Membranous E-cadherin Research Articles

Abstract C054: Hypoxia promotes a durable epithelial-mesenchymal transition in pancreas cancer through a histone methylation-MAPK signaling axis

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumors are poorly vascularized and exhibit regions of hypoxia. Here, we demonstrate that this feature of the tumor microenvironment promotes epithelial-mesenchymal transition (EMT), which occurs early in PDAC and drives chemoresistance, and we identify the underlying signaling mechanism. Analysis of publicly-available human transcriptomics and proteomics demonstrated that PDAC cells or tumors enriched in mesenchymal markers were also enriched in markers of hypoxia or HIF activity. Furthermore, in lineage-traced autochthonous, orthotopic patient-derived xenograft, and orthotopic or subcutaneous implanted cell line models of PDAC, hypoxic tumor tissue regions were enriched for neoplastic cells that had undergone EMT. In cell culture experiments, PDAC cells from human and mouse tumors exhibited an ability to undergo EMT in response to 1% O2, with loss of membranous E-cadherin, increased vimentin protein expression, and transcriptional changes indicative of both hypoxia and EMT. Moreover, EMT in response to hypoxia was substantially more persistent than that observed in response to growth factors, and a hypoxia fate mapping system revealed that once-hypoxic cells could retain mesenchymal characteristics outside hypoxic tumor regions. To understand the mechanism for EMT in response to low oxygen tension, we constructed a multivariable linear regression model of the dependence of the hypoxic gene signature on different gene sets in PDAC ductal cells, which identified MAPK signaling as the most important feature. Consistent with the model inference, in both in vitro and in vivo settings, hypoxic cells showing evidence of EMT displayed elevated MAPK signaling. We further demonstrated that MAPK activation in hypoxia was potentiated by suppressed activity of a histone demethylase and concomitant loss of protein phosphatase expression, which reinforced the mechanism by stabilizing the expression of a histone methyltransferase. Thus, this study identifies a tumor microenvironment-initiated mechanism leading to EMT and nominates several potential drug targets whose antagonism may promote PDAC chemoresponse. Citation Format: Brooke A. Brown, Paul J. Myers, Sara J. Adair, Jason R. Pitarresi, Shiv Sah Teli, Peppi Karppinen, Ben Z. Stanger, Todd W. Bauer, Matthew J. Lazzara. Hypoxia promotes a durable epithelial-mesenchymal transition in pancreas cancer through a histone methylation-MAPK signaling axis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C054.

Cell adhesion inhibiting peptides exhibit potent anticancer activity and modulate intestinal microbiota

Membrane E-cadherin is one of the proteins whose expression affects cancer cell migration and adhesion. The design of peptide drugs that target such cancer cell-specific characteristics have received great attention in recent years and cysteine derivatives in particular have been shown to enhance anticancer effects. Herein, we set out to investigate the anticancer activity of a cell adhesion inhibiting peptide comprised of l-lysine (Lys) and Benzyl-l-cysteine (BnCys) and its influence on intestinal microbiota. The peptide inhibits cell migration and adhesion by interfering E-cadherin packaging, perturbing the integrity of cell membrane, and triggering caspase-3 and PARP related apoptotic pathways. It is highly possible that the cationic Lys segment would bind with the negatively charged cadherin molecules and the bulky BnCys segment would act as a spacing moiety to prevent the packing of E-cadherin. The peptide mixed with low-dose cisplatin effectively inhibits tumor growth and metastasis without incurring weight loss, prolonging the survival of tumor-bearing mice. The peptide treatment group increases the relative proportion of intestinal Akkermansia muciniphila (Akk), suggesting at prebiotic properties. These results paint a promising picture for designing cell migration/adhesion inhibiting peptides to target membrane E-cadherin for anticancer therapy.

