Rationale: KRAS is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. Methods: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the KRAS G12S mutant allele or repress its transcription, respectively, with the goal of treating KRAS-driven cancers. Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. Conclusion: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic KRAS mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.

Alternative splicing (AS) has emerged as a key event in tumor development and microenvironment formation. However, comprehensive analysis of AS and its clinical significance in head and neck squamous cell carcinoma (HNSC) is urgently required. Methods: Genome-wide profiling of AS events using RNA-Seq data from The Cancer Genome Atlas (TCGA) program was performed in a cohort of 464 patients with HNSC. Cancer-associated AS events (CASEs) were identified between paired HNSC and adjacent normal tissues and evaluated in functional enrichment analysis. Splicing networks and prognostic models were constructed using bioinformatics tools. Unsupervised clustering of the CASEs identified was conducted and associations with clinical, molecular and immune features were analyzed. Results: We detected a total of 32,309 AS events and identified 473 CASEs in HNSC; among these, 91 were validated in an independent cohort (n = 15). Functional protein domains were frequently altered, especially by CASEs affecting cancer drivers, such as PCSK5. CASE parent genes were significantly enriched in pathways related to HNSC and the tumor immune microenvironment, such as the viral carcinogenesis (FDR < 0.001), Human Papillomavirus infection (FDR < 0.001), chemokine (FDR < 0.001) and T cell receptor (FDR < 0.001) signaling pathways. CASEs enriched in immune-related pathways were closely associated with immune cell infiltration and cytolytic activity. AS regulatory networks suggested a significant association between splicing factor (SF) expression and CASEs and might be regulated by SF methylation. Eighteen CASEs were identified as independent prognostic factors for overall and disease-free survival. Unsupervised clustering analysis revealed distinct correlations between AS-based clusters and prognosis, molecular characteristics and immune features. Immunogenic features and immune subgroups cooperatively depict the immune features of AS-based clusters. Conclusion: This comprehensive genome-wide analysis of the AS landscape in HNSC revealed novel AS events related to carcinogenesis and immune microenvironment, with implications for prognosis and therapeutic responses.

Objectives: Sorafenib is the only FDA-approved first-line target drug for HCC patients. However, sorafenib merely confers 3-5 months of survival benefit with less than 30% of HCC patients sensitive to sorafenib therapy. Thus, it's necessary to develop a sensitizer for hepatocellular carcinoma (HCC) to sorafenib. Methods: The principal component analysis, gene ontology, and KEGG analysis are utilized following RNA-sequencing. The mass spectrometry analysis following immunoprecipitation is performed to discover the phosphatase targets. Most importantly, both the cell line-derived xenograft (CDX) and the patient-derived xenograft (PDX) mouse model are used to determine the effect of 3-HAA on sorafenib-resistant HCC in vivo. Results: In nude mice carrying HCC xenograft, tumor growth is inhibited by sorafenib or 3-HAA alone. When used in combination, the treatment particularly prevents the xenograft from growing. Combined treatment also suppresses the growth of sorafenib-resistant (≥30mg/kg) PDXs. In a set of mechanistic experiments, we find enhanced AKT activation and decreased apoptotic cells in de novo and acquired sorafenib-resistant HCC cells and tissues. 3-HAA decreases AKT phosphorylation and increases the apoptosis of HCC in both cultured cells and mouse xenografts by upregulation of phosphatases PPP1R15A/DUSP6. PPP1R15A/PPP1α directly reduces Akt phosphorylation while DUSP6 decreases Akt activity through inhibiting PDK1. The AKT activator abolishes 3-HAA inhibition of HCC growth in vitro and in mice. Conclusion: This study demonstrates that 3-HAA sensitizes HCC cells to sorafenib by upregulation of phosphatases, suggesting it as a promising molecule for HCC therapy.

