Bioinformatics Prediction and in vivo Verification Identify SLC7A5 as Immune Infiltration Related Biomarker in Breast Cancer.

Purpose: Solute carrier family 7, member 5 (SLC7A5) is reportedly a key gene in various tumors. However, the relationship between SLC7A5 and immune cell infiltration in breast cancer remains elusive. In this study, we investigated the expression levels and clinical value of SLC7A5 using the R package in breast cancer through the TCGA database. Methods: Public transcript data and clinical information of patients were downloaded from the TCGA database. Prognosis analysis was performed to explore the clinical value of SLC7A5 as well as drug sensitivity prediction and Gene Set Enrichment Analysis (GSEA). We also explored the correlations between SLC7A5 and immune infiltration as well as immune markers. Results: We observed that high SLC7A5 expression levels were related to poor survival. Our GSEA demonstrated that G protein-coupled receptor-ligand binding, interleukin signaling, neutrophil degranulation, amino acid and derivative metabolism, and Rho GTPase signaling were differentially enriched in the SLC7A5 high-expression phenotype. Patients in the low SLC7A5 expression group showed sensitivity to paclitaxel and doxorubicin. Conclusion: SLC7A5 could serve as a biomarker for breast cancer prognosis.

Gamma synuclein promotes cancer metastasis through the MKK3/6-p38MAPK cascade.

Gamma synuclein (SNCG) is a neuronal protein that is also aberrantly overexpressed in various types of human cancer. SNCG overexpression promotes cancer invasion and metastasis. However, the mechanisms that drive cancer metastasis upon SNCG expression remain elusive. Elucidation of the mechanisms underlying the promotion of cancer metastasis by SNCG may help discover therapeutic avenues for SNCG-overexpressed cancer. Here, we show that SNCG promotes transforming growth factor-ß (TGF-ß)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Mechanistically, SNCG promotes p38MAPK phosphorylation by interacting with the MAPK kinase 3/6 (MKK3/6) and prevents their degradation. SNCG knockdown leads to a decrease in TGF-ß-induced phosphorylation of MKK3/6; and abrogates the induction of matrix metalloproteinase (MMP)-9 expression by TGF-ß and its target gene Twist1. Furthermore, p38MAPK inhibition abrogates the promotion of MMP-9 expression and cancer cell invasion by SNCG. Both p38MAPK and MMP inhibitors can suppress the promotion of cancer cell invasion by SNCG. Finally, overexpression of SNCG in liver cancer cells promotes lung metastasis, which can be suppressed by the p38MAPK inhibitor. Together, our data uncover a previously unknown role of SNCG in promoting TGF-ß-MKK3/6-p38MAPK signaling. This study highlights the critical role of p38MAPK in the promotion of cancer metastasis by SNCG, and indicates that p38MAPK inhibitor may serve as a potential therapeutic for SNCG-overexpressed cancer.

LncRNA PCAT6 activated by SP1 facilitates the progression of breast cancer by the miR-326/LRRC8E axis.

Breast cancer is an aggressive malignancy with high morbidity in females worldwide. Extensive studies reveal that long noncoding RNAs (lncRNAs) are abnormally expressed and act as key regulators in various cancers, including breast cancer. In this work, we investigated the role and regulatory mechanism of lncRNA prostate cancer-associated transcript 6 (PCAT6) in breast cancer progression. Our findings revealed that PCAT6 was overexpressed in breast cancer tissues and cell lines. Furthermore, elevation of PCAT6 reflected an adverse prognosis of patients. Functional experiments indicated that PCAT6 knockdown hampered cell proliferation, facilitated apoptosis and cell cycle arrest in vitro, and inhibited tumor growth in vivo. We also found that the transcription factor SP1 could bind to the PCAT6 promoter and promoted its expression. Subsequently, it was verified that PCAT6 was a molecular sponge for microRNA-326 (miR-326), and the leucine-rich repeat containing the eight family member E (LRRC8E) was a direct target of miR-326. Rescue assays revealed that LRRC8E overexpression attenuated the suppressive effect of PCAT6 knockdown on cellular progression of breast cancer. In summary, this study demonstrated that SP1-activated PCAT6 promoted the malignant behaviors of breast cancer cells by regulating the miR-326/LRRC8E axis.

A dendritic cell-targeted chimeric hepatitis B virus immunotherapeutic vaccine induces both cellular and humoral immune responses in vivo.

