Alternative Reading Frame Protein Research Articles

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

A Proteomic Approach to Study the Biological Role of Hepatitis C Virus Protein Core+1/ARFP.

Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein—known as ARFP (alternative reading frame protein) or Core+1 protein—is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.

The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNβ-mediated Lyme arthritis.

Type I interferon (IFN) has been identified in patients with Lyme disease, and its abundant expression in joint tissues of C3H mice precedes development of Lyme arthritis. Forward genetics using C3H mice with severe Lyme arthritis and C57BL/6 (B6) mice with mild Lyme arthritis identified the Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1) on chromosome 4 (Chr4) as a regulator of B. burgdorferi-induced IFNβ expression and Lyme arthritis severity. B6 mice introgressed with the C3H allele for Bbaa1 (B6.C3-Bbaa1 mice) displayed increased severity of arthritis, which is initiated by myeloid lineage cells in joints. Using advanced congenic lines, the physical size of the Bbaa1 interval has been reduced to 2 Mbp, allowing for identification of potential genetic regulators. Small interfering RNA (siRNA)-mediated silencing identified Cdkn2a as the gene responsible for Bbaa1 allele-regulated induction of IFNβ and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). The Cdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNβ induction in BMDMs as shown by siRNA silencing and overexpression of ARF. In vivo studies demonstrated that p19ARF contributes to joint-specific induction of IFNβ and arthritis severity in B. burgdorferi-infected mice. p19ARF regulates B. burgdorferi-induced IFNβ in BMDMs by stabilizing the tumor suppressor p53 and sequestering the transcriptional repressor BCL6. Our findings link p19ARF regulation of p53 and BCL6 to the severity of IFNβ-induced Lyme arthritis in vivo and indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.

Sero-reactivity to three distinct regions within the hepatitis C virus alternative reading frame protein (ARFP/core+1) in patients with chronic HCV genotype-3 infection.

Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5 % of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three −20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47 % for ARFP-P2, 5.9 % for ARFP-P3 and 100 % for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.

Anticancer and Antimicrobial Evaluations on Alternative Reading Frame (ARF) Peptides and their Derivatives.

Alternative reading frame (ARF) protein up-regulates the intracellular level of a tumour suppressor protein, p53, by blocking MDM2 mediated p53 ubiquitination. The two homologous forms of ARF proteins are p19ARF in mice and p14ARF in humans. In our study, p19ARF-derived peptide ARF (26-44) and its cell-penetrating peptide conjugate Tat-ARF (26-44), p14ARF-derived peptide ARF (1-22), and its NrLS conjugate ARF (1-22)-NrLS were designed, and their anticancer properties were investigated. Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. Peptides synthesized using solid-phase peptide synthesis (SPPS) were purified using RPHPLC and characterized using Bruker MALDI-TOF mass spectrometry. Cytotoxicity was evaluated on HeLa and BE(2)-C cells by cell viability IC50 determination. Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Morphological studies were carried out using SEM and TEM techniques, live/dead staining, ROS and Hoest staining. Peptides Tat-ARF (1-22) and ARF (1-22)-NrLS exhibited potent cytotoxic effects, comparable to the known standard cisplatin. Cellular morphological studies showed signs of apoptosis which were confirmed by reactive oxygen species (ROS) generation and Hoechst nuclear staining. ARF peptides showed potent antimicrobial activities at low micromolar concentrations without haemolysis. Tat modification improved the activity of ARF (26-44) by 9 folds against HeLa and 5 folds against BE(2)-C cells. NrLS modification of ARF (1-22) imparted 12 fold potency against HeLa and 2-fold potency against BE(2)-C cells. This study helps to further understand the effect of these peptides on MDM2 proteins and their role in the apoptosis signalling pathway.

Spherical Nucleic Acid Vaccine Structure Markedly Influences Adaptive Immune Responses of Clinically Utilized Prostate Cancer Targets.