Circular RNA (circRNA) possesses great pre-clinical diagnostic and therapeutic potentials in multiple cancers. It has been reported playing roles in multiple malignant behaviors including proliferation, migration, metastasis and chemoresistance. However, the underlying correlation between circRNAs and cancer stem cells (CSCs) has not been reported yet. Methods: circZKSCAN1 level was detected in HCC tissue microarrays to clarify its prognostic values. Gain and loss function experiments were applied to investigate the role of circZKSCAN1 in HCC stemness. Bioinformatic analysis was used to predict the possible downstream RNA binding protein and further RNA immunoprecipitation sequencing was carried out to identify the RBP-regulated genes. Results: The absence of circZKSCAN1 endowed several malignant properties including cancer stemness and tightly correlated with worse overall and recurrence-free survival rate in HCC. Bioinformatics analysis and RNA immunoprecipitation-sequencing (RIP-seq) results revealed that circZKSCAN1 exerted its inhibitive role by competitively binding FMRP, therefore, block the binding between FMRP and β-catenin-binding protein-cell cycle and apoptosis regulator 1 (CCAR1) mRNA, and subsequently restrain the transcriptional activity of Wnt signaling. In addition, RNA-splicing protein Quaking 5 was found downregulated in HCC tissues and responsible for the reduction of circZKSCAN1. Conclusion: Collectively, this study revealed the mechanisms underlying the regulatory role of circZKSCAN1 in HCC CSCs and identified the newly discovered Qki5-circZKSCAN1-FMRP-CCAR1-Wnt signaling axis as a potentially important therapeutic target for HCC treatment.

Rationale: The present study reports the multifunctional anticancer activity against B16F10 melanoma cancer cells and the bioimaging ability of fluorescent nitrogen-phosphorous-doped carbon dots (NPCDs). Methods: The NPCDs were synthesized using a single-step, thermal treatment and were characterized by TEM, XPS, fluorescence and UV-Vis spectroscopy, and FTIR analysis. The anticancer efficacy of NPCDs was confirmed by using cell viability assay, morphological evaluation, fluorescent live-dead cell assay, mitochondrial potential assay, ROS production, RT-PCR, western-blot analysis, siRNA transfection, and cellular bioimaging ability. Results: The NPCDs inhibited the proliferation of B16F10 melanoma cancer cells after 24 h of treatment and induced apoptosis, as confirmed by the presence of fragmented nuclei, reduced mitochondrial membrane potential, and elevated levels of reactive oxygen species. The NPCDs treatment further elevated the levels of pro-apoptotic factors and down-regulated the level of Bcl2 (B-cell lymphoma 2) that weakened the mitochondrial membrane, and activated proteases such as caspases. Treatment with NPCDs also resulted in dose-dependent cell cycle arrest, as indicated by reduced cyclin-dependent kinase (CDK)-2, -4, and -6 protein levels and an enhanced level of p21. More importantly, the NPCDs induced the activation of autophagy by upregulating the protein expression levels of LC3-II and ATG-5 (autophagy-related-5) and by downregulating p62 level, validated by knockdown of ATG-5. Additionally, owing to their excellent luminescence property, these NPCDs were also applicable in cellular bioimaging, as evidenced by the microscopic fluorescence imaging of B16F10 melanoma cells. Conclusion: Based on these findings, we conclude that our newly synthesized NPCDs induced cell cycle arrest, autophagy, and apoptosis in B16F10 melanoma cells and presented good cellular bioimaging capability.