Chimigen® HBV Immunotherapeutic Vaccine (C-HBV), a recombinant chimeric fusion protein comprising hepatitis B virus (HBV) S1 and S2 surface antigen fragments, Core antigen and a murine monoclonal antibody heavy chain fragment (Fc), was designed and produced in Sf9 insect cells. C-HBV targets the host immune system through specific receptors present on dendritic cells (DCs) which facilitates antigen internalization, processing, and presentation on MHC class I and II to induce both cellular and humoral immune responses against HBV antigens. T cell responses, previously assessed by ex vivo antigen presentation assays using human peripheral blood mononuclear cell (PBMC)-derived DCs and T cells from uninfected and HBV chronic-infected donors, demonstrated that C-HBV was highly immunogenic. A vaccine dose response study was performed in sheep to analyze the immunogenicity of C-HBV in vivo. Sheep (n = 8/group) received three consecutive subcutaneous injections of each dose of C-HBV at four-week intervals. Analysis of serum antibody levels confirmed C-HBV induced a dose-dependent antibody response to C-HBV and S1/S2-Core. Kinetics of the S1/S2-Core specific antibody response was similar to hepatitis B surface antigen (HBsAg)-specific antibody responses induced by ENGERIX-B. Analysis of cell-mediated immune responses (CMI) confirmed C-HBV induced both dose-dependent S1/S2-Core-specific lymphocyte proliferative responses and IFN-γ secretion. These responses were stronger with blood lymphocytes than with cells isolated from the lymph node draining the vaccination site. No correlation was seen between antibody titers and CMI. The results confirm C-HBV is an effective delivery vehicle for the induction of T cell responses and may be an appropriate candidate for immunotherapy for chronic HBV infections.

A dendritic cell receptor-targeted chimeric immunotherapeutic protein (C-HBV) for the treatment of chronic hepatitis B.

In chronic Hepatitis B Virus (HBV) infections HBV-specific T cells are functionally impaired. Immunotherapy may restore HBV-specific T cell responses essential for sustained disease remission off-treatment and induction of a functional cure. Chimigen® Molecules are fusion proteins of antigen(s) with the Fc fragment of a xenotypic antibody designed to target specific receptors on dendritic cells (DCs). Here we describe the production and pre-clinical evaluation of Chimigen® HBV (C-HBV), containing HBV PreS1 and PreS2 peptide fragments, HBV core and murine Fc, produced in insect cells. C-HBV binding to immature DCs and internalization by endocytosis was FcγRII (CD32) and mannose receptor (CD206) dependent and led to increased MHC I and MHC II surface expression. Upon exposure of human T cells isolated from HBV un-infected healthy and chronically HBV-infected donors to C-HBV-pulsed mature DCs ex vivo, C-HBV induced vigorous T cell proliferation and enhanced expression of IFN-γ, TNF-α, perforin and granzyme B in both CD4+ and CD8+ T cell subsets. Re-stimulation of C-HBV-activated T cells from chronically infected donors with HBV PreS1/PreS2 and core overlapping peptides induced IFN-γ production in both CD4+ and CD8+ populations. C-HBV-activation of peripheral blood mononuclear cells (PBMCs) from chronically HBV-infected patients stimulated granzyme B production by CD4+CD25- T responder (Tresp) cells, accompanied by an increase in Annexin V staining on CD4+CD25+ T regulatory (Treg) cell phenotype, consistent with apoptosis. The observed HBV-specific cellular responses induced by C-HBV ex vivo suggest that C-HBV is a promising immunotherapeutic candidate for the treatment of chronic HBV infections.

4-Hydroxy estrogen metabolite, causing genomic instability by attenuating the function of spindle-assembly checkpoint, can serve as a biomarker for breast cancer.

Sex hormone metabolism is altered during mammary gland tumorigenesis, and different metabolites may have different effects on mammary epithelial cells. This study aimed to evaluate associations between urinary sexual metabolite levels and breast cancer risk among premenopausal women of Mainland China. The molecular metabolism of the cancer-related metabolites was also explored based on the clinical data. The sex hormone metabolites in the urine samples of patients with breast cancer versus normal healthy women were analyzed comprehensively. Among many alterations of sex hormone metabolisms, 4-hydroxy estrogen (4-OH-E) metabolite was found to be significantly increased in the urine samples of patients with breast cancer compared with the normal healthy controls. This was the most important risk factor for breast cancer. Several experiments were conducted in vitro and in vivo to probe this mechanism. 4-Hydroxyestradiol (4-OH-E2) was found to induce malignant transformation of breast cells and tumorigenesis in nude mice. At the molecular level, 4-OH-E2 compromised the function of spindle-assembly checkpoint and rendered resistance to the anti-microtubule drug. Further, transgenic mice with high expression of CYP1B1, a key enzyme of 4-hydroxy metabolites, were established and stimulated with estrogen. Cancerous tissue was found to appear in the mammary gland of transgenic mice.

miR-942 promotes tumor migration, invasion, and angiogenesis by regulating EMT via BARX2 in non-small-cell lung cancer.

