Proteogenomic Characterization Reveals Estrogen Signaling as a Target for Never-Smoker Lung Adenocarcinoma Patients without EGFR or ALK Alterations.

Never-smoker lung adenocarcinoma (NSLA) is prevalent in Asian populations, particularly in women. EGFR mutations and anaplastic lymphoma kinase (ALK) fusions are major genetic alterations observed in NSLA, and NSLA with these alterations have been well studied and can be treated with targeted therapies. To provide insights into the molecular profile of NSLA without EGFR and ALK alterations (NENA), we selected 141 NSLA tissues and performed proteogenomic characterization, including whole genome sequencing (WGS), transcriptomic, methylation EPIC array, total proteomic, and phosphoproteomic analyses. Forty patients with NSLA harboring EGFR and ALK alterations and seven patients with NENA with microsatellite instability were excluded. Genome analysis revealed that TP53 (25%), KRAS (22%), and SETD2 (11%) mutations and ROS1 fusions (14%) were the most frequent genetic alterations in NENA patients. Proteogenomic impact analysis revealed that STK11 and ERBB2 somatic mutations had broad effects on cancer-associated genes in NENA. DNA copy number alteration analysis identified 22 prognostic proteins that influenced transcriptomic and proteomic changes. Gene set enrichment analysis revealed estrogen signaling as the key pathway activated in NENA. Increased estrogen signaling was associated with proteogenomic alterations, such as copy number deletions in chromosomes 14 and 21, STK11 mutation, and DNA hypomethylation of LLGL2 and ST14. Finally, saracatinib, an Src inhibitor, was identified as a potential drug for targeting activated estrogen signaling in NENA and was experimentally validated in vitro. Collectively, this study enhanced our understanding of NENA NSLA by elucidating the proteogenomic landscape and proposed saracatinib as a potential treatment for this patient population that lacks effective targeted therapies. SIGNIFICANCE: The proteogenomic landscape in never-smoker lung cancer without known driver mutations reveals prognostic proteins and enhanced estrogen signaling that can be targeted as a potential therapeutic strategy to improve patient outcomes.

FOXM1 Inhibition Enhances the Therapeutic Outcome of Lung Cancer Immunotherapy by Modulating PD-L1 Expression and Cell Proliferation.

Programmed death-ligand 1 (PD-L1) is a major target to cancer immunotherapy, and anti-PD-L1 and anti-PD-1 antibody-mediated immunotherapy are being increasingly used. However, immune checkpoint inhibitors (ICIs) are ineffective in treating large tumors and cause various immune-related adverse events in nontarget organs, including life-threatening cardiotoxicity. Therefore, the development of new therapeutic strategies to overcome these limitations is crucial. The focus of this study is the forkhead box protein M1 (FOXM1), which is identified as a potential therapeutic target for cancer immunotherapy and is associated with the modulation of PD-L1 expression. Selective small interfering RNA knockdown of FOXM1 or treatment with thiostrepton (TST) significantly reduces PD-L1 expression in non-small-cell lung cancer (NSCLC) cells and inhibits proliferation. Chromatin immunoprecipitation-PCR reveals that FOXM1 selectively upregulates PD-L1 expression by binding directly to the PD-L1 promoter. In vivo animal studies have shown that TST treatment significantly downregulates PD-L1 expression in human NSCLC tumors, while greatly reducing tumor size without side effects on normal tissues. Combined treatment with TST and anti-4-1BB antibody in the LLC-1 syngeneic tumor model induces synergistic therapeutic outcomes against immune resistant lung tumors as well as 2.72-folds higher CD3+ T cells in tumor tissues compared to that in the anti-4-1BB antibody treatment group.

ELF3 Is a Target That Promotes Therapeutic Efficiency in EGFR Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer Cells via Inhibiting PKCί.

