Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.

The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.

Cytokinesis failure triggers hippo tumor suppressor pathway activation.

Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets.

Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9­a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

Pediatric Emergency Medicine Point-of-Care Ultrasound for the Diagnosis of Intussusception.

BACKGROUND: Delayed diagnosis of intussusception can lead to air enema failure and increased morbidity. There are limited studies reporting the accuracy of pediatric emergency medicine (PEM) physician point-of-care ultrasound (POCUS) in diagnosing intussusception. OBJECTIVES: The primary objective was to evaluate the accuracy of PEM POCUS in identifying ileocolic intussusception. The secondary objective was to identify factors associated with air enema failure. METHODS: This was a retrospective study of children who underwent POCUS for suspected intussusception in a pediatric emergency department between January 2001 and December 2015. Patients were included if a pediatric radiologist confirmed the POCUS examination interpretation by image review, radiology department ultrasound, or air enema. Age, symptom duration, recurrent intussusception, and location of intussusception were examined as factors for air enema failure. RESULTS: One hundred and two POCUS examinations were completed on 101 patients who met the inclusion criteria. The mean age of patients was 22 months. Of 75 patients with intussusception, 72 were detected with POCUS. PEM POCUS had a sensitivity of 96.0% (95% confidence interval [CI] 91.6-100.0%), specificity of 92.6% (95% CI 82.7-100.0%), positive predictive value of 97.3% (95% CI 93.6-100.0%), and negative predictive value of 89.3% (95% CI 77.8-100.0%). Air enema failure was associated with intussusception distal to the splenic flexure (odds ratio = 10.00 [95% CI 2.81-35.61]; p < 0.01) and age <6 months (OR = 6.83 [95% CI 1.94-24.09]; p < 0.01). CONCLUSION: PEM POCUS identifies intussusception with high sensitivity and specificity. Patients <6 months old or with intussusception distal to the splenic flexure had a higher risk of air enema failure.

Global burden of diseases, injuries, and risk factors for young people's health during 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013.

BACKGROUND: Young people's health has emerged as a neglected yet pressing issue in global development. Changing patterns of young people's health have the potential to undermine future population health as well as global economic development unless timely and effective strategies are put into place. We report the past, present, and anticipated burden of disease in young people aged 10-24 years from 1990 to 2013 using data on mortality, disability, injuries, and health risk factors. METHODS: The Global Burden of Disease Study 2013 (GBD 2013) includes annual assessments for 188 countries from 1990 to 2013, covering 306 diseases and injuries, 1233 sequelae, and 79 risk factors. We used the comparative risk assessment approach to assess how much of the burden of disease reported in a given year can be attributed to past exposure to a risk. We estimated attributable burden by comparing observed health outcomes with those that would have been observed if an alternative or counterfactual level of exposure had occurred in the past. We applied the same method to previous years to allow comparisons from 1990 to 2013. We cross-tabulated the quantiles of disability-adjusted life-years (DALYs) by quintiles of DALYs annual increase from 1990 to 2013 to show rates of DALYs increase by burden. We used the GBD 2013 hierarchy of causes that organises 306 diseases and injuries into four levels of classification. Level one distinguishes three broad categories: first, communicable, maternal, neonatal, and nutritional disorders; second, non-communicable diseases; and third, injuries. Level two has 21 mutually exclusive and collectively exhaustive categories, level three has 163 categories, and level four has 254 categories. FINDINGS: The leading causes of death in 2013 for young people aged 10-14 years were HIV/AIDS, road injuries, and drowning (25·2%), whereas transport injuries were the leading cause of death for ages 15-19 years (14·2%) and 20-24 years (15·6%). Maternal disorders were the highest cause of death for young women aged 20-24 years (17·1%) and the fourth highest for girls aged 15-19 years (11·5%) in 2013. Unsafe sex as a risk factor for DALYs increased from the 13th rank to the second for both sexes aged 15-19 years from 1990 to 2013. Alcohol misuse was the highest risk factor for DALYs (7·0% overall, 10·5% for males, and 2·7% for females) for young people aged 20-24 years, whereas drug use accounted for 2·7% (3·3% for males and 2·0% for females). The contribution of risk factors varied between and within countries. For example, for ages 20-24 years, drug use was highest in Qatar and accounted for 4·9% of DALYs, followed by 4·8% in the United Arab Emirates, whereas alcohol use was highest in Russia and accounted for 21·4%, followed by 21·0% in Belarus. Alcohol accounted for 9·0% (ranging from 4·2% in Hong Kong to 11·3% in Shandong) in China and 11·6% (ranging from 10·1% in Aguascalientes to 14·9% in Chihuahua) of DALYs in Mexico for young people aged 20-24 years. Alcohol and drug use in those aged 10-24 years had an annual rate of change of >1·0% from 1990 to 2013 and accounted for more than 3·1% of DALYs. INTERPRETATION: Our findings call for increased efforts to improve health and reduce the burden of disease and risks for diseases in later life in young people. Moreover, because of the large variations between countries in risks and burden, a global approach to improve health during this important period of life will fail unless the particularities of each country are taken into account. Finally, our results call for a strategy to overcome the financial and technical barriers to adequately capture young people's health risk factors and their determinants in health information systems. FUNDING: Bill & Melinda Gates Foundation.

