Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the United States (1, 2). Over the past decade, morbidity and lethality from CDI have increased (3, 4), and the need for new treatment options has become a priority.The pathology associated with CDI is associated with the activities of two large, glucosylating toxins, TcdA and TcdB (5). Upon binding to the colonic epithelium, these toxins induce the fluid secretion, immune cell influx, and tissue damage associated with clinical manifestations of CDI (5). TcdA and TcdB have four functional domains: an N-terminal glucosyltransferase domain (GTD), an autoprotease domain, a pore-forming and delivery domain, and a combined repetitive oligopeptides (CROPS) domain, which extends from around residue 1830 to the C terminus and has been implicated in receptor binding. The toxins enter cells by receptor-mediated endocytosis (6). Acidification of the endosome is thought to trigger a structural change in the delivery domain, allowing for pore formation and translocation of the GTD into the cytosol (7, 8). Activation of the autoprocessing domain by eukaryotic inositol hexakisphosphate results in the release of the GTD into the cell, allowing access to substrates (8). The GTD transfers a glucose from UDP glucose onto the switch I region of Rho family GTPases such as Rho, Rac1, and Cdc42 (9, 10). These modifications cause a cytopathic effect resulting from rearrangement of the actin cytoskeleton and can lead to apoptosis (11). At higher concentrations, TcdB is also capable of inducing the production of reactive oxygen species, resulting in cell death by a necrotic mechanism (12, 13). We speculate that both mechanisms are important in the context of disease; the cytopathic effects promote inflammation and disruption of the tight junctions, whereas the TcdB-induced necrosis contributes to the colonic tissue damage observed in severe cases of CDI.Although TcdA and TcdB are homologs, they appear to perform separate, nonredundant functions (14, 15). TcdA and TcdB are thought to have different receptors, based on sensitivity differences among cell types in vitro (16–19). Multiple receptors for TcdA have been proposed including Gal alpha 1–3Gal beta 1–4GlcNAc, blood antigens I, X, and Y, rabbit sucrase isomaltase, and gp96 (18, 20–22). The TcdA CROPS domain is thought to play a role in binding cell surface carbohydrates (18, 23, 24). Antibodies against the CROPS domains of TcdA and TcdB can block intoxication (25, 26), and excess TcdA CROPS domain can compete with TcdA holotoxin for cell binding (27). At the same time, truncations of TcdA and TcdB that lack the CROPS domains are still capable of intoxicating cells (7, 28, 29) and a homologous toxin from Clostridium perfringens, TpeL, lacks a CROPS domain entirely (29, 30). A receptor for TpeL has been identified (29), suggesting that a receptor-binding site for other large clostridial toxins could exist outside of the CROPS. A recent report indicates that chondroitin sulfate proteoglycan 4 (CSPG4) mediates TcdB-induced cytopathic and apoptotic events in HeLa and HT29 cells (31). CSPG4 does not mediate the necrotic effects that occur at higher TcdB concentrations and CSPG4 binds TcdB outside the CROPS; these observations are consistent with a dual receptor hypothesis. This study represents an independent effort to define the cellular factor(s) responsible for TcdB binding and toxicity.     

Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth,invasiveness, and inflammatory responses

The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2–associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.Prostate cancer is the most common noncutaneous cancer of men in the United States, affecting one in six men (1). Although the causes of prostate cancer are poorly understood, inflammation has been implicated in both initiation and progression of the disease (2, 3). The origin of inflammation in prostate cancer is unclear, although chronic infections are believed to promote and establish a tumor-enhancing proinflammatory environment.Trichomonas vaginalis is the causative agent of the most common nonviral sexually transmitted infection, infecting ∼275 million people worldwide (4). T. vaginalis is a flagellated, protozoan parasite that infects the prostate epithelium (5, 6). Over 75% of men harboring T. vaginalis are asymptomatic and may not seek treatment, resulting in chronic inflammation (5). Several studies have positively associated T. vaginalis infection with increased incidence and severity of prostate cancer, as well as benign prostate hyperplasia (2, 6–9). The magnitude of the association between T. vaginalis seropositivity and overall prostate cancer risk is between 1.23 and 1.43 based on two large, nested case–control studies (7, 8). Additionally there is a statistically significant increase in risk of extraprostatic cancer [odds ratio (OR) = 2.17] or cancer-specific death (OR = 2.69) with T. vaginalis seropositive status (7).Our research focuses on the potential contribution of a proinflammatory protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), to prostate cancer, because the human homolog has a role in the growth and invasion of prostate cancer (10, 11). Human macrophage migration inhibitory factor (HuMIF) has been implicated in a broad array of conditions associated with inflammation, including autoimmunity, cell proliferation, angiogenesis, and tumorigenesis (12–15). Increased expression of HuMIF has been reported in several cancers, including prostate cancer (16–18). Studies show high expressers of HuMIF have a heightened risk of prostate cancer, as well as a significant increase in prostate cancer progression and drug resistance (17–23). Several clinical studies have shown HuMIF production correlates with both tumor aggressiveness and metastatic potential (23, 24). Additionally, the expression of the HuMIF receptor CD74 is increased in prostate cancer (10, 25).HuMIF induces ERK1/2, MAPK, and Akt activation via binding with the extracellular domain of the MIF receptor CD74 (26). HuMIF has multiple functions with regard to regulating the immune system, including protecting monocytes and macrophages from activation-induced apoptosis, which results in sustained inflammation (27, 28). Consequently, HuMIF is implicated in the pathogenesis of several inflammatory and autoimmune diseases in addition to cancer (29, 30).Recent studies have shown that several parasitic eukaryotes encode MIF-like proteins with considerable structural and biological similarity to their mammalian hosts (31–34). These parasite MIF proteins have been shown to modulate host immune responses and regulate pathways to promote parasite survival. Here, we characterize TvMIF and show that it can act as a molecular mimic of HuMIF. We find that TvMIF binding to the human CD74 receptor activates extracellular signal-regulated kinases (ERK)1/2 and Akt protein kinase/proapoptotic Bcl-2–associated death promoter (BAD) pathways as well as secretion of proinflammatory IL-8 from monocytes, reduces monocyte migration, and increases growth and invasiveness of benign prostate hyperplasia (BPH-1) and prostate cancer (PC3) cells. This research is, to our knowledge, the first to identify a human inflammatory cytokine mimic in T. vaginalis and to begin to explore the link between this sexually transmitted infection and prostate cancer.     