(1) Background: Mutations in epidermal growth factor receptor (EGFR) proteins account for many non-small cell lung cancers (NSCLCs), and EGFR tyrosine kinase inhibitors (TKIs) are being used as targeted therapeutics. However, resistance to TKIs continues to increase owing to additional mutations in more than half of the patients receiving EGFR TKI therapy. In addition to targeting new mutations with next-generation therapeutics, it is necessary to find an alternative target to overcome the challenges associated with resistance. (2) Methods: To identify potential alternative targets in patients with NSCLC undergoing targeted therapy, putative targets were identified by transcriptome profiling and validated for their biological and therapeutic effects in vitro and in vivo. (3) Results: ELF3 was found to be differentially expressed in NSCLC, and ELF3 knockdown significantly increased cell death in K-Ras mutant as well as in EGFR L858R/T790M mutation harboring lung cancer cells. We also found that auranofin, an inhibitor of protein kinase C iota (PKCί), a protein upstream of ELF3, effectively induced cell death. (4) Conclusions: Our study suggests that blocking ELF3 is an effective way to induce cell death in NSCLC with K-Ras and EGFR T790M/L858R mutations and thus advocates the use of auranofin as an effective alternative drug to overcome EGFR TKI resistance.

Comparison between MGI and Illumina sequencing platforms for whole genome sequencing.

BACKGROUND: Illumina next generation sequencing (NGS) systems are the major sequencing platform in worldwide next-generation sequencing market. On the other hand, MGI Tech launched a series of new NGS equipment that promises to deliver high-quality sequencing data faster and at lower prices than Illumina's sequencing instruments. OBJECTIVE: In this study, we compared the performance of the two platform's major sequencing instruments-Illumina's NovaSeq 6000 and MGI's MGISEQ-2000 and DNBSEQ-T7-to test whether the MGISEQ-2000 and DNBSEQ-T7 sequencing instruments are also suitable for whole genome sequencing. METHODS: We sequenced two pairs of normal and tumor tissues from Korean lung cancer patients using the three platforms. Then, we called single nucleotide variants (SNVs) and insertion and deletion (indels) for somatic and germline variants to compare the performance among the three platforms. RESULTS: In quality control analysis, all of the three platforms showed high-quality scores and deep coverages. Comparison among the three platforms revealed that MGISEQ-2000 is most concordant with NovaSeq 6000 for germline SNVs and indels, and DNBSEQ-T7 is most concordant with NovaSeq 6000 for somatic SNVs and indels. CONCLUSIONS: These results suggest that the performances of the MGISEQ-2000 and DNBSEQ-T7 platforms are comparable to that of the Illumina NovaSeq 6000 platform and support the potential applicability of the MGISEQ-2000 and DNBSEQ-T7 platforms in actual genome analysis fields.

Fibronectin regulates anoikis resistance via cell aggregate formation.

The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.

FAM188B Downregulation Sensitizes Lung Cancer Cells to Anoikis via EGFR Downregulation and Inhibits Tumor Metastasis In Vivo.

Anoikis is a type of apoptosis induced by cell detachment from the extracellular matrix (ECM), which removes mislocalized cells. Acquisition of anoikis resistance is critical for cancer cells to survive during circulation and, thus, metastasize at a secondary site. Although the sensitization of cancer cells to anoikis is a potential strategy to prevent metastasis, the mechanism underlying anoikis resistance is not well defined. Although family with sequence similarity 188 member B (FAM188B) is predicted as a new deubiquitinase (DUB) member, its biological function has not been fully studied. In this study, we demonstrated that FAM188B knockdown sensitized anoikis of lung cancer cell lines expressing WT-EGFR (A549 and H1299) or TKI-resistant EGFR mutant T790M/L858R (H1975). FAM188B knockdown using si-FAM188B inhibited the growth of all three human lung cancer cell lines cultured in both attachment and suspension conditions. FAM188B knockdown resulted in EGFR downregulation and thus decreased its activity. FAM188B knockdown decreased the activities of several oncogenic proteins downstream of EGFR that are involved in anoikis resistance, including pAkt, pSrc, and pSTAT3, with little changes to their protein levels. Intriguingly, si-FAM188B treatment increased EGFR mRNA levels but decreased its protein levels, which was reversed by treatment with the proteasomal inhibitor MG132, indicating that FAM188B regulates EGFR levels via the proteasomal pathway. In addition, cells transfected with si-FAM188B had decreased expression of FOXM1, an oncogenic transcription factor involved in cell growth and survival. Moreover, FAM188B downregulation reduced metastatic characteristics, such as cell adhesion, invasion, and migration, as well as growth in 3D culture conditions. Finally, tail vein injection of si-FAM188B-treated A549 cells resulted in a decrease in lung metastasis and an increase in mice survival in vivo. Taken together, these findings indicate that FAM188B plays an important role in anoikis resistance and metastatic characteristics by maintaining the levels of various oncogenic proteins and/or their activity, leading to tumor malignancy. Our study suggests FAM188B as a potential target for controlling tumor malignancy.