Cancer vaccines, which activate the immune system against a target antigen, are attractive for prostate cancer, where multiple upregulated protein targets are identified. However, many clinical trials implementing peptides targeting these proteins have yielded suboptimal results. Using spherical nucleic acids (SNAs), we explore how precise architectural control of vaccine components can activate a robust antigen-specific immune response in comparison to clinical formulations of the same targets. The SNA vaccines incorporate peptides for human prostate-specific membrane antigen (PSMA) or T-cell receptor γ alternate reading frame protein (TARP) into an optimized architecture, resulting in high rates of immune activation and cytolytic ability in humanized mice and human peripheral blood mononuclear cells (hPBMCs). Specifically, administered SNAs elevate the production and secretion of cytokines and increase polyfunctional cytotoxic T cells and effector memory. Importantly, T cells raised from immunized mice potently kill targets, including clinically relevant cells expressing the whole PSMA protein. Treatment of hPBMCs increases costimulatory markers and cytolytically active T cells. This work demonstrates the importance of vaccine structure and its ability to reformulate and elevate clinical targets. Moreover, it encourages the field to reinvestigate ineffective peptide targets and repackage them into optimally structured vaccines to harness antigen potency and enhance clinical outcomes.

TARP as antigen in cancer immunotherapy.

In recent decades, immunotherapy has become a pivotal element in cancer treatment. A remaining challenge is the identification of cancer-associated antigens suitable as targets for immunotherapeutics with potent on-target and few off-tumor effects. The T-cell receptor gamma (TCRγ) chain alternate reading frame protein (TARP) was first discovered in the human prostate and androgen-sensitive prostate cancer. Thereafter, TARP was also identified in breast and endometrial cancers, salivary gland tumors, and pediatric and adult acute myeloid leukemia. Interestingly, TARP promotes tumor cell proliferation and migration, which is reflected in an association with worse survival. TARP expression in malignant cells, its role in oncogenesis, and its limited expression in normal tissues raised interest in its potential utility as a therapeutic target, and led to development of immunotherapeutic targeting strategies. In this review, we provide an overview of TARP expression, its role in different cancer types, and currently investigated TARP-directed immunotherapeutic options.

MiR-6838-5p facilitates the proliferation and invasion of renal cell carcinoma cells through inhibiting the DMTF1/ARF-p53 axis.

Renal cell carcinoma (RCC) is one of the most common renal malignancies in the urinary system. Numerous studies have demonstrated that miRNAs can regulate tumorigenesis and progression. This study aims to investigate the role and regulatory mechanism of miR-6838-5p in RCC. Our study confirmed that miR-6838-5p was upregulated in human RCC tissues (30/42, 77.43%, P < 0.01) and RCC cell lines (P < 0.05) compared to adjacent non-neoplastic tissues and normal renal epithelial cells. In vitro, overexpression of miR-6838-5p enhanced cell proliferation and invasion in human RCC cell lines (ACHN and 786-O), which were detected by CCK-8, Transwell and Colony formation assays (P < 0.05), and knockdown of miR-6838-5p suppressed cell proliferation and invasion (P < 0.05). Results of Bioinformatics analysis combined with Dual-luciferase reporter gene assay demonstrated that miR-6838-5p could bind to Cyclin D binding myb-like transcription factor 1 (DMTF1). In addition, RT-qPCR and Western blotting confirmed that DMTF1 was downregulated in RCC tissues and cell lines. Meanwhile, it was demonstrated that overexpression of miR-6838-5p inhibited DMTF1 level in ACHN cells. Next, we confirmed that DMTF1 overexpression reversed the inhibitory effects of overexpression of miR-6838-5p on phosphatase and tensin homolog (PTEN), tumor protein 53(p53), murine double minute 2 (MDM2) and alternative reading frame (ARF) protein levels in the ARF-p53 signaling pathway. In conclusion, our research showed that miR-6838-5p enhanced the proliferation and invasion of RCC cells by inhibiting the DMTF1/ARF-p53 axis.

High expression of TARP correlates with inferior FLT3 mutations in non-adolescents and young adults with acute myeloid leukaemia

ABSTRACT Objectives Acute myeloid leukaemia (AML) is a haematopoietic malignancy with a dismal outcome. Consequently, risk stratification based on more effective prognostic biomarkers is crucial to make accurate therapy decisions. T cell receptor gamma alternative reading frame protein (TARP) has been reported in prostate and breast cancers, but its correlation with AML remains unclear. Methods Differential expression of TARP mRNA in different AML subtypes was analysed using the UALCAN online platform. Its relationship with baseline clinical attributes, survival and efficacy were analysed based on three GSE1159, GSE425 and GSE6891 microarray datasets downloaded from Gene Expression Omnibus (GEO) and Oncomine databases. Quantitative real-time PCR was performed to determine mRNA levels of TARP in bone marrow mononuclear cells (BMMCs) isolated from AML patients. Results TARP was significantly overexpressed in AML patients. In AML, relatively low TARP expression was associated with the CBFβ-MYH11 fusion gene. The proportion of FLT3 mutations was significantly higher in non-adolescent and young adult (non-AYA, >39 years of age) AML patients who had high TARP levels but not in AYA (15–39 years) patients. High expression of TARP was related to poor outcome by univariate analysis but not by multivariate analysis and unsatisfactory therapeutic effects, which could be overcome by haematopoietic stem cell transplantation (HSCT). Conclusion Our findings suggest that TARP might be a potential prognostic marker of AML and serve as a promising immunotherapeutic target.

