IGF2BP2 promotes cancer progression by degrading the RNA transcript encoding a v-ATPase subunit.

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

Oncogenic RAS promotes MYC protein stability by upregulating the expression of the inhibitor of apoptosis protein family member Survivin.

The small GTPase KRAS is frequently mutated in pancreatic cancer and its cooperation with the transcription factor MYC is essential for malignant transformation. The key to oncogenic KRAS and MYC working together is the stabilization of MYC expression due to KRAS activating the extracellular signal-regulated kinase 1/2, which phosphorylates MYC at serine 62 (Ser 62). This prevents the proteasomal degradation of MYC while enhancing its transcriptional activity. Here, we identify how this essential signaling connection between oncogenic KRAS and MYC expression is mediated by the inhibitor of apoptosis protein family member Survivin. This discovery stemmed from our finding that Survivin expression is downregulated upon treatment of pancreatic cancer cells with the KRASG12C inhibitor Sotorasib. We went on to show that oncogenic KRAS increases Survivin expression by activating extracellular signal-regulated kinase 1/2 in pancreatic cancer cells and that treating the cells either with siRNAs targeting Survivin or with YM155, a small molecule that potently blocks Survivin expression, downregulates MYC and strongly inhibited their growth. We further determined that Survivin protects MYC from degradation by blocking autophagy, which then prevents cellular inhibitor of protein phosphatase 2A from undergoing autophagic degradation. Cellular inhibitor of protein phosphatase 2A, by inhibiting protein phosphatase 2A, helps to maintain MYC phosphorylation at Ser 62, thereby ensuring its cooperation with oncogenic KRAS in driving cancer progression. Overall, these findings highlight a novel role for Survivin in mediating the cooperative actions of KRAS and MYC during malignant transformation and raise the possibility that targeting Survivin may offer therapeutic benefits against KRAS-driven cancers.

Tissue transglutaminase 2 regulates tumor cell tensional homeostasis by increasing contractility.

Abnormal tensional cellular homeostasis is now considered a hallmark of cancer. Despite this, the origin of this abnormality remains unclear. In this work, we investigated the role of tissue transglutaminase 2 (TG2, also known as TGM2), a protein associated with poor prognosis and increased metastatic potential, and its relationship to the EGF receptor in the regulation of the mechanical state of tumor cells. Remarkably, we observed a TG2-mediated modulation of focal adhesion composition as well as stiffness-induced FAK activation, which was linked with a distinctive increase in cell contractility, in experiments using both pharmacological and shRNA-based approaches. Additionally, the increased contractility could be reproduced in non-malignant cells upon TG2 expression. Moreover, the increased cell contractility mediated by TG2 was largely due to the loss of EGFR-mediated inhibition of cell contractility. These findings establish intracellular TG2 as a regulator of cellular tensional homeostasis and suggest the existence of signaling switches that control the contribution of growth factor receptors in determining the mechanical state of a cell.

New insights into extracellular vesicle biogenesis and function.

It is becoming increasingly evident that most cell types are capable of forming and releasing multiple distinct classes of membrane-enclosed packages, referred to as extracellular vesicles (EVs), as a form of intercellular communication. Microvesicles (MVs) represent one of the major classes of EVs and are formed by the outward budding of the plasma membrane. The second major class of EVs, exosomes, are produced as components of multivesicular bodies (MVBs) and are released from cells when MVBs fuse with the cell surface. Both MVs and exosomes have been shown to contain proteins, RNA transcripts, microRNAs and even DNA that can be transferred to other cells and thereby trigger a broad range of cellular activities and biological responses. However, EV biogenesis is also frequently de-regulated in different pathologies, especially cancer, where MVs and exosomes have been suggested to promote tumor cell growth, therapy resistance, invasion and even metastasis. In this Review, we highlight some of the recent advances in this rapidly emerging and exciting field of cell biology, focusing on the underlying mechanisms that drive MV and exosome formation and release, with a particular emphasis on how EVs potentially impact different aspects of cancer progression and stem cell biology.

Extracellular vesicles and their roles in stem cell biology.

