L1CAM Expression Research Articles

RELA Fusion in Supratentorial Extraventricular Ependymomas: A Morphologic, Immunohistochemical, and Molecular Study of 43 Cases.

Supratentorial extraventricular ependymomas (STEEs) are relatively rare ependymomas, and their pathologic and genetic characteristics are still poorly understood. The aim of this study was to determine the histologic, immunohistochemical, and RELA fusion features, as well as to clarify in more detail the clinical courses of STEEs. Data from a total of 43 patients with STEEs was analyzed retrospectively. The status of RELA fusion was evaluated using fluorescence in situ hybridization. The expression levels of L1CAM, p65, cyclin D1, and p53 were assessed using immunohistochemistry. Progression-free survival and overall survival were calculated via Kaplan-Meier estimation using the log-rank test. Among all 43 STEEs, 65.1% (28/43) are positive for RELA fusion. Interestingly, almost half of the patients with RELA fusion-positive ependymomas are adults (13/28), and 89.3% (25/28) cases are anaplastic ependymomas, which suggests that RELA fusion testing is necessary in adults with STEEs. We investigated the immunohistochemical status of p65, L1CAM and CCND1 protein expression for their ability to predict RELA fusion status. RELA fusion-positive STEEs are frequently associated with expression of p65 (85.2%), L1CAM (85.2%), and CCND1 (81.5%). The accuracy of predicting RELA fusion status was much higher when the expression of p65 and L1CAM was combined, that is, when both were immunopositive. The status of RELA fusion, p53 overexpression, and extent of tumor resection are significantly associated with prognosis.

L1CAM expression in colorectal cancer identifies patients prone to metastasis already in early-stage disease

L1CAM expression in colorectal cancer identifies patients prone to metastasis already in early-stage disease

Addition of IMP3 to L1CAM for discrimination between low- and high-grade endometrial carcinomas: a European Network for Individualised Treatment of Endometrial Cancer collaboration study

Discrimination between low- and high-grade endometrial carcinomas (ECs) is clinically relevant but can be challenging for pathologists, with moderate interobserver agreement. Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is an oncofoetal protein that is associated with nonendometrioid endometrial carcinomas but has been limited studied in endometrioid carcinomas. The aim of this study is to investigate the diagnostic and prognostic value of IMP3 in the discrimination between low- and high-grade ECs and its added value to L1CAM. IMP3 and L1CAM expression was assessed in tumors from 378 patients treated for EC at 1 of 9 participating European Network for Individualised Treatment of Endometrial Cancer centers. IMP3 was expressed in 24.6% of the tumors. In general, IMP3 was more homogeneously expressed than L1CAM. IMP3 expression was significantly associated with advanced stage, nonendometrioid histology, grade 3 tumors, deep myometrial invasion, lymphovascular space invasion, distant recurrences, overall mortality, and disease-related mortality. Simultaneous absence of IMP3 and L1CAM expression showed the highest accuracy for identifying low-grade carcinomas (area under the curve 0.766), whereas simultaneous expression of IMP3 and L1CAM was strongly associated with high-grade carcinomas (odds ratio 19.7; 95% confidence interval 9.2-42.2). Even within endometrioid carcinomas, this combination remained superior to IMP3 and L1CAM alone (odds ratio 8.6; 95% confidence interval 3.4-21.9). In conclusion, IMP3 has good diagnostic value and together with L1CAM represents the optimal combination of diagnostic markers for discrimination between low- and high-grade ECs compared to IMP3 and L1CAM alone. Because of the homogenous expression of IMP3, this marker might be valuable in preoperative biopsies when compared to the more patchy L1CAM expression.

Rare inherited missense variants of POGZ associate with autism risk and disrupt neuronal development