Quenched cetuximab conjugate for fast fluorescence imaging of EGFR-positive lung cancers.

Cetuximab-dye conjugates have shown great potential for image-guided surgery of epidermal growth factor receptor (EGFR)-positive cancers in clinical trials. However, their long circulation half-life and prolonged generation of high background signals require the injection of antibody conjugates several days prior to imaging, which limits the clinical applications. Herein, we developed a cetuximab-ATTO655 conjugate (i.e., Q-Cetuximab) for fast and real-time fluorescence imaging of EGFR-positive lung cancers. The fluorescence intensity of Q-Cetuximab was quenched to just 6.9% of that of the unconjugated dye when only 2.14 ATTO655 dyes were conjugated to cetuximab. In vitro real-time cell imaging showed that EGFR-positive A549 cells emitted strong fluorescence at 10 min after Q-Cetuximab treatment in the absence of the washing step, implying target-specific activation of quenched Q-Cetuximab fluorescence upon binding with EGFR-positive cancer cells. When mice with orthotropic A549 tumors received intravenous injection of Q-Cetuximab, scattered microsized tumors in the lungs could be clearly identified from near-infrared fluorescence imaging with a tumor-to-background ratio of 4.28 at 8 h post-injection. For comparison, the cetuximab-Alexa647 conjugate (i.e., ON-Cetuximab), which does not show fluorescence quenching, was synthesized as an always-on type of probe. The ON-Cetuximab-treated mice expressed strong fluorescence throughout their body at 8 h post-injection; therefore, lung tumor sites could not be discriminated using fluorescence imaging. These results confirm the benefits of Q-Cetuximab for image-guided precision surgery of EGFR-positive lung cancers.

FAM188B Expression Is Critical for Cell Growth via FOXM1 Regulation in Lung Cancer.

Although family with sequence similarity 188 member B (FAM188B) is known to be a member of a novel putative deubiquitinase family, its biological role has not been fully elucidated. Here, we demonstrate the oncogenic function of FAM188B via regulation of forkhead box M1 (FOXM1), another oncogenic transcription factor, in lung cancer cells. FAM188B knockdown induced the inhibition of cell growth along with the downregulation of mRNA and protein levels of FOXM1. FAM188B knockdown also resulted in downregulation of Survivin and cell cycle-related proteins, which are direct targets of FOXM1. Interestingly, FOXM1 co-immunoprecipitated with FAM188B, and the levels of FOXM1 ubiquitination increased with FAM188B knockdown but decreased with FAM188B overexpression. In addition, in vivo xenograft of FAM188B siRNA (siFAM188B) RNA-treated cells resulted in the retardation of tumor growth compared with that in the control. Furthermore, protein levels of FAM188B and FOXM1 were elevated in the human lung cancer tissues, and FAM188B expression was negatively correlated with the overall survival of lung cancer patients. These results indicate that FAM188B exerts its oncogenic effects by regulating FOXM1 deubiquitination and thus its stability. Therefore, FAM188B might be a potential therapeutic target to control lung cancer progression.

The clinical impact of family history of cancer in female never-smoker lung adenocarcinoma.