Invasive lobular carcinoma with extracellular mucin (ILCEM): clinicopathologic and molecular characterization of a rare entity

AbstractInvasive lobular carcinoma with extracellular mucin (ILCEM) is a rare histologic subtype of breast cancer. Little is known about the pathologic or genomic signatures that distinguish ILCEM from classic invasive lobular carcinoma (ILC) or mucinous carcinoma. We studied 17 breast cancers with lobular morphology and extracellular mucin. Thirteen tumors with sufficient tissue for DNA extraction were analyzed by a next generation sequencing (NGS) assay that interrogates 447 genes for mutations and copy number variations (CNVs). Median patient age was 66 yrs (range: 31–77 yrs). Sixteen patients presented with masses, 7 of which were >2 cm. Seven patients had lymph node metastases. The cases of ILCEM were moderately (n = 13) or poorly differentiated (n = 4), frequently exhibiting variant morphology that has not been previously described or emphasized, including grade 3 nuclei (n = 11), diffuse signet ring cells (n = 10), solid growth (n = 4), tumor necrosis (n = 3) or apocrine features (n = 2). All tumors showed absent or reduced membranous E-cadherin expression. Concurrent lobular carcinoma in situ (LCIS) was seen in 11/17 cases, 1 of which was a striking example of signet ring cell LCIS with extracellular mucin. Receptor profiles were ER+/HER2− (n = 15) and ER+/HER2+ (n = 2). With a median follow-up of 83.5 months (range: 3–171 months) in 12 patients with available information, 8 patients had recurrences resulting in 4 cancer-related deaths. The most common CNVs were 16q loss (n = 11) and 1q gain (n = 9). CDH1 gene-level alterations were detected in all but one case, including frameshift (n = 7), nonsense (n = 2), and donor splice site (n = 1) mutations and indels (n = 2). Recurrent mutations were also seen in PIK3CA (n = 3), POLQ (n = 3), TP53 (n = 3), ERBB3 (n = 3), ERBB2 (n = 2), and RUNX1 (n = 2). Genes with recurrent amplifications included GATA3 (n = 4), FOXA1 (n = 3), CCND1 (n = 2). Our data highlights ILCEM as a distinct variant of ILC that often presents with higher-grade and variant morphologic features and is associated with an aggressive clinical course. NGS data support an overall lobular-type molecular profile and reveal potentially targetable alterations in a subset of cases with recurrence.

Abstract 5914: E-cadherin and beta-catenin in oral dysplasia progression

Abstract Objectives: Epithelial-mesenchymal transition (EMT), a biological process characterized by a progressive decrease in epithelial features and increase in mesenchymal traits, has been suggested as a critical mechanism in cancer development. A defining feature of EMT is the loss of E-cadherin. Together with beta-catenin, alpha-catenin, and p120-catenin, E-cadherin forms the adherens junctions, which are involved in cell-cell adhesion of epithelial cells. As beta-catenin has both a structural and signaling role, E-cadherin potentially contributes to malignant progression via beta-catenin signaling through the Wnt pathway. E-cadherin and beta-catenin expression is altered from normal oral tissue, oral epithelial dysplasia (OED), to oral squamous cell carcinoma (OSCC), but there is no longitudinal research on the role of these biomarkers in malignant progression. The objective of this study is to explore the expression of E-cadherin and beta-catenin in OED and to determine if these expression patterns predict malignant progression. Methods: 28 progressors (PR) and 56 non-progressors (NPR) were included in this case-control study. Samples that progressed to severe OED, carcinoma in situ, or OSCC were categorized as PRs, and samples that did not progress were NPRs. Patient samples with a baseline biopsy of low-grade (mild or moderate) OED and at least 5 years of follow-up for NPRs were obtained from the Oral Cancer Prediction Longitudinal study, which has been prospectively following patients with OED for over 20 years. Immunohistochemistry (IHC) is performed on formalin-fixed paraffin-embedded tissue samples to assess for reduced membranous E-cadherin, and reduced membranous and increased cytoplasmic and/or nuclear beta-catenin expression in PRs compared to NPRs. We hypothesized that OED with this expression pattern will show greater likelihood of progression. Fisher’s exact test was used. Logistic regression will be performed to predict progression. Results: There were no significant differences in age, sex, risk of lesion site, and grade of dysplasia between PRs and NPRs. There were significant differences in length of follow-up time and smoking history. IHC has been completed on 27 samples to date, which show decreased membranous beta-catenin expression in PRs compared to NPRs (p=0.042). IHC for additional samples will be performed to assess for cytoplasmic and nuclear beta-catenin expression, and to compare expression patterns between PRs and NPRs to determine prediction of malignant progression. Conclusion: Early results suggest that membranous beta-catenin expression may be decreased in OED that progressed compared to OED that did not. This research enhances the understanding of EMT’s role in malignant progression and hopes to potentially aid in the intervention of oral lesions at risk of cancer. Citation Format: Ilena Yim, Iris Lin, Leigha Rock, Lewei Zhang, Miriam Rosin, Denise Laronde. E-cadherin and beta-catenin in oral dysplasia progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5914.