Rational: Intracellular bacterial survival is a major factor causing chronic or recurrent infection, leading to the failure of both host defense and/or antibiotic treatment. However, the elimination of intracellular bacteria is challenging as they are protected from antibiotics and host immune attack. Recent studies have indicated that iron helps macrophages against intracellular bacteria, contradictory to traditional "nutritional immunity", in which iron is considered a key nutrient for bacterial survival in host cells. However, how iron facilitates intracellular bacterial death has not been fully clarified. In this study, we found that ferroptotic stress can help macrophages suppress intracellular bacteria by reversing the importation of ferrous iron into bacterial vacuoles via ferroportin and thereby inducing in situ ferroptosis-like bacterial death. Methods: A macrophage model of bacterial invasion was established to monitor dynamic changes in ferroptotic hallmarks, including ferrous iron and lipid peroxidation. Ferroptosis inducers and inhibitors were added to the model to evaluate the relationship between ferroptotic stress and intracellular bacterial survival. We then determined the spatiotemporal distributions of ferroportin, ferrous iron, and lipid peroxidation in macrophages and intracellular bacteria. A bacterial infection mouse model was established to evaluate the therapeutic effects of drugs that regulate ferroptotic stress. Results: Ferrous iron and lipid peroxidation increased sharply in the early stage of bacterial infection in the macrophages, then decreased to normal levels in the late stage of infection. The addition of ferroptosis inducers (ras-selective lethal small molecule 3, sulfasalazine, and acetaminophen) in macrophages promoted intracellular bacterial suppression. Further studies revealed that ferrous iron could be delivered to the intracellular bacterial compartment via inward ferroportin transportation, where ferrous iron induced ferroptosis-like death of bacteria. In addition, ferroptotic stress declined to normal levels in the late stage of infection by regulating iron-related pathways in the macrophages. Importantly, we found that enhancing ferroptotic stress with a ferroptosis inducer (sulfasalazine) successfully suppressed bacteria in the mouse infection models. Conclusions: Our study suggests that the spatiotemporal response to ferroptosis stress is an efficient pathway for macrophage defense against bacterial invasion, and targeting ferroptosis may achieve therapeutic targets for infectious diseases challenged by intracellular pathogens.

Emerging evidence is revealing that microRNAs (miRNAs) play essential roles in mechanosensing for regulating osteogenesis. However, no mechanoresponsive miRNAs have been identified in human bone specimens. Methods: Bedridden and aged patients, hindlimb unloaded and aged mice, and Random Positioning Machine and primary aged osteoblasts were adopted to simulate mechanical unloading conditions at the human, animal and cellular levels, respectively. Treadmill exercise and Flexcell cyclic mechanical stretching were used to simulate mechanical loading in vivo and in vitro, respectively. Results: Here, we found increased miR-138-5p levels with a lower degree of bone formation in bone specimens from bedridden and aged patients. Loss- and gain-of-function studies showed that miR-138-5p directly targeted microtubule actin crosslinking factor 1 (MACF1) to inhibit osteoblast differentiation under different mechanical conditions. Regarding translational medicine, bone-targeted inhibition of miR-138-5p attenuated the decrease in the mechanical bone anabolic response in hindlimb unloaded mice. Moreover, bone-targeted inhibition of miR-138-5p sensitized the bone anabolic response to mechanical loading in both miR-138-5p transgenic mice and aged mice to promote bone formation. Conclusion: These data suggest that miR-138-5p as a mechanoresponsive miRNA accounts for the mechanosensitivity of the bone anabolic response and that inhibition of miR-138-5p in osteoblasts may be a novel bone anabolic sensitization strategy for ameliorating disuse or senile osteoporosis.