OBJECTIVES: Accumulating evidence reveals the association between the risk of never-smoker lung cancer and family history of cancer. However, the clinicogenomic effect of family history of cancer in never-smoker lung cancer remains unknown. MATERIAL AND METHODS: We screened 3,241 lung cancer patients who (a) underwent curative resection at National Cancer Center (Goyang, Korea) between 2001-2014, and (b) completed a pre-designed interview about family/smoking history at the time of diagnosis and identified 604 female never smoker lung adenocarcinoma. A positive family history of cancer [categorized as pulmonary cancer (FH-PC) or non-pulmonary cancer (FH-NPC)] was defined as a self-reported history of cancer in first-degree relatives. Survival data were followed up until January 2017. Multiplexed targeted next-generation sequencing was performed for genetic profiling. RESULTS: Of 604 patients, 29.1% (n = 176) had a FH, including 132 (21.9%) with FH-NPC and 44 (7.3%) with FH-PC. Patients with the FH-NPC had a higher proportion of young patients (≤45 years) than those without the FH-NPC (FH-NPC, FH-PC, and no FH; 13.6%, 2.3%, and 8.2%, respectively; P = 0.032). Patients with the FH-NPC had an increased risk of recurrence (hazard ratio [HR]: 1.90; 95% confidence interval [CI]: 1.40-2.56; P<0.001) and death (HR: 1.67; 95% CI: 1.18-2.37; P=0.004). In contrast, the FH-PC had no prognostic effect on recurrence (HR: 1.23; 95% CI: 0.71-2.15; P = 0.456) and death (HR: 0.93; 95% CI: 0.45-1.91; P=0.838). Among three driver oncogene alterations, EGFR mutation was significantly associated with the FH-PC (53.8%, 84.1%, and 65.8%, respectively; P = 0.016), ALK/ROS1/RET fusions was significantly associated with the FH-NPC (13.7%, 0.0%, and 5.0%, respectively; P = 0.004), but KRAS mutation was not associated with any type of the FH (13.8% vs. 6.0% vs. 7.8%, respectively; P = 0.288). CONCLUSION: The type of family history of cancer was associated with distinct clinocogenomic subtypes and prognosis of never-smoker lung adenocarcinoma.

Modified U1 snRNA and antisense oligonucleotides rescue splice mutations in SLC26A4 that cause hereditary hearing loss.

One of most important factors for messenger RNA (mRNA) transcription is the spliceosomal component U1 small nuclear RNA (snRNA), which recognizes 5' splicing donor sites at specific regions in pre-mRNA. Mutations in these sites disrupt U1 snRNA binding and cause abnormal splicing. In this study, we investigated mutations at splice sites in SLC26A4 (HGNC 8818), one of the major causative genes of hearing loss, which may result in the synthesis of abnormal pendrin, the channel protein encoded by the gene. Seventeen SLC26A4 variants with mutations in the U1 snRNA binding sites were assessed by minigene splicing assays, and 11 were found to result in abnormal splicing. Interestingly, eight of the 11 pathogenic mutations were intronic, suggesting the importance of conserved sequences at the intronic splice site. The application of modified U1 snRNA effectively rescued the abnormal splicing for most of these mutations. Although three were cryptic mutations, they were rescued by cotransfection of modified U1 snRNA and modified antisense oligonucleotides. Our results demonstrate the important role of snRNA in SLC26A4 mutations, suggesting the therapeutic potential of modified U1 snRNA and antisense oligonucleotides for neutralizing the pathogenic effect of the splice-site mutations that may result in hearing loss.

Anti-cancer effect of doxorubicin is mediated by downregulation of HMG-Co A reductase via inhibition of EGFR/Src pathway.

Doxorubicin is a widely used DNA damage-inducing anti-cancer drug. However, its use is limited by its dose-dependent side effects, such as cardiac toxicity. Cholesterol-lowering statin drugs increase the efficacy of some anti-cancer drugs. Cholesterol is important for cell growth and a critical component of lipid rafts, which are plasma membrane microdomains important for cell signaling. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-CR) is a critical enzyme in cholesterol synthesis. Here, we show that doxorubicin downregulated HMG-CR protein levels and thus reduced levels of cholesterol and lipid rafts. Cholesterol addition attenuated doxorubicin-induced cell death, and cholesterol depletion enhanced it. Reduction of HMG-CR activity by simvastatin, a statin that acts as an HMG-CR inhibitor, or by siRNA-mediated HMG-CR knockdown enhanced doxorubicin cytotoxicity. Doxorubicin-induced HMG-CR downregulation was associated with inactivation of the EGFR-Src pathway. Furthermore, a high-cholesterol-diet attenuated the anti-cancer activity of doxorubicin in a tumor xenograft mouse model. In a multivulva model of Caenorhabditis elegans expressing an active-EGFR mutant, doxorubicin decreased hyperplasia more efficiently in the absence than in the presence of cholesterol. These data indicate that EGFR/Src/HMG-CR is a new pathway mediating doxorubicin-induced cell death and that cholesterol control could be combined with doxorubicin treatment to enhance efficacy and thus reduce side effects.