Human ARF Specifically Inhibits Epimorphic Regeneration in the Zebrafish Heart.

The Alternative Reading Frame (ARF) protein is a tumor suppressor encoded by the Cyclin Dependent Kinase Inhibitor 2A gene in mammals but not lower regenerative vertebrates, and has been previously implicated as a context-sensitive suppressor of regeneration in murine skeletal muscle and humanized ARF-expressing zebrafish fins. This study extends our investigation of the role of ARF in the regeneration of other solid tissues, including the zebrafish heart and the mammalian digit. Heart regeneration after cryoinjury was used to mimic massive myocardial infarction. ARF gene expression was upregulated during the cardiac regenerative process and slowed the rate of morphological recovery. ARF specifically impacts cardiomyocytes, neovascularization, and the endothelial-mesenchymal transition, while not affecting epicardial proliferation. This suggests that in the context of regeneration, ARF is specifically expressed in cells undergoing dedifferentiation. To investigate ARF as a suppressor of epimorphic regeneration in mammalian systems, we also tested whether the absence of ARF was permissive for murine digit regeneration, but found that ARF absence alone was insufficient to significantly alter digit restoration. These findings provide additional evidence that ARF suppresses epimorphic regeneration, but suggests that modulation of ARF alone is insufficient to permit regeneration.

Hepatitis C virus alternative reading frame protein (ARFP): Production, features, and pathogenesis.

Earlier observation suggests that hepatitis C virus (HCV) is a single-stranded RNA virus which encodes at least 10 viral proteins. F protein is a novel protein which has been discovered recently. These studies suggest three mechanisms for the production of this protein concerning ribosomal frameshift at codon 10, initial translation at codons 26 and 85 or 87. In this study, the association between protein F and chronicity of hepatocellular carcinoma (HCC) has been reviewed. Evidence suggests that humoral immune system can recognize this protein and produce antibodies against it. By detecting antibodies in infected people, investigators found that F protein might have a role in HCV infection causing chronic cirrhosis and HCC as higher prevalence was found in patients with mentioned complications. The increment of CD4+, CD25+, and FoxP3+ T cells, along with CD8+ T cells with low expression of granzyme B, also leads to weaker responses of the immune system which helps the infection to become chronic. Moreover, it contributes to the survival of the virus in the body through affecting the production of interferon. F protein also might play roles in the disease development, resulting in HCC. The existence of F protein affects cellular pathways through upregulating p53, c-myc, cyclin D1, and phosphorylating Rb. This review will summarize these effects on immune system and related mechanisms in cellular pathways.

R-DOTAP (Versamune): A novel enantiospecific cationic lipid nanoparticle that induces CD4 and CD8 cellular immune responses to whole protein and tumor-specific peptide antigens.

