ICG-labeled PD-L1-antagonistic affibody dimer for tumor imaging and enhancement of tumor photothermal-immunotherapy.

In clinical practice, tumor-targeting diagnosis and immunotherapy against programmed death ligand 1 (PD-L1) have a significant impact. In this research, a PD-L1-antagonistic affibody dimer (ZPD-L1) was successfully prepared through Escherichia coli expression system, and conjugated with the photosensitizer of ICG via N-hydroxysuccinimide (NHS) ester to develop a novel tumor-targeting agent (ICG-ZPD-L1) for both tumor imaging diagnosis and photothermal-immunotherapy simultaneously. In vitro, ZPD-L1 could specifically bind to PD-L1-positive LLC and MC38 tumor cells, and ICG-ZPD-L1-mediated photothermal therapy (PTT) also showed excellent phototoxicity to these tumor cells. In vivo, ICG-ZPD-L1 selectively enriched into the PD-L1-positive MC38 tumor tissues, and the high-contrast optical imaging of tumors was obtained. ICG-ZPD-L1-mediated PTT exhibited a potent anti-tumor effect in vivo due to its remarkable photothermal properties. Furthermore, ICG-ZPD-L1-mediated PTT significantly induced the immunogenic cell death (ICD) of primary tumors, promoted maturation of dendritic cells (DCs), up-regulated anti-tumor immune response, enhanced immunotherapy, and superiorly inhibited the growth of metastatic tumors. In addition, ICG-ZPD-L1 showed favorable biosafety throughout the brief duration of treatment. In summary, these results suggest that ICG-ZPD-L1 is a multifunctional tumor-targeting drug integrating tumor imaging diagnosis and photothermal-immunotherapy, and has great guiding significance for the diagnosis and treatment of clinical PD-L1-positive tumor patients.

PDGFRß-Antagonistic Affibody-Mediated Tumor-Targeted Tumor Necrosis Factor-Alpha for Enhanced Radiotherapy in Lung Cancer.

The morbidity and mortality of lung cancer are still the highest among all malignant tumors. Radiotherapy plays an important role in clinical treatment of lung cancer. However, the effect of radiotherapy is not ideal due to the radiation resistance of tumor tissues. Abnormalities in tumor vascular structure and function affect blood perfusion, and oxygen transport is impeded, making tumor microenvironment hypoxic. Tumor hypoxia is the major cause of radiotherapy resistance. By promoting tumor vessel normalization and enhancing vascular transport function, tumor hypoxia can be relieved to reduce radiotherapy resistance and increase tumor radiotherapy sensitivity. In our previous study, a pericytes-targeted tumor necrosis factor alpha (named Z-TNFα) was first constructed and produced by genetically fusing the platelet-derived growth factor receptor ß (PDGFRß)-antagonistic affibody (ZPDGFRß) to the TNFα, and the Z-TNFα induced normalization of tumor vessels and improved the delivery of doxorubicin, enhancing tumor chemotherapy. In this study, the tumor vessel normalization effect of Z-TNFα in lung cancer was further clarified. Moreover, the tumor hypoxia improvement and radiosensitizing effect of Z-TNFα were emphatically explored in vivo. Inspiringly, Z-TNFα specifically accumulated in Lewis lung carcinoma (LLC) tumor graft and relieved tumor hypoxia as well as inhibited HIF-1α expression. As expected, Z-TNFα significantly increased the effect of radiotherapy in mice bearing LLC tumor graft. In conclusion, these results demonstrated that Z-TNFα is also a promising radiosensitizer for lung cancer radiotherapy.

Dual effect of radiotherapy related concomitant cardiovascular diseases in non-small cell lung cancer.

