Search

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 CDK9—a 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.

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. L1CAM high cells partially overlap with LGR5 high 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 L1CAM low to an L1CAM high state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells., Competing Interests: Competing interests J.M. is a science advisor for and owns company stock in Scholar Rock.

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.

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.

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.

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 ., (©2017 American Association for Cancer Research.)

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.

In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro .

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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9(D10A) (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Summary: High bone mineral density on routine dual energy X-ray absorptiometry (DXA) may indicate an underlying skeletal dysplasia. Two hundred fifty-eight individuals with unexplained high bone mass (HBM), 236 relatives (41% with HBM) and 58 spouses were studied. Cases could not float, had mandible enlargement, extra bone, broad frames, larger shoe sizes and increased body mass index (BMI). HBM cases may harbour an underlying genetic disorder., Introduction: High bone mineral density is a sporadic incidental finding on routine DXA scanning of apparently asymptomatic individuals. Such individuals may have an underlying skeletal dysplasia, as seen in LRP5 mutations. We aimed to characterize unexplained HBM and determine the potential for an underlying skeletal dysplasia., Methods: Two hundred fifty-eight individuals with unexplained HBM (defined as L1 Z-score ≥ +3.2 plus total hip Z-score ≥ +1.2, or total hip Z-score ≥ +3.2) were recruited from 15 UK centres, by screening 335,115 DXA scans. Unexplained HBM affected 0.181% of DXA scans. Next 236 relatives were recruited of whom 94 (41%) had HBM (defined as L1 Z-score + total hip Z-score ≥ +3.2). Fifty-eight spouses were also recruited together with the unaffected relatives as controls. Phenotypes of cases and controls, obtained from clinical assessment, were compared using random-effects linear and logistic regression models, clustered by family, adjusted for confounders, including age and sex., Results: Individuals with unexplained HBM had an excess of sinking when swimming (7.11 [3.65, 13.84], p < 0.001; adjusted odds ratio with 95% confidence interval shown), mandible enlargement (4.16 [2.34, 7.39], p < 0.001), extra bone at tendon/ligament insertions (2.07 [1.13, 3.78], p = 0.018) and broad frame (3.55 [2.12, 5.95], p < 0.001). HBM cases also had a larger shoe size (mean difference 0.4 [0.1, 0.7] UK sizes, p = 0.009) and increased BMI (mean difference 2.2 [1.3, 3.1] kg/m(2), p < 0.001)., Conclusion: Individuals with unexplained HBM have an excess of clinical characteristics associated with skeletal dysplasia and their relatives are commonly affected, suggesting many may harbour an underlying genetic disorder affecting bone mass.

The pathophysiological relationship between scleroderma and malignancy remains poorly understood. Although some previous studies have demonstrated an increased malignancy risk in patients with scleroderma, others have been inconclusive. We aimed to determine if patients with scleroderma had an increased risk of malignancy compared to an age- and sex-matched local South West England population, and if there were any important differences between scleroderma patients with and without malignancy. Methods of this study are as follows. Notes were obtained on all local scleroderma patients (n = 68) locally, and those diagnosed with malignancy verified by contacting each patient's general practitioner. Expected malignancy figures were obtained from age- and sex-stratified regional prevalence data provided by the South West Cancer Intelligence Service registry. Among the patients, 22.1% with scleroderma were identified with concurrent malignancy. Affected sites were of the breast (n = 5), haematological system (n = 5), skin (n = 4), and unknown primary (n = 1). Overall, malignancy risk was found to be increased in scleroderma (RR = 3.15, 95% CI 1.77-5.20, p = 0.01). In particular, this risk was the highest for haematological malignancies (RR = 18.5, 95% CI 6-43, p = 0.03), especially for non-Hodgkin's lymphoma (RR = 25.8, 95% CI 5-75, p = 0.10). The majority of patients (86.7%) developed malignancy after the onset of scleroderma (mean = 6.9 years). Age of >70 and patients with limited scleroderma were significant risk factors for a patient with scleroderma to have a concurrent malignancy; however, no increased risk was found in patients with any particular pattern of organ involvement, cytotoxic usage or serology. To conclude, in this small patient cohort, we have found that scleroderma is associated with an increased risk of malignancy. This risk is statistically significant in patients with limited scleroderma. Patients who are elderly and those with limited disease should be closely scrutinized at follow-up appointments.

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.

Leg pain is a common presentation to the outpatient department. Bilateral calf chronic compartment syndrome is a rare cause of bilateral calf pain. Although this condition has been well documented in young athletes, it has rarely been reported in the elderly. We present the case of a 68-year-old male bodybuilder with bilateral calf chronic compartment syndrome, describe the presentation and evaluation of the condition, and provide a review of the literature herewith.

Lymphotoxin-Beta (LT-Beta) is implicated in lymphoid follicle development, production of pro-inflammatory cytokines, and can enhance the proliferation of fibroblasts and synoviocytes. The objective of this study was to investigate LT-Beta and LT-BetaReceptor (LT-BetaR) gene expression in RA patient synovium and blood samples compared with control individuals, and correlate with LT-Alpha and TNF-Alpha gene expression and disease parameters. RT-PCR was used to investigate the gene expression of LT-Beta, LT-BetaR, TNF-Alpha and LT-Alpha in the blood and synovium of RA patients and a control group of individuals. LT-Beta gene expression was significantly higher in RA patient synovium compared to control synovium (P = 0.005). There was a significant positive correlation between LT-Beta and LT-Alpha gene expression in both the synovium (P = 0.001) and blood (P = 0.002) of RA patients. LT-Beta gene expression was significantly higher in RA patient synovial samples that were inflamed to a moderately severe degree compared to those inflamed to a minimal degree (P = 0.02). Analysis of clinical variables revealed a significant positive correlation between LT-BetaR gene expression in RA patient synovium and Pain VAS Score (P = 0.01) and also HAQ Score (P = 0.01). Increased LT-Beta gene expression occurs in RA synovium and correlates with the degree of inflammation. LT-Beta may play a role in RA disease pathogenesis by contributing to a more intense inflammatory reaction in the synovium.