Ultrasound for the Evaluation of Skin and Soft Tissue Infections.

Skin and sott tissue infections, including abscesses and cellulitides, are common problems seen by physicians. The treatment of soft tissue infections varies depending on the depth of infection or the presence of a fluid collection requiring incision and drainage. Ultrasound is a valuable tool in the evaluation of skin and soft tissue infections, enhancing our ability to diagnose an abscess cavity or deeper infection and has been shown to be more reliable than clinical exam alone. The judicious use of ultrasound allows for more appropriate patient care and management of their underlying infection. It can prevent an unnecessary procedure or identify occult abscesses that may go on to develop a more severe infection requiring hospitalization. In this article,we discuss the utility of ultrasound as a diagnostic tool for skin and soft tissue infections, techniques to optimize scanning, and the potential drawbacks of its use.

KRAS Mutants Upregulate Integrin ß4 to Promote Invasion and Metastasis in Colorectal Cancer.

KRAS mutation in colorectal cancer is associated with aggressive tumor behavior through increased invasiveness and higher rates of lung metastases, but the biological mechanisms behind these features are not fully understood. In this study, we show that KRAS-mutant colorectal cancer upregulates integrin α6ß4 through ERK/MEK signaling. Knocking-out integrin ß4 (ITGB4) specifically depleted the expression of integrin α6ß4 and this resulted in a reduction in the invasion and migration ability of the cancer cells. We also observed a reduction in the number and area of lung metastatic foci in mice that were injected with ITGB4 knockout KRAS-mutant colorectal cancer cells compared with the mice injected with ITGB4 wild-type KRAS-mutant colorectal cancer cells, while no difference was observed in liver metastases. Inhibiting integrin α6ß4 in KRAS-mutant colorectal cancer could be a potential therapeutic target to diminish the KRAS-invasive phenotype and associated pulmonary metastasis rate. IMPLICATIONS: Knocking-out ITGB4, which is overexpressed in KRAS-mutant colorectal cancer and promotes tumor aggressiveness, diminishes local invasiveness and rates of pulmonary metastasis.

Four great Asian trade collapses.

This paper introduces a new dataset of commodity-specific, bilateral import data for four large Asian economies in the interwar period: China, the Dutch East Indies, India and Japan. It uses these data to describe the interwar trade collapses in the economies concerned. These resembled the post-2008 Great Trade Collapse in some respects but not in others: they occurred along the intensive margin, imports of cars were particularly badly affected, and imports of durable goods fell by more than those of non-durables, except in China and India which were rapidly industrialising. On the other hand the import declines were geographically imbalanced, while prices were more important than quantities in driving the overall collapse.

KRAS mutant rectal cancer cells interact with surrounding fibroblasts to deplete the extracellular matrix.

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS-mt) and KRAS-wild-type (KRAS-wt) patients. We found that KRAS-mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment.

Genome-wide characterization of the SloR metalloregulome in Streptococcus mutans.

Streptococcus mutans is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the sloABCR metal ion transport operon in S. mutans. In the present study, we demonstrate that S. mutans coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the S. mutans chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional "class I" genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204 S. mutans genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of S. mutans virulence genes that are subject to transcriptional upregulation by SloR and noted that such "class II"-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating S. mutans-induced caries formation.

L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer.

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin-REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.

A rectal cancer organoid platform to study individual responses to chemoradiation.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients' tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.

A genome-wide microRNA screen identifies regulators of tetraploid cell proliferation.

Tetraploid cells, which are most commonly generated by errors in cell division, are genomically unstable and have been shown to promote tumorigenesis. Recent genomic studies have estimated that ∼40% of all solid tumors have undergone a genome-doubling event during their evolution, suggesting a significant role for tetraploidy in driving the development of human cancers. To safeguard against the deleterious effects of tetraploidy, nontransformed cells that fail mitosis and become tetraploid activate both the Hippo and p53 tumor suppressor pathways to restrain further proliferation. Tetraploid cells must therefore overcome these antiproliferative barriers to ultimately drive tumor development. However, the genetic routes through which spontaneously arising tetraploid cells adapt to regain proliferative capacity remain poorly characterized. Here, we conducted a comprehensive gain-of-function genome-wide screen to identify microRNAs (miRNAs) that are sufficient to promote the proliferation of tetraploid cells. Our screen identified 23 miRNAs whose overexpression significantly promotes tetraploid proliferation. The vast majority of these miRNAs facilitate tetraploid growth by enhancing mitogenic signaling pathways (e.g., miR-191-3p); however, we also identified several miRNAs that impair the p53/p21 pathway (e.g., miR-523-3p), and a single miRNA (miR-24-3p) that potently inactivates the Hippo pathway via down-regulation of the tumor suppressor gene NF2. Collectively, our data reveal several avenues through which tetraploid cells may regain the proliferative capacity necessary to drive tumorigenesis.