Rationale: Protein arginine methyltransferase 5 (PRMT5) is an oncogene that promotes tumor cell proliferation, invasion and metastasis. However, the underlying mechanisms by which PRMT5 contributes to the progression of cervical cancer and especially the tumor microenvironment remain poorly understood. Methods: PRMT5 expression level was analyzed by Q-PCR, western blot, immunohistochemistry, and TCGA database. The role of PRMT5 in tumor growth was observed by transplanted tumor models, and the function of T cells in tumor microenvironment and in vitro co-culture system was investigated through flow cytometry. The transcriptional regulation of PRMT5 was analyzed using luciferase reporter and chromatin immunoprecipitation (ChIP) assay. The therapeutic effect of PRMT5 inhibitor was evaluated in a cervical cancer cell line transplanted tumor model. Results: We observed that the mRNA and protein expression levels of PRMT5 were increased in cervical cancer tissues, and the high expression of PRMT5 was associated with poor outcomes in cervical cancer patients. The absence of PRMT5 significantly inhibited tumor growth in a cervical cancer transplanted tumor model, and importantly, PRMT5 absence in tumors led to increase the number and enhance the function of tumor infiltrating T cells. Mechanistically, PRMT5 enhanced the transcription of STAT1 through symmetric dimethylation of histone H3R2 and thus promoted PD-L1 expression in cervical cancer cells. Moreover, in an in vitro co-culture system, knockdown of PRMT5 in tumor cells could directly enhance the expression of IFN-γ, TNF-α and granzyme B in T cells. These results suggested that PRMT5 promoted the development of cervical cancer by the crosstalk between tumor cells and T cells. Furthermore, the PRMT5 inhibitor EPZ015666 treatment could suppress tumor growth in a cervical cancer transplanted tumor model. Conclusion: Our results clarify a new mechanism which PRMT5 knockdown in cervical cancer cells drives an antitumor function via reprogramming T cell-mediated response and regulating PD-L1 expression. Thus, our study highlights that PRMT5 may be a potential target for cervical cancer therapy.

Background: Platinum (II) (Pt(II))-based anticancer drugs dominate the chemotherapy field of ovarian cancer. However, the patient's quality of life has severely limited owing to dose-limiting toxicities and the advanced disease at the time of diagnosis. Multifunctional tumor-targeted nanosized ultrasound contrast agents (glutathione (GSH)-sensitive platinum (IV) (Pt(IV)) prodrug-loaded phase-transitional nanoparticles, Pt(IV) NP-cRGD) were developed for precise theranostics against ovarian cancer. Methods: Pt(IV) NP-cRGD were composed of a perfluorohexane (PFH) liquid core, a hybrid lipid-polymer shell with PLGA12k-PEG2k and DSPE-PEG1k-Pt(IV), and an active targeting ligand, the cRGD peptide (PLGA: poly(lactic-co-glycolic acid), PEG: polyethylene glycol, DSPE: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, cRGD: cyclic Arg-Gly-Asp). Pt(IV), a popular alternative to Pt(II), was covalently attached to DSPE-PEG1k to form the prodrug, which fine-tuned lipophilicity and improved cellular uptake. The potential of Pt(IV) NP-cRGD as contrast agents for ultrasound (US) imaging was assessed in vitro and in vivo. Moreover, studies on the antitumor efficiency and antitumor mechanism of Pt(IV) NP-cRGD assisted by US were carried out. Results: Pt(IV) NP-cRGD exhibited strong echogenic signals and excellent echo persistence under an US field. In addition, the GSH-sensitive and US-triggered drug delivery system maximized the therapeutic effect while reducing the toxicity of chemotherapy. The mechanistic studies confirmed that Pt(IV) NP-cRGD with US consumed GSH and enhanced reactive oxy gen species (ROS) levels, which further causes mitochondria-mediated apoptosis. Conclusion: A multifunctional nanoplatform based on phase-transitional Pt(IV) NP-cRGD with US exhibited excellent echogenic signals, brilliant therapeutic efficacy and limited side effect, suggesting precise theranostics against ovarian cancer.