e15211 Background: Immunotherapy approaches are limited in their ability to induce antigen-specific CD8+ T cells in vivo able to recognize and kill tumor cells. We developed a novel immunotherapy approach using enantiomerically pure, R-DOTAP cationic lipid nanoparticles and tumor-derived T cell antigens, and previously demonstrated that R-DOTAP formulations efficiently prime cytotoxic T cells through enhanced cross presentation and induction of type I interferons.[1] A phase I clinical trial of a R-DOTAP HPV16 peptide formulation confirmed induction of strong in vivo HPV-specific CD8+ cytolytic T-cells without associated systemic toxicities. In this study, we assessed R-DOTAP nanoparticle formulations containing whole protein (ovalbumin) or long multi-epitope peptides from the tumor antigen TARP (T-cell alternate reading frame protein): a 58-residue protein overexpressed in prostate and breast cancers, documented to be immunogenic in humans. Methods: R-DOTAP formulations were prepared containing ovalbumin (OVA) or TARP peptides. C57BL/6K mice were immunized with 10 μg/mouse of OVA plus R-DOTAP, CFA or sucrose on Days 0, 15 and 30. OVA-specific cellular and humoral responses following vaccination were assessed by measuring splenic CD4 and CD8 T cell IFN-γ production and circulating OVA-specific antibodies in serum. HLA-A2 transgenic mice (AAD mice) were vaccinated with long, multi-epitope TARP peptides delivered as an R-DOTAP admixture or with CFA or sucrose on Days 0 and 7. Antigen-specific T cell responses were measured by IFN-γ ELISpot assay. Results: OVA R-DOTAP formulations induced strong antigen-specific effector CD4 and CD8 immune and memory responses detected 7 and 30 days, respectively, following vaccination as well as OVA-specific antibody responses. In TARP peptide vaccinated mice, R-DOTAP formulations were able to present multiple CD8 T cell epitopes and stimulate responses that were superior to CFA. Conclusions: Our results suggest that R-DOTAP is a versatile immune activating therapy that can be formulated with long, multi-epitope tumor-derived peptides or whole proteins. R-DOTAP formulations induce quantitatively robust antigen-specific CD4 and CD8 T cells in vivo compared to well-established immune stimulants. Reference: 1.Gandhapudi SK, Ward M, Bush JP et al. Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response. J Immunol 2019;202:3524-3536

Loss of ARF/INK4A Promotes Liver Progenitor Cell Transformation Toward Tumorigenicity Supporting Their Role in Hepatocarcinogenesis.

Liver progenitor cells (LPCs) contribute to liver regeneration during chronic damage and are implicated as cells of origin for liver cancers including hepatocellular carcinoma (HCC). The CDKN2A locus, which encodes the tumor suppressors alternate reading frame protein (ARF) and INK4A, was identified as one of the most frequently altered genes in HCC. This study demonstrates that inactivation of CDKN2A enhances tumorigenic transformation of LPCs. The level of ARF and INK4A expression was determined in a panel of transformed and nontransformed wild-type LPC lines. Moreover, the transforming potential of LPCs with inactivated CDKN2A was shown to be enhanced in LPCs derived from Arf-/- and CDKN2Afl/fl mice and in wild-type LPCs following CRISPR-Cas9 suppression of CDKN2A. ARF and INK4A abundance is consistently reduced or ablated following LPC transformation. Arf-/- and CDKN2A-/- LPCs displayed hallmarks of transformation such as anchorage-independent and more rapid growth than control LPC lines with unaltered CDKN2A. Transformation was not immediate, suggesting that the loss of CDKN2A alone is insufficient. Further analysis revealed decreased p21 expression as well as reduced epithelial markers and increased mesenchymal markers, indicative of epithelial-to-mesenchymal transition, following inactivation of the CDKN2A gene were required for tumorigenic transformation. Loss of ARF and INK4A enhances the propensity of LPCs to undergo a tumorigenic transformation. As LPCs represent a cancer stem cell candidate, identifying CDKN2A as a driver of LPC transformation highlights ARF and INK4A as viable prognostic markers and therapeutic targets for HCC.

Antibody Development to HCV Alternate Reading Frame Protein in Liver Transplant Candidate and its Computational Analysis

Background:: HCV Alternate Reading Frame Protein (ARFP) is a frameshift product of HCV-core encoding. Here, we characterized specific anti-ARFP antibodies in Liver Transplant Candidate (LTC) and chronic HCV-infected patients. Methods:: The ARFP gene was cloned and the recombinant protein was purified using Nickel chromatography and confirmed by western blotting. ELISA was developed using recombinant core-1a, core- 1b, ARFP-1a protein, and 99-residue synthetic ARFP 1b peptide. By several Bioinformatics tools, general properties, immunogenic epitopes, and structures of these proteins were obtained. Results:: The seroprevalence of anti-core and anti-ARFP antibodies was 100% in LTC patients, but only 75.2% and 94.3% of chronic patients had evidence of anti-ARFP and anti-core antibodies, respectively. In-silico results demonstrated physicochemical features, antigen properties and potential interactors that could describe progression toward advanced liver disease. Conclusion:: As the first report, the prevalence of anti-ARFP antibodies in LTC patients is of the order of 100% and titer of anti-ARFP antibody was significantly higher in LTC patients compared to chronic individuals, suggesting the possible role of ARFP in the progression toward advanced liver disease. In addition, docking analysis determined several interactor proteins such as prefoldin 2, cathepsin B, vitronectin, and angiotensinogen that have an important role in progression to chronic infection and liver disease development.

