A dynamic transcriptome technique for transcriptional profiling and gene regulatory network involving the helicase antigen (HAGE)

Increased knowledge into the molecular pathways disrupted in tumours has led to the development of various therapies that can target specific mediators of these cascades. Such therapies have proven successful in patients or demonstrate significant potential for clinical use. However, this better understanding is undermined by the continued prevalence of cancer and the limitations of these drugs. Therefore, it is possible signalling networks could be influenced by as yet unknown molecules or known mediators with function that have not yet been described. As a result of this, work must continue to improve knowledge yet further to allow the design of novel therapies to aid in the treatment of cancer patients. In the present study, work was performed on the helicase antigen (HAGE), a cancer/testis (CT) antigen and DEAD-box protein found to be present in numerous types of malignancy. As with the majority of CT antigens, the role of HAGE remains unclear. In this instance, studies were carried to discover the function of HAGE in malignant cells. Preliminary in vitro proliferation studies following HAGE gene knockdown or cDNA transfection strongly indicated an association between HAGE expression and increased tumour cell proliferation. This was supported by results gained from in vivo work performed within an immuno-compromised murine model. Expression profiling analysis of data gained from using the Genechip oligonucleotide microarray platform found significant changes to genes linked not only with proliferation but other cell processes altered during tumorigenesis. Confirmation using real-time qPCR suggested change in expression of certain genes could be recognised in other HAGE-expressing tumour cell lines. This analysis also indicated a possible interaction between HAGE and the oncogene N-RAS. Subsequent genetic and protein studies implicated HAGE acting upstream of N-RAS, markedly increasing the N-RAS level in cells. Very preliminary work has begun to demonstrate a role not just in proliferation, but in immune escape, apoptosis inhibition and metastasis, all processes potentially influenced by the RAS oncogenes. The data presented here strongly supports the hypothesis of HAGE having a significant role in malignant biology and warrant continued investigation to further confirm its role in cancer and possibly use as a target for malignancy in the future

HAGE, a cancer/testis antigen expressed at the protein level in a variety of cancers

The search for novel tumour antigens that are either uniquely expressed or over-expressed in a wide variety of tumours is still ongoing. Because of their expression in a broad spectrum of cancers and limited expression in normal tissues, cancer/testis antigens are considered to be potentially reliable targets for immunotherapy of cancer in general. The helicase antigen HAGE has been identified as a cancer/testis antigen. However, little is known about its expression in normal and cancer tissues. Using a newly developed antibody against HAGE, specific staining of its expression by immunohistochemistry was validated and optimised on murine tumours transfected to express the HAGE protein. The antibody was subsequently used to determine HAGE expression in normal human and cancer tissue microarrays. HAGE protein expression was confirmed in 75% (12/16) of carcinomas as compared to normal tissues, which either did not express HAGE at all or expressed HAGE at very low levels with the exception of testis. Interestingly, discrepancies were also found between mRNA analysis by real time quantitative PCR (RT-qPCR) and protein analysis by immunohistochemistry, emphasising the need to validate the expression of cancer/testis antigens at the protein level prior to the development of new vaccine strategies. HAGE is therefore proposed to be a valid candidate for designing a broad spectrum vaccine against cancer

The helicase HAGE prevents interferon-a-induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5 + malignant melanoma-initiating cells (ABCB5 + MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK-STAT (janus kinase-signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK-STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro

HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer

Background: HAGE protein is a known immunogenic cancer-specific antigen. Methods: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. Results: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGEþ) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGEþexpression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+ did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+ and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+ residual disease (P=0.0003). Conclusions: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC

Kinobead Profiling Reveals Reprogramming of BCR Signaling in Response to Therapy within Primary CLL Cells

Purpose: B-cell receptor (BCR) signaling is critical for the pathogenesis of chronic lymphocytic leukemia (CLL), promoting both malignant cell survival and disease progression. Although vital, understanding of the wider signaling network associated with malignant BCR stimulation is poor. This is relevant with respect to potential changes in response to therapy, particularly involving kinase inhibitors. In the current study, we describe a novel high-resolution approach to investigate BCR signaling in primary CLL cells and track the influence of therapy on signaling response. Experimental Design: A kinobead/mass spectrometry–based protocol was used to study BCR signaling in primary CLL cells. Longitudinal analysis of samples donated by clinical trial patients was used to investigate the impact of chemoimmunotherapy and ibrutinib on signaling following surface IgM engagement. Complementary Nanostring and immunoblotting analysis was used to verify our findings. Results: Our protocol isolated a unique, patient-specific signature of over 30 kinases from BCR-stimulated CLL cells. This signature was associated with 13 distinct Kyoto Encyclopedia of Genes and Genomes pathways and showed significant change in cells from treatment-naïve patients compared with those from patients who had previously undergone therapy. This change was validated by longitudinal analysis of clinical trials samples where BCR-induced kinome responses in CLL cells altered between baseline and disease progression in patients failing chemoimmunotherapy and between baseline and treatment in patients taking ibrutinib. Conclusions: These data comprise the first comprehensive proteomic investigation of the BCR signaling response within CLL cells and reveal unique evidence that these cells undergo adaptive reprogramming of this signaling in response to therapy

Antibody Persistence After Primary SARS-CoV-2 Infection and Protection Against Future Variants Including Omicron in Adolescents: National, Prospective Cohort Study.

