Critical Role of the cGAS-STING Pathway in Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin is an effective chemotherapy drug for treating various types of cancer. However, lethal cardiotoxicity severely limits its clinical use. Recent evidence has indicated that aberrant activation of the cytosolic DNA-sensing cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING (stimulator of interferon genes) pathway plays a critical role in cardiovascular destruction. Here, we investigate the involvement of this mechanism in doxorubicin-induced cardiotoxicity (DIC). METHODS: Mice were treated with low-dose doxorubicin to induce chronic DIC. The role of the cGAS-STING pathway in DIC was evaluated in cGAS-deficiency (cGAS-/-), Sting-deficiency (Sting-/-), and interferon regulatory factor 3 (Irf3)-deficiency (Irf3-/-) mice. Endothelial cell (EC)-specific conditional Sting deficiency (Stingflox/flox/Cdh5-CreERT) mice were used to assess the importance of this pathway in ECs during DIC. We also examined the direct effects of the cGAS-STING pathway on nicotinamide adenine dinucleotide (NAD) homeostasis in vitro and in vivo. RESULTS: In the chronic DIC model, we observed significant activation of the cGAS-STING pathway in cardiac ECs. Global cGAS, Sting, and Irf3 deficiency all markedly ameliorated DIC. EC-specific Sting deficiency significantly prevented DIC and endothelial dysfunction. Mechanistically, doxorubicin activated the cardiac EC cGAS-STING pathway and its target, IRF3, which directly induced CD38 expression. In cardiac ECs, the cGAS-STING pathway caused a reduction in NAD levels and subsequent mitochondrial dysfunction via the intracellular NAD glycohydrolase (NADase) activity of CD38. Furthermore, the cardiac EC cGAS-STING pathway also regulates NAD homeostasis and mitochondrial bioenergetics in cardiomyocytes through the ecto-NADase activity of CD38. We also demonstrated that pharmacological inhibition of TANK-binding kinase 1 or CD38 effectively ameliorated DIC without compromising the anticancer effects of doxorubicin. CONCLUSIONS: Our findings indicate a critical role of the cardiac EC cGAS-STING pathway in DIC. The cGAS-STING pathway may represent a novel therapeutic target for preventing DIC.

Dynamic immunoediting by macrophages in homologous recombination deficiency-stratified pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, notably resistant to existing therapies. Current research indicates that PDAC patients deficient in homologous recombination (HR) benefit from platinum-based treatments and poly-ADP-ribose polymerase inhibitors (PARPi). However, the effectiveness of PARPi in HR-deficient (HRD) PDAC is suboptimal, and significant challenges remain in fully understanding the distinct characteristics and implications of HRD-associated PDAC. We analyzed 16 PDAC patient-derived tissues, categorized by their homologous recombination deficiency (HRD) scores, and performed high-plex immunofluorescence analysis to define 20 cell phenotypes, thereby generating an in-situ PDAC tumor-immune landscape. Spatial phenotypic-transcriptomic profiling guided by regions-of-interest (ROIs) identified a crucial regulatory mechanism through localized tumor-adjacent macrophages, potentially in an HRD-dependent manner. Cellular neighborhood (CN) analysis further demonstrated the existence of macrophage-associated high-ordered cellular functional units in spatial contexts. Using our multi-omics spatial profiling strategy, we uncovered a dynamic macrophage-mediated regulatory axis linking HRD status with SIGLEC10 and CD52. These findings demonstrate the potential of targeting CD52 in combination with PARPi as a therapeutic intervention for PDAC.

Antinuclear antibody (ANA) status predicts immune-related adverse events in liver cancer patients undergoing anti-PD-1 therapy.

