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Patient Derived Explants (PDEs) represent the direct culture of fragments of freshly-resected tumour tissue under conditions that retain the original architecture of the tumour. PDEs have advantages over other preclinical cancer models as platforms for predicting patient-relevant drug responses in that they preserve the tumour microenvironment and tumour heterogeneity. At endpoint, PDEs may either be processed for generation of histological sections or homogenised and processed for “omic” evaluation of biomarker expression. A significant advantage of spatial profiling is the ability to co-register drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. Spatial profiling of PDEs relies on the utilisation of robust immunostaining approaches for validated biomarkers and incorporation of appropriate image analysis methods to quantitatively and qualitatively monitor changes in biomarker expression in response to anti-cancer drugs. Automation of immunostaining and image analysis would provide a significant advantage for the drug discovery pipeline and therefore, here, we have sought to optimise digital pathology approaches. We compare three image analysis software platforms (QuPath, ImmunoRatio and VisioPharm) for evaluating Ki67 as a marker for proliferation, cleaved PARP (cPARP) as a marker for apoptosis and pan-cytokeratin (CK) as a marker for tumour areas and find that all three generate comparable data to the views of a histomorphometrist. We also show that Virtual Double Staining of sequential sections by immunohistochemistry results in imperfect section alignment such that CK-stained tumour areas are over-estimated. Finally, we demonstrate that multi-immunofluorescence combined with digital image analysis is a superior method for monitoring multiple biomarkers simultaneously in tumour and stromal areas in PDEs.

Objective To undertake a pilot study to develop a novel Patient-Derived-Explant (PDE) model system for use in endometrial cancer (EC) that is capable of monitoring differential drug responses in a pre-clinical setting. Methods Fresh tumour was obtained post-hysterectomy from 27 patients with EC. Tumours were cut into 1–3 mm3 explants that were cultured at the air-liquid interface for 16–24 h in culture media. Explants were cultured in different media conditions to optimise viability. Explants were also treated with carboplatin/paclitaxel or pembrolizumab for 24 h and processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed followed by image analysis and quantitation of biomarker expression. Results EC samples are amenable to PDE culture with preserved histological architecture and PDE viability for up to 48 h, with the addition of autologous serum in culture media facilitating EC-PDE viability. Our PDE platform provides evidence of differential drug-response to conventional chemotherapeutics and immune checkpoint inhibition, and these responses can be assessed in the context of a preserved tumour microenvironment. Conclusions Our PDE platform represents a rapid, low-cost pre-clinical model which can be easily integrated into drug development pipelines. PDE culture preserves original tumour architecture and enables evaluation of spatial relationships in the tumour microenvironment. PDE culture has the potential for personalised drug-testing in a pre-clinical setting which is increasingly important in an era of personalised medicine in the treatment of EC.

Preclinical models that can accurately predict outcomes in the clinic are much sought after in the field of cancer drug discovery and development. Existing models such as organoids and patient-derived xenografts have many advantages, but they suffer from the drawback of not contextually preserving human tumour architecture. This is a particular problem for the preclinical testing of immunotherapies, as these agents require an intact tumour human-specific microenvironment for them to be effective. In this review, we explore the potential of patient-derived explants (PDEs) for fulfilling this need. PDEs involve the ex vivo culture of fragments of freshly resected human tumours that retain the histological features of original tumours. PDE methodology for anti-cancer drug testing has been in existence for many years, but the platform has not been widely adopted in translational research facilities, despite strong evidence for its clinical predictivity. By modifying PDE endpoint analysis to include the spatial profiling of key biomarkers by using multispectral imaging, we argue that PDEs offer many advantages, including the ability to correlate drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. As such, PDEs are a powerful model of choice for cancer drug and biomarker discovery programmes.

The majority of endometrial cancers are detected early with a favourable prognosis. However, for patients with advanced disease, chemotherapy response rates and overall survival remains poor. The endometrial cancer population is typically elderly with multiple co-morbidities and aggressive cytotoxic therapy may be hazardous. Therefore, there is an urgent need to define optimal treatment strategies for advanced and recurrent disease and personalise therapy based on individual tumour and patient characteristics. Three-dimensional (3D) models that preserve the tumour microenvironment and tumour-stromal interactions are increasingly important for translational research with the advent of immunotherapy and molecularly targeted agents. 3D patient-relevant pre-clinical models in endometrial cancer include spheroids, patient-derived organoids, microfluidic systems, patient-derived xenografts and patient-derived explants. Here we present a review of available 3D modelling systems in endometrial cancers, highlighting their current use, advantages, disadvantages and applications to translational research with a focus on the power of the patient-derived explant platform.