Excess de novo likely gene-disruptive and missense variants within dozens of genes have been identified in autism spectrum disorder (ASD) and other neurodevelopmental disorders. However, many rare inherited missense variants of these high-risk genes have not been thoroughly evaluated. In this study, we analyzed the rare missense variant burden of POGZ in a large cohort of ASD patients from the Autism Clinical and Genetic Resources in China (ACGC) and further dissected the functional effect of disease-associated missense variants on neuronal development. Our results showed a significant burden of rare missense variants in ASD patients compared to the control population (P = 4.6 × 10−5, OR = 3.96), and missense variants in ASD patients showed more severe predicted functional outcomes than those in controls. Furthermore, by leveraging published large-scale sequencing data of neurodevelopmental disorders (NDDs) and sporadic case reports, we identified 8 de novo missense variants of POGZ in NDD patients. Functional analysis revealed that two inherited, but not de novo, missense variants influenced the cellular localization of POGZ and failed to rescue the defects in neurite and dendritic spine development caused by Pogz knockdown in cultured mouse primary cortical neurons. Significantly, L1CAM, an autism candidate risk gene, is differentially expressed in POGZ deficient cell lines. Reduced expression of L1cam was able to partially rescue the neurite length defects caused by Pogz knockdown. Our study showed the important roles of rare inherited missense variants of POGZ in ASD risk and neuronal development and identified the potential downstream targets of POGZ, which are important for further molecular mechanism studies.

A Novel Approach to Preoperative Risk Stratification in Endometrial Cancer: The Added Value of Immunohistochemical Markers.

Background: The current model used to preoperatively stratify endometrial cancer (EC) patients into low- and high-risk groups is based on histotype, grade, and imaging method and is not optimal. Our study aims to prove whether a new model incorporating immunohistochemical markers, L1CAM, ER, PR, p53, obtained from preoperative biopsy could help refine stratification and thus the choice of adequate surgical extent and appropriate adjuvant treatment.Materials and Methods: The following data were prospectively collected from patients operated for EC from January 2016 through August 2018: age, pre- and post-operative histology, grade, lymphovascular space invasion, L1CAM, ER, PR, p53, imaging parameters obtained from ultrasound, CT chest/abdomen, final FIGO stage, and current decision model (based on histology, grade, imaging method).Results: In total, 132 patients were enrolled. The current model revealed 48% sensitivity and 89% specificity for high-risk group determination. In myometrial invasion >50%, lower levels of ER (p = 0.024), PR (0.048), and higher levels of L1CAM (p = 0.001) were observed; in cervical involvement a higher expression of L1CAM (p = 0.001), lower PR (p = 0.014); in tumors with positive LVSI, higher L1CAM (p = 0.014); in cases with positive LN, lower expression of ER/PR (p < 0.001), higher L1CAM (p = 0.002) and frequent mutation of p53 (p = 0.008).Cut-offs for determination of high-risk tumors were established: ER <78% (p = 0.001), PR <88% (p = 0.008), and L1CAM ≥4% (p < 0.001). The positive predictive values (PPV) for ER, PR, and L1CAM were 87% (60.8–96.5%), 63% (52.1–72.8%), 83% (70.5–90.8%); the negative predictive values (NPV) for each marker were as follows: 59% (54.5–63.4%), 65% (55.6–74.0%), and 77% (67.3–84.2%). Mutation of p53 revealed PPV 94% (67.4–99.1%) and NPV 61% (56.1–66.3%). When immunohistochemical markers were included into the current diagnostic model, sensitivity improved (48.4 vs. 75.8%, p < 0.001). PPV was similar for both methods, while NPV (i.e., the probability of extremely low risk in negative test cases) was improved (66 vs. 78.9%, p < 0.001).Conclusion: We proved superiority of new proposed model using immunohistochemical markers over standard clinical practice and that new proposed model increases accuracy of prognosis prediction. We propose wider implementation and validation of the proposed model.

The prognostic significance of positive peritoneal cytology in endometrial cancer and its correlations with L1-CAM biomarker

BackgroundThe aim of this study was to evaluate the prognostic role of positive peritoneal cytology (PPC) in a cohort of patients with endometrial cancer (EC). The secondary objective was to correlate the PPC and the expression of L1CAM in a group of patients with recurrence endometrial disease. MethodsAll women diagnosed with EC and who performed a peritoneal cytology at “Regina Elena” National Cancer Institute of Rome from 2001 to 2013 were included in the study. Patients were divided into two groups according to positivity at peritoneal cytology. Moreover, patients with a recurrence disease and whose a tissue microarray (TMA) tumor sample was available underwent a L1CAM analysis. ResultsSeven hundred sixty six patients underwent to EC staging in our Institute: 696 (90.8%) with negative and 70 (9.2%) with positive cytology. Five-year recurrence rate was higher in women with PPC (46.9% vs 18.4%, p = 0 < 0.0001) and, in particular, distant recurrence (86.7% vs 53.4%, p = 0.03). Moreover, we found an interesting pattern of recurrence disease in the group of early stage of EC with NPC and positive L1CAM. ConclusionsOur results support the data that PPC may be a potential prognostic factor in early EC, due to its significant association with other risk factors and its significant influence on survival. Our findings confirm the need for large studies that point out the role of PPC and new prognostic factors, including biomarkers as L1CAM.