Rationale: Glioblastoma (GBM) is the most common and aggressive brain tumor, characterized by its propensity to invade the surrounding brain parenchyma. The effect of extracellular high-mobility group box 1 (HMGB1) protein on glioblastoma (GBM) progression is still controversial. p62 is overexpressed in glioma cells, and has been associated with the malignant features and poor prognosis of GBM patients. Hence, this study aimed to clarify the role of p62 in HMGB1-induced epithelial-mesenchymal transition (EMT) of GBM both in vitro and in vivo. Methods: Immunoblotting, immunofluorescence and qRT-PCR were performed to evaluate EMT progression in both human GBM cell line and primary GBM cells. Transwell and wound healing assays were used to assess the invasion and migration of GBM cells. shRNA technique was used to investigate the role of p62 in HMGB1-induced EMT both in vitro and in vivo orthotopic tumor model. Co-immunoprecipitation assay was used to reveal the interaction between p62 and GSK-3β (glycogen synthase kinase 3 beta). Immunohistochemistry was performed to detect the expression levels of proteins in human GBM tissues. Results: In this study, GBM cells treated with recombinant human HMGB1 (rhHMGB1) underwent spontaneous EMT through GSK-3β/Snail signaling pathway. In addition, our study revealed that rhHMGB1-induced EMT of GBM cells was accompanied by p62 overexpression, which was mediated by the activation of TLR4-p38-Nrf2 signaling pathway. Moreover, the results demonstrated that p62 knockdown impaired rhHMGB1-induced EMT both in vitro and in vivo. Subsequent mechanistic investigations showed that p62 served as a shuttling factor for the interaction of GSK-3β with proteasome, and ultimately activated GSK-3β/Snail signaling pathway by augmenting the degradation of GSK-3β. Furthermore, immunohistochemistry analysis revealed a significant inverse correlation between p62 and GSK-3β, and a combination of the both might serve as a more powerful predictor of poor survival in GBM patients. Conclusions: This study suggests that p62 is an effector for HMGB1-induced EMT, and may represent a novel therapeutic target in GBM.

Rationale: Currently, for locoregionally advanced nasopharyngeal carcinoma (LA-NPC), there is no effective blood-based method to predict distant metastasis. We aimed to detect plasma protein profiles to identify biomarkers that could distinguish patients with NPC who are at high risk of posttreatment distant metastasis. Methods: A high-throughput antibody array was initially applied to detect 1000 proteins in pretreatment plasma from 16 matched LA-NPC patients with or without distant metastasis after radical treatment. Differentially expressed proteins were further examined using a low-throughput array to construct a plasma protein-based signature for distant metastasis (PSDM) in a cohort of 226 patients. Results: Fifty circulating proteins were differentially expressed between metastatic and non-metastatic patients and 18 were proven to be strongly correlated with distant metastasis-free survival (DMFS) in NPC. A PSDM signature consisting of five proteins (SLAMF5, ESM-1, MMP-8, INSR, and Serpin A5) was established to assign patients with NPC into a high-risk group and a low-risk group. Patients in the high-risk group had shorter DMFS (P < 0.001), disease-free survival (DFS) (P < 0.001) and overall survival (OS) (P < 0.001). Moreover, the PSDM performed better than N stage and Epstein-Barr virus (EBV) DNA load at effectively identifying patients with NPC at high risk of metastasis. For patients in the high-risk group, induction chemotherapy significantly improved DMFS, DFS, and OS. Conclusions: The PSDM could be a useful liquid biopsy tool to effectively predict distant metastasis and the benefit of induction chemotherapy in patients with LA-NPC.

Split thickness skin graft (STSG) implantation is one of the standard therapies for full thickness wound repair when full thickness autologous skin grafts (FTG) or skin flap transplants are inapplicable. Combined transplantation of STSG with dermal substitute could enhance its therapeutic effects but the results remain unsatisfactory due to insufficient blood supply at early stages, which causes graft necrosis and fibrosis. Human mesenchymal stem cell (hMSC) sheets are capable of accelerating the wound healing process. We hypothesized that pre-vascularized hMSC sheets would further improve regeneration by providing more versatile angiogenic factors and pre-formed microvessels. In this work, in vitro cultured hMSC cell sheets (HCS) and pre-vascularized hMSC cell sheets (PHCS) were implanted in a rat full thickness skin wound model covered with an autologous STSG. Results demonstrated that the HCS and the PHCS implantations significantly reduced skin contraction and improved cosmetic appearance relative to the STSG control group. The PHCS group experienced the least hemorrhage and necrosis, and lowest inflammatory cell infiltration. It also induced the highest neovascularization in early stages, which established a robust blood micro-circulation to support grafts survival and tissue regeneration. Moreover, the PHCS grafts preserved the largest amount of skin appendages, including hair follicles and sebaceous glands, and developed the smallest epidermal thickness. The superior therapeutic effects seen in PHCS groups were attributed to the elevated presence of growth factors and cytokines in the pre-vascularized cell sheet, which exerted a beneficial paracrine signaling during wound repair. Hence, the strategy of combining STSG with PHCS implantation appears to be a promising approach in regenerative treatment of full thickness skin wounds.

