ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

A Potential "Anti-Warburg Effect" in Circulating Tumor Cell-mediated Metastatic Progression?

Metabolic reprogramming is a defining hallmark of cancer metastasis, warranting thorough exploration. The tumor-promoting function of the "Warburg Effect", marked by escalated glycolysis and restrained mitochondrial activity, is widely acknowledged. Yet, the functional significance of mitochondria-mediated oxidative phosphorylation (OXPHOS) during metastasis remains controversial. Circulating tumor cells (CTCs) are considered metastatic precursors that detach from primary or secondary sites and harbor the potential to seed distant metastases through hematogenous dissemination. A comprehensive metabolic characterization of CTCs faces formidable obstacles, including the isolation of these rare cells from billions of blood cells, coupled with the complexities of ex vivo-culturing of CTC lines or the establishment of CTC-derived xenograft models (CDX). This review summarized the role of the "Warburg Effect" in both tumorigenesis and CTC-mediated metastasis. Intriguingly, bioinformatic analysis of single-CTC transcriptomic studies unveils a potential OXPHOS dominance over Glycolysis signature genes across several important cancer types. From these observations, we postulate a potential "Anti-Warburg Effect" (AWE) in CTCs-a metabolic shift bridging primary tumors and metastases. The observed AWE could be clinically important as they are significantly correlated with therapeutic response in melanoma and prostate patients. Thus, unraveling dynamic metabolic regulations within CTC populations might reveal an additional layer of regulatory complexities of cancer metastasis, providing an avenue for innovative anti-metastasis therapies.

FLOT2 promotes nasopharyngeal carcinoma progression through suppression of TGF-ß pathway via facilitating CD109 expression.

In nasopharyngeal carcinoma (NPC), the TGF-ß/Smad pathway genes are altered with inactive TGF-ß signal, but the mechanisms remain unclear. RNA-sequencing results showed that FLOT2 negatively regulated the TGF-ß signaling pathway via up-regulating CD109 expression. qRT-PCR, western blot, ChIP, and dual-luciferase assays were used to identify whether STAT3 is the activating transcription factor of CD109. Co-IP immunofluorescence staining assays were used to demonstrate the connection between FLOT2 and STAT3. In vitro and in vivo experiments were used to detect whether CD109 could rescue the functional changes of NPC cells resulting from FLOT2 alteration. IHC and Spearman correlation coefficients were used to assay the correlation between FLOT2 and CD109 expression in NPC tissues. Our results found that FLOT2 promotes the development of NPC by inhibiting TGF-ß signaling pathway via stimulating the expression of CD109 by stabilizing STAT3, which provides a potential therapeutic strategy for NPC treatment.

Deep Multi-Magnification Similarity Learning for Histopathological Image Classification.

Precise classification of histopathological images is crucial to computer-aided diagnosis in clinical practice. Magnification-based learning networks have attracted considerable attention for their ability to improve performance in histopathological classification. However, the fusion of pyramids of histopathological images at different magnifications is an under-explored area. In this paper, we proposed a novel deep multi-magnification similarity learning (DSML) approach that can be useful for the interpretation of multi-magnification learning framework and easy to visualize feature representation from low-dimension (e.g., cell-level) to high-dimension (e.g., tissue-level), which has overcome the difficulty of understanding cross-magnification information propagation. It uses a similarity cross entropy loss function designation to simultaneously learn the similarity of the information among cross-magnifications. In order to verify the effectiveness of DMSL, experiments with different network backbones and different magnification combinations were designed, and its ability to interpret was also investigated through visualization. Our experiments were performed on two different histopathological datasets: a clinical nasopharyngeal carcinoma and a public breast cancer BCSS2021 dataset. The results show that our method achieved outstanding performance in classification with a higher value of area under curve, accuracy, and F-score than other comparable methods. Moreover, the reasons behind multi-magnification effectiveness were discussed.

Nasopharyngeal carcinoma ecology theory: cancer as multidimensional spatiotemporal "unity of ecology and evolution" pathological ecosystem.

