Silencing of tumoral carbohydrate sulfotransferase 15 reactivates lymph node pancreatic cancer T cells in mice.

Limited intratumoral T-cell infiltration in pancreatic ductal adenocarcinoma (PDAC) is an obstacle to immunotherapy, yet the efficient approach to enhance tumor-infiltrating T cells is not fully established. Here, we show that tumor-specific knockdown of carbohydrate sulfotransferase 15 (CHST15), a tumor stromal proteoglycan-synthetic enzyme, suppresses tumor growth in a T-cell-dependent manner in a murine model of PDAC. Silencing of tumoral CHST15 unexpectedly expanded CD4+ and CD8+ T cells in tumor draining LN (TDLN), leading to accelerated accumulation of EdU+ proliferating CD4+ and CD8+ T cells and granzyme B+ CD8+ T cells in the tumor. RNA expression analysis indicated that tumoral CHST15 knockdown (KD) downregulated matrix remodeling-related genes, while upregulated anti-tumor T-cell activity-related genes in both tumor and TDLN. CHST15 KD significantly diminished intratumoral and TDLN Ly6C/G+ myeloid-derived suppressor cells prior to TDLN T-cell expansion, suggesting that tumoral CHST15 remotely regulated myeloid-derived suppressor cell mediated T-cell suppression in the TDLN. Our findings illustrate a novel immunotherapeutic potential of tumoral CHST15 blockage by reactivating T cells in immune suppressive TDLN of PDAC.

Phosphorylation of hTERT at threonine 249 is a novel tumor biomarker of aggressive cancer with poor prognosis in multiple organs.

Recent evidence indicates that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates expression of target genes and is directly involved in tumor formation in a telomere-independent manner. Non-canonical function of hTERT has been considered as a therapeutic target for cancer therapy. We have previously shown that hTERT phosphorylation at threonine 249 (p-hTERT), which promotes RdRP activity, is an indicator of an aggressive phenotype and poor prognosis in liver and pancreatic cancers, using two cohorts with small sample sizes with polyclonal p-hTERT antibody. To clarify the clinical relevance of p-hTERT, we developed a specific monoclonal antibody and determined the diagnostic and prognostic value of p-hTERT in cancer specimens using a large cohort. A monoclonal antibody for phosphorylated hTERT (p-hTERT) at threonine 249 was developed and validated. The antibody was used for the immunohistochemical staining of formalin-fixed, paraffin-embedded specimens from 1523 cases of lung, colon, stomach, pancreatic, liver, breast, and kidney cancers. We detected elevated p-hTERT expression levels in cases with a high mitotic activity, high pathological grade, and high nuclear pleomorphism. Elevated p-hTERT expression was an independent prognostic factor for lung, pancreatic, and liver cancers. Furthermore, p-hTERT expression was associated with immature and aggressive features, such as adenosquamous carcinoma (lung and pancreas), invasive type of cancer (lung), high serum alpha-fetoprotein level (liver), and triple-negative status (breast). In conclusion, RdRP activity indicated by p-hTERT expression predicts aggressive cancer phenotypes in various types of cancer. Thus, p-hTERT is a novel biomarker for the diagnosis of aggressive cancers with a poor prognosis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Reproducibility and prognostic significance of area of residual tumor (ART) in post-neoadjuvant resections of pancreatic ductal adenocarcinoma.

BACKGROUND: The pathologic assessments of tumor response after neoadjuvant chemoradiotherapy (NACRT) are critical to improving the prognostic stratification for patients with pancreatic ductal adenocarcinoma (PDAC). Here we clarified the utility of our new grading system based on the area of residual tumor (ART) as compared to existing systems, such as the College of American Pathologists (CAP) and MD Anderson (MDA) score. METHODS: Eight reviewers individually evaluated the tumor regression grade of 30 patients with PDAC based on three types of grading systems. The interobserver concordance and clinicopathological characteristics were compared between the three systems. RESULTS: The interobserver concordance (kappa value) of the ART, CAP, and MDA score were 0.61, 0.48, and 0.53, respectively. Discrepant cases, which were 27% of the cases, exhibited smaller tumor and tumor bed sizes than concordant cases. The reduction in tumor size evaluated by microscopy showed a correlation with the rate of change in carcinoembryonic antigen (CEA) level, CA19-9 level, and tumor size on computed tomography (CT). The ART score was correlated with the tumor size on CT before and after NACRT and disease-free survival. The CAP and MDA scores were not associated with prognosis. CONCLUSION: The ART grading system may be the most practical system to assess the tumor response in post-NACRT resections of PDAC.