Upregulation of transforming growth factor-beta type I receptor by interferon consensus sequence-binding protein in osteosarcoma cells.

Transforming growth factor-beta (TGF-ß) is a known tumor suppressor, which also exerts a tumor promoting activity at an advanced stage of cancer. Previously, we reported that expression of interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, is positively correlated with TGF-ß type I receptor (TGF-ß RI) expression in osteosarcoma patient tissues. In this study, we demonstrated that ICSBP upregulated TGF-ß RI and induced epithelial-to-mesenchymal transition-like phenomena in human osteosarcoma cell lines. As determined by soft agar growth of osteosarcoma cells and xenografted mouse models, ICSBP increased tumorigenicity, which was reversed by ICSBP knock-down or a TGF-ß RI inhibitor. To test whether ICSBP directly regulates the promoter activity of TGF-ß RI, we performed a TGF-ß RI promoter assay, an electro mobility shift assay, and a chromatin immunoprecipitation assay. We observed that TGF-ß RI promoter was activated in ICSBP-overexpressing osteosarcoma cells. Exploiting serial deletions and mutations of the TGF-ß RI promoter, we found a putative ICSBP-binding site at nucleotides -216/-211 (GGXXTC) in the TGF-ß RI promoter. Our data suggest that ICSBP upregulates TGF-ß RI expression by binding to this site, causing ICSBP-mediated tumor progression in osteosarcoma cells. In addition, we found a positive correlation between ICSBP and TGF-ß RI expression in several types of tumors using the cBioportal database. SUMMARY: We demonstrated that interferon consensus sequence-binding protein upregulates transforming growth factor-beta type I receptor (TGF-ß RI) expression by binding to nucleotides -216/-211 (GGXXTC) in the TGF-ß RI promoter, which resulted in increased tumorigenicity and tumor progression in human osteosarcoma cells.

Porous platinum nanoparticles as a high-Z and oxygen generating nanozyme for enhanced radiotherapy in vivo.

Radiotherapy (RT), along with surgery and chemotherapy, is a major modality of cancer therapy. Nevertheless, insufficient deposition of radiation energy in tumors and hypoxia-associated radioresistance remain the greatest challenges in RT. Here, we propose porous platinum nanoparticles as a new nanomedicine platform for solving these two problems at the same time using a single agent. Because of the combined advantages of a high-Z element and oxygen generation capability, porous platinum nanoparticles can significantly increase radiation-induced DNA damage, ROS stress, and cell cycle arrest by effectively depositing X-ray radiation energy within the cancer cells. Further, porous platinum nanoparticles increase tumor oxygenation by converting endogenic H2O2 to O2, thus greatly enhancing RT with no apparent in vivo toxicity to animals. This study presents a new nanomedicine strategy based on the use of porous high-Z metal nanoparticles with oxygen generation function for the synergistic enhancement of RT in cancer treatment.

FAM188B enhances cell survival via interaction with USP7.

We have previously reported that FAM188B showed significant differential exon usage in cancers (NCBI GEO GSE30727), but the expression and function of FAM188B is not well characterized. In the present study, we explored the functions of FAM188B by a knockdown strategy, using siRNAs specific for FAM188B in colon cancer cell lines. FAM188B is a novel gene that encodes a protein that is evolutionarily conserved among mammals. Its mRNA has been found to be highly expressed in most solid tumors, including colorectal cancer. FAM188B knockdown induced cell growth inhibition due to an increase in apoptosis in colon cancer cell lines. Interestingly, siFAM188B treatment induced the upregulation and activation of p53, and consequently increased p53-regulated pro-apoptotic proteins, PUMA and BAX. Proteomic analysis of FAM188B immunocomplexes revealed p53 and USP7 as putative FAM188B-interacting proteins. Deletion of the putative USP7-binding motif in FAM188B reduced complex formation of FAM188B with USP7. It is noteworthy that FAM188B knockdown resulted in a decrease in overall ubiquitination in the p53 immunocomplexes, as well as p53 ubiquitination, because USP7 is involved in p53 deubiquitination. FAM188B knockdown inhibited both colony formation and anchorage-independent growth in vitro. In addition, FAM188B knockdown by siRNA reduced tumor growth in xenografted mice, with an increase in p53 proteins. Taken together, our data suggest that FAM188B is a putative oncogene that functions via interaction with USP7. Therefore, control of FAM188B could be a possible target to inhibit tumor growth.

