ProGlycProt V3.0: updated insights into prokaryotic glycoproteins and their glycosyltransferases.

ProGlycProt is a comprehensive database of experimentally validated information about protein glycosylation in prokaryotes, including the glycoproteins, glycosyltransferases, and their accessory enzymes. The first release of ProGlycProt featured experimentally validated information on glycoproteins only. For the second release in 2019, the size and scope of the database were expanded twofold, and experimental data on cognate glycosyltransferases and their accessory proteins was incorporated. The growing research and technology interest in microbial glycoproteins and their enzymes is evident from the steady rise in academic publications and patents in this area. Accordingly, the third update comprises a new section on patents related to glycosylation methods, novel glycosyltransferases, and technologies developed therefrom. The structure gallery is reorganized, wherein the number and quality of the models are upgraded with the help of AlphaFold2. Over the years, the influx of experimental proteomics data into public repositories like PRIDE has surged. Harnessing this legacy data for in-silico glycoprotein identification is a smart approach. Version 3.0 adds 45 N-glycoprotein entries annotated from MS datasets available on PRIDE and reviewed by independent research groups. With a 67% rise in entries corresponding to 119 genera of prokaryotes, the ProGlycProt continues to be the exclusive database of experimentally validated comprehensive information about protein glycosylation in prokaryotes.

Cancer immunotherapy with check point inhibitor can cause autoimmune adverse events due to loss of Treg homeostasis.

Regulatory T-cells (Tregs) can facilitate immune evasion by tumor cells by dampening anti-tumor immunity. Reduced Teff/Treg ratio and enhanced Treg functional activity have been observed in patients suffering from different types of cancers, and attenuated Treg numbers/functions can serve as prognostic indicators. Normally, Tregs play an essential role in the maintenance of immune tolerance and prevention of autoimmunity. The most common immune checkpoint blockers (ICB) targeting co-inhibitory receptors such as anti-CTLA4 (ipilimumab and tremelimumab) and anti-PD1 (pembrolizumab and nivolumab)/anti-PD-L1 (atezolizumab) have achieved unprecedented success in cancer treatment by facilitating an effective anti-tumor immune response, at least in part, by blocking Treg mediated immunosuppression. While ICBs have shown remarkable success in cancer immunotherapy, immune-related adverse events (IRAEs) arising from ICB have forced consideration of ways to maintain immune homeostasis post ICB treatment. Preclinical models of IRAEs have shown a negative correlation between Treg numbers and IRAEs. Therefore, understanding the "ying-yang" role of Tregs in the regulation of autoimmunity and anti-tumor immunity is critical to provoking an effective anti-tumor response while maintaining immune homeostasis. Studies aimed at developing effective approaches to minimize IRAEs without compromising anti-tumor immunity are underway. Herein, we discuss 1) the critical role of key co-inhibitory receptors on Treg homeostasis and tumor tolerance; 2) how co-receptor blockade by cancer immunotherapy can lead to autoimmune adverse events; and 3) recently emerging management strategies to minimize autoimmune adverse events arising from ICB.

Restoring self-tolerance in autoimmune diseases by enhancing regulatory T-cells.

Self-tolerance, the state of unresponsiveness to self-tissues/antigens, is maintained through central and peripheral tolerance mechanisms, and a breach of these mechanisms leads to autoimmune diseases. Foxp3 + T-regulatory cells (Tregs) play an essential role in suppressing autoimmune response directed against self-antigens and thereby regulate self-tolerance. Natural Tregs are differentiated in the thymus on the basis of their higher TCR-affinity to self-antigens and migrate to the periphery where they maintain peripheral tolerance. In addition, extra-thymic differentiation of induced Tregs can occur in the periphery which can control abrupt immune responses under inflammatory conditions. A defect in Treg cell numbers and/or function is found to be associated with the development of autoimmune disease in several experimental models and human autoimmune diseases. Moreover, augmentation of Tregs has been shown to be beneficial in treating autoimmunity in preclinical models, and Treg based cellular therapy has shown initial promise in clinical trials. However, emerging studies have identified an unstable subpopulation of Tregs which expresses pro-inflammatory cytokines under both homeostatic and autoimmune conditions, as well as in ex vivo cultures. In addition, clinical translation of Treg cellular therapy is impeded by limitations such as lack of easier methods for selective expansion of Tregs and higher cost associated with GMP-facilities required for cell sorting, ex vivo expansion and infusion of ex vivo expanded Tregs. Here, we discuss the recent advances in molecular mechanisms regulating Treg differentiation, Foxp3 expression and lineage stability, the role of Tregs in the prevention of various autoimmune diseases, and critically review their clinical utility for treating human autoimmune diseases.