ROCK Inhibitor Y27632 Induced Morphological Shift and Enhanced Neurite Outgrowth-Promoting Property of Olfactory Ensheathing Cells via YAP-Dependent Up-Regulation of L1-CAM.

Olfactory ensheathing cells (OECs) are heterogeneous in morphology, antigenic profiles and functions, and these OEC subpopulations have shown different outcomes following OEC transplantation for central nervous system (CNS) injuries. Morphologically, OECs are divided into two subpopulations, process-bearing (Schwann cells-like) and flattened (astrocytes-like) OECs, which could switch between each other and are affected by extracellular and intracellular factors. However, neither the relationship between the morphology and function of OECs nor their molecular mechanisms have been clarified. In the present study, we first investigated morphological and functional differences of OECs under different cytokine exposure conditions. It demonstrated that OECs mainly displayed a process-bearing shape under pro-inflammatory conditions (lipopolysaccharide, LPS), while they displayed a flattened shape under anti-inflammatory conditions [interleukin-4 (IL-4) and transforming growth factor-β1 (TGF-β1)]. The morphological changes were partially reversible and the Rho-associated coiled-coil-containing protein kinase (ROCK)/F-actin pathway was involved. Functionally, process-bearing OECs under pro-inflammatory conditions showed increased cellular metabolic activity and a higher migratory rate when compared with flattened OECs under anti-inflammatory conditions and significantly promoted neurite outgrowth and extension. Remarkably, the morphological shift towards process-bearing OECs induced by ROCK inhibitor Y27632 enhanced the neurite outgrowth-promoting property of OECs. Furthermore, as the downstream of the ROCK pathway, transcriptional co-activator Yes-associated protein (YAP) mediated morphological shift and enhanced the neurite outgrowth-promoting property of OECs through upregulating the expression of the neural adhesion molecule L1-CAM. Our data provided evidence that OECs with specific shapes correspond to specific functional phenotypes and opened new insights into the potential combination of OECs and small-molecule ROCK inhibitors for the regeneration of CNS injuries.

Ten-year results of the PORTEC-2 trial for high-intermediate risk endometrial carcinoma: improving patient selection for adjuvant therapy

BackgroundPORTEC-2 was a randomised trial for women with high-intermediate risk (HIR) endometrial cancer, comparing pelvic external beam radiotherapy (EBRT) with vaginal brachytherapy (VBT). We evaluated long-term outcomes combined with the results of pathology review and molecular analysis.Methods427 women with HIR endometrial cancer were randomised between 2002–2006 to VBT or EBRT. Primary endpoint was vaginal recurrence (VR). Pathology review was done in 97.4%, combined with molecular analysis.ResultsMedian follow-up was 116 months; 10-year VR was 3.4% versus 2.4% for VBT vs. EBRT (p = 0.55). Ten-year pelvic recurrence (PR) was more frequent in the VBT group (6.3% vs. 0.9%, p = 0.004), mostly combined with distant metastases (DM). Ten-year isolated PR was 2.5% vs. 0.5%, p = 0.10, and DM 10.4 vs. 8.9% (p = 0.45). Overall survival for VBT vs. EBRT was 69.5% vs. 67.6% at 10 years (p = 0.72). L1CAM and p53-mutant expression and substantial lymph-vascular space invasion were risk factors for PR and DM. EBRT reduced PR in cases with these risk factors.ConclusionLong-term results of the PORTEC-2 trial confirm VBT as standard adjuvant treatment for HIR endometrial cancer. Molecular risk assessment has the potential to guide adjuvant therapy. EBRT provided better pelvic control in patients with unfavourable risk factors.

