Tamoxifen has been commonly used in endocrinotherapy for estrogen receptor (ER)-positive breast cancer. In recent years, the immunomodulatory effects of high-dose tamoxifen have been discovered. The immunosuppressive tumor microenvironment (TME), however, remains a major obstacle to the efficacy of radiotherapy. Whether high-dose tamoxifen can reprogram TME to synergistically enhance radiation efficacy is still ambiguous. Here, we have found high-dose tamoxifen could dramatically enhance radiation-induced antitumor effects without significant side effects in immunocompetent mice. Flow cytometry and multiplex immunofluorescence experiments revealed that radiation combined with tamoxifen resulted in significant enrichment of effector CD8+ T cells and M1 tumor associated macrophages (TAMs). Depletion of TAMs and CD8+ T cells impaired the synergistic antitumor effects mediated by combination treatment of radiation and tamoxifen. Furthermore, spatial proximity analysis demonstrated a dramatically reduced nearest-neighbor distance between CD8+ T cells and M1-like TAMs in the combination group, indicating enhanced cellular interaction within the TME. In vitro experiments demonstrated that tamoxifen directly acted on TAMs, rather than CD8+ T cells, to promote M1 polarization and subsequently enhanced the activation and effector function of CD8+ T cells, regardless of ER expression in tumor cells and macrophages. Mechanistically, RNA-sequencing and experimental validation uncovered that tamoxifen and tumor cell-derived TNF-α and IL-1β after irradiation synergistically activated the JNK/c-JUN pathway and promoted M1 polarization of TAMs. In conclusion, our study revealed the immunomodulatory effects of high-dose tamoxifen in the context of radiation and provided preclinical evidence for combination therapy of high-dose tamoxifen and radiotherapy in both ER-positive and ER-negative cancers.

Radiotherapy is a critical modality in cancer treatment, not only to eradicate cancer cells but also to trigger antitumor immunity. IL-21, an immunomodulatory cytokine with potential in cancer therapy, has unexplored synergy with radiotherapy. Our study, leveraging human cancer databases and tissue microarrays, identified a positive correlation between IL-21 and radiotherapy outcomes, particularly in tumor microenvironment (TME) activation. In mouse tumor models, IL-21 combined with radiation significantly enhanced the TME, boosting CD8+ T cell activation and function, reducing tumor burden, and extending survival. Single-cell transcriptome sequencing revealed that the combination of IL-21 and radiation increased the cytotoxicity of effector and memory CD8+T cells and prevented their exhaustion. These effects were further validated in humanized mice, where IL-21 combined with radiation reduced A549 tumor growth and enhanced CD8+ T cell function. Post-neoadjuvant radiotherapy samples from patients with esophageal cancer showed a positive correlation between IL-21 levels and CD8+ T cell infiltration. Our findings suggest that IL-21 is a promising adjuvant to radiotherapy, potentially improving treatment efficacy through TME enhancement. This study provides a foundation for future clinical exploration of IL-21 for enhancing radiotherapy.

Emerging evidence implicates the significant influence of gut microbiome and its metabolic byproducts in the non-small cell lung cancer (NSCLC). Here, we identify distinct disparity in the gut microbiota composition between patients with NSCLC and healthy controls, with Bifidobacterium animalis being markedly decreased in NSCLC. B. animalis suppresses tumor progression in two NSCLC mouse models and NSCLC cell lines. Integrative metabolomic analysis identifies indole-3 acetic acid (IAA) as the pivotal metabolite of B. animalis, with significant anti-NSCLC properties. Mechanistically, B. animalis and its derived IAA activate aryl hydrocarbon receptor (AHR) in lung to suppress METTL3 and the m6A methylation of STAT3. Moreover, B. animalis and IAA diminish polarization of M2 macrophage and enhance CD8+ T cell functions by suppressing interleukin-6 (IL-6). B. animalis and its derived IAA protect against NSCLC by modulating AHR/METTL3/STAT3 and bolstering antitumor immunity through gut-lung axis. B. animalis and IAA supplementation represent a promising prophylactic for NSCLC prevention.

