CD109 drives pro-tumorigenic cell properties in human non-small cell lung cancer through interaction with desmoglein-2.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein, expressed on epithelial and endothelial cells, CD4+ and CD8+ T-cells, and premature lymphocytes. CD109 interacts with different cell surface receptors and thereby modulates intracellular signaling pathways, which ultimately changes cellular functions. One well-studied example is the interaction of CD109 with the TGFß/TGFß-receptor complex at the cell surface. CD109 silences intracellular SMAD2/3 signaling and targets TGFß/TGFß-receptor to the endosomal/lysosomal compartment. In recent years, CD109 emerged as a tumor marker for different tumor entities and expression of CD109 could be linked to adverse outcome in patients. In this study, we show that silencing of CD109 in human non-small cell lung cancer (NSCLC) cells, returns these cells to an epithelial like growth phenotype. On the transcriptional level, we describe changes in cell-cell contact and epithelial-mesenchymal transition associated gene clusters. At the cell surface, we identify desmoglein-2 (DSG2) as a new interaction partner of CD109 and demonstrate CD109 dependent targeting of DSG2 to the apical cell surface, where it forms desmosomes between apical and basal cell poles. Both, CD109 and DSG2 are genetic risk factors, linked to reduced overall survival in lung adenocarcinoma patients (subtype of NSCLC). In this study, we show the expression of both proteins in the same tumor and suggest a new CD109-DSG2 axis in NSCLC patients that could present a targetable therapeutic option in the future.

DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.

Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

FOXA1 Regulation Turns Benzamide HDACi Treatment Effect-Specific in BC, Promoting NIS Gene-Mediated Targeted Radioiodine Therapy.

Human sodium iodide symporter (NIS) gene mediated radio-ablation is a successful procedure in thyroid cancer clinics. In recent years, natural expression of NIS is reported in breast cancer (BC) cases but is yet to make its mark as a therapeutic procedure in BC clinics. A pre-exposure to histone deacetylase (HDAC) inhibitors to amplify endogenous NIS expression was attempted, but achieving cancer tissue-specific enhancement of NIS in patients is an important challenge to win. Here, for the first time, we show that a benzamide class of HDACi (bHDACi) can significantly induce NIS gene expression and function (p < 0.05) in BC cells with minimal off-target effects. Transcription factor (TF) profiler and promoter binding array reveals 22 TFs differentially activated by CI-994, of which FOXA1 is identified as a unique and positive regulator of NIS. Clonogenic assay shows reduced survival with bHDACi + 131I combination treatment. Further, AR-42 and MS-275 treatment shows enhanced NIS expression in an orthotopic breast tumor model. Combining bHDACi with 1 mCi 131I shows 40% drop in signal (p < 0.05), indicating enhanced radio-ablation effect. Cerenkov imaging revealed higher accumulation of 131I in MS-275-treated tumors. Thus, bHDACi-mediated selective enhancement ensuring minimal off-target effect is a step further toward using NIS as a therapeutic target for BC.

Mannose glycosylation is an integral step for NIS localization and function in human breast cancer cells.

Chasing an intriguing biological question on the disparity of sodium iodide symporter (NIS, officially known as SLC5A5) expression and function in the clinical scenario of breast cancer, this study addresses key molecular defects involved. NIS in cancer patients has primarily been recorded to be a cytoplasmic protein, thus limiting the scope for targeted radio-iodine therapy. We developed NIS transgene-overexpressing MCF-7 breast cancer cells, and found a few clonal derivatives that show predominant expression of NIS in the plasma membrane. The majority of clones, however, showed cytosolic NIS expression over long passages. Cells expressing membranous NIS show unperturbed dynamic trafficking of NIS through secretory pathway organelles when compared to cells expressing cytoplasmic NIS or to parental cells. Further, treatment of cells expressing membranous NIS with specific glycosylation inhibitors highlighted the importance of inherent glycosylation processing and an 84 gene signature glycosylation RT-Profiler array revealed that clones expressing NIS in their membrane cluster separately compared to the other cells. We further confirm a role of three differentially expressed genes, i.e. MAN1B1, MAN1A1 and MAN2A1, in regulating NIS localization by RNA interference. Thus, this study shows the important role of mannosidase in N-glycosylation processing in order to correctly traffic NIS to the plasma membrane in breast cancer cells.This article has an associated First Person interview with the first author of the paper.

Targeting stem cells in the realm of drug-resistant breast cancer.

Since its first documentation, breast cancer (BC) has been a conundrum that ails millions of women every year. This cancer has been well studied by researchers all over the world, which has improved the patient outcome significantly. There are many diagnostic markers to identify the disease, but early detection and then subclassification of this cancer remain dubious. Even after the correct diagnosis, more than half the patients come back with a more aggressive and metastatic tumor. The underpinning mechanism that governs the resistance includes over-amplification of receptors, mutations in key gene targets, and activation of different signaling. A plethora of drugs have been devised that have shown promising results in clinical settings. However, in recent times, the role played by cancer stem cells in disease progression and their interaction in mediating the resistance to cellular insults have come into the limelight. As breast cancer stem cells (BCSCs) are dormant in nature, it is highly likely that they fail to directly respond to the cytotoxic drugs which are meant for ablating rapidly proliferating cells. Furthermore, the absence of well-characterized, drug-able surface markers to date, has limited the application of targeted therapies in complete eradication of the disease. In this review, our intent is to discuss versatile therapeutics in practice followed by discussing the upcoming therapy strategies in the pipeline for BC. Furthermore, we focus on the roles played by BCSCs in mediating the resistance, and therefore, the aspects of new therapeutics against BCSCs under development that may ease the burden in future has also been discussed.

Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo.

Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10-100 Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.