Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.As a genomic disease, cancer involves a series of changes in the genome, starting from primary tumors, via circulating tumor cells (CTCs), to metastases that cause the majority of mortalities (1–3). These genomic alterations include copy number variations (CNVs), single-nucleotide variations (SNVs), and insertions/deletions (INDELs). Regardless of the concentrated efforts in the past decades, the key driving genomic alterations responsible for metastases are still elusive (1).For noninvasive prognosis and diagnosis of cancer, it is desirable to monitor genomic alterations through the circulatory system. Genetic analyses of cell-free DNA fragments in peripheral blood have been reported (4–6) and recently extended to the whole-genome scale (7–9). However, it may be advantageous to analyze CTCs, as they represent intact functional cancer cells circulating in peripheral blood (10). Although previous studies have shown that CTC counting was able to predict progression and overall survival of cancer patients (11, 12), genomic analyses of CTCs could provide more pertinent information for personalized therapy (13). However, it is difficult to probe the genomic changes in DNA obtainable from the small number of captured CTCs. To meet this challenge, a single-cell whole-genome amplification (WGA) method, multiple annealing and looping-based amplification cycles (MALBAC) (14), has been developed to improve the amplification uniformity across the entire genome over previous methods (15, 16), allowing precise determination of CNVs and detection of SNVs with a low false-positive rate in a single cell. Here, we present genomic analyses of CTCs from 11 patients (SI Appendix, Table S1) with lung cancer, the leading cause of worldwide cancer-related deaths. CTCs were captured with the CellSearch platform using antibodies enrichment after fixation, further isolated with 94% specificity (Materials and Methods), and then subjected to WGA using MALBAC before next-generation sequencing.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispair-containing DNA substrates. A mixture of MutS homolog 2 (Msh2)–MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2–MutS homolog 3 mispair recognition protein could substitute for the Msh2–Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1–postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA mismatch repair (MMR) is a critical DNA repair pathway that is coupled to DNA replication in eukaryotes where it corrects misincorporation errors made during DNA replication (1–9). This pathway prevents mutations and acts to prevent the development of cancer (10, 11). MMR also contributes to gene conversion by repairing mispaired bases that occur during the formation of recombination intermediates (3, 4, 12). Finally, MMR acts to suppress recombination between divergent but homologous DNA sequences, thereby preventing the formation of genome rearrangements that can result from nonallelic homologous recombination (4, 13–15).Our knowledge of the mechanism of eukaryotic MMR comes from several general lines of investigation (3–9). Studies of bacterial MMR have provided a basic mechanistic framework for comparative studies (5). Genetic and cell-biology studies, primarily in Saccharomyces cerevisiae, have identified eukaryotic MMR genes, provided models for how their gene products define MMR pathways, and elucidated some of the details of how MMR pathways interact with replication (1–4). Reconstitution studies, primarily in human systems, have identified some of the catalytic features of eukaryotic MMR (7–9, 16, 17). Biochemical and structural studies of S. cerevisiae and human MMR proteins have provided information about the function of individual MMR proteins (6–9).In eukaryotic MMR, mispairs are bound by MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) and Msh2–MutS homolog 3 (Msh3), two partially redundant complexes of MutS-related proteins (3, 4, 18, 19). These complexes recruit a MutL-related complex, called MutL homoloh 1 (Mlh1)–postmeiotic segregation 1 (Pms1) in S. cerevisiae and Mlh1–postmeiotic segregation 2 (Pms2) in human and mouse (3, 4, 20–23). The Mlh1–Pms1/Pms2 complex has an endonuclease activity suggested to play a role in the initiation of the excision step of MMR (24, 25). Downstream of mismatch recognition is a mispair excision step that can be catalyzed by Exonuclease 1 (Exo1) (26–28); however, defects in both S. cerevisiae and mouse Exo1 result in only a partial MMR deficiency, suggesting the existence of additional excision mechanisms (26, 27, 29). DNA polymerase δ, the single-strand DNA binding protein replication protein A (RPA), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC) are also required for MMR at different steps, including activation of Mlh1–Pms1/Pms2, stimulation of Exo1, potentially in Exo1-independent mispair excision, and in the gap-filling resynthesis steps of MMR (3, 16, 17, 24, 27, 30–36). Although much is known about these core MMR proteins, it is not well understood how eukaryotic MMR is coupled to DNA replication (1, 2), how excision is targeted to the newly replicated strand (1, 25, 37–39), or how different MMR mechanisms such as Exo1-dependent and -independent subpathways are selected or how many such subpathways exist (1, 24, 27, 29).S. cerevisiae has provided a number of tools for studying MMR, including forward genetic screens for mutations affecting MMR, including dominant and separation-of-function mutations, the ability to evaluate structure-based mutations in vivo, cell biological tools for visualizing and analyzing MMR proteins in vivo, and overproduction of individual MMR proteins for biochemical analysis. However, linking these tools with biochemical systems that catalyze MMR reactions in vitro for mechanistic studies has not yet been possible. Here, we describe the development of MMR reactions reconstituted using purified proteins for the analysis of MMR mechanisms.     

