Multi-Omic Analysis of Two Common P53 Mutations: Proteins Regulated by Mutated P53 as Potential Targets for Immunotherapy.

The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein-protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53.

A Proteomic Approach Reveals That miR-423-5p Modulates Glucidic and Amino Acid Metabolism in Prostate Cancer Cells.

Recently, we have demonstrated that miR-423-5p modulates the growth and metastases of prostate cancer (PCa) cells both in vitro and in vivo. Here, we have studied the effects of miR-423-5p on the proteomic profile in order to identify its intracellular targets and the affected pathways. Applying a quantitative proteomic approach, we analyzed the effects on the protein expression profile of miR-423-5p-transduced PCa cells. Moreover, a computational analysis of predicted targets of miR-423-5p was carried out by using several target prediction tools. Proteomic analysis showed that 63 proteins were differentially expressed in miR-423-5-p-transfected LNCaP cells if compared to controls. Pathway enrichment analysis revealed that stable overexpression of miR-423-5p in LNCaP PCa cells induced inhibition of glycolysis and the metabolism of several amino acids and a parallel downregulation of proteins involved in transcription and hypoxia, the immune response through Th17-derived cytokines, inflammation via amphorin signaling, and ion transport. Moreover, upregulated proteins were related to the S phase of cell cycle, chromatin modifications, apoptosis, blood coagulation, and calcium transport. We identified seven proteins commonly represented in miR-423-5p targets and differentially expressed proteins (DEPs) and analyzed their expression and influence on the survival of PCa patients from publicly accessible datasets. Overall, our findings suggest that miR-423-5p induces alterations in glucose and amino acid metabolism in PCa cells paralleled by modulation of several tumor-associated processes.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy.

BACKGROUND: Homocitrullination is the post-translational modification of lysine that is recognized by T cells. METHODS: This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry. RESULTS: Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment. CONCLUSION: We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

ß2-Adrenergic Signalling Promotes Cell Migration by Upregulating Expression of the Metastasis-Associated Molecule LYPD3.

Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and ß2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-ß2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-ß2 inhibition. This study also highlights the first association between ADRß2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRß2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

PYK2 promotes HER2-positive breast cancer invasion.

BACKGROUND: Metformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer. METHODS: The effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER-/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER-/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER-/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER-/PR- patients' cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER-/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays. RESULTS: Our study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44. CONCLUSIONS: We provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients.

Generation of In Vitro Model of Epithelial Mesenchymal Transition (EMT) Via the Expression of a Cytoplasmic Mutant Form of Promylocytic Leukemia Protein (PML).

Epithelial Mesenchymal Transition (EMT) is a key event in cancer progression. During this event, epithelial cancer cells undergo molecular and cellular changes leading to their trans-differentiation into mesenchymal cancer cells that are capable of migration, invasion, and metastasis to other tissues and organs. Here, we present a method for in vitro induction of EMT in prostate cancer cell lines using lentiviral expression of a PMLI isoform mutant construct.

The localization of pre mRNA splicing factor PRPF38B is a novel prognostic biomarker that may predict survival benefit of trastuzumab in patients with breast cancer overexpressing HER2.

Cancer biomarkers that can define disease status and provide a prognostic insight are essential for the effective management of patients with breast cancer (BC). The prevalence, clinicopathological and prognostic significance of PRPF38B expression in a consecutive series of 1650 patients with primary invasive breast carcinoma were examined using immunohistochemistry. Furthermore, the relationship(s) between clinical outcome and PRPF38B expression was explored in 627 patients with ER-negative (oestrogen receptor) disease, and 322 patients with HER2-overexpressing disease. Membranous expression of PRPF38B was observed in 148/1388 (10.7%) cases and was significantly associated with aggressive clinicopathological features, including high grade, high mitotic index, pleomorphism, invasive ductal carcinoma of no specific type (IDC-NST), ER-negative, HER2-overexpression and p53 mutational status (all p < 0.01). In patients with ER-negative disease receiving chemotherapy, nuclear expression of PRPF38B was significantly associated with a reduced risk of relapse (p = 0.0004), whereas membranous PRPF38B expression was significantly associated with increased risk of relapse (p = 0.004; respectively) at a 5 year follow-up. When patients were stratified according to ER-negative/HER2-positive status, membranous PRPF38B expression was associated with a higher risk of relapse in those patients that did not receive trastuzumab therapy (p = 0.02), whereas in those patients with ER-negative/HER2-positive disease that received trastuzumab adjuvant therapy, membranous PRPF38B expression associated with a lower risk of relapse (p = 0.00018). Nuclear expression of PRPF38B is a good prognostic indicator in both ER-negative patients and ER-negative/HER2-positive BC (breast cancer) patients, whereas membranous localisation of PRPF38B is a poor prognostic biomarker that predicts survival benefit from trastuzumab therapy in patients with ER-negative/HER2-overexpressing BC.