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase–, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion.Cofilin is one crucial mediator of actin cytoskeletal dynamics during cell motility (1–5). At the cell edge, cofilin severs F-actin filaments, generating substrates for Arp2/3-mediated branching activity and contributing to F-actin depolymerization by creating a new pointed end and F-actin assembly by increasing the pool of polymerization-competent actin monomers (G-actin) (6, 7). Because of its ability to sever actin filaments and thus, modulate actin dynamics, the precise spatial and temporal regulation of cofilin activity at the cell leading edge is crucial to cell protrusion, chemotaxis, and motility both in vitro and in vivo (2, 8–13). Misregulation of cofilin activity and/or expression is directly related to diseases, including tumor metastasis (14–18) and Alzheimer’s disease (19).Several mechanisms regulate tightly the activation of cofilin in response to upstream stimuli, including interaction with phosphatidylinositol (4,5)-bisphosphate (20–22), local pH changes (23, 24), and phosphorylation at a single regulatory serine (Ser3) (8, 25). The phosphorylation of cofilin, leading to its inactivation, is catalyzed by two kinase families: the LIM-kinases [LIMKs(Lin11, Isl-1, and Mec-3 domain)] and the testicular kinases (25–27). Two primary families of ser/thr phosphatases dephosphorylate and reactivate the actin-depolymerizing and -severing functions of cofilin: slingshot (SSH) (28) and chronophin (CIN) (29).SSH was identified as a cofilin phosphatase through genetic studies in Drosophila (28). The most active and abundant SSH isoform, SSH-1L, has been implicated in such biological processes as cell division, growth cone motility/morphology, neurite extension, and actin dynamics during membrane protrusion (30). SSH dephosphorylates a number of actin regulatory proteins in addition to cofilin, including LIMK1 (31) and Coronin 1B (32). CIN is a haloacid dehydrogenase-type phosphatase, a family of enzymes with activity in mammalian cells that has been poorly characterized. CIN dephosphorylates a very limited number of substrates (33) and as opposed to SSH, has little phosphatase activity toward LIMK both in vitro and in vivo; thus, it seems to be the more specific activator of cofilin (29, 30). CIN exhibits several predicted interaction motifs potentially linking it to regulation by PI3-kinase and phospholipase Cγ (PLCγ), both of which have been implicated in signaling to cofilin activation in vivo in MTLn3 adenocarcinoma cells (10, 34). CIN has been involved in cell division (29), cofilin–actin rod formation in neurons (35), and chemotaxing leukocytes (36, 37). The molecular mechanisms that control the activity and localization of CIN in cells are still not well-understood. In neutrophils, CIN mediates cofilin dephosphorylation downstream of Rac2 (36), and stimulation of protease-activated receptor2 results in recruitment of CIN and cofilin at the cell edge by β-arrestins to promote localized generation of free actin barbed ends, membrane protrusion, and chemotaxis (37). Chemotaxis to EGF by breast tumor cells is directly correlated with cancer cell invasion and metastasis (38, 39). Although cofilin activity is required for tumor cell migration, the contribution(s) of CIN to the regulation of actin dynamics at the leading edge has not yet been investigated.The importance of cofilin in regulating tumor cell motility has been extensively studied using MTLn3 mammary carcinoma cells as a model system. The initial step of MTLn3 cell chemotaxis to EGF consists of a biphasic actin polymerization response resulting from two peaks of free actin barbed end formation (34, 40, 41). The first or early peak of actin polymerization occurs at 1 min after EGF stimulation and requires both cofilin and PLCγ activities (34), but it is not dependent on cofilin dephosphorylation (42). This first transient allows the cells to sense EGF gradients and initiate small-membrane protrusions (11). The second or late peak of actin polymerization occurs at 3 min and is dependent on both cofilin and PI3-kinase activities (43, 44). Cofilin activity in this late transient has been associated with full protrusion of lamellipodia (34). The mechanism by which cofilin becomes activated at the 3-min peak has not been identified, although it is likely to involve the phosphoregulation of Ser3 (42, 45).In this work, we determine the molecular mechanisms involved in the full protrusion of the leading edge upon EGF stimulation. We have identified CIN as a critical regulator of cofilin activation to coordinate leading edge dynamics. Our results yield insights into how CIN controls cell protrusion, a key step in the process of cell migration and metastasis.     