Epithelial-mesenchymal transition (EMT) has an important function in cancer. Recently, microRNAs have been reported to be involved in EMT by regulating target genes. miR-942 is considered a novel oncogene in esophageal squamous cell carcinoma. However, its role in non-small-cell lung cancer (NSCLC) has not been investigated. In this study, the expression of miR-942 in NSCLC patients tumor and paired adjacent tissues were assessed by quantitative real-time polymerase chain reaction and in situ hybridization. Transwell, wound healing, tube formation, and tail vein xenograft assays were conducted to assess miR-942's function in NSCLC. Potential miR-942 targets were confirmed using dual-luciferase reporter assays, immunohistochemistry, immunoblot, and rescue experiments. The results showed miR-942 is relatively highly expressed in human NSCLC tissues and cells. In vitro assays demonstrated that overexpression of miR-942 promoted cell migration, invasion, and angiogenesis. Tail vein xenograft assays suggested that miR-942 contributed to NSCLC metastasis in vivo. Three bioinformatics software was searched, and BARX2 was predicted as a downstream target of miR-942. Direct interaction between them was validated by dual-luciferase assays. Rescue experiments further confirmed that BARX2 overexpression could reverse functional changes caused by miR-942. Moreover, miR-942 increased EMT-associated proteins N-cadherin and vimentin by inhibiting BARX2, while E-cadherin expression is reduced. In summary, this study reveals that miR-942 induces EMT-related metastasis by directly targeting BARX2, which may provide a potential therapeutic strategy for NSCLC.

EZH2-Mediated Epigenetic Suppression of GDF15 Predicts a Poor Prognosis and Regulates Cell Proliferation in Non-Small-Cell Lung Cancer.

Growth differentiation factor 15 (GDF15), a member of the TGF-ß superfamily of cytokines, has been reported to exert very heterogeneous functions in various tumors. However, its expression and roles in mediating non-small-cell lung cancer (NSCLC) progression remain unknown. In this study, we found that GDF15 is downregulated in paired NSCLC tissues and correlated with poor clinical outcomes in NSCLC. A functional experiment demonstrated that overexpression of GDF15 significantly repressed NSCLC proliferation both in vitro and in vivo. Mechanistic studies reveal that inhibition of EZH2 expression prevented its binding to the GDF15 promoter region and reduced the trimethylation modification pattern of H3K27. Together, our data uncover that GDF15 is a direct target of EZH2 and, as a regulator of proliferation, might serve as a candidate prognostic biomarker and target for new therapies in human NSCLC.

Iodine stimulates estrogen receptor singling and its systemic level is increased in surgical patients due to topical absorption.

Iodine is crucial for thyroid hormone production. However, recent epidemiologic studies have shown that breast cancer patients have an elevated risk of developing thyroid cancer and vice versa. A notable finding in this study is that iodine stimulated the transcriptional activity of estrogen receptor-α (ER-α) in breast cancer cells. Iodine stimulated expression of several ER-α regulated gene including PS2, Cathepsin D, CyclinD1, and PR both in vitro and in nude mice, which is consistent with its stimulation of both anchorage-dependent and -independent growth of ER-α positive breast cancer cells and the effect to dampen tumor shrinkage of MCF-7 xenograft in ovariectomized nude mice. Analyses of clinical urine samples from breast cancer patients undergoing surgery demonstrated that urinary iodine levels were significantly higher than that in controls; and this increased level is due to the antiseptic use of iodine during breast surgery. The present study indicates that excess iodine intake may be an unfavorable factor in breast cancer by stimulation of ER-α transcriptional activity.

Sex Hormone Metabolism and Threatened Abortion.