Colorectal cancer (CRC) is the second leading cause of cancer death in the UK. Novel therapeutic prevention strategies to inhibit the development and progression of CRC would be invaluable. Potential contenders include low toxicity agents such as dietary-derived agents or repurposed drugs. However, in vitro and in vivo models used in drug development often do not take into account the heterogeneity of tumours or the tumour microenvironment. This limits translation to a clinical setting. Our objectives were to develop an ex vivo method utilizing CRC and adenoma patient-derived explants (PDEs) which facilitates screening of drugs, assessment of toxicity, and efficacy. Our aims were to use a multiplexed immunofluorescence approach to demonstrate the viability of colorectal tissue PDEs, and the ability to assess immune cell composition and interactions. Using clinically achievable concentrations of curcumin, we show a correlation between curcumin-induced tumour and stromal apoptosis (P < .001) in adenomas and cancers; higher stromal content is associated with poorer outcomes. B cell (CD20+ve) and T cell (CD3+ve) density of immune cells within tumour regions in control samples correlated with curcumin-induced tumour apoptosis (P < .001 and P < .05, respectively), suggesting curcumin-induced apoptosis is potentially predicted by baseline measures of immune cells. A decrease in distance between T cells (CD3+ve) and cytokeratin+ve cells was observed, indicating movement of T cells (CD3+ve) towards the tumour margin (P < .001); this change is consistent with an immune environment associated with improved outcomes. Concurrently, an increase in distance between T cells (CD3+ve) and B cells (CD20+ve) was detected following curcumin treatment (P < .001), which may result in a less immunosuppressive tumour milieu. The colorectal tissue PDE model offers significant potential for simultaneously assessing multiple biomarkers in response to drug exposure allowing a greater understanding of mechanisms of action and efficacy in relevant target tissues, that maintain both their structural integrity and immune cell compartments.

Malignant pleural mesothelioma (MPM) is an incurable cancer with a dismal prognosis and few effective treatment options. Nonetheless, recent positive phase III trial results for immune checkpoint blockade (ICB) in MPM herald a new dawn in the fight to advance effective treatments for this cancer. Tumor mutation burden (TMB) has been widely reported to predict ICB in other cancers, but MPM is considered a low-TMB tumor. Similarly, tumor programmed death-ligand 1 (PD-L1) expression has not been proven predictive in phase III clinical trials in MPM. Consequently, the precise mechanisms that determine response to immunotherapy in this cancer remain unknown. The present review therefore aimed to synthesize our current understanding of the tumor immune microenvironment in MPM and reflects on how specific cellular features might impact immunotherapy responses or lead to resistance. This approach will inform stratified approaches to therapy and advance immunotherapy combinations in MPM to improve clinical outcomes further.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

Purpose Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor dedifferentiation occurs, leading to RAI resistance. Digoxin has been demonstrated to restore iodide uptake capacity in vitro in poorly differentiated and anaplastic NMTC cells, termed redifferentiation. The aim of the present study was to investigate the in vivo effects of digoxin in TPO-Cre/LSL-BrafV600E mice and digoxin-treated NMTC patients. Methods Mice with thyroid cancer were subjected to 3D ultrasound for monitoring tumor growth and 124I PET/CT for measurement of intratumoral iodide uptake. Post-mortem analyses on tumor tissues comprised gene expression profiling and measurement of intratumoral autophagy activity. Through PALGA (Dutch Pathology Registry), archived tumor material was obtained from 11 non-anaplastic NMTC patients who were using digoxin. Clinical characteristics and tumor material of these patients were compared to 11 matched control NMTC patients never treated with digoxin. Results We found that in mice, tumor growth was inhibited and 124I accumulation was sustainably increased after short-course digoxin treatment. Post-mortem analyses revealed that digoxin treatment increased autophagy activity and enhanced expression of thyroid-specific genes in mouse tumors compared to vehicle-treated mice. Digoxin-treated NMTC patients exhibited significantly higher autophagy activity and a higher differentiation status as compared to matched control NMTC patients, and were associated with favourable clinical outcome. Conclusions These in vivo data support the hypothesis that digoxin may represent a repositioned adjunctive treatment modality that suppresses tumor growth and improves RAI sensitivity in patients with RAI-refractory NMTC.