Distribution of E- and N-cadherin in subgroups of non-functioning pituitary neuroendocrine tumours

PurposeClinically non-functioning pituitary neuroendocrine tumours (NF-PitNETs) present a varying degree of aggressiveness, and reliable prognostic markers are lacking. We aimed to characterise the distribution of E- and N-cadherin in corticotroph, PIT1 and null-cell NF-PitNETs, and link it to the course of the tumours.MethodsThe distribution of E- and N-cadherin was investigated by immunohistochemistry in a retrospective cohort of 30 tumours of the less common NF-PitNETs (corticotroph (N = 18), PIT1 (N = 8) and null-cell PitNETs (N = 4)). Immunoreactive scores (IRS) were compared to previously presented cohorts of gonadotroph NF-PitNETs (N = 105) and corticotroph functioning PitNETs (N = 17).ResultsWe found a low IRS for the extra-cellular domain of E-cadherin (median 0 (IQR 0–0, N = 135)), a medium to high IRS for the intra-cellular domain of E-cadherin (median 6 (IQR 4–9)) and a high IRS for N-cadherin (median 12 (IQR 10.5–12)) throughout the cohort of NF-PitNETs. The corticotroph NF-PitNETs presented a higher IRS for both the extra- and intra-cellular domain of E-cadherin (median 0 (IQR 0–1) and median 9 (IQR 6–12), respectively) than the gonadotroph NF-PitNETs (p < 0.001 for both comparisons). Presence of nuclear E-cadherin was associated with a weaker staining for the intra-cellular domain of E-cadherin (median 4 (IQR 0.5–6) and median 9 (IQR 9–12), for tumours with and without nuclear E-cadherin, respectively), and with a lower rate of re-intervention (p = 0.03).ConclusionsConsidering our results and the benign course of NF-PitNETs, we suggest that a high N-cadherin and downregulation of membranous E-cadherin are not associated with a more aggressive tumour behaviour in these subgroups of NF-PitNETs.

A meiotic switch in lysosome activity supports spermatocyte development in young flies but collapses with age

SummaryGamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recently, we found that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, we report that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging.

Inhibiting ADAM10‐MMP14 Complex Blocks Cystogenesis of Kidney Epithelial Cells

BackgroundPolycystic kidney disease (PKD) is the most common genetic kidney disorder. It is characterized by the aberrant renal tubule epithelial cells proliferation leads to the formation of multiple fluid‐filled cysts, and it causes renal failure. Currently, kidney transplant or dialysis is the only effective treatment. A disintegrin‐like metalloproteinase 10 (ADAM10) and matrix metalloproteinase 14 (MMP14) are two major members in the superfamily of metalloproteinases that primarily cleave several cell membrane proteins and growth factors. Inhibition of ADAM10 activity reduces cystic growth of renal epithelial cells, and inhibition of MMP14 also reduces the number of cysts in the kidneys of rats with PKD. In our study, we propose to elucidate the unclear relationship between ADAM10 and MMP14 in the development of cystogenesis in kidney.MethodsMadin‐Darby Canine Kidney (MDCK) cells with stable expression of MMP14 and its mutants were developed and used in our study. MTT proliferation assay was used to measure cell growth. Immunoprecipitation and Western blots were used to determine physical association and proteinase activity. 3‐dimensional (3D) culture of MDCK cells was used to examine cell growth patterns.ResultsWe found that a unique ADAM10‐MMP14 complex forms in cell membrane. The hemopexin domain of MMP14 is essential for its association with ADAM10. Their interaction causes differential cleavage of cell membrane proteins, E‐cadherin and L1 cell adhesion molecule protein (L1CAM). In MDCK cells, only ADAM10 cleaves E‐cadherin but its cleavage is dependent on the active form of MMP14. In addition, both ADAM10 and MMP14 cleave L1CAM but inactive MMP14 blocks the ADAM10 cleavage of L1CAM. We tested the effects of ADAM10 and MMP14 interaction on cell proliferation and cystic growth. Ectopic expression of MMP14 increases cell proliferation and cystic growth of MDCK cells in a 3D culture matrix. Further, use of MMP14‐specific inhibitor NSC405020 or ectopic expression of a dominant‐negative form of MMP14 mutant also results in proper contact inhibition, and further promotes tubular growth of MDCK cells in 3D culture. Inhibition of ADAM10 with siRNA or enzymatic inhibitor GI254023X significantly reduces cell proliferation and promotes tubular growth. Combined use of GI254023X and NSC405020 results in a 73.4 ±8.23% decrease in cell proliferation compared to GI254023X, and 68.6 ± 6.84% decrease compared to NSC405020 alone. In 3D culture, MDCK‐MMP14 (ectopic expression of MMP14) cells form cystic growth only, but addition of both ADAM10 and MMP14 inhibitors leads to tubular growth only.ConclusionOur data identified the solid physical association of ADAM10 and MMP14. Their functional interaction causes differential cleavage of cell membrane proteins, E‐cadherin and L1CAM. Inhibition of ADAM10‐MMP14 complex blocks proliferation and the cystogenesis of of kidney epithelial cells. Individual ADAM10 or MMP14 inhibitors have been used in clinical trials for certain tumors, however, they have never been used together nor have been used in PKD treatments. Our results suggest the combination of ADAM10 and MMP14 inhibitors could be served as a novel effective therapeutic strategy of PKD.

