: Background: Immunotherapy has changed the options for the treatment of various cancer types, but not colon cancer. Current checkpoint blockade approaches are ineffective in a large proportion of colon cancer cases, necessitating studies to elucidate its mechanisms and to identify new targets and strategies against it. Methods: Here, we examined Programmed Death-Ligand 1(PD-L1), cytokine and receptor responses of colon cancer cells exposed to camptothecin (CPT), a clinically used topoisomerase inhibitor. Colon cancer cells were treated with CPT at concentrations of up to 10 µM, and the expressions of PD-L1 and immunoregulatory cytokine genes and receptors were analyzed. Results: PD-L1, a current immunotherapy target for various cancers, was shown to be upregulated in colon cancer cells independent of the cellular p53 status. In metastasis-derived SW620 cells, CPT most extensively upregulated cytokines with T-cell attraction or growth factor functions. Of those modulated genes, SPP1, IL1RN, IL1A, TNFSF13B, OSM, and CSF3 had the most clinical relevance, as their high expression was associated with poor cancer patient overall survival. Conclusions: These findings highlight the need to examine, in preclinical and clinical situations, the potential benefits of combining topoisomerase inhibitors with immune-checkpoint inhibitors.
The molecular mechanisms involved in breast cancer progression and metastasis still remain unclear to date. It is a heterogeneous disease featuring several different phenotypes with consistently different biological characteristics. Neuroligins are neural cell adhesion molecules that have been implicated in heterotopic cell adhesion. In humans, alterations in neuroligin genes are implicated in autism and other cognitive diseases. Until recently, neuroligins have been shown to be abundantly expressed in blood vessels and also play a role implicated in the growth of glioma cells. Here we report increased expression of neuroligin 4X (NLGN4X) in breast cancer. We found NLGN4X was abundantly expressed in breast cancer tissues. NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) was analyzed. Correlation between NLGN4X levels and clinicopathologic parameters were analyzed within Oncomine datasets. Evaluation of these bioinfomatic datasets results revealed that NLGN4X expression was higher in triple negative breast cancer cells, particularly the basal subtype and tissues versus non-triple-negative sets. Its level was also observed to be higher in metastatic tissues. RT-PCR, flow cytometry and immunofluorescence study of MDA-MB-231 and MCF-7 breast cancer cells validated that NLGN4X was increased in MDA-MB-231. Knockdown of NLGN4X expression by siRNA decreased cell proliferation and migration significantly in MDA-MB-231 breast cancer cells. NLGN4X knockdown in MDA-MB-231 cells resulted in induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. High NLGN4X expression highly correlated with decrease in relapse free-survival in TNBC. NLGN4X might represent novel biomarkers and therapeutic targets for breast cancer. Inhibition of NLGN4X may be a new target for the prevention and treatment of breast cancer.
Summary: Black in Cancer was designed to strengthen networks and highlight Black excellence in cancer research and medicine. Here we expound on our actions to increase the representation of Black people in cancer-related fields.
Epithelial to mesenchymal transition (EMT) is believed to be crucial for primary tumors to escape their original residence and invade and metastasize. To properly define EMT, there is a need for ligands that can identify this phenomenon in tumor tissue and invivo. A phage-display selection screening was performed to select novel binding phage peptides for identification of EMT in breast cancer. Epithelial breast cancer cell line, MCF-7 was transformed to mesenchymal phenotype by TGF-β treatment and was used for selection. Breast fibroblasts were used for subtractive depletion and breast cancer metastatic cell lines MDA-MB-231, T47D-shNMI were used for specificity assay. The binding peptides were identified, and their binding capacities were confirmed by phage capture assay, phage-based ELISA, immunofluorescence microscopy. The phage peptide bearing the 7-amino acid sequence, LGLRGSL, demonstrated selective binding to EMT phenotypic cells (MCF-7/TGF-β and MDA-MB-231) as compared to epithelial subtype, MCF-7, T47D and breast fibroblasts (Hs578T). The selected phage was also able to identify metastatic breast cancer tumor in breast cancer tissue microarray (TMA). These studies suggest that the selected phage peptide LGLRGSL identified by phage-display library, showed significant ability to bind to mesenchymal-like breast cancer cells/ tissues and can serve as a novel probe/ligand for metastatic breast cancer diagnostic and imaging.
Purpose: Neuroligins are neural cell adhesion molecules that are implicated in heterotopic cell adhesion. Our previous studies using a selection from a combinatorial random peptide library against breast and pancreatic cancer cell lines identified several peptides mimicking neuroligin (NLGN-1, 3 and NLGN4X respectively). In this study, we investigated the expression, relevance and functional significance of Neuroligin 4X in human breast cancer. Methods: NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia were analyzed. The correlation between NLGN4X levels and clinicopathologic parameters were established within Oncomine datasets. As a proof-of- principle, we evaluated survival by generating Kaplan–Meier plots using publicly available microarray datasets. To examine the expression of NLGN4X, immunohistochemistry of breast cancer tissue arrays was conducted. Additionally, flow cytometry and immunofluorescence staining were performed to investigate NLGN4X expression in MDA-MB-231, MCF-7, and MCF-7 treated with TGFβ. To observe the effect of NLGN4X gene knockdown, MDA-MB-231 cells were transfected with NLGN4X-specific siRNA. NLGN4X gene expression was analyzed by RT-PCR, western blot and flow cytometry. Post transfection, wound healing and cell viability assays were performed to determine the effect of NLGN4X knockdown on migration and proliferation. Apoptotic outcomes were examined through detection of caspase activation and Annexin V-FITC methods by using flow cytometry. Results: NLGN4X showed abundant expression in breast cancer tissues. The evaluation of bioinfomatic datasets revealed that NLGN4X expression was higher in triple negative breast cancer (TNBC) and in metastatic tissues. Interestingly, high NLGN4X expression correlated with a decrease in relapse free-survival in TNBC. RT-PCR, flow cytometry and immunofluorescence validated that NLGN4X expression was high in MDA-MB-231 as well as MCF-7 TGFβ, which suggests that NLGN4X is associated with the mesenchymal phenotype. Knockdown of NLGN4X expression by siRNA significantly decreased cell proliferation(P<0.001) and migration(P<0.05) in MDA-MB-231 in conjunction with the induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. Conclusion: Our findings suggest that NLGN4X could represent a novel biomarker and therapeutic target. This is the first study to link the expression of neuronal cell adhesion molecules, neuroligins, to breast cancer. We intend to continue the investigation of the role of NLGN4X to elucidate the mechanistic role of this adhesion protein in breast cancer progression and metastasis. Citation Format: Henry J. Henderson, Karanam Balasubramanyam, Rajeev Samant, Komal Vig, Shree R. Singh, Clayton Yates, Deepa Bedi. Neuroligin 4X: A neural cell adhesion molecule, in breast cancer [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 25.
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