Antibody Development to HCV Alternate Reading Frame Protein in Liver Transplant Candidate and its Computational Analysis

Antibody Development to HCV Alternate Reading Frame Protein in Liver Transplant Candidate and its Computational Analysis

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

PF204 TARP AS IMMUNOTHERAPEUTIC TARGET IN AML EXPRESSED IN THE LSC COMPARTMENT

Background:Acute myeloid leukemia (AML) accounts for 80% of adult and 20% of pediatric leukemias. Despite frequent initial remission, patients exhibit a high relapse risk, probably due to therapy‐resistant leukemic stem cells (LSCs). Particularly the prognosis of FLT3‐ITD harboring patients remains extremely poor.Aims:To identify LSC‐specific targets and develop immunotherapeutic strategies for LSC elimination while assuring salvage of normal hematopoietic stem cells (HSCs).Methods:The GSE 17054 micro‐array dataset was used for transcriptome analysis of CD34+CD38‐ populations sorted from de novo adult AML (LSCs, n = 9) and healthy adults (HSCs, n = 4). We next evaluated transcript expression in CD34+CD38‐ and CD34+CD38+ populations sorted from de novo pediatric AML (pedAML) and healthy controls. An 8x60K human gene expression micro‐array was performed in four pediatric AML (pedAML) (2 FLT3 WT, 2 FLT3‐ITD) and 3 cord blood samples. In addition, qPCR was performed in 13 pedAML, 17 adult AML, and 15 controls (cord blood, normal bone marrow and mobilized blood stem cells). Finally, cell lines of various origin, including 9 AML cell lines, were examined. Protein expression was evaluated by Western blotting and confocal microscopy. Viral transduction was used to generate T‐cell receptor (TCR)‐engineered cytotoxic T‐cells (CTLs), next to HLA‐A∗0201 expressing, TARP overexpression and TARP knockdown AML cell lines. Killing by TCR‐engineered CTLs was evaluated in vitro by cytokine assays, flow cytometry‐ and bioluminescence‐based killing assays.Results:Re‐analysis of the GSE 17054 dataset revealed that the TCRγ chain alternate reading frame protein (TARP) was the most differentially expressed gene between LSC and HSC. Until now, TARP is known as a prostate‐specific marker, also expressed in breast adenocarcinoma. Subsequently, micro‐array and qPCR analysis showed aberrantly high TARP expression within pedAML and adult AML leukemic blasts and LSCs, while absent in normal counterparts. Noteworthy, all FLT3‐ITD harboring pedAML patients (n = 8) showed high TARP expression (P &lt; 0.01), whereas this association was not significant for FLT3‐ITD adult AML (n = 9). No TARP transcripts were identified in B‐ALL, CML nor B‐cell lines, while expression in AML cell lines was significantly higher (P &lt; 0.001). Aberrant TARP expression in cell lines and primary patient samples was confirmed at the protein level and confocal microscopy further corroborated these data. Importantly, cell lines and primary patient samples co‐expressing TARP and HLA‐A∗0201 were efficiently killed by TARP‐TCR engineered CTLs, providing proof‐of‐concept that TARP qualifies as a physiologically relevant target for T‐cell therapy in AML.Summary/Conclusion:We showed that TARP is highly expressed in AML leukemic cells, including LSCs, while absent in normal counterparts. High TARP expression is significantly associated with FLT3‐ITD in pedAML, a marker for poor prognosis. TARP‐TCR engineered CTLs effectively killed TARP+ HLA‐A∗0201+ co‐expressing AML cell lines and primary leukemic cells in vitro. To the best of our knowledge, this is the first report indicating that TARP‐TCR engineered CTLs hold great promise for immunotherapeutic T‐cell therapy in AML.

COMMD1 regulates cell proliferation and cell cycle progression by modulating p21 Cip1 levels.