Nanostructures based on metal-organic frameworks (MOFs) have promising potential as theragnostic nanoplatforms for phototherapy of cancer cells. However, the MOFs alone are seldom reported to be used as photothermal agents mainly due to their poor near-infrared (NIR) light absorption. Methods: Ultrathin copper-tetrakis (4-carboxyphenyl) porphyrin (Cu-TCPP) MOF nanosheets were prepared by a facile solvothermal route. The photothermal therapy (PTT), photodynamic therapy (PDT), and T1 -weighted magnetic resonance (MR) imaging capabilities and the high biocompatibility of these composite nanosheets were evaluated in vitro as well as in vivo in a mouse tumor model. Results: The ultrathin Cu-TCPP MOF nanosheets exhibited 1) strong NIR absorption because of the d-d energy band transition of Cu2+ and the ultrathin characteristic translating into excellent photothermal performance, 2) ability to produce singlet oxygen because of the inherent characteristic of TCPP, and 3) capability for MR imaging because of the unpaired 3d electrons of copper. Conclusion: Our study demonstrated that the Cu-TCPP MOF nanosheets are a promising phototherapy nanoplatform with the synergistic ability for PTT and PDT of cancer, guided by MR and infrared thermal imaging.

Gold nanoparticle-coated Pluronic-b-poly(L-lysine) nanoparticles (Pluronic-PLL@Au NPs) were synthesized via an easy one-step method and employed as carriers for the delivery of paclitaxel (PTX) in chemo-photothermal therapy, in which Pluronic-PLL acts as the reductant for the formation of AuNPs without the need for an additional reducing agent.

X-ray excited persistent luminescence (XEPL) imaging has attracted increasing attention in biomedical imaging due to elimination of autofluorescence, high signal-to-noise ratio and repeatable activation with high penetration. However, optical imaging still suffers from limited for high spatial resolution. Methods: Herein, we report Mn3+-rich manganese oxide (MnOx)-coated chromium-doped zinc gallogermanate (ZGGO) nanoparticles (Mn-ZGGOs). Enhanced XEPL and magnetic resonance (MR) imaging were investigated by the decomposition of MnOx shell in the environment of tumors. We also evaluated the tumor cell-killing mechanism by detection of reactive oxygen (ROS), lipid peroxidation and mitochondrial membrane potential changes in vitro. Furthermore, the in vivo biodistribution, imaging and therapy were studied by U87MG tumor-bearing mice. Results: In the tumor region, the MnOx shell is quickly decomposed to produce Mn3+ and oxygen (O2) to directly generate singlet oxygen (1O2). The resulting Mn2+ transforms endogenous H2O2 into highly toxic hydroxyl radical (·OH) via a Fenton-like reaction. The Mn2+ ions and ZGGOs also exhibit excellent T1-weighted magnetic resonance (MR) imaging and ultrasensitive XEPL imaging in tumors. Conclusion: Both the responsive dual-mode imaging and simultaneous self-supplied O2 for the production of 1O2 and oxygen-independent ·OH in tumors allow for more accurate diagnosis of deep tumors and more efficient inhibition of tumor growth without external activation energy.