Immune-related adverse events (irAEs) clinically resemble autoimmune diseases, indicating autoantibodies could be potential biomarkers for the prediction of irAEs. This study aimed to assess the predictive value of peripheral blood antinuclear antibody (ANA) status for irAEs, considering the time and severity of irAEs, as well as treatment outcome in liver cancer patients administered anti-PD-1 therapy. Ninety-three patients with advanced primary liver cancer administered anti-PD-1 treatment were analyzed retrospectively. They were divided into the ANA positive (ANA+, titer ≥ 1:100) and negative (ANA-, titer < 1:100) groups. Development of irAEs, progression-free survival (PFS), and overall survival (OS) were assessed. Compared with ANA- patients, ANA+ cases were more prone to develop irAEs (43.3% vs. 19.2%, P = 0.031). With the increase of ANA titers, the frequency of irAEs increased. The time interval between anti-PD-1 therapy and the onset of irAEs was significantly shorter in ANA+ patients compared with the ANA- group (median, 1.7 months vs. 5.0 months, P = 0.022). Moreover, the time between anti-PD-1 therapy and irAE occurrence decreased with increasing ANA titer. In addition, PFS and OS were decreased in ANA+ patients compared with the ANA- group (median PFS, 2.8 months vs. 4.2 months, P = 0.043; median OS, 21.1 months vs. not reached, P = 0.041). IrAEs occur at higher frequency in ANA+ liver cancer patients undergoing anti-PD-1 therapy. ANA titer could help predict irAE development and treatment outcome in these patients.

Deep dissection of stemness-related hierarchies in hepatocellular carcinoma.

BACKGROUND: Increasing evidence suggests that hepatocellular carcinoma (HCC) stem cells (LCSCs) play an essential part in HCC recurrence, metastasis, and chemotherapy and radiotherapy resistance. Multiple studies have demonstrated that stemness-related genes facilitate the progression of tumors. However, the mechanism by which stemness-related genes contribute to HCC is not well understood. Here, we aim to construct a stemness-related score (SRscores) model for deeper analysis of stemness-related genes, assisting with the prognosis and individualized treatment of HCC patients.Further, we found that the gene LPCAT1 was highly expressed in tumor tissues by immunohistochemistry, and sphere-forming assay revealed that knockdown of LPCAT1 inhibited the sphere-forming ability of hepatocellular carcinoma cells. METHODS: We used the TCGA-LIHC dataset to screen stemness-related genes of HCC from the MSigDB database. Prognosis, tumor microenvironment, immunological checkpoints, tumor immune dysfunction, rejection, treatment sensitivity, and putative biological pathways were examined. Random forest created the SRscores model. The anti-PD-1/anti-CTLA4 immunotherapy, tumor mutational burden, medication sensitivity, and cancer stem cell index were compared between the high- and low-risk score groups. We also examined risk scores for different cell types using single-cell RNA sequencing data and correlated transcription factor activity in cancer stem cells with SRscores genes. Finally, we tested core marker expression and biological functions. RESULTS: Patients can be divided into two subtypes (Cluster1 and Cluster2) based on the TCGA-LIHC dataset's identification of 11 stemness-related genes. Additionally, a SRscores was developed based on subtypes. Cluster2 and the group with the lowest SRscores had superior survival and immunotherapy response than Cluster1 and the group with the highest SRscores. The group with a high SRscores was significantly more enriched in classical tumor pathways than the group with a low SRscores. Multiple transcription factors and SRscores genes are correlated. The core gene LPCAT1 is highly expressed in rat liver cancer tissues and promotes tumor cell sphere formation. CONCLUSION: A SRscores model can be utilized to predict the prognosis of HCC patients as well as their response to immunotherapy.

Integration of glucose and cardiolipin anabolism confers radiation resistance of HCC.

BACKGROUND AND AIMS: Poor response to ionizing radiation (IR) due to resistance remains a clinical challenge. Altered metabolism represents a defining characteristic of nearly all types of cancers. However, how radioresistance is linked to metabolic reprogramming remains elusive in hepatocellular carcinoma (HCC). APPROACH AND RESULTS: Baseline radiation responsiveness of different HCC cells were identified and cells with acquired radio-resistance were generated. By performing proteomics, metabolomics, metabolic flux, and other functional studies, we depicted a metabolic phenotype that mediates radiation resistance in HCC, whereby increased glucose flux leads to glucose addiction in radioresistant HCC cells and a corresponding increase in glycerophospholipids biosynthesis to enhance the levels of cardiolipin. Accumulation of cardiolipin dampens the effectiveness of IR by inhibiting cytochrome c release to initiate apoptosis. Mechanistically, mammalian target of rapamycin complex 1 (mTORC1) signaling-mediated translational control of hypoxia inducible factor-1α (HIF-1α) and sterol regulatory element-binding protein-1 (SREBP1) remodels such metabolic cascade. Targeting mTORC1 or glucose to cardiolipin synthesis, in combination with IR, strongly diminishes tumor burden. Finally, activation of glucose metabolism predicts poor response to radiotherapy in cancer patients. CONCLUSIONS: We demonstrate a link between radiation resistance and metabolic integration and suggest that metabolically dismantling the radioresistant features of tumors may provide potential combination approaches for radiotherapy in HCC.