The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Summary The tumor microenvironment (TME) consists of different cell types, including tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs). How these cells interact and contribute to lung carcinogenesis remains elusive. Using G12DKRAS- and V600EBRAF-driven mouse lung models, we identify the pleiotropic glycoprotein stanniocalcin-1 (STC1) as a regulator of TAM-TAF interactions. STC1 is secreted by TAFs and suppresses TAM differentiation, at least in part, by sequestering the binding of GRP94, an autocrine macrophage-differentiation-inducing factor, to its cognate scavenger receptors. The accumulation of mature TAMs in the Stc1-deficient lung leads to enhanced secretion of TGF-β1 and, thus, TAF accumulation in the TME. Consistent with the mouse data, in human lung adenocarcinoma, STC1 expression is restricted to myofibroblasts, and a significant increase of naive macrophages is detected in STC1-high compared with STC1-low cases. This work increases our understanding of lung adenocarcinoma development and suggests new approaches for therapeutic targeting of the TME., Graphical Abstract, Highlights • STC1 is expressed and secreted from TAFs • STC1 depletion results in an accumulation of mature TAMs and TAFs in mouse lung models • Fibroblast-derived STC1 binds to GRP94, preventing macrophage differentiation • STC1high human lung adenocarcinomas have increased unpolarized TAMs, The tumor microenvironment contains heterogeneous cell types, but how these cells interact to regulate tumor progression is unknown. Kamata et al. identify the fibroblast-derived, secreted glycoprotein STC1 as a paracrine regulator of tumor-associated macrophage differentiation in lung adenocarcinoma.

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong β-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

// Kristiaan Lenos 1, 2 , Jeroen A.C.M. Goos 3 , Ilona M. Vuist 1, 4 , Sjoerd H. den Uil 3, 5 , Pien M. Delis-van Diemen 3, 6 , Eric J.Th. Belt 7 , Hein B.A.C. Stockmann 5 , Herman Bril 8 , Meike de Wit 9, 6 , Beatriz Carvalho 3, 6 , Susan Giblett 10 , Catrin A. Pritchard 10 , Gerrit A. Meijer 3, 6 , Yvette van Kooyk 1 , Remond J.A. Fijneman 3, 6 , Sandra J. van Vliet 1 1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands 2 Current address: Laboratory of Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands 3 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 4 Current address: Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands 5 Department of Surgery, Spaarne Gasthuis, Haarlem, The Netherlands 6 Current address: Netherlands Cancer Institute, Amsterdam, The Netherlands 7 Department of Surgery, Erasmus Medical Center, Rotterdam, The Netherlands 8 Department of Pathology, Spaarne Gasthuis, Haarlem, The Netherlands 9 Department of Medical Oncology, VU University Medical Center Amsterdam, The Netherlands 10 Department of Biochemistry, University of Leicester, Leicester, UK Correspondence to: Sandra J. van Vliet, e-mail: s.vanvliet@vumc.nl Keywords: colorectal cancer, BRAF, glycosylation, MGL, C-type lectin Received: May 06, 2015 Accepted: June 18, 2015 Published: July 02, 2015 ABSTRACT Colorectal cancer (CRC) is the third most prevalent cancer type worldwide with a mortality rate of approximately 50%. Elevated cell-surface expression of truncated carbohydrate structures such as Tn antigen (GalNAcα-Ser/Thr) is frequently observed during tumor progression. We have previously demonstrated that the C-type lectin macrophage galactose-type lectin (MGL), expressed by human antigen presenting cells, can distinguish healthy tissue from CRC through its specific recognition of Tn antigen. Both MGL binding and oncogenic BRAF mutations have been implicated in establishing an immunosuppressive microenvironment. Here we aimed to evaluate whether MGL ligand expression has prognostic value and whether this was correlated to BRAF V600E mutation status. Using a cohort of 386 colon cancer patients we demonstrate that high MGL binding to stage III tumors is associated with poor disease-free survival, independent of microsatellite instability or adjuvant chemotherapy. In vitro studies using CRC cell lines showed an association between MGL ligand expression and the presence of BRAF V600E . Administration of specific BRAF V600E inhibitors resulted in decreased expression of MGL-binding glycans. Moreover, a positive correlation between induction of BRAF V600E and MGL binding to epithelial cells of the gastrointestinal tract was found in vivo using an inducible BRAF V600E mouse model. We conclude that the BRAF V600E mutation induces MGL ligand expression, thereby providing a direct link between oncogenic transformation and aberrant expression of immunosuppressive glycans. The strong prognostic value of MGL ligands in stage III colon cancer patients, i.e . when tumor cells disseminate to lymph nodes, further supports the putative immune evasive role of MGL ligands in metastatic disease.