Causes of death among children aged 5-14 years in the WHO European Region: a systematic analysis for the Global Burden of Disease Study 2016.

BACKGROUND: The mortality burden in children aged 5-14 years in the WHO European Region has not been comprehensively studied. We assessed the distribution and trends of the main causes of death among children aged 5-9 years and 10-14 years from 1990 to 2016, for 51 countries in the WHO European Region. METHODS: We used data from vital registration systems, cancer registries, and police records from 1980 to 2016 to estimate cause-specific mortality using the Cause of Death Ensemble model. FINDINGS: For children aged 5-9 years, all-cause mortality rates (per 100 000 population) were estimated to be 46·3 (95% uncertainty interval [UI] 45·1-47·5) in 1990 and 19·5 (18·1-20·9) in 2016, reflecting a 58·0% (54·7-61·1) decline. For children aged 10-14 years, all-cause mortality rates (per 100 000 population) were 37·9 (37·3-38·6) in 1990 and 20·1 (18·8-21·3) in 2016, reflecting a 47·1% (43·8-50·4) decline. In 2016, we estimated 10 740 deaths (95% UI 9970-11 542) in children aged 5-9 years and 10 279 deaths (9652-10 897) in those aged 10-14 years in the WHO European Region. Injuries (road injuries, drowning, and other injuries) caused 4163 deaths (3820-4540; 38·7% of total deaths) in children aged 5-9 years and 4468 deaths (4162-4812; 43·5% of total) in those aged 10-14 years in 2016. Neoplasms caused 2161 deaths (1872-2406; 20·1% of total deaths) in children aged 5-9 years and 1943 deaths (1749-2101; 18·9% of total deaths) in those aged 10-14 years in 2016. Notable differences existed in cause-specific mortality rates between the European subregions, from a two-times difference for leukaemia to a 20-times difference for lower respiratory infections between the Commonwealth of Independent States (CIS) and EU15 (the 15 member states that had joined the European Union before May, 2004). INTERPRETATION: Marked progress has been made in reducing the mortality burden in children aged 5-14 years over the past 26 years in the WHO European Region. More deaths could be prevented, especially in CIS countries, through intervention and prevention efforts focusing on the leading causes of death, which are road injuries, drowning, and lower respiratory infections. The findings of our study could be used as a baseline to assess the effect of implementation of programmes and policies on child mortality burden. FUNDING: WHO and Bill & Melinda Gates Foundation.

Chromosome 20q Amplification Defines a Subtype of Microsatellite Stable, Left-Sided Colon Cancers with Wild-type RAS/RAF and Better Overall Survival.

Here, comprehensive analysis was performed on the molecular and clinical features of colorectal carcinoma harboring chromosome 20q amplification. Tumor and normal DNA from patients with advanced colorectal carcinoma underwent next-generation sequencing via MSK-IMPACT, and a subset of case samples was subjected to high-resolution microarray (Oncoscan). Relationships between genomic copy number and transcript expression were assessed with The Cancer Genome Atlas (TCGA) colorectal carcinoma data. Of the colorectal carcinoma patients sequenced (n = 401) with MSK-IMPACT, 148 (37%) had 20q gain, and 30 (7%) had 20q amplification. In both the MSK-IMPACT and TCGA datasets, BCL2L1 was the most frequently amplified 20q oncogene. However, SRC was the only recognized 20q oncogene with a significant inverse relationship between mRNA upregulation and RAS/RAF mutation (OR, -0.4 ± 0.2, P = 0.02). In comparison with 20q diploid colorectal carcinoma, 20q gain/amplification was associated with wild-type KRAS (P < 0.001) and BRAF (P = 0.01), microsatellite stability (P < 0.001), distal primary tumors (P < 0.001), and mutant TP53 (P < 0.001), but not stage. On multivariate analysis, longer overall survival from the date of metastasis was observed with chromosome 20q gain (P = 0.02) or amplification (P = 0.04) compared with diploid 20q.Implications: 20q amplification defines a subset of colorectal cancer patients with better overall survival from the date of metastasis, and further studies are warranted to assess whether the inhibition of 20q oncogenes, such as SRC, may benefit this subset of patients. Mol Cancer Res; 15(6); 708-13. ©2017 AACR.

Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation.

Reflections on the papal allocution concerning care for persistent vegetative state patients.

This article critically examines the recent papal allocution on patients in a persistent vegetative state with regard to the appropriate conditions for considering "reformable statements." In the first part of the article, the purpose and meaning of the allocution are assessed. O'Rourke concludes that give consideration of the individual patient's best interest, prolonging artificial nutrition and hydration is not, in every case, the best option. Although he stresses favorability for preservation of the life of the patient through artificial nutrition and hydration, costs and benefits to the patient should be weighed. Ultimately, he argues in favor of leaving the decision to the patient, his caregivers, and others immediately involved in the case.

Immunofluorescent Staining of Mouse Intestinal Stem Cells.

Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.