L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial\u2013mesenchymal transition

Uterine carcinosarcoma (UCS) has been proposed as a model for epithelial–mesenchymal transition (EMT), a process characterized by a functional change facilitating migration and metastasis in many types of cancer. L1CAM is an adhesion molecule that has been involved in EMT as a marker for mesenchymal phenotype. We examined expression of L1CAM in UCS in a cohort of 90 cases from four different centers. Slides were immunohistochemically stained for L1CAM and scored in four categories (0%, < 10%, 10–50%, and > 50%). A score of more than 10% was considered positive for L1CAM. The median age at presentation was 68.6 years, and half of the patients (53.3%) presented with FIGO stage 1 disease. Membranous L1CAM expression was positive in the epithelial component in 65.4% of cases. Remarkably, expression was negative in the mesenchymal component. In cases where both components were intermingled, expression limited to the epithelial component was confirmed by a double stain for L1CAM and keratin. Expression of L1CAM did not relate to overall or disease-free survival. Our findings suggest L1CAM is either not a marker for the mesenchymal phenotype in EMT, or UCS is not a good model for EMT.

Abstract B42: The cell adhesion molecule, L1CAM, is important for the dissemination and metastasis of fallopian tube precursor lesions

Abstract One of the hallmarks during the progression of high-grade serous ovarian cancer (HGSOC) is the dissemination and metastasis of cells from fallopian tube precursor lesions, namely serous tubal in situ carcinoma (STIC), to other sites of the fallopian tube, ovary, or peritoneal cavity. We, and others, have previously demonstrated that HGSOC originates from benign-appearing lesions in the fallopian tube, harboring TP53 mutations (P53 signatures), that progress into STIC lesions, and show an increase in DNA damage and proliferation. While studies have analyzed those precursor lesions at the distal end of the fallopian tube, little is known about the mechanism that triggers the dissemination of cells from fallopian tube precursor lesions to other sites. The L1 cell adhesion molecule (L1CAM, CD171) has been shown to play an important role in many types of cancer, including ovarian carcinoma. In vitro studies have shown that L1CAM enhances proliferation, cell migration, epithelial-to-mesenchymal transition (EMT), and chemoresistance in ovarian carcinoma cells. In HGSOC, high expression of L1CAM is associated with poor overall survival and confers chemoresistance in this setting. It was further shown that L1CAM expression and the presence of its soluble form in ovarian carcinoma patient ascites correlates with shorter patient survival. Current knowledge is limited on the role of L1CAM during the initiation or the early stages of HGSOC. Therefore, we investigated precursor STIC lesions for L1CAM expression. We found expression of L1CAM in most of the analyzed STIC lesions, but also in scattered normal fallopian epithelial cells. Interestingly, the expression in STIC lesions was not homogenous but showed a gradient with its lowest expression in cells attached to the basement membrane and its highest expression in pseudo-stratified cells that appear to be in the process of detaching. To investigate whether L1CAM contributes to detachment, we used the CRISPR/CAS9 system to knock down L1CAM in the ovarian cancer cell line OVCAR8. Importantly, the knockdown of L1CAM decreased the ability of cells to grow and survive under anchorage-independent culture conditions and to build 3-dimensional spheres. Conversely, overexpression of L1CAM in primary fallopian tube cells led to a strong increase in their ability to form spheres. We found that the expression of L1CAM in ovarian cancer and primary fallopian tube cell lines led to an increase in integrin alpha-5 and integrin beta-1 expression. This ultimately leads to an increased recruitment and organization of fibronectin and assembly of a basement membrane under anchorage-independent culture conditions. These data indicate that in precursor lesions L1CAM expression may be necessary for the tumor to disseminate and metastasize to other sites of the fallopian tube, ovary, or peritoneal cavity. Finally, we found a strong link between the noncanonical WNT pathway and L1CAM expression, which might explain the high L1CAM expression at the apical side of the STIC lesions. However, further studies must be performed to analyze which factors regulate the expression gradient of L1CAM and whether the inhibition of those factors or L1CAM itself delay the onset of the malignant disease. Citation Format: Kai Doberstein, Marcin Iwanicki, Mina Fogel, Peter Altevogt, Ronny Drapkin. The cell adhesion molecule, L1CAM, is important for the dissemination and metastasis of fallopian tube precursor lesions. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B42.

Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas.

