Impacts of cytoplasmic p53 aggregates on the prognosis and the transcriptome in lung squamous cell carcinoma.

The tumor suppressor TP53 gene, the most frequently mutated gene in human cancers, produces the product tumor protein p53, which plays an essential role in DNA damage. p53 protein mutations may contribute to tumorigenesis by loss of tumor suppressive functions and malignancy of cancer cells via gain-of-oncogenic functions. We previously reported that mutant p53 proteins form aggregates and that cytoplasmic p53 aggregates were associated with poor prognosis in human ovarian cancer. However, the prognostic impact of p53 aggregation in other tumors including lung squamous cell carcinoma (SCC) is poorly understood. Here, we demonstrated that lung SCC cases with cytoplasmic p53 aggregates had a significantly poor clinical prognosis. Analysis via patient-derived tumor organoids (PDOs) established from lung SCC patients and possessing cytoplasmic p53 aggregates showed that eliminating cytoplasmic p53 aggregates suppressed cell proliferation. RNA sequencing and transcriptome analysis of p53 aggregate-harboring PDOs indicated multiple candidate pathways involved in p53 aggregate oncogenic functions. With lung SCC-derived cell lines, we found that cytoplasmic p53 aggregates contributed to cisplatin resistance. This study thus shows that p53 aggregates are a predictor of poor prognosis in lung SCC and suggests that detecting p53 aggregates via p53 conventional immunohistochemical analysis may aid patient selection for platinum-based therapy.

Calnexin Is Involved in Forskolin-Induced Syncytialization in Cytotrophoblast Model BeWo Cells.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and ß-human chorionic gonadotropin (ß-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a ß-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of ß-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-ß.

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, constitute the consensus motifs for this glycosylation modification and are commonly found in thrombospondin type 1 repeats that regulate molecular functions of thrombospondin 1 in binding and activation of transforming growth factor ß (TGF-ß). TGF-ß plays critical roles in the control of the central nervous system including synaptogenesis. Here, we investigated whether C-mannosylation of the synthetic Trp-Ser-Pro-Trp (WSPW) peptide may confer certain functions to this peptide in TGF-ß-mediated synaptogenesis. By using primary cultured rat astrocytes and cortical neurons, we found that the C-mannosylated WSPW (C-Man-WSPW) peptide, but not non-mannosylated WSPW peptide, suppressed astrocyte-conditioned medium (ACM)-stimulated synaptogenesis. C-Man-WSPW peptide inhibited both ACM- and recombinant mature TGF-ß1-induced activations of Smad 2, an important mediator in TGF-ß signaling. Interactions of recombinant mature TGF-ß with the C-Man-WSPW peptide were similar to those with non-C-mannosylated WSPW peptide. Taken together, our results reveal a novel function of C-mannosylation of the WxxW motif in signaling and synaptogenesis mediated by TGF-ß. Molecular details of how C-mannosylation affects the biological functions of WxxW motifs deserve future study for clarification.

Cytoplasmic p53 aggregates accumulated in p53-mutated cancer correlate with poor prognosis.

Recent studies suggested that aggregates of mutant p53 proteins may propagate and impair normal p53 functioning in recipient cells. Our previous study showed that cancer cell-derived p53 aggregates that cells internalized interfered with p53-dependent apoptosis in recipient cells. However, involvement of p53 aggregate propagation in cancer pathology has not been fully elucidated. Here, we screened patients with high-grade serous ovarian carcinoma, which is characterized by an extremely high frequency of TP53 gene mutations, to show that patients with cytoplasmic p53 deposits have a poor prognosis compared with patients with complete p53 absence or strong nuclear p53 positivity. Cytoplasmic p53 in the patients with poor prognosis consisted of protein aggregates, which suggests that p53 aggregates are oncogenic drivers. Indeed, an inhibitor of p53 aggregation restored cellular apoptosis, a proper p53 function, in p53 aggregate-bearing patient-derived tumor organoids. In cell-based assays, endogenous and exogenous mutant p53 aggregates hindered chemotherapeutic activity of cisplatin, which depends on normal p53 functions. This inhibition was reduced by blocking p53 aggregation or internalization of p53 aggregates. Our study, thus indicates the involvement of p53 aggregate transmission in poor prognosis and in chemotherapy resistance in cancers.

