Amanda K. Miles

Identification of tumour antigens by serological analysis of cDNA expression cloning

The identification of antigens that distinguish normal cells from cancer cells is an important challenge in the field of tumour immunology and immunotherapy. The immunoscreening of cDNA expression libraries constructed from human tumour tissues with antibodies in sera from cancer patents (SEREX: serological identification of antigens by recombinant expression cloning) provides a powerful approach to identify immunogenic tumour antigens. To date, over 2,000 tumour antigens have been identified from a variety of malignancies using SEREX. These antigens can be classified into several categories, of which the cancer/testis (CT) antigens appear to be the most attractive candidates for vaccine development. The SEREX-defined tumour antigens facilitate the identification of epitopes (antigenic peptides) recognised by antigen-specific cytotoxic T lymphocytes (CTLs) and provide a basis for peptide vaccine and gene therapy in a wide variety of human cancers. Moreover, some of these antigens seem to play a functional role in the pathogenesis of cancer.

Diagnostic biomarkers differentiating metastatic melanoma patients from healthy controls identified by an integrated MALDI-TOF mass spectrometry/bioinformatic approach

The prognosis of advanced metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) remains dismal with a 5‐year survival rate of 6–18%. In the present study, an integrated MALDI mass spectrometric approach combined with artificial neural networks (ANNs) analysis and modeling has been used for the identification of biomarker ions in serum from stage IV melanoma patients allowing the discrimination of metastatic disease from healthy status with high specificities of 92% for protein ions and 100% for peptide biomarkers. Our ANNs model also correctly classified 98% of a blind validation set of AJCC stage I melanoma samples as nonstage IV samples, emphasizing the power of the newly defined biomarkers to identify patients with late‐stage metastatic melanoma. Sequence analysis identified peptides derived from metastasis‐associated proteins; alpha 1‐acid glycoprotein precursor‐1/2 (AAG‐1/2) and complement C3 component precursor‐1 (CCCP‐1). Furthermore, quantitation of serum AAG by an immunoassay showed a significant (p<0.001) increase in AAG serum concentration in stage IV patients in comparison with healthy volunteers; moreover; the quantity of AAG plotted against MALDI‐MS peak intensity classified the groups into two distinct clusters. Ongoing studies of other disease stages will provide evidence whether our strategy is sufficiently robust to give rise to stage‐specific protein/peptide signatures in melanoma.

Identification of SPARC-like 1 protein as part of a biomarker panel for Alzheimer's disease in cerebrospinal fluid.

We have used proteomic fingerprinting to investigate diagnosis of Alzheimers disease (AD). Samples of lumbar cerebrospinal fluid (CSF) from clinically-diagnosed AD cases (n = 33), age-matched controls (n = 20), and mild cognitive impairment (MCI) patients (n = 10) were used to obtain proteomic profiles, followed by bioinformatic analysis that generated a set of potential biomarkers in CSF samples that could discriminate AD cases from controls. The identity of the biomarker ions was determined using mass spectroscopy. The panel of seven peptide biomarker ions was able to discriminate AD patients from controls with a median accuracy of 95% (sensitivity 85%, specificity 97%). When this model was applied to an independent blind dataset from MCI patients, the intensity of signals was intermediate between the control and AD patients implying that these markers could potentially predict patients with early neurodegenerative disease. The panel were identified, in order of predictive ability, as SPARC-like 1 protein, fibrinogen alpha chain precursor, amyloid-β, apolipoprotein E precursor, serum albumin precursor, keratin type I cytoskeletal 9, and tetranectin. The 7 ion ANN model was further validated using an independent cohort of samples, where the model was able to classify AD cases from controls with median accuracy of 84.5% (sensitivity 93.3%, specificity 75.7%). Validation by immunoassay was performed on the top three identified markers using the discovery samples and an independent sample cohort which was from postmortem confirmed AD patients (n = 17).