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.Solid tumors represent a complex mass of tissue composed of multiple distinct cell types (1, 2). Cells within the tumor produce a range of soluble factors to create a complex of signaling networks within the tumor microenvironment (3–7). One of the outcomes of this crosstalk is tumor-promoting inflammation (TPI) (8, 9). TPI can modulate the functions of tumor-infiltrating myeloid lineage cells including macrophages (10–12). Tumor-associated macrophages (TAMs) consistently display an alternatively activated phenotype (M2) commonly found in sites of wound healing (13–18). These macrophages promote tumor growth while suppressing the host immune response locally (19–22). Polarization and subversion of tumor-infiltrating macrophages is accomplished via immune mediators in the tumor microenvironment (23, 24). Adding to the complexity of solid tumors is the heterogeneity of the cancer cells (2). Tumor cells of varying differentiation states and different characteristics coexist within a tumor (25–29). However, the different roles of each tumor cell subset during cancer progression remain undefined.Bladder cancer (BC) represents a growing number of solid tumors characterized by the infiltration of a significant number of myeloid cells in the neoplastic lesion (30, 31). We have previously determined that keratin 14 (KRT14) expression marks the most primitive differentiation state in BC cells (32). KRT14 expression is significantly associated with poor overall patient survival. However, the mechanisms used by KRT14-expressing cells to promote tumor growth remain unclear. In the current study, we found that KRT14+ basal BC cells also express higher levels of CD14. Here, we investigate the strategies used by KRT14+ CD14-high BC cells to promote tumor growth.     

Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients

Circulating tumor cells (CTCs) are promising biomarkers for diagnosis and therapy in systemic cancer. However, their infrequent and unreliable detection, especially in nonmetastatic cancer, currently impedes the clinical use of CTCs. Because leukapheresis (LA) targets peripheral blood mononuclear cells, which have a similar density to CTCs, and usually involves processing the whole circulating blood, we tested whether LA could substantially increase CTC detection in operable cancer patients. Therefore, we screened LA products generated from up to 25 L of blood per patient in two independent studies, and found that CTCs can be detected in more than 90% of nonmetastatic breast cancer patients. Interestingly, complete white blood cell sampling enabled determining an upper level for total CTC numbers of about 100,000 cells (median, 7,500 CTCs) per patient and identified a correlation of CTC numbers with anatomic disease spread. We further show that diagnostic leukapheresis can be easily combined with the US Food and Drug Administration-approved CellSearch system for standardized enumeration of CTCs. Direct comparison with 7.5 mL of blood revealed a significantly higher CTC frequency in matched LA samples. Finally, genomic single-cell profiling disclosed highly aberrant CTCs as therapy-escaping variants in breast cancer. In conclusion, LA is a clinically safe method that enabled a reliable detection of CTCs at high frequency even in nonmetastatic cancer patients, and might facilitate the routine clinical use of CTCs as in the sense of a liquid biopsy. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring of early systemic cancer.Circulating tumor cells (CTCs) hold promise as relevant biomarkers for early detection of systemic cancer spread, surrogate markers for therapy monitoring, and for direct access to the molecular characteristics of early and advanced metastatic disease (1, 2). However, in the past 20 y, their infrequent and unreliable detection has prevented clinical routine use of CTCs. The search for CTCs in 1–10 mL of peripheral blood by various methods has generated controversial results. For example, CTCs were detected in nonmetastatic patients [Union for International Cancer Control (UICC) stage M0 (nonmetastatic)] between 1 and 100,000 cells per mL of blood in 5–100% of patients (3–8). Although reliable methods for single-cell genomics have been available for more than 10 y (9), most of the studies did not provide direct genetic proof of malignancy for the detected cells, raising doubts about some of the reported results. Successful experiments using immunomagnetic enrichment of cancer cells expressing the membranous epithelial cell adhesion molecule (EpCAM) protein (10) led to the development of the US Food and Drug Administration (FDA)-approved CellSearch system, which has become the most widely used standard for CTC detection (11). With this method, baseline CTC counts (≥3 or ≥5 CTCs per 7.5-mL blood sample, depending on the tumor type) are observed in 26–49% of patients with metastatic cancer (12). The detection rate is even lower in nonmetastatic cancer (5–24%), usually with a median count of only one CTC per 7.5 mL (5, 9, 11, 13). Although 20–30 mL of blood was used in some CellSearch studies in nonmetastatic cancer (13–15) and more recently developed CTC detection devices claimed higher detection rates (16), all available systems failed to detect CTCs at an acceptable rate and yield in a large fraction of patients, especially in M0 cancer patients. Therefore, the clinical use of CTCs is currently limited.We reasoned that this problem could be overcome by substantially increasing the analyzed peripheral blood (PB) volume. To this end, leukapheresis (LA) is a standard clinical method that is frequently used to isolate mononuclear cells (MNCs) from blood for various applications including stem cell harvest. LA enables the extracorporeal continuous density-based cell separation of MNCs with a density of 1.055–1.08 g/mL from several liters of total processed blood (TPB) volume. Because one to three blood volumes are commonly processed in adults and because the density of epithelial cells falls in the optimal range of LA, we reasoned that CTCs could be collected together with MNCs and enriched in LA products. The high processed blood volume would then reduce sampling error of venipuncture. To test our hypothesis, we analyzed two independent LA sample sets for CTC prevalence: a historical sample collective from M0 breast cancer patients, and LA samples from a prospective validation study in gastrointestinal cancer and breast cancer of different disease stages.     