Oxygen vacancy mediated cubic phase stabilization at room temperature in pure nano-crystalline zirconia films: a combined experimental and first-principles based investigation.

We report here the stabilization of the cubic phase under ambient conditions in the thin films of zirconia synthesized by electron beam evaporation. The cubic phase stabilization was achieved without the use of chemical stabilizers and/or concurrent ion beam bombardment. Films of two different thicknesses (660 nm and 140 nm) were deposited. While the 660 nm as-deposited films were in the cubic phase, as indicated by X-ray diffraction and Raman spectroscopy, the 140 nm as-deposited films were amorphous and the transformation to the cubic phase was obtained after thermal annealing. Extended X-ray absorption fine structure measurements revealed the existence of oxygen vacancies in the local structure surrounding zirconium for all films. However, the amount of these oxygen vacancies was found to be significantly higher for the amorphous films as compared to that for the films in the cubic phase (660 nm as-deposited and 140 nm annealed films). The stabilization of the cubic phase is attributed to the breaking of the oxygen-zirconium bonds due to the presence of the oxygen vacancies, which results in the suppression of the soft X2- mode of vibration of the oxygen sub-lattice. Our first-principles modeling under the framework of density functional theory shows that the cubic structure with oxygen vacancies is indeed more stable under ambient conditions than its pristine (without vacancies) counterpart due to breaking of the oxygen bonds. The requirement of a critical amount of these vacancies for cubic phase stabilization is discussed.

Therapeutic advances in anaplastic thyroid cancer: a current perspective.

Thyroid cancer incidence is increasing at an alarming rate, almost tripling every decade. In 2017, it was the fifth most common cancer in women. Although the majority of thyroid tumors are curable, about 2-3% of thyroid cancers are refractory to standard treatments. These undifferentiated, highly aggressive and mostly chemo-resistant tumors are phenotypically-termed anaplastic thyroid cancer (ATC). ATCs are resistant to standard therapies and are extremely difficult to manage. In this review, we provide the information related to current and recently emerged first-line systemic therapy (Dabrafenib and Trametinib) along with promising therapeutics which are in clinical trials and may be incorporated into clinical practice in the future. Different categories of promising therapeutics such as Aurora kinase inhibitors, multi-kinase inhibitors, epigenetic modulators, gene therapy using oncolytic viruses, apoptosis-inducing agents, and immunotherapy are reviewed. Combination treatment options that showed synergistic and antagonistic effects are also discussed. We highlight ongoing clinical trials in ATC and discuss how personalized medicine is crucial to design the second line of treatment. Besides using conventional combination therapy, embracing a personalized approach based on advanced genomics and proteomics assessment will be crucial to developing a tailored treatment plan to improve the chances of clinical success.

Expression and humoral response of A-kinase anchor protein 4 in cervical cancer.

BACKGROUND: Cervical cancer is one of the major gynecologic cancers. In developing countries, because of a lack of medical support and infrastructure, cervical cancer is the leading cause of cancer-related deaths. Therefore, there is a need to identify novel biomarkers for cervical cancers. In this context, cancer-testis (CT) antigens represent a unique class of tumor antigens that have been shown to be associated with various solid tumors. These antigens have restricted expression in testis and no expression in somatic tissues. Because of their restricted expression, CT antigens are novel candidate molecules for early detection and diagnosis and immunotherapy. In the present study, novel CT antigen A-kinase anchor protein 4 (AKAP4) expression and humoral response were investigated in patients with cervical cancer. METHODS: In this study, 74 cervical cancer tissue specimens, which included different tumor stages (stage I [n = 35], stage II [n = 39]) and histologic grades (grade 1 [n = 17], grade 2 [n = 46], and grade 3[n = 11]) and 62 adjacent noncancerous tissue specimens were investigated for AKAP4 gene expression by using reverse transcriptase polymerase chain reaction and in situ RNA hybridization. Furthermore, AKAP4 protein expression was determined by immunohistochemistry. In addition, humoral response against purified recombinant AKAP4 protein was determined in available sera of 70 patients with cervical cancer by enzyme-linked immuno assay (ELISA). FINDINGS: Our study revealed that AKAP4 gene and protein expression was detected in 86% of total patients with cervical cancer. Based on the AKAP4 immunoreactivity score, most of stage I (n = 22/29) and stage II (n = 30/35) specimens revealed high AKAP4 expression (≥50% AKAP4-positive cells). A-kinase anchor protein 4 expression was significantly associated with early grades tumor specimens (P = 0.023). In addition, humoral response was detected in 53% of patients irrespective of stages, lymph node positivity, and grades. CONCLUSIONS: Collectively, our data indicate the putative role of AKAP4 in early tumorigenesis and may be implicated as a biomarker and immunotherapeutic target for cervical cancer.

