Antonio Cappuccio

Research on Early-Stage Carcinogenesis: Are We Approaching Paradigm Instability?

In a recent article in the New York Times, Gina Kolata reported that a major change is occurring in the way researchers view early-stage carcinogenesis. We offer a more detailed perspective on this change and discuss implications for research funding. The prevailing paradigm for early-stage carcinogenesis is the somatic mutation theory, which states that “cancer results from an accumulation of mutations and other heritable changes in susceptible cells.” The somatic mutation theory has changed over time, and multiple variations have occurred in recent years. In the early 1900s, Theodor Boveri postulated that imperfect or irregular division of the chromosome leads to cancer, and Ernest Tyzzer first used the term somatic mutation in connection with cancer. The discovery of DNA as the genetic material and the observation that cancerous changes are transmitted from one generation of cells to the next pointed to DNA as the critical target of carcinogens. This viewpoint was so ingrained in the research community that, by 1959, future Nobel Laureate Peyton Rous wrote that “numerous workers on cancer are now content to think it [cancer] results from somatic mutations. Hence they see no other reason to seek in other directions to learn its nature.’’ Not surprisingly, in light of the comment of Peyton Rous, the seminal discovery in 1981 that DNA from human cancers introduced into mouse cells resulted in tumors and later experiments that induced human tumors in transgenic mice carrying an oncogene were widely viewed as supporting the somatic mutation theory, and there was little serious consideration of alternative explanations. Since then, an ever-growing list of somatic mutations has been associated with cancer; these are now frequently classified as either driver mutations that confer growth advantage or passenger mutations that do not. Recently, researchers have proposed variations on the somatic mutation theory that share the assumption that carcinogens directly alter the DNA structure or function in cells in the tissue from which cancer arises. These variations, which include epigenetic, chromosomal, and cancer stem-cell theories, differ in how the alteration occurs and in what types of cells are involved. Under the epigenetic theory, heritable changes in gene expression that are not caused by an alteration in the DNA sequence are postulated to contribute to carcinogenesis by increasing chromosomal instability, by reactivating transposons (sequences of DNA that move around the genome), or by loss of imprinting (ie, loss of a silenced genetic locus that leads to monoallelic gene expression). The link between epigenetics and cancer began with the observation of hypomethylation of human tumors and was followed by the identification of hypermethylated tumor-suppressor genes and inactivation of microRNA genes by DNA methylation. However, the epigenetic theory cannot explain unpredictable effects, such as an experiment in which hypomethylation led to fewer tumors than expected. According to the chromosomal theory, a carcinogen induces random aneuploidy, which slowly leads to chromosomal variations and eventual expansion of the most adaptable cells. The chromosomal theory offers an explanation for nonmutagenic carcinogens, the strong association between aneuploidy and cancer, and long latency periods. The more-or-less repeatable patterns of chromosomal changes seen particularly in hematologic cancers is consistent with this theory. According to the stem-cell theory, carcinogens induce cancers by altering those cells that possess characteristics associated with normal stem cells, such as self-renewal and generation of mature cells through differentiation. The stem-cell theory explains experimental results that only a small fraction of injected leukemia cells produce spleen colonies. Support for the stem-cell theory also comes from the apparent identification of stem cells in solid tumors; however, this conclusion was later challenged by findings of substantial genetic differences between the purported stem cells and their descendents. A common feature of the somatic mutation theory and the related epigenetic, chromosomal, and stem-cell theories of cancer is the underlying notion that cancer originates at the cellular level of biologic organization. A central problem for the somatic mutation theory and its variations derives from experiments in which various observations remain unexplained. For example, tumors arise when filters with small holes are inserted subcutaneously in mice but not when filters composed of the same material, but with large holes, are similarly inserted. Equally paradoxical is the observation that tumors arise in epithelial cells at a much higher rate than in controls when normal rat mammary epithelial cells are transplanted adjacent to stroma that had previously been exposed to a chemical carcinogen after clearing out the local epithelial cells. It has been observed that just transplanting normal cells into another (untreated but inappropriate) stromal environment (eg, testis cells to kidney capsule) is enough to induce carcinoma predictably and that, despite the abnormal phenotype of the subsequent cancer JOURNAL OF CLINICAL ONCOLOGY COMMENTS AND CONTROVERSIES VOLUME 28 NUMBER 20 JULY 1

Toll-like receptor control of glucocorticoid-induced apoptosis in human plasmacytoid predendritic cells (pDCs).

