Embracing cancer complexity: Hallmarks of systemic disease.

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, aging, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumor-related thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene-environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes.

K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling.

K-Ras and H-Ras share identical effectors and have similar properties; however, the high degree of tumor-type specificity associated with K-Ras and H-Ras mutations suggests that they have unique roles in oncogenesis. Here, we report that oncogenic K-Ras, but not H-Ras, suppresses non-canonical Wnt/Ca(2+) signaling, an effect that contributes strongly to its tumorigenic properties. K-Ras does this by binding to calmodulin and so reducing CaMKii activity and expression of Fzd8. Restoring Fzd8 in K-Ras mutant pancreatic cells suppresses malignancy, whereas depletion of Fzd8 in H-Ras(V12)-transformed cells enhances their tumor initiating capacity. Interrupting K-Ras-calmodulin binding using genetic means or by treatment with an orally active protein kinase C (PKC)-activator, prostratin, represses tumorigenesis in K-Ras mutant pancreatic cancer cells. These findings provide an alternative way to selectively target this "undruggable" protein.

KRAS4A directly regulates hexokinase 1.

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region1. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation2. No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells3 in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen4 (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

The mutational landscapes of genetic and chemical models of Kras-driven lung cancer.

Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (Kras(LA2)). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras(LA2) model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras(LA2) model. By contrast, the Kras(LA2) tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

Inflammation and Hras signaling control epithelial-mesenchymal transition during skin tumor progression.

Epithelial-mesenchymal transition (EMT) is thought to be an important, possibly essential, component of the process of tumor dissemination and metastasis. About 20%-30% of Hras mutant mouse skin carcinomas induced by chemical initiation/promotion protocols have undergone EMT. Reduced exposure to TPA-induced chronic inflammation causes a dramatic reduction in classical papillomas and squamous cell carcinomas (SCCs), but the mice still develop highly invasive carcinomas with EMT properties, reduced levels of Hras and Egfr signaling, and frequent Ink4/Arf deletions. Deletion of Hras from the mouse germline also leads to a strong reduction in squamous tumor development, but tumors now acquire activating Kras mutations and exhibit more aggressive metastatic properties. We propose that invasive carcinomas can arise by different genetic and biological routes dependent on exposure to chronic inflammation and possibly from different target cell populations within the skin. Our data have implications for the use of inhibitors of inflammation or of Ras/Egfr pathway signaling for prevention or treatment of invasive cancers.

Fine-tuning p53 activity through C-terminal modification significantly contributes to HSC homeostasis and mouse radiosensitivity.

Cell cycle regulation in hematopoietic stem cells (HSCs) is tightly controlled during homeostasis and in response to extrinsic stress. p53, a well-known tumor suppressor and transducer of diverse stress signals, has been implicated in maintaining HSC quiescence and self-renewal. However, the mechanisms that control its activity in HSCs, and how p53 activity contributes to HSC cell cycle control, are poorly understood. Here, we use a genetically engineered mouse to show that p53 C-terminal modification is critical for controlling HSC abundance during homeostasis and HSC and progenitor proliferation after irradiation. Preventing p53 C-terminal modification renders mice exquisitely radiosensitive due to defects in HSC/progenitor proliferation, a critical determinant for restoring hematopoiesis after irradiation. We show that fine-tuning the expression levels of the cyclin-dependent kinase inhibitor p21, a p53 target gene, contributes significantly to p53-mediated effects on the hematopoietic system. These results have implications for understanding cell competition in response to stresses involved in stem cell transplantation, recovery from adverse hematologic effects of DNA-damaging cancer therapies, and development of radioprotection strategies.

Identification of Hipk2 as an essential regulator of white fat development.

