BH-3 like motif containing inducer of cell death (BLID) is a known prognostic factor in breast cancer. The aim of the present study was to determine the significance of BLID in the outcomes of chemotherapy and mechanisms affected in BLID-deficient breast cancer cells. Reverse transcription-PCR, reverse transcription-quantitative PCR, dual-luciferase reporter and chromatin immunoprecipitation assays were used to determine the effects of drugs on BLID expression and binding of forkhead box protein O3a (FOXO3a) to the BLID promoter. RNA arrays, antibody microarrays and microRNA arrays were used to illuminate the omics features of BLID knockdown vs. isogenic control breast cancer cell lines. Kaplan-Meier plotter and receiver operating characteristic plotter tools were used to determine the prognostic and therapy response benefits of BLID expression using publicly available clinical datasets. BLID expression was induced in response to several chemotherapeutic drugs. Drug treatment resulted in increased binding of FOXO3a to the BLID promoter, and FOXO3a knockdown was associated with decreased expression of BLID. BLID depletion led to a decrease in the cytotoxicity of chemotherapeutic drugs. Through multi-omics profiling, three functionally distinct classes of effectors that were predominately influenced in BLID knockdown cells were identified: i) Genes and proteins associated with cell death and survival, including cellular inhibitor of apoptosis protein 2/baculoviral IAP repeat containing 3, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α/p110α and DFNA5/gasdermin E; ii) members of the aldo-ketoreductase family 1, specifically aldo-keto reductase family 1 member C3 implicated in drug metabolism; and iii) effectors of the interferon response, including IFNβ1, interferon-induced protein with tetratricopeptide repeats 2 and interferon-induced protein with tetratricopeptide repeats 3. Finally, higher BLID expression was associated with improved overall survival in several types of cancer and the response of breast cancer to anthracyclines. The results of the present study demonstrated that BLID is a target of FOXO3a, and BLID-deficiency in breast cancer cells was associated with modulation of cell death, survival and proliferation, chemoresistance, drug potency, and the interferon response. These findings highlighted BLID as a promising biomarker of drug response and offer a novel framework of integrative mechanisms of therapeutic resistance and disease progression.

Abstract SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air–liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.

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.

Dynamic post-translational processes regulate protein expression in eukaryotic cells. However, the processes are difficult to assess on a proteomic scale because protein levels actually reflect the sum of individual biosynthesis and degradation rates. These rates are presently hidden from the conventional proteomic technologies. We present here a novel and dynamic, antibody microarray-based time-resolved approach to simultaneously measure not only the total protein changes but also the rates of biosynthesis of low abundance proteins in the proteome of lung epithelial cells. In this chapter, we describe the feasibility of this technique by investigating the complete proteomic kinetics of 507 low abundance proteins in cultured cystic fibrosis (CF) lung epithelial cells using 35[S] methionine or 32[P] and the consequences of repair by gene therapy with [wildtype] CFTR. This novel antibody microarray-based technology identifies relevant, hidden proteins whose regulation by the CF genotype would never have been detected by simple measurements of total proteomic masses.

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/β/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the β [E484K], Mink [Y453F] and α/β/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.

IntroductionBiopsy 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 MethodsWe 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.ResultsWe 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.ConclusionALP 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.

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.

African American (AA) women are often diagnosed with more aggressive breast cancers and have worse survival outcomes than their Caucasian American (CA) counterparts. However, a comprehensive understanding of this disparity remains unclear. In this study, we attempted to identify the race-specific non-invasive protein biomarkers that may particularly benefit interventions aimed at reducing the risk of recurrence and metastasis in breast cancers (BrCa). Our technical strategy has been to discover candidate protein biomarkers in patient sera using a high throughput antibody microarray platform. A total of 240 subjects were selected, composed of controls and all immunohistochemistry-based subtypes of breast cancer cases, subdivided by pre- and post-menopausal status and by race. A global Wilcoxon analysis comparing no-cancer controls and cancer patients identified Pyk2, SAPK/JNK, and phosphatase and tensin homolog as present in higher concentrations in cancer patient serum. A paired t-test revealed that c-kit and Rb are significantly over-represented in AA cancer serum when compared to CA cancer serum. Interestingly, VEGFR2, a protein linked to BrCa metastasis and poor prognosis, was significantly over-represented in AA cancer serum compared to AA controls; however, this was not found in CA cancer serum compared to CA controls, suggesting a possible explanation for the higher incidence of aggressive BrCa in AA versus CA patients. Through examining race-specific differences in the protein landscape of BrCa patient serum, the identified proteins could lay the groundwork for the development of an all-inclusive liquid mammogram test.

