Tumor MK2 transcript levels are associated with improved response to chemotherapy and patient survival in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.

MK2 nonenzymatically promotes nuclear translocation of caspase-3 and resultant apoptosis.

We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.

A Dominant-Negative Mutant of ANXA7 Impairs Calcium Signaling and Enhances the Proliferation of Prostate Cancer Cells by Downregulating the IP3 Receptor and the PI3K/mTOR Pathway.

Annexin A7/ANXA7 is a calcium-dependent membrane fusion protein with tumor suppressor gene (TSG) properties, which is located on chromosome 10q21 and is thought to function in the regulation of calcium homeostasis and tumorigenesis. However, whether the molecular mechanisms for tumor suppression are also involved in the calcium- and phospholipid-binding properties of ANXA7 remain to be elucidated. We hypothesized that the 4 C-terminal endonexin-fold repeats in ANXA7 (GX(X)GT), which are contained within each of the 4 annexin repeats with 70 amino acids, are responsible for both calcium- and GTP-dependent membrane fusion and the tumor suppressor function. Here, we identified a dominant-negative triple mutant (DNTM/DN-ANXA7J) that dramatically suppressed the ability of ANXA7 to fuse with artificial membranes while also inhibiting tumor cell proliferation and sensitizing cells to cell death. We also found that the [DNTM]ANA7 mutation altered the membrane fusion rate and the ability to bind calcium and phospholipids. In addition, in prostate cancer cells, our data revealed that variations in phosphatidylserine exposure, membrane permeabilization, and cellular apoptosis were associated with differential IP3 receptor expression and PI3K/AKT/mTOR modulation. In conclusion, we discovered a triple mutant of ANXA7, associated with calcium and phospholipid binding, which leads to the loss of several essential functions of ANXA7 pertinent to tumor protection and highlights the importance of the calcium signaling and membrane fusion functions of ANXA7 for preventing tumorigenesis.

A randomized, phase 1, placebo-controlled trial of APG-157 in oral cancer demonstrates systemic absorption and an inhibitory effect on cytokines and tumor-associated microbes.

BACKGROUND: Although curcumin's effect on head and neck cancer has been studied in vitro and in vivo, to the authors' knowledge its efficacy is limited by poor systemic absorption from oral administration. APG-157 is a botanical drug containing multiple polyphenols, including curcumin, developed under the US Food and Drug Administration's Botanical Drug Development, that delivers the active components to oromucosal tissues near the tumor target. METHODS: A double-blind, randomized, placebo-controlled, phase 1 clinical trial was conducted with APG-157 in 13 normal subjects and 12 patients with oral cancer. Two doses, 100 mg or 200 mg, were delivered transorally every hour for 3 hours. Blood and saliva were collected before and 1 hour, 2 hours, 3 hours, and 24 hours after treatment. Electrocardiograms and blood tests did not demonstrate any toxicity. RESULTS: Treatment with APG-157 resulted in circulating concentrations of curcumin and analogs peaking at 3 hours with reduced IL-1ß, IL-6, and IL-8 concentrations in the salivary supernatant fluid of patients with cancer. Salivary microbial flora analysis showed a reduction in Bacteroidetes species in cancer subjects. RNA and immunofluorescence analyses of tumor tissues of a subject demonstrated increased expression of genes associated with differentiation and T-cell recruitment to the tumor microenvironment. CONCLUSIONS: The results of the current study suggested that APG-157 could serve as a therapeutic drug in combination with immunotherapy. LAY SUMMARY: Curcumin has been shown to suppress tumor cells because of its antioxidant and anti-inflammatory properties. However, its effectiveness has been limited by poor absorption when delivered orally. Subjects with oral cancer were given oral APG-157, a botanical drug containing multiple polyphenols, including curcumin. Curcumin was found in the blood and in tumor tissues. Inflammatory markers and Bacteroides species were found to be decreased in the saliva, and immune T cells were increased in the tumor tissue. APG-157 is absorbed well, reduces inflammation, and attracts T cells to the tumor, suggesting its potential use in combination with immunotherapy drugs.

Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells.

Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-κB kinase-ß (IKKß)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKKß on Hsp90. Interestingly, IKKß binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKKß to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKKß. The pathophysiological relevance of the IKKß-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2(Akita) in vivo model. Our study further defines the preferential involvement of α- vs. ß-isoforms of Hsp90 in the IKKß-eNOS-Hsp90 interaction, even though both Hsp90α and Hsp90ß stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKKß within the cell system that regulates NO production, but they also confirm that the IKKß-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes.

Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment. METHODS: We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand. RESULTS: We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models. CONCLUSIONS: Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation. TRIAL REGISTRATION NUMBER: NCT00068003, NCT01174121, and NCT03412877.

Integrated In Silico Analysis of Proteogenomic and Drug Targets for Pancreatic Cancer Survival.

Pancreatic cancer remains a major health concern, being among the deadliest forms of cancer with over 80% of the patients presenting with metastatic disease. According to the American Cancer Society, for all stages of pancreatic cancer combined, the 5-year survival rate is less than 10%. Genetic research on pancreatic cancer has generally been focused on familial pancreatic cancer, which is only 10% of all pancreatic cancer patients. This study focuses on finding genes that impact the survival of pancreatic cancer patients which can be used as biomarkers and potential targets to develop personalized treatment options. We used cBioPortal platform using NCI-initiated The Cancer Genome Atlas (TCGA) dataset to find genes that were altered differently in different ethnic groups which can serve as potential biomarkers and analyzed the genes' impact on patient survival. MD Anderson Cell Lines Project (MCLP) and genecards.org were also utilized to identify potential drug candidates that can target the proteins encoded by the genes. The results showed that there are unique genes that are associated with each race category which may influence the survival outcomes of patients, and their potential drug candidates were identified.

Cell surface marker-based capture of neoantigen-reactive CD8+ T-cell receptors from metastatic tumor digests.

BACKGROUND: Cellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive T-cell receptors (TCRs) comes to the forefront of clinical investigation, the rapid, scalable, and cost-effective detection of patient-specific neoantigen-reactive TIL remains a top priority. METHODS: We analyzed the single-cell transcriptomic states of 31 neoantigen-specific T-cell clonotypes to identify cell surface dysfunction markers that best identified the metastatic transcriptional states enriched with antitumor TIL. We developed an efficient method to capture neoantigen-reactive TCRs directly from resected human tumors based on cell surface co-expression of CD39, programmed cell death protein-1, and TIGIT dysfunction markers (CD8+ TILTP). RESULTS: TILTP TCR isolation achieved a high degree of correlation with single-cell transcriptomic signatures that identify neoantigen-reactive TCRs, making it a cost-effective strategy using widely available resources. Reconstruction of additional TILTP TCRs from tumors identified known and novel antitumor TCRs, showing that at least 39.5% of TILTP TCRs are neoantigen-reactive or tumor-reactive. Despite their substantial enrichment for neoantigen-reactive TCR clonotypes, clonal dynamics of 24 unique antitumor TILTP clonotypes from four patients indicated that most in vitro expanded TILTP populations failed to demonstrate neoantigen reactivity, either by loss of neoantigen-reactive clones during TIL expansion, or through functional impairment during cognate neoantigen recognition. CONCLUSIONS: While direct usage of in vitro-expanded CD8+ TILTP as a source for cellular therapy might be precluded by profound TIL dysfunction, isolating TILTP represents a streamlined effective approach to rapidly identify neoantigen-reactive TCRs to design engineered cellular immunotherapies against cancer.

Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers.

Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ T cells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCRPBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

Circulating Cell-free DNA in Serum as a Marker for the Early Detection of Tumor Recurrence in Breast Cancer Patients.

BACKGROUND/AIM: Circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. The aim of this study was to implement a simple, yet effective quantitative method for measuring the cfDNA in serum and to investigate the relationship between cfDNA and the occurrence of recurrence in breast cancer (BrCa) patients. PATIENTS AND METHODS: A total of 240 cases were selected, which comprised different subtypes of BrCa patients and control individuals. We selected 20 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. SYBR green was used as a reporter molecule to estimate the amount of cfDNA in these serum samples. RESULTS: A global Wilcoxon analysis was performed to compare the cfDNA abundance between non-recurrent and recurrent patients. The amount of cfDNA was higher in recurrent patients (recurrent vs. non-recurrent ratio=1.3; p=0.03; AUC=0.76) compared to non-recurrent patients. The data between normal/healthy controls and non-recurrent patients indicated no significant differences (n=20 in each group, healthy to non-recurrent ratio=1.03; p=0.20; AUC=0.61). CONCLUSION: We implemented a straightforward one-step technique to measure the amount of cfDNA in serum, which can translate into a clinical diagnostic tool in the near future. The high levels of cfDNA in the serum of recurrent BrCa patients compared to non-recurrent BrCa patients indicates a possible uncovered role for circulating genetic information, which either contributes to the cancer recurrence phenomenon or at the very least, serves as an identifier for the potential of recurrence.

Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo-expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53-reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02-restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919.

Expression of the HSV-1 capsid protein VP19C in Escherichia coli: a single amino acid change overcomes an expression block of the full-length polypeptide.

The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.

Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells.

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/ß/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the ß [E484K], Mink [Y453F] and α/ß/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

Liver Function Enzymes are Potential Predictive Markers for Kidney Allograft Dysfunction.

INTRODUCTION: Biopsy of the allograft is the gold standard for assessing kidney allograft dysfunction. The aim of our pilot study was to identify serum biomarkers that could obviate the need for biopsy. MATERIALS AND METHODS: We conducted a study to identify the biomarkers in the serum from different groups of chronic kidney disease (CKD) patients and kidney transplanted patients vs. healthy individuals. The four groups (n=25 in each group) were as follows: 1) Patients with unstable kidney allograft transplants requiring biopsy for cause, 2) Patients with stable kidney allograft transplants, 3) Patients with CKD not on immunosuppressive therapy and, 4) healthy subjects. We measured the activity and level of serum alkaline phosphatase (ALP) and other liver enzymes (alanine transaminase (ALT) and aspartate transaminase (AST)) as potential serum biomarkers in acute allograft dysfunction. RESULTS: We found that ALP correlated with allograft biopsy findings, liver function, and clinical outcomes and possibly graft survival. Additionally, AST and ALT were higher in patients with graft rejection compared to non-rejected and stable kidney transplants. Moreover, the low Pearson correlations (r- values) between ALP level with age (r=0.179), gender, body mass index (r=0.236), creatinine (r=0.044) or estimated glomerular filtration rate (r=0.048) suggest that ALP may be an independent biomarker which is relatively unaffected by other individual-level variables. CONCLUSION: ALP may be a putative biomarker to predict kidney allograft function and rejection. Data also indicated that liver function plays an important role for the overall success of kidney transplantation.

Functional role of vitronectin in breast cancer.

Breast Cancer is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Identification of new biomarkers could be key for early diagnosis and detection. Vitronectin, a glycoprotein that is abundantly found in serum, extracellular matrix, and bone, binds to integrin αvß3, and promotes cell adhesion and migration. Current studies indicate that patients with amplified vitronectin levels have lower survival rates than patients without amplified vitronectin levels. In this study, we focused on the role of vitronectin in breast cancer survival and its functional role as a non-invasive biomarker for early stage and stage specific breast cancer detection. To confirm that the expression of vitronectin is amplified in breast cancer, a total of 240 serum samples (n = 240), 200 from breast cancer patients and 40 controls were analyzed using the Reverse Phase Protein Array (RPPA) technique. Of the 240 samples, 120 samples were of African American (AA) descent, while the other 120 were of White American (WA) descent. Data indicated that there were some possible racial disparities in vitronectin levels and, differences also seen in the recurrent patient samples. Next, we tried to uncover the underlying mechanism which plays a critical role in vitronectin expression. The cellular data from four different breast cancer cell lines- MCF7, MDA-MB-231, MDA-MB-468, and HCC1599 indicated that the PI3K/AKT axis is modulating the expression of vitronectin. We believe that vitronectin concentration levels are involved and connected to the metastasis of breast cancer in certain patients, specifically based on recurrence or ethnicity, which is detrimental for poor prognosis. Therefore, in this current study we showed that the serum vitronectin levels could be an early marker for the breast cancer survival and we also determine the cellular signaling factors which modulate the expression and concentration of vitronectin.

Proteomic Analysis of Inflammatory Biomarkers Associated With Breast Cancer Recurrence.

