Marc A. Antonyak

Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells.

Tumor progression involves the ability of cancer cells to communicate with each other and with neighboring normal cells in their microenvironment. Microvesicles (MV) derived from human cancer cells have received a good deal of attention because of their ability to participate in the horizontal transfer of signaling proteins between cancer cells and to contribute to their invasive activity. Here we show that MV may play another important role in oncogenesis. In particular, we demonstrate that MV shed by two different human cancer cells, MDAMB231 breast carcinoma cells and U87 glioma cells, are capable of conferring onto normal fibroblasts and epithelial cells the transformed characteristics of cancer cells (e.g., anchorage-independent growth and enhanced survival capability) and that this effect requires the transfer of the protein cross-linking enzyme tissue transglutaminase (tTG). We further demonstrate that tTG is not sufficient to transform fibroblasts but rather that it must collaborate with another protein to mediate the transforming actions of the cancer cell-derived MV. Proteomic analyses of the MV derived from MDAMB231 and U87 cells indicated that both these vesicle preparations contained the tTG-binding partner and cross-inking substrate fibronectin (FN). Moreover, we found that tTG cross-links FN in MV from cancer cells and that the ensuing MV-mediated transfers of cross-linked FN and tTG to recipient fibroblasts function cooperatively to activate mitogenic signaling activities and to induce their transformation. These findings highlight a role for MV in the induction of cellular transformation and identify tTG and FN as essential participants in this process.

Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease

Caused by a polyglutamine expansion in the huntingtin protein, Huntingtons disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de‐repressed two established regulators of mitochondrial function, PGC‐1α and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3‐nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC‐independent epigenetic strategy for treating neurodegeneration.

Elevated JNK activation contributes to the pathogenesis of human brain tumors

The ERK pathway is typically associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. An analogous signaling module, the JNK pathway, has not been shown to be consistently activated by the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines. The function of the JNK pathway in primary tumors is unclear as it has been implicated in both promoting apoptosis and cell growth in vitro, which may be a reflection of the cell lines chosen. Primary human brain tumors frequently show overexpression of the EGF receptor. To clarify the role of JNK in tumorigenesis, we have investigated the role of JNK in a large panel of primary human brain tumors and tumor derived cell lines. Here we present evidence that JNK has a major role in promoting tumorigenesis both in vivo and in vitro. Western blot analysis demonstrated that 86% (18 of 21) of primary brain tumors showed evidence of JNK activation but only 38% (8 of 21) showed evidence of ERK activation. Kinase assays revealed that 77% of brain tumor cell lines activated JNK in response to EGF (7 of 13) or had high levels of basal activity (3 of 13), whereas none of six normal cell lines analysed, including astrocytes, had these properties. Of several growth factors examined, EGF produced the highest level of JNK induction in tumor cell lines and the duration of activation was greater than that seen for ERK. Expression of a dominant-negative (dn) form of JNK potently inhibited EGF mediated anchorage independent growth and protection from cell death in two glial tumor cell lines. These findings demonstrate that enhanced JNK activation is frequently found in primary brain tumors and that this activation contributes to phenotypes related to transformation.

RhoA triggers a specific signaling pathway that generates transforming microvesicles in cancer cells

Vesicular structures called microvesicles (MVs) that are shed from the surfaces of cancer cells are capable of transferring oncogenic cargo to recipient cancer cells, as well as to normal cells, sending mitogenic signals that greatly enhance tumor growth. Because MVs are stable in the circulation, they also may have a key role in secondary colonization and metastasis. Thus, understanding how MVs are generated could have important consequences for interfering with cancer progression. Here we report that the small GTPase RhoA triggers a specific signaling pathway essential for MV biogenesis in various human cancer cells. Inhibiting the activity of different proteins comprising this pathway blocks MV biogenesis in the donor cancer cells and prevents oncogenic transformation in cell culture as well as tumor growth in mice. Although RhoA has often been implicated in human cancer, these findings now highlight a previously unappreciated role for this GTPase in malignant transformation, and demonstrate that blocking MV biogenesis may offer novel approaches for interfering with malignant transformation.

Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease - eScholarship

Caused by a polyglutamine expansion in the huntingtin protein, Huntingtons disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de‐repressed two established regulators of mitochondrial function, PGC‐1α and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3‐nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC‐independent epigenetic strategy for treating neurodegeneration.

Two isoforms of tissue transglutaminase mediate opposing cellular fates

Opposing cellular responses are typically regulated by distinct sets of genes. However, tissue transglutaminase (TGase) provides an interesting example of a single gene product that has been implicated both in affording protection against cellular insults as well as in promoting cell death. Here, we shed some light on how these conflicting activities might be manifested by demonstrating that alternative transcripts of TGase differentially affect cell viability. We show that although the full-length TGase protein affords strong protection against cell death signals, a shorter version of TGase that is truncated at the 3′ end, and thus called TGase-short (TGase-S), is cytotoxic. The apoptotic activity of TGase-S is not dependent on its transamidation activity because the mutation of a cysteine residue that is essential for catalyzing this reaction does not compromise the ability of TGase-S to induce cell death. Intriguingly, TGase-S undergoes inappropriate oligomer formation in cells before cell death, suggesting a novel mechanism for the apoptotic effects of this protein.

Tissue Transglutaminase Is an Essential Participant in the Epidermal Growth Factor-stimulated Signaling Pathway Leading to Cancer Cell Migration and Invasion

Epidermal growth factor (EGF) exerts pleiotropic effects during oncogenesis, including the stimulation of cell migration and invasiveness. Although a number of traditional signaling proteins (e.g. Ras and Rho GTPases) have been implicated in EGF-stimulated cancer cell migration, less is known about the identity of those proteins functioning further downstream in this growth factor pathway. Here we have used HeLa carcinoma cells as a model system for investigating the role of tissue transglutaminase (TGase), a protein that has been linked to oncogenesis, in EGF-stimulated cancer cell migration and invasion. Treatment of HeLa cells with EGF resulted in TGase activation and its accumulation at their leading edges, whereas knocking down TGase expression, or treating cells with a TGase inhibitor, blocked EGF-stimulated cell migration and invasion. We show that EGF signaling through Ras and c-Jun N-terminal kinase is responsible for targeting TGase to the leading edges of cells and activating it. The requirement for EGF to properly localize and activate TGase can be circumvented by the expression of oncogenic Ras (G12V), whose ability to stimulate migration is also dependent on TGase. We further show that, in the highly aggressive breast cancer cell line MDAMB231, where EGF stimulation is unnecessary for migration and invasive activity, TGase is already at the leading edge and activated. These findings demonstrate that TGase plays a key role in cancer cell motility and invasiveness and represents a previously unappreciated participant in the EGF pathway that stimulates these processes in cancer cells.

Extracellular Vesicles: Satellites of Information Transfer in Cancer and Stem Cell Biology

The generation and shedding of extracellular vesicles (EVs), including exosomes and microvesicles (MVs), by cells has emerged as a form of intercellular communication with important roles in several physiological processes and diseases such as cancer. These membrane-enclosed packets can transfer specific proteins, RNA transcripts, microRNAs, and even DNA to target cells, thereby altering their function. Despite the exponential growth of the EV field, a great deal remains unclear about the mechanisms that regulate exosome and MV biogenesis, as well as about how to isolate different classes of EVs and how to best take advantage of them for clinical applications.

Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations

Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer.

Phosphoinositide 3-Kinase Activity Is Required for Retinoic Acid-induced Expression and Activation of the Tissue Transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [α-32P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.