The goal of this work was to clarify the effect of carbon-ion beams on reduction of the metastatic potential of malignant melanoma using in vitro and in vivo techniques. We utilized a 290 MeV/u carbon beam with a 6-cm spread-out Bragg-peak (SOBP), 137Cs γ rays or 200 kVp X rays for irradiation, and in vitro murine melanoma B16/BL6 cells that were implanted into C57BL/6J mice. The metastatic abilities (migration, invasion and adhesion) were suppressed by carbon ion treatment at all doses that were tested, whereas invasion and migration tended to increase after X-ray irradiation at low dose. Biological effects of carbon ions increased with linear energy transfer (LET) for both cell killing and metastatic abilities, although the effects were more pronounced for migration and invasion. mRNA expression of E-cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expression of Mel-CAM and L1-CAM was upregulated after low-dose photon exposure, but decreased with dose, especially after carbon-ion treatment. Conversely, these molecules showed a reversal in expression changes, especially after low-dose photon exposure. Cell-cell adhesion may be an important contributor to the antimetastatic effect of carbon ion treatment. The number of lung metastases after local tumor irradiation significantly decreased with increased dose and LET, with carbon ions being more effective than γ rays. Integrating dose-response curves to examine the relationship between cell killing and lung metastasis clearly showed that carbon ions inhibit lung metastasis more efficiently than photons at the iso-effective level of cell killing. Thus, carbon ions were more effective than photon beams, not only at killing tumor cells, but also at inhibiting metastatic spread caused by tumor cells that survived irradiation.

Endometrial cancer prognosis correlates with the expression of L1CAM and miR34a biomarkers

BackgroundPatients with endometrial cancer (EC) and presumably with good prognosis may develop a recurrence indicating that the classification of this tumor is still not definitive and that new markers are needed to identify a subgroup at risk of relapse. The cell adhesion molecule L1CAM is highly expressed in several human carcinomas and has recently been described as a new marker for endometrial and ovarian carcinomas. The aim of this study was to determine the relevance of L1CAM in recurrent EC.MethodsIn this work we have analyzed, by immunohistochemical and RT-qPCR analysis, the expression of L1CAM in a cohort of 113 endometrial cancers at different stages, which 50% have relapsed. As a predictor of good outcome, the tumors were also analyzed for the expression of miR-34a, a post-transcriptional regulator of L1CAM.ResultsAmong metastatic EC, the highest levels (60%) and the median level (24%) of L1CAM in tumors correlate with the progression, suggesting that the expression of this molecule is linked to the tumor component most involved in metastatic processes. We also found an inverse correlation between miR-34a and L1CAM protein expression, suggesting that miR-34a is a positive prognostic marker of EC.ConclusionsOur results demonstrate the expression of L1CAM and miR-34a in EC as prognostic factors that identify subgroup of patients at high risk of recurrence suggesting for them more aggressive schedules of treatment.

Abstract 4990: Regenerative origin of colorectal metastasis stem cells

Abstract Metastatic cancers invariably relapse due to the emergence of resistant tumor clones capable of self-renewal, entry into and exit from quiescence, tumor re-initiation and therapy resistance. The origins of such metastasis propagating cells (MPCs), which ultimately cause cancer death, are not well-understood. To directly scrutinize MPCs in patient metastases, we established ex vivo organoid cultures from surgically resected, chemoresistant residual colorectal cancer (CRC) liver metastases. We show that the neuronal cell-adhesion molecule L1CAM, which is ectopically expressed in many cancer types and strongly associated with poor prognosis, is a marker of MPCs. L1CAM+ cells are largely quiescent in structured neoplastic glands in tumors, but when dissociated from their epithelial niche, proliferate to regenerate heterogeneous organoids or xenografts containing both L1CAM+ and L1CAM- progeny. To define the relationship between L1CAM+ MPCs and Lgr5+ intestinal stem cells, we performed single cell mRNA sequencing on ~15,000 CRC organoid-derived cells from four patients. We identified only partial overlap between Lgr5+ and L1CAM+ cells. Lgr5high cells consistent with homeostatic stem cells, have low L1CAM levels, while Lgr5low transit amplifying progenitor-like cells have high L1CAM levels. In addition, we identify a separate population of L1CAMhighLgr5- cells. The data suggest that human CRC metastases are derived from an L1CAM+ population of transit-amplifying, partially differentiated cells. L1CAM is not expressed in intact human or mouse intestinal crypts during homeostasis. However, when the intestinal epithelium is disrupted by dextran sodium sulfate-mediated colitis, L1CAM is strongly induced in cells in the middle of regenerating crypts. Intestinal epithelium specific deletion of L1CAM causes profound weight loss, poor tissue healing and reduces survival in DSS-treated mice. In turn, L1CAM knockdown/knockout in mouse or human CRC cells inhibits regeneration of organoids in vitro, subcutaneous tumors and orthotopic liver metastases in vivo. Mechanistically, L1CAM RNA expression is normally silenced in non-neuronal cells by the transcriptional repressor REST. We show that disruption of epithelial integrity by organoid dissociation or E-cadherin knockdown reduces REST binding to an L1CAM intronic enhancer, thus inducing L1CAM expression. Our results suggest that L1CAM is dispensable for epithelial homeostasis, but is required for normal and neoplastic epithelial regeneration when tissue integrity is disrupted. During cancer progression, disseminated tumor cells at the invasion front of primary tumors, in the circulation, or in isolated residual disease following therapy, induce and depend on L1CAM for survival and eventual regrowth. Thus, L1CAM represents a crucial vulnerability of disseminated and residual MPCs that could be exploited therapeutically to treat patients with metastatic cancer. Citation Format: Karuna Ganesh, Harihar Basnet, Kevin P. O'Rourke, Ashley M. Laughney, Lan He, Eduard Batlle, Scott W. Lowe, Dana Pe'er, Jinru Shia, Joan Massague. Regenerative origin of colorectal metastasis stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4990.

