LMP2 and TAP2 impair tumor growth and metastasis by inhibiting Wnt/ß-catenin signaling pathway and EMT in cervical cancer.

BACKGROUND: The roles of low molecular mass polypeptide 2 (LMP2) and transporter-associated with antigen processing (TAP2) in tumorigenesis are controversial. Here we aimed to explore the effect of LMP2 and TAP2 on the oncogenesis and metastasis of cervical cancer cells. METHODS: The expressions of LMP2 and TAP2 in cervical cancer and normal tissues were determined by qPCR. Plate colony formation, cell counting kit-8 analysis and in vivo tumor xenograft assays were used to detect the tumor growth. Wound healing and transwell assays were used to detect the metastasis of cervical cancer. Gelatin zymography and western blotting assays were used to detect the effect of LMP2 and TAP2 on the EMT and Wnt/ß-catenin pathway in cervical cancer cells. RESULTS: In the present study, we reported that LMP2 and TAP2 levels were overexpressed in cervical cancer. Overexpression of LMP2 and TAP2 impaired the proliferation of Hela cells. In vivo studies substantiated that LMP2 and TAP2 antagonized tumor growth. Likewise, LMP2 and TAP2 overexpression decreased the migration and invasion ability of Hela cells by regulating the process of epithelial-mesenchymal transition (EMT). Mechanically, LMP2 and TAP2 subverted the protein abundance of Wnt1 and ß-catenin, thereby downregulating their downstream targets Cyclin D1 and c-Myc. In addition, Wnt1 overexpression partially rescued the observed consequences of ectopic expression of LMP2 and TAP2 in cervical cancer cells. Taken together, our study revealed that LMP2 and TAP2 suppress the oncogenesis and metastasis of cervical cancer cells by Wnt/ß-catenin pathway and altering EMT. CONCLUSION: LMP2 and TAP2 may inhibit the oncogenesis and metastasis of cervical cancer cells by inhibiting the process of EMT and the Wnt/ß-catenin signaling pathway, which may provide important insight into prospective targets for the treatment of cervical cancer.

Spotlight on TAP and its vital role in antigen presentation and cross-presentation.

In the late 1980s and early 1990s, the hunt for a transporter molecule ostensibly responsible for the translocation of peptides across the endoplasmic reticulum (ER) membrane yielded the successful discovery of transporter associated with antigen processing (TAP) protein. TAP is a heterodimer complex comprised of TAP1 and TAP2, which utilizes ATP to transport cytosolic peptides into the ER across its membrane. In the ER, together with other components it forms the peptide loading complex (PLC), which directs loading of high affinity peptides onto nascent major histocompatibility complex class I (MHC-I) molecules that are then transported to the cell surface for presentation to CD8+ T cells. TAP also plays a crucial role in transporting peptides into phagosomes and endosomes during cross-presentation in dendritic cells (DCs). Because of the critical role that TAP plays in both classical MHC-I presentation and cross-presentation, its expression and function are often compromised by numerous types of cancers and viruses to evade recognition by cytotoxic CD8 T cells. Here we review the discovery and function of TAP with a major focus on its role in cross-presentation in DCs. We discuss a recently described emergency route of noncanonical cross-presentation that is mobilized in DCs upon TAP blockade to restore CD8 T cell cross-priming. We also discuss the various strategies employed by cancer cells and viruses to target TAP expression or function to evade immunosurveillance - along with some strategies by which the repertoire of peptides presented by cells which downregulate TAP can be targeted as a therapeutic strategy to mobilize a TAP-independent CD8 T cell response. Lastly, we discuss TAP polymorphisms and the role of TAP in inherited disorders.

Chemotherapy agents stimulate dendritic cells against human colon cancer cells through upregulation of the transporter associated with antigen processing.

Single immunotherapy fails to demonstrate efficacy in patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Research on immune reactions before and after systemic agents for mCRC is warranted. Our study examined cell line models to compare the expression of immune surface markers on colon cancer cells before and after chemotherapy agents. We also elucidated mechanisms underlying the effects of chemotherapy agents on immune surface markers. We used real-world clinical samples with NanoString analysis and the Perkin-Elmer Opal multiplex system. We established that chemotherapy agents, particularly 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, stimulated the expression of stimulatory MHC class I alleles through stimulation the pathway of transporters associated with antigen processing 1 and 2 (TAP1 and TAP2) in cell line models. Application of infected cell protein 47 (ICP-47), a specific inhibitor of the TAP1/TAP2, significantly inhibited expression of TAP1/TAP2 and also inhibited the expression of the downstream MHC class I. In the functional assay, SN-38 significantly promoted the phagocytosis of colon cancer cells by monocyte-derived dendritic cells (MoDCs). We confirmed that the expression of major histocompatibility complex (MHC) class I, significantly increased after first-line chemotherapy and targeted therapy in the samples of real-world patients with de novo mCRC. Our study provides new insights for novel immunotherapy combinations.

Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter.

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

Activation of the immunoproteasome protects SH-SY5Y cells from the toxicity of rotenone.