Stem cells use a variety of mechanisms to help maintain their pluripotency and promote self-renewal, as well as, at the appropriate time, to differentiate into specialized cells. One such mechanism that is attracting significant attention from the stem cell, development, and regenerative medicine research communities involves a form of intercellular communication, specifically, the ability of cells to form and release nontraditional membrane-enclosed structures, referred to as extracellular vesicles (EVs). There are two major classes of EVs, microvesicles (MVs), which are generated through the outward budding and fission of the plasma membrane, and exosomes, which are formed as multivesicular bodies (MVBs) in the endo-lysosomal pathway that fuse with the cell surface to release their contents. Although they differ in how they are formed, both MVs and exosomes have been shown to contain a diverse array of bioactive cargo, such as proteins, RNA transcripts, microRNAs, and even DNA, which can be transferred to other cells and promote phenotypic changes. Here, we will describe what is currently known regarding EVs and the roles they play in stem cell biology and different aspects of early development. We will also highlight how the EVs produced by stem cells are being aggressively pursued for clinical applications, including their potential use as therapeutic delivery systems and for their regenerative capabilities.

Extracellular vesicle docking at the cellular port: Extracellular vesicle binding and uptake.

Extracellular vesicles (EVs), lipid bilayer-enclosed structures that contain a variety of biological molecules shed by cells, are increasingly becoming appreciated as a major form of cell-to-cell communication. Indeed, EVs have been shown to play important roles in several physiological processes, as well as diseases such as cancer. EVs dock on to the surfaces of recipient cells where they transmit signals from the cell surface and/or transfer their contents into cells to elicit functional responses. EV docking and uptake by cells represent critical, but poorly understood processes. Here, we focus on the mechanisms by which EVs dock and transfer their contents to cells. Moreover, we highlight how these findings may provide new avenues for therapeutic intervention.

Matrix stiffness regulates microvesicle-induced fibroblast activation.

Extracellular vesicles released by cancer cells have recently been implicated in the differentiation of stromal cells to their activated, cancer-supporting states. Microvesicles, a subset of extracellular vesicles released from the plasma membrane of cancer cells, contain biologically active cargo, including DNA, mRNA, and miRNA, which are transferred to recipient cells and induce a phenotypic change in behavior. While it is known that microvesicles can alter recipient cell phenotype, little is known about how the physical properties of the tumor microenvironment affect fibroblast response to microvesicles. Here, we utilized cancer cell-derived microvesicles and synthetic substrates designed to mimic the stiffness of the tumor and tumor stroma to investigate the effects of microvesicles on fibroblast phenotype as a function of the mechanical properties of the microenvironment. We show that microvesicles released by highly malignant breast cancer cells cause an increase in fibroblast spreading, α-smooth muscle actin expression, proliferation, cell-generated traction force, and collagen gel compaction. Notably, our data indicate that these phenotypic changes occur only on stiff matrices mimicking the stiffness of the tumor periphery and are dependent on the cell type from which the microvesicles are shed. Overall, these results show that the effects of cancer cell-derived microvesicles on fibroblast activation are regulated by the physical properties of the microenvironment, and these data suggest that microvesicles may have a more robust effect on fibroblasts located at the tumor periphery to influence cancer progression.

Cool-associated Tyrosine-phosphorylated Protein 1 Is Required for the Anchorage-independent Growth of Cervical Carcinoma Cells by Binding Paxillin and Promoting AKT Activation.

Cool-associated tyrosine-phosphorylated protein 1 (Cat-1) is a signaling scaffold as well as an ADP-ribosylation factor-GTPase-activating protein. Although best known for its role in cell migration, we recently showed that the ability of Cat-1 to bind paxillin, a major constituent of focal complexes, is also essential for the anchorage-independent growth of HeLa cervical carcinoma cells. Here we set out to learn more about the underlying mechanism by which Cat-paxillin interactions mediate this effect. We show that knocking down paxillin expression in HeLa cells promotes their ability to form colonies in soft agar, whereas ectopically expressing paxillin in these cells inhibits this transformed growth phenotype. Although knocking down Cat-1 prevents HeLa cells from forming colonies in soft agar, when paxillin is knocked down together with Cat-1, the cells are again able to undergo anchorage-independent growth. These results suggest that the requirement of Cat-1 for this hallmark of cellular transformation is coupled to its ability to bind paxillin and abrogate its actions as a negative regulator of anchorage-independent growth. We further show that knocking down Cat-1 expression in HeLa cells leads to a reduction in Akt activation, which can be reversed by knocking down paxillin. Moreover, expression of constitutively active forms of Akt1 and Akt2 restores the anchorage-independent growth capability of HeLa cells depleted of Cat-1 expression. Together, these findings highlight a novel mechanism whereby interactions between Cat-1 and its binding partner paxillin are necessary to ensure sufficient Akt activation so that cancer cells are able to grow under anchorage-independent conditions.