Olaparib enhances radiation-induced systemic anti-tumor effects via activating STING-chemokine signaling in hepatocellular carcinoma.

Although Poly (ADP-ribose) polymerase (PARP) inhibitors have been clinically approved for cancers with BRCA mutations and are known to augment radiotherapy responses, their roles in promoting the abscopal effect and mediating immunotherapy in BRCA-proficient hepatocellular carcinoma (HCC) remain underexplored. Our study elucidates that olaparib enhances the radio-sensitivity of HCC cells. Coadministration of olaparib and irradiation induces significant DNA damage by generating double-strand breaks (DSBs), as revealed both in vitro and in immune-deficient mice. These DSBs activate the cGAS-STING pathway, initiating immunogenic cell death in abscopal tumors. STING activation reprograms the immune microenvironment in the abscopal tumors, triggering the release of type I interferon and chemokines, including CXCL9, CXCL10, CXCL11, and CCL5. This in turn amplifies T cell priming against tumor neoantigens, leading to an influx of activated, neoantigen-specific CD8+ T-cells within the abscopal tumors. Furthermore, olaparib attenuated the immune exhaustion induced by radiation and enhances the responsiveness of HCC to immune checkpoint inhibitors. Collectively, our data advocate that a synergistic regimen of PARP inhibitors and radiotherapy can strategically reinforce both local (primary) and systemic (abscopal) tumor control, bolstering HCC susceptibility to immunotherapy.

METTL3-Mediated STING Upregulation and Activation in Kupffer Cells Contribute to Radiation-Induced Liver Disease via Pyroptosis.

PURPOSE: Radiation therapy is a vital adjuvant treatment for liver cancer, although the challenge of radiation-induced liver diseases (RILDs) limits its implementation. Kupffer cells (KCs) are a crucial cell population of the hepatic immune system, and their biologic function can be modulated by multiple epigenetic RNA modifications, including N6-methyladenosine (m6A) methylation. However, the mechanism for m6A methylation in KC-induced inflammatory responses in RILD remains unclear. The present study investigated the function of m6A modification in KCs contributing to RILD. METHODS AND MATERIALS: Methylated RNA-immunoprecipitation sequencing and RNA transcriptome sequencing were used to explore the m6A methylation profile of primary KCs isolated from mice after irradiation with 3 × 8 Gy. Western blotting and quantitative real-time PCR were used to evaluate gene expression. DNA pulldown and chromatin immunoprecipitation assays were performed to verify target gene binding and identify binding sites. RESULTS: Methylated RNA-immunoprecipitation sequencing revealed significantly increased m6A modification levels in human KCs after irradiation, suggesting the potential role of upregulated m6A in RILD. In addition, the study results corroborated that methyltransferase-like 3 (METTL3) acts as a main modulator to promote the methylation and gene expression of TEAD1, leading to STING-NLRP3 signaling activation. Importantly, it was shown that IGF2BP2 functions as an m6A "reader" to recognize methylated TEAD1 mRNA and promote its stability. METTL3/TEAD1 knockdown abolished the activation of STING-NLRP3 signaling, protected against RILD, and suppressed inflammatory cytokines and hepatocyte apoptosis. Moreover, clinical human normal liver tissue samples collected after irradiation showed increased expression of STING and interleukin-1ß in KCs compared with nonirradiated samples. Notably, STING pharmacologic inhibition alleviated irradiation-induced liver injury in mice, indicating its potential therapeutic role in RILD. CONCLUSIONS: The results of our study reveal that TEAD1-STING-NLRP3 signaling activation contributes to RILD via METTL3-dependent m6A modification.

Radiotherapy enhances efficacy of PD-1 inhibitors in advanced hepatocellular carcinoma: A propensity-matched real-world study.