Nasopharyngeal carcinoma (NPC) is a particular entity of head neck cancer that is generally regarded as a genetic disease with diverse intertumor and intratumor heterogeneity. This perspective review mainly outlines the up-to-date knowledge of cancer ecology and NPC progression, and presents a number of conceptual stepping-stones. At the beginning, I explicitly advocate that the nature of NPC (cancer) is not a genetic disease but an ecological disease: a multidimensional spatiotemporal "unity of ecology and evolution" pathological ecosystem. The hallmarks of cancer is proposed to act as ecological factors of population fitness. Subsequently, NPC cells are described as invasive species and its metastasis as a multidirectional ecological dispersal. The foundational ecological principles include intraspecific relationship (e.g. communication) and interspecific relationship (e.g. competition, predation, parasitism and mutualism) are interpreted to understand NPC progression. "Mulberry-fish-ponds" model can well illustrate the dynamic reciprocity of cancer ecosystem. Tumor-host interface is the ecological transition zone of cancer, and tumor buddings should be recognized as ecological islands separated from the mainland. It should be noted that tumor-host interface has a significantly molecular and functional edge effect because of its curvature and irregularity. Selection driving factors and ecological therapy including hyperthermia for NPC patients, and future perspectives in such field as "ecological pathology", "multidimensional tumoriecology" are also discussed. I advance that "nothing in cancer evolution or ecology makes sense except in the light of the other". The cancer ecology tree is constructed to comprehensively point out the future research direction. Taken together, the establishment of NPC ecology theory and cancer ecology tree might provide a novel conceptual framework and paradigm for our understanding of cancer complex causal process and potential preventive and therapeutic applications for patients.

ESRG is critical to maintain the cell survival and self-renewal/pluripotency of hPSCs by collaborating with MCM2 to suppress p53 pathway.

The mechanisms of self-renewal and pluripotency maintenance of human pluripotent stem cells (hPSCs) have not been fully elucidated, especially for the role of those poorly characterized long noncoding RNAs (lncRNAs). ESRG is a lncRNA highly expressed in hPSCs, and its functional roles are being extensively explored in the field. Here, we identified that the transcription of ESRG can be directly regulated by OCT4, a key self-renewal factor in hPSCs. Knockdown of ESRG induces hPSC differentiation, cell cycle arrest, and apoptosis. ESRG binds to MCM2, a replication-licensing factor, to sustain its steady-state level and nuclear location, safeguarding error-free DNA replication. Further study showed that ESRG knockdown leads to MCM2 abnormalities, resulting in DNA damage and activation of the p53 pathway, ultimately impairs hPSC self-renewal and pluripotency, and induces cell apoptosis. In summary, our study suggests that ESRG, as a novel target of OCT4, plays an essential role in maintaining the cell survival and self-renewal/pluripotency of hPSCs in collaboration with MCM2 to suppress p53 signaling. These findings provide critical insights into the mechanisms underlying the maintenance of self-renewal and pluripotency in hPSCs by lncRNAs.

Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

TBL1X and Flot2 form a positive feedback loop to promote metastasis in nasopharyngeal carcinoma.

Metastasis is the main cause of death in patients with nasopharyngeal carcinoma (NPC). The molecular mechanisms underlying the metastasis of NPC remain to be elucidated. TBL1X has been shown abnormally expressed in diverse cancers. However, the role and mechanism of TBL1X in NPC remain unknown. Here, we showed TBL1X expression was significantly higher in metastatic NPC tissues compared to non-metastatic tissues and significantly correlated with TNM stage and metastasis of NPC patients. In addition, NPC patients with high TBL1X expression had a poor prognosis. TBL1X interacted with TCF4 to trans-activate Flot2 expression. TBL1X promoted NPC cell migration and invasion in vitro and in vivo through Flot2. Moreover, Flot2 increased the expression of TBL1X by upregulating c-myc, which was identified to be a positively regulatory transcription factor of TBL1X. TBL1X could restore the functional changes of NPC cells resulting from Flot2 alteration. TBL1X and Flot2 were positively correlated in NPC. Patients with high expression of both TBL1X and Flot2 possessed poorer overall survival (OS) and disease-free survival (DFS) compared to patients with high expression of any single one of the two proteins. Our findings demonstrate that TBL1X and Flot2 positively regulate each other to promote NPC metastasis, which provides novel potential molecular targets for NPC treatment.

Kindlin-2 Acts as a Key Mediator of Lung Fibroblast Activation and Pulmonary Fibrosis Progression.