Emergence of spatial structure in the tumor microenvironment due to the Warburg effect

Drastic metabolic alterations, such as the Warburg effect, are found in most if not all types of malignant tumors. Emerging evidence shows that cancer cells benefit from these alterations, but little is known about how they affect noncancerous stromal cells within the tumor microenvironment. Here we show that cancer cells are better adapted to metabolic changes in the microenvironment, leading to the emergence of spatial structure. A clear example of tumor spatial structure is the localization of tumor-associated macrophages (TAMs), one of the most common stromal cell types found in tumors. TAMs are enriched in well-perfused areas, such as perivascular and cortical regions, where they are known to potentiate tumor growth and invasion. However, the mechanisms of TAM localization are not completely understood. Computational modeling predicts that gradients—of nutrients, gases, and metabolic by-products such as lactate—emerge due to altered cell metabolism within poorly perfused tumors, creating ischemic regions of the tumor microenvironment where TAMs struggle to survive. We tested our modeling prediction in a coculture system that mimics the tumor microenvironment. Using this experimental approach, we showed that a combination of metabolite gradients and differential sensitivity to lactic acid is sufficient for the emergence of macrophage localization patterns in vitro. This suggests that cancer metabolic changes create a microenvironment where tumor cells thrive over other cells. Understanding differences in tumor-stroma sensitivity to these alterations may open therapeutic avenues against cancer.Cancer cells in tumors display pronounced metabolic alterations (1–10). The genetic and biochemical mechanisms behind these changes are under intensive investigation, but the question of how metabolic changes affect noncancerous cells in the tumor microenvironment remains largely unanswered. The Warburg effect—or oxidative glycolysis, a process whereby cells exhibit a high glycolytic rate even in the presence of oxygen—is arguably the best-known metabolic alteration in cancer (1). Due to a lower yield of glucose to ATP associated with glycolysis, the Warburg effect was initially viewed as a detrimental aberration (1, 5). However, it is now clear that ATP is not a limiting resource for cell growth (4, 9) and that glycolytic alterations increase glucose and glutamine uptake, enhance reductive power, and favor anabolism by retaining carbon-rich macromolecules (4, 7, 9). Thus, rather than being detrimental, metabolic alterations in tumor cells can be required to sustain the high proliferation rate that characterizes malignant cancers (4, 7, 9). In fact, similar metabolic changes occur in healthy processes with rapid population growth such as pluripotent stem-cell proliferation (11), T-cell activation (12), embryonic development (13), and wound healing (14), suggesting that cancer cells have co-opted conserved metabolic processes used by rapidly proliferating cells (4, 7, 9).Despite their beneficial effect for cell proliferation, metabolic changes have dramatic consequences on the extracellular milieu. Alterations in tumor metabolism were first identified by studying how cancer cells alter their culture media (1, 5). Chaotic vascularization can be a feature in tumors in vivo, which intensifies the effect of cancer cells on their microenvironment and causes damaging processes such as acidosis, hypoxia, and nutrient deprivation (15, 16). Thus, cancer cells must balance the benefits of an altered metabolism with its potentially toxic extracellular consequences.Cancer is a disease of clonal evolution where different cell lineages compete (17, 18). Mathematical models in the literature suggest that metabolic modifications can be advantageous for lineages competing within tumors (16, 19–21). Nonetheless, how stromal cells within tumors cope with these changes has been largely neglected. Thus, it is possible that a toxic microenvironment created by metabolic alterations may be a mechanism for cancer cells to gain a selective advantage.We focused our study on how tumor metabolism affects macrophages. Tumor-associated macrophages (TAMs) are one of the most common stromal cell types found within tumors, and their number is directly correlated with poor patient prognosis in the majority of cancers analyzed to date (22–26). TAMs are well adapted to, and recruited toward, low-oxygen-tension regions (22, 27, 28). However, TAMs in vivo are also enriched in well-perfused regions of the tumor—such as the invasive edge and perivascular areas—where they potentiate cancer progression and invasion (29–31). Other tumor-associated stromal cells, live, or even dying cancer cells are known to recruit macrophages to the tumor (32–34). Nonetheless, why resident and recruited macrophages do not infiltrate the tumor homogenously remains poorly understood. An intriguing hypothesis then is that TAMs may be precluded from poorly perfused regions because metabolic alterations generate a toxic environment where only adapted tumor cells can survive.Here we show that metabolically altered microenvironments can indeed provide cancer cells with a selective advantage. In particular, these cancer cells are more resistant than macrophages to high levels of lactic acid produced by their glycolytic metabolism. We combine computational modeling with a custom-made cell culture system that allows the emergence of spatially graded microenvironments ranging from well-perfused to ischemic regions. With this approach we show that differential sensitivity to lactic acid between cancer cells and macrophages is sufficient to generate localization patterns that resemble in vivo observations.     