Redox-Responsive Manganese Dioxide Nanoparticles for Enhanced MR Imaging and Radiotherapy of Lung Cancer.

In this study, we synthesized manganese dioxide nanoparticles (MnO2 NPs) stabilized with biocompatible polymers (polyvinylpyrrolidone and polyacrylic acid) and analyzed their effect on non-small cell lung cancer (NSCLC) cells with or without gefitinib resistance in vitro. MnO2 NPs showed glutathione (GSH)-responsive dissolution and subsequent enhancement in magnetic resonance (MR) imaging. Of note, treatment with MnO2 NPs induced significant cytotoxic effects on NSCLC cells, and additional dose-dependent therapeutic effects were obtained upon X-ray irradiation. Normal cells treated with MnO2 NPs were viable at the tested concentrations. In addition, increased therapeutic efficacy could be achieved when the cells were treated with MnO2 NPs in hypoxic conditions. Therefore, we conclude that the use of MnO2 NPs in MR imaging and combination radiotherapy may be an efficient strategy for the imaging and therapy of NSCLC.

CD44v8-10 as a potential theranostic biomarker for targeting disseminated cancer cells in advanced gastric cancer.

Gastric cancer is the third most common cause of cancer mortality, and the survival rate of stage IV advanced gastric cancer (AGC) patients with distant metastasis is very low. Thus, the detection and eradication of disseminated cancer cells by targeting cell surface molecules in AGC would improve patient survival. The hyaluronic acid receptor, CD44, has various isoforms generated by alternative splicing, and some isoforms are known to be correlated to gastric cancer. In this study, to find out the most appropriate CD44v for targeting AGC, we analysed the expression differences of CD44 isoforms at the mRNA level in stomach cancer cell lines as well as in 74 patients with AGC by using exon-specific qRT-PCR. Among the CD44v isoforms, CD44v8-10 was determined as the most promising biomarker for the development of theranostic agents of gastric cancer. Next, we synthesised the conjugate of anti-CD44v9 antibody with near-infrared fluorophore or photosensitiser, and then demonstrated its feasibility for target cell-specific imaging and photoimmunotherapy in gastric cancer. As a result, these conjugates have clearly demarcated the surface of CD44v8-10 expressing cancer cells and showed efficient phototoxic effects. Therefore, this study revealed that CD44v8-10 is the efficient theranostic biomarker to target disseminated cancer cells in AGC.

CHK2 is involved in the p53-independent radiosensitizing effects of valproic acid.

Radiotherapy is an effective treatment for the majority of types of localized solid cancer. However, the risk of side effects to the surrounding normal tissues limits radiotherapeutic approaches. Whilst the mechanism of action of valproic acid, an inhibitor of histone deacetylase, remains unknown, the inhibitor is a potential antineoplastic radiosensitizer. The present study demonstrated the in vitro radiosensitizing effects of valproic acid on the human breast cancer MCF7 cell line, and revealed that valproic acid increased the level of DNA breakage, apoptosis and senescence. In addition, western blot analyses revealed that valproic acid induced tumor suppressor protein (p)53 and p21 expression, and activated checkpoint kinase 2 (CHK2) in MCF7 cells and primary mouse embryonic fibroblasts. Notably, treatment with valproic acid also induced increases in the level of p21 protein levels and CHK2 activity in p53-null colon cancer HCT116 cells. Furthermore, the present study demonstrated that valproic acid-induced radiosensitization was largely dependent on the activity of CHK2. The results of the present study reveal that valproic acid may exhibit clinical utility with respect to increasing the anticancer efficacy of radiotherapy by affecting the level of p53.