Pancreatic cancer is the eighth leading cause of cancer-related deaths worldwide. Chemotherapy including gemcitabine, 5-fluorouracil, adriamycin and cisplatin, immunotherapy with immune checkpoint inhibitors and targeted therapy have been demonstrated to significantly improve prognosis of pancreatic cancer patients with advanced diseases. However, most patients developed drug resistance to these therapeutic agents, which leading to shortened patient survival. The detailed molecular mechanisms contributing to pancreatic cancer drug resistance remain largely unclear. The growing evidences have shown that noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), are involved in pancreatic cancer pathogenesis and development of drug resistance. In the present review, we systematically summarized the new insight on of various miRNAs, lncRNAs and circRNAs on drug resistance of pancreatic cancer. These results demonstrated that targeting the tumor-specific ncRNA may provide novel options for pancreatic cancer treatments.

Esophageal cancer is the eighth most common malignancy and the sixth leading cause of cancer-related deaths worldwide. Chemotherapy based on platinum drugs, 5-fluorouracil, adriamycin, paclitaxel, gemcitabine, and vinorelbine, as well as targeted treatment and immunotherapy with immune checkpoint inhibitors improved the prognosis in a portion of patients with advanced esophageal cancer. Unfortunately, a number of esophageal cancer patients develop drug resistance, resulting in poor outcomes. Multiple mechanisms contributing to drug resistance of esophageal cancer have been reported. Notably, non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), have been identified to play crucial roles in modulating esophageal cancer drug resistance. In the present review, we highlight the underlying mechanisms how miRNAs, lncRNAs, and circRNAs impact the drug resistance of esophageal cancer. Several miRNAs, lncRNAs, and circRNAs may have potential clinical implications as novel biomarkers and therapeutic targets for esophageal cancer.

Gastric cancer is the fourth most common malignancy and the third leading cause of cancer-related deaths worldwide. Advanced gastric cancer patients can notably benefit from chemotherapy including adriamycin, platinum drugs, 5-fluorouracil, vincristine, and paclitaxel as well as targeted therapy drugs. Nevertheless, primary drug resistance or acquisition drug resistance eventually lead to treatment failure and poor outcomes of the gastric cancer patients. The detailed mechanisms involved in gastric cancer drug resistance have been revealed. Interestingly, different noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), are critically involved in gastric cancer development. Multiple lines of evidences demonstrated that ncRNAs play a vital role in gastric cancer resistance to chemotherapy reagents and targeted therapy drugs. In this review, we systematically summarized the emerging role and detailed molecular mechanisms of ncRNAs impact drug resistance of gastric cancer. Additionally, we propose the potential clinical implications of ncRNAs as novel therapeutic targets and prognostic biomarkers for gastric cancer.

Background Colorectal cancer (CRC) is the third most prevalent cancer in the world and one of the most lethal human malignancies. Chemotherapy with 5-fluorouracil, platinum, hydroxycamptothecin, vincristine, methotrexate, irinotecan, paclitaxel and/or cetuximab has significantly improved the survival of CRC patients. However, most CRC patients eventually develop chemoresistance, resulting in a poor prognosis. The mechanisms involved in CRC chemoresistance are complex and, as yet, inconclusive. Noncoding RNAs (ncRNAs), such as small nucleolar RNAs (snoRNAs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), represent transcripts without protein-coding potential. Accumulating evidence indicates that multiple deregulated ncRNAs, including miRNAs and lncRNAs, play pivotal roles in the development of chemoresistance in CRC. This notion has potential clinical implications. Conclusions In this review, we highlight the emerging roles and the regulatory mechanisms by which miRNAs and lncRNAs affect CRC chemoresistance. Tumor-specific miRNAs and lncRNAs may serve as novel therapeutic targets and prognostic biomarkers for CRC.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the second most lethal human cancer. A portion of patients with advanced HCC can significantly benefit from treatments with sorafenib, adriamycin, 5-fluorouracil and platinum drugs. However, most HCC patients eventually develop drug resistance, resulting in a poor prognosis. The mechanisms involved in HCC drug resistance are complex and inconclusive. Human transcripts without protein-coding potential are known as noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), long noncoding RNAs (lncRNAs) and circular RNA (circRNA). Accumulated evidences demonstrate that several deregulated miRNAs and lncRNAs are important regulators in the development of HCC drug resistance which elucidates their potential clinical implications. In this review, we summarized the detailed mechanisms by which miRNAs and lncRNAs affect HCC drug resistance. Multiple tumor-specific miRNAs and lncRNAs may serve as novel therapeutic targets and prognostic biomarkers for HCC.