BACKGROUND The aim of this study was to evaluate changes in sex hormone metabolism in patients with threatened miscarriage. MATERIAL AND METHODS We recruited 73 women in early pregnancy (6-8 weeks of gestation) and divided them into the following 2 groups based on whether they had vaginal bleeding: group A (n=34), the threatened abortion group; and group B (n=39), the normal pregnancy group. Human chorionic gonadotrophin (hCG), estradiol (E2), progesterone (P4), and testosterone (T) serum levels were tested and sex hormone metabolites in the urine were detected using gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). As the control, data for sex hormones and their metabolites were obtained in normal women of childbearing age without pregnancy (group C: n=23). RESULTS E2 and T serum levels were lower in women with threatened miscarriage (group A). Estrone (E1), E2, estriol (E3), 16α-hydroxyestrone (16α-OHE1), 4-methoxyestrone (4-MeOE1), 2-hydroxyestradiol (2-OHE2), and 4-methoxyestradiol (4-MeOE2) levels were significantly lower in group A (P=0.001, 0.003, 0.009, 0.001, 0.012, 0.032, and 0.047, respectively.). Urine levels of dehydroepiandrosterone (DHEA), androstenedione (A2), and the metabolite of (A2) were also significantly lower in group A (P=0.007, 0.009, and 0.011, respectively). The 2-OHE1/E1, 4-OHE1/E1, 2-MeOE1/E1, and 2-MeOE2/E2 ratios were lower in group B, whereas the 2-OHE2/E2, 4-OHE2/E2, and 4-MeOE2/E2 ratios were dramatically lower in all pregnant women (groups A and B) than in group C. CONCLUSIONS Deficiency in DHEA and abnormal levels of sex hormone metabolites may cause a reduction in the activity of estrogens in women with threatened abortion. These alterations may result in bleeding during the first trimester of pregnancy.

Synuclein γ compromises spindle assembly checkpoint and renders resistance to antimicrotubule drugs.

Defects in the spindle assembly checkpoint (SAC) have been proposed to contribute to the chromosomal instability in human cancers. One of the major mechanisms underlying antimicrotubule drug (AMD) resistance involves acquired inactivation of SAC. Synuclein γ (SNCG), previously identified as a breast cancer-specific gene, is highly expressed in malignant cancer cells but not in normal epithelium. Here, we show that SNCG is sufficient to induce resistance to AMD-caused apoptosis in breast cancer cells and cancer xenografts. SNCG binds to spindle checkpoint kinase BubR1 and inhibits its kinase activity. Specifically, the C-terminal (Gln106-Asp127) of SNCG binds to the N-terminal TPR (tetratricopeptidelike folds) motif of BubR1. SNCG-BubR1 interaction induces a structure change of BubR1, attenuates its interaction with other key checkpoint proteins of Cdc20, and thus compromises SAC function. SNCG expression in breast cancers from patients with a neoadjuvant clinical trial showed that SNCG-positive tumors are resistant to chemotherapy-induced apoptosis. These data show that SNCG renders AMD resistance by inhibiting BubR1 activity and attenuating SAC function.

Vitexin 6, a novel lignan, induces autophagy and apoptosis by activating the Jun N-terminal kinase pathway.

Previous studies have reported that vitexins induce cytotoxic effects. In the present study, we investigate a new native lignan vitexin 6 (VB6) in vitro to determine the molecular mechanism underlying its cytotoxicity. We screened and cultured several tumor cell lines and subsequently analyzed VB6 cytotoxicity against 14 different tumor cell lines using a 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The expression of proteins that regulate apoptosis and autophagy was determined using western blot analysis. VB6 showed an excellent cytotoxic effect against various cancer cell lines in vitro. It induced apoptosis and autophagy of cancer cells. VB6-induced apoptosis showed a time-dependent and concentration-dependent relationship with cleaved poly (ADP-ribose) polymerase, cleaved caspase-3, Bax upregulation, and Bcl-2 downregulation. The levels of Beclin-1 and LC3-II, which are markers for cell autophagy, gradually increased after VB6 treatment. Jun N-terminal kinase (JNK) phosphorylation was increased after VB6 treatment, accompanied by upregulation of P-Bcl-2 and P-C-Jun expression. Cotreatment with a JNK inhibitor significantly decreased VB6-induced cell death and downregulated P-Bcl-2, and cleaved PARP and Beclin-1 expression. The new native lignan VB6 inhibits cancer cell proliferation by activating the JNK pathway. We believe that VB6 could be a valuable chemotherapeutic drug after further evaluation.