BACKGROUND: Nowadays, cancer and cardiac diseases are two of the most causes of death, so cancer treatment-related cardiac death cannot be ignored. For lung cancer, chest radiotherapy (RT) is essential, but the related cardiotoxicity has not been fully studied. METHODS: We reviewed the data of 11,455 patients with non-small cell lung cancer (NSCLC) from the Surveillance, Epidemiology, and End Results database from 2001 to 2015. The change trend for concomitant cardiovascular diseases (CVD)-specific death was calculated and graphically demonstrated. Univariate and multivariate analyses for survival were performed using Cox risk regression model. RESULTS: In our analysis, the overall incidence and mortality from NSCLC declined, but CVD-specific death increased. Both chemoradiotherapy and radiotherapy alone played a significant role in CVD-specific death. Analyzed longitudinally from diagnosis, we found that the effect of RT in CVD-specific death increased continuously over the third years and the hazard ratio for CVD-specific death was 1.386 times between RT and non-RT group (HR = 1.386, 95% CI 1.322-1.452; p < 0.0001). On the other hand, RT played a protective role in CVD-specific death before the second years, especially in recent years from 2013 to 2015 (HR = 0.843, 95% CI 0.740-0.959; p = 0.009). CONCLUSIONS: Although the mortality from NSCLC decreased, but radiotherapy-related CVD-specific mortality cannot be ignored. In the long-term over 3 years, RT significantly promoted CVD-specific death. However, RT turned to be a protective role in the short-term within 2 years. In clinical practice, we need to comprehensively consider the dual effects of radiotherapy on the side effect of heart.

Aberrant ROS Served as an Acquired Vulnerability of Cisplatin-Resistant Lung Cancer.

Lung cancer has become a global health issue in recent decades. Approximately 80-85% of cases are non-small-cell lung cancer (NSCLC). Despite the high rate of resistance, cisplatin-base chemotherapy is still the main treatment for NSCLC patients. Thus, overcoming cisplatin resistance is urgently needed in NSCLC therapy. In this study, we identify NADPH metabolism and reactive oxygen species (ROS) levels as the main causes accounting for cisplatin resistance. Based on a small panel consisting of common chemotherapy drugs or compounds, APR-246 is proved to be an effective compound targeting cisplatin-resistant NSCLC cells. APR-246 specially inhibits proliferation and colony formation of cisplatin-resistant cells. In details, APR-246 can significantly cause G0/G1 accumulation and S phase arrest of cisplatin resistant cells and gives rise to severe mitochondria dysfunction as well as elevated apoptosis. Further study proves that it is the aberrant ROS levels as well as NRF2/SLC7A11/GSH axis dysfunction accounting for the specific antitumor effects of APR-246. Scavenging ROS with N-acetylcysteine (NAC) disrupts the inhibitory effect of APR-246 on cisplatin-resistant cells. Mechanistically, NRF2 is specifically degraded by the proteasome following its own ubiquitylation in APR-246-treated cisplatin-resistant cells, which in turn decreases NRF2/SLC7A11/GSH axis activity. Our study provides new insights into the biology driving cisplatin resistance of lung cancer and highlights APR-246 as a potential therapeutic reagent for overcoming cisplatin resistance.

Her3-specific affibody mediated tumor targeting delivery of ICG enhanced the photothermal therapy against Her3-positive tumors.

Photothermal therapy (PTT), mediated by tumor-targeted drug delivery of indocyanine green (ICG), is a promising strategy for cancer therapy. Human epidermal growth factor receptor 3 (Her3) is highly expressed in several solid tumors and is an ideal target for tumor diagnosis and therapy. This study prepared a Her3-specific dimeric affibody (ZHer3) using an Escherichia coli expression system. The affibody could bind explicitly to Her3-positive MCF7 and LS174T cells, rather than to Her3-negative SKOV-3 cells in vitro. ICG was coupled with the ZHer3 affibody (ICG-ZHer3) through an N-hydroxysuccinimide (NHS) ester reactive group for tumor-targeted delivery. As expected, Her3-positive cells were selectively and efficiently killed by ICG-ZHer3-mediated PTT in vitro. In vivo, ICG-ZHer3 preferentially accumulated in Her3-positive LS174T tumor grafts because of the tumor-targeting ability of the ZHer3 affibody. As a result of the local generation of cytotoxic reactive oxygen species and hyperthermia, the growth rates of LS174T tumor grafts were significantly inhibited by ICG-ZHer3-mediated PTT, and ICG-ZHer3 showed good safety performance during short-term treatment. In conclusion, these results demonstrated that ICG-ZHer3 is a promising photosensitizer for PTT against Her3-positive tumors.

Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal tumors, accounting for almost half a million deaths per year. Cancer-associated fibroblasts (CAFs) are the major constituent of the tumor microenvironment (TME) and dramatically impact ESCC progression. Recent evidence suggests that exosomes derived from CAFs are able to transmit regulating signals and promote ESCC development. In this study, we compared different the component ratios of miRNAs in exosomes secreted by CAFs in tumors and with those from normal fibroblasts (NFs) in precancerous tissues. The mRNA level of hsa-miR-3656 was significantly upregulated in the former exosomes. Subsequently, by comparing tumor cell development in vitro and in vivo, we found that the proliferation, migration and invasion capabilities of ESCC cells were significantly improved when miR-3656 was present. Further target gene analysis confirmed ACAP2 was a target gene regulated by miR-3656 and exhibited a negative regulatory effect on tumor proliferation. Additionally, the downregulation of ACAP2 triggered by exosomal-derived miR-3656 further promotes the activation of the PI3K/AKT and ß-catenin signaling pathways and ultimately improves the growth of ESCC cells both in vitro and in xenograft models. These results may represent a potential therapeutic target for ESCC and provide a new basis for clinical treatment plans.

Oncogenic SNORD12B activates the AKT-mTOR-4EBP1 signaling in esophageal squamous cell carcinoma via nucleus partitioning of PP-1α.

Esophageal cancer is a complex malignancy and the sixth leading cause of cancer death worldwide. In Eastern Asia including China, about 90% of all incident cases have esophageal squamous cell carcinoma (ESCC). Mounting evidence elucidates that aberrant expression of various non-coding RNAs (ncRNAs) contributes to ESCC progression, but it remains unclear how small nucleolar RNAs (snoRNAs) are involved in ESCC development. We systemically screened clinically relevant snoRNAs in ESCC via integrative analyses of The Cancer Genome Atlas (TCGA) data and validation in ESCC tissues. We found that snoRNA SNORD12B was one of the most evidently upregulated snoRNAs in ESCC specimens and its high expression was significantly associated with poor prognosis of patients. SNORD12B profoundly promoted proliferation, migration, invasion, and metastasis of ESCC cells in vitro and in vivo, indicating its oncogene nature. In particular, SNORD12B could interact with PP-1α, one of the three catalytic subunits of serine/threonine protein phosphatase 1, which is a major phosphatase that directly dephosphorylates AKT to suppress its activation. Interestingly, high levels of SNORD12B in ESCC cells could break interactions between 14-3-3ζ and PP-1α, abolish the retention of PP-1α in the cytosol by 14-3-3ζ and relocate PP-1α from the cytosol to the nucleus. This led to sequestered PP-1α in the nucleus, enhanced phosphorylation of AKT in the cytosol, activated AKT-mTOR-4EBP1 signaling, and, thus, ESCC progression. These insights would improve our understanding of how snoRNAs contribute to tumorigenesis and highlight the potential of snoRNAs as future therapeutic targets against cancers.

miR-671-5p Blocks The Progression Of Human Esophageal Squamous Cell Carcinoma By Suppressing FGFR2.

Esophageal cancer is the eighth most common malignant tumor worldwide, of which esophageal squamous cell carcinoma (ESCC) is the dominant histological subtype. A drug shortage for ESCC therapy triggered us to explore the roles of fibroblast growth factor receptor 2 (FGFR2) and its upstream regulator miR-671-5p in ESCC progression. We compared the levels of FGFR2 and miR-671-5p between human ESCC tissues and their matched normal esophageal tissues and found an association between higher levels of FGFR2 and lower levels of miR-671-5p in ESCC tissues. High levels of FGFR2 resulted in the activation of the ERK and AKT pathways and a promotion of ESCC progression. High levels of miR-671-5p specifically reduced the expression of FGFR2 and suppressed ESCC progression in both in vitro and in vivo models. Therefore, suppressing FGFR2 and enhancing miR-671-5p expression may be the right approaches for ESCC therapy.

CACNA2D3 Enhances the Chemosensitivity of Esophageal Squamous Cell Carcinoma to Cisplatin via Inducing Ca2+-Mediated Apoptosis and Suppressing PI3K/Akt Pathways.

Resistance to platinum-based combination chemotherapy is the main cause of poor prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC). Previously, we showed that CACNA2D3 (voltage-dependent subunit alpha 2 delta 3 of a calcium channel complex) was significantly downregulated and functioned as a tumor suppressor in ESCC, but its role in the chemosensitivity of ESCC to cisplatin remained unknown. Here, we found that the expression of CACNA2D3 was significantly associated with poor platinum response in ESCC patients from the Gene Expression Omnibus database. Overexpression of CACNA2D3 increased sensitivity to cisplatin in ESCC in vitro, whereas knockdown of CACNA2D3 increased cisplatin resistance. CACNA2D3 promoted cisplatin-induced apoptosis by modulating intracellular Ca2+ stores. In vivo experiments further showed that overexpression of CACNA2D3 enhanced cisplatin anti-tumor effects in a xenograft mouse model. CACNA2D3 overexpression also resulted in the attenuation of PI3K and Akt phosphorylation. Treatment with the PI3K/Akt inhibitor LY294002 restored the chemosensitivity of CACAN2D3-knockdown cells to cisplatin. In conclusion, the results of the current study indicate that CACAN2D3 enhances the chemosensitivity of ESCC to cisplatin via inducing Ca2+-mediated apoptosis and suppressing PI3K/Akt pathways. Therefore, regulating the expression of CACNA2D3 is a potential new strategy to increase the efficacy of cisplatin in ESCC patients.