Microbial infection triggers the endogenous production of immunosuppressive glucocorticoid (GC) hormones and simultaneously activates innate immunity through toll-like receptors (TLRs). How innate immune cells integrate these 2 opposing signals in dictating immunity or tolerance to infection is not known. In this study, we show that human plasmacytoid predendritic cells (pDCs) were highly sensitive to GC-induced apoptosis. Strikingly, they were protected by microbial stimulation through TLR-7 and TLR-9, but not by microbial-independent stimuli, such as interleukin-3, granulocyte macrophage colony-stimulating factor, or CD40-ligand. This protection was dependent on TLR-induced autocrine tumor necrosis factor-α and interferon-α, which collectively increased the expression ratio between antiapoptotic genes (Bcl-2, Bcl-xL, BIRC3, CFLAR) versus proapoptotic genes (Caspase-8, BID, BAD, BAX). In particular, virus-induced Bcl-2 up-regulation was dependent on autocrine interferon-α. Using small interfering RNA technology, we demonstrated that Bcl-2 and CFLAR/c-flip were essential for TLR-induced protection of pDCs from GC-induced caspase-8-mediated apoptosis. Our results demonstrate a novel property of the TLR pathway in regulating the interface between GC and innate immunity and reveal a previously undescribed mechanism of GC resistance.

Plausibility of stromal initiation of epithelial cancers without a mutation in the epithelium: a computer simulation of morphostats

BackgroundThere is experimental evidence from animal models favoring the notion that the disruption of interactions between stroma and epithelium plays an important role in the initiation of carcinogenesis. These disrupted interactions are hypothesized to be mediated by molecules, termed morphostats, which diffuse through the tissue to determine cell phenotype and maintain tissue architecture.MethodsWe developed a computer simulation based on simple properties of cell renewal and morphostats.ResultsUnder the computer simulation, the disruption of the morphostat gradient in the stroma generated epithelial precursors of cancer without any mutation in the epithelium.ConclusionThe model is consistent with the possibility that the accumulation of genetic and epigenetic changes found in tumors could arise after the formation of a founder population of aberrant cells, defined as cells that are created by low or insufficient morphostat levels and that no longer respond to morphostat concentrations. Because the model is biologically plausible, we hope that these results will stimulate further experiments.

Multiple-checkpoint inhibition of thymic stromal lymphopoietin–induced TH2 response by TH17-related cytokines

BACKGROUND The interplay between allergy and autoimmunity has been a matter of long debate. Epidemiologic studies point to a decreased frequency of allergy in patients with autoimmune diseases. However, recent studies suggest that IL-17 and related cytokines, which play a central role in autoimmunity, might also promote allergy. OBJECTIVE To address this controversy, we systematically studied the interactions between T(H)17-related cytokines and the thymic stromal lymphopoietin (TSLP)-mediated proallergic pathway. METHODS We used human primary dendritic cells (DCs), T cells, and skin explants. A novel geometric representation and multivariate ANOVA were used to analyze the T(H) cytokine profile. RESULTS We show that IL-17A specifically inhibits TSLP production but increases proinflammatory IL-8 production in human skin explants exposed to TNF-α and IL-4. This inhibitory activity was confirmed in cultured skin explants of atopic dermatitis lesions. At the T-cell level, T(H)17-polarizing cytokines (IL-1β, IL-6, TGF-β, and IL-23) inhibited T(H)2 differentiation induced by TSLP-activated DCs. This led to a global dominance of a T(H)17-polarizing environment over TSLP-activated DCs, as revealed by clustering and computational analysis. CONCLUSIONS Our data indicate that T(H)17-related cytokines are negative regulators of the TSLP immune pathway. This might explain the decreased frequency of allergy in patients with autoimmunity and suggests new means of manipulating proallergic responses.

A one-mutation mathematical model can explain the age incidence of acute myeloid leukemia with mutated nucleophosmin (NPM1).

Acute myeloid leukemia with mutated NPM1 gene and aberrant cytoplasmic expression of nucleophosmin shows distinctive biological and clinical features. Based on the use of a one-mutation mathematical model, this study supports the hypothesis that a single genetic event, the NPM1 mutation, is sufficient to cause this type of leukemia. Acute myeloid leukemia with mutated NPM1 gene and aberrant cytoplasmic expression of nucleophosmin (NPMc+ acute myeloid leukemia) shows distinctive biological and clinical features. Experimental evidence of the oncogenic potential of the nucleophosmin mutant is, however, still lacking, and it is unclear whether other genetic lesion(s), e.g. FLT3 internal tandem duplication, cooperate with NPM1 mutations in acute myeloid leukemia development. An analysis of age-specific incidence, together with mathematical modeling of acute myeloid leukemia epidemiology, can help to uncover the number of genetic events needed to cause leukemia. We collected data on age at diagnosis of acute myeloid leukemia patients from five European Centers in Germany, The Netherlands and Italy, and determined the age-specific incidence of AML with mutated NPM1 (a total of 1,444 cases) for each country. Linear regression of the curves representing age-specific rates of diagnosis per year showed similar slopes of about 4 on a double logarithmic scale. We then adapted a previously designed mathematical model of hematopoietic tumorigenesis to analyze the age incidence of acute myeloid leukemia with mutated NPM1 and found that a one-mutation model can explain the incidence curve of this leukemia entity. This model fits with the hypothesis that NPMc+ acute myeloid leukemia arises from an NPM1 mutation with haploinsufficiency of the wild-type NPM1 allele.

