The Human EKC/KEOPS Complex Is Recruited to Cullin2 Ubiquitin Ligases by the Human Tumour Antigen PRAME

The human tumour antigen PRAME (preferentially expressed antigen in melanoma) is frequently overexpressed during oncogenesis, and high PRAME levels are associated with poor clinical outcome in a variety of cancers. However, the molecular pathways in which PRAME is implicated are not well understood. We recently characterized PRAME as a BC-box subunit of a Cullin2-based E3 ubiquitin ligase. In this study, we mined the PRAME interactome to a deeper level and identified specific interactions with OSGEP and LAGE3, which are human orthologues of the ancient EKC/KEOPS complex. By characterizing biochemically the human EKC complex and its interactions with PRAME, we show that PRAME recruits a Cul2 ubiquitin ligase to EKC. Moreover, EKC subunits associate with PRAME target sites on chromatin. Our data reveal a novel link between the oncoprotein PRAME and the conserved EKC complex and support a role for both complexes in the same pathways.     

Identification of novel HLA-restricted preferentially expressed antigen in melanoma peptides to facilitate off-the-shelf tumor-associated antigen-specific T-cell therapies

Background aimsPreferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized.MethodsPeptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes.ResultsPRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes.ConclusionsTAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.     

Expression and prognostic relevance of PRAME in primary osteosarcoma

The preferentially expressed antigen of melanoma (PRAME), a cancer-testis antigen with unknown function, is expressed in many human malignancies and is considered an attractive potential target for tumor immunotherapy. However, studies of its expression and function in osteosarcoma have rarely been reported. In this study, we found that PRAME is expressed in five osteosarcoma cell lines and in more than 70% of osteosarcoma patient specimens. In addition, an immunohistochemical analysis showed that high PRAME expression was associated with poor prognosis and lung metastasis. Furthermore, PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. Our results suggest that PRAME plays an important role in cell proliferation and disease progression in osteosarcoma. However, the detail mechanisms of PRAME function in osteosarcoma require further investigation.     

Treating cancer with genetically engineered T cells

Administration of ex vivo cultured, naturally occurring tumor-infiltrating lymphocytes (TILs) has been shown to mediate durable regression of melanoma tumors. However, the generation of TILs is not possible in all patients and there has been limited success in generating TIL in other cancers. Advances in genetic engineering have overcome these limitations by introducing tumor-antigen-targeting receptors into human T lymphocytes. Physicians can now genetically engineer lymphocytes to express highly active T-cell receptors (TCRs) or chimeric antigen receptors (CARs) targeting a variety of tumor antigens expressed in cancer patients. In this review, we discuss the development of TCR and CAR gene transfer technology and the expansion of these therapies into different cancers with the recent demonstration of the clinical efficacy of these treatments.     

Increased PRAME-Specific CTL Killing of Acute Myeloid Leukemia Cells by Either a Novel Histone Deacetylase Inhibitor Chidamide Alone or Combined Treatment with Decitabine

As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8⁺ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201⁺ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME^100–108 or PRAME^300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.     

Biomarkers for the lung cancer diagnosis and their advances in proteomics

Over a last decade, intense interest has been focused on biomarker discovery and their clinical uses. This interest is accelerated by the completion of human genome project and the progress of techniques in proteomics. Especially, cancer biomarker discovery is eminent in this field due to its anticipated critical role in early diagnosis, therapy guidance, and prognosis monitoring of cancers. Among cancers, lung cancer, one of the top three major cancers, is the one showing the highest mortality because of failure in early diagnosis. Numerous potential DNA biomarkers such as hypermethylations of the promoters and mutations in K-ras, p53, and protein biomarkers; carcinoembryonic antigen (CEA), CYFRA21-1, plasma kallikrein B1 (KLKB1), Neuron-specific enolase, etc. have been discovered as lung cancer biomarkers. Despite extensive studies thus far, few are turned out to be useful in clinic. Even those used in clinic do not show enough sensitivity, specificity and reproducibility for general use. This review describes what the cancer biomarkers are for, various types of lung cancer biomarkers discovered at present and predicted future advance in lung cancer biomarker discovery with proteomics technology.     

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer

Background: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. Methods: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. Results: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. Conclusions: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.     

Unravelling the complexity of lncRNAs in autophagy to improve potential cancer therapy

Autophagy is well-known as an internal catabolic process that is evolutionarily conserved and performs the key biological function in maintaining cellular homeostasis. It is tightly controlled by several autophagy-related (ATG) proteins, which are closely associated with many types of human cancers. However, what has remained controversial is the janus roles of autophagy in cancer progression. Interestingly, the biological function of long non-coding RNAs (lncRNAs) in autophagy has been gradually understood in different types of human cancers. More recently, numerous studies have demonstrated that several lncRNAs may regulate some ATG proteins and autophagy-related signaling pathways to either activate or inhibit the autophagic process in cancer. Thus, in this review, we summarize the latest advance in the knowledge of the complicated relationships between lncRNAs and autophagy in cancer. Also, the in-depth dissection of the lncRNAs-autophagy-cancers axis involved in this review would shed new light on discovery of more potential cancer biomarkers and therapeutic targets in the future.     

Hodgkin’s lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.     

