Glycans unique to the relapse-prone subset within triple-negative breast cancer as revealed by lectin array-based analysis of surgical specimens.

IntroductionMolecular and cellular characteristics of the relapse-prone subset within triple-negative breast cancer (TNBC) remain unclear. Aberrant glycosylation is involved in the malignant behavior of cancer cells. In the present study, we aimed to reveal glycan profiles unique to relapsed TNBC patients.MethodsThirty TNBC patients who did not undergo neoadjuvant chemotherapy but postoperative standard adjuvant therapy from 2009 through 2016 at Juntendo Hospital were investigated. TNBC cells were resected from primary breast cancer sections of formalin-fixed surgical specimens using laser-assisted microdissection. The binding intensities of the extracted glycoproteins to 45 lectins were quantified using lectin microarray and compared between relapsed and non-relapsed patients. Immunohistochemical staining with TJA-II lectin in specimen sections was performed.ResultsFive patients relapsed during the follow-up (range 37-123 months). Lectin microarray analysis revealed that 7 out of 45 lectins showed significant differences in binding intensity between the relapsed and the non-relapsed group. TJA-II, ACA, WFA, and BPL showed stronger binding in the relapsed group. PNGase F treatment of TNBC cell lysates suggested that TJA-II and ACA bind O-glycans. TJA-II staining of tissue sections revealed strong binding to cell surface membranes and to the cytoplasm of TNBC cells, but not to other types of cells. Significantly more TNBC cells were stained in tissue sections from relapsed than non-relapsed patients.ConclusionsTNBC cells from relapsed patients showed a unique lectin reactivity, with higher levels of TJA-II (also WFA and BPL) binding than in non-relapsed patients. The results are potentially useful to develop new prognostic and therapeutic tools

Possible correlation of apical localization of MUC1 glycoprotein with luminal A-like status of breast cancer

Abstract Adjuvant chemotherapy has played a major role in the treatment of hormone receptor-positive breast cancer for many years. To better determine which patient subsets need adjuvant chemotherapy, various gene expression analyses have been developed, but cost-effective tools to identify such patients remain elusive. In the present report, we retrospectively investigated immunohistochemical expression and subcellular localization of MUC1 in primary tumors and examined their relationship to tumor malignancy, chemotherapy effect and patient outcomes. We retrospectively examined three patient cohorts with hormone receptor-positive/human epidermal growth factor receptor 2-negative invasive breast cancer: 51 patients who underwent 21-gene expression analysis (multi-gene assay-cohort), 96 patients who received neoadjuvant chemotherapy (neoadjuvant chemotherapy-cohort), and 609 patients whose tumor tissue was used in tissue-microarrays (tissue-microarray-cohort). The immunohistochemical staining pattern of the anti-MUC1 monoclonal antibody, Ma695, was examined in cancer tissues, and subcellular localization was determined as apical, cytoplasmic or negative. In the multi-gene assay-cohort, tumors with apical patterns had the lowest recurrence scores, reflecting lower tumor malignancy, and were significantly lower than MUC1-negative tumors (P = 0.038). In the neoadjuvant chemotherapy-cohort, there was no correlation between MUC1 staining patterns and effects of chemotherapy. Finally, in the tissue-microarray-cohort, we found that patients with apical MUC1 staining patterns had significantly longer disease-free-survival and overall survival than other patterns (P = 0.020 and 0.039, respectively). Our data suggest that an apical MUC1 staining pattern indicates luminal A-likeness. Assessment of the subcellular localization of MUC1 glycoprotein may be useful for identifying patients who can avoid adjuvant chemotherapy

Lethality of mice bearing a knockout of the <i>Ngly1</i>-gene is partially rescued by the additional deletion of the <i>Engase</i> gene

<div><p>The cytoplasmic peptide:<i>N</i>-glycanase (Ngly1 in mammals) is a de-<i>N</i>-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the <i>NGLY1</i> gene exhibited severe systemic symptoms (<i>NGLY1</i>-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed <i>Ngly1</i>-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-<i>N</i>-acetylglucosaminidase (<i>Engase</i>), which is another de-<i>N</i>-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the <i>Ngly1</i>-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of <i>Ngly1</i>-deficient mice. Viable <i>Ngly1</i>-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of <i>NGLY1</i>-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of <i>Engase</i> in the C57BL/6 and ICR mixed background. The defects observed in <i>Ngly1/Engase</i>-deficient mice (C57BL/6 background) and <i>Ngly1</i>-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an <i>NGLY1</i>-deficiency. These observations strongly suggest that the <i>Ngly1</i>- or <i>Ngly1/Engase</i>-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the <i>NGLY1-</i>deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.</p></div

Genotyping results of pups by the crossing of <i>Ngly1</i>^<i>−/+</i> mice or <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>} mice in C57BL/6 and ICR mixed background.