C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells.

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or ß-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.

Lipid Droplet Accumulation Independently Predicts Poor Clinical Prognosis in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is an epithelial cancer that accounts for most ovarian cancer deaths. Metabolic abnormalities such as extensive aerobic glycolysis and aberrant lipid metabolism are well-known characteristics of cancer cells. Indeed, accumulation of lipid droplets (LDs) in certain types of malignant tumors has been known for more than 50 years. Here, we investigated the correlation between LD accumulation and clinical prognosis. In 96 HGSOC patients, we found that high expression of the LD marker adipophilin was associated with poor progression-free and overall survival (p = 0.0022 and p = 0.014, respectively). OVCAR-3 ovarian carcinoma cells accumulated LDs in a glucose-dependent manner, which suggested the involvement of aerobic glycolysis and subsequently enhanced lipogenesis, with a result being LD accumulation. The acyl-CoA: cholesterol acyltransferase 1 inhibitor K604 and the hydroxymethylglutaryl-CoA reductase inhibitor pitavastatin blocked LD accumulation in OVCAR-3 cells and reduced phosphorylation of the survival-related kinases Akt and ERK1/2, both of which have been implicated in malignancy. Our cell-based assays thus suggested that enhanced aerobic glycolysis resulted in LD accumulation and activation of survival-related kinases. Overall, our results support the idea that cancers with lipogenic phenotypes are associated with poor clinical prognosis, and we suggest that adipophilin may serve as an independent indicator of a poor prognosis in HGSOC.

Cell-to-cell transmission of p53 aggregates: a novel player in oncology?

The mutants of the tumor suppressor protein p53 form protein aggregates. It has been proposed that these aggregates propagate like prions, albeit the detailed mechanism of the propagation is unclear. Our recent study revealed that sulfated glycosaminoglycans, especially highly sulfated domains of heparan sulfate (heparan sulfate S-domains), participate in cancer pathology by mediating transcellular propagation of p53 aggregates.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Hsc70 is required for E-cadherin expression in epithelial-like NRK-52E cells.

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone, is involved in multiple cellular functions. We previously demonstrated that Hsc70 is required for TGF-ß-induced Smad signaling in mesenchymal-like NRK-49F cells. In the present study, to compare the Hsc70 functions in TGF-ß-related signaling between epithelial and mesenchymal cells, we examined the effect of Hsc70 downregulation on TGF-ß-induced signaling in epithelial-like NRK-52E cells. TGF-ß-induced Smad signaling was suppressed in cells treated with small interfering RNA (siRNA) for Hsc70. Interestingly, despite interference with TGF-ß signaling, TGF-ß-induced suppression of E-cadherin expression was not affected by Hsc70 knockdown. Instead, Hsc70 knockdown itself caused the suppression of E-cadherin expression at the transcription level in cells treated with Hsc70 siRNA. We also examined the effects of Hsc70 knockdown on the level of E-cadherin-gene repressors, such as Snail1, Slug, Zeb1, Zeb2, and Twist1, and found that transcription of the repressors was upregulated after 24- or 36-h treatment with Hsc70 siRNA. Collectively, these results indicate that, in addition to a supportive role in TGF-ß-induced signaling, Hsc70 supports E-cadherin expression through downregulation of the E-cadherin-gene repressors in NRK-52E cells, suggesting that Hsc70 plays a functional role to maintain the epithelial cell phenotype.

Calreticulin protects insulin against reductive stress in vitro and in MIN6 cells.