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo. Core support from the MRC and grants from MRCT, CRUK and the AICR financially supported the research in J.G's laboratory. J.G. is also supported by the EMBO Young Investigator Programme

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development. These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis., Cutaneous squamous cell carcinomas is a growing problem but the driver genes causing this remain poorly defined. Here, the authors demonstrate that inactivating driver mutations in TGFBR1 and TGFBR2 occur in vemurafenib-induced and sporadic cutaneous squamous cell carcinomas.

L597VBRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline L597VBRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of L597VBraf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined L597VBraf with G12DKras and found that L597VBraf modified G12DKras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity V600EBraf mutant. Mek/Erk activation levels were comparable with those driven by V600EBraf in the double-mutant cells, and the gene expression signature was more similar to that induced by V600EBraf than G12DKras. However, unlike V600EBraf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, L597VBraf epistatically modifies the transforming effects of driver oncogenes.

Mutations of BRAF are found in ∼45% of papillary thyroid cancers and are enriched in tumors with more aggressive properties. We developed mice with a thyroid-specific knock-in of oncogenic Braf ( LSL-Braf V600E /TPO-Cre ) to explore the role of endogenous expression of this oncoprotein on tumor initiation and progression. In contrast to other Braf -induced mouse models of tumorigenesis (i.e., melanomas and lung), in which knock-in of Braf V600E induces mostly benign lesions, Braf-expressing thyrocytes become transformed and progress to invasive carcinomas with a very short latency, a process that is dampened by treatment with an allosteric MEK inhibitor. These mice also become profoundly hypothyroid due to deregulation of genes involved in thyroid hormone biosynthesis and consequently have high TSH levels. To determine whether TSH signaling cooperates with oncogenic Braf in this process, we first crossed LSL-Braf V600E /TPO-Cre with TshR knockout mice. Although oncogenic Braf was appropriately activated in thyroid follicular cells of these mice, they had a lower mitotic index and were not transformed. Thyroid-specific deletion of the Gsα gene in LSL-Braf V600E /TPO-Cre/Gnas-E1 fl/fl mice also resulted in an attenuated cancer phenotype, indicating that the cooperation of TshR with oncogenic Braf is mediated in part by cAMP signaling. Once tumors were established in mice with wild-type TshR , suppression of TSH did not revert the phenotype. These data demonstrate the key role of TSH signaling in Braf-induced papillary thyroid cancer initiation and provide experimental support for recent observations in humans pointing to a strong association between TSH levels and thyroid cancer incidence.

We show here that inducible expression of Braf(V600E) off the endogenous Braf gene in mouse melanocytes stimulates skin hyperpigmentation and the appearance of nevi harboring senescent melanocytes. Additionally, approximately 70% of Braf(V600E) mice develop melanomas that reproduce many of the cardinal histological and molecular features of human melanoma and whose cells can colonize the lungs of nude mice. We show that the tumor suppressor p16(INK4a) is not required to induce melanocyte senescence and that its loss is not required for tumor progression, although it does regulate tumor penetrance and latency. Thus, we have developed a mouse model of melanoma driven by Braf(V600E) expressed at physiological levels that reflects the genetics and pathology of the human disease.

SummaryTo investigate the role of signaling by the small GTPase Ral, we have generated mice deficient for RalGDS, a guanine nucleotide exchange factor that activates Ral. We show that RalGDS is dispensable for mouse development but plays a substantial role in Ras-induced oncogenesis. Lack of RalGDS results in reduced tumor incidence, size, and progression to malignancy in multistage skin carcinogenesis, and reduced transformation by Ras in tissue culture. RalGDS does not appear to participate in the regulation of cell proliferation, but instead controls survival of transformed cells. Experiments performed in cells isolated from skin tumors suggest that RalGDS mediates cell survival through the activation of the JNK/SAPK pathway. These studies identify RalGDS as a key component in Ras-dependent carcinogenesis in vivo.

ERK pathway activation in cells expressing wild-type BRAF is a well-reported, clinically-relevant adverse effect of the otherwise impressive response of BRAF V600E -mutated melanomas to RAF inhibitors. In this issue of Cancer Cell , Holderfield and colleagues show that RAF autoinhibition underpins this paradox, further complicating therapeutic strategies centered around RAF.