Rationale: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid tumor, with extremely low survival rates. Identifying key signaling pathways driving PDAC progression is crucial for the development of therapies to improve patient response rates. Kindlin-2, a multi-functional protein, is involved in numerous biological processes including cell proliferation, apoptosis and migration. However, little is known about the functions of Kindlin-2 in pancreatic cancer progression in vivo. Methods: In this study, we employ an in vivo PDAC mouse model to directly investigate the role of Kindlin-2 in PDAC progression. Then, we utilized RNA-sequencing, the molecular and cellular assays to determine the molecular mechanisms by which Kindlin-2 promotes PDAC progression. Results: We show that loss of Kindlin-2 markedly inhibits KrasG12D-driven pancreatic cancer progression in vivo as well as in vitro. Furthermore, we provide new mechanistic insight into how Kindlin-2 functions in this process, A fraction of Kindlin-2 was localized to the endoplasmic reticulum and associated with the RNA helicase DDX3X, a key regulator of mRNA translation. Loss of Kindlin-2 blocked DDX3X from binding to the 5'-untranslated region of c-Myc and inhibited DDX3X-mediated c-Myc translation, leading to reduced c-Myc-mediated glucose metabolism and tumor growth. Importantly, restoration of the expression of either the full-length Kindlin-2 or c-Myc, but not that of a DDX3X-binding-defective mutant of Kindlin-2, in Kindlin-2 deficient PDAC cells, reversed the inhibition of glycolysis and pancreatic cancer progression induced by the loss of Kindlin-2. Conclusion: Our studies reveal a novel Kindlin-2-DDX3X-c-Myc signaling axis in PDAC progression and suggest that inhibition of this signaling axis may provide a promising therapeutic approach to alleviate PDAC progression.

In the war on cancer marked by personalized medicine, positron emission tomography (PET)-based theranostic strategy is playing an increasingly important role. Well-designed clinical trials are of great significance for validating the PET applications and ensuring evidence-based cancer care. This study aimed to provide a comprehensive landscape of the characteristics of PET clinical trials using the substantial resource of ClinicalTrials.gov database. We identified 25,599 oncology trials registered with ClinicalTrials.gov in the last ten-year period (October 2005-September 2015). They were systematically reviewed to validate classification into 519 PET trials and 25,080 other oncology trials used for comparison. We found that PET trials were predominantly phase 1-2 studies (86.2%) and were more likely to be single-arm (78.9% vs. 57.9%, P <0.001) using non-randomized assignment (90.1% vs. 66.7%, P <0.001) than other oncology trials. Furthermore, PET trials were small in scale, generally enrolling fewer than 100 participants (20.3% vs. 25.7% for other oncology trials, P = 0.014), which might be too small to detect a significant theranostic effect. The funding support from industry or National Institutes of Health shrunk over time (both decreased by about 5%), and PET trials were more likely to be conducted in only one region lacking international collaboration (97.0% vs. 89.3% for other oncology trials, P <0.001). These findings raise concerns that clinical trials evaluating PET imaging in oncology are not receiving the attention or efforts necessary to generate high-quality evidence. Advancing the clinical application of PET imaging will require a concerted effort to improve the quality of trials.

Rationale: Enhancer RNA (eRNA) bi-directionally expresses from enhancer region and sense eRNA regulates adjacent mRNA in cis and in trans. However, it has remained unclear whether antisense eRNAs in different direction are functional or merely a reflection of enhancer activation. Methods: Strand-specific, ribosome-minus RNA sequencing (RNA-seq) were performed in AR positive prostate cancer cells. RNA-seq, GRO-seq, ChIP-seq, 4C-seq and DNA-methylation-seq that published in our and other labs were re-analyzed to define bi-directional enhancer RNA and DNA methylation regions. Molecular mechanisms were demonstrated by 3C, ChIP, ChIRP, CLIP, RT-PCR and western blot assays. The biological functions of antisense-eRNA were assessed using mice xenograft model and RT-PCR analysis in human tissues. Results: In this study, we identified that antisense eRNA was regulated by androgen receptor (AR) activity in prostate cancer cells. Antisense eRNA negatively regulated antisense ncRNA in AR-related target genes' loci, through recruiting DNMT1 on the antisense enhancer in the gene-ending regions and elevating DNA methylation. Importantly, the chromatin exhibited a double looping manner that facilitated sense-eRNA to promoter and antisense-eRNA to gene-ending region in cis. Depletion of antisense eRNA impaired its neighbor mRNA expression, cancer growth and invasion. The expressions of antisense eRNA were correlated with biochemical recurrence and clinical marker PSA's levels in patients' tissues. Conclusions: The findings indicated that antisense eRNA was a functional RNA and may be a novel target that when suppressed improved prostate cancer therapy and diagnosis. New chromatin interaction among enhancer, promoter and gene-ending region might provide new insight into the spatiotemporal mechanism of the gene transcription and acting of bi-directional eRNAs.