Objectives: Children presenting with sport related injuries (SRIs) as a result of soccer, rugby and gaelic football are frequently seen in an emergency medicine (EM) setting in Ireland. A comparison of the demographics of injuries in these three sports has however not previously been performed. The purpose of this study was to provide up-to-date data on the nature of these SRIs., Method: Data was collected retrospectively on all children (<17 years of age), injured in these three sports, presenting to an emergency medicine department over 6 months, and was entered into a database for analysis., Results: Retrospective analysis was performed on 23,000 charts, and 409 SRIs were identified over a 6-month period. None of the children reported using any form of protective gear, and 27% reported a previous presentation to the emergency department with a SRI. Most injuries were as a result of soccer (56%), with 24% occurring in gaelic football, and 20% occurring in rugby. The predominant mechanism of injury was different in each sport, in soccer-falls (38%), in gaelic football-collisions with objects (balls) (37%), and in rugby-collision with persons (55%). Although the predominant type of injury in soccer and gaelic football was a fracture, accounting for 50% and 42% of injuries, respectively, in rugby however, skin/soft tissue injuries presented more commonly, accounting for 44% of injuries. When the general site of injury was investigated, the upper limb accounted for the majority of SRIs in each sport. In the management of SRIs, oral analgesics were prescribed in 50%, however, it was observed that no use was made of topical, intramuscular or rectal analgesic routes of administration. In addition it was observed that RICE/general injury advice was given in only 27%, physiotherapy was requested in 2%, and no injury prevention advice was given to any child. Overall, 8% required admission., Conclusions: The data provided from this study may raise awareness of the nature of SRIs affecting children in each of these three sports, and may be useful in formulating much needed injury prevention strategies.

Background: Direct current cardioversion (DCC) is a method to control persistent AF, to facilitate a reduction in stroke risk. Although this is a frequently performed procedure, there are no available published data regarding its outcome in an Irish setting., Aims: To determine the short- and long-term outcome of DCC, factors predicting a successful outcome, and its safety., Methods: Data relating to each DCC were collected retrospectively from patient notes over a 6.3 year-period, and subsequently entered into a Microsoft Access database before subsequent statistical analysis., Results: Forty-five consecutive unselected patients were identified, in which 59 DCCs were performed. Sinus rhythm (SR) was achieved immediately after DCC in 54/59 (91%) patients. There was a significant positive correlation between patient body weight and the energy level required to achieve SR (p=0.0001). No thromboembolic complications were noted. After a mean follow-up time of 12 +/- 13.7months, 30/45 (67%) had maintained SR. After univariate analysis, a number of important factors predictive of maintenance of SR at follow-up were identified., Conclusion: DCC was found to be an effective method for short- and long-term control of AF, without thromboembolic complications, and patients with a favourable long-term outcome after DCC could conceivably be predicted on the basis of a methodical history, careful examination, simple investigations and pharmacological variables.

Many genes in the central region of the major histocompatibility complex (MHC) encode proteins involved in immune and inflammatory responses. In this study, we have further characterized two genes in the MHC class IV region, leucocyte-specific transcript (LST) 1 and natural cytotoxicity-triggering receptor 3 (NCR3) (also known as 1C7 and natural killer (NK)p30). The specific function of LST1 is not known, although expression analysis and functional data suggest an immunomodulatory role. The LST1 gene undergoes extensive alternative splicing, giving rise to both membrane-bound (encoded by exon 3) and soluble isoforms. The NCR3 protein is involved in NK-mediated cytotoxicity and plays a role in NK/dendritic cell crosstalk. Expression of these genes was examined, by real-time reverse transcriptase-polymerase chain reaction, in autoimmune-induced inflammation, specifically rheumatoid-arthritis-affected blood and synovium, and in response to stimulation with inflammatory mediators and bacterial agents. The expression of LST1, specifically splice variants encoding soluble isoforms and NCR3, was increased in rheumatoid-arthritis-affected blood and synovium and was associated with more severe inflammation in the synovium. Furthermore, both genes were significantly up-regulated in response to lipopolysaccharide, interferon (IFN)-gamma and bacterial infection. These findings suggest that NCR3 and soluble isoforms of LST1 may play a role in inflammatory and infectious diseases.

Unlabelled: The purpose of this study was to provide up-to-date data on the nature of sport related injury (SRI) presenting to a large emergency department in Ireland. Data were collected retrospectively on all children under 17 years of age with a SRI, presenting to the emergency department of a major teaching hospital, over a 6-month period, and entered into a Microsoft Access database. A total of 1143 SRIs were identified which had occurred over a 6-month period, from 53 different sports. There was a high proportion of humerus and back SRIs in females, and a higher proportion of falls in females. Males were more frequently involved in collisions. Children with SRI were not using protective equipment in 94% of cases. Advice regarding rest, ice, compression and elevation (RICE)/general injury advice was given to 25% of patients and regarding injury preventive measures in less than 0.1% of cases. Of children, 28% had previously attended with a SRI. We also observed a lower rate of analgesia prescription to children under age 4, compared to children of an older age, and rarity of topical analgesic prescription. Overall, 10% of SRIs required admission, with 65% of these cases needing orthopaedic intervention., Conclusion: The data provided from this study should raise awareness of the different aspects of sport related injuries affecting children, and may help to provide the impetus for suggesting direction and guidance for reducing such events.