Members of the Raf family of serine/threonine protein kinases have been well studied in a variety of organisms ranging from Drosophila to humans. Three raf homologues (raf-1, B-raf, and A-raf) exist in mammals, while a single prototypic homologue exists in lower organisms. A wealth of genetic and biochemical data have indicated that Raf family members are signaling kinases that are integral components of the conserved Ras/Raf/MEK/ERK signaling cascade. Following activation by Ras-dependent mechanisms, Raf protein kinases act as mitogen-activated protein (MAP) kinase kinase kinases, which phosphorylate and activate the type 1/2 MAP kinase kinases, also known as MEK1/2. These dual-specificity threonine and tyrosine kinases in turn phosphorylate and activate the ERK1/2 members of the MAP kinase family. This highly conserved Ras/Raf/MEK/ERK pathway serves to relay signals from the extracellular membrane to cellular effectors and has a profound influence on cell fate by regulating patterns of proliferation, differentiation, apoptosis, and motility (7, 25). All three mammalian Raf proteins have been shown to bind to MEK1/2 and lead to the sequential activation of MEK and ERK in vitro (12, 24, 33). However, there is accumulating evidence that B-Raf may be the more important physiological MEK activator. When endogenous Raf isotypes were immunoprecipitated and their activities were measured by using the MEK/ERK kinase cascade assay, B-Raf was shown to have considerably stronger kinase activity than the other two isotypes (14, 16). B-Raf is known to have a higher affinity for MEK1 than Raf-1 (29) and also to have a higher level of basal activity toward its substrate (24, 26). Gene targeting studies have shown that ERK phosphorylation and activation are not disrupted in mice or mouse embryonic fibroblasts (MEFs) containing an A-raf knockout mutation (27), a raf-1 knockout mutation (14, 28), or a MEK kinase-inactive version of Raf-1 with the Y340FY341F mutation (14). However, preliminary studies of B-raf−/− MEFs have shown that ERK phosphorylation is significantly reduced, if not absent, following stimulation with epidermal growth factor (EGF) (40). The appropriate regulation of the phosphorylation status and activity of the ERKs, as imposed by the Ras/Raf/MEK pathway, is absolutely critical to maintaining cell homeostasis (39). If ERK activity is inappropriately regulated, then cell transformation can arise (4), leading to tumourigenesis (6, 13). Oncogenic Ras alleles are detected in over 30% of human cancers (3) and are thought, at least in part, to mediate their effects through the deregulation of ERK activation (8). Activating mutations of the BRAF gene were detected recently in 70% of human malignant melanomas (6), 30% of thyroid cancers (18), and 15% of colon cancers. A total of 82% of BRAF mutations encode the V599E mutant, which has basal kinase activity 12.5-fold higher than wild-type B-Raf activity and stimulates constitutive ERK phosphorylation (6). Oncogenic BRAF and RAS alleles are rarely present in the same cancer samples, but they are present in the same cancer types and are thought to transform cells in a similar way through their ability to induce constitutive ERK phosphorylation (6). While the function of ERKs has been best characterized with regard to their ability to translocate to the nucleus and phosphorylate transcription factor complexes, ERKs also have a number of cytoplasmic substrates that can influence cell growth (39), apoptosis (20), and motility (2). With regard to cell motility, Klemke et al. (19) showed that the phosphorylation of myosin light chain (MLC20) kinase (MLCK) is high in cells expressing constitutively active MEK but is reduced in cells treated with MEK inhibitors (9, 19). MLCK also contains multiple MAP kinase consensus phosphorylation sites, and both ERK1 and ERK2 are able to directly phosphorylate MLCK, leading to enhanced MLCK activity. MLCK-mediated phosphorylation of serine 19 and threonine 18 of MLC is critical in myosin function, since it promotes myosin ATPase activity and the contractility of actomyosin. Consistent with these findings, it has been shown that ERK is involved in the potentiation of force development in vascular smooth muscle, most likely through the regulation of MLCK (5). ERK-mediated MLCK or myosin potentiation may also be important for targeting active ERK to newly formed focal adhesions (9). The expression of oncogenic alleles in fibroblasts is associated with MEK/ERK-dependent disruption of the actin cytoskeleton (30, 31, 35, 42). The sustained activation of ERK induced by oncogenic Ras leads to posttranscriptional down-regulation of the expression of ROCKI and Rho kinase (ROCKII), two Rho effectors required for actin stress fiber formation (31, 35). This down-regulation leads to reduced signaling through the LIM kinase (LIMK)/cofilin pathway but is functionally restored by MEK inhibition or by the overexpression of ROCK (31, 35). Similarly, v-src-induced disruption of the actin cytoskeleton of fibroblasts is mediated by sustained MEK/ERK signaling and leads to the down-regulation of ROCK expression and dephosphorylation of the actin-depolymerizing protein cofilin (30). In order to begin to assess how deregulated B-Raf activity may contribute to the motile phenotype of normal and tumor cells, we have analyzed MEFs derived from mice bearing a null mutation for B-raf (40, 41). These B-raf−/− mice die in midgestation at embryonic day 12.5 (E12.5) due to increased levels of spontaneous apoptosis of the endothelial cell lineage. However, the phenotype of B-raf−/− MEFs derived from these mice has not been investigated to date. Our studies now show that the most profound defect is one of altered motility associated with a collapsed actin cytoskeleton. B-raf−/− cells have significantly reduced levels of ERK1/2 phosphorylation. Contrary to what might be expected from the suggested role of ERK in controlling MLCK activity (19), however, the levels of phospho-MLC are not significantly changed. Instead, we provide evidence for a role of B-Raf in regulating a ROCKII/LIMK/cofilin signaling pathway leading to actin polymerization.

