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A select group of patients with hepatocellular carcinomas (HCC) benefit from surgical, radiologic, and systemic therapies that include a combination of anti-angiogenic and immune-checkpoint inhibitors. However, because HCC is generally asymptomatic in its early stages, this not only leads to late diagnosis, but also to therapy resistance. The nucleoside analogue 6-thio-dG (THIO) is a first-in-class telomerase-mediated telomere-targeting anticancer agent. In telomerase expressing cancer cells, THIO is converted into the corresponding 5′-triphosphate, which is efficiently incorporated into telomeres by telomerase, activating telomere damage responses and apoptotic pathways. Here, we show how THIO is effective in controlling tumor growth and, when combined with immune checkpoint inhibitors, is even more effective in a T-cell-dependent manner. We also show telomere stress induced by THIO increases both innate sensing and adaptive antitumor immunity in HCC. Importantly, the extracellular high-mobility group box 1 protein acts as a prototypical endogenous DAMP (Damage Associated Molecular Pattern) in eliciting adaptive immunity by THIO. These results provide a strong rationale for combining telomere-targeted therapy with immunotherapy.

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Supplementary Data

Exosomes from Nischarin Tumors Reduce Focal Adhesions and Cell Spreading

Characterization of Exosomes in Human Cell Lines

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Caspase 3 Staining of Mouse Tumors From Exosome Studies

Nischarin Decreases the Attachment of Breast Cancer Cells

Schematic Representation of the Effects of Nischarin on Breast Cancer Cell Motility through Exosomes

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology.Significance:Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.See related commentary by McAndrews and Kalluri, p. 2099

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7-10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) oncogene. The MMTV-PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild-type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin-deficient breast cancers.

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. Significance: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration. See related commentary by McAndrews and Kalluri, p. 2099

Background During metastasis, tumor cells move through the tracks of extracellular matrix (ECM). Focal adhesions (FAs) are the protein complexes that link the cell cytoskeleton to the ECM and their presence is necessary for cell attachment. The tumor suppressor Nischarin interacts with a number of signaling proteins such as Integrin α5, PAK1, LIMK1, LKB1, and Rac1 to prevent cancer cell migration. Although previous findings have shown that Nischarin exerts this migratory inhibition by interacting with other proteins, the effects of these interactions on the entire FA machinery are unknown. Methods RT-PCR, Western Blotting, invadopodia assays, and immunofluorescence were used to examine FA gene expression and determine whether Nischarin affects cell attachment, as well as the proteins that regulate it. Results Our data show that Nischarin prevents cell migration and invasion by altering the expression of key focal adhesion proteins. Furthermore, we have found that Nischarin-expressing cells have reduced ability to attach the ECM, which in turn leads to a decrease in invadopodia-mediated matrix degradation. Conclusions These experiments demonstrate an important role of Nischarin in regulating cell attachment, which adds to our understanding of the early events of the metastatic process in breast cancer. Electronic supplementary material The online version of this article (10.1186/s12943-018-0764-6) contains supplementary material, which is available to authorized users.

Tumor suppressor genes regulate cell growth and prevent spontaneous proliferation that could lead to aberrant tissue function. Deletions and mutations of these genes typically lead to progression through the cell-cycle checkpoints, as well as increased cell migration. Studies of these proteins are important as they may provide potential treatments for breast cancers. In this review, we discuss a comprehensive overview on Nischarin, a novel protein discovered by our laboratory. Nischarin, or imidazoline receptor antisera-selected protein, is a protein involved in a vast number of cellular processes, including neuronal protection and hypotension. The NISCH promoter experiences hypermethylation in several cancers, whereas some highly aggressive breast cancer cells exhibit genomic loss of the NISCH locus. Furthermore, we discuss data illustrating a novel role of Nischarin as a tumor suppressor in breast cancer. Analysis of this new paradigm may shed light on various clinical questions. Finally, the therapeutic potential of Nischarin is discussed. Cancer Res; 75(20); 4252–9. ©2015 AACR.