BACKGROUND: To address the need for immunotherapy in patients with advanced primary hepatocellular carcinoma (HCC), combination with radiotherapy (RT) has emerged as a promising strategy. In preclinical studies, irradiated tumors released tumor antigens to synergistically increase the antitumor effect of immunotherapy. Hence, we investigated whether RT enhances the efficacy of anti-programmed death receptor-1 (PD-1) inhibitors in advanced HCC in real-world practice. METHODS: Between August 2018 and June 2021, 172 patients with advanced primary HCC were enrolled in the tertiary center (Zhongshan Hospital of Fudan University); 95 were treated with a combination of RT and the inhibitor of PD-1 (RT-PD1 cohort), and 77 were administered anti-PD-1 therapy (PD1 cohort). The first cycle of PD-1 inhibitors was administered within 60 days or concurrently with RT. Propensity score matching for bias reduction was used to evaluate the clinical outcomes. RESULTS: Among 71 propensity-matched pairs, median progression-free survival was 5.7 months in the RT-PD1 cohort vs. 2.9 months in the PD1 cohort ( P  <0.001). Median overall survival was 20.9 months in the RT-PD1 cohort vs. 11.2 months in the PD1 cohort ( P  = 0.018). Compared with patients in the PD1 cohort, patients in the RT-PD1 cohort had significantly higher objective response rates (40.8%, 29/71 vs. 19.7%, 14/71, P  = 0.006) and disease control rates (62.0%, 44/71 vs. 31.0%, 22/71, P  <0.001). The incidences of toxic effects were not significantly different between the two cohorts. CONCLUSIONS: RT plus anti-PD-1 therapy is well tolerated. RT enhances the efficacy of anti-PD-1 therapy in patients with advanced primary HCC by improving survival outcomes without increased toxic effects.

Deciphering the immune modulation through deep transcriptomic profiling and therapeutic implications of DNA damage repair pattern in hepatocellular carcinoma.

AIMS: DNA damage repair (DDR) plays a pivotal role in hepatocellular carcinoma (HCC), driving oncogenesis, progression, and therapeutic response. However, the mechanisms of DDR mediated immune cells and immuno-modulatory pathways in HCC are yet ill-defined. METHODS: Our study introduces an innovative deep machine learning framework for precise DDR assessment, utilizing single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data. Single-cell RNA sequencing data were obtained and in total 85,628 cells of primary or post-immunotherapy cases were analyzed. Large-scale HCC datasets, including 1027 patients in house together with public datasets, were used for 101 machine-learning models and a novel DDR feature was derived at single-cell resolution (DDRscore). Druggable targets were predicted using the reverse phase protein array (RPPA) proteomic profiling of 169 HCC patients and RNA-seq data from 22 liver cancer cell lines. RESULTS: Our investigation reveals a dynamic interplay of DDR with natural killer cells and B cells in the primary HCC microenvironment, shaping a tumor-promoting immune milieu through metabolic programming. Analysis of HCC post-immunotherapy demonstrates elevated DDR levels that induces epithelial-mesenchymal transition and fibroblast-like transformation, reshaping the fibrotic tumor microenvironment. Conversely, attenuated DDR promotes antigen cross-presentation by dendritic cells and CD8+ T cells, modulating the inflammatory tumor microenvironment. Regulatory network analysis identifies the CXCL10-CXCR3 axis as a key determinant of immunotherapeutic response in low DDR HCC, potentially regulated by transcription factors GATA3, REL, and TBX21. Using machine learning techniques by combining bulk RNA-seq data in house together with public datasets, we introduce DDRscore, a robust consensus DDR scoring system to predict overall survival and resistance to PD-1 therapy in HCC patients. Finally, we identify BRAF as a potential therapeutic target for high DDRscore patients. CONCLUSION: Our comprehensive findings advance our understanding of DDR and the tumor microenvironment in HCC, providing insights into immune regulatory mechanisms mediated via DDR pathways.

Mechanism of immune activation mediated by genomic instability and its implication in radiotherapy combined with immune checkpoint inhibitors.

Various genetic and epigenetic changes associated with genomic instability (GI), including DNA damage repair defects, chromosomal instability, and mitochondrial GI, contribute to development and progression of cancer. These alterations not only result in DNA leakage into the cytoplasm, either directly or through micronuclei, but also trigger downstream inflammatory signals, such as the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Apart from directly inducing DNA damage to eliminate cancer cells, radiotherapy (RT) exerts its antitumor effects through intracellular DNA damage sensing mechanisms, leading to the activation of downstream inflammatory signaling pathways. This not only enables local tumor control but also reshapes the immune microenvironment, triggering systemic immune responses. The combination of RT and immunotherapy has emerged as a promising approach to increase the probability of abscopal effects, where distant tumors respond to treatment due to the systemic immunomodulatory effects. This review emphasizes the importance of GI in cancer biology and elucidates the mechanisms by which RT induces GI remodeling of the immune microenvironment. By elucidating the mechanisms of GI and RT-induced immune responses, we aim to emphasize the crucial importance of this approach in modern oncology. Understanding the impact of GI on tumor biological behavior and therapeutic response, as well as the possibility of activating systemic anti-tumor immunity through RT, will pave the way for the development of new treatment strategies and improve prognosis for patients.