Pulmonary fibrosis is a progressive and fatal lung disease characterized by activation of lung fibroblasts and excessive deposition of collagen matrix. We show here that the concentrations of kindlin-2 and its binding partner PYCR1, a key enzyme for proline synthesis, are significantly increased in the lung tissues of human patients with pulmonary fibrosis. Treatment of human lung fibroblasts with TGF-ß1 markedly increased the expression of kindlin-2 and PYCR1, resulting in increased kindlin-2 mitochondrial translocation, formation of the kindlin-2-PYCR1 complex, and proline synthesis. The concentrations of the kindlin-2-PYCR1 complex and proline synthesis were markedly reduced in response to pirfenidone or nintedanib, two clinically approved therapeutic drugs for pulmonary fibrosis. Furthermore, depletion of kindlin-2 alone was sufficient to suppress TGF-ß1-induced increases of PYCR1 expression, proline synthesis, and fibroblast activation. Finally, using a bleomycin mouse model of pulmonary fibrosis, we show that ablation of kindlin-2 effectively reduced the concentrations of PYCR1, proline, and collagen matrix and alleviate the progression of pulmonary fibrosis in vivo. Our results suggest that kindlin-2 is a key promoter of lung fibroblast activation, collagen matrix synthesis, and pulmonary fibrosis, underscoring the therapeutic potential of targeting the kindlin-2 signaling pathway for control of this deadly lung disease.

Distinct Disease Severity Between Children and Older Adults With Coronavirus Disease 2019 (COVID-19): Impacts of ACE2 Expression, Distribution, and Lung Progenitor Cells.

BACKGROUND: Children and older adults with coronavirus disease 2019 (COVID-19) display a distinct spectrum of disease severity yet the risk factors aren't well understood. We sought to examine the expression pattern of angiotensin-converting enzyme 2 (ACE2), the cell-entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the role of lung progenitor cells in children and older patients. METHODS: We retrospectively analyzed clinical features in a cohort of 299 patients with COVID-19. The expression and distribution of ACE2 and lung progenitor cells were systematically examined using a combination of public single-cell RNA-seq data sets, lung biopsies, and ex vivo infection of lung tissues with SARS-CoV-2 pseudovirus in children and older adults. We also followed up patients who had recovered from COVID-19. RESULTS: Compared with children, older patients (>50 years.) were more likely to develop into serious pneumonia with reduced lymphocytes and aberrant inflammatory response (P = .001). The expression level of ACE2 and lung progenitor cell markers were generally decreased in older patients. Notably, ACE2 positive cells were mainly distributed in the alveolar region, including SFTPC positive cells, but rarely in airway regions in the older adults (P < .01). The follow-up of discharged patients revealed a prolonged recovery from pneumonia in the older (P < .025). CONCLUSIONS: Compared to children, ACE2 positive cells are generally decreased in older adults and mainly presented in the lower pulmonary tract. The lung progenitor cells are also decreased. These risk factors may impact disease severity and recovery from pneumonia caused by SARS-Cov-2 infection in older patients.

Modeling COVID-19 with Human Pluripotent Stem Cell-Derived Cells Reveals Synergistic Effects of Anti-inflammatory Macrophages with ACE2 Inhibition Against SARS-CoV-2.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19) from mild to severe stages including fatality, with pro-inflammatory macrophages as one of the main mediators of lung hyper-inflammation. Therefore, there is an urgent need to better understand the interactions among SARS-CoV-2 permissive cells, macrophage, and the SARS-CoV-2 virus, thereby offering important insights into new therapeutic strategies. Here, we used directed differentiation of human pluripotent stem cells (hPSCs) to establish a lung and macrophage co-culture system and model the host-pathogen interaction and immune response caused by SARS-CoV-2 infection. Among the hPSC-derived lung cells, alveolar type II and ciliated cells are the major cell populations expressing the viral receptor ACE2 and co-effector TMPRSS2, and both were highly permissive to viral infection. We found that alternatively polarized macrophages (M2) and classically polarized macrophages (M1) had similar inhibitory effects on SARS-CoV-2 infection. However, only M1 macrophages significantly up-regulated inflammatory factors including IL-6 and IL-18, inhibiting growth and enhancing apoptosis of lung cells. Inhibiting viral entry into target cells using an ACE2 blocking antibody enhanced the activity of M2 macrophages, resulting in nearly complete clearance of virus and protection of lung cells. These results suggest a potential therapeutic strategy, in that by blocking viral entrance to target cells while boosting anti-inflammatory action of macrophages at an early stage of infection, M2 macrophages can eliminate SARS-CoV-2, while sparing lung cells and suppressing the dysfunctional hyper-inflammatory response mediated by M1 macrophages.

