of ionizing radiation activated NRF2 in lung cancer radioresistance

Radiotherapies are commonly used to target remaining tumor niches after surgery of solid tumors but are restricted due to therapeutic resistance. Several pathways of radioresistance have been reported in various cancers. This study investigates the pivotal role of Nuclear factor-erythroid 2–related factor 2 (NRF2) in the activation of DNA damage repair in lung cancer cells after x-rays exposure. To explore the NRF2 activation after ionizing irradiations, this study uses a knockdown of NRF2, which shows potential DNA damage after x-rays irradiation in lung cancers. This work further shows that NRF2 knockdown disrupts damaged DNA repair by inhibiting DNA-dependent protein kinase catalytic subunit. At the same time, NRF2 knockdown by shRNA considerably disparate homologous recombination by interfering with Rad51 expression. Further investigation of the associated pathway reveals that NRF2 activation mediates DNA damage response via the mitogen-activated protein kinase (MAPK) pathway as the knockout of NRF2 directly enhances intracellular MAPK phosphorylation. Similarly, both N-acetylcysteineand constitutive knockout of NRF2 disrupt DNA-dependent protein kinase catalytic subunit, while NRF2 knockout failed to upregulate Rad51 expression after irradiation in-vivo. Taken together, these findings advocate NRF2 plays a critical role in the development of radioresistance by upregulating DNA damage response via the MAPK pathway, which can be of great significance.

Abstract 5793: Met confers radioresistance-associated aggressiveness through enhancing PLXDC2-mediated cancer stem cell plasticity

Abstract The development of radioresistance in head and neck squamous cell carcinoma (HNSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in the past decades. One of the clinical challenges is that radioresistance often promotes tumor aggressiveness. However, the underlying mechanisms and molecular determinants are largely unknown. We report here that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but can be suppressed by its genetic knockdown and pharmacological inactivation. Through unbiased RNAseq data, we further uncovered that the resulting upregulation of c-Met signaling increases the expression of PLXDC2. a critical gene associated with the tumor microenvironment. Mechanistically, PLXDC2 is upregulated in radioresistant HNSCC cells through c-Met mediated activation of ERK1/2-ELK1 signaling cascade. Most importantly, PLXDC2 modulates cancer cell plasticity by inducing epithelial-mesenchymal transition (EMT) and the emergence of cancer stem cell (CSC) subpopulation. Additionally, depletion of PLXDC2 overcomes c-Met-mediated radioresistance by reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, a combination of the c-Met selective inhibitor SU11274 with radiation remarkably induces tumor shrinkage and constrains tumor metastasis to lymph nodes in vivo. Our study is novel in being the first to explore the role and mechanism of the c-Met-PLXDC2 axis in radioresistance-associated HNSCC aggressiveness and the first to our knowledge to evaluate how to take advantage of blocking this signaling to overcome radioresistance in preclinical mouse models of HNSCC. Citation Format: Fanghui Chen, Liwei Lang, Chloe Shay, Georgia Chen, Nabil Saba, Yong Teng. Met confers radioresistance-associated aggressiveness through enhancing PLXDC2-mediated cancer stem cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5793.

Insights into the characteristics of primary radioresistant cervical cancer using single-cell transcriptomics

Radioresistance is a major cause of radiotherapy failure among patients with cervical cancer (CC), the fourth most common cause of cancer mortality in women worldwide. Traditional CC cell lines lose intra-tumoral heterogeneity, posing a challenge for radioresistance research. Meanwhile, conditional reprogramming (CR) maintains intra-tumoral heterogeneity and complexity, as well as the genomic and clinical characteristics of original cells and tissues. Three radioresistant and two radiosensitive primary CC cell lines were developed under CR conditions from patient specimens, and their characteristics were verified via immunofluorescence, growth kinetics, clone forming assay, xenografting, and immunohistochemistry. The CR cell lines had homogenous characteristics with original tumor tissues and maintained radiosensitivity in vitro and in vivo, while also maintaining intra-tumoral heterogeneity according to single-cell RNA sequencing analysis. Upon further investigation, 20.83% of cells in radioresistant CR cell lines aggregated in the G2/M cell cycle phase, which is sensitive to radiation, compared to 38.1% of cells in radiosensitive CR cell lines. This study established three radioresistant and two radiosensitive CC cell lines through CR, which will benefit further research investigating radiosensitivity in CC. Our present study may provide an ideal model for research on development of radioresistance and potential therapeutic targets in CC.