Phenelzine, a cell adhesion molecule L1 mimetic small organic compound, promotes functional recovery and axonal regrowth in spinal cord-injured zebrafish

Injury to the spinal cord initiates a cascade of cellular and molecular events that contribute to the tissue environment that is non-permissive for cell survival and axonal regrowth/sprouting in the adult mammalian central nervous system. The endogenous repair response is impaired in this generally inhibitory environment. Previous studies indicate that homophilic interactions of the neural cell adhesion molecule L1 (L1CAM) promote recovery after spinal cord injury and ameliorate neurodegenerative processes in experimental rodent and zebrafish models. In light of reports that phenelzine, a small organic compound that mimics L1, stimulates neuronal survival, neuronal migration, neurite outgrowth, and Schwann cell proliferation in vitro in a L1-dependent manner, we examined the restorative potential of phenelzine in a zebrafish model of spinal cord injury. Addition of phenelzine into the aquarium water immediately after spinal cord injury accelerated locomotor recovery and promoted axonal regrowth and remyelination in larval and adult zebrafish. Phenelzine treatment up-regulated the expression and proteolysis of L1.1 (a homolog of the mammalian recognition molecule L1) and phosphorylation of Erk in the spinal cord caudal to lesion site. By combining the results of the present study with those of other studies, we propose that phenelzine bears hopes for therapy of nervous system injuries.

Glioblastoma Cells Respond Directionally to L1CAM Protein Ectodomain

Glioblastoma Multiforme (GBM) is the most common form of brain cancer and is also the most malignant. Part of what makes this cancer so insidious is the invariable tendency for tumors to regrow and kill patients after tumor removal. Currently, there is no definitive treatment for such cases, nor is there a consensus on the cause. It is our hypothesis that a major reason is the expression and release of L1CAM (L1), a neural cell adhesion protein, by GBM cells. Previously, our lab has used Super Scratch assays to show that L1 increases motility and proliferation in glioblastoma cells. However, until now, there has not been research into a possible attractive chemotactic effect of L1 on glioblastoma cells to guide their motility. In this study, we investigated the possibility of the released L1 ectodomain (L1ecto) guiding glioblastoma cells up a concentration gradient of L1ecto released by neighboring cells, and the ability of patterned immobilized L1ecto to stimulate GBM cell motility. This was done in vitro by modifying existing scratch assay protocols to examine not only the velocity of GBM cells, but also the preferential direction of their movement toward other GBM cells either releasing, or not releasing, L1ecto. The results of these experiments showed a significant chemotactic effect of released L1ecto. Responding cells moved significantly faster and directionally toward cells releasing L1ecto compared to their motility toward cells not releasing L1ecto. This suggests cells were responding to a gradient of L1ecto originating from the L1ecto releasing cells. These experiments suggest a possible leader‐follower relationship between L1 releasing and responding GBM cells during brain tumor invasion into surrounding tissue. To test this, we also studied a possible chemotactic relationship in vivo by injecting a mixture of fluorescently labelled red L1 positive and green L1 negative glioblastoma cells into the optic tectum of chick embryos. The embryos developed for ten days, after which the brain was dissected, sectioned with a vibratome, and then optically sectioned using a confocal microscope. 3D models then were rendered to examine the invasion pathways of the cancerous cells. Results thus far suggest a strong “leader‐follower” relationship between L1ecto releasing cells and non‐L1 producing cells. We conclude that 1) released L1ecto can form a soluble gradient that can be detected by GBM cells and guide their direction and stimulate their velocity; 2) immobilized L1ecto also can stimulate and guide GBM cell motility in vitro; and 3) GBM cells releasing L1ecto in brain appear to be able to influence other GBM cells to follow them.Support or Funding InformationWe thank the University of Delaware's Summer Scholars Program, Undergraduate Research Program, Dept. of Biological Sciences Undergraduate Research Fund, ASBMB, and graduate student Reetika Dutt.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunohistochemical Profiles of Endometrioid Endometrial Carcinomas With and Without Metastatic Disease.