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.     

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.     

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.     

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF–CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.The RAF serine/threonine protein kinase is a component of the three-tiered MAPK signaling pathway that regulates cell growth and many other essential biological processes (1, 2). In normal cells, extracellular mitogenic stimuli are transmitted to the nucleus via the receptor–RAS–RAF–MAPK cascade (2). Abnormal activation of this pathway is a central event in many human cancers and results from activating mutations in BRAF itself or in upstream factors (such as the RAS genes) (3, 4). As the RAS proteins so far are intractable pharmacologic targets (5), attention has shifted to development of small-molecule RAF inhibitors (RAFis) as antitumor therapeutic agents (6). To date, RAFi clinical efficacy has been demonstrated only for BRAF^V600E melanoma (6–8). In tumors with WT BRAF or mutant RAS, most RAFis paradoxically promote growth, at times malignant in nature (6). Moreover, BRAF mutant melanomas that are initially sensitive to RAFi rapidly become resistant by using a variety of compensatory mechanisms including RAF isoform switching and activation of other pathways including RTKs, RAS, or PI3K (9). In addition, some RAFis (e.g., vemurafenib) accelerate the occurrence of secondary squamous cell carcinomas (10).Several lines of investigation suggest that multimer formation plays an important role in RAF activation and tumor responses to RAFi (11–16). RAF dimerization-mediated signaling was first suggested by the observation that artificial dimerization activates RAF (17, 18). Next, immunoprecipitation (IP) suggested that formation of homo- and heterotypic RAF “dimers” is associated with active RAS (15, 16, 19). X-ray crystallography of the BRAF catalytic domain (CatB) identified critical residues postulated to enable CatB–CatB dimer formation (11). Mutations of these residues (e.g., R509H in BRAF and, equivalently, R401H in CRAF) profoundly diminished dimerization and kinase activity of RAF (11–13). Recently, dimerization also was implicated in RAFi-mediated activation of RAF in BRAF^WT tumors (12, 13), in acquired resistance of BRAF^V600E tumors to RAFi (14), and in the development of RAFi-induced secondary squamous carcinomas (10).Although these studies implicate RAF multimer formation in the regulation of signaling in some circumstances, they do not provide direct characterization of the nature of these multimers, nor the information about their intracellular locations. This is in a large part because of the lack of techniques with sufficient spatial and stoichiometric (protein counting) resolution to visualize RAF multimers inside an intact cell. For example, X-ray crystallography studies used purified and truncated RAF proteins (11, 20). IP measures protein–protein interactions but does not provide stoichiometric or cellular localization information of the protein complexes (12, 13).The foregoing studies underscore the need for better tools to study the RAF complexes involved in cell signaling. In the present work, we show that recently introduced single-molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM) (21, 22) can be used for direct, quantitative analysis of RAF multimer formation inside an intact mammalian cell. We first demonstrate that PALM provides sufficient spatial and stoichiometric resolution to distinguish artificial protein dimers and higher-order multimers from monomers when using a suitable fluorescent probe and combined with quantitative spatial analysis. We then apply the quantitative PALM imaging approach to study CRAF multimerization under resting and various activating conditions, including the presence of active mutant RAS, N-terminal truncation, and artificial membrane localization. Our results clearly indicate the formation and significance of RAF dimers in cell signaling. The biological and therapeutic implications of these results are discussed.