microRNA-193b protects against myocardial ischemia-reperfusion injury in mouse by targeting mastermind-like 1.

The current study aimed to explore the functions and roles of microRNA-193b (miR-193b) in the myocardium with ischemia-reperfusion (I/R) injury and a potential therapeutic method for myocardial I/R injury. The mice were subjected to myocardial I/R with or without miR-193b pretreatment. The infarct size and myocardial enzymes were detected. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was conducted to investigate the effect of miR-193b on cardiomyocyte apoptosis. The expression levels of miR-193b and mastermind-like 1 (MAML1) were validated by quantitative real-time polymerase chain reaction and Western blot analysis. The results suggested that the miR-193b expression level was significantly downregulated in the myocardium with I/R injury compared with control group. miR-193b overexpression is able to reduce infarct size and myocardial enzymes after myocardial I/R injury. Furthermore, overexpression of miR-193b could alleviate the apoptosis level after myocardial I/R injury. Taken together, the present study demonstrated that upregulated miRNA-193b alleviated myocardial I/R injury via targeting MAML1.

Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer.

Small interfering RNAs (siRNA)/microRNAs (miRNA) have promising therapeutic potential, yet their clinical application has been hampered by the lack of appropriate delivery systems. Herein, we employed extracellular vesicles (EVs) as a targeted delivery system for small RNAs. EVs are cell-derived small vesicles that participate in cell-to-cell communication for protein and RNA delivery. We used the aptamer AS1411-modified EVs for targeted delivery of siRNA/microRNA to breast cancer tissues. Tumor targeting was facilitated via AS1411 binding to nucleolin, which is highly expressed on the surface membrane of breast cancer cells. This delivery vesicle targeted let-7 miRNA delivery to MDA-MB-231 cells in vitro as confirmed with fluorescent microscopic imaging and flow cytometry. Also, intravenously delivered AS1411-EVs loaded with miRNA let-7 labeled with the fluorescent marker, Cy5, selectively targeted tumor tissues in tumor-bearing mice and inhibited tumor growth. Importantly, the modified EVs were well tolerated and showed no evidence of nonspecific side effects or immune response. Thus, the RNAi nanoplatform is versatile and can deliver siRNA or miRNA to breast cancer cells both in vitro and in vivo. Our results suggest that the AS1411-EVs have a great potential as drug delivery vehicles to treat cancers.

Urokinase plasminogen activator secreted by cancer-associated fibroblasts induces tumor progression via PI3K/AKT and ERK signaling in esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) are believed to influence tumor behavior and clinical outcomes. We previously showed that conditioned medium (CM) from CAFs induces proliferation and motility of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the molecular mechanisms by which the CAF-secreted proteins induce ESCC development and progression. Using antibody arrays, we identified urokinase plasminogen activator (uPA) as one of the main proteins whose release was increased in CAFs compared to normal fibroblasts (NFs). Immunohistochemical analysis of pathological sections showed that uPA-positive cells were localized at the boundaries of tumor and stroma tissues, in stroma between tumor nests, and within the tumors. Increased stromal uPA levels (132/146 cases) correlated with tumor invasion (p < 0.05) and overall survival of ESCC patients (p < 0.05). In vitro assays showed that uPA promotes ESCC cell proliferation, migration, and invasion via PI3K/AKT and ERK signaling pathways. In vivo, anti-uPA antibody suppressed tumor growth in ESCC xenografts. These results suggest that uPA released from stroma, and especially from CAFs, might be a predictive marker for ESCC diagnosis and prognosis, as well as an effective therapeutic target.

GSK3ß activity is essential for senescence-associated heterochromatin foci (SAHF) formation induced by HMGA2 in WI38 cells.