Unravelling the reactivity of metastable molybdenum carbide nanoclusters in the C-H bond activation of methane, ethane and ethylene.

C-H bond activation steps in non-oxidative methane dehydroaromatization (MDA), constitute a key functionalization of the reactant and adsorbed species to form aromatics. Previous studies have focused on studying the energetics of these steps at the most stable active sites involving molybdenum carbide species. Herein, a different paradigm is presented via studying the reactivity of a metastable molybdenum carbide (Mo2C6) nanocluster for the C-H bond activation of methane, ethane, and ethylene and comparing it with the reactivity of the lowest energy Mo2C6 nanocluster. Interestingly, the metastable nanocluster is observed to result in a consistent reduction (by half) in the C-H bond activation barrier of the respective alkane and alkene molecules compared to the global minimum isomer. This specific metastable form of the nanocluster is identified from a cascade genetic algorithm search, which facilitated a rigorous scan of the potential energy surface. We attribute this significant lowering of the C-H bond activation barrier to unique co-planar orbital overlap between the reactant molecule and active centers on the metastable nanocluster. Based on geometrical and orbital analysis of the transition states arising during the C-H bond activation of methane, ethane, and ethylene, a proton-coupled electron transfer mechanism is proposed that facilitated C-H bond cleavage. Motivated by the high reactivity for C-H bond activation observed on the metastable species, a contrasting framework to analyze the elementary-step rate contributions is presented. This is based on the statistical ensemble analysis of nanocluster isomers, where the calculated rates on respective isomers are normalized with respect to the Boltzmann probability distribution. From this framework, the metastable isomer is observed to provide significant contributions to the ensemble average representations of the rate constants calculated for C-H bond activation during the MDA reaction.

Synthesis and characterization of ultra-fine Y2O3:Eu3+ nanophosphors for luminescent security ink applications.

We report a simple method for the synthesis of ultra-fine Eu(3+)-doped yttria (Y(2)O(3)) nanophosphors with an average diameter of approximately 5 nm for development of a transparent colloid that could be used as a luminescent security ink. This has been achieved by suitably substituting Eu(3+) ions at the favorable C(2) symmetry sites of Y(3+) ions and quantum mechanically confining the growth of the nanophosphor using a novel acid-catalyzed sol-gel technique. This is one of the few reports that depict the development of a transparent aqueous-stable Y(2)O(3):Eu(3+) colloidal solution for strategic applications related to security codes. High resolution transmission electron microscopy images showed excellent lattice fringes that in turn support the presence of better crystal quality and enhanced photoluminescence (PL) emission from the Y(1.9)O(3)Eu(0.1)(3+) nanophosphor system. Time resolved emission spectroscopy measurement indicated a PL decay time in the range of a few milliseconds, suitable for making luminescent security ink and other advanced applications in optoelectronic devices and bio-labeling.

Theoretical insights of codoping to modulate electronic structure of [Formula: see text] and [Formula: see text] for enhanced photocatalytic efficiency.