A minority of endometrial carcinomas present at an advanced stage with a poor prognosis, and should be identified to individualize treatment. Immunohistochemical markers have been studied, but most have not been directly linked to metastasis. This study analyzes the immunohistochemical profile of endometrioid endometrial carcinomas (EECs) with and without metastases, and corresponding metastases. Tissue microarray slides from stage I EECs, stage III-IV EECs, and corresponding metastases were stained and scored for expression of β-catenin, E-cadherin, ER, PR, PTEN, p16, MLH1, PMS2, L1CAM, p53, p21, and MIB1. Scores were compared between primary stage I and III-IV EECs, stage III-IV EECs, and the corresponding metastases, and between intra-abdominal and distant metastases. Primary tumors with distant metastases had a significantly lower ER expression than those without metastases or with intra-abdominal metastases. Distant metastases had a significantly lower PR expression than the corresponding primary tumor and intra-abdominal metastases. In contrast, p16 and PTEN expression was significantly higher in intra-abdominal metastases compared with corresponding primary tumors. Immunohistochemistry predicts both presence and location of EEC metastases. Loss of ER and PR was related to distant spread, and increased expression of PTEN and p16 was related to intra-abdominal spread. Additional research should assess the use of these markers in the diagnostic workup as well as the possibility to target metastases through these markers.

Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by down-regulating STAT3 / NF-kB and L1CAM activities

ObjectiveTo explore the molecular mechanisms of celecoxib-induced pancreatic cancer suppression in vivo and in vitro. MethodsThe anti-pancreatic cancer activities of celecoxib (0, 20, 60 and 100 μmol/L) were investigated by cell viability and migration of Panc-1 and Bxpc-3 cells in vitro. The expression of L1CAM in pancreatic cancer and adjacent tissues was compared using immunohistochemistry. The expressions of L1CAM, STAT3, p-STAT3, NF-κB, p-NF-κB were determined by western blotting, and cell invasive ability was determined by wound healing assay in L1CAM-silenced and over-expressed Panc-1and Bxpc-3 cells. ResultsThe expression of L1CAM in pancreatic carcinoma was stronger than that in the adjacent tissues and L1CAM could increase the growth and invasion of pancreatic cancer cells. Over-expression of L1CAM activated the STAT3/NF-κB signaling pathway in Panc-1 and Bxpc-3 pancreatic cancer cells and celecoxib inhibited their viability and the expressions of STAT3, p-STAT3, NF-κB, p-NF-κB as well as full length L1CAM in a concentration dependent manner. ConclusionsL1CAM was highly expressed in pancreatic cancer tissue and positively correlated with age, TNM staging and tumor differentiation. L1CAM activated the STAT/NF-κB signaling pathway and celecoxib could inhibit the activity of L1CAM, STAT3 and the NF-κB signaling pathway resulting in decreased growth and invasion of pancreatic cancer cells.