<p>Genotyping results of pups by the crossing of <i>Ngly1</i>^<i>−/+</i> mice or <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>} mice in C57BL/6 and ICR mixed background.</p

Genotyping results of pups by crossing of <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^<i>−/+</i> mice or <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>} mice in C57BL/6 background.

<p>Genotyping results of pups by crossing of <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^<i>−/+</i> mice or <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>} mice in C57BL/6 background.</p

Loss of Ngly1 causes ventricular septal defects (VSD) and the additional <i>Engase</i> deletion rescues the VSD phenotypes.

<p><b>(A, B, G)</b> Maximum intensity projection of heart μ-CT images of E16.5 embryo of wild-type (A), <i>Ngly1</i>^{<i>−/−</i>} (B), and <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} (G). White arrows in (B) indicates VSD. <b>(C, D, H)</b> Transverse section of wild-type (C), <i>Ngly1</i>^{<i>−/−</i>} (D), and <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} embryo (H) at E16.5 were stained with H&E. White arrows in (D) indicate VSD. Shown are representative sections (n = 3). Scale bar in (C), (D) and (H) indicate 200 μm. RA: right atrium, RV: right ventricle, LA: left atrium, LV: left ventricle. <b>(E)</b> Anemia was observed in <i>Ngly1</i>^{<i>−/−</i>} embryo at E16.5 (left panel). Right panel shows <i>Ngly1</i>^<i>+/+</i> embryo at E16.5 (littermate of the left panel). <b>(F)</b> Edema was observed in <i>Ngly1</i>^{<i>−/−</i>} embryo at E16.5 (left panel). Right panel shows <i>Ngly1</i>^<i>+/+</i> embryo at E16.5 (littermate of the left panel). Black arrowhead indicates edema. <b>(I)</b> Anemia was observed in <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} embryo at E16.5 (left panel). Right panel shows <i>Ngly1</i>^<i>+/+</i><i>;Engase</i>^{<i>−/−</i>} embryo at E16.5 (littermate of the left panel). Representative images were shown.</p

<i>N</i>-GlcNAc hypothesis and the accumulation of <i>N</i>-glycoproteins in the cytoplasm of <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} MEF cells.

<p><b>(A)</b> Schematic representation of an <i>N</i>-GlcNAc hypothesis and therapeutic treatment of <i>NGLY1</i>-deficiency based on ENGase inhibition. In the absence of Ngly1, ENGase acts on some portions of unfolded glycoproteins to form <i>N</i>-GlcNAc proteins. The presence of an excess of <i>N</i>-GlcNAc proteins somehow results in detrimental effects on cells/mice. (B) Relative peak area intensity (%) of glycopeptides observed in <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} MEF cells. The peak area of each glycopeptide in wild-type MEF cells was identified as 100% and the relative ratio of the glycopeptide peak area in <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} MEF cells were calculated. Shown are the average values for two samples that were independently prepared.</p

<i>Engase</i>-deletion improves the % survival and partially rescues the body weight loss of viable <i>Ngly1</i>^{<i>−/−</i>} mice.

<p><b>(A)</b> Survival curve for <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} mice (n = 9) and <i>Ngly1</i>^{<i>−/−</i>} mice (n = 29) in the C57BL/6 and ICR mixed background. <b>(B, C)</b> Change of body weight of <i>Ngly1</i>^<i>−/+</i> mice and <i>Ngly1</i>^{<i>−/−</i>} mice in C57BL/6 and ICR mixed background. (B) shows the results of male mice (<i>Ngly1</i>^<i>−/+</i>:n = 3~4, <i>Ngly1</i>^{<i>−/−</i>}:n = 2~5) and (C) shows the results of female mice (<i>Ngly1</i>^<i>−/+</i>:n = 5~11, <i>Ngly1</i>^{<i>−/−</i>}:n = 2~5). <b>(D)</b> Hind-limb clasping of 4 weeks-old <i>Ngly1</i>^{<i>−/−</i>} mice in the C57BL/6 and ICR mixed background. <b>(E, F)</b> Change in body weight of <i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>} mice and <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>} mice in the C57BL/6 and ICR mixed background. (E) shows the results of male mice (<i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>}:n = 4~8, <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>}:n = 2~4) and (F) shows the results of female mice (<i>Ngly1</i>^<i>−/+</i><i>;Engase</i>^{<i>−/−</i>}:n = 7~11, <i>Ngly1</i>^{<i>−/−</i>}<i>;Engase</i>^{<i>−/−</i>}:n = 2~3). For statistical analysis, Student’s t-test was used. ***:p<0.001. The number in panel B, C, E, and F indicates the relative ratio of weight of <i>Ngly1</i>^{<i>−/−</i>} mice to <i>Ngly1</i>^<i>−/+</i> mice at 20 weeks of age.</p