Synuclein gamma stimulates membrane-initiated estrogen signaling by chaperoning estrogen receptor (ER)-alpha36, a variant of ER-alpha.

Synuclein gamma (SNCG), previously identified as a breast cancer-specific gene, is highly expressed in malignant cancer cells but not in normal epithelium. The molecular targets of SNCG during breast cancer progression have not been fully identified. Here we analyzed the effect of SNCG on stimulation of membrane-initiated estrogen signaling. While SNCG expression enhanced estrogen-induced activation of ERK1/2 and mammalian target of rapamycin, knockdown of endogenous SNCG decreased membrane-initiated estrogen signaling. SNCG functions as a molecular chaperone protein for estrogen receptor (ER)-alpha36, a membrane-based variant of ER-alpha. SNCG bound to ER-alpha36 in the presence and absence of functional molecular chaperone heat shock protein 90. Disruption of heat shock protein 90 with 17-AAG significantly reduced ER-alpha36 expression and membrane-initiated estrogen signaling. However, expression of SNCG prevented ER-alpha36 degradation and completely recovered 17-AAG-mediated down-regulation of estrogen signaling. The function of SNCG in ER-alpha36-mediated estrogen signaling is consistent with its ability to stimulate cell growth in response to estrogen. Expression of SNCG also renders tamoxifen resistance, which is consistent with the clinical observation on the association of ER-alpha36 expression and tamoxifen resistance. The present study indicates that ER-alpha36 is a new member of the ER-alpha family that mediates membrane-initiated estrogen signaling and that SNCG can replace the function of heat shock protein 90, chaperone ER-alpha36 activity, stimulate ligand-dependent cell growth, and render tamoxifen resistance.

Neuronal protein synuclein gamma predicts poor clinical outcome in breast cancer.

Synuclein gamma (SNCG), previously identified as a breast cancer-specific gene (BCSG1), is highly expressed in breast carcinomas but not in normal epithelium. SNCG regulates many pathways in growth and progression of breast cancer. To determine if SNCG is a biomarker for clinical prognosis of breast cancer, we generated a panel of murine monoclonal antibodies (mAbs) against human SNCG and correlated SNCG protein expression in 358 clinical breast cancer specimens with clinical outcome. A panel of 14 mAbs was characterized by ELISA, immunoprecipitation (IP), Western blot, immunocytochemistry and immunohistochemistry. SNCG protein expression was determined in 438 clinical breast specimens by immunohistochemical analysis using mAb 5C5. Expression of SNCG was strongly correlated with the stage, lymph node involvement, metastasis, tumor size and Her-2 status, but its expression was not associated with ER and PR expression status. While 71.4% of advanced breast cancers were positive for SNCG expression, only 26.8% of Stage I/II breast cancers were positive for SNCG expression and 5.2% of benign hyperplasia expressed SNCG. SNCG protein was not detectable in normal tissue adjacent to breast cancer. After a median follow-up of 64 months, patients with an SNCG-positive tumor had a significantly shorter disease-free survival and overall survival and a high probability of death compared no expression of SNCG. Multivariate analysis demonstrated that SNCG was a strong independent prognostic variable. SNCG is a new unfavorable prognostic marker for breast cancer progression and a potential target for breast cancer treatment.

Stage-specific expression of breast cancer-specific gene gamma-synuclein.

Gamma-synuclein (SNCG), also referred as breast cancer-specific gene 1, is the third member of a neuronal protein family synuclein. SNCG is highly expressed in human-infiltrating breast carcinomas but not expressed in normal or benign breast tissues. To evaluate the clinical relevance of SNCG expression in breast cancer progression and its correlation with clinical parameters, we analyzed SNCG expression in 79 clinical breast specimens from primary breast cancer, hyperplasia, and fibroadenoma patients by reverse transcription-PCR. The status of estrogen receptor, progesterone receptor, proliferating cell nuclear antigen, and C-erBb2 was also analyzed by immunohistochemistry. Overall SNCG mRNA expression was detectable in 38.8% of breast cancers. However, 79% of stage III/IV breast cancers were positive for SNCG expression, whereas only 15% of stage I/II breast cancers were positive for SNCG expression. In contrast, the expression of SNCG was undetectable in all benign breast lesions. The expression of SNCG was strongly correlated to the stage of breast cancer (P=0.000). This study suggests that the expression of SNCG is stage specific for breast cancer. SNCG is expected to be a useful marker for breast cancer progression and a potential target for breast cancer treatment.