S2 Peptide-Conjugated SARS-CoV-2 Virus-like Particles Provide Broad Protection against SARS-CoV-2 Variants of Concern.

Approved COVID-19 vaccines primarily induce neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. However, the emergence of variants of concern with RBD mutations poses challenges to vaccine efficacy. This study aimed to design a next-generation vaccine that provides broader protection against diverse coronaviruses, focusing on glycan-free S2 peptides as vaccine candidates to overcome the low immunogenicity of the S2 domain due to the N-linked glycans on the S antigen stalk, which can mask S2 antibody responses. Glycan-free S2 peptides were synthesized and attached to SARS-CoV-2 virus-like particles (VLPs) lacking the S antigen. Humoral and cellular immune responses were analyzed after the second booster immunization in BALB/c mice. Enzyme-linked immunosorbent assay revealed the reactivity of sera against SARS-CoV-2 variants, and pseudovirus neutralization assay confirmed neutralizing activities. Among the S2 peptide-conjugated VLPs, the S2.3 (N1135-K1157) and S2.5 (A1174-L1193) peptide-VLP conjugates effectively induced S2-specific serum immunoglobulins. These antisera showed high reactivity against SARS-CoV-2 variant S proteins and effectively inhibited pseudoviral infections. S2 peptide-conjugated VLPs activated SARS-CoV-2 VLP-specific T-cells. The SARS-CoV-2 vaccine incorporating conserved S2 peptides and CoV-2 VLPs shows promise as a universal vaccine capable of generating neutralizing antibodies and T-cell responses against SARS-CoV-2 variants.

Novel S2 subunit-specific antibody with broad neutralizing activity against SARS-CoV-2 variants of concern.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), had a major impact on both the global health and economy. Numerous virus-neutralizing antibodies were developed against the S1 subunit of SARS-CoV-2 spike (S) protein to block viral binding to host cells and were authorized for control of the COVID-19 pandemic. However, frequent mutations in the S1 subunit of SARS-CoV-2 enabled the emergence of immune evasive variants. To address these challenges, broadly neutralizing antibodies targeting the relatively conserved S2 subunit and its epitopes have been investigated as antibody therapeutics and universal vaccines. Methods: We initiated this study by immunizing BALB/c mice with ß-propiolactone-inactivated SARS-CoV-2 (IAV) to generate B-cell hybridomas. These hybridomas were subsequently screened using HEK293T cells expressing the S2-ECD domain. Hybridomas that produced anti-S2 antibodies were selected, and we conducted a comprehensive evaluation of the potential of these anti-S2 antibodies as antiviral agents and versatile tools for research and diagnostics. Results: In this study, we present a novel S2-specific antibody, 4A5, isolated from BALB/c mice immunized with inactivated SARS-CoV-2. 4A5 exhibited specific affinity to SARS-CoV-2 S2 subunits compared with those of other ß-CoVs. 4A5 bound to epitope segment F1109-V1133 between the heptad-repeat1 (HR1) and the stem-helix (SH) region. The 4A5 epitope is highly conserved in SARS-CoV-2 variants, with a significant conformational feature in both pre- and postfusion S proteins. Notably, 4A5 exhibited broad neutralizing activity against variants and triggered Fc-enhanced antibody-dependent cellular phagocytosis. Discussion: These findings offer a promising avenue for novel antibody therapeutics and insights for next-generation vaccine design. The identification of 4A5, with its unique binding properties and broad neutralizing capacity, offers a potential solution to the challenge posed by SARS-CoV-2 variants and highlights the importance of targeting the conserved S2 subunit in combating the COVID-19.