INTRODUCTION: Breast cancer is the most frequent cancer detected for women, and while our ability to treat breast cancer has improved substantially over the years, recurrence remains a major obstacle. Standard screening for new and recurrent breast cancer involves clinical breast imaging. However, there is no clinically approved noninvasive body fluid test for the early detection of recurrent breast cancer. Materials and Method: In this study, we analyzed serum samples from both recurrent and nonrecurrent breast cancer patients by different proteomics methods to identify biomarkers in patients with recurrence of disease. RESULTS: Comparative data analysis identified several histone deacetylase (HDAC) proteins, which were found at significantly higher levels in the serum of recurrent breast cancer patients: HDAC9 (C-term) (P = 0.0035), HDAC5 (C-term) (P = 0.013), small ubiquitin-like modifier 1 (N-term) (P = 0.017), embryonic stem cell-expressed Ras (inter) (P = 0.018), and HDAC7 (C-term) (P = 0.020). Chronic inflammation plays a critical role in the development of the breast cancer recurrence, and we identified several proinflammatory cytokines that were present at elevated levels only in recurrent breast cancer patient serum. CONCLUSIONS: Our data indicated that the epigenetic regulation of inflammatory processes plays a critical role in breast cancer recurrence. The identified proteins could lay the groundwork for the development of a serum-based breast cancer recurrence assay.

A quantitative characterization of interaction between prion protein with nucleic acids.

Binding of recombinant prion protein with small highly structured RNAs, prokaryotic and eukaryotic prion protein mRNA pseudoknots, tRNA and polyA has been studied by the change in fluorescence anisotropy of the intrinsic tryptophan groups of the protein. The affinities of these RNAs to the prion protein and the number of sites where the protein binds to the nucleic acids do not vary appreciably although the RNAs have very different compositions and structures. The binding parameters do not depend upon pH of the solution and show a poor co-operativity. The reactants form larger nucleoprotein complexes at pH 5 compared to that at neutral pH. The electrostatic force between the protein and nucleic acids dominates the binding interaction at neutral pH. In contrast, nucleic acid interaction with the incipient nonpolar groups exposed from the structured region of the prion protein dominates the reaction at pH 5. Prion protein of a particular species forms larger complexes with prion protein mRNA pseudoknots of the same species. The structure of the pseudoknots and not their base sequences probably dominates their interaction with prion protein. Possibilities of the conversion of the prion protein to its infectious form in the cytoplasm by nucleic acids have been discussed.

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis.

High ANXA7 Potentiates Eucalyptol Toxicity in Hormone-refractory Prostate Cancer.

BACKGROUND/AIM: Our studies showed that ANXA7 is a novel tumor suppressor gene that is lost in various aggressive forms of prostate cancer. However, little is known about the role of ANXA7 in the anticancer drug treatment towards different cancers. MATERIALS AND METHODS: The expression of ANXA7 was measured in the 60 cancer cell lines of the NCI-60 ADS project and correlated with the enhanced sensitivity to over 30,000 natural and synthetic compounds. RESULTS: Eucalyptol showed a high positive correlation with ANXA7 expression and castration-resistant prostate cancer cell death occurred very effectively in response to the combination of eucalyptol and overexpressed wt-ANXA7 than either agent alone. The synergistic effects of ANXA7 and eucalyptol resulted in concordant changes in gene expression profiles particularly of Ras family members, MDM4, NF-ĸB and VEGF. CONCLUSION: Overexpression of ANXA7 enhances eucalyptol cytotoxicity in prostate cancer cell lines.

Tissue microarray analysis delineate potential prognostic role of Annexin A7 in prostate cancer progression.

BACKGROUND: Annexin A7 (ANXA7) is a member of the multifunctional calcium or phospholipid-binding annexin gene family. While low levels of ANXA7 are associated with aggressive types of cancer, the clinical impact of ANXA7 in prostate cancer remains unclear. Tissue microarrays (TMA) have revealed several new molecular markers in human tumors. Herein, we have identified the prognostic impact of ANXA7 in a prostate cancer using a tissue microarray containing 637 different specimens. METHODS: The patients were diagnosed with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival data (median 5.9 years) were available. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), syndecan-1 (CD-138) and ANXA7 were analyzed by immunohistochemistry. RESULTS: A bimodal distribution of ANXA7 was observed. Tumors expressing either high or no ANXA7 were found to be associated with poor prognosis. However, ANXA7 at an optimal level, in between high and no ANXA7 expression, had a better prognosis. This correlated with low Ki67, Bcl-2, p53 and high syndecan-1 which are known predictors of early recurrence. At Gleason grade 3, ANXA7 is an independent predictor of poor overall survival with a p-value of 0.003. Neoadjuvant hormonal therapy, which is known to be associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, was found to be associated with under-expression of ANXA7. CONCLUSIONS: The results of this TMA study identified ANXA7 as a new prognostic factor and indicates a bimodal correlation to tumor progression.