Identification of novel breast cancer-associated transcripts by UniGene database mining and gene expression analysis in normal and malignant cells.

Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation.

The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo.

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.

Expression of the tumour antigen T21 is up-regulated in prostate cancer and is associated with tumour stage.

OBJECTIVES: • To define the expression pattern of the tumour antigen T21 at the protein level in prostate tissues, prostate cell lines and a panel of normal tissues. • To correlate the expression pattern of T21 in prostate cancer with clinical parameters. PATIENTS AND METHODS: • Tissue samples were collected from 79 patients presenting at clinic with either prostate cancer (63 patients) or benign prostatic hyperplasia (BPH, 16 patients). • A tissue microarray (TMA) was constructed from 44 of the prostate cancer tissues and areas of benign disease (43 patients) from these tissues were also included on the TMA. The remaining tissues (prostate cancer 19 patients and BPH 16 patients) were mounted fresh frozen onto cork boards and sectioned. • Full ethical approval was granted for all aspects of the study and informed patient consent was taken before tissue collection. • Immunohistochemistry was used on the prostate tumour TMA, the normal tissue TMA and the fresh-frozen prostate tissues. Fluorescent microscopy and flow cytometry was performed on prostate cell lines. RESULTS: • Expression of T21 was highly restricted within normal tissues with only the stomach, ovary, breast and prostate having detectable T21 expression. • T21 was significantly over-expressed in prostate cancer glands compared with benign tissue and was present in >80% of the malignant specimens analysed. • Increased expression was positively correlated to pathological stage of prostate tumours. • Additionally, T21 was associated with Gleason grade and prostate-specific antigen recurrence, although statistical significance was not reached in this restricted cohort of patients. CONCLUSION: • Taken together these results show that T21 is a potential new biomarker for advanced disease and that elevated levels of T21 appear relevant to prostate cancer development.

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.

Identification of a novel prostate cancer-associated tumor antigen.

BACKGROUND: The identification of antigens that distinguish cancer cells from normal cells is of major importance for the definition of therapeutic targets in human malignancies. Using sera from cancer patients, we have previously reported on the identification of immunologically recognized proteins that belong to the family of cancer testis antigens (CTAs). METHODS: A normal testicular cDNA library was screened with pooled allogeneic sera from patients with prostate cancer using a modified SEREX approach. Subsequently we have identified and characterized a novel antigen, T21, with an expression pattern similar to that of CTAs. mRNA expression of T21 was determined using a panel of whole tissues and prostate cell lines using Q-RT-PCR. For laser microdissection, fresh prostate cancer and benign tissue was obtained using our novel validated harvesting technique. Protein expression and cellular localization of T21 were assessed in prostate cell lines using Western blotting, confocal microscopy and flow cytometry. RESULTS: T21 showed tissue-restricted mRNA expression in gastric, kidney and prostate cancers, and in normal testis and prostate tissues. Following laser microdissection, T21 was significantly over-expressed in malignant compared to benign prostatic epithelium. We have demonstrated expression of T21 at the protein level and confocal microscopy on PC3 cells probed with a T21-monospecific antibody revealed cytoplasmic localization of T21 protein. CONCLUSIONS: The highly restricted expression pattern of T21 makes it an attractive vaccine target for prostate cancer. Several CTAs reportedly induce cytotoxic T-lymphocyte responses, therefore it is reasonable to assume that T21 will be a valuable target for cancer immunotherapy.

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.

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.