The Helicase HAGE Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Cell Proliferation in Vivo

Background: ABCB5+ MMIC are a population of chemoresistant cancer stem cell-like cells responsible for melanoma initiation, growth, and progression. Results: HAGE promotes ABCB5+ MMIC-dependent tumorigenesis by enhancing RAS protein expression. Conclusion: ABCB5+ MMIC require the presence of HAGE for their tumorigenic activity. Significance: HAGE is expressed only by tumor cells. Hence, targeting HAGE helicase may have broad therapeutic applications. Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types.

Immunotherapy of prostate cancer: should we be targeting stem cells and EMT?

Cancer stem cells have been implicated in a number of solid malignancies including prostate cancer. In the case of localised prostate cancer, patients are often treated with surgery (radical prostatectomy) and/or radiotherapy. However, disease recurrence is an issue in about 30% of patients, who will then go on to receive hormone ablation therapy. Hormone ablation therapy is often palliative in a vast proportion of individuals, and for hormone-refractory patients, there are several immunotherapies targeting a number of prostate tumour antigens which are currently in development. However, clinical responses in this setting are inconsistent, and it is believed that the failure to achieve full and permanent tumour eradication is due to a small, resistant population of cells known as ‘cancer stem cells’ (CSCs). The stochastic and clonal evolution models are among several models used to describe cancer development. The general consensus is that cancer may arise in any cell as a result of genetic mutations in oncogenes and tumour suppressor genes, which consequently result in uncontrolled cell growth. The cancer stem cell theory, however, challenges previous opinion and proposes that like normal tissues, tumours are hierarchical and only the rare subpopulation of cells at the top of the hierarchy possess the biological properties required to initiate tumourigenesis. Furthermore, where most cancer models infer that every cell within a tumour is equally malignant, i.e. equally capable of reconstituting new tumours, the cancer stem cell theory suggests that only the rare cancer stem cell component possess tumour-initiating capabilities. Hence, according to this model, cancer stem cells are implicated in both tumour initiation and progression. In recent years, the role of epithelial–mesenchymal transition (EMT) in the advancement of prostate cancer has become apparent. Therefore, CSCs and EMT are both likely to play critical roles in prostate cancer tumourigenesis. This review summarises the current immunotherapeutic strategies targeting prostate tumour antigens taking into account the need to consider treatments that target cancer stem cells and cells involved in epithelial–mesenchymal transition.

Cytoplasmic PML promotes TGF-β-associated epithelial-mesenchymal transition and invasion in prostate cancer.

Epithelial–mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-β signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-β signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-β canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-β signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.

The identification of human tumour antigens: current status and future developments

The biggest challenge facing us today in cancer control and prevention is the identification of novel biomarkers for detection and improved therapeutic interventions to reduce mortality and morbidity rates. Biomarkers are important indicators to inform us of the physiological state of the cell at a specific time. It is now clear that malignant transformation occurs by changes in cellular DNA and protein expression with subsequent clonal proliferation of the altered cells. The affected genes and their expressed protein products or biomarkers are those involved in the normal growth and maintenance of the cancerous cells. These biomarkers could prove pivotal for the identification of early cancer and people at risk of developing cancer. Altered proteins or changes in gene expression in malignant cells may lead to the expression of tumour antigens recognised by host immune system. In this review we discuss current research into the molecular technologies making possible the global genomic-wide analysis of changes in DNA (genotyping), RNA expression (transcriptomics) and protein expression (proteomics) that have accelerated the rate of new biomarker/tumour antigen discovery. To gain a comprehensive understanding of the physiology and pathophysiology of cancer an approach that harmoniously integrates the various ‘omic’ platforms are key to unraveling the complexity ‘needle-in-a-haystack’ quality of biomarker/tumour antigen discovery.

The helicase HAGE prevents interferon-α-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. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.

Serum biomarkers which correlate with failure to respond to immunotherapy and tumor progression in a murine colorectal cancer model

Purpose: To advance our understanding of mechanisms involved in tumor progression/regression, a CT26 colorectal mouse model treated intra‐tumorally with DISC‐herpes simplex virus as immunotherapy was used in the discovery and validation phases to investigate and ultimately identify biomarkers correlating with the failure to respond to immunotherapy.

Immunity to Tumour Antigens

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.