[Formula: see text] and [Formula: see text] are well known materials in the field of photocatalysis due to their exceptional electronic structure, high chemical stability, non-toxicity and low cost. However, owing to the wide band gap, these can be utilized only in the UV region. Thus, it's necessary to expand their optical response in visible region by reducing their band gap through doping with metals, nonmetals or the combination of different elements, while retaining intact the photocatalytic efficiency. We report here, the codoping of a metal and a nonmetal in anatase [Formula: see text] and [Formula: see text] for efficient photocatalytic water splitting using hybrid density functional theory and ab initio atomistic thermodynamics. The latter ensures to capture the environmental effect to understand thermodynamic stability of the charged defects at a realistic condition. We have observed that the charged defects are stable in addition to neutral defects in anatase [Formula: see text] and the codopants act as donor as well as acceptor depending on the nature of doping (p-type or n-type). However, the most stable codopants in [Formula: see text] mostly act as donor. Our results reveal that despite the response in visible light region, the codoping in [Formula: see text] and [Formula: see text] cannot always enhance the photocatalytic activity due to either the formation of recombination centers or the large shift in the conduction band minimum or valence band maximum. Amongst various metal-nonmetal combinations, [Formula: see text] (i.e. Mn is substituted at Ti site and S is substituted at O site), [Formula: see text] in anatase [Formula: see text] and [Formula: see text], [Formula: see text] in [Formula: see text] are the most potent candidates to enhance the photocatalytic efficiency of anatase [Formula: see text] and [Formula: see text] under visible light irradiation.

Electronic Effect in a Ruthenium Catalyst Designed in Nanoporous N-Functionalized Carbon for Efficient Hydrogenation of Heteroarenes.

Active metal catalysts are the key in chemical industry for sustainable production of multitude of chemical resources. Here, we report a new ruthenium (Ru) composite with a synergistically controlled nanostructure and electronic properties as a highly efficient hydrogenation catalyst which comprises stable small Ru nanoparticles (mean particle size, ca. 0.9 nm) in situ generated into a nanoporous N-functionalized carbon with high surface area (ca. 650 m2 g-1) and has strong electron-donating power of Ru sites of nanoparticles. The scalable and highly reusable catalyst, prepared from a self-assembled Ru complex, performs actively with low per metal usage under mild conditions (60-80 °C and 0.5-1.0 MPa H2) for selective hydrogenation of various quinolines and pyridines. The role of electron-donating properties of the new Ru nanohybrid for highly efficient catalysis was characterized by both experiments and computational studies. Density functional theory calculations reveal that weak adsorption energies of quinoline at the electron-rich Ru surface prevents poisoning caused by its strong coordination and provides excellent reusability of the catalyst, while low activation barriers for the hydrogenation steps of the N-heterocyclic ring correlate with high catalytic activity. Our catalyst exhibits 5-24-fold higher turnover frequency up to ca. 167 h-1 among the efficient noble metal catalysts reported for selective hydrogenation of quinoline to 1,2,3,4-tetrahydroquinoline.

Stability of non-metal dopants to tune the photo-absorption of TiO2 at realistic temperatures and oxygen partial pressures: A hybrid DFT study.

TiO2 anatase is considered to play a significant importance in energy and environmental research. However, for developing artificial photosynthesis with TiO2, the major drawback is its large bandgap of 3.2 eV. Several non-metals have been used experimentally for extending the TiO2 photo-absorption to the visible region of the spectrum. It's therefore of paramount importance to provide theoretical guidance to experiment about the kind of defects that are thermodynamically stable at a realistic condition (e.g. Temperature (T), oxygen partial pressure ([Formula: see text]), doping). However, disentangling the relative stability of different types of defects (viz. substitution, interstitial, etc.) as a function of charge state and realistic T, [Formula: see text] is quite challenging. We report here using state-of-the-art first-principles based methodologies, the stability and meta-stability of different non-metal dopants X (X = N, C, S, Se) at various charge states and realistic conditions. The ground state electronic structure is very accurately calculated via density functional theory with hybrid functionals, whereas the finite T and [Formula: see text] effects are captured by ab initio atomistic thermodynamics under harmonic approximations. On comparing the defect formation energies at a given T and [Formula: see text] (relevant to the experiment), we have found that Se interstitial defect (with two hole trapped) is energetically most favored in the p-type region, whereas N substitution (with one electron trapped) is the most abundant defect in the n-type region to provide visible region photo-absorption in TiO2. Our finding validates that the most stable defects in X doped TiO2 are not the neutral defects but the charged defects. The extra stability of [Formula: see text] is carefully analyzed by comparing the individual effect of bond-making/breaking and the charge carrier trapping energies.

Molecular aberrations and signaling cascades implicated in the pathogenesis of anaplastic thyroid cancer.