Florid Lymphovascular Invasion of Inflammatory Breast Cancer is Due to Geometric Embolic Budding

The sina qua non of inflammatory breast cancer (IBC) is numerous tumor emboli especially within overlying dermal lymphatics. The explanation remains a mystery. In human studies comparing IBC with non‐IBC, although common tumor parameters such as Ki‐67 index, mitotic count and nuclear size show a close overlap, there is a dramatic exponential difference in embolic density in IBC v non‐IBC. Using contrasting properties of two IBC xenografts, Mary‐X and Karen‐X, this study offers a novel explanation and proposed mechanism to explain the very high embolic numbers in IBC. The explanation is geometric budding of emboli. Mary‐X exhibits florid lymphovascular invasion in vivo which give rise to high numbers of CTCs and pulmonary metastases whereas Karen‐X lacks these features. Mary‐X also gives rise to compact spheroids in vitrowhich exhibit dramatic budding whereas Karen‐X exhibits only loose non‐budding aggregates. Furthermore Mary‐X emboli also bud into daughter emboli in vivo. The mechanism that regulates the compactness of the spheroids as well as the emboli involves the generation of E‐cad/NTF1, a calpain‐mediated cleavage product of membrane E‐cadherin. Inhibiting the generation of E‐cad/NTF1 by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides both reduces spheroid compactness and decreases budding. Since E‐cad/NFT1 retains the p120ctn binding site but loses both the β‐catenin and α‐binding sites, promoting its 360° distribution around the cell’s membrane, the varying levels of expression of this truncated molecule may trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of IBC tumor emboli seen in patients.

Prognostic value of combining E-cadherin, p53, Bcl-2 and Bcl-xL expression and survival in Tunisian colorectal adenocarcinoma patients.

Colorectal cancer (CRC) is a commonhealth issue worldwide with an extremely low survival rate after relapse. This study aims to evaluate the immunohistochemical expression of p53, E-cadherin, Bcl-2 and Bcl-xL and find a potential correlation between these markers, clinicopathological factors and overall survival of colorectal cancer patients. Marker expression was immunohistochemically determined in 105 patients with colorectal adenocarcinoma from southern Tunisia, followed by statistical analysis. Positivity rate of nuclear p53, membranous E-cadherin and cytoplasmic Bcl-2 - Bcl-xL was 85.71%, 76.47%, 59.8%, and 85.71% respectively. Spearman correlation showed that p53 was significantly and positively related to E-cadherin, Bcl-2, Bcl-xL and distant metastasis. A positive significant correlation between E-cadherin and anti-apoptotic proteins was also seen. Membranous E-cadherin expression was significantly and negatively associated to poor prognosis factors including lymph node metastasis, lymph invasion, venous invasion and distant metastasis. Bcl-2 expression was significantly correlated to distant metastasis. Multivariate analysis showed a significant association between dependent variable E-cadherin and covariates including differentiation, lymph invasion, venous invasion, distant metastasis, Bcl-2 and Bcl-xL. Poor 3-years OS and 5-years OS were significantly related to p53, Bcl-2 expression and E-cadherin loss. Positive E-cadherin combined with negative p53 and Bcl-2 as well as double-positive for E-cadherin and Bcl-xL were associated to best overall survival. Although each protein can be an independent prognostic factor, Simultaneous E-cadherin, p53, Bcl-2, Bcl-xL expression could be a crucial prognostic and overall survival marker to CRC patients. Multivariate analysis confirmed a positive correlation between membranous E-cadherin loss and colorectal cancer severity.