ECHIDNA-mediated post-Golgi trafficking of auxin carriers for differential cell elongation

The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)–mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development.Polar auxin transport (PAT) plays a key role in plant development (1–5). PAT is mediated by plasma membrane localized auxin influx and efflux carriers of the auxin-resistant (AUX)/like-AUX (LAX), pin-formed (PIN), and ABCB families (6–12). Highly regulated tissue, cellular localization, and amount of auxin carriers at the plasma membrane (PM) provide directionality to the auxin transport and underlies the creation of auxin concentration gradient that is essential for controlling several aspects of plant development (13–18). One of the developmental programs in which auxin concentration gradient plays a central role is the formation of apical hook, a bending in the embryonic stem during early seedling germination (19). Hook formation involves differential elongation of cells on the two opposite sides of the hypocotyl. This process is mediated by the formation of an auxin maximum at the concave side of the hook, leading to the inhibition of cell elongation (20–25). A model based on mutational analysis shows that auxin carriers including polarly localized auxin efflux and influx facilitators PIN3 and AUX1/LAX3, respectively, are important for hook development (23, 24). The amount of auxin carriers at the PM is important for the regulation of auxin concentration, and this depends on the balance between secretion, endocytosis, and recycling. The analysis of PIN efflux carriers has revealed how cell wall anchoring, endocytosis, targeted degradation, and also posttranslational modifications strongly influence the location and amount of these carriers at the PM (15, 17, 26–29). In contrast, little is known about the mechanisms and molecular components underlying the deposition of auxin carriers at the PM. Post-Golgi secretion to the PM occurs via the trans-Golgi network (TGN), a post-Golgi compartment (30). The TGN is a complex tubulo-vesicular membrane network maturing from the trans-most cisternae of the Golgi apparatus to become a highly dynamic independent structure from which secretory vesicles (SVs) and CLATHRIN-coated vesicles (CCVs) originate (31–34). Although auxin carriers traffic via TGN, components and mechanisms specifically involved in trafficking to the PM of de novo-synthesized auxin carriers remain largely undefined (35, 36). Importantly, it is not known whether auxin carriers traffic through SV or CCV sites of the TGN on their way to the PM. We have used apical hook development as a model system to investigate the mechanisms that link post-Golgi trafficking of auxin carriers to the PM with control of differential cell elongation. We previously identified the transmembrane TGN-localized protein ECHIDNA (ECH) that is required for cell elongation (37). We discovered that the ech mutant is defective in hook development and is insensitive to ethylene like the aux1 mutant. These data prompted us to investigate the role of ECH and the TGN in post-Golgi trafficking of auxin carriers during hook development. Using genetic, pharmacological, and cell biological approaches, we show that distinct mechanisms/components underlie post-Golgi trafficking of influx and efflux carriers. We show that post-Golgi trafficking of de novo-synthesized AUX1 occurs via an ECH-dependent SV-based pathway, whereas that of PIN3 and LAX3 are largely independent of ECH at the TGN. Thus, these results reveal the complexity of trafficking from the TGN to PM as shown by the differential trafficking of influx carriers AUX1 versus LAX3 and the efflux carrier PIN3. Hence, our results reveal an additional layer of regulatory control to auxin transport.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution

The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 “polymorphic” SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, M_ALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors.The level of genetic diversity in a natural population is determined by several evolutionary forces, including mutation, genetic drift, migration, and natural selection (1–3). Tumors can be regarded as asexual populations of cells, so they are subjected to similar forces to those of natural populations (4–7). Therefore, the genetic diversity in tumors of the same patient is informative about how various forces drive their evolution. The level of diversity may also influence how tumors respond to environmental perturbations, either natural or medical (5–7). In the prevailing view, Darwinian selection for and against new mutations is the main driving force of intratumor diversity (4, 8–18). Because selection generally reduces genetic diversity within populations (19–21), studies assuming Darwinian evolution usually described M_ALL (the total number of coding region mutations within