BackgroundBoth quantitative and qualitative aspects of plasma cell‐free DNA (plasma cfDNA, pcfDNA) have been well‐studied as potential biomarkers in non‐small cell lung cancer (NSCLC). Accumulating evidence has proven that saliva also has the potential for the detection and analysis of circulating free DNA (saliva cfDNA, scfDNA).MethodsIn the current study, we aimed to explore the potential application of scfDNA in NSCLC diagnostics and consistency of epidermal growth factor receptor (EGFR) mutation detection in paired pcfDNA and scfDNA using droplet digital PCR (ddPCR) and analyze the relationship between EGFR mutations and clinical treatment response.ResultsIn the quantitative cohort study, scfDNA concentration in NSCLC patients was no different from that in healthy donors, or in benign patients. ScfDNA concentration was significantly lower than pcfDNA concentration, yet they were not statistically significant in relevance (Spearman's rank correlation r = −0.123, P = 0.269). In the qualitative cohort study, the overall concordance rate of EGFR mutations between pcfDNA and scfDNA was 83.78% (31 of 37; k = 0.602; P < 0.001). EGFR mutation detection in paired pcfDNA and scfDNA was significantly correlated with the clinical treatment response (Spearman's rank correlation r = 0.664, P = 0.002).ConclusionsOur results demonstrated that saliva might not be the idea material for a cfDNA quantitative test, and scfDNA concentration is not applicable for NSCLC diagnostics. Conversely, scfDNA was capable of acting as the supplement for EGFR mutations due to the coincidence rate of EGFR mutation detection between scfDNA and pcfDNA.

The Wilms' tumor-associated gene WT1 encodes a tumor suppressor gene, which is implicated in renal differentiation and development of adult urogenital system. Wilms' tumor 1-associating protein (WTAP) is initially identified as a nuclear protein that specifically interacts with WT1 in both in vitro and in vivo assays. WTAP is ubiquitously expressed in different tissues and various growth periods, and its expression is involved in cell cycle, RNA splicing and stabilization, N6-methyladenosine RNA modification, cell proliferation, and apoptosis as well as embryonic development. In the present review, we aimed to summarize the functions of WTAP in various physiological and pathological processes, in particular with regard to the current knowledge about the role of WTAP in tumorigenesis of different cancers.

Genotype-directed targeted therapy has become one of the standard treatment options for non-small cell lung cancer (NSCLC). There have been numerous limitations associated with mutation analysis of tissue samples. Consequently, mutational profile analysis of circulating cell-free DNA (cfDNA) by highly sensitive droplet digital PCR (ddPCR) assay has been developed. Possibly due to differences in cfDNA concentrations, previous studies have shown numerous discrepancies in mutation detection consistency between tissue and cfDNA. In order to rigorously analyze the amount of cfDNA needed, we constructed 72 athymic nude mice xenografted with NCI-H1975 (harboring a EGFR T790M mutation) or NCI-H460 (harboring a KRAS Q61H mutation) human NSCLC. We thoroughly investigated the relationship between plasma cfDNA using Q-PCR targeting human long interspersed nuclear element-1 ( LINE-1 ) retrotransposon and the mouse ACTB gene, and the accuracy of mutation detection by ddPCR at different times post-graft. Our results show that the concentration and fragmentation of human (tumor) derived cfDNA (hctDNA) were positively correlated with tumor weight, but not with mouse-derived cfDNA (mcfDNA). Quantification of cfDNA by Q-PCR depends on the amplified target length. Mutation copies in plasma of per milliliter were positively linked to tumor weight, hctDNA level and hctDNA/mcfDNA ratio, respectively. Furthermore, tumor weight, hctDNA level and ratio of hctDNA/mcfDNA were significantly higher in cfDNA mutation-positive mice than in negative mice. Also, our data indicate that when plasma hctDNA level and hctDNA/mcfDNA ratio reach a certain level in xenografted mice, plasma cfDNA mutation can be detected. In summary, the present study suggests that determination of ctDNA levels may be essential for reliable mutation detection by analysis of cfDNA.