A human Barrett's esophagus organoid system reveals epithelial-mesenchymal plasticity induced by acid and bile salts.

The pathogenesis of subsquamous intestinal metaplasia (SSIM), in which glands of Barrett's esophagus (BE) are buried under esophageal squamous epithelium, is unknown. In a rat model of reflux esophagitis, we found that columnar-lined esophagus developed via a wound-healing process involving epithelial-mesenchymal plasticity (EMP) that buried glands under ulcerated squamous epithelium. To explore a role for reflux-induced EMP in BE, we established and characterized human Barrett's organoids and sought evidence of EMP after treatment with acidic bile salts (AB). We optimized media to grow human BE organoids from immortalized human Barrett's cells and from BE biopsies from seven patients, and we characterized histological, morphological, and molecular features of organoid development. Features and markers of EMP were explored following organoid exposure to AB, with and without a collagen I (COL1) matrix to simulate a wound-healing environment. All media successfully initiated organoid growth, but advanced DMEM/F12 (aDMEM) was best at sustaining organoid viability. Using aDMEM, organoids comprising nongoblet and goblet columnar cells that expressed gastric and intestinal cell markers were generated from BE biopsies of all seven patients. After AB treatment, early-stage Barrett's organoids exhibited EMP with loss of membranous E-cadherin and increased protrusive cell migration, events significantly enhanced by COL1. Using human BE biopsies, we have established Barrett's organoids that recapitulate key histological and molecular features of BE to serve as high-fidelity BE models. Our findings suggest that reflux can induce EMP in human BE, potentially enabling Barrett's cells to migrate under adjacent squamous epithelium to form SSIM.NEW & NOTEWORTHY Using Barrett's esophagus (BE) biopsies, we established organoids recapitulating key BE features. During early stages of organoid development, a GERD-like wound environment-induced features of epithelial-mesenchymal plasticity (EMP) in Barrett's progenitor cells, suggesting that reflux-induced EMP can enable Barrett's cells to migrate underneath squamous epithelium to form subsquamous intestinal metaplasia, a condition that may underlie Barrett's cancers that escape detection by endoscopic surveillance, and recurrences of Barrett's metaplasia following endoscopic eradication therapy.

Hepatitis A Virus Cellular Receptor 1 (HAVcr-1) Initiates Prostate Cancer Progression in Human Cells via Hepatocyte Growth Factor (HGF)-Induced Changes in Junctional Integrity.

Background: HAVcR-1 has been linked to cancer aetiology and may regulate junctional complexes, with its role in prostate cancer still unexplored. This study aims to investigate the expression of HAVcR-1 in prostate cancer samples and the exploration of the cellular/molecular impact of HAVcR-1. Methods: Levels of HAVcR-1 ectodomain in the serum of prostate cancer patients were compared to healthy controls, and assessed as the total protein and gene expression of HAVcR-1 and tissues sections. The manipulation of HAVcR-1 levels within prostate cancer cell lines determined changes in cell behaviour using in vitro cell models and barrier function assays. Protein/phosphoprotein levels were assessed using Western blotting. Results: Levels of HAVcR-1 ectodomain from serum were decreased in patients with prostate cancer. Ectodomain levels correlated with the Gleason score. Histologically, the total protein/gene expression of HAVcR-1 was overexpressed in prostate cancer. The overexpression of HAVcR-1 in prostate cancer cell lines resulted in key changes in cell behaviour and the phosphorylation of β-catenin with a concurrent decrease in membranous E-cadherin, increased nuclear β-catenin and increased cyclin D1 protein expression, which were associated with HGF-promoted changes in the barrier function. Conclusions: HAVcR-1 expression and ectodomain release coincides with the presence of prostate cancer; thus, indicating HAVcR-1 as a potential biomarker to aid in diagnostics, and implicating HAVcR-1 in the dysregulation of junctional complexes.