the whole tumor) in the range of tens to hundreds of coding mutations (22, 23).Despite its wide acceptance, the Darwinian view has never been subjected to hypothesis testing, by which the observed diversity is compared with quantitative predictions. This study is to our knowledge the first one that uses high-density sampling in a single tumor and compares the observations with theoretical predictions. In this test, we consider a null model of non-Darwinian evolution in which M_ALL is a function of N (population size), u (mutation rate per generation), and growth parameters. In tumors, N is large, generally ≫ 10⁶, and u is the mutation rate of the entire functional portion of the genome (at the level of 10⁻² per cell division) (18, 24). Hence, the expected genetic diversity of tumors by non-Darwinian evolution would be large, probably on the order of millions of mutations, most of which are present at low frequencies (25).We ask whether the observed intratumor genetic diversity can be largely explained by non-Darwinian forces and we invoke positive selection only when the null model of non-Darwinian evolution is rejected. There was a controversy in molecular evolution generally known as the neutralism–selectionism debate (1, 26, 27). In the postdebate modern view, genetic polymorphisms in natural populations are largely consistent with the non-Darwinian model (1–3, 26–28). There are further reasons to question the efficacy of selection within populations of cells that make up tumors (Discussion). For instance, although selection against nonsynonymous mutations is nearly universal in natural species (1, 3, 27), selection against such mutations in tumors is not apparently stronger than against synonymous ones (29).In the recent literature, there has been increasingly more attention on assessing the non-Darwinian model of tumor evolution vs. the prevailing Darwinian view (30, 31). Tao et al. (31) studied 12 cases of multitumor hepatocellular carcinomas (HCCs) and concluded that competition often occurs between tumors large enough to be visible. In contrast, the genetic diversity contained within the same tumor does not deviate from the predictions of the non-Darwinian model. A caveat is that whereas the number of population samples used in testing Darwinian selection in natural populations is often in the hundreds, the sample number rarely exceeds 10 in intratumor studies (12, 13, 15–18, 30, 31). Therefore, the power to reject the null model in tumor studies might have been too low. Clearly, there is a need to sample a large number of regions in one single tumor. In this study we sampled close to 300 regions to examine the spatial distribution of single-nucleotide variants and to estimate the amount of genetic diversity in the tumor. We used these data to give a rigorous test of the null hypothesis of non-Darwinian evolution.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses.Classical feed-forward inhibition (FFI) involves a sequence of excitation rapidly terminated by inhibition. This temporal sequence narrows the time window for synaptic integration and enforces precise spike timing (1–7). FFI is thought to be important for regulating the temporal fidelity of spike responses in many neural systems, including the motor system, where rapid and adaptable changes in muscle activity are essential for coordinated motor control (8–10). The cerebellum plays a central role in fine sculpting of movements, and damage to the cerebellum produces severe motor deficits, most notably enhanced temporal variability of voluntary movements (11, 12). These findings suggest that cerebellar circuits have the ability to preserve precise timing information during behavior (5, 6, 13), and in vitro studies have shown that feed-forward inhibitory networks in the input layer of the cerebellum provide a mechanism for maintaining the temporal fidelity of information transmission (6, 14, 15).Synaptic inhibition in the granule cell layer is generated by Golgi cells, GABAergic interneurons that provide direct inhibitory input to granule cells (6, 15–17). The prevailing view is that, when mossy fibers are activated, granule cells receive both monosynaptic excitation and disynaptic FFI from Golgi cells, providing temporally precise inhibitory input that narrows the window for the temporal summation of discrete mossy fiber inputs (6, 14, 18). This classical excitation–inhibition sequence forms the basis of a variety of contemporary cerebellar models (7, 9, 18, 19). However, the exact temporal relationship between sensory-evoked excitation and inhibition in granule cells has never been determined in vivo. Here, we combined in vivo whole-cell voltage-clamp recordings from granule cells and in vitro dynamic clamp experiments to investigate both the temporal dynamics of Golgi-cell–mediated inhibition and its importance for shaping sensory responses in the input layer of the cerebellum.     