Anaplastic Thyroid Cancer (ATC) accounts for >40% thyroid cancer-related deaths and has a dismal prognosis. In the past decade, significant efforts have been made towards understanding the pathogenesis of this disease and developing novel therapeutics. Unfortunately, effective treatment is still lacking and a more thorough understanding of ATC pathogenesis may provide new opportunities to improve ATC therapeutics. This review provides insights into ATC clinical presentation and pathology, and the putative role of genetic aberrations and alterations in molecular signaling pathways in ATC pathogenesis. We reviewed prevalent mutations, chromosomal abnormalities and fusions, epigenetic alterations and dysregulations in ATC, and highlighted several signaling cascades which appeared to be integral to ATC pathogenesis. Moreover, these features offer insights into de-differentiated, aggressive and drug-resistant phenotype of ATC, and thus may help in exploring potential new molecular targets for developing novel therapeutics.

MADD silencing enhances anti-tumor activity of TRAIL in anaplastic thyroid cancer.

ATC is an aggressive disease with limited therapeutic options due to drug resistance. TRAIL is an attractive anti-cancer therapy that can trigger apoptosis in a cancer cell-selective manner. However, TRAIL resistance is a major clinical obstacle for its use as a therapeutic drug. Previously, we demonstrated that MADD is a cancer cell pro-survival factor that can modulate TRAIL resistance. However, its role, if any, in overcoming TRAIL resistance in ATC is unknown. First, we characterized ATC cell lines as either TRAIL resistant, TRAIL sensitive or moderately TRAIL sensitive and evaluated MADD expression/cellular localization. We determined the effect of MADD siRNA on cellular growth and investigated its effect on TRAIL treatment. We assessed the effect of combination treatment (MADD siRNA and TRAIL) on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels. The effect of combination treatment on tumor growth was assessed in vivo. We found increased levels of MADD in ATC cells relative to Nthy-ori 3-1. MADD protein localizes in the cytosol (endoplasmic reticulum and Golgi body) and membrane. MADD knockdown resulted in spontaneous cell death that was synergistically enhanced when combined with TRAIL treatment in otherwise resistant ATC cells. Combination treatment resulted in a significant reduction in MMP and enhanced generation of ROS indicating the putative mechanism of action. In an orthotopic mouse model of TRAIL-resistant ATC, treatment with MADD siRNA alone reduced tumor growth that, when combined with TRAIL, resulted in significant tumor regressions. We demonstrated the potential clinical utility of MADD knockdown in sensitizing cells to TRAIL-induced apoptosis in ATC.

Loss of MADD expression inhibits cellular growth and metastasis in anaplastic thyroid cancer.

Anaplastic Thyroid Cancer (ATC) is an aggressive malignancy with limited therapeutic options and dismal patient survival. We have previously shown MADD to be differentially overexpressed in multiple cancer histologies and to contribute to tumor cell growth and survival. Therefore, we targeted MADD by gene silencing, explored its effect on cellular proliferation and metastases and examined its therapeutic potential in an orthotopic ATC model in athymic nude mice. When compared to untreated control and scramble siRNA, MADD siRNA treatment inhibited the proliferative capacity of 8505C, C643 and HTH7 cells in vitro and 8505C-derived-orthotopic tumor growth in vivo. MADD ablation caused a significant reduction in cellular migration and invasion potential; clonogenic capacity; as well as, mitochondrial length and potential in vitro. This MADD siRNA-induced anti-migratory/invasive effect corresponded with inhibition of epithelial-mesenchymal transition (EMT) and Wnt signaling. Mechanistically, MADD siRNA inhibited TNFα induced activation of pERK, pGSK3ß and ß-catenin, suggesting that MADD knockdown might exert its anti-migratory/invasive effects, by blocking TNFα/ERK/GSK3ß axis. MADD siRNA can inhibit ß-catenin nuclear translocation and consequently, the expression of its target genes in ATC cells. In in vivo experiments, along with tumor regression, MADD siRNA treatment also decreased evidence of lung metastases. Immunohistochemically, MADD siRNA-treated tumor tissues exhibited a reduction in Ki67 and N-Cadherin expression, and an increase in E-Cadherin expression. In conclusion, we show the crucial role of MADD in ATC tumorigenesis and metastasis and its potential implications as a molecular target for ATC therapy.

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation.

Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40-/- mice had significantly reduced numbers of CD25-Foxp3low tTreg precursors and CD25+Foxp3+ mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.

Targeting cancer testis antigens for biomarkers and immunotherapy in colorectal cancer: Current status and challenges.