Cancer testis antigen PRAME: An anti-cancer target with immunomodulatory potential

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re-expression in poor prognostic solid tumours. PRAME has been extensively investigated as a target for immunotherapy, however, its role in modulating the anti-tumour immune response remains largely unknown. Here, we show that PRAME tumour expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune-unfavourable tumours. Using direct and indirect co-culture models, we found that PRAME overexpressing MDA-MB-468 breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduced expression of several immune checkpoints and their ligands, including PD-1, LAG3, PD-L1, CD86, Gal-9 and VISTA. Interestingly, silencing of PRAME induced cancer cell killing to levels similar to anti-PD-L1 atezolizumab treatment. Comprehensive analysis of soluble inflammatory mediators and cancer cell expression of immune-related genes showed that PRAME tumour expression can suppress the expression and secretion of multiple pro-inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. Together, our data indicate that targeting of PRAME offers a potential, novel dual therapeutic approach to specifically target tumour cells and regulate immune activation in the tumour microenvironment.     

gp78 is specifically expressed in human prostate cancer rather than normal prostate tissue

Elevated expression of gp78 has been observed in many types of cancers including lung, stomach, colon, liver and skin cancer. But there is no report about its expression in prostate cancers. In this study, using immunohistochemical staining we found gp78 is highly expressed in prostate cancers especially early stage tumors, but not in normal prostate tissues. gp78 protein expression is heterogeneous. In some tumors it was expressed in basal cells, while others in stromal cells. For gp78 is a ubiquitin E3 ligase, we then investigated the expression pattern of its cognate E2 (ubiquitin conjugating enzyme)-Ube2g2 in prostate cancers. We found it was expressed in both cancerous and normal tissues of prostate without significant differences in expression level. And unlike gp78, it exhibited a homogeneous expression pattern in different cell types in prostate tissues. In conclusion, our results indicate that gp78 is expressed specifically in human prostate cancer rather than normal prostate tissues, it could be a putative biomarker for prostate cancer diagnosis.     

Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.     

STEAP proteins: from structure to applications in cancer therapy

The human 6-transmembrane epithelial antigen of prostate (STEAP) family comprises STEAP1, STEAP2, STEAP3, and STEAP4. All of these proteins are unique to mammals and share an innate activity as metalloreductases, indicating their importance in metal metabolism. Overall, they participate in a wide range of biologic processes, such as molecular trafficking in the endocytic and exocytic pathways and control of cell proliferation and apoptosis. STEAP1 and STEAP2 are overexpressed in several types of human cancers, namely prostate, bladder, colon, pancreas, ovary, testis, breast, cervix, and Ewing sarcoma, but their clinical significance and role in cancer cells are not clear. Still, their localization in the cell membrane and differential expression in normal and cancer tissues make STEAP proteins potential candidates as biomarkers of several cancers, as well as potential targets for new immunotherapeutic strategies for disease attenuation or treatment. This review brings together the current knowledge about each STEAP protein, giving an overview of the roles of this family of proteins in human physiology and disease, and analyzes their potential as immunotherapeutic agents in cancer research.     

Targeting cancer stem cells by using chimeric antigen receptor-modified T cells: a potential and curable approach for cancer treatment

Cancer stem cells (CSCs), a subpopulation of tumor cells, have self-renewal and multi-lineage differentiation abilities that play an important role in cancer initiation, maintenance, and metastasis. An accumulation of evidence indicates that CSCs can cause conventional therapy failure and cancer recurrence because of their treatment resistance and self-regeneration characteristics. Therefore, approaches that specifically and efficiently eliminate CSCs to achieve a durable clinical response are urgently needed. Currently, treatments with chimeric antigen receptor-modified T (CART) cells have shown successful clinical outcomes in patients with hematologic malignancies, and their safety and feasibility in solid tumors was confirmed. In this review, we will discuss in detail the possibility that CART cells inhibit CSCs by specifically targeting their cell surface markers, which will ultimately improve the clinical response for patients with various types of cancer. A number of viewpoints were summarized to promote the application of CSC-targeted CART cells in clinical cancer treatment. This review covers the key aspects of CSC-targeted CART cells against cancers in accordance with the premise of the model, from bench to bedside and back to bench.     

Implications of hedgehog signaling antagonists for cancer therapy

The hedgehog (Hh) pathway, initially discovered in Drosophila by two Nobel laureates, Dr. Eric Wieschaus and Dr. Christiane Nusslein-Volhard, is a major regulator for cell differentiation, tissue polarity and cell proliferation. Studies from many laboratories, including ours, reveal activation of this pathway in most basal cell carcinomas and in approximately 30% of extracutaneous human cancers, including medulloblastomas, gastrointestinal, lung, breast and prostate cancers. Thus, it is believed that targeted inhibition of Hh signaling may be effective in treating and preventing many types of human cancers. Even more exciting is the discovery and synthesis of specific signaling antagonists for the Hh pathway, which have significant clinical implications in novel cancer therapeutics. This review discusses the major advances in the current understanding of Hh signaling activation in different types of human cancers, the molecular basis of Hh signaling activation, the major antagonists for Hh signaling inhibition and their potential clinical application in human cancer therapy.