Genomic characterization of sarcomatoid transformation in clear cell renal cell carcinoma

The presence of sarcomatoid features in clear cell renal cell carcinoma (ccRCC) confers a poor prognosis and is of unknown pathogenesis. We performed exome sequencing of matched normal-carcinomatous-sarcomatoid specimens from 21 subjects. Two tumors had hypermutation consistent with mismatch repair deficiency. In the remainder, sarcomatoid and carcinomatous elements shared 42% of somatic single-nucleotide variants (SSNVs). Sarcomatoid elements had a higher overall SSNV burden (mean 90 vs. 63 SSNVs, P = 4.0 × 10⁻⁴), increased frequency of nonsynonymous SSNVs in Pan-Cancer genes (mean 1.4 vs. 0.26, P = 0.002), and increased frequency of loss of heterozygosity (LOH) across the genome (median 913 vs. 460 Mb in LOH, P < 0.05), with significant recurrent LOH on chromosomes 1p, 9, 10, 14, 17p, 18, and 22. The most frequent SSNVs shared by carcinomatous and sarcomatoid elements were in known ccRCC genes including von Hippel–Lindau tumor suppressor (VHL), polybromo 1 (PBRM1), SET domain containing 2 (SETD2), phosphatase and tensin homolog (PTEN). Most interestingly, sarcomatoid elements acquired biallelic tumor protein p53 (TP53) mutations in 32% of tumors (P = 5.47 × 10⁻¹⁷); TP53 mutations were absent in carcinomatous elements in nonhypermutated tumors and rare in previously studied ccRCCs. Mutations in known cancer drivers AT-rich interaction domain 1A (ARID1A) and BRCA1 associated protein 1 (BAP1) were significantly mutated in sarcomatoid elements and were mutually exclusive with TP53 and each other. These findings provide evidence that sarcomatoid elements arise from dedifferentiation of carcinomatous ccRCCs and implicate specific genes in this process. These findings have implications for the treatment of patients with these poor-prognosis cancers.Sarcomatoid transformation is common in various epithelial malignancies, featuring further loss of differentiation and acquisition of characteristics typical of a sarcoma. In renal cell carcinoma (RCC), sarcomatoid features are observed in 5% of tumors. However, among individuals with stage IV disease, it occurs in 15% (1, 2). Although once believed to represent a distinct subtype of RCC, it is now considered a specific histologic feature (3). Whereas sarcomatoid features are found in all forms of kidney cancer, >65% of cases are found with clear cell RCC (ccRCC) (4, 5). When sarcomatoid features are present, renal tumors generally are large (median size 10 cm), invasive (20%), and/or metastatic (50%) at presentation (4, 5). Whereas all such tumors are considered to be Fuhrman grade IV (6), their prognosis is significantly worse compared with other high-grade tumors (7). These tumors, when metastatic, have among the poorest survival of all genitourinary malignancies, with a median survival of only 6 mo (1, 2, 4). Even with resected localized disease, nearly 75% recur and have a median survival of <2 y (4, 8). The response to systemic therapy is poor, with rare durable responses occurring with any therapeutic strategy (9–12).Although there have been major breakthroughs in the understanding of kidney cancer, progress in the characterization of the genetic events associated with sarcomatoid kidney tumors has been limited (9). Various theories have been proposed regarding the origins of sarcomatoid features in renal tumors. Given that they virtually always occur in conjunction with typical epithelial RCC elements, the terminology of a “mixed malignancy” appeared a half century ago (13). Proposals have included independent occurrences of tumor types in close proximity, as has been observed in various genitourinary malignancies (14), and the influence of tumor microenvironment (15). The current prevailing theory is that sarcomatoid features represents a subclonal dedifferentiation or transformation from an incident carcinomatous component (16). However, the current theory is based on limited evidence. Evidence of common cell of origin is suggested by the shared patterns of X chromosome inactivation (17). Although there is limited evidence that the epithelial component transforms into the sarcomatoid element, groups have considered sarcomatoid features to result from a final common dedifferentiation pathway in RCC (16). This hypothesis is based on the sarcomatoid component more frequently metastasizing, possessing higher tumor grade, increased proliferative index, and frequent reduced expression of epithelial adhesion molecules such as E-cadherin (18–20). Because some tumors demonstrate increased expression of N-cadherin, it has been suggested that epithelial-mesenchymal transformation (EMT) may be involved in the development of sarcomatoid elements (21). However, what could be driving this process has escaped elucidation.To address the molecular basis for sarcomatoid elements in RCC, we performed whole exome sequencing of distinct regions of clear cell and sarcomatoid morphology from the same tumors.