Probing the mechanisms of extracellular vesicle biogenesis and function in cancer.

Tumor cells interact with each other, and their surroundings, using a variety of mechanisms to promote virtually all aspects of cancer progression. One such form of intercellular communication that has been attracting considerable attention from the cancer community and the pharmaceutical industry in recent years involves the ability of cancer cells to generate multiple distinct types of non-classical secretory vesicles, generally referred to as extracellular vesicles (EVs). Microvesicles (MVs) represent one of the major classes of EVs and are formed as a result of the outward budding and fission of the plasma membrane. The other main class of EVs is exosomes, which are generated when multivesicular bodies fuse with the cell surface and release their contents into the extracellular space. Both MVs and exosomes have been shown to contain bioactive cargo, including proteins, metabolites, RNA transcripts, microRNAs, and DNA that can be transferred to other cancer cells and stimulate their growth, survival, and migration. However, cancer cell-derived EVs also play important roles in helping re-shape the tumor microenvironment to support tumor expansion and invasive activity, dampen immune responses, as well as enter the circulation to help promote metastatic spread. Here, we provide an overview of what is currently known regarding how the different classes of EVs are generated and contribute to various cancer cell phenotypes. Moreover, we highlight how some of the unique properties of EVs are being used for the development of novel diagnostic and clinical applications.

Identifying the functional contribution of the defatty-acylase activity of SIRT6.

Mammalian sirtuin 6 (SIRT6) exhibits many pivotal functions and multiple enzymatic activities, but the contribution of each activity to the various functions is unclear. We identified a SIRT6 mutant (G60A) that possesses efficient defatty-acylase activity but has substantially decreased deacetylase activity in vitro and no detectable deacetylase activity in cells. The G60A mutant has a decreased ability to bind NAD(+), but the presence of fatty-acyl lysine peptides restores NAD(+) binding, explaining the retention of the defatty-acylase activity. Using this mutant, we found that the defatty-acylase activity of SIRT6 regulates the secretion of numerous proteins. Notably, many ribosomal proteins were secreted via exosomes from Sirt6 knockout mouse embryonic fibroblasts, and these exosomes increased NIH 3T3 cell proliferation compared with control exosomes. Our data indicate that distinct activities of SIRT6 regulate different pathways and that the G60A mutant is a useful tool to study the contribution of defatty-acylase activity to SIRT6's various functions.

The Different Conformational States of Tissue Transglutaminase Have Opposing Affects on Cell Viability.

Tissue transglutaminase (tTG) is an acyltransferase/GTP-binding protein that contributes to the development of various diseases. In human cancer cells, tTG activates signaling pathways that promote cell growth and survival, whereas in other disorders (i.e. neurodegeneration), overexpression of tTG enhances cell death. Therefore, it is important to understand how tTG is differentially regulated and functioning to promote diametrically distinct cellular outcomes. Previous structural studies revealed that tTG adopts either a nucleotide-bound closed conformation or a transamidation-competent open conformation. Here we provide evidence showing that these different conformational states determine whether tTG promotes, or is detrimental to, cell survival, with the open conformation of the protein being responsible for inducing cell death. First, we demonstrate that a nucleotide binding-defective form of tTG, which has previously been shown to induce cell death, assumes an open conformation in solution as assessed by an enhanced sensitivity to trypsin digestion and by small angle x-ray scattering (SAXS) analysis. We next identify two pairs of intramolecular hydrogen bonds that, based on existing x-ray structures, are predicted to form between the most C-terminal ß-barrel domain and the catalytic core domain of tTG. By disrupting these hydrogen bonds, we are able to generate forms of tTG that constitutively assume an open conformation and induce apoptosis. These findings provide important insights into how tTG participates in the pathogenesis of neurodegenerative diseases, particularly with regard to the actions of a C-terminal truncated form of tTG (TG-Short) that has been linked to such disorders and induces apoptosis by assuming an open-like conformation.