Signal-anchor sequences are an essential factor for the Golgi-plasma membrane localization of type II membrane proteins.

Despite studies of the mechanism underlying the intracellular localization of membrane proteins, the specific mechanisms by which each membrane protein localizes to the endoplasmic reticulum, Golgi apparatus, and plasma membrane in the secretory pathway are unclear. In this study, a discriminant analysis of endoplasmic reticulum, Golgi apparatus and plasma membrane-localized type II membrane proteins was performed using a position-specific scoring matrix derived from the amino acid propensity of the sequences around signal-anchors. The possibility that the sequence around the signal-anchor is a factor for identifying each localization group was evaluated. The discrimination accuracy between the Golgi apparatus and plasma membrane-localized type II membrane proteins was as high as 90%, indicating that, in addition to other factors, the sequence around signal-anchor is an essential component of the selection mechanism for the Golgi and plasma membrane localization. These results may improve the use of membrane proteins for drug delivery and therapeutic applications.

Partially induced transition from horizontal to vertical orientation of helical peptides at the air-water interface and the structure of their monolayers transferred on the solid substrates.

To apply the Langmuir-Blodgett (LB) technique as a platform for investigating the fundamental properties of amphiphilic peptides (APs), we have investigated the structure of LB films using the APs. To vertically orient the helical APs like transmembrane proteins in the membrane, the primary structure of the APs was designed to have two domains: a hydrophilic domain (three amino acids) and a hydrophobic domain (ca. 20 amino acids). However, we are still far from having full control of their orientation. This study reports the contribution of the subphase temperature to the change in the orientation of helical APs. When the surface pressure-area isotherm of AP was observed at the subphase temperature at 41.5°C, the isotherm exhibited a plateau, implying that a phase transition of the monolayer at the air-water interface occurred. Circular dichroism (CD) spectra of the monolayers transferred on the solid substrates revealed that the orientation of the helices changed at the pressure, where the plateau of the isotherm was observed. This change was not observed at 21.5°C, i.e., the horizontal alignment of helixes was maintained. Atomic force microscopy (AFM) was used to systematically investigate the surface structure of the monolayers transferred at different surface pressures. A structural model of the monolayer that did not contradict with the results obtained by the three different techniques (the isotherm, CD spectroscopy, and AFM) was derived, and it was concluded that the horizontally oriented helices partially changed their orientation to vertical upon compression in the plateau region of the isotherm.

Discrepancies Between Morphological and Immunohistochemical Classifications Are Associated With Prognoses and Subtypes of Lung Cancer.

BACKGROUND/AIM: Immunohistochemical (IHC) staining has been routinely used to distinguish adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the lungs; however, it is challenging to obtain an accurate diagnosis, especially for cases with discrepancies between IHC and hematoxylin and eosin (H&E) staining results. This study aimed to clarify the clinicopathological characteristics of these discrepant cases. PATIENTS AND METHODS: Tissue microarray specimens from 321 patients with ADC and SCC were used for H&E and IHC staining of thyroid transcription factor 1 (TTF-1), Napsin A, cytokeratin 5/6 (CK5/6), p40, and p63. The pathological diagnosis was made based on (1) H&E, (2) IHC, and (3) both H&E and IHC results. Discrepant cases were defined as those with different diagnoses based on the H&E and IHC results. RESULTS: A total of 32 (10%) discrepant cases were identified. ADC (3.9%) showed fewer discrepant cases than SCC (51%). Discrepant cases of ADC had a significantly higher proportion of poorly differentiated tumors and subtypes of solid and invasive mucinous ADC, and they also had shorter overall and disease-free survival than concordant cases. Solid and invasive mucinous ADC cases showed low positivity for TTF-1 (84% and 40%, respectively) and Napsin A (88% and 80%, respectively), and invasive mucinous ADC cases showed high positivity for CK5/6 (80%). The sensitivity and specificity of TTF-1+Napsin A for ADC were 91% and 83%, respectively, whereas those of CK5/6+p40 for SCC cases were 90% and 96%, respectively. CONCLUSION: Discrepant cases of ADC are associated with solid and invasive mucinous subtypes and shorter survival.

Intra-tumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer.