BACKGROUND Cell-free DNA (cfDNA) is an important marker for cancer diagnosis and monitoring. Extraction of cfDNA from serum is indispensable for its quantification, and the DNA isolation procedure can significantly influence the quantification and qualification of DNA. RESULTS A cfDNA-extraction-free (EF) method and a SuperGreen quantitative PCR assay for determining total cfDNA in serum were developed. The cfDNA from the EF method did not affect the specificity and sensitivity of quantitative polymerase chain reaction (qPCR) assay. It was found that the median amount of spike-in cfDNA quantified by different isolation methods varied from 3.01 to 83.81 ng mL(-1). The efficiency of DNA extraction by the EF method was significantly better than that in the case of other cfDNA extraction kits. The serum samples from the patients with nonsmall cell lung cancer (NSCLC) contained a higher concentration of cfDNA than that in healthy people, and cfDNA showed a better prognostic ability for diagnosing NSCLC. CONCLUSION The cfDNA-EF method gives a more reliable, easy, and low-cost estimate of total cell-free DNA quantity in serum. (c) 2018 Society of Chemical Industry

Hypoxia promotes tumor invasion and metastasis via multiple mechanisms, including epithelial-mesenchymal transition (EMT). Twist, an EMT regulator, has been disclosed to associate with invasion and metastasis as well as poor prognosis of many malignancies. However, it remains undefined whether Twist is involved in invasion and metastasis of hypoxic non-small cell lung cancer (NSCLC). In this study, protein levels of Twist, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin and vimentin) were examined by immunohistochemistry in 76 lung cancer tissues from NSCLC patients. Expression of Twist and its correlation with HIF-1α, E-cadherin, and vimentin were analyzed. Small interfering RNA (siRNA) against Twist was used to knockdown Twist expression in hypoxic NSCLC cells, A549 and NCI-H460. Cellular invasion and protein levels of Twist, E-cadherin, and vimentin were evaluated by matrigel invasion assay and Western blot, respectively. Our results showed that in clinical samples, there was a significant association between Twist expression and differentiation degree, lymph node metastasis, and TNM stage. Correlation analysis demonstrated that expression of Twist was negatively correlated with E-cadherin expression, but positively associated with HIF-1α and vimentin expression. In cultured NSCLC cells, Twist messenger RNA (mRNA) and protein levels were upregulated under hypoxia, while knockdown of Twist suppressed potentiated invasion and expression of mesenchymal marker vimentin induced by hypoxia. Protein level of increased epithelial marker E-cadherin was shown along with Twist downregulation. These findings suggest that Twist promoting hypoxic invasion and metastasis of NSCLC may be associated with altered expression of EMT markers. Inhibition of Twist may be of therapeutic significance.

Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy without efficient chemotherapeutic agents for it. In our current study, we demonstrated the cytotoxicity effects of a newly patented compound temozolomide–perillyl alcohol (TMZ-POH) on NPC in vitro and in vivo, and the possible mechanisms involved. Human NPC cell lines CNE1, CNE2, HNE2, and SUME-α were treated with control (DMSO), TMZ, POH, TMZ plus POH, and TMZ-POH. Our data indicated that TMZ-POH could inhibit NPC cell proliferation, cause G2/M arrest and DNA damage. TMZ-POH triggered apoptosis in NPC cells via significant activation of caspase-3 and poly(ADP-ribose) polymerase (PARP). Importantly, TMZ-POH-induced cell death was found to be associated with (i) the loss of inner mitochondrial membrane potential (ΔΨm) and release of mitochondrial Cytochrome c, (ii) the increase in ROS generation, and (iii) the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinases (JNK) signaling pathway. The generation of ROS in response to TMZ-POH seems to play a crucial role in the cell death process since the blockage of ROS production using the antioxidant N-acetyl-L-cysteine or catalase reversed the TMZ-POH-induced JNK activation, DNA damage, and cancer cell apoptosis. These results provide the rationale for further research and preclinical investigation of the antitumor effect of TMZ-POH against human NPC.

Mediator 19 (Med19) is a component of the mediator complex which is a coactivator for DNA-binding factors that activate transcription via RNA polymerase II. Accumulating evidence has shown that Med19 plays important roles in cancer cell proliferation and tumorigenesis. The involvement of Med19 in sensitivity to the chemotherapeutic agent cisplatin was here investigated. We employed RNA interference to reduce Med19 expression in human non-small cell lung cancer (NSCLC) cell lines and analyzed their phenotypic changes. The results showed that after Med19 siRNA transfection, expression of Med19 mRNA and protein was dramatically reduced (p<0.05). Meanwhile, impaired growth potential, arrested cell cycle at G0/G1 phase and enhanced sensitivity to cisplatin were exhibited. Apoptosis and caspase-3 activity were increased when cells were exposed to Med19 siRNA and/or cisplatin. The present findings suggest that Med19 facilitates tumorigenic properties of NSCLC cells and knockdown of Med19 may be a rational therapeutic tool for lung cancer cisplatin sensitization.

目的 探讨Snail、乏氧诱导因子-1a (HIF-1α)、E-钙黏素(E-cadherin)在非小细胞肺癌(NSCLC)中表达的临床意义及相关性.方法 采用免疫组织化学法检测62例NSCLC患者肺癌组织和20例癌旁组织Snail、HIF-1α、E-cadherin蛋白表达,并分析与临床病理因素的关系.结果 肺癌组织中Snail、HIF-1α及E-cadherin阳性表达率分别为64.5％、59.7％和54.8％,癌旁组织分别为15％、10％和100％.三种蛋白在肺癌组织及癌旁组织表达差异有统计学意义(P＜0.05).Snail和HIF-1α表达升高及E-cadherin降低与肺癌临床分期和淋巴结转移有关(P＜0.05).HIF-1α和Snail表达均与E-cadherin表达呈负相关性(P＜0.05),但HIF-1α和Snail表达未显示相关性(P＞0.05).结论 Snail和HIF-1α表达升高、E-cadherin表达降低与肺癌发生发展和转移有关,Snail、HIF-1α和E-cadherin检测有助于评估肺癌恶性程度及预后.

目的 探讨缺氧诱导因子1α(hypoxia inducible factor 1 alpha,HIF-1α)RNA干扰与内皮抑素(endostatin,ET)基因联合对人肺癌裸鼠移植瘤的生长抑制作用.方法 SPCA1/HIF-1α(-)人肺癌细胞的裸鼠,给予脂质体-内皮抑素基因转染,观察转然瘤组织7、14、21天的HIF-1α、内皮生长因子(vascular endothelial growth factor,VEGF)蛋白表达及微血管生成数量.结果 HIF-1α RNA干扰明显降低瘤组织内VEGF蛋白表达;HIF-1α RNA干扰、ET及联合治疗均显著抑制肿瘤微血管生成;至接种后21天,HIF-1α RNA干扰ET联合组肿瘤体积为(312.7±78.6)mm3,抑瘤率为64.3％,与对照组比(P＜0.01)显著高于HIF-1α RNA干扰组(39.8％)和单用内皮抑素组(43.1％).结论 HIF-1α RNA干扰与ET基因联合应用,显著抑制人肺癌裸鼠移植瘤生长.