BACKGROUND: Antibodies are a measure of immunity after primary infection, which may help protect against further SARS-CoV-2 infections. They may also provide some cross-protection against SARS-CoV-2 variants. There are limited data on antibody persistence and, especially, cross-reactivity against different SARS-CoV-2 variants after primary infection in children. METHODS: We initiated enhanced surveillance in 18 secondary schools to monitor SARS-CoV-2 infection and transmission in September 2020. Students and Staff provided longitudinal blood samples to test for variant-specific SARS-CoV-2 antibodies using in-house receptor binding domain assays. We recruited 1189 students and 1020 staff; 160 (97 students, 63 staff) were SARS-CoV-2 nucleocapsid-antibody positive at baseline and had sufficient serum for further analysis. RESULTS: Most participants developed sustained antibodies against their infecting [wild-type (WT)] strain as well as cross-reactive antibodies against the Alpha, Beta and Delta variants but at lower titers than WT. Staff had significantly lower antibodies titers against WT as cross-reactive antibodies against the Alpha, Beta and Delta variants than students (all P < 0.01). In participants with sufficient sera, only 2.3% (1/43) students and 17.2% (5/29) staff had cross-reactive antibodies against the Omicron variant; they also had higher antibody titers against WT (3042.5; 95% confidence interval: 769.0-12,036.2) than those who did not have cross-reactive antibodies against the Omicron variant (680.7; 534.2-867.4). CONCLUSIONS: We found very high rates of antibody persistence after primary infection with WT in students and staff. Infection with WT induced cross-reactive antibodies against Alpha, Beta and Delta variants, but not Omicron. Primary infection with WT may not be cross-protective against the Omicron variant

Positive functioning inventory: initial validation of a 12-item self-report measure of well-being

Background: This paper describes the validation of the Positive Functioning Inventory (PFI-12). This is a 12-item self-report tool developed to assess a spectrum of functioning ranging from states of mental distress to states of well-being. Method: Two samples (Sample 1: N = 242, mean age = 20 years. Sample 2: N = 301, mean age = 20 years) completed self-report measures of personality and social, physical and psychological functioning. Results: Evidence is provided for internal-consistency reliability, test-retest reliability, incremental validity, and convergent and discriminant validity in relation to a number of other measures of personality, social, physical and psychological functioning. Conclusion: The tool promises to be useful to practitioners and researchers who wish to assess positive psychological functioning

SARS-CoV-2 infection, antibody positivity and seroconversion rates in staff and students following full reopening of secondary schools in England: A prospective cohort study, September-December 2020.

Background: Older children have higher SARS-CoV-2 infection rates than younger children. We investigated SARS-CoV-2 infection, seroprevalence and seroconversion rates in staff and students following the full reopening of all secondary schools in England. Methods: Public Health England (PHE) invited secondary schools in six regions (East and West London, Hertfordshire, Derbyshire, Manchester and Birmingham) to participate in SARS-CoV-2 surveillance during the 2020/21 academic year. Participants had nasal swabs for RT-PCR and blood samples for SARS-CoV-2 antibodies at the beginning (September 2020) and end (December 2020) of the autumn term. Multivariable logistic regression was used to assess independent risk factors for seropositivity and seroconversion. Findings: Eighteen schools in six regions enrolled 2,209 participants, including 1,189 (53.8%) students and 1,020 (46.2%) staff. SARS-CoV-2 infection rates were not significantly different between students and staff in round one (5/948; [0.53%] vs. 2/876 [0.23%]; p = 0.46) or round two (10/948 [1.05%] vs. 7/886 [0.79%]; p = 0.63), and similar to national prevalence. None of four and 7/15 (47%) sequenced strains in rounds 1 and 2 were the highly transmissible SARS-CoV-2 B.1.1.7 variant. In round 1, antibody seropositivity was higher in students than staff (114/893 [12.8%] vs. 79/861 [9.2%]; p = 0.016), but similar in round 2 (117/893 [13.1%] vs.117/872 [13.3%]; p = 0.85), comparable to local community seroprevalence. Between the two rounds, 8.7% (57/652) staff and 6.6% (36/549) students seroconverted (p = 0.16). Interpretation: In secondary schools, SARS-CoV-2 infection, seropositivity and seroconversion rates were similar in staff and students, and comparable to local community rates. Ongoing surveillance will be important for monitoring the impact of new variants in educational settings

Antibody persistence and neutralising activity in primary school students and staff: Prospective active surveillance, June to December 2020, England.

Background: Prospective, longitudinal SARS-CoV-2 sero-surveillance in schools across England was initiated after the first national lockdown, allowing comparison of child and adult antibody responses over time. Methods: Prospective active serological surveillance in 46 primary schools in England tested for SARS-CoV-2 antibodies during June, July and December 2020. Samples were tested for nucleocapsid (N) and receptor binding domain (RBD) antibodies, to estimate antibody persistence at least 6 months after infection, and for the correlation of N, RBD and live virus neutralising activity. Findings: In June 2020, 1,344 staff and 835 students were tested. Overall, 11.5% (95%CI: 9.4-13.9) and 11.3% (95%CI: 9.2-13.6; p = 0.88) of students had nucleoprotein and RBD antibodies, compared to 15.6% (95%CI: 13.7-17.6) and 15.3% (95%CI: 13.4-17.3; p = 0.83) of staff. Live virus neutralising activity was detected in 79.8% (n = 71/89) of nucleocapsid and 85.5% (71/83) of RBD antibody positive children. RBD antibodies correlated more strongly with neutralising antibodies (rs=0.7527; p6 months) but further work is required to understand the extent to which this protects against reinfection