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The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway., Competing Interests: Declaration of interests All authors except C.B., E.B., K.T.D., W.-Y.L., S.J.F., B.H., M.T.D., P.-A.C., and P.L. are or were employees of Novartis Pharma AG., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies., (© 2023 Bhin et al.)

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response., (© 2023. The Author(s).)

The roof plate-specific spondin-leucine-rich repeat-containing G-protein coupled receptor 4/5 (LGR4/5)-zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation. However, its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. The current study investigated whether hepatic epithelial cell-specific loss of the Wnt/β-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice fed with normal diet (ND) or high-fat diet (HFD). Six-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis but the control mice did not. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with those in control mice. An ex vivo primary hepatocyte culture assay and a comprehensive bile acid (BA) characterization in liver, plasma, bile, and feces demonstrated that ND-fed Lgr4/5dLKO mice had impaired BA secretion, predisposing them to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in the livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Abstract: Eighty-five percent of nurses report incivility in healthcare. Promoting a culture of civility-from nursing school to the workplace-is vital to ensuring healthy learning and work environments and ultimately safe patient care. This article presents a nursing program's journey in addressing incivility among the nurse faculty and the results achieved., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The limited efficacy of immune checkpoint inhibitor treatment in triple-negative breast cancer (TNBC) patients is attributed to sparse or unresponsive tumor-infiltrating lymphocytes, but the mechanisms that lead to a therapy resistant tumor immune microenvironment are incompletely known. Here we show a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of TNBC proficient or deficient of breast cancer type 1 susceptibility gene (BRCA1), MYC overexpression dramatically decreases lymphocyte infiltration in tumors, along with immune signature remodelling. MYC-mediated suppression of inflammatory signalling induced by BRCA1/2 inactivation is confirmed in human TNBC cell lines. Moreover, MYC overexpression prevents the recruitment and activation of lymphocytes in both human and mouse TNBC co-culture models. Chromatin-immunoprecipitation-sequencing reveals that MYC, together with its co-repressor MIZ1, directly binds promoters of multiple interferon-signalling genes, resulting in their downregulation. MYC overexpression thus counters tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist via suppressing induction of interferon signalling. Together, our data reveal that MYC suppresses innate immunity and facilitates tumor immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs., (© 2022. The Author(s).)

WNT/β-catenin signaling plays pivotal roles during liver development, homeostasis, and regeneration. Likewise, its deregulation disturbs metabolic liver zonation and is responsible for the development of a large number of hepatic tumors. Liver fibrosis, which has become a major health burden for society and a hallmark of NASH, can also be promoted by WNT/β-catenin signaling. Upstream regulatory mechanisms controlling hepatic WNT/β-catenin activity may constitute targets for the development of novel therapies addressing these life-threatening conditions. The R-spondin (RSPO)-leucine-rich repeat-containing G protein-coupled receptor (LGR) 4/5-zinc and ring finger (ZNRF) 3/ring finger 43 (RNF43) module is fine-tuning WNT/β-catenin signaling in several tissues and is essential for hepatic WNT/β-catenin activity. In this review article, we recapitulate the role of the RSPO-LGR4/5-ZNRF3/RNF43 module during liver development, homeostasis, metabolic zonation, regeneration, and disease. We further discuss the controversy around LGR5 as a liver stem cell marker., (© 2022 American Association for the Study of Liver Diseases.)

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer 1 . However, clinical responses to FGFR inhibitors have remained variable 1-9 , emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening 10,11 and tumour modelling in mice 12,13 , and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2 ΔE18 ). Functional in vitro and in vivo examination of a compendium of FGFR2 ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2 ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

When locally advanced breast cancer is treated with neoadjuvant chemotherapy, the recurrence risk is significantly higher if no complete pathologic response is achieved. Identification of the underlying resistance mechanisms is essential to select treatments with maximal efficacy and minimal toxicity. Here we employed gene expression profiles derived from 317 HER2-negative treatment-naïve breast cancer biopsies of patients who underwent neoadjuvant chemotherapy, deep whole exome, and RNA-sequencing profiles of 22 matched pre- and post-treatment tumors, and treatment outcome data to identify biomarkers of response and resistance mechanisms. Molecular profiling of treatment-naïve breast cancer samples revealed that expression levels of proliferation, immune response, and extracellular matrix (ECM) organization combined predict response to chemotherapy. Triple negative patients with high proliferation, high immune response and low ECM expression had a significantly better treatment response and survival benefit (HR 0.29, 95% CI 0.10-0.85; p = 0.02), while in ER+ patients the opposite was seen (HR 4.73, 95% CI 1.51-14.8; p = 0.008). The characterization of paired pre-and post-treatment samples revealed that aberrations of known cancer genes were either only present in the pre-treatment sample (CDKN1B) or in the post-treatment sample (TP53, APC, CTNNB1). Proliferation-associated genes were frequently down-regulated in post-treatment ER+ tumors, but not in triple negative tumors. Genes involved in ECM were upregulated in the majority of post-chemotherapy samples. Genomic and transcriptomic differences between pre- and post-chemotherapy samples are common and may reveal potential mechanisms of therapy resistance. Our results show a wide range of distinct, but related mechanisms, with a prominent role for proliferation- and ECM-related genes., (© 2022. The Author(s).)