Oxidative folding of proinsulin in the endoplasmic reticulum (ER) is critical for the proper sorting and secretion of insulin from pancreatic ß-cells. Here, by using non-cell-based insulin aggregation assays and mouse insulinoma-derived MIN6 cells, we searched for a candidate molecular chaperone for (pro)insulin when its oxidative folding is compromised. We found that interaction between insulin and calreticulin (CRT), a lectin that acts as an ER-resident chaperone, was enhanced by reductive stress in MIN6 cells. Co-incubation of insulin with recombinant CRT prevented reductant-induced aggregation of insulin. Furthermore, lysosomal degradation of proinsulin, which was facilitated by dithiothreitol-induced reductive stress, depended on CRT in MIN6 cells. Together, our results suggest that CRT may be a protective molecule against (pro)insulin aggregation when oxidative folding is defective, e.g. under reductive stress conditions, in vitro and in cultured cells. Because CRT acts as a molecular chaperone for not only glycosylated proteins but also non-glycosylated polypeptides, we also propose that (pro)insulin is a novel candidate client of the chaperone function of CRT.

Calreticulin Regulates Syncytialization Through Control of the Synthesis and Transportation of E-Cadherin in BeWo Cells.

During placental development, mononuclear cytotrophoblasts differentiate and fuse to syncytiotrophoblasts (STBs) to form syncytia, which secrete human chorionic gonadotropin (hCG). Decreased maternal perfusion of the placenta, which leads to placental dysfunction, induces changes in trophoblast syncytialization. Our previous study showed that calreticulin (CRT), a Ca2+-binding molecular chaperone found in the endoplasmic reticulum, is expressed in the human placenta and is involved in regulating extravillous trophoblast invasion, although its role in villous trophoblasts remains unclear. In this study, we investigated the functional role of CRT in trophoblast differentiation using the human trophoblast-like cell line BeWo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in human third-trimester placentas and mainly localized in STBs. The fetal growth restriction group exhibited significantly lower CRT expression levels than did the control group. In BeWo cells, CRT knockdown markedly suppressed forskolin-induced cell fusion and ß-hCG secretion. As for the mechanism responsible for these effects, the cell surface expression of E-cadherin, a key adhesion molecule related to syncytialization, was decreased, and E-cadherin accumulated adjacent to the Golgi apparatus in the CRT-knockdown cells, which led to dysfunctional cell-to-cell adhesion. Additionally, metabolic labeling and a pulse-chase study revealed that the protein expression of E-cadherin was suppressed at the translational level in the CRT-knockdown cells. Collectively, these results demonstrate that CRT regulates syncytialization by ensuring appropriate control of both the synthesis and transportation of E-cadherin, suggesting that CRT expression is important for placental development during pregnancy.

Calreticulin Is Involved in Invasion of Human Extravillous Trophoblasts Through Functional Regulation of Integrin ß1.

Calreticulin (CRT), a molecular chaperone in the endoplasmic reticulum (ER), plays a variety of roles in cell growth, differentiation, apoptosis, immunity, and cancer biology. It has been reported that CRT is expressed in the human placenta, although its function in placental development is poorly understood. Appropriate invasion of extravillous trophoblasts (EVTs) into the maternal decidua is necessary for successful pregnancy. The objective of the present study was to investigate the expression and functional role of CRT in EVTs using the human EVT cell line HTR8/SVneo, in which CRT gene expression was knocked down. We found that CRT was highly expressed in the human placenta in the early stage of pregnancy and localized to the EVTs. CRT knockdown markedly suppressed the invasion ability of HTR8/SVneo cells. Furthermore, the adhesion to fibronectin was suppressed in the CRT-knockdown cells via the dysfunction of integrin α5ß1. In the CRT-knockdown cells, terminal sialylation and fucosylation were decreased, and the core galactose-containing structure was increased in the N-glycans of integrin ß1. In addition, the expression levels of several critical glycosyltransferases were changed in the CRT-knockdown cells, consistent with the changes in the N-glycans. These results showed that CRT regulates the function of integrin ß1 by affecting the synthesis of N-glycans in HTR8/SVneo cells. Collectively, the results of the present study demonstrate that the ER chaperone CRT plays a regulatory role in the invasion of EVTs, suggesting the importance of CRT expression in placental development during early pregnancy.

C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells.