The dense stroma is one cause of poor efficacy of T cell-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC). Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan-synthetic enzyme responsible for remodeling tumor stroma. Intra-tumoral injection of CHST15 small interfering RNA (siRNA) has been shown to increase the tumor-infiltrating T cells (TILs) in patients with unresectable PDAC. However, the mechanism underlying the enhanced accumulation of TILs is not fully explored. Here, we demonstrate that intra-tumoral injection of CHST15 siRNA locally and remotely diminishes myeloid-derived suppressor cells (MDSCs) and enhances TILs in mice. CHST15 was expressed by tumor cells and MDSCs in both tumor and tumor-draining lymph nodes (TDLNs), and CHST15 siRNA repressed stromal density, neutrophil extracellular traps, and Ly6C/G+ MDSCs in vivo. Remarkably, tumor growth inhibition was only observed in the immunocompetent KPC model, which is associated with enhanced TILs. In vitro, CHST15 siRNA significantly downregulated the levels of CHST15 and indoleamine 2,3-dioxygenase mRNA in CD33+ MDSCs derived from human peripheral blood mononuclear cells. These results suggest a dual role for intra-tumorally injected CHST15 siRNA on modulating the tumor immune microenvironment for T cell entry and remotely diminishing CHST15+ MDSCs, decreasing T cell suppression and expanding T cells in the TDLN, ultimately leading to an enhanced accumulation of TILs.

Discrimination of mammalian GPI-anchored proteins by hydropathy and amino acid propensities.

The glycosylphosphatidylinositol (GPI) attachment is a most important post-translational modification of proteins that plays essential roles in promoting the biochemical activities of eukaryotic cells. Described here is an analysis of the amino acid properties of mammalian GPI-anchored proteins (GPI-APs) and the development of an innovative method of detecting them. GPI-APs are characterized by two high-hydropathy regions: the signal peptide, located inside the Endoplasmic Reticulum (ER), and the GPI attachment signal, a sequence adjacent to the GPI-anchoring site (the ω-site). Especially in sequence analysis of known GPI-APs, there were some distinct aspects of the amino acid propensities around the ω-sites. Therefore, a method of detecting GPI-APs was developed based on hydropathy profiles and a position-specific scoring matrix (PSSM) calculated by position-specific amino acid propensities. First, sequences of GPI-APs and negative controls, determined by screening based on hydropathy and residue volume profiles, were aligned based on residue volume profiles in the C-terminal region, and the position-specific amino acid propensities of each group were calculated according to their alignment positions. Then, a PSSM was devised using the amino acid propensities of GPI-APs and negative controls, and discrimination scores were estimated for each dataset. Based on these scores at a threshold was fixed for each dataset. GPI-APs were detected with 81.1% sensitivity and a 0.818 success rate in an optimized calculation region determined by adjusting the window size of this region using a 5-fold dataset. The results indicate that a PSSM around the ω-site can effectively discriminate GPI-APs.

Association of longer telomere length in cancer cells and cancer-associated fibroblasts with worse prognosis.

BACKGROUND: Telomere dysfunction has been reported to be directly involved in carcinogenesis owing to chromosomal instability and immortalization; however, the clinicopathological significance of telomeres remains controversial. We have shown that telomere shortening occurs in normal-appearing duct cells at initiation and then continues during the progression of pancreatic cancer. In this study, we determined the clinicopathological and prognostic value of telomere length (TL) in cancer progression. METHODS: TL in both cancer cells and cancer-associated fibroblasts (CAFs) was analyzed by high-throughput quantitative fluorescence in situ hybridization using a previously reported cohort comprising 1434 cases of adenocarcinoma (ADC), squamous cell carcinoma (SCC), adenosquamous carcinoma, hepatocellular carcinoma, and renal cell carcinoma (RCC), which are known cancers with a statistically significantly low incidence of alternative lengthening of telomeres. Cases were divided into 2 groups as follows: longer and shorter telomeres, according to the median TL of cancer cells and CAFs. The statistical significance of TL in cancer cells and CAFs on clinicopathological characteristics and prognosis was analyzed. RESULTS: There was a close association between TL in cancer cells and CAFs. Longer telomeres in cancer cells and CAFs were associated with aggressive features such as advanced stage, high mitosis score and nuclear score, poorly differentiated cancer, and desmoplastic stroma in ADC. Furthermore, a longer TL was an independent prognostic factor for ADC, SCC, and RCC. CONCLUSIONS: Longer telomeres are associated with worse prognosis in ADC, SCC, and RCC. Thus, TL is a novel biomarker for the diagnosis of aggressive cancers with poor prognoses.

Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system.

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.

Fabrication of Langmuir-Blodgett films using amphiphilic peptides.

To introduce self-organization ability of transmembrane proteins into Langmuir (L) and Langmuir-Blodgett (LB) techniques, we focused on "amphiphilic peptide" (AP) which is composed of two distinct hydrophilic and hydrophobic domains. Three types of APs of different average hydropathies were used to prepare the AP/lipid mixed L and LB films. According to the circular dichroism spectra, the secondary structures of APs were not uniform but were a mixture of alpha-helix, beta-strand and random coil. The fraction of alpha-helix was higher for lower hydropathy AP. The interaction between AP and lipid in the L film and the structure of the LB film were also depended on the APs used.

Telomere length was associated with grade and pathological features of meningioma.

Telomeres are tandem repeats of the TTAGGG sequence at chromosomal ends and afford protection against chromosomal instability. To investigate the contribution of telomere dysfunction in meningiomas, here we estimate the associations between telomere length, tumor grade, and proliferation index in a series of 14 archived samples, using quantitative-fluorescence in situ hybridization, Ki67 immunostaining, and pathological analysis. The number of mitoses per 10 high-power fields (HPF) and Ki67 index was higher in grade III cases than in grade I or grade II cases. Telomere length was negatively associated with both the number of mitoses/10HPF and Ki67 index. Meningioma cases with atypical mitosis, a morphological marker of chromosomal instability, exhibited shortened telomeres. Among telomere-shortened meningioma cases, 40% were grade I, 20% were grade II, and 100% were grade III. In grade I or II meningiomas, shortened telomeres lacked high proliferation activity and atypical mitosis. In conclusion, telomere shortening might be pivotal in the development of high-grade meningioma. Analysis of telomere length might be a selective marker for meningiomas with high-grade malignant potential.

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl ß-D-maltoside.

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl ß-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.

Discrimination of Golgi type II membrane proteins based on their hydropathy profiles and the amino acid propensities of their transmembrane regions.

Membrane proteins in the Golgi apparatus play important roles in biological functions, predominantly as catalysts related to post-translational modification of protein oligosaccharides. We succeeded in extracting the characteristics of Golgi type II membrane proteins computationally by comparison with those of Golgi no retention proteins, which are mainly localized in the plasma membrane. Golgi type II membrane proteins were detected by combining hydropathy alignment and a position-specific score matrix of the amino acid propensities around the transmembrane region. We achieved 96.2% sensitivity, 93.5% specificity, and a 0.949 success rate in a self-consistency test. In a 5-fold cross-validation test, 88.0% sensitivity, 85.5% specificity, and a 0.867 success rate were achieved.

Relationship between Lung Carcinogenesis and Chronic Inflammation in Rodents.

Lung cancer remains the leading cause of cancer-related deaths, with an estimated 1.76 million deaths reported in 2018. Numerous studies have focused on the prevention and treatment of lung cancer using rodent models. Various chemicals, including tobacco-derived agents induce lung cancer and pre-cancerous lesions in rodents. In recent years, transgenic engineered rodents, in particular, those generated with a focus on the well-known gene mutations in human lung cancer (KRAS, EGFR, and p53 mutations) have been widely studied. Animal studies have revealed that chronic inflammation significantly enhances lung carcinogenesis, and inhibition of inflammation suppresses cancer progression. Moreover, the reduction in tumor size by suppression of inflammation in animal experiments suggests that chronic inflammation influences the promotion of tumorigenesis. Here, we review rodent lung tumor models induced by various chemical carcinogens, including tobacco-related carcinogens, and transgenics, and discuss the roles of chronic inflammation in lung carcinogenesis.

A Case of Adult Pancreatoblastoma With Novel APC Mutation and Genetic Heterogeneity.