RECQL4 Inhibits Radiation-Induced Tumor Immune Awakening via Suppressing the cGAS-STING Pathway in Hepatocellular Carcinoma.

Many patients with hepatocellular carcinoma (HCC) respond poorly to radiotherapy despite remarkable advances in treatment. A deeper insight into the mechanism of sensitivity of HCC to this therapy is urgently required. It is demonstrated that RECQL4 is upregulated in the malignant cells of patients with HCC. Elevated RECQL4 levels reduce the sensitivity of HCC to radiotherapy by repairing radiation-induced double-stranded DNA (dsDNA) fragments. Mechanistically, the inhibitory effect of RECQL4 on radiotherapy is due to the reduced recruitment of dendritic cells and CD8+ T cells in the tumor microenvironment (TME). RECQL4 disrupts the radiation-induced transformation of the TME into a tumoricidal niche by inhibiting the cGAS-STING pathway in dendritic cells. Knocking out STING in dendritic cells can block the impact of RECQL4 on HCC radiosensitivity. Notably, high RECQL4 expressions in HCC is significantly associated with poor prognosis in multiple independent cohorts. In conclusion, this study highlights how HCC-derived RECQL4 disrupts cGAS-STING pathway activation in dendritic cells through DNA repair, thus reducing the radiosensitivity of HCC. These findings provide new perspectives on the clinical treatment of HCC.

Efficacy and Toxicity of Moderately Hypofractionated Radiation Therapy with Helical TomoTherapy Versus Conventional Radiation Therapy in Patients with Unresectable Stage III Non-Small Cell Lung Cancer Receiving Concurrent Chemotherapy: A Multicenter, Randomized Phase 3 Trial.

PURPOSE: The standard treatment schedule for unresectable stage III non-small cell lung cancer (NSCLC) is chemotherapy with concurrent radiation therapy (60 Gy delivered in 30 fractions), although moderately hypofractionated radiation therapy (Hypo-RT) has also been considered as an alternative strategy. This study aimed to compare the efficacy and toxicity of moderately Hypo-RT with helical TomoTherapy versus conventionally fractionated radiation therapy (Con-RT) in patients with unresectable stage III NSCLC receiving concurrent chemotherapy. METHODS AND MATERIALS: In this randomized, multicenter, nonblinded phase 3 clinical trial, eligible patients were randomised at a 1:1 ratio to either the Hypo-RT group (60 Gy in 20 fractions) or Con-RT group (60 Gy in 30 fractions). All patients received 2 cycles of concurrent platinum-based chemotherapy plus 2 cycles of consolidation therapy. The primary endpoint was 3-year overall survival (OS) in the intention-to-treat population. The secondary endpoints were progression-free survival and treatment-related adverse events. RESULTS: A total of 146 patients were enrolled from July 27, 2018, to November 1, 2021. The median follow-up was 46 months. The 3-year OS rates in the Hypo-RT and Con-RT groups were 58.4% and 38.4%, respectively (P = .02). The median OS from randomisation was 41 months in the Hypo-RT group and 30 months in the Con-RT group (hazard ratio, 0.61; 95% confidence interval, 0.40-0.94; P = .02). There was no significant difference in the rates of grade ≥2 treatment-related adverse events between the 2 groups. CONCLUSIONS: Moderately Hypo-RT using helical TomoTherapy may improve OS in patients with unresectable stage III NSCLC, while maintaining toxicity rates.

Melanoma Derived Exosomes Amplify Radiotherapy Induced Abscopal Effect via IRF7/I-IFN Axis in Macrophages.