Castration Resistant Prostate Cancer (CRPC) is thought to be driven by a collaborative mechanism between TNFα/NFκB and TGFβ signaling, leading to inflammation, Epithelial-to-Mesenchymal-Transition (EMT), and metastasis. Initially, TGFβ is a tumor suppressor, but in advanced metastatic disease it switches to being a tumor promoter. TGFBR2 may play a critical role in this collaboration, as its expression is driven by NFκB and it is the primary receptor for TGFβ. We have previously reported that the cardenolide drug digitoxin blocks TNFα/NFκB-driven proinflammatory signaling. We therefore hypothesized that digitoxin might break the collaborative process between NFκB and TGFβ by also inhibiting expression of TGFBR2. We therefore tested whether TGFβ-driven EMT and resulting metastases would be suppressed. Here we show, in vitro, that digitoxin inhibits NFκB-driven TGFBR2 expression, as well as Vimentin, while elevating E-cadherin expression. Digitoxin also significantly reduces HSPB1 mRNA and the HSPB1/RBFOX2 mRNA ratio in PC3 cells. In vivo, in a syngeneic, immune competent rat model of metastatic CRPC, we show that digitoxin also suppresses Tgfbr2 expression, as well as expression of other genes classically driven by NFκB, and of multiple EMT genes associated with metastasis. Concurrently, digitoxin suppresses tumor growth and metastasis in these animals, and prolongs survival. Gross tumor recurrence following tumor resection also appears prevented in ca 30% of cases. While the existence of a collaboration between NFκB and TGFβ to drive EMT and metastasis has previously been appreciated, we show here, for the first time, that chronic, low concentrations of digitoxin are able to block CRPC tumor progression, EMT and the ensuing metastatic disease.

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.

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.

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity at 10q, are commonly observed in most human tumors, including prostate, breast, and kidney cancers. The ANXA7-GTPase is a tumor suppressor, which is frequently inactivated by genomic alterations at 10q21. In the last few years, considerable amounts of data have accumulated describing inactivation of ANXA7-GTPase in a variety of human malignancies and demonstrating the tumor suppressor potential of ANXA7-GTPase. ANXA7-GTPase contains a calcium binding domain that classifies it as a member of the annexin family. The cancer-specific expression of ANXA7-GTPase, coupled with its importance in regulating cell death, cell motility, and invasion, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Recently, emerging evidence suggests that ANXA7-GTPase is a critical factor associated with the metastatic state of several cancers and can be used as a risk biomarker for HER2 negative breast cancer patients. Cross talk between ANXA7, PTEN, and EGFR leads to constitutive activation of PI3K-AKT signaling, a central pathway of tumor cell survival and proliferation. This review focuses on the recent progress in understanding the tumor suppressor functions of ANXA7-GTPase emphasizing the role of this gene in Ca2+ metabolism, and exploring opportunities for function as an example of a calcium binding GTPase acting as a tumor suppressor and opportunities for ANXA7-GTPase gene cancer therapy.