Homeodomain-interacting protein kinase 2 (Hipk2) has previously been implicated in the control of several transcription factors involved in embryonic development, apoptosis, cell proliferation, and tumor development, but very little is understood about the exact mechanisms through which Hipk2 influences these processes. Analysis of gene expression in normal tissues from genetically heterogeneous mouse or human populations can reveal network motifs associated with the structural or functional components of the tissue, and may predict roles for genes of unknown function. Here we have applied this network strategy to uncover a role for the Hipk2 gene in the transcriptional system controlling adipogenesis. Both in vitro and in vivo models were used to show that knockdown or loss of Hipk2 specifically inhibits white adipose cell differentiation and tissue development. In addition, loss of Hipk2 leads to induction of pockets of multilocular brown fat-like cells in remaining white adipose depots, which express markers of brown and beige fat such as uncoupling protein 1 and transmembrane protein 26. These changes are accompanied by increased insulin sensitivity in Hipk2 knockout mice and reduced high-fat diet-induced weight gain, highlighting a potential role for this kinase in diseases such as diabetes and obesity. Our study underscores the versatility and power of a readily available tissue, such as skin, for network modeling of systemic transcriptional programs involved in multiple pathways, including lipid metabolism and adipogenesis.

Cellular reprogramming in skin cancer.

Early primitive stem cells have long been viewed as the cancer cells of origin (tumor initiating target cells) due to their intrinsic features of self-renewal and longevity. However, emerging evidence suggests a surprising capacity for normal committed cells to function as reserve stem cells upon reprogramming as a consequence of tissue damage resulting in inflammation and wound healing. This results in an alternative concept positing that tumors may originate from differentiated cells that can re-acquire stem cell properties due to genetic or epigenetic reprogramming. It is likely that both models are correct, and that a continuum of potential cells of origin exists, ranging from early primitive stem cells to committed progenitor or even terminally differentiated cells. A combination of the nature of the target cell and the specific types of gene mutations introduced determine tumor cell lineage, as well as potential for malignant conversion. Evidence from mouse skin models of carcinogenesis suggests that initiated cells at different stages within a stem cell hierarchy have varying degrees of requirement for reprogramming (e.g. inflammation stimuli), depending on their degree of differentiation. This article will present evidence in favor of these concepts that has been developed from studies of several mouse models of skin carcinogenesis.

The Trp53 delta proline (Trp53ΔP) mouse exhibits increased genome instability and susceptibility to radiation-induced, but not spontaneous, tumor development.

The tumor suppressor TP53 can initiate a plethora of anti-proliferative effects to maintain genomic integrity under conditions of genotoxic stress. The N-terminal proline-rich domain (PRD) of TP53 is important in the regulation of TP53 activity and stability. A common polymorphism at codon 72 in this region has been associated with altered cancer risk in humans. The Trp53ΔP mouse, which carries a germline homozygous deletion of a region of the PRD, does not develop spontaneous tumors in a mixed 129/Sv and C57BL/6 genetic background, but is highly susceptible to a broad range of tumor types following total body exposure to 4 Gy gamma (γ) radiation. This contrasts with the tumor spectrum in Trp53 null (-/-) mice, which mainly develop thymic lymphomas and osteosarcomas. Analysis of genomic instability in tissues and cells from Trp53ΔP mice demonstrated elevated basal levels of aneuploidy, but this is not sufficient to drive spontaneous tumorigenesis, which requires an additional DNA damage stimulus. Levels of genomic instability did not increase significantly in Trp53ΔP mice following irradiation exposure, suggesting that other radiation effects including tissue inflammation, altered metabolism or autophagy, may play an important role. The Trp53ΔP mouse is a novel model to dissect the mechanisms of tumor development induced by radiation exposure. © 2015 Wiley Periodicals, Inc.

Keratin 16 regulates innate immunity in response to epidermal barrier breach.