Radiotherapy (RT) can induce tumor regression outside the irradiation field, known as the abscopal effect. However, the detailed underlying mechanisms remain largely unknown. A tumor-bearing mouse model is successfully constructed by inducing both subcutaneous tumors and lung metastases. Single-cell RNA sequencing, immunofluorescence, and flow cytometry are performed to explore the regulation of tumor microenvironment (TME) by RT. A series of in vitro assays, including luciferase reporter, RNA Pulldown, and fluorescent in situ hybridization (FISH) assays, are performed to evaluate the detailed mechanism of the abscopal effect. In addition, in vivo assays are performed to investigate combination therapy strategies for enhancing the abscopal effect. The results showed that RT significantly inhibited localized tumor and lung metastasis progression and improved the TME. Mechanistically, RT promoted the release of tumor-derived exosomes carrying circPIK3R3, which is taken up by macrophages. circPIK3R3 promoted Type I interferon (I-IFN) secretion and M1 polarization via the miR-872-3p/IRF7 axis. Secreted I-IFN activated the JAK/STAT signaling pathway in CD8+ T cells, and promoted IFN-γ and GZMB secretion. Together, the study shows that tumor-derived exosomes promote I-IFN secretion via the circPIK3R3/miR-872-3p/IRF7 axis in macrophages and enhance the anti-tumor immune response of CD8+ T cells.

Single High-Dose Irradiation-Induced iRhom2 Upregulation Promotes Macrophage Antitumor Activity Through cGAS/STING Signaling.

PURPOSE: The clinical application of stereotactic body radiation therapy (SBRT) allows a high dose of radiation to be safely delivered to extracranial targets within the body; however, a high dose per fraction (hypofractionation) has opened the radiation oncology field to new questions on a variety of dose-fractionation schedules, especially the immunomodulatory effects of radiation therapy, which can change after various dose-fractionation schedules. We investigated the immunomodulatory effects of different fractionation schedules. METHODS AND MATERIALS: We established a subcutaneous tumor model in wild-type C57BL/6J mice and STING (stimulator of interferon genes)-deficient mice. We then compared the tumor control efficacy of 3 different fractionation schedules: 2 Gy × 8, 4.5 Gy × 3, and 10 Gy × 1, which are similar biologically effective doses. RESULTS: We found the fractionation schedule of 10 Gy × 1 had a significantly higher antitumor effect, suggesting that a single high dose induced enhanced antitumor immunity compared with conventional fractionation (2 Gy × 8) and moderate hypofractionation (4.5 Gy × 3). However, in STING-deficient mice, differential tumor control was not observed among the 3 dose-fractionation schedules, suggesting that cGAS (cyclic GMP-AMP synthase)/STING signaling is involved in the antitumor immune effects of single high-dose schedules. Mechanistically, we found that conventional fractionation induced apoptosis; by comparison, a single high dose was more attuned to induced necroptosis, leading to the release of intracellular irradiation-induced double-stranded DNA (dsDNA) due to the loss of plasma membrane integrity, which then activated the dsDNA sensing signaling cGAS/STING in the recruited macrophage. Furthermore, iRhom2, a member of the conserved family of inhibitory rhomboid-like pseudoproteases, was upregulated in infiltrated macrophages in the single high-dose irradiation microenvironment. Therefore, iRhom2 positively regulates STING and directly promotes tumor necrosis factor α secretion. This exacerbates necroptosis of irradiated tumor cells, leading to continuous dsDNA release and enhancement of cGAS/STING signaling antitumor immunity in a positive feedback loop. CONCLUSIONS: iRhom2 amplifies antitumor signaling in a positive feedback loop mediated by cGAS/STING signaling and tumor necrosis factor-driven necroptosis after single high-dose radiation.

The miR-34a-5p-c-MYC-CHK1/CHK2 Axis Counteracts Cancer Stem Cell-Like Properties and Enhances Radiosensitivity in Hepatocellular Cancer Through Repression of the DNA Damage Response.

Radiotherapy has become an increasingly widespread modality for treating hepatocellular cancer (HCC); however, the development of radioresistance significantly limits its effectiveness and invariably leads to tumor recurrence. Cancer stem cell (CSC) theory offers a potential explanation for tumor relapse and radioresistance, but the underlying mechanism remains unknown. Herein we investigate the role of miRNA in molecular regulation of stemness and radioresistance in HCC. Two HCC radiation-resistant cell lines (Huh7-RR and SMMC-7721-RR) were established by selecting the radioresistant subpopulation from HCC cells via clonogenic survival assays. MiRNA Sequencing was used to identify potential radiosensitivity involved miRNA in HCC-RR cells. Xenograft tumor mouse model was established for in vivo study. CSC properties were assessed using sphere formation assay and side population (SP) cells analysis. We found that miR-34a-5p was significantly downregulated in HCC-RR cells. Overexpression of miR-34a-5p counteracts CSC properties and enhances radiosensitivity in HCC. Mechanistic investigation revealed that c-MYC is the direct target of miR-34a-5p. Overexpression of miR-34a-5p reversed c-MYC-induced radioresistance. Moreover, we found that the specific molecular mechanism was that c-MYC activated CHK1 and CHK2, which are two key DNA damage checkpoint kinases, and facilitated the DNA damage response to radiation. Repression of the miR-34a-5p-cMYC-CHK1/CHK2 axis contributes to the acquisition of radioresistance in HCC cells. In summary, the miR-34a-5p-c-MYC-CHK1/CHK2 axis counteracts cancer stem cell-like properties and enhances radiosensitivity in hepatocellular cancer through repression of the DNA damage response.