BACKGROUND: Pancreatoblastoma is a rare malignant epithelial neoplasm of the pancreas that mainly occurs in children and involves abnormalities in the WNT/ß-catenin pathway, such as CTNNB1 mutation. However, the molecular abnormalities in adult pancreatoblastoma are not well known. CASE PRESENTATION: An elderly man, who underwent elective distal pancreatectomy and splenectomy, was referred to our hospital with a mass in the tail of the pancreas. Histologically, the lesion revealed proliferation of clear, basophilic, and cartilaginous tumor cells with lymphatic metastasis. Each of the morphologically distinct tumor components showed different immunohistochemical patterns, indicating heterogeneous differentiation, including epithelial (both acinar and ductal), mesenchymal, and neuroendocrine differentiation. All tumor components showed nuclear expression of ß-catenin and cyclin D1. Per next-generation sequencing (NGS), the clear and basophilic tumor cells shared mutations in APC, GRM8, LAMP1, and AKA9. Among the mutations, APC, c.1816_1817insA showed the highest frequency in both cell types, indicating that APC mutation was a driver mutation of the tumor. A diagnosis of PB was rendered. SUMMARY: In conclusion, the clear and basophilic cells of the tumor were supposedly derived from the same clone and subsequently acquired additional mutations. This is the first report of clonal evolution in pancreatoblastoma.

Large-scale analysis of human alternative protein isoforms: pattern classification and correlation with subcellular localization signals.

We investigated human alternative protein isoforms of >2600 genes based on full-length cDNA clones and SwissProt. We classified the isoforms and examined their co-occurrence for each gene. Further, we investigated potential relationships between these changes and differential subcellular localization. The two most abundant patterns were the one with different C-terminal regions and the one with an internal insertion, which together account for 43% of the total. Although changes of the N-terminal region are less common than those of the C-terminal region, extension of the C-terminal region is much less common than that of the N-terminal region, probably because of the difficulty of removing stop codons in one isoform. We also found that there are some frequently used combinations of co-occurrence in alternative isoforms. We interpret this as evidence that there is some structural relationship which produces a repertoire of isoformal patterns. Finally, many terminal changes are predicted to cause differential subcellular localization, especially in targeting either peroxisomes or mitochondria. Our study sheds new light on the enrichment of the human proteome through alternative splicing and related events. Our database of alternative protein isoforms is available through the internet.

A computational study of structure-reactivity relationships in Na-adduct oligosaccharides in collision-induced dissociation reactions.

Elucidating the fragmentation mechanisms in oligosaccharides using theoretical calculations is useful in analyzing the experimentally obtained mass spectra. Semi-empirical and ab initio quantum mechanics calculations were used to study the relationship between the structure and reactivity and the chemical properties of oligosaccharides. In these calculations, sodium-cationized oligosaccharides were investigated to determine Na+ ion affinity at several binding positions; in addition, the dependence of the glycosidic bond cleavage on the Na+ position was examined. The calculated structures reported in this study are directed at interpreting experimentally observed fragment ions, resulting from the cleavage of the glycosidic bonds. The calculated results for oligosaccharides containing between three and five monosaccharide units (27 oligosaccharides) were compared with experimental data generated by matrix-assisted laser-desorption/ionization (MALDI) using a quadrupole ion trap (QIT) with a time-of-flight (TOF) mass spectrometer (MS).

Cleavage of unactivated amide bonds by ammonium salt-accelerated hydrazinolysis.

Hydrazinolysis of unactivated amide bonds is significantly accelerated by the addition of ammonium salts. The reactions proceed at 50-70 °C to give amines with broad substrate scope that outperforms existing amide bond cleavage reactions. Application to peptide and amino sugar derivatives is also demonstrated.

MIDAS/GPP34, a nuclear gene product, regulates total mitochondrial mass in response to mitochondrial dysfunction.

To investigate the regulatory system in mitochondrial biogenesis involving crosstalk between the mitochondria and nucleus, we found a factor named MIDAS (mitochondrial DNA absence sensitive factor) whose expression was enhanced by the absence of mitochondrial DNA (mtDNA). In patients with mitochondrial diseases, MIDAS expression was increased only in dysfunctional muscle fibers. A majority of MIDAS localized to mitochondria with a small fraction in the Golgi apparatus in HeLa cells. To investigate the function of MIDAS, we stably transfected HeLa cells with an expression vector carrying MIDAS cDNA or siRNA. Cells expressing the MIDAS protein and the siRNA constitutively showed an increase and decrease in the total mass of mitochondria, respectively, accompanying the regulation of a mitochondria-specific phospholipid, cardiolipin. In contrast, amounts of the mitochondrial DNA, RNA and proteins did not depend upon MIDAS. Thus, MIDAS is involved in the regulation of mitochondrial lipids, leading to increases of total mitochondrial mass in response to mitochondrial dysfunction.