A recent report has shown that activating mutations in the BRAF gene are present in a large percentage of human malignant melanomas and in a proportion of colon cancers. The vast majority of these mutations represent a single nucleotide change of T-A at nucleotide 1796 resulting in a valine to glutamic acid change at residue 599 within the activation segment of B-Raf. This exciting new discovery is the first time that a direct association between any RAF gene and human cancer has been reported. Raf proteins are also indirectly associated with cancer as effectors of activated Ras proteins, oncogenic forms of which are present in approximately one-third of all human cancers. BRAF and RAS mutations are rarely both present in the same cancers but the cancer types with BRAF mutations are similar to those with RAS mutations. This has been taken as evidence that the inappropriate regulation of the downstream ERKs (the p42/p44 MAP kinases) is a major contributing factor in the development of these cancers. Recent studies in mice with targeted mutations of the raf genes have confirmed that B-Raf is a far stronger activator of ERKs than its better studied Raf-1 homologue, even in cell types in which the protein is barely expressed. The explanation for this lies in a number of key differences in the regulation of B-Raf and Raf-1 activity. Constitutive phosphorylation of serine 445 of B-Raf leads to this protein having a higher basal kinase activity than Raf-1. Phosphorylation of threonine 598 and serine 601 within the activation loop of B-Raf at the plasma membrane also regulates its activity. The V599E mutation is thought to mimic these phosphorylations, resulting in a protein with high activity, leading to constitutive ERK activation. B-Raf now provides a critical new target to which drugs for treating malignant melanoma can be developed and, with this in mind, it is now important to gain clear insight into the biochemical properties of this relatively little characterised protein.

TRACERx, a prospective study of patients with primary non-small cell lung cancer, aims to map the genomic landscape of lung cancer by tracking clonal heterogeneity and tumour evolution from diagnosis to relapse., The importance of intratumour genetic and functional heterogeneity is increasingly recognised as a driver of cancer progression and survival outcome. Understanding how tumour clonal heterogeneity impacts upon therapeutic outcome, however, is still an area of unmet clinical and scientific need. TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]), a prospective study of patients with primary non-small cell lung cancer (NSCLC), aims to define the evolutionary trajectories of lung cancer in both space and time through multiregion and longitudinal tumour sampling and genetic analysis. By following cancers from diagnosis to relapse, tracking the evolutionary trajectories of tumours in relation to therapeutic interventions, and determining the impact of clonal heterogeneity on clinical outcomes, TRACERx may help to identify novel therapeutic targets for NSCLC and may also serve as a model applicable to other cancer types.