STING-Dependent Sensing of Self-DNA Driving Pyroptosis Contributes to Radiation-Induced Lung Injury.

PURPOSE: Radiation therapy (RT) is indispensable for managing thoracic carcinomas. However, its application is limited by radiation-induced lung injury (RILI), one of the most common and fatal complications of thoracic RT. Nonetheless, the exact molecular mechanisms of RILI remain poorly understood. METHODS AND MATERIALS: To elucidate the underlying mechanisms, various knockout mouse strains were subjected to 16 Gy whole-thoracic RT. RILI was assessed by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, histology, western blot, immunohistochemistry, and computed tomography examination. To perform further mechanistic studies on the signaling cascade during the RILI process, pulldown, chromatin immunoprecipitation assay, and rescue assays were conducted. RESULTS: We found that the cGAS-STING pathway was significantly upregulated after irradiation exposure in both the mouse models and clinical lung tissues. Knocking down either cGAS or STING led to attenuated inflammation and fibrosis in mouse lung tissues. NLRP3 is hardwired to the upstream DNA-sensing cGAS-STING pathway to trigger of the inflammasome and amplification of the inflammatory response. STING deficiency suppressed the expressions of the NLRP3 inflammasome and pyroptosis-pertinent components containing IL-1ß, IL-18, GSDMD-N, and cleaved caspase-1. Mechanistically, interferon regulatory factor 3, the essential transcription factor downstream of cGAS-STING, promoted the pyroptosis by transcriptionally activating NLRP3. Moreover, we found that RT triggered the release of self-dsDNA in the bronchoalveolar space, which is essential for the activation of cGAS-STING and the downstream NLRP3-mediated pyroptosis. Of note, Pulmozyme, an old drug for the management of cystic fibrosis, was revealed to have the potential to mitigate RILI by degrading extracellular dsDNA and then inhibiting the cGAS-STING-NLRP3 signaling pathway. CONCLUSIONS: These results delineated the crucial function of cGAS-STING as a key mediator of RILI and described a mechanism of pyroptosis linking cGAS-STING activation with the amplification of initial RILI. These findings indicate that the dsDNA-cGAS-STING-NLRP3 axis might be potentially amenable to therapeutic targeting for RILI.

An improved 3D KiU-Net for segmentation of liver tumor.

It is a challenging task to accurately segment liver tumors from Computed Tomography (CT) images. The widely used U-Net and its variants generally suffer from the issue to accurately segment the detailed edges of small tumors, because the progressive down sampling operations in the encoder module will gradually increase the receptive fields. These enlarged receptive filed have limited ability to learn the information about tiny structures. KiU-Net is a newly proposed dual-branch model that can effectively perform image segmentation for small targets. However, the 3D version of KiU-Net has high computational complexity, which limits its application. In this work, an improved 3D KiU-Net (named TKiU-NeXt) is proposed for liver tumor segmentation from CT images. In TKiU-NeXt, a Transformer-based Kite-Net (TK-Net) branch is proposed to build the over-complete architecture to learn more detailed features for small structures, and an extended 3D version of UNeXt is developed to replace the original U-Net branch, which can effectively reduce computational complexity but still with superior segmentation performance. Moreover, a Mutual Guided Fusion Block (MGFB) is designed to effectively learn more features from two branches and then fuse the complementary features for image segmentation. The experimental results on two public CT datasets and a private dataset demonstrate that the proposed TKiU-NeXt outperforms all the compared algorithms, and it also has less computational complexity. It suggests the effectiveness and efficiency of TKiU-NeXt.

Early complete tumor response as a survival predictor in hepatocellular carcinoma patients receiving stereotactic body radiation therapy.