Expression of HER2/neu in gastric adenocarcinoma and its correlation with serum HER2/neu level and E-cadherin expression.

: To assess HER2/neu expressions and correlate with E-cadherin and Serum HER2 level in gastric carcinoma. 31 gastric biopsies and 1 resected specimen were taken in the study with patient details and stained with H and E for histopathological details following Lauren's classification. Immunohistochemistry for HER2 and E-cadherin expression was conducted followed by serum HER2/neu ELISA. Adenocarcinoma with 61% diffuse, 29% intestinal, and 10% other type were observed with predominant HER2 immunoexpression in intestinal-type than in diffuse-type adenocarcinoma. Other observations marked 44% as 3+/positive and 56% as 2+/equivocal in intestinal type while 26% cases as 3+/positive, 69% as 2+/equivocal, and 1% as 1+/negative were observed in diffuse type. The data presented 33% membranous positivity and 67% both membranous + cytoplasmic positivity in intestinal type while 2% showed membranous positivity, 47% both membranous + cytoplasmic, and 42% only cytoplasmic positivity in diffused type. On comparing the localization pattern of HER2 and E-cadherin, 25% of cases showed membranous staining while 50% of cases showed membranous with cytoplasmic staining for both. No cytoplasmic HER2 staining as well as no any staining for E-cadherin was shown by 6% cases. Thus, it can be concluded that cytoplasmic expression of HER2 in gastric adenocarcinoma (mainly diffuse type) may be due to shedding of its extracellular domain, leading to loss of membranous E-cadherin expression on immunohistochemistry. The loss of membranous expression of E-cadherin and increased serum HER2 ELISA were correlated well with these findings.

E-Cadherin Modulation and Inter-Cellular Trafficking in Tubular Gastric Adenocarcinoma: A High-Resolution Microscopy Pilot Study.

Despite the numerous advances in tumor molecular biology and chemotherapy options, gastric adenocarcinoma is still the most frequent form of gastric cancer. One of the core proteins that regulates inter-cellular adhesion, E-cadherin plays important roles in tumorigenesis as well as in tumor progression; however, the exact expression changes and modulation that occur in gastric cancer are not yet fully understood. In an attempt to estimate if the synthesis/degradation balance matches the final membrane expression of this adhesion molecule in cancer tissue, we assessed the proportion of E-cadherin that is found in the Golgi vesicles as well as in the lysosomal pathway We utilized archived tissue fragments from 18 patients with well and poorly differentiated intestinal types of gastric cancer and 5 samples of normal gastric mucosa, by using high-magnification multispectral microscopy and high-resolution fluorescence deconvolution microscopy. Our data showed that E-cadherin is not only expressed in the membrane, but also in the cytoplasm of normal and tumor gastric epithelia. E-cadherin colocalization with the Golgian vesicles seemed to be increasing with less differentiated tumors, while co-localization with the lysosomal system decreased in tumor tissue; however, the membrane expression of the adhesion molecule clearly dropped from well to poorly differentiated tumors. Thus E-cadherin seems to be more abundantly synthetized than eliminated via lysosomes/exosomes in less differentiated tumors, suggesting that post-translational modifications, such as cleavage, conformational inactivation, or exocytosis, are responsible for the net drop of E-cadherin at the level of the membrane in more anaplastic tumors. This behavior is in perfect accordance with the concept of partial epithelial-to-mesenchymal transition (P-EMT), when the E-cadherin expression of tumor cells is in fact not downregulated but redistributed away from the membrane in recycling vesicles. Moreover, our high-resolution deconvolution microscopy study showed for the first time, at the tissue level, the presence of Lysosome-associated membrane glycoprotein 1 (LAMP1)-positive exosomes/multivesicular bodies being trafficked across the membranes of tumor epithelial cells. Altogether, a myriad of putative modulatory pathways is available as a treatment turning point, even if we are to only consider the metabolism of membrane E-cadherin regulation. Future super-resolution microscopy studies are needed to clarify the extent of lysosome/exosome exchange between tumor cells and with the surrounding stroma, in histopathology samples or even in vivo.