OBJECTIVE:To investigate the different expressions of ER and ER gene status between primary and relapsed/metastatic lesions and their clinical significance.METHODS:ER and ER gene status of primary and relapse/metastatic breast cancer masked in 70 metastatic breast cancer patients were assessed by determination of methylation status by immunohistochemistry (IHC) and methylation specific polymerase chain reaction (MSP), respectively.RESULTS:Positive rate of ER in the primary breast cancers was 64.3%, and in the relapse/metastatic lesions was 41.4% (P < 0.05). There were six patients whose positive ER status was changed to negative, among them the ER gene status was changed from demethylation to hypermethylation in four cases. Another four patients with negative ER status changed to positive, and their ER gene hypermethylation changed to ER demethylation status.CONCLUSIONS:The discordance of ER expression status in primary and relapse/metastatic lesions of breast cancer might be related to DNA methylation status.

目的:观察乏氧状态下干扰锌指转录因子Snail对人肺腺癌SPCA1细胞侵袭的影响及其上皮-间质转化(epithelial mesenehymal transition,EMT)相关分子机制.方法:应用靶向人Snail基因的小干扰RNA (Snail siRNA)转染常氧(19％O2)肺癌细胞,48 h后将细胞置乏氧(0.5％O2)培养箱培养,乏氧24 h后采用Real-Time PCR检测细胞Snail mRNA表达,蛋白质印迹法检测Snail和EMT表型分子E-钙黏素蛋白表达,Transwell小室评价细胞侵袭能力.结果:与常氧细胞(设为1)相比,乏氧诱导肺癌SPCA1细胞Snail mRNA(5.31±0.73)和蛋白(4.82±0.67)表达均显著增加,P均＜0.05.与乏氧对照siRNA组(设为1)比较,LOXsiRNA转染后乏氧SPCA1细胞SnailmRNA和蛋白表达分别为0.26±0.08和0.28±0.03.将常氧细胞侵袭力和E-钙黏素表达设为1,则乏氧细胞侵袭力和E-钙黏素表达分别为1.85±0.13和0.45±0.04,与常氧细胞存在显著差异,P值均＜0.05.下调Snail表达后,乏氧细胞侵袭力和E-钙黏素表达分别为0.90±0.08和1.81±0.09,与乏氧对照siRNA组存在显著差异,P值均＜0.05.将乏氧对照siRNA细胞侵袭力和E-钙黏素表达设为1,则Snail siRNA组侵袭力和E-钙黏素分别为0.50±0.03和2.04±0.17,P值均＜0.05.结论:乏氧状态下干扰Snail降低肺癌细胞侵袭,其机制可能与增加E-钙黏素蛋白表达有关.

To determine the effect of the down-regulation of osteopontin (OPN) expression on the sensitivity of breast cancer cells to doxorubicin, a chemotherapeutic drug, OPN shRNA was used to transfect the OPN-positive breast cancer cell line MDA-MB-231. The expression of OPN mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell cycle distribution and apoptosis were analyzed by flow cytometry. Transfected MDA-MB-231 cells were treated with different concentrations of doxorubicin. Growth of MDA-MB-231 cells and IC50 of doxorubicin were determined by MTT assay. Expression of OPN mRNA and OPN protein of cells transfected with OPN shRNA was significantly decreased when compared to the negative controls (P<0.05). The number of MDA-MB-231 cells in the S phase was significantly increased, from 22.77 to 43.67%. Transfection increased apoptosis from 3.61 to 4.91%. Furthermore, the proliferation of MDA-MB-231 cells transfected with OPN shRNA was significantly decreased when compared to the negative control (transfected with empty vector). Sensitivity of MDA-MB-231 cells to doxorubicin was enhanced after treatment with OPN shRNA. The IC50 values of doxorubicin of the OPN-transfected group, the MDA-MB-231 cells and the negative transfected group were 0.13588, 0.83869 and 0.79652 µg/ml, respectively. Down-regulation of OPN significantly inhibits expression of OPN protein in MDA-MB-231 breast cancer cells, reduces cell proliferation and increases their sensitivity to doxorubicin.