Mutational activation of BRAF leading to expression of the BRAFV600E oncoprotein was recently identified in a high percentage of specific hematopoietic neoplasms in monocyte/histiocyte and mature B-cell lineages. Although BRAFV600E is a driver oncoprotein and pharmacologic target in solid tumors such as melanoma, lung, and thyroid cancer, it remains unknown whether BRAFV600E is an appropriate therapeutic target in hematopoietic neoplasms. To address this critical question, we generated a mouse model expressing inducible BRAFV600E in the hematopoietic system, and evaluated the efficacy of pathway-targeted therapeutics against primary hematopoietic cells. In this model, BRAFV600E expression conferred cytokine-independent growth to monocyte/macrophage-lineage progenitors leading to aberrant in vivo and in vitro monocyte/macrophage expansion. Furthermore, transplantation of BRAFV600E-expressing bone marrow cells promoted an in vivo pathology most notable for monocytosis in hematopoietic tissues and visceral organs. In vitro analysis revealed that MAP–ERK kinase inhibition, but not RAF inhibition, effectively suppressed cytokine-independent clonal growth of monocyte/macrophage-lineage progenitors. However, combined RAF and phosphoinositide 3-kinase (PI3K) inhibition effectively inhibited cytokine-independent colony formation, suggesting autocrine PI3K pathway activation. Taken together, these results provide evidence that constitutively activated BRAFV600E drives aberrant proliferation of monocyte-lineage cells. Implications: This study supports the development of pathway-targeted therapeutics in the treatment of BRAFV600E-expressing hematopoietic neoplasms in the monocyte/histiocyte lineage. Mol Cancer Res; 11(12); 1530–41. ©2013 AACR.

The strength and duration of intracellular signalling pathway activation is a key determinant of the biological outcome of cells in response to extracellular cues. This has been particularly elucidated for the Ras/Raf/MEK [mitogen-activated growth factor/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathway with a number of studies in fibroblasts showing that sustained ERK signalling is a requirement for S-phase entry, whereas transient ERK signalling does not have this capability. A major unanswered question, however, is how a cell can sustain ERK activation, particularly when ERK-specific phosphatases are transcriptionally up-regulated by the pathway itself. A major point of ERK regulation is at the level of Raf, and, to sustain ERK activation in the presence of ERK phosphatases, sustained Raf activation is a requirement. Three Raf proteins exist in mammals, and the activity of all three is induced following growth factor stimulation of cells, but only B-Raf activity is maintained at later time points. This observation points to B-Raf as a regulator of sustained ERK activation. In the present review, we consider evidence for a link between B-Raf and sustained ERK activation, focusing on a potential role for the subcellular localization of B-Raf in this key physiological event.

Talin binds to and activates integrins and is thought to couple them to cytoskeletal actin. However, functional studies on talin have been restricted by the fact that most cells express two talin isoforms. Here we show that human umbilical vein endothelial cells (HUVEC) express only talin1, and that talin1 knockdown inhibited focal adhesion (FA) assembly preventing the cells from maintaining a spread morphology, a phenotype that was rescued by GFP-mouse talin1. Thus HUVEC offer an ideal model system in which to conduct talin structure/function studies. Talin contains an N-terminal FERM domain (comprised of F1, F2 and F3 domains) and a C-terminal flexible rod. The F3 FERM domain binds β-integrin tails, and mutations in F3 that inhibited integrin binding (W359A) or activation (L325R) severely compromised the ability of GFP-talin1 to rescue the talin1 knockdown phenotype despite the presence of a second integrin-binding site in the talin rod. The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs. The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly. Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.

SummaryWe describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS. We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK–ERK signaling. This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS. Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice. Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression. They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.PaperClip

The RAF-mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase 1/2 (RAF-MEK1/2-ERK1/2) pathway is activated in many human tumours and can protect cells against growth factor deprivation; however, most such studies have relied upon overexpression of RAF or MEK constructs that are not found in tumours. Here we show that expression of the endogenous BRAF(V600E) allele in mouse embryonic fibroblasts from conditional knock-in transgenic mice activates ERK1/2, represses the BH3-only protein BIM and protects cells from growth factor withdrawal. Human colorectal cancer (CRC) cell lines harbouring BRAF(V600E) are growth factor independent for the activation of ERK1/2 and survival. However, treatment with the MEK1/2 inhibitors U0126, PD184352 or the novel clinical candidate AZD6244 (ARRY-142886) overcomes growth factor independence, causing CRC cell death. BIM is de-phosphorylated and upregulated following MEK1/2 inhibition in all CRC cell lines studied and knockdown of BIM reduces cell death, indicating that repression of BIM is a major part of the ability of BRAF(V600E) to confer growth factor-independent survival. We conclude that a single endogenous BRAF(V600E) allele is sufficient to repress BIM and prevent death arising from growth factor withdrawal, and CRC cells with BRAF(V600E) mutations are addicted to the ERK1/2 pathway for repression of BIM and growth factor-independent survival.