Colorectal cancer ranks third among the estimated cancer cases and cancer related mortalities in United States in 2014. Early detection and efficient therapy remains a significant clinical challenge for this disease. Therefore, there is a need to identify novel tumor associated molecules to target for biomarker development and immunotherapy. In this regard, cancer testis antigens have emerged as a potential targets for developing novel clinical biomarkers and immunotherapy for various malignancies. These germ cell specific proteins exhibit aberrant expression in cancer cells and contribute in tumorigenesis. Owing to their unique expression profile and immunogenicity in cancer patients, cancer testis antigens are clinically referred as the most promising tumor associated antigens. Several cancer testis antigens have been studied in colorectal cancer but none of them could be used in clinical practice. This review is an attempt to address the promising cancer testis antigens in colorectal cancer and their possible clinical implications as biomarkers and immunotherapeutic targets with particular focus on challenges and future interventions.

A novel cancer testis antigen, A-kinase anchor protein 4 (AKAP4) is a potential biomarker for breast cancer.

BACKGROUND: Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma. METHODOLOGY/FINDINGS: We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein. CONCLUSIONS: Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use.

Down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells: possible implications in targeted therapy.

BACKGROUND: Recently, we reported an association of a novel cancer testis (CT) antigen, sperm-associated antigen 9 (SPAG9) expression in breast cancer clinical samples, indicating its potential role in carcinogenesis. Around 15% breast cancers are designated as triple-negative for which treatment modalities are limited. Therefore, in the present study, we assessed the role of SPAG9 in triple-negative breast cancer cells. METHODS: SPAG9 mRNA and protein expression was investigated in various breast cancer cells of different hormone receptor status and different subtypes by employing reverse transcriptase-polymerase chain reaction (RT-PCR), real time PCR, Western blotting, indirect immunofluorescence (IIF) and fluorescence activated cell sorting (FACS). Employing plasmid-based small interfering RNA (siRNA) approach, knockdown of SPAG9 was carried out in triple-negative breast cancer cells, MDA-MB-231, to assess its role on various malignant properties in vitro and in vivo. RESULTS: SPAG9 mRNA and protein expression was detected in all breast cancer cells. Further, IIF results showed that SPAG9 was predominantly localized in the cytoplasm of breast cancer cells. FACS analysis revealed distinct SPAG9 surface localization in breast cancer cells. Gene silencing of SPAG9 resulted in significant reduction in cellular proliferation, colony forming ability, migration, invasion and cellular motility of MDA-MB-231 cells. Further, ablation of SPAG9 expression resulted in reduction in the tumor growth of human breast cancer xenograft in nude mice in vivo. CONCLUSIONS: In summary, our data indicated that down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells, suggesting that SPAG9 may be a potential target for therapeutic use.

The novel cancer-testis antigen A-kinase anchor protein 4 (AKAP4) is a potential target for immunotherapy of ovarian serous carcinoma.

Ovarian cancer is one of the neoplasms affecting the reproductive tract associated with high mortality rate because of limited therapeutic options and an elevated incidence of chemoresistance and recurrence. In this context, immunotherapy may constitute a promising approach to improve survival rates and clinical outcome, raising the need for specific target antigens. Cancer-testis antigens (CTAs) are considered promising candidates in this sense because they are aberrant expressed by various malignancies but not by non-transformed tissue, with the exception of testes. Here, we examined the expression and potential to promote humoral immune responses of a novel CTA, A-kinase anchor protein 4 (AKAP4), among 38 ovarian carcinoma patients. Our results reveal that AKAP4 was expressed at both the mRNA and protein levels in 89% (34/38) of ovarian carcinoma tissue specimens but not in 21 matched adjacent non-cancerous tissues. In addition, a humoral response against AKAP4 was detected in 58% (22/38) of ovarian carcinoma patients by ELISA. In particular, 65% (22/34) patients bearing an AKAP4-expressing tumor exhibited circulating anti-AKAP4 antibodies. Interestingly, the majority of specimens were categorized as ovarian serous adenocarcinoma and serous papillary carcinoma, of which 93% (28/30) and 100% (6/6), respectively, expressed AKAP4. A humoral response against AKAP4 was detected in 79% (19/24) and 67% (4/6) of ovarian serous adenocarcinoma and serous papillary carcinoma patients, respectively. The presence of circulating anti-AKAP4 antibodies suggests the AKAP4 is highly immunogenic in ovarian serous carcinoma patients. Our study lays the foundations for exploring AKAP4 as a potential target for the immunotherapy of ovarian cancer.