The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites., (© 2022. The Author(s).)

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target., Competing Interests: Declaration of interests G.C.M.S. is an employee and shareholder of ArtiosPharma Ltd. and of AstraZeneca PLC. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation., Competing Interests: Declaration of interests All authors except L.M.T., P.L., L.C.M., V.K., and S.P. are or were employees of Novartis Pharma AG., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types., (© 2020 The Authors.)

The defect in homologous recombination (HR) found in BRCA1-associated cancers can be therapeutically exploited by treatment with DNA-damaging agents and PARP inhibitors. We and others previously reported that BRCA1-deficient tumors are initially hypersensitive to the inhibition of topoisomerase I/II and PARP, but acquire drug resistance through restoration of HR activity by the loss of end-resection antagonists of the 53BP1/RIF1/REV7/Shieldin/CST pathway. Here, we identify radiotherapy as an acquired vulnerability of 53BP1;BRCA1-deficient cells in vitro and in vivo . In contrast to the radioresistance caused by HR restoration through BRCA1 reconstitution, HR restoration by 53BP1 pathway inactivation further increases radiosensitivity. This highlights the relevance of this pathway for the repair of radiotherapy-induced damage. Moreover, our data show that BRCA1-mutated tumors that acquire drug resistance due to BRCA1-independent HR restoration can be targeted by radiotherapy. SIGNIFICANCE: These findings uncover radiosensitivity as a novel, therapeutically viable vulnerability of BRCA1-deficient mouse mammary cells that have acquired drug resistance due to the loss of the 53BP1 pathway., (©2018 American Association for Cancer Research.)

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini 1,2 . This function of 53BP1 requires interactions with PTIP 3 and RIF1 4-9 , the latter of which recruits REV7 (also known as MAD2L2) to break sites 10,11 . How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases 12,13 , and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice., (© 2016 Annunziato et al.; Published by Cold Spring Harbor Laboratory Press.)

The cancer genomics revolution has rapidly expanded the inventory of somatic mutations characterizing human malignancies, highlighting a previously underappreciated extent of molecular variability between and within patients. Also in breast cancer, the most commonly diagnosed malignancy in women, this heterogeneity complicates the understanding of the stepwise sequence of pathogenic events and the design of effective and long-lasting target therapies. To disentangle this complexity and pinpoint which molecular perturbations are crucial to hijack the cellular machinery and lead to tumorigenesis and drug resistance, functional studies are needed in model systems that faithfully and comprehensively recapitulate all the salient aspects of their cognate human counterparts. Mouse models of breast cancer have been instrumental for the study of tumor initiation and drug response but also involve cost and time limitations that represent serious bottlenecks in translational research. To keep pace with the overwhelming amount of hypotheses that warrant in vivo testing, continuous refinement of current breast cancer models and implementation of new technologies is crucial. In this review, we summarize the current state of the art in modeling human breast cancer in mice, and we put forward our vision for future developments., (© 2016 Annunziato et al; Published by Cold Spring Harbor Laboratory Press.)

Insertional mutagenesis has been used as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. Retroviruses have been used for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using retrovirus-based approaches. Most recently, lentiviral vectors have appeared on the scene for use in cancer gene screens. Lentiviral vectors are replication-defective integrating vectors that have the advantage of being able to infect nondividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations, and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future.

Purpose: To evaluate the interobserver and intraobserver reliability of radiographic assessment of tunnel placement in anterior cruciate ligament reconstruction., Methods: Seven sports fellowship-trained orthopaedic surgeons in the Multicenter Orthopaedic Outcomes Network (MOON) group participated in the study. We prospectively enrolled 54 consecutive patients after primary anterior cruciate ligament reconstruction. Postoperative plain radiographs were obtained including a full-extension anteroposterior view of the knee, a lateral view of the knee in full extension, and a notch view at 45° of flexion (Rosenberg view). Three blinded reviewers performed 8 different radiographic measurements including those of Harner and Aglietti/Jonsson. Intraclass correlation coefficients were used to determine reliability of the measurements. Intrarater reliability was assessed by repeated measurements of a subset of 20 patient images from 1 institution, and inter-rater reliability was assessed by use of all 54 sets of films from a total of 4 institutions., Results: Intraobserver reliability for femoral measures ranged from none to substantial, with notch height having the worst results. Intraobserver reliability was moderate to almost perfect for tibial measures. Interobserver reliability ranged from slight to moderate for femoral measures. The Harner method for determining tunnel depth was more reliable than the Aglietti/Jonsson method. Interobserver reliability for tibial measures ranged from fair to substantial. The presence of metal interference screws did not improve reliability of measurements., Conclusions: Postoperative radiographs are easily obtained, but our results show that radiographic measurements are of quite variable reliability, with most of the results falling into the fair to moderate categories., (Published by Elsevier Inc.)