We performed a comprehensive immuno-genomic analysis of tumor microenvironment immune types (TMITs), which is classified into four groups based on PD-L1+CD8A or PD-L1+cytolytic activity (CYT) expression, across a broad spectrum of solid tumors in order to help identify patients who will benefit from anti- PD-1/PD-L1 therapy. The mRNA sequencing data from The Cancer Genome Atlas (TCGA) of 14 solid cancer types representing 6,685 tumor samples was analyzed. TMIT was classified only for those tumor types that both PD-L1 and CD8A/CYT could prefict mutation and/or neoantigen number. The mutational and neoepitope features of the tumor were compared according to the four TMITs. We found that PD-L1/CD8A/CYT subgroups could not distinguish different mutation and neoantigen numbers in certain tumor types such as glioblastoma multiforme, prostate adenocarcinoma, and head and neck and lung squamous cell carcinoma. For the remaining tumor types, compared with TIMT II (low PD-L1 and CD8A/CYT), TIMT I (high PD-L1 and CD8A/CYT) had a significantly higher number of mutations or neoantigens in bladder urothelial carcinoma, breast and cervical cancer, colorectal, stomach and lung adenocarcinoma, and melanoma. In contrast, TMIT I of kidney clear cell, liver hepatocellular, and thyroid carcinoma were negatively correlated with mutation burden or neoantigen numbers. Our findings show that the TMIT stratification proposed could serve as a favorable approach for tailoring optimal immunotherapeutic strategies in certain tumor types. Going forward, it will be important to test the clinical practicability of TMIT based on quantification of immune infiltrates using mRNA-seq to predict clinical response to these and other immunotherapeutic strategies in more different tumors.

High aggressiveness and recurrence of melanoma tumors require multiple systemic drug administrations, causing discomfort and severe side effects to the patients. Topical treatment strategies that provide repetitively controllable and precise drug administrations will greatly improve treatment effects. Methods: In this study, a spatiotemporally controlled pulsatile release system, which combined dissolving microneedles (DMNs) and thermal-sensitive solid lipid nanoparticles (SLNs), was constructed to realize multiple doses of dual-modal chemo-photothermal therapy in a single administration. Paclitaxel (PTX) and photothermal agent IR-780 were encapsulated into SLNs and were concentrated in the tips of DMNs (PTX/IR-780 SLNs @DMNs). Equipped with several needles, the DMN patch could be directly inserted into the tumor site and provide a stable "Zone accumulation" to constrain the PTX/IR-780 SLNs at the tumor site with uniform distribution. Results:In vitro experiments showed that after irradiation with near-infrared light, the PTX/IR-780 SLNs gradually underwent phase transition, thereby accelerating the release of PTX. When irradiation was switched off, the PTX/IR-780 SLNs cooled to re-solidify with limited drug release. Compared with intravenous and intratumoral injections, very few SLNs from PTX/IR-780 SLNs @DMNs were distributed into other organs, resulting in enhanced bioavailability at the tumor site and good safety. In vivo analysis revealed that PTX/IR-780 SLNs @DMNs exhibited significant anti-tumor efficacy. In particular, the primary tumor was completely eradicated with a curable rate of 100% in 30 days and the highest survival rate of 66.67% after 100 days of treatment. Conclusion: Herein, we developed a DMN system with a unique spatiotemporally controlled pulsatile release feature that provides a user-friendly and low-toxicity treatment route for patients who need long-term and repeat treatments.