The thrombospondin type 1 repeat (TSR) is a functional module of proteins called TSR superfamily proteins (e.g., thrombospondin, F-spondin, mindin, etc.) and includes a conserved Trp-x-x-Trp (W-x-x-W) motif, in which the first Trp residue is preferably modified by C-mannosylation. We previously reported that synthesized C-mannosylated TSR-derived peptides (e.g., C-Man-WSPW) specifically enhanced lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells. In this study, we searched for the proteins that bind to C-mannosylated TSR-derived peptides in RAW264.7 cells and identified heat shock cognate protein 70 (Hsc70). The binding affinity of Hsc70 for C-mannosylated peptides in solution was higher than that for the peptides without C-mannose. The binding was influenced by a nucleotide-induced conformational change of Hsc70, and C-mannosylated peptides preferred the substrate-binding domain of Hsc70. Furthermore, in RAW264.7 cells, addition of Hsc70 stimulated cellular signaling to produce tumor necrosis factor-alpha, via transforming growth factor-beta-activated kinase 1, and the Hsc70-induced signaling was enhanced more in the presence of the peptides with C-mannose than that without C-mannose, suggesting functional interaction between Hsc70 and the C-mannosylated peptides in the cells. Together, these results demonstrate a novel function of the C-mannosylation of TSR-derived peptides in terms of interaction with Hsc70 to regulate cellular signaling.

Dehydroepiandrosterone augments sensitivity to gamma-ray irradiation in human H4 neuroglioma cells through down-regulation of Akt signaling.

Dehydroepiandrosterone (DHEA) modulates sensitivity to radiation-induced injury in human neuroglioma cells (H4) through effects on Akt signalling by glutathione (GSH)-dependent redox regulation. Previous treatment of H4 cells with DHEA for 18 h reduced the gamma-ray-induced phosphorylation of Akt, activated p21(waf1) synthesis and up-regulated phosphorylation of Rb independent of p53. These reactions were followed by a decrease in cell number and an increase in apoptosis and G(2)/M checkpoint arrest. The suppression of phosphorylation of Akt by DHEA was due to regulation of the dephosphorylation by protein phosphatase 2A (PP2A). DHEA up-regulated the expression of gamma-glutamylcysteine synthetase, a rate-limiting enzyme of glutathione (GSH) synthesis, and the levels of GSH to maintain PP2A activity. The results suggested that DHEA increases the sensitivity of cells to gamma-ray irradiation by inducing apoptosis and cell cycle arrest through GSH-dependent regulation of the reduced form of PP2A to down-regulate the Akt signalling pathway.

C-Mannosylated peptides derived from the thrombospondin type 1 repeat enhance lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells.

C-Mannosylation is a unique type of glycosylation occurring at the first Trp (W) in the WXXW motif, which is found in the thrombospondin type 1 repeat (TSR) of proteins. However, the biological function of C-mannosylation is not fully understood. In this study, we investigated the effect of C-mannosylated TSR-derived peptides on lipopolysaccharide (LPS)-induced signaling in macrophage-like RAW264.7 cells. The cells were stimulated with LPS in the presence or absence of chemically synthesized peptides with or without C-mannose (e.g., (C-Man)-Trp-Ser-Pro-Trp [C-Man-WSPW], C-Man-W, WSPW, etc.), then the effects of the peptides on cellular viability and signaling were examined. We found a cytotoxic effect in the cells treated with LPS and C-Man-WSPW, but not in the cells solely treated with LPS or C-Man-WSPW. We also found that production of tumor necrosis factor-alpha (TNF-alpha) was upregulated more in response to LPS plus C-Man-WSPW, than in response to LPS plus WSPW or LPS alone. Among the LPS-induced signaling pathways that induce production of TNF-alpha, the activation of c-Jun N-terminal kinase (JNK) was greatly enhanced by LPS and C-Man-WSPW, and the production of TNF-alpha was suppressed by an inhibitor for JNK. Together, these results demonstrate a novel function of the C-mannosylated TSR-derived peptide motif, to promote LPS-induced JNK signaling, and this leads to an enhancement of cytotoxicity via the upregulation of TNF-alpha production.