Genetic linkage studies have mapped the Huntington disease (HD) mutation to the distal region of the short arm of human chromosome 4. Analysis of recombination events in this region has produced contradictory locations for HD. One possible location is in the region distal to the D4S90 marker, which is located within 300 kilobases of the telomere. Other crossover events predict a more centromeric position for HD. Here we analyze the telomeric region of 4p in detail. Cloned DNA segments were derived from this region by utilizing a radiation-induced somatic cell hybrid as a source of DNA combined with preparative pulsed-field gel electrophoresis to enrich for the telomeric fraction. Additional DNA was obtained by using the cloned segments as multiple start points for cosmid walks. This strategy proved to be an effective method for cloning 250 kilobases of DNA in the region telomeric to D4S90. Hybridization analysis with the cloned DNA did not provide any evidence for the presence of rearrangements of 100 base pairs or greater in the DNA of individuals affected with HD. We also found no change in the size or structure of the 4p telomere in these samples.

Cellular senescence is a terminal differentiation state that has been proposed to have a role in both tumour suppression and ageing. This view is supported by the fact that accumulation of senescent cells can be observed in response to oncogenic stress as well as a result of normal organismal ageing. Thus, identifying senescent cells in in vivo and in vitro has an important diagnostic and therapeutic potential. The molecular pathways involved in triggering and/or maintaining the senescent phenotype are not fully understood. As a consequence, the markers currently utilized to detect senescent cells are limited and lack specificity. In order to address this issue, we screened for plasma membrane-associated proteins that are preferentially expressed in senescent cells. We identified 107 proteins that could be potential markers of senescence and validated 10 of them (DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A and PLD3). We demonstrated that a combination of these proteins can be used to specifically recognize senescent cells in culture and in tissue samples and we developed a straightforward fluorescence-activated cell sorting-based detection approach using two of them (DEP1 and B2MG). Of note, we found that expression of several of these markers correlated with increased survival in different tumours, especially in breast cancer. Thus, our results could facilitate the study of senescence, define potential new effectors and modulators of this cellular mechanism and provide potential diagnostic and prognostic tools to be used clinically.

We have utilised genomic and EST databases to assemble the sequence of the human talin2 (TLN2) gene. Talin2 protein is similar in size and sequence to talin1 throughout its length (74% identity, 86% similarity). The major differences are in (i) the size of the genes, the TLN2 gene is >200 kb compared with approximately 30 kb for TLN1 due to a difference in intron size, although intron/exon boundaries, with the exception of two, are strictly conserved; (ii) the expression patterns, TLN1 gives rise to an approximately 8-kb mRNA which is observed in all tissues, whereas TLN2 gives rise to multiple transcripts with the highest levels in heart.

We have used differential display PCR to search for mRNAs induced by delta Raf-1:ER, an estradiol-dependent form of Raf-1 kinase. Through this approach the gene encoding heparin-binding epidermal growth factor (HB-EGF) was identified as an immediate-early transcriptional target of oncogenic Raf kinases. Activation of delta Raf-1:ER and a conditional oncogenic form of B-Raf, delta B-RAF:ER, resulted in rapid and sustained induction of HB-EGF mRNA expression and secretion of mature HB-EGF from cells. Neutralizing anti-HB-EGF antisera prevented the delayed activation of the c-Jun amino-terminal kinases that is observed in cells transformed by delta Raf-1:ER. These results demonstrate that distinct signaling pathways can cross talk via the secretion of polypeptide growth factors. Furthermore, cells transformed by oncogenic Ras, which also induced HB-EGF expression, demonstrated a marked increase in sensitivity to the cytotoxic action of diphtheria toxin, for which the membrane anchored HB-EGF precursor acts as a cell-surface receptor.

The Huntington disease (HD) mutation has been localized to human chromosome 4p16, in a 6-Mb region between the D4S10 locus and the 4p telomere. In a report by Robbins et al., a family was identified in which an affected individual failed to inherit three alleles within the 6-Mb region originating from the parental HD chromosome. To explain these results, it was suggested that the HD locus (HD) lies close to the telomere and that a recombination event took place between HD and the most telomeric marker examined, D4S90. As a test of this telomere hypothesis, we examined six members of this family, five of whom are affected with HD, for the segregation of 12 polymorphic markers from 4p16, including D4S169, which lies within 80 kb of the 4p telomere