Regulation of anoikis resistance by NADPH oxidase 4 and epidermal growth factor receptor.

BACKGROUND: Normal cells are sensitive to anoikis, which is a cell detachment-induced apoptosis. However, cancer cells acquire anoikis resistance that is essential for successful metastasis. This study aimed to demonstrate the function and potential mechanism of NADPH oxidase 4 (NOX4) and EGFR activation in regulating anoikis resistance in lung cancer. METHODS: Cells were cultured either in the attached or suspended condition. Cell viability was measured by cell counting and live and dead cell staining. Expression levels of NOX4 and EGFR were measured by PCR and immunoblotting. Reactive oxygen species (ROS) levels were measured by flow cytometry. Effects of NOX4 overexpression or NOX4 knockdown by si-NOX4 on anoikis sensitivity were explored. Levels of NOX4 and EGFR in lung cancer tissues were evaluated by IHC staining. RESULTS: NOX4 was upregulated but EGFR decreased in suspended cells compared with attached cells. Accordingly, ROS levels were increased in suspended cells, resulting in the activation of Src and EGFR. NOX4 knockdown decreased activation of Src and EGFR, and thus sensitised cells to anoikis. NOX4 overexpression increased EGFR levels and attenuated anoikis. NOX4 expression is upregulated and is positively correlated with EGFR levels in the lung cancer patient tissues. CONCLUSIONS: NOX4 upregulation confers anoikis resistance by ROS-mediated activation of EGFR and Src, and by maintaining EGFR levels, which is critical for cell survival.

Overexpression of KLHL23 protein from read-through transcription of PHOSPHO2-KLHL23 in gastric cancer increases cell proliferation.

Gene fusion, as a prototypical pathognomonic mutation, contributes to genome complexity, and the cis-transcription-induced gene fusions generated by read-through transcription of adjacent genes have been found to be important for tumor development. We screened read-through transcription events from stomach adenocarcinoma RNA-seq data and selected three candidates PHOSPHO2-KLHL23, RPL17-C18orf32, and PRR5-ARHGAP8, to assess their biological role in gastric cancer. The expression of all three read-through fusion transcripts was confirmed in gastric cancer cell lines and paired normal/tumor gastric cancer tissues by real-time quantitative reverse transcription polymerase chain reaction and their expression was found to be significantly higher in the tumor (P < 0.05; n = 75). The correlation between the expression level and clinicopathological information was statistically analyzed. The level of the PHOSPHO2-KLHL23 read-through fusion transcript correlated with the Lauren classification and was significantly associated with the presence of perineural invasion. Overexpression of KLHL23 from PHOSPHO2-KLHL23 read-through transcript led to a significant increase in cell proliferation and resistance to anticancer drug treatment. Silencing of KLHL23 expression decreased cyclin D1 levels. The expression of KLHL23 from prevalent read-through transcripts of PHOSPHO2-KLHL23 in gastric cancer may undermine the efficacy of anticancer drug treatment.

Photosensitizer-conjugated tryptophan-containing peptide ligands as new dual-targeted theranostics for cancers.

Here we report that new dual-targeted theranostic anti-cancer agents can be produced by simple conjugation of photosensitizers with tryptophan-containing peptide ligands via cyclic disulfide linkages. In the proof-of-concept study, photosensitizers conjugated with EGFR-targeting peptide GE11 (C-EGFR) were in close proximity with tryptophan residues in the conjugate, resulting in quenching of its fluorescence and singlet oxygen generation. C-EGFR specifically binds to target receptors on the cancer cell surface, after which it is internalized via receptor-mediated endocytosis. Intracellular cleavage of cyclic disulfide bonds allows separation of the photosensitizers from the tryptophan residue, after which they emit near-infrared (NIR) fluorescence and produce a phototoxic effect in the target cells. This strategy enabled us to accomplish simultaneous real-time NIR fluorescence imaging of EGFR-overexpressing cancer cells with high contrast and selective photodynamic therapy.