Computer-Aided Pathologic Diagnosis of Nasopharyngeal Carcinoma Based on Deep Learning.

The pathologic diagnosis of nasopharyngeal carcinoma (NPC) by different pathologists is often inefficient and inconsistent. We have therefore introduced a deep learning algorithm into this process and compared the performance of the model with that of three pathologists with different levels of experience to demonstrate its clinical value. In this retrospective study, a total of 1970 whole slide images of 731 cases were collected and divided into training, validation, and testing sets. Inception-v3, which is a state-of-the-art convolutional neural network, was trained to classify images into three categories: chronic nasopharyngeal inflammation, lymphoid hyperplasia, and NPC. The mean area under the curve (AUC) of the deep learning model is 0.936 based on the testing set, and its AUCs for the three image categories are 0.905, 0.972, and 0.930, respectively. In the comparison with the three pathologists, the model outperforms the junior and intermediate pathologists, and has only a slightly lower performance than the senior pathologist when considered in terms of accuracy, specificity, sensitivity, AUC, and consistency. To our knowledge, this is the first study about the application of deep learning to NPC pathologic diagnosis. In clinical practice, the deep learning model can potentially assist pathologists by providing a second opinion on their NPC diagnoses.

Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB inductions and conversions induced by therapeutic proton beams.

Purpose: This study evaluated the DNA double strand breaks (DSBs) induced by indirect actions and its misrepairs to estimate the relative biological effectiveness (RBE) of proton beams.Materials and methods: From experimental data, DSB induction was evaluated in cells irradiated by 62 MeV proton beams in the presence of dimethylsulphoxide (DMSO) and under hypoxic conditions. The DNA damage yields for calculating the RBE were estimated using Monte Carlo Damage Simulation (MCDS) software. The repair outcomes (correct repairs, mutations and DSB conversions) were estimated using Monte Carlo Excision Repair (MCER) simulations.Results: The values for RBE of 62 MeV protons (LET = 1.051 keV/µm) for DSB induction and enzymatic DSB under aerobic condition (21% O2) was 1.02 and 0.94, respectively, as comparing to 60Co γ-rays (LET = 2.4 keV/µm). DMSO mitigated the inference of indirect action and reduced DSB induction to a greater extent when damaged by protons rather than γ-rays, resulting in a decreased RBE of 0.86. DMSO also efficiently prevented enzymatic DSB yields triggered by proton irradiation and reduced the RBE to 0.83. However, hypoxia (2% O2) produced a similar level of DSB induction with respect to the protons and γ-rays, with a comparable RBE of 1.02.Conclusions: The RBE values of proton beams estimated from DSB induction and enzymatic DSB decreased by 16% and 12%, respectively, in the presence of DMSO. Our findings indicate that the overall effects of DSB induction and enzymatic DSB could intensify the tumor killing, while alleviate normal tissue damage when indirect actions are effectively interrupted.

Matrix metalloproteinase 2 contributes to aggressive phenotype, epithelial-mesenchymal transition and poor outcome in nasopharyngeal carcinoma.

BACKGROUND: Though matrix metalloproteinase 2 (MMP-2) involvement in tumor aggressiveness and invasion is well-known, its prognostic impacts still remain largely controversial. Furthermore, the correlations between MMP-2 and epithelial-mesenchymal transition (EMT) have not been directly established in nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: The purpose of this study was to investigate MMP-2 expression in NPC. Tissue microarrays from 144 patients with NPC and 45 non-cancerous pharynx tissues were analyzed for MMP-2 expression by immunohistochemistry. MMP-2 expression in relation to clinicopathological characteristics and EMT were assessed in NPC. Tumor-invasive potential affected by exogenous expression of MMP-2 in NPC cells was also detected in vitro. RESULTS: Compared to normal nasopharyngeal epithelium, high expression of tumoral MMP-2 was detected in 47.9% of NPC samples. Significant association was found between MMP-2 expression and various aggressive features including T classification, M classification and tumor stage (P<0.05). Of note, high expression of MMP-2 was prominently observed at tumor invasive front, neoplastic spindle cells migrating into the stroma and vessel invasion. Importantly, high MMP-2 expression predicted worse survival in patients with stage III-IV (P=0.039). Overexpression of MMP-2 could decrease cell-cell adhesion, promote tumor invasion and EMT including downregulation of E-cadherin and upregulation of N-cadherin, Fibronectin and Slug of NPC cells. CONCLUSION: Our findings demonstrate that MMP-2 expression contributes to tumor aggressiveness and poor prognosis, and induces the occurrence of EMT in NPC.