Copper metabolism MURR1 domain-containing 1 (COMMD1) is a protein that participates in multiple cellular processes, including copper homeostasis and nuclear factor kappa B (NF-κB) and hypoxia-inducible factor 1α (HIF-1α) signaling. The COMMD1 upstream regulators X-linked inhibitor of apoptosis protein (XIAP) and p300 and downstream targets such as NF-κB and HIF-1α are involved in the regulation of cell proliferation and cell cycle progression. However, whether COMMD1 regulates cell proliferation and the cell cycle remains unclear. In the present study, we demonstrated that both overexpression and knockdown of COMMD1 affected the proliferation of HEK293 cells, and the cell cycle assay revealed that ectopic expression of COMMD1 arrested the cell cycle at the G1 phase. Furthermore, western blot analysis showed that COMMD1 affected p21 Cip1 levels. Taken together, these results suggest that COMMD1 regulates cell proliferation and cell cycle progression by modulating p21 Cip1 levels. Abbreviations COMMD1: Copper metabolism MURR1 domain containing 1; XIAP: X chromosome-linked inhibitor of apoptosis protein; FCS: Fetal calf serum; WCE: Whole cell extracts; RT-PCR: Reverse transcription-polymerase chain reaction; HEK293: Human embryonic kidney 293; ShRNA: Short hairpin RNA; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; ARF: Alternate reading frame protein product of the CDKN2A locus.

The silencing of long non-coding RNA ANRIL suppresses invasion, and promotes apoptosis of retinoblastoma cells through the ATM-E2F1 signaling pathway.

As one of the most common primary intraocular carcinomas, retinoblastoma generally stems from the inactivation of the retinoblastoma RB1 gene in retinal cells. Antisense non-coding RNA in the INK4 locus (ANRIL), a long non-coding RNA (lncRNA), has been reported to affect tumorigenesis and progression of various cancers, including gastric cancer and non-small cell lung cancer. However, limited investigations emphasized the role of ANRIL in human retinoblastoma. Hence, the current study was intended to investigate the effects of ANRIL on the proliferation, apoptosis, and invasion of retinoblastoma HXO-RB44 and Y79 cells. The lentivirus-based packaging system was designed to aid the up-regulation of ANRIL and ATM expressions or employed for the down-regulation of ANRIL in human retinoblastoma cells. Afterward, ANRIL expression, mRNA and protein expression of ATM and E2F1, and protein expression of INK4b, INK4a, alternate reading frame (ARF), p53 and retinoblastoma protein (pRB) were determined in order to elucidate the regulation effect associated with ANRIL on the ATM-E2F1 signaling pathway. In addition, cell viability, apoptosis, and invasion were detected accordingly. The results indicated that the down-regulation of ANRIL or up-regulation of ATM led to an increase in the expressions of ATM, E2F1, INK4b, INK4a, ARF, p53, and pRB. The silencing of ANRIL or up-regulation of ATM exerted an inhibitory effect on the proliferation and invasion while improving the apoptosis of HXO-RB44 and Y79 cells. In conclusion, the key observations of our study demonstrated that ANRIL depletion could act to suppress retinoblastoma progression by activating the ATM-E2F1 signaling pathway. These results provide a potentially promising basis for the targetted intervention treatment of human retinoblastoma.

The application of prostate specific membrane antigen in CART‑cell therapy for treatment of prostate carcinoma (Review).

Adoptive cell transfer (ACT) is an emerging immunotherapy technique that restricts tumor growth and invasion in cancer patients. Among the different types of ACT, chimeric antigen receptor (CAR)T‑cell therapy is considered to be the most advanced and a potentially powerful technique for the treatment of cancer in clinical trials. The primary aim of CART‑cell therapy is to destroy cancer cells and therefore, it serves an important role in tumor immunotherapy. CART‑cell therapy has been demonstrated to mainly treat blood cancer by targeting cluster of differentiation (CD)‑19, CD20, CD22, CD33 and CD123. However, the use of CART‑cell therapy for treating solid tumors is currently under extensive investigation. With respect to prostate cancer, prostatic acid phosphatase, prostate‑specific antigen, prostate‑specific membrane antigen (PSMA), prostate stem cell antigen, T‑cell receptorγ alternate reading frame protein, transient receptor potential‑p8 and six‑transmembrane epithelial antigen of the prostate 1 are among the identified target antigens for prostate tumors. However, mesothelin, fibroblast activation protein, epidermal growth factor receptor, carcinoembryonic antigen, disialoganglioside‑2 and human epidermal growth factor 2 are among the main targets of CART‑cell therapy in the case of other types of solid tumors. The main challenges in CART‑cell therapy are the selection of the target antigens and the modulation of the ideal tumor microenvironment for T‑cells to fight against the cancer. The present review focuses on the 1st, 2nd, 3rd and 4th generations of anti‑PSMA CARs and their application for combating prostate carcinoma.