“Soldier’s Heart,” is an American Civil War term linking post-traumatic stress disorder (PTSD) with increased propensity for cardiovascular disease (CVD). We have hypothesized that there might be a quantifiable genetic basis for this linkage. To test this hypothesis we identified a comprehensive set of candidate risk genes for PTSD, and tested whether any were also independent risk genes for CVD. A functional analysis algorithm was used to identify associated signaling networks.We identified 106 PTSD studies that report one or more polymorphic variants in 87 candidate genes in 83,463 subjects and controls. The top upstream drivers for these PTSD risk genes are predicted to be the glucocorticoid receptor (NR3C1) and Tumor Necrosis Factor alpha (TNFA). We find that 37 of the PTSD candidate risk genes are also candidate independent risk genes for CVD. The association between PTSD and CVD is significant by Fisher’s Exact Test (P= 3*10-54). We also find 15 PTSD risk genes that are independently associated with Type 2 Diabetes Mellitus (T2DM; also significant by Fisher’s Exact Test (P= 1.8*10-16). Our findings offer quantitative evidence for a genetic link between post-traumatic stress and cardiovascular disease, Computationally, the common mechanism for this linkage between PTSD and CVD is innate immunity and NFκB-mediated inflammation.

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Tocopherol succinate (TS) has been shown to protect mice against acute radiation syndrome, however, its exact mechanism of action and its possible use in humans has not yet been evaluated. Our approach has been to test the radioprotectant properties of TS on CD34-positive stem cells from healthy volunteers. We hypothesize that a radioproteomics strategy can identify a drug-dependent, personalized proteomics signature for radioprotection. To directly test the radioproteomics hypothesis, we treated human CD34-positive stem cells with 20 μM TS for 24 h, and then exposed the cells to 2 Gy of cobalt-60 gamma-radiation. We isolated protein from all cultures and used a high throughput Antibody Microarray (AbMA) platform to measure concentrations of 725 low abundance proteins. As an in vivo control, we also tested mouse CD34-positive stem cells using the same preemptive TS paradigm on progenitor colony forming units. TS pretreatment of in vitro or in vivo CD34-positive stem cells rescued radiation-induced loss of colony-forming potential of progenitors. We identified 50 of 725 proteins that could be preemptively rescued from radiation-induced reduction by pretreatment with TS. Ingenuity Pathway Analysis (IPA) reveals that the modified proteins fall into categories dominated by epigenetic regulation, DNA repair, and inflammation. Our results suggest that radioproteomics can be used to develop personalized medicine for radioprotection using protein signatures from primary CD34-positive progenitors derived from the patient or victim prior to radiation exposure. The protective effect of TS may be due to its ability to preemptively activate epigenetic mechanisms relevant to radioprotection and to preemptively activate the programs for DNA repair and inflammation leading to cell survival.

A serum proteomics platform enabling expression Profiling in transplantation-associated clinical subsets gives an opportunity to identify non-invasive biomarkers that can accurately predict transplant outcome. In this study, we attempted to identify candidate serum biomarkers that could predict kidney allograft rejection/injury, regardless of its etiological and therapeutic heterogeneity. Using serum samples collected from kidney transplantation patients and healthy controls, we first employed Clontech-500 Ab microarrays to Profile acute rejection (AR) and chronic graft injury (CGI) versus stable graft function (SF) and normal kidneys (NK). Using GenePattern analysis of duplicate arrays on pooled samples, we identified gender-independent biomarkers PARP1, MAPK1, SRP54, DP1, and p57 (FDR ≈ 25%), the concordant downregulation of which represented a detrimental Profile common for both rejection/ injury types (AR-CGI). The reverse phase arrays qualified a 2-fold upregulation of PARP1 with an ROC of 0.87 in individual samples from patients with SF vs. AR-CGI rendering serum PARP1 as a biomarker for early prognosis. Ingenuity Pathways Analysis (IPA) connected PARP1 to some other markers (MAPK1), elucidating their possible interactions and connections to the immune response and graft-versus-host disease signaling. The downregulation of serum PARP1 in the damaged graft tissues, represents a perspective non-invasive marker, predicting the failing kidney graft, regardless of rejection/injury causes or gender. Thus, the successful identification of PARP1 as a bio-marker in limited patient cohorts demonstrates that serum proteomics platform empowered by the GenePattern- and IPA-based Bioinformatics algorithm can guarantee a successful development of the clinically applicable prognostic biomarker panel.