FoxM1 Promotes Cell Proliferation, Invasion, and Stem Cell Properties in Nasopharyngeal Carcinoma.

Background: The self-renewal and tumourigenicity of FoxM1 in nasopharyngeal carcinoma (NPC) remain largely unknown. In this study, we attempt to investigate the self-renewal and tumourigenicity of FoxM1 and its clinical significance in nasopharyngeal carcinoma (NPC). Methods: Several assays including cell counting Kit-8 (CCK-8) assays, colony formation, flow cytometry, immunofluorescence, tumor spheres, and mice model were used to detect the biological function of FoxM1 in NPC. The association between FoxM1 and clinical pathological features, and stem cell markers was analyzed using immunohistochemistry. Results: High expression of FoxM1 was prominently present in the T4 stages, cancer cells migrating into the stroma and vasculature. Overexpression of FoxM1 enhanced tumor proliferation, cell cycle progression, migration and stress fibers formation in vitro. In NPC tissues, FoxM1 correlated significantly with stem cells-related clinical pathological features including late clinical stage, tumor recurrence and distant metastasis. Meanwhile, FoxM1 linked closely with the expression levels of stem cell markers including Nanog, Sox2, and OCT4 in tumor samples, and also promoted the expression of these stemness-related genes in vitro. Moreover, FoxM1 conferred the self-renewal properties of cancer cells by increasing side populations (SP) cells and formed larger and more tumor spheres. Importantly, FoxM1 enhanced the ability of tumourigenicity of NPC cell lines in mice xenograft. Conclusions: We demonstrate that FoxM1 greatly induces cancer progression and cancer stem cell (CSC) features in NPC.

Klf4 reduces stemness phenotype, triggers mesenchymal-epithelial transition (MET)-like molecular changes, and prevents tumor progression in nasopharygeal carcinoma.

The reprogramming factor Krüppel-like factor 4 (Klf4), one of the Yamanaka's reprogramming factors, plays an essential role in reprogramming somatic cells into induced pluripotent stem cells (iPSCs). Klf4 is dysregulated and displays divergent functions in multiple malignancies, but the biological roles of Klf4 in nasopharyngeal carcinoma (NPC) remain unknown. The present study revealed that Klf4 downregulation in a cohort of human NPC biopsies is significantly associated with invasive and metastatic phenotypes of NPC. Our results showed exogenous expression of Klf4 significantly inhibited cell proliferation, decreased stemness, triggered mesenchymal-epithelial transition (MET)-like molecular changes, and suppressed migration and invasion of NPC cells, whereas depletion of endogeneous Klf4 by RNAi reversed the aforementioned biological behaviors and characheristics. Klf4 silencing significantly enhanced the metastatic ability of NPC cells in vivo. In addition, CHIP assay confirmed that E-cadherin is a transcriptional target of Klf4 in NPC cells. Additional studies demonstrated that Klf4-induced MET-like cellular marker alterations, and reduced motility and invasion of NPC cells were mediated by E-cadherin. This study revealed the clinical correlation between Klf4 expression and epithelial-mesenchymal transition (EMT) biomarkers (including its target gene E-cadherin) in a cohort of NPC biopsies. Taken together, our findings suggest, for what we believe is the first time, that Klf4 functions as a tumor suppressor in NPC to decrease stemness phenotype, inhibit EMT and prevent tumor progression, suggesting that restoring Klf4 function may provide therapeutic benefits in NPC.

5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells.