Mutations in the type I keratin 16 (Krt16) and its partner type II keratin 6 (Krt6a, Krt6b) cause pachyonychia congenita (PC), a disorder typified by dystrophic nails, painful hyperkeratotic calluses in glabrous skin, and lesions involving other epithelial appendages. The pathophysiology of these symptoms and its relationship to settings in which Krt16 and Krt6 are induced in response to epidermal barrier stress are poorly understood. We report that hyperkeratotic calluses arising in the glabrous skin of individuals with PC and Krt16 null mice share a gene expression signature enriched in genes involved in inflammation and innate immunity, in particular damage-associated molecular patterns. Transcriptional hyper-activation of damage-associated molecular pattern genes occurs following de novo chemical or mechanical irritation to ear skin and in spontaneously arising skin lesions in Krt16 null mice. Genome-wide expression analysis of normal mouse tail skin and benign proliferative lesions reveals a tight, context-dependent coregulation of Krt16 and Krt6 with genes involved in skin barrier maintenance and innate immunity. Our results uncover a role for Krt16 in regulating epithelial inflammation that is relevant to genodermatoses, psoriasis, and cancer and suggest a avenue for the therapeutic management of PC and related disorders.

Systems genetics analysis of cancer susceptibility: from mouse models to humans.

Genetic studies of cancer susceptibility have shown that most heritable risk cannot be explained by the main effects of common alleles. This may be due to unknown gene-gene or gene-environment interactions and the complex roles of many genes at different stages of cancer. Studies using mouse models of cancer suggest that methods that integrate genetic analysis and genomic networks with knowledge of cancer biology can help to extend our understanding of heritable cancer susceptibility.

Crosstalk between Aurora-A and p53: frequent deletion or downregulation of Aurora-A in tumors from p53 null mice.

The Aurora-A kinase gene is amplified in a subset of human tumors and in radiation-induced lymphomas from p53 heterozygous mice. Normal tissues from p53-/- mice have increased Aurora-A protein levels, but lymphomas from these mice exhibit heterozygous deletions of Aurora-A and/or reduced protein expression. A similar correlation between low p53 levels and Aurora-A gene deletions and expression is found in human breast cancer cell lines. In vitro studies using mouse embryo fibroblasts demonstrate that inhibition of Aurora-A can have either positive or negative effects on cell growth as a function of p53 status. These data have implications for the design of approaches to targeted cancer therapy involving the crosstalk between Aurora-A kinase and p53 pathways.

Genetic architecture of mouse skin inflammation and tumour susceptibility.

Germline polymorphisms in model organisms and humans influence susceptibility to complex trait diseases such as inflammation and cancer. Mice of the Mus spretus species are resistant to tumour development, and crosses between M. spretus and susceptible Mus musculus strains have been used to map locations of genetic variants that contribute to skin cancer susceptibility. We have integrated germline polymorphisms with gene expression in normal skin from a M. musculus x M. spretus backcross to generate a network view of the gene expression architecture of mouse skin. Here we demonstrate how this approach identifies expression motifs that contribute to tissue organization and biological functions related to inflammation, haematopoiesis, cell cycle control and tumour susceptibility. Motifs associated with inflammation, epidermal barrier function and proliferation are differentially regulated in backcross mice susceptible or resistant to tumour development. The intestinal stem cell marker Lgr5 is identified as a candidate master regulator of the hair follicle, and the vitamin D receptor (Vdr) is linked to coordinated control of epidermal barrier function, inflammation and tumour susceptibility.

A functional switch from lung cancer resistance to susceptibility at the Pas1 locus in Kras2LA2 mice.

Pulmonary adenoma susceptibility 1 (Pas1) is the major mouse lung cancer susceptibility locus on chromosome 6 (ref. 1). Kras2 is a common target of somatic mutation in chemically induced mouse lung tumors and is a candidate Pas1 gene. M. spretus mice (SPRET/Ei) carry a Pas1 resistance haplotype for chemically induced lung tumors. We demonstrate that the SPRET/Ei Pas1 allele is switched from resistance to susceptibility by fixation of the parental origin of the mutant Kras2 allele. This switch correlates with low expression of endogenous Kras2 in SPRET/Ei. We propose that the Pas1 modifier effect is due to Kras2, and that a sensitive balance between the expression levels of wild-type and mutant alleles determines lung tumor susceptibility. These data demonstrate that cancer predisposition should also be considered in the context of somatic events and could have major implications for the design of human association studies to identify cancer susceptibility genes.