Microvesicle Cargo and Function Changes upon Induction of Cellular Transformation.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), have emerged as a major form of intercellular communication, playing important roles in several physiological processes and diseases, including cancer. EVs generated by cancer cells contain a variety of proteins and RNA species that can be transferred between cancer cells as well as between cancer and non-transformed (normal) cells, thereby impacting a number of aspects of cancer progression. Here we show how oncogenic transformation influences the biogenesis and function of EVs using a mouse embryonic fibroblast (MEF) cell line that can be induced to express an oncogenic form of diffuse B cell lymphoma (Dbl). Although MEFs induced to express onco-Dbl generated a similar amount of MVs as uninduced control cells, we found that MVs isolated from onco-Dbl-transformed cells contain a unique signaling protein, the ubiquitously expressed non-receptor tyrosine kinase focal adhesion kinase. The addition of MVs isolated from MEFs expressing onco-Dbl to cultures of fibroblasts strongly promoted their survival and induced their ability to grow under anchorage-independent conditions, outcomes that could be reversed by knocking down focal adhesion kinase and depleting it from the MVs or by inhibiting its kinase activity using a specific inhibitor. We then showed the same to be true for MVs isolated from aggressive MDAMB231 breast cancer cells. Together, these findings demonstrate that the induction of oncogenic transformation gives rise to MVs, which uniquely contain a signaling protein kinase that helps propagate the transformed phenotype and thus may offer a specific diagnostic marker of malignant disease.

An Essential Role for Cdc42 in the Functioning of the Adult Mammary Gland.

The Rho family small GTPase Cdc42 has been implicated in a wide range of cellular functions including the establishment of cell polarity and the remodeling of the actin cytoskeletal architecture, resulting in the tight regulation of cell growth and survival during developmental processes. The complete knock-out of Cdc42 in the mouse is embryonic-lethal, and its targeted deletion in various tissues has been shown to disrupt tissue homeostasis. Thus far, in most studies, the targeted deletion of Cdc42 occurred during embryogenesis. Here, we have used a conditional gene deletion strategy in mice to probe the specific role of Cdc42 during adult mammary gland function. Cdc42 conditional-knock-out females were unable to adequately nourish their pups, due to a disorganized epithelial compartment within their mammary glands. A closer examination showed that their mammary epithelial cells were not able to maintain functional alveolar lumens, due to an inability to establish normal apical/basal epithelial polarity, as well as proper cell-cell contacts. Loss of these essential epithelial characteristics led to a premature sloughing off of the Cdc42-null epithelial cells. Overall our findings demonstrate that Cdc42 plays essential roles in mammary gland function post pregnancy, where it helps to establish proper epithelial cell polarity and tissue homeostasis during lactation.

Ras and the FAK paradox.

In a recent issue of Molecular Cell, Zheng et al. (2009) describe a surprising set of findings that highlight an unexpected negative regulation of FAK by oncogenic Ras and its consequences for cancer cell migration and invasion.

A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival.

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Simultaneously targeting tissue transglutaminase and kidney type glutaminase sensitizes cancer cells to acid toxicity and offers new opportunities for therapeutic intervention.

Most cancer cells undergo characteristic metabolic changes that are commonly referred to as the Warburg effect, with one of the hallmarks being a dramatic increase in the rate of lactic acid fermentation. This leads to the production of protons, which in turn acidifies the microenvironment surrounding tumors. Cancer cells have acquired resistance to acid toxicity, allowing them to survive and grow under these detrimental conditions. Kidney type glutaminase (GLS1), which is responsible for the conversion of glutamine to glutamate, produces ammonia as part of its catalytic activities and has been shown to modulate cellular acidity. In this study, we show that tissue, or type 2, transglutaminase (TG2), a γ-glutamyl transferase that is highly expressed in metastatic cancers and produces ammonia as a byproduct of its catalytic activity, is up-regulated by decreases in cellular pH and helps protect cells from acid-induced cell death. Since both TG2 and GLS1 can similarly function to protect cancer cells, we then proceeded to demonstrate that treatment of a variety of cancer cell types with inhibitors of each of these proteins results in synthetic lethality. The combination doses of the inhibitors induce cell death, while individual treatment with each compound shows little or no ability to kill cells. These results suggest that combination drug treatments that simultaneously target TG2 and GLS1 might provide an effective strategy for killing cancer cells.

Mutations in the polybasic juxtamembrane sequence of both plasma membrane- and endoplasmic reticulum-localized epidermal growth factor receptors confer ligand-independent cell transformation.

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.

Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition.

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell-cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

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.

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.