This study investigated the expression and role of immunoproteasome (i-proteasome) in a cell model of Parkinson's disease (PD). The cytotoxicity of rotenone was measured by CCK-8 assay. The i-proteasome ß1i subunit PSMB9 was suppressed by a specific shRNA or transfected with an overexpression plasmid in the SH-SY5Y cells. Under the exposure to rotenone or not, the expression of constitutive proteasome ß subunits, i-proteasome ßi subunits, antigen presentation related proteins, α-syn and TH were detected by Western blot in PSMB9-silenced or -overexpressed cells, and the proteasomal activities were detected by fluorogenic peptide substrates. The location of i-proteasome ßi subunits and α-syn were detected by immunofluorescence staining. The levels of ROS, GSH and MDA were measured by commercial kits. Cell apoptosis was detected by flow cytometry. Besides impairing the constitutive proteasomes, rotenone induced the expression of ßi subunits of i-proteasome and antigen presentation related proteins such as TAP1, TAP2 and MHC-I. Silencing or overexpressing PSMB9 had no obvious effect on the levels of other subunits, but could regulate the chymotrypsin-like activity of 20S proteasome and the expression of TAP1, TAP2 and MHC-I. Three ßi subunits (PSMB9, PSMB10, PSMB8) of i-proteasome were all co-localized with α-syn. PSMB9 knockdown aggravated accumulation of α-syn, degradation of TH, release of ROS, increased level of MDA, decreased level of GSH and eventually promoted apoptosis in SH-SY5Y cells after rotenone treatment, while over-expression of PSMB9 could attenuate these toxic effects of rotenone. I-proteasome is activated in SH-SY5Y cells treated with rotenone and may play a neuroprotective role.

Sld5 Ensures Centrosomal Resistance to Congression Forces by Preserving Centriolar Satellites.

The migration of chromosomes during mitosis is mediated primarily by kinesins that bind to the chromosomes and move along the microtubules, exerting pulling and pushing forces on the centrosomes. We report that a DNA replication protein, Sld5, localizes to the centrosomes, resisting the microtubular pulling forces experienced during chromosome congression. In the absence of Sld5, centriolar satellites, which normally cluster around the centrosomes, are dissipated throughout the cytoplasm, resulting in the loss of their known function of recruiting the centrosomal protein, pericentrin. We observed that Sld5-deficient centrosomes lacking pericentrin were unable to endure the CENP-E- and Kid-mediated microtubular forces that converge on the centrosomes during chromosome congression, resulting in monocentriolar and acentriolar spindle poles. The minus-end-directed kinesin-14 motor protein, HSET, sustains the traction forces that mediate centrosomal fragmentation in Sld5-depleted cells. Thus, we report that a DNA replication protein has an as yet unknown function of ensuring spindle pole resistance to traction forces exerted during chromosome congression.

Structure of the human MHC-I peptide-loading complex.

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide-MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt's lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

Downregulation of TAP1 and TAP2 in early stage breast cancer.

TAP1-TAP2 heterodimeric complexes are recognized as the transporter associated with antigen processing of major histocompatibility complex class I peptides for recognition by tumor-specific cytotoxic T lymphocytes. In this study, we investigated the immunohistochemical expression of TAP1 and TAP2 in 160 patients with breast cancer and correlated their expression levels with clinicopathologic parameters. The median age of the patient cohort was 52.5 years (range, 30-86 years). Both TAP1 and TAP2 immunohistochemical expression levels correlated significantly with breast cancer characteristics (P < .001). TAP1 expression levels were low to negative in stage I breast tumors. TAP1 and TAP2 levels were significantly higher in grade 3 tumors than low-grade (grade 1 and 2) tumors. TAP1 and TAP2 expression levels were not significantly different among different levels of HER2-expressing tumors and did not vary by estrogen and progesterone receptor status or patient age. Both TAP1 and TAP2 overexpression in breast cancer might be an indicator of an aggressive breast tumor.

A novel spontaneous mutation in the TAP2 gene unravels its role in macrophage survival.

We report a new mouse strain with a single point mutation in the type 2 transporter associated with antigen processing (TAP2). This strain randomly arose in one of our C57BL/6J mouse colonies and was initially discovered because of the lack of CD8+ T cells in the periphery. Following our observation, we subsequently revealed a lack of cell surface MHC-I expression, derived from TAP2 protein deficiency. Our strain, named eightless, has a C to T substitution in exon 5 resulting in a glutamine to stop codon substitution at position 285 in the TAP2 protein. Interestingly, in addition to the expected lack of CD8+ T cell phenotype, eightless mice have a diminished number of macrophages in their peritoneum. Moreover, following peritoneal inflammation, elicited eightless macrophages showed impaired survival both in vivo and ex vivo. Our study describes the first ever TAP2 complete knockout mouse strain and provides a possible explanation for why patients with TAP2 deficiency syndrome present clinical manifestations that would suggest a phagocyte defect rather than a lack of CD8+ T cells.

An Ancient Fecundability-Associated Polymorphism Switches a Repressor into an Enhancer of Endometrial TAP2 Expression.

Variation in female reproductive traits, such as fertility, fecundity, and fecundability, is heritable in humans, but identifying and functionally characterizing genetic variants associated with these traits has been challenging. Here, we explore the functional significance and evolutionary history of a T/C polymorphism of SNP rs2071473, which we have previously shown is an eQTL for TAP2 and significantly associated with fecundability (time to pregnancy). We replicated the association between the rs2071473 genotype and TAP2 expression by using GTEx data and demonstrated that TAP2 is expressed by decidual stromal cells at the maternal-fetal interface. Next, we showed that rs2071473 is located within a progesterone-responsive cis-regulatory element that functions as a repressor with the T allele and an enhancer with the C allele. Remarkably, we found that this polymorphism arose before the divergence of modern and archaic humans, segregates at intermediate to high frequencies across human populations, and has genetic signatures of long-term balancing selection. This variant has also previously been identified in genome-wide association studies of immune-related disease, suggesting that both alleles are maintained as a result of antagonistic pleiotropy.