Figure S1. A) WT and Null MEFs were seeded onto Fibronectin coated coverslip (10 μg/ml) overnight then stained with VASP. # FA per cell and % area covered by FA was acquired using CellProfiler. B) Co-Immunoprecipitation of Myc-Nischarin and VASP in HEK293 cells. (PDF 190 kb)

Adherent cells produce cellular traction force (CTF) on a substrate to maintain their physical morphologies, sense external environment, and perform essential cellular functions. Precise characterization of the CTF can expand our knowledge of various cellular processes as well as lead to the development of novel mechanical biomarkers. However, current methods that measure CTF require special substrates and fluorescent microscopy, rendering them less suitable in a clinical setting. Here, we demonstrate a rapid and direct approach to measure the combined CTF of a large cell population using thin polydimethylsiloxane (PDMS) cantilevers. Cells attached to the top surface of the PDMS cantilever produce CTF, which causes the cantilever to bend. The side view of the cantilever was imaged with a low-cost camera to extract the CTF. We characterized the CTF of fibroblasts and breast cancer cells. In addition, we were able to directly measure the contractile force of a suspended cell sheet, which is similar to the CTF of the confluent cell layer before detachment. The demonstrated technique can provide rapid and real-time measurement of the CTF of a large cell population and can directly characterize its temporal dynamics. The developed thin film PDMS cantilever can be fabricated affordably and the CTF extraction technique does not require expensive equipment. Thus, we believe that the developed method can provide an easy-to-use and affordable platform for CTF characterization in clinical settings and laboratories.

The epithelial to mesenchymal transition (EMT) is a biological process in which a non-motile epithelial cell changes to a mesenchymal phenotype with invasive capacities. This phenomenon has been well documented in multiple biological processes including embryogenesis, fibrosis, tumor progression and metastasis. The hallmark of EMT is the loss of epithelial surface markers, most notably E-cadherin, and the acquisition of mesenchymal markers including vimentin and N-cadherin. The downregulation of E-cadherin during EMT can be mediated by its transcriptional repression through the binding of EMT transcription factors (EMT-TFs) such as SNAIL, SLUG and TWIST to E-boxes present in the E-cadherin promoter. Additionally, EMT-TFs can also cooperate with several enzymes to repress the expression of E-cadherin and regulate EMT at the epigenetic and post- translational level. In this review, we will focus on epigenetic and post- translational modifications that are important in EMT. In addition, we will provide an overview of the various therapeutic approaches currently being investigated to undermine EMT and hence, the metastatic progression of cancer as well.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

Women with denser breasts, have a higher risk of developing breast cancer. Breast density reflects the amount of connective tissue and collagen present. During metastasis, tumor cells move through tracks of extracellular matrix (ECM). A stiff and ordered tissue microenvironment is necessary for tumor cell invasiveness. Matrix stiffness is mediated by ECM fiber organization and cell-matrix adhesions. Our lab is investigating a tumor suppressor that contributes to ECM organization. The protein Nischarin interacts with a number of signaling proteins such as integrin α5, PAK1, LIMK1, LKB1, MLCK, ERK and Rac1 to prevent cancer cell migration. Our data shows that spontaneous breast tumors from wild-type (WT) animals have reduced ECM fiber expression and organization compared to those from homozygous Nischarin Null mice. Furthermore, we have found Nischarin to alter integrin and focal adhesion signaling, therefore affecting cell-matrix adhesions. We hypothesize that the absence of Nischarin in the breast tumor leads to increased matrix stiffness, through increased fiber organization, and altered cell-matrix adhesions. Since no breast tumor suppressors have been linked to collagen and fibronectin ECM remodeling, we present a novel role for Nischarin in organizing the ECM. By integrating cell signaling with optical and computational instrumentation, we will assess the role of Nischarin in the bidirectional cellular/matrix crosstalk, which will add to our understanding of the metastatic process in breast cancer. Citation Format: Mazvita Maziveyi, Shengli Dong, Suresh K. Alahari. The role of nischarin in the breast tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1177.