Cellular senescence is an irreversible form of cell cycle arrest, which is often characterized by domains of facultative heterochromatin substructures also known as senescence-associated heterochromatin foci (SAHF). SAHF assembly is likely mediated through the downregulation of the Wnt pathway, which inhibits Glycogen Synthase Kinase 3 Beta (GSK3ß) in cells undergoing replicative senescence. Alternatively, High Mobility Group AT-Hook 2 (HMGA2) can also induce SAHF formation in primary cells, through a process in which the involved cell signaling pathway is unknown. Accordingly, it is important to determine whether GSK3ß and the Wnt pathway are necessary during HMGA2-induced SAHF formation. In this study, we developed a senescence model for SAHF assembly in WI38 cell through ectopic expression of HMGA2. In this model, typical senescent features were identified, including elevated SA-ß-galactosidase staining and the downregulation of the Wnt pathway. We also showed that the GSK3ß inhibitor LiCl can partly disable SAHF formation through the HMGA2 overexpression in WI38 cells. However, the disabled SAHF formation resulting from the inactivity of GSK3ß in our senescence model cannot be restored through ectopic overexpression of Catenin Beta 1 (CTNNB1), a downstream transcription factor of the Wnt pathway. This indicates that the GSK3ß activity alone, and not those of downstream target genes, is crucial for the HMGA2-induced SAHF formation following the downregulation of the Wnt pathway.

A novel anti-proliferative role of HMGA2 in induction of apoptosis through caspase 2 in primary human fibroblast cells.

The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that γH2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.

Rb protein is essential to the senescence-associated heterochromatic foci formation induced by HMGA2 in primary WI38 cells.

Cellular senescence is an irreversible form of cell cycle arrest that provides a barrier to neoplastic transformation. The integrity of the Rb (Retinoblastoma) pathway is necessary for the formation of the senescence-associated heterochromatin foci (SAHF) that offers a molecular basis for the stability of the senescent state. Surprisingly, although high mobility group A2 protein (HMGA2) can promote tumorigenesis and inhibit Rb function in tumor cells, high-level expression of HMGA2 is sufficient to induce SAHF formation in primary cells. It therefore becomes significant to determine whether Rb protein is necessary in HMGA2-induced SAHF formation. In this study, we established the cellular senescence and SAHF assembly WI38 cell model by ectopic expression of HMGA2, in which typical senescent markers were seen, including notable upregulation of p53, p21 and p16, and elevated SA-ß-galactosidase staining together with downregulation of E2F target genes. We then showed that the Rb pathway inhibitor E7 protein was able to partly abolish the ability of SAHF formation after HMGA2 expression in WI38 cells, indicating that Rb is a crucial factor for HMGA2-induced SAHF formation. However, Rb depletion did not completely rescue the cell growth arrest induced by HMGA2, suggesting that Rb is not an exclusive pathway for HMGA2-induced senescence in WI38 cells.

Mitogen-activated protein kinase p38 and retinoblastoma protein signalling is required for DNA damage-mediated formation of senescence-associated heterochromatic foci in tumour cells.

DNA-damaging agents are able to induce irreversible cell growth arrest and senescence in some types of tumour cells, thus contributing to the static feature of cancer. However, senescent tumour cells may re-enter the cell cycle, leading to tumour relapse. Understanding the mechanisms that control the viability of senescent cells may be critical for tumour suppression. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which contribute to the stability of the senescent state. However, it is unclear whether SAHF formation is universal in tumour cells. We report that the DNA-damaging agents doxorubicin and 7-ethyl-10-hydroxycamptothecin were able to induce the formation of SAHF in some tumour cell types, and this induction was accompanied by activation of the retinoblastoma protein pathway. By contrast, tumour cells in which the retinoblastoma protein pathway could not be activated by doxorubicin or 7-ethyl-10-hydroxycamptothecin failed to form SAHF. In parallel, tumour cells with deficient retinoblastoma protein were also unable to form SAHF. In addition, we show that the mitogen-activated protein kinase p38 pathway was involved in tumour cell SAHF formation in response to doxorubicin and 7-ethyl-10-hydroxycamptothecin. Furthermore, HMG box transcription factor 1 (HBP1), a downstream target of the mitogen-activated protein kinase p38-mediated senescence pathway, was required for SAHF formation. Taken together, the results of the present study highlight the roles of the mitogen-activated protein kinase p38/retinoblastoma protein pathway in tumour cell SAHF formation in response to DNA-damaging agents, and provide new insights into the mechanisms of DNA damage-mediated tumour suppression.