PTP-PEST Regulated Membranous/Cytoplasmic Translocation of p120ctn in the Lung Cancer Resistance to Tyrosine Kinase Inhibitor.

Our previous studies indicate that resistance induction using first-generation tyrosine kinase inhibitors (TKIs) in lung cancer is accompanied with p120-catenin (p120ctn) cytoplasmic translocation from the membrane. However, the molecular mechanism underlying p120ctn intracytoplasmic translocation has not yet been reported. We performed immunohistochemistry to detect the correlation of p120ctn distribution with protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) and p120ctn Y335 phosphorylation levels in non-small cell lung cancer (NSCLC) patients. After resistance induction using first-generation TKIs in lung cancer cells, Western blotting and substrate trapping were used to assess PTP-PEST expression and its influence on p120ctn Y335 phosphorylation, as well as the role of p120ctn Y335 phosphorylation on the association of p120ctn with E-cadherin and p120ctn membrane/cytoplasm translocation. In 197 samples collected from NSCLC patients, cytoplasmic p120ctn and enhanced p120ctn Y335 phosphorylation were associated with decreased PTP-PEST. After resistance induction using gefitinib, decreased PTP-PEST expression was accompanied by enhanced phosphorylation of p120ctn Y335 and p120ctn translocated to the cytoplasm. In gefitinib-resistant cells, PTP-PEST overexpression restrained p120ctn Y335 phosphorylation and restored membrane p120ctn expression. PTP-PEST enhanced the interaction of p120ctn with E-cadherin and elevated p120ctn membrane expression. However, increased p120ctn-Y335F mutant had no effect on p120ctn interaction with E-cadherin and membrane/cytoplasm translocation compared with the control group. In conclusion, resistance to first-generation TKIs inhibited PTP-PEST expression, which promoted p120ctn-Y335 phosphorylation and reduced the interaction of p120ctn with E-cadherin, resulting in p120ctn cytoplasmic translocation.

Study of expression of epithelial cadherin in benign and malignant epithelial lesions of uterine cervix

Context: Epithelial cadherin (E-cadherin) is inversely proportional to the histologic grade of squamous cell carcinoma (SCC) of the cervix and is reduced with the increasing grade of cervical intraepithelial neoplasia, with downregulation upon transition to cervical intraepithelial neoplasia and further to invasive cancer. Aims: The aim of the study was to study the expression of E-cadherin in benign and malignant epithelial cervical lesions and to compare the results statistically. Settings and Design: The cross-sectional study was hospital based conducted in the department of pathology from January 2018 to March 2019. Methods: The study was carried out on sixty biopsy specimen of cervix (33 malignant, 4 premalignant, and 23 benign lesions). Two sections were taken; one for hematoxylin and eosin and other for E-cadherin immunohistochemical staining. Statistical Analysis Used: Statistical analysis was done using Chi-square test and Fisher's exact test. Results: In well-differentiated SCC, the majority cases (40%) revealed membranous staining while in moderately differentiated SCC and poorly differentiated SCC, the most common staining pattern was membranous and cytoplasmic (70% and 45.4%, respectively). In adenocarcinoma, 66.6% of cases had membranous pattern of staining, whereas two cases (33.3%) were negative. In High-grade Squamous Intraepithelial Lesion (HSIL), 75% of cases had membranous staining. Pure membranous pattern of staining of E-cadherin showed a progressive decline with a loss of differentiation while pure cytoplasmic revealed a reverse pattern. Conclusions: We concluded that, in malignant lesions, there is a loss of membranous E-cadherin pattern as compared to benign lesions. Furthermore, there is a progressive decline in pure membranous staining of E-cadherin with increase in shift to cytoplasmic staining as differentiation decreases.