Relapse and metastasis of nasopharyngeal carcinoma (NPC) are presumably attributed to cancer stem cells (CSCs). In recent years, chimeric antigen receptor (CAR)-modified immune effector cells have been shown to have impressive antitumour efficacy. In this study, we aimed to identify appropriate tumour-associated antigens predominantly expressed on NPC stem cells (NPCSCs) and determine their suitability for CAR-engineered cytokine-induced killer (CIK) cell therapy against NPC. By investigating the expression patterns of potential target antigens (ROR1, 5T4 and CAIX) in NPC, we found that the oncofetal antigen 5T4 was predominately expressed in NPC cell lines and tissues but absent in non-cancerous nasopharyngeal tissues. Moreover, significantly enhanced expression of 5T4 in NPC spheroids revealed its relationship with putative NPCSCs. Hence, we designed a CAR construct (5T4-28Z) specific for 5T4 and generated CAR-transduced CIK cells. Our results showed that the artificial CAR was efficiently expressed on the surface of CIK cells and that no native phenotypes were altered by the gene transduction. Functional assays revealed that 5T4-28Z-CIK cells possessed both CAR-mediated and CAR-independent anti-NPC activity and were capable of efficiently attacking NPC cells, especially NPCSC-like cells in vitro, suggesting that they might serve as an attractive tool for developing efficient therapies against NPC.

LATS2 as a poor prognostic marker regulates non-small cell lung cancer invasion by modulating MMPs expression.

Large tumor suppressor 2 (LATS2) plays significant roles in tumorigenesis and cancer progression. This study was aimed to analyze the correlation between LATS2 expression and clinicopathologic features and its prognostic significance in non-small cell lung cancer (NSCLC). LATS2 expression was examined in 73 NSCLC clinical specimens and 22 normal lung tissues using immunohistochemistry. Low levels of LATS2 protein were inversely associated with the T classification (P=0.001), N classification (P=0.005) and clinical stage (P=0.001) in NSCLC patients. Patients with lower LATS2 expression had a significantly shorter overall survival than patients with high LATS2 expression. Multivariate analysis suggested that low expression of LATS2 was an independent prognostic indicator (P=0.002) for the survival of patients with NSCLC. Furthermore, overexpression of LATS2 resulted in mobility inhibition in NSCLC cell lines A549 and H1299, and reduced protein level of matrix metalloproteinase-2 (MMP-2) and MMP-9. On the contrary, LATS2 siRNA treatment enhanced cell mobility and increased MMP-2 and MMP-9 protein expression level. In conclusion, low expression of LATS2 is a potential unfavorable prognostic factor and promoted cell invasion and migration in NSCLC.

Hes1 triggers epithelial-mesenchymal transition (EMT)-like cellular marker alterations and promotes invasion and metastasis of nasopharyngeal carcinoma by activating the PTEN/AKT pathway.

Overexpression of the transcriptional factor Hes1 (hairy and enhancer of split-1) has been observed in numerous cancers, but the precise roles of Hes1 in epithelial-mesenchymal transition (EMT), cancer invasion and metastasis remain unknown. Our current study firstly revealed that Hes1 upregulation in a cohort of human nasopharyngeal carcinoma (NPC) biopsies is significantly associated with the EMT, invasive and metastatic phenotypes of cancer. In the present study, we found that Hes1 overexpression triggered EMT-like cellular marker alterations of NPC cells, whereas knockdown of Hes1 through shRNA reversed the EMT-like phenotypes, as strongly supported by Hes1-mediated EMT in NPC clinical specimens described above. Gain-of-function and loss-of-function experiments demonstrated that Hes1 promoted the migration and invasion of NPC cells in vitro. In addition, exogenous expression of Hes1 significantly enhanced the metastatic ability of NPC cells in vivo. Chromatin immunoprecipitation (ChIP) assays showed that Hes1 inhibited PTEN expression in NPC cells through binding to PTEN promoter region. Increased Hes1 expression and decreased PTEN expression were also observed in a cohort of NPC biopsies. Additional studies demonstrated that Hes1-induced EMT-like molecular changes and increased motility and invasion of NPC cells were mediated by PTEN. Taken together, our results suggest, for what we believe is the first time, that Hes1 plays an important role in the invasion and metastasis of NPC through inhibiting PTEN expression to trigger EMT-like phenotypes.