P4 Diallyl Trisulfides, a garlic-derived organosulfur compound, attenuates vascular metastasis of hypoxic breast cancer cells by targeting the thioredoxin system

Metastasis is the leading cause of high mortality in breast cancer patients. As is shown by numerous clinical studies and research data, intra-tumor hypoxia is closely related to the fatality rate of breast cancer patients, which may suggest that HIF-1α expression plays a contributory role in breast cancer metastasis. DATS was screened out from the garlic fat-soluble components on the basis of HIF-1α transcriptional activity. DATS not only demonstrated that it could effectively inhibit HIF-1α transcriptional activity, but that it also affected the mRNA levels of its direct target genes LOX, ANGPTL4 and LOXL-4 to varying degrees. DATS attenuated the metastatic potential of MDA-MB-231 cells in hypoxia-induced embryonic zebrafish assays, xenograft and orthotopic tumor assays. Those results also showed that DATS could dose-dependently inhibit the migration and angiogenesis of MDA-MB-231 cells using EC-cancer cell adhesion, wound-healing, transwell and tube formation in vitro experiments. Inhibition of L1CAM, EMT-related proteins (Slug, Snail, MMP-2) and VEGF-A protein expression was also induced by DATS. DATS modulates HIF-1α not only at the protein level but also via direct inhibition of its activity through its effects on upstream Trx-1 protein level. DATS also inhibits the enzyme activity of Trx reductase in breast cancer cells. In keeping with reduced Trx-1 nuclear translocation from cytoplasm by DATS, the production of the reduced form of Trx-1 was dramatically decreased. Collectively, this present study indicates that targeting the Trx system with DATS may provide a promising strategy for treating metastasis in breast cancer.

Disseminated tumor cells are not associated with established risk factors, L1CAM immunoreactivity and outcome in endometrial carcinoma.

The presence of disseminated tumor cells (DTC) in the bone marrow of endometrial carcinoma patients has been demonstrated previously. In contrast to breast cancer, no prognostic significance or association with clinicopathological features was revealed for endometrial carcinoma so far. The aim of this study was to investigate DTC in a large patient cohort with in-depth pathology review data available and to study DTC occurrence in the context of L1CAM and long-term disease specific follow-up. Patients treated for endometrial carcinoma at the Tuebingen University Women's hospital between 2003 and 2013 were identified. Cases with previous expert central pathology review including L1CAM immunohistochemistry and bone marrow aspirates available were selected. The presence of DTC and L1CAM expression was studied immunohistochemically. In 395 cases with a confirmed diagnosis of endometrial carcinoma, bone marrow aspirates were available. DTC were detected in 17.2%. The presence of DTC was independent from tumor histology, grade, lymphovascular space involvement (LVSI), FIGO stage, myoinvasion, L1CAM immunoreactivity, and nodal metastasis. DTC occurred less frequently in cases with a microcystic elongated and fragmented (MELF) pattern of invasion (2.2 vs. 21.8%, p=0.0003). Disease progression was distributed equally among patients with and without DTC present. We were able to confirm previous findings of DTC presence in a large well-characterized cohort of endometrial carcinoma patients. DTC are detectable in almost one-fifth of endometrial carcinoma and occur less frequently with a MELF pattern of invasion. Further studies investigating the role of DTC in endometrial carcinoma are warranted.

L1CAM and HER2 Expression in Early Endometrioid Uterine Cancer.

Recently L1CAM was shown to be a promising biomarker for early-stage endometrial carcinoma (EC). As L1CAM expression was found to be related to serous ECs and areas of serous differentiation in endometrioid carcinomas, there is evidence that L1CAM-positive cancers more likely resemble type II carcinomas. Furthermore, expression of growth factor receptor HER2 has been found to be closely associated with serous ECs. We conducted a retrospective study on 142 patients in FIGO stages I and II with endometrioid EC and analyzed L1CAM and HER2 expression by double-staining immunohistochemistry. The association between these 2 transmembrane molecules and their impact on patient outcome was analyzed. Both L1CAM and HER2 showed a significant association with recurrent disease (P<0.001 and P=0.007, respectively). We found 39 (27%) L1CAM-positive cases and 17 (12%) HER2-positive cases. About 6.3% of cases were positive for both biomarkers. Survival of L1CAM-positive patients showed a significant difference between HER2-positive and HER2-negative patients (P=0.019) regarding disease-free survival. The most unfavorable disease-free survival and overall survival was found for patients with L1CAM and HER2 double-positive tumors (P<0.001). Double immunostaining revealed a mutually exclusive staining pattern for L1CAM and HER2 expression on the level of tumor cells. In early endometrioid uterine carcinoma, an additional expression of HER2 to L1CAM seems to further worsen disease-free survival and overall survival. In terms of "personalized medicine," detection of these molecules in endometrioid ECs may open new avenues for targeted therapies with the newly available anti-HER2 drugs and/or with the upcoming humanized anti-L1CAM antibodies.