Sequential mutations in Notch1, Fbxw7, and Tp53 in radiation-induced mouse thymic lymphomas.

T-cell acute lymphoblastic lymphomas commonly demonstrate activating Notch1 mutations as well as mutations or deletions in Fbxw7. However, because Fbxw7 targets Notch1 for degradation, genetic alterations in these genes are expected to be mutually exclusive events in lymphomagenesis. Previously, by using a radiation-induced Tp53-deficient mouse model for T-cell acute lymphoblastic lymphoma, we reported that loss of heterozygosity at the Fbxw7 locus occurs frequently in a Tp53-dependent manner. In the current study, we show that these thymic lymphomas also commonly exhibit activating Notch1 mutations in the proline-glutamic acid-serine-threonine (PEST) domain. Moreover, concurrent activating Notch1 PEST domain mutations and single-copy deletions at the Fbxw7 locus occur with high frequency in the same individual tumors, indicating that these changes are not mutually exclusive events. We further demonstrate that although Notch1 PEST domain mutations are independent of Tp53 status, they are completely abolished in mice with germline Fbxw7 haploinsufficiency. Therefore, Notch1 PEST domain mutations only occur when Fbxw7 expression levels are intact. These data suggest a temporal sequence of mutational events involving these important cancer-related genes, with Notch1 PEST domain mutations occurring first, followed by Fbxw7 deletion, and eventually by complete loss of Tp53.

Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects.

BACKGROUND: Atopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency, with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype. OBJECTIVE: We sought to identify the matted mutant gene in mice and further define whether mutations in the human gene were associated with AD. METHODS: A mouse genetics approach was used to separate the matted and Flg mutations to produce congenic single-mutant strains for genetic and immunologic analysis. Next-generation sequencing was used to identify the matted gene. Five independently recruited AD case collections were analyzed to define associations between single nucleotide polymorphisms (SNPs) in the human gene and AD. RESULTS: The matted phenotype in flaky tail mice is due to a mutation in the Tmem79/Matt gene, with no expression of the encoded protein mattrin in the skin of mutant mice. Matt(ft) mice spontaneously have dermatitis and atopy caused by a defective skin barrier, with mutant mice having systemic sensitization after cutaneous challenge with house dust mite allergens. Meta-analysis of 4,245 AD cases and 10,558 population-matched control subjects showed that a missense SNP, rs6684514, [corrected] in the human MATT gene has a small but significant association with AD. CONCLUSION: In mice mutations in Matt cause a defective skin barrier and spontaneous dermatitis and atopy. A common SNP in MATT has an association with AD in human subjects.

Mutations, Bottlenecks, and Clonal Sweeps: How Environmental Carcinogens and Genomic Changes Shape Clonal Evolution during Tumor Progression.

The transition from a single, initiated cell to a full-blown malignant tumor involves significant genomic evolution. Exposure to carcinogens-whether directly mutagenic or not-can drive progression toward malignancy, as can stochastic acquisition of cancer-promoting genetic events. Mouse models using both carcinogens and germline genetic manipulations have enabled precise inquiry into the evolutionary dynamics that take place as a tumor progresses from benign to malignant to metastatic stages. Tumor progression is characterized by changes in somatic point mutations and copy-number alterations, even though any single tumor can itself have a high or low burden of genomic alterations. Further, lineage-tracing, single-cell analyses and CRISPR barcoding have revealed the distinct clonal dynamics within benign and malignant tumors. Application of these tools in a range of mouse models can shed unique light on the patterns of clonal evolution that take place in both mouse and human tumors.