Background and Purpose: To evaluate the different response patterns after Stereotactic Body Radiation Therapy (SBRT) and their predictive value in local control and progression of hepatocellular carcinoma (HCC). Materials and methods: Seventy-two HCC patients who were treated with SBRT during 2015-2020 were included in this retrospective study. The assessment was made using MRI, CT, and PET-CT. Local and systemic responses were determined according to modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria during follow up. Patients were categorized as early responders (complete response during 6 months after radiotherapy) or non-early responders (the rest of the patients). Prognostic factors were determined using multivariate logistic models. Results: The median follow-up was 24.0 months (range, 7.7-74.5 months). We found that 84.7%（61/72) of patients achieved a complete response. Early responses occurred in 45 patients (45/72, 62.5%), and they had 1-, 2-, and 5- year intrahepatic outfield-free survival (OutFFS) rates of 86.2%, 80.3%, and 76.3% vs. 55.3%, 44.7%, and 33.5% in non-early responses patients, whereas the 1-, 2-, and 5- year distant metastasis-free survival (DMFS) were 95.5%, 84.5% and 79.5% and 74.1%, 56.2% and 56.2%, respectively. The 1-, 2-, and 5-year overall survival (OS) were 97.7%, 92.1%, 79.1%, and 85.2%, 53.8%, and 40.3%, respectively. Multivariate analysis revealed that early tumor response was an independent predictor of OutFFS, DMFS, and OS. Conclusions: Early complete tumor response within 6 months after radiotherapy predicted better intrahepatic outfield-free survival, distant metastasis-free survival, and overall survival outcomes. Confirmation is warranted for early response on SBRT to guide decision making.

Maternal embryonic leucine zipper kinase in tumor cells and tumor microenvironment: An emerging player and promising therapeutic opportunity.

Maternal embryonic leucine zipper kinase (MELK) is a member of the AMPK (AMP-activated protein kinase) protein family, which is widely and highly expressed in multiple cancer types. Through direct and indirect interactions with other targets, it mediates various cascades of signal transduction processes and plays an important role in regulating tumor cell survival, growth, invasion and migration and other biological functions. Interestingly, MELK also plays an important role in the regulation of the tumor microenvironment, which can not only predict the responsiveness of immunotherapy, but also affect the function of immune cells to regulate tumor progression. In addition, more and more small molecule inhibitors have been developed for the target of MELK, which exert important anti-tumor effects and have achieved excellent results in a number of clinical trials. In this review, we outline the structural features, molecular biological functions, potential regulatory mechanisms and important roles of MELK in tumors and tumor microenvironment, as well as substances targeting MELK. Although many molecular mechanisms of MELK in the process of tumor regulation are still unknown, it is worth affirming that MELK is a potential tumor molecular therapeutic target, and its unique superiority and important role provide clues and confidence for subsequent basic research and scientific transformation.

Comparison of long-term outcomes of stereotactic body radiotherapy (SBRT) via Helical tomotherapy for early-stage lung cancer with or without pathological proof.

BACKGROUND: Stereotactic body radio therapy (SBRT) has emerged as a standard treatment option for nonsurgical candidates with early-stage non-small cell lung cancer (NSCLC). Pathological proof is sometimes difficult to obtain in patients with solitary pulmonary nodules (SPNs). We aimed to compare the clinical outcomes of stereotactic body radiotherapy via helical tomotherapy (HT-SBRT) for early-stage lung cancer patients with or without a pathological diagnosis. METHODS: Between June 2011 and December 2016, we treated 119 lung cancer patients with HT-SBRT, including 55 with a clinical diagnosis and 64 with a pathological diagnosis. Survival outcomes, including local control (LC), progression-free survival (PFS), cancer-specific survival (CSS), and overall survival (OS), were compared between two cohorts with and without a pathological diagnosis. RESULTS: The median follow-up for the whole group was 69 months. Patients with a clinical diagnosis were significantly older (p = 0.002). No significant differences were observed between the clinical and pathological diagnosis cohorts in terms of the long-term outcome, with 5-year LC, PFS, CSS, and OS of 87% versus 83% (p = 0.58), 48% versus 45% (p = 0.82), 87% versus 84% (p = 0.65), and 60% versus 63% (p = 0.79), respectively. Recurrence patterns and toxicity were also similar. CONCLUSIONS: Empiric SBRT appears to be a safe and effective treatment option in a multidisciplinary setting when patients with SPNs highly suggestive of malignancy are unable/refuse to obtain a definitive pathological diagnosis.