Angiotensin II type 2 receptor agonist, compound 21, prevents tubular epithelial cell damage caused by renal ischemia

During ischemic acute kidney injury (AKI), loss of cytoskeletal integrity and disruption of intercellular junctions are rapid events in response to ATP depletion. Angiotensin II type 2 receptor (AT2R) is overexpressed in injury situations and its stimulation by angiotensin II (AngII) is related to beneficial renal effects. Its role on ischemic AKI has not been deeply studied. The aim of the present study was to investigate whether pretreatment with the AT2R agonist, C21, prevents ischemic renal epithelial cell injury. Studies in a model of 40 min of renal ischemia followed by 24 h of reperfusion (IR) in rats demonstrated that C21 pretreatment attenuated renal dysfunction and induced better preservation of tubular architecture. In addition, we studied the expression of Rho GTPases, RhoA and Cdc42, since they are key proteins in the regulation of the actin cytoskeleton and the stability of epithelial intercellular junctions. IR downregulated RhoA and Cdc42 abundance in rat kidneys. C21 pretreatment prevented RhoA reduction and increased Cdc42 abundance compared to controls. We also used an in vitro model of ATP depletion in MDCK cells grown on filter support. Using immunofluorescence we observed that in MDCK cells, C21 pretreatment prevented the ATP depletion-induced reduction of actin in brush border microvilli and in stress fibers. Moreover, C21 prevented membrane E-cadherin reduction, and RhoA and Cdc42 downregulation. The present study describes for the first time a renoprotective effect of the AT2R agonist, C21, against AKI, and provides evidence supporting that stimulation of AT2R triggers cytoprotective mechanisms against an ischemic event.

Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer-Like Phenotype.

Age is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer‐like phenotype, while promoting motility and invasiveness in MDA‐MB‐231 cells. Decellularized breast matrix from aged mice leads to loss of E‐cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer‐related proteins. The aged matrix upregulates cancer‐related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial‐mesenchymal transition‐related genes. Lysyl oxidase knockdown reverts the aged matrix‐induced changes to the young levels; it relocalizes E‐cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer‐like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.

The RhoA dependent anti-metastatic function of RKIP in breast cancer

Raf-1 kinase inhibitor protein was initially discovered as a physiological kinase inhibitor of the MAPK signaling pathway and was later shown to suppress cancer cell invasion and metastasis. Yet, the molecular mechanism through which RKIP executes its effects is not completely defined. RhoA has both a pro- and anti-metastatic cell-context dependent functions. Given that Rho GTPases primarily function on actin cytoskeleton dynamics and cell movement regulation, it is possible that one way RKIP hinders cancer cell invasion/metastasis is by targeting these proteins. Here we show that RKIP inhibits cancer cell invasion and metastasis by stimulating RhoA anti-tumorigenic functions. Mechanistically, RKIP activates RhoA in an Erk2 and GEF-H1 dependent manner to enhance E-cadherin membrane localization and inhibit CCL5 expression.

Spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis: a case report

BackgroundSpindle cell squamous cell carcinoma is an uncommon variant of squamous cell carcinoma; its diagnosis is sometimes challenging because it histopathologically resembles neoplastic or reactive spindle cell lesions of mesenchymal origins. Here, we report a rare case of spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis.Case presentationA 69-year-old Japanese man presented with pain and a polypoid mass on the lower left gingiva. He had received chemoradiotherapy for squamous cell carcinoma of the buccal mucosa 15 years prior to this consultation. In addition, he was treated for mandibular osteonecrosis 6 years after chemoradiotherapy without evidence of cancer recurrence. A biopsy revealed atypical spindle or pleomorphic cells scattered in the edematous and fibrin-rich stroma; however, no malignant squamous components were apparent. These atypical cells frequently contained neutrophils within their cytoplasm that formed cell-in-cell figures. Immunohistochemically, the atypical cells were negative for cytokeratins, epithelial membrane antigen, and E-cadherin, but positive for p63, vimentin, and p53. Although these findings suggested spindle cell squamous cell carcinoma, it was difficult to reach a definitive diagnosis. Based on a clinical diagnosis of a malignant tumor, the patient underwent a hemimandibulectomy. The surgically resected specimen had a typical spindle cell squamous cell carcinoma histology consisting of biphasic spindle cells and conventional squamous cell carcinoma components. Moreover, the surgical specimen also exhibited spindle tumor cells that frequently included neutrophils, around which intense staining for lysosomal-associated membrane protein 1 and cathepsin B was observed. This suggested that the cell-in-cell figures represent active neutrophil phagocytosis by tumor cells, and not emperipolesis.ConclusionThe presence of neutrophil phagocytosis may be a potent indicator of malignancy.

Calcium signaling induces a partial EMT.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.