Multiscale modelling in immunology: a review

One of the greatest challenges in biomedicine is to get a unified view of observations made from the molecular up to the organism scale. Towards this goal, multiscale models have been highly instrumental in contexts such as the cardiovascular field, angiogenesis, neurosciences and tumour biology. More recently, such models are becoming an increasingly important resource to address immunological questions as well. Systematic mining of the literature in multiscale modelling led us to identify three main fields of immunological applications: host-virus interactions, inflammatory diseases and their treatment and development of multiscale simulation platforms for immunological research and for educational purposes. Here, we review the current developments in these directions, which illustrate that multiscale models can consistently integrate immunological data generated at several scales, and can be used to describe and optimize therapeutic treatments of complex immune diseases.

Combinatorial flexibility of cytokine function during human T helper cell differentiation

In an inflammatory microenvironment, multiple cytokines may act on the same target cell, creating the possibility for combinatorial interactions. How these may influence the system-level function of a given cytokine is unknown. Here we show that a single cytokine, interferon (IFN)-alpha, can generate multiple transcriptional signatures, including distinct functional modules of variable flexibility, when acting in four cytokine environments driving distinct T helper cell differentiation programs (Th0, Th1, Th2 and Th17). We provide experimental validation of a chemokine, cytokine and antiviral modules differentially induced by IFN-α in Th1, Th2 and Th17 environments. Functional impact is demonstrated for the antiviral response, with a lesser IFN-α-induced protection to HIV-1 and HIV-2 infection in a Th17 context. Our results reveal that a single cytokine can induce multiple transcriptional and functional programs in different microenvironments. This combinatorial flexibility creates a previously unrecognized diversity of responses, with potential impact on disease physiopathology and cytokine therapy.

Combinatorial code governing cellular responses to complex stimuli

Cells adapt to their environment through the integration of complex signals. Multiple signals can induce synergistic or antagonistic interactions, currently considered as homogenous behaviours. Here, we use a systematic theoretical approach to enumerate the possible interaction profiles for outputs measured in the conditions 0 (control), signals X, Y, X+Y. Combinatorial analysis reveals 82 possible interaction profiles, which we biologically and mathematically grouped into five positive and five negative interaction modes. To experimentally validate their use in living cells, we apply an original computational workflow to transcriptomics data of innate immune cells integrating physiopathological signal combinations. Up to 9 of the 10 defined modes coexisted in context-dependent proportions. Each interaction mode was preferentially used in specific biological pathways, suggesting a functional role in the adaptation to multiple signals. Our work defines an exhaustive map of interaction modes for cells integrating pairs of physiopathological and pharmacological stimuli.

Systems approaches to unravel innate immune cell diversity, environmental plasticity and functional specialization.

Innate immune cells are generated through central and peripheral differentiation pathways, and receive multiple signals from tissue microenvironment. The complex interplay between immune cell state and environmental signals is crucial for the adaptation and efficient response to pathogenic threats. Here, we discuss how systems biology approaches have brought global view and high resolution to the characterization of (1) immune cell diversity, (2) phenotypic, transcriptional and functional changes in response to environmental signals, (3) integration of multiple stimuli. We will mostly focus on systems level studies in dendritic cells and macrophages. Generalization of these approaches should elucidate innate immune cell diversity and plasticity, and may be used in the human to generate hypothesis on cell filiation and novel strategies for immunotherapy.

Using Transcriptional Signatures to Assess Immune Cell Function: From Basic Mechanisms to Immune-Related Disease

Assessing human immune response remains a challenge as it involves multiple cell types in specific tissues. The use of microarray-based expression profiling as a tool for assessing the immune response has grown increasingly over the past decade. Transcriptome analyses provide investigators with a global perspective of the complex molecular and cellular events that unfold during the development of an immune response. In this review, we will detail the broad use of gene expression profiling to decipher the complexity of immune responses from disease biomarkers identification to cell activation, polarisation or functional specialisation. We will also describe how such data-driven strategies revealed the flexibility of immune function with common and specific transcriptional programme under multiple stimuli.