Development of nucleocapsid-specific monoclonal antibodies for SARS-CoV-2 and their ELISA diagnostics on an automatic microfluidic device.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has threatened public health globally, and the emergence of viral variants has exacerbated an already precarious situation. To prevent further spread of the virus and determine government action required for virus control, accurate and rapid immunoassays for SARS-CoV-2 diagnosis are urgently needed. In this study, we generated monoclonal antibodies (mAbs) against the SARS-CoV-2 nucleocapsid protein (NP), compared their reactivity using an enzyme-linked immunosorbent assay (ELISA), and selected four mAbs designated 1G6, 3E10, 3F10, and 5B6 which have higher reactivity to NP and viral lysates of SARS-CoV-2 than other mAbs. Using an epitope mapping assay, we identified that 1G6 detected the C-terminal domain of SARS-CoV-2 NP (residues 248-364), while 3E10 and 3F10 bound to the N-terminal domain (residues 47-174) and 3F10 detected the N-arm region (residues 1-46) of SARS-CoV-2 NP. Based on the epitope study and sandwich ELISA, we selected the 1G6 and 3E10 Abs as an optimal Ab pair and applied them for a microfluidics-based point-of-care (POC) ELISA assay to detect the NPs of SARS-CoV-2 and its variants. The integrated and automatic microfluidic system could operate the serial injection of the sample, the washing solution, the HRP-conjugate antibody, and the TMB substrate solution simply by controlling air purge via a single syringe. The proposed Ab pair-equipped microsystem effectively detected the NPs of SARS-CoV-2 variants as well as in clinical samples. Collectively, our proposed platform provides an advanced protein-based diagnostic tool for detecting SARS-CoV-2.

Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide­conjugated BSA.

Tumor­associated (TA) autoantibodies are considered to be promising biomarkers for the early detection of cancer, prior to the development of clinical symptoms. In the present study, a novel TA autoantibody was detected, which may prove to be useful as a diagnostic marker of human HCC using an HBx­transgenic (HBx­tg) hepatocellular carcinoma (HCC) mouse model. Its target antigen was identified as the bromodomain­containing protein 2 (BRD2), a transcriptional regulator that plays a pivotal role in the transcriptional control of diverse genes. BRD2 was upregulated in HCC tissues of the H­ras12V­tg mouse and human subjects, as demonstrated using western blotting or immunohistochemical analysis, with the BRD2 autoantibody. In addition, the truncated BRD2 reactive to the BRD2 autoantibody was detected in tumor cell­derived exosomes, which possibly activated TA immune responses and the generation of autoantibodies. For the detection of the serum BRD2 autoantibody, epitope mimicries of autoantigenic BRD2 were screened from a random cyclic peptide CX7C library with the BRD2 autoantibody. A mimotope with the sequence of CTSVFLPHC, which was cyclized by one pair of cysteine residues, exhibited high affinity to the BRD2 autoantibody and competitively inhibited the binding of the autoantibody to the cellular BRD2 antigen. The use of this cyclic peptide as a capture antigen in human serum enzyme­linked immunosorbent assay allowed the distinction of patients with HCC from healthy subjects with 64.41% sensitivity and 82.42% specificity (area under the ROC curve, 0.7761), which is superior to serum alpha­fetoprotein (AFP; 35.83% sensitivity; 100% specificity; area under the ROC curve, 0.5337) for the diagnosis of HCC. In addition, the detection of the BRD2 autoantibody combined with other autoantibody biomarkers or AFP has increased the accuracy of HCC diagnosis, suggesting that the combinational detection of cancer biomarkers, including the BRD2 autoantibody, is a promising assay for HCC diagnosis.

Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop.

Cancer stem cells (CSCs) are regarded as essential targets to overcome tumor progression and therapeutic resistance; however, practical targeting approaches are limited. Here, we identify testis-specific Y-like protein 5 (TSPYL5) as an upstream regulator of CSC-associated genes in non-small cell lung cancer cells, and suggest as a therapeutic target for CSC elimination. TSPYL5 elevation is driven by AKT-dependent TSPYL5 phosphorylation at threonine-120 and stabilization via inhibiting its ubiquitination. TSPYL5-pT120 also induces nuclear translocation and functions as a transcriptional activator of CSC-associated genes, ALDH1 and CD44. Also, nuclear TSPYL5 suppresses the transcription of PTEN, a negative regulator of PI3K signaling. TSPYL5-pT120 maintains persistent CSC-like characteristics via transcriptional activation of CSC-associated genes and a positive feedback loop consisting of AKT/TSPYL5/PTEN signaling pathway. Accordingly, elimination of TSPYL5 by inhibiting TSPYL5-pT120 can block aberrant AKT/TSPYL5/PTEN cyclic signaling and TSPYL5-mediated cancer stemness regulation. Our study suggests TSPYL5 be an effective target for therapy-resistant cancer.

Cyclic Peptide Mimotopes for the Detection of Serum Anti-ATIC Autoantibody Biomarker in Hepato-Cellular Carcinoma.

Tumor-associated (TA) autoantibodies have been identified at the early tumor stage before developing clinical symptoms, which holds hope for early cancer diagnosis. We identified a TA autoantibody from HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) model mouse, characterized its target antigen, and examined its relationship to human HCC. The mimotopes corresponding to the antigenic epitope of TA autoantibody were screened from a random cyclic peptide library and used for the detection of serum TA autoantibody. The target antigen of the TA autoantibody was identified as an oncogenic bi-functional purine biosynthesis protein, ATIC. It was upregulated in liver cancer tissues of HBx-tg mouse as well as human HCC tissues. Over-expressed ATIC was also secreted extracellularly via the cancer-derived exosomes, which might cause auto-immune responses. The cyclic peptide mimotope with a high affinity to anti-ATIC autoantibody, CLPSWFHRC, distinguishes between serum samples from HCC patients and healthy subjects with 70.83% sensitivity, 90.68% specificity (AUC = 0.87). However, the recombinant human ATIC protein showed a low affinity to anti-ATIC autoantibody, which may be incompatible as a capture antigen for serum TA autoantibody. This study indicates that anti-ATIC autoantibody can be a potential HCC-associated serum biomarker and suggests that autoantibody biomarker's efficiency can be improved by using antigenic mimicry to native antigens present in vivo.

HSPA1L Enhances Cancer Stem Cell-Like Properties by Activating IGF1Rß and Regulating ß-Catenin Transcription.

Studies have shown that cancer stem cells (CSCs) are involved in resistance and metastasis of cancer; thus, therapies targeting CSCs have been proposed. Here, we report that heat shock 70-kDa protein 1-like (HSPA1L) is partly involved in enhancing epithelial-mesenchymal transition (EMT) and CSC-like properties in non-small cell lung cancer (NSCLC) cells. Aldehyde dehydrogenase 1 (ALDH1) is considered a CSC marker in some lung cancers. Here, we analyzed transcriptional changes in genes between ALDH1high and ALDH1low cells sorted from A549 NSCLC cells and found that HSPA1L was highly expressed in ALDH1high cells. HSPA1L played two important roles in enhancing CSC-like properties. First, HSPA1L interacts directly with IGF1Rß and integrin αV to form a triple complex that is involved in IGF1Rß activation. HSPA1L/integrin αV complex-associated IGF1Rß activation intensified the EMT-associated cancer stemness and γ-radiation resistance through its downstream AKT/NF-κB or AKT/GSK3ß/ß-catenin activation pathway. Secondly, HSPA1L was also present in the nucleus and could bind directly to the promoter region of ß-catenin to function as a transcription activator of ß-catenin, an important signaling protein characterizing CSCs by regulating ALDH1 expression. HSPA1L may be a novel potential target for cancer treatment because it both enhances IGF1Rß activation and regulates γß-catenin transcription, accumulating CSC-like properties.

Serum anti-EIF3A autoantibody as a potential diagnostic marker for hepatocellular carcinoma.

Tumor-associated autoantibodies are promising diagnostic biomarkers for early detection of tumors. We have screened a novel tumor-associated autoantibody in hepatocellular carcinoma (HCC) model mice. Its target antigen was identified as eukaryotic translation initiation factor 3 subunit A (EIF3A) by proteomic analysis, and the elevated expression of EIF3A in HCC tissues of tumor model mice as well as human patients was shown. Also, its existence in tumor-derived exosomes was revealed, which seem to be the cause of tumor-associated autoantibody production. To use serum anti-EIF3A autoantibody as biomarker, ELISA detecting anti-EIF3A autoantibody in human serum was performed using autoantibody-specific epitope. For the sensitive detection of serum autoantibodies its specific conformational epitopes were screened from the random cyclic peptide library, and a streptavidin antigen displaying anti-EIF3A autoantibody-specific epitope, XC90p2(-CPVRSGFPC-), was used as capture antigen. It distinguished patients with HCC (n = 102) from healthy controls (n = 0285) with a sensitivity of 79.4% and specificity of 83.5% (AUC = 0.87). Also, by simultaneously detecting with other HCC biomarkers, including alpha-fetoprotein, HCC diagnostic sensitivity improved from 79.4% to 85%. Collectively, we suggest that serum anti-EIF3A autoantibody is a useful biomarker for the diagnosis of HCC and the combinational detection of related biomarkers can enhance the accuracy of the cancer diagnosis.

Hypoxia-inducible transgelin 2 selects epithelial-to-mesenchymal transition and γ-radiation-resistant subtypes by focal adhesion kinase-associated insulin-like growth factor 1 receptor activation in non-small-cell lung cancer cells.

Microenvironment, such as hypoxia common to cancer, plays a critical role in the epithelial-to-mesenchymal transition (EMT) program, which is a major route of cancer metastasis and confers γ-radiation resistance to cells. Herein, we showed that transgelin 2 (TAGLN2), an actin-binding protein, is significantly induced in hypoxic lung cancer cells and that Snail1 is simultaneously increased, which induces EMT by downregulating E-cadherin expression. Forced TAGLN2 expression induced severe cell death; however, a small population of cells surviving after forced TAGLN2 overexpression showed γ-radiation resistance, which might promote tumor relapse and recurrence. These surviving cells showed high metastatic activity with an increase of EMT markers including Snail1. In these cells, TAGLN2 activated the insulin-like growth factor 1 receptor ß (IGF1Rß)/PI3K/AKT pathway by recruitment of focal adhesion kinase to the IGF1R signaling complex. Activation of the IGF1Rß/PI3K/AKT pathway also induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which is involved in Snail1 stabilization. Therefore, both the IGF1Rß inhibitor (AG1024) and the PI3K inhibitor (LY294002) or AKT inactivation with MK2206 lower the cellular level of Snail1. Involvement of GSK3ß was also confirmed by treatment with lithium chloride, the inducer of GSK3ß phosphorylation, or MG132, the 26S proteasomal inhibitor, which also stabilized Snail1. In conclusion, the present study provides important evidence that hypoxia-inducible TAGLN2 is involved in the selection of cancer cells with enhanced EMT properties to overcome the detrimental environment of cancer cells.

Tescalcin/c-Src/IGF1Rß-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1.

Tescalcin (TESC; also known as calcineurin B homologous protein 3, CHP3) has recently reported as a regulator of cancer progression. Here, we showed that the elevation of TESC in non-small cell lung cancer (NSCLC) intensifies epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties, consequently enhancing the cellular resistance to γ-radiation. TESC expression and the phosphorylation (consequent activation) of signal transducer and activator of transcription 3 (STAT3) were upregulated in CSC-like ALDH1high cells than in ALDH1low cells sorted from A549 NSCLC cells. Knockdown of TESC suppressed CSC-like properties as well as STAT3 activation through inhibition of insulin-like growth factor 1 receptor (IGF1R), a major signaling pathway of lung cancer stem cells. TESC activated IGF1R by the direct recruitment of proto-oncogene tyrosine kinase c-Src (c-Src) to IGF1Rß complex. Treatment of IGF1R inhibitor, AG1024, also suppressed c-Src activation, implicating that TESC mediates the mutual activation of c-Src and IGF1R. STAT3 activation by TESC/c-Src/IGF1R signaling pathway subsequently upregulated ALDH1 expression, which enhanced EMT-associated CSC-like properties. Chromatin immunoprecipitation and luciferase assay demonstrated that STAT3 is a potential transcription activator of ALDH1 isozymes. Ultimately, targeting TESC can be a potential strategy to overcome therapeutic resistance in NSCLC caused by augmented EMT and self-renewal capacity.

Identification of anti-SF3B1 autoantibody as a diagnostic marker in patients with hepatocellular carcinoma.

BACKGROUND: Tumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients' serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum. METHODS: To overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens. RESULTS: In this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081). CONCLUSIONS: ELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.

Development and Characterization of Monoclonal Antibodies against Nucleoprotein for Diagnosis of Influenza A Virus.

Influenza, which is a highly contagious disease caused by the influenza A virus, continues to be a major health concern worldwide. Although the accurate and early diagnosis of influenza virus infection is important for controlling the spread of this disease and rapidly initiating antiviral therapy, the current influenza diagnostic kits are limited by their low sensitivity. In this study, we developed several new influenza nucleoprotein (NP)-specific monoclonal antibodies (mAbs) and compared their sensitivity and specificity of those with commercially available anti-NP mAbs. Three mAbs, designated M24.11, M34.3, and M34.33, exhibited higher reactivities to recombinant NPs and A/Puerto Rico/8/1934 (H1N1) viral lysates compared with the commercial mAbs, as assessed using enzyme-linked immunosorbent assays. M34.3 and M34.33 showed higher reactivities with A/California/04/09 (pandemic H1N1) and A/Philippines/2/82 (H3N2) viral lysates than the commercial mAbs. In contrast, M24.11 had marked reactivity with H3N2 but not with pandemic H1N1. Immunofluorescent confocal microscopy showed that the three mAbs effectively detected the presence of influenza virus in lung tissues of mice infected with A/Puerto Rico/8/1934. These results indicate that the newly developed M34.3 and M34.33 mAbs could be useful for the development of influenza diagnostics.

Osteopontin production by TM4SF4 signaling drives a positive feedback autocrine loop with the STAT3 pathway to maintain cancer stem cell-like properties in lung cancer cells.

Transmembrane 4 L6 family proteins have been known to promote cancer. In this study, we demonstrated that transmembrane 4 L6 family member 4 (TM4SF4), which is induced by γ-radiation in non-small cell lung cancer (NSCLC) cells, is involved in epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) properties of NSCLC through the regulation of osteopontin (OPN). Forced TM4SF4 overexpression in A549 cells increased the secretion of OPN, which activates CD44 or integrin signaling and thus maintains EMT-associated CSC-like properties. OPN, known as a downstream target of ß-catenin/T-cell factor 4 (TCF-4), was induced by up-regulated ß-catenin via TM4SF4-driven phosphorylation of glycogen synthase kinase 3b (GSK3ß). TCF4 complexed to promoter regions of OPN in TM4SF4-overexpressing A549 cells was also confirmed by chromatin immunoprecipitation. Knockout of either ß-catenin or TCF4-suppressed OPN expression, demonstrating that both factors are essential for OPN expression in NSCLC cells. OPN secreted by TM4SF4/GSK3ß/ß-catenin signaling activated the JAK2/STAT3 or FAK/STAT3 pathway, which also up-regulates OPN expression in an autocrine manner and consequently maintains the self-renewal and metastatic capacity of cancer cells. Neutralizing antibody to OPN blocked the autocrine activation of OPN expression, consequently weakened the metastatic and self-renewal capacity of cancer cells. Collectively, our findings indicate that TM4SF4-triggered OPN expression is involved in the persistent reinforcement of EMT or cancer stemness by creating a positive feedback autocrine loop with JAK2/STAT3 or FAK/STAT3 pathways.

Enhanced expression of cell-surface B-cell receptor-associated protein 31 contributes to poor survival of non-small cell lung carcinoma cells.

B-cell receptor-associated protein 31 (BAP31) is an endoplasmic reticulum (ER) membrane protein which plays a role as a molecular chaperone for the newly synthesized transmembrane proteins. BAP31 is also an important apoptosis regulator for extrinsic apoptosis induction in the ER membrane. Recent studies have shown that BAP31 is also expressed on the surface of embryonic stem cells. However, the function of cell surface BAP31 (csBAP31) still remains unclarified. In an attempt to search for surface markers on tumorspheres, here, we generated monoclonal antibodies (MAbs) against the sphere cells from the non-small cell lung carcinoma cell (NSCLC) line A549. SP1-B7, one of the MAbs, recognized csBAP31 whose expression was further increased on A549 sphere cells, as compared with A549 adherent cells. To investigate the role of csBAP31 in A549 cells, A549 adherent and sphere cells were stained with annexin V, propidium iodide, and SP1-B7. Interestingly, annexin V-high cells showed increased expression of csBAP31 as compared with annexin V-low cells. Caspase-3/7 activity was also increased in csBAP31-high cells as compared with csBAP31-low cells, suggesting that csBAP31-high cells are more sensitive to apoptosis. To further demonstrate the survival of csBAP31-positive A549 cells, csBAP31-positive and -negative A549 cells were sorted and subjected to the clonogenic survival assay. The colony number of csBAP31-positive A549 cells was decreased by approximately 1.7-fold, as compared that of csBAP31-negative A549 cells, suggesting that csBAP31-positve cells are sensitive to cell death indeed. The results suggest that enhanced expression of csBAP31 contributes to poor survival of NSCLC cells.

APBB1 reinforces cancer stem cell and epithelial-to-mesenchymal transition by regulating the IGF1R signaling pathway in non-small-cell lung cancer cells.

Amyloid ß precursor protein binding family B member 1(APBB1) was first identified as a binding partner of amyloid precursor protein during brain development, but its function in the context of cancer remain unclear. Here we show for the first time that APBB1 is partly associated with intensifying cancer stem cell(CSC) and epithelial-to-mesenchymal transition (EMT) and enhancing radiation-resistant properties of lung cancer cells. We found that APBB1 was highly expressed in ALDH1high CSC-like cells sorted from A549 lung cancer cells. In APBB1-deficient H460 cells with forced overexpression of APBB1, the protein directly interacted with IGF1Rß, enhanced phosphorylation of IGF1Rß/PI3K/AKT pathway(activation) and subsequently induced the phosphorylation of GSK3ß(inactivation). This phosphorylation stabilized Snail1, a negative regulator of E-cadherin expression, and regulated ß-catenin-mediated ALDH1 expression, which are representative markers for EMT and CSCs, respectively. In contrast, suppression of APBB1 expression with siRNA yielded the opposite effects in APBB1-rich A549 cells. We concluded that APBB1 partly regulates the expression of ALDH1. We also found that APBB1 regulates activation of nuclear factor-κB, which is involved in reducing various stresses including oxidative stress, which suggests that APBB1 is associated with γ-radiation sensitivity. Our findings imply that APBB1 plays an important role in the maintenance of EMT-associated CSC-like properties and γ-radiation resistance via activation of IGF1Rß/AKT/GSK3ß pathway in lung cancer cells, highlighting APBB1 as a potential target for therapeutic cancer treatment.

Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines.

Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.

Genistein decreases cellular redox potential, partially suppresses cell growth in HL­60 leukemia cells and sensitizes cells to γ­radiation­induced cell death.

Various mechanisms have been proposed to underlie the cellular activity of genistein, based on biological experiments and epidemiological studies. The present study demonstrated that genistein inhibited the expression of cytoplasmic nicotinamide adenine dinucleotide phosphate (NADP)­dependent isocitrate dehydrogenase (cICDH), thus increasing levels of intracellular reactive oxygen species (ROS) in human promyeloid leukemia HL­60 cells. In genistein­treated cells, the cellular redox potential (GSH/GSSG) was significantly decreased. This decrease in redox potential was caused by significant downregulation of the cICDH gene, generating the reducing equivalents (NADPH) for maintenance of cellular redox potential and cellular ROS level, which may regulate cell growth and cell death. Genistein­induced ROS partially induced rapid transition into the G2/M phase by upregulation of p21wap1/cip1 and apoptotic cell death. Treatment of cells with N­acetylcysteine, a well­known antioxidant (ROS scavenger), not only partially restored cell growth and inhibited cell cycle arrest in G2/M, but also prevented apoptotic cell death. By contrast, normal lymphocytes did not significantly progress into the G2/M phase and radiation­induced cell death was inhibited by genistein treatment. Therefore, genistein and γ­irradiation together synergistically cause cell death in leukemia cells, however, genistein has a radioprotective effect in normal human lymphocytes. In conclusion, it was suggested that genistein selectively functions, not as an antioxidant, but as a pro­oxidant in HL­60 cells. This property can increase ionizing radiation­induced cell cycle arrest and sensitivity to apoptotic cell death in human promyeloid leukemia HL­60 cells, but does not cause significant damage to normal cells.

TM4SF4 overexpression in radiation-resistant lung carcinoma cells activates IGF1R via elevation of IGF1.

Transmembrane 4 L six family member 4 (TM4SF4) is a member of the tetraspanin L6 domain family. Other members of this family, TM4SF1 (also known as L6-Ag) and TM4SF5, have been shown to be upregulated in multiple tumors and involved in epithelial-to-mesenchymal transition and cell migration. However, unlike its homologs, little is known about TM4SF4. Here, we show that TM4SF4 was highly expressed in radiation-resistant lung adenocarcinoma cells, such as A549 and Calu-3 cells, and its expression activated cell growth, migration, and invasion. Overexpression of TM4SF4 in A549 cells increased the activation of PI3K, AKT, and NF-kappaB and the expression of PTEN. IGF1R was clearly activated by overexpression of TM4SF4, although EGFR was also slightly activated. TM4SF4 expression was correlated with the increased expression of IGF1, consequently resulting in IGF1R activation. Tumorigenic activity of TM4SF4 in lung adenocarcinoma cells was also demonstrated by xenograft assay; however, this activity was almost completely suppressed by treatment with anti-TM4SF4 antibody. Our results suggest that TM4SF4 overexpression in lung carcinoma cells results in resistance to radiotherapy via IGF1-induced IGF1R activation and blocking the activity of TM4SF4 using specific antibody can be a promising therapeutics against TM4SF4-overexpressing lung adenocarcinoma.

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity.

Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cells than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial-mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.

Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer.

A novel circulating tumor-associated autoantibody, K94, obtained from a hepatocellular carcinoma (HCC) mouse model was characterized. The target antigen of K94 autoantibody was expressed in various tumor cell lines including liver cancer, and its secretion was detectable using MCF-7 breast carcinoma cells. Proteomic analysis revealed that the protein bands reactive to K94 included cytokeratin (CK) 8 and 18, which are known to be related to tumorigenesis and form a heterotypic complex with each other. However, K94 showed no activity toward CK8 or CK18 separately. The epitope of the K94 antibody was only presented by a complex between CK8 and CK18, which was confirmed by analysis using recombinant CK8 and CK18 proteins. To formulate an assay for anti-CK8/18 complex autoantibody, a mimotope peptide reactive to K94 was selected from loop-constrained heptapeptide (-CX7C-) display phage library, of which sequence was CISPDAHSC (K94p1). A mimotope enzyme-linked immunosorbent assay (ELISA) using phage-displayed K94p1 peptide as a coating antigen was able to discriminate breast cancer (n=30) patients from normal subjects (n=30) with a sensitivity of 50% and a specificity of 82.61%. CA15.3 was detected at very low levels in the same breast cancer subjects and did not discriminate breast cancer patients from normal subjects, although it is a conventional biomarker of breast cancer. These results suggest that a mimotope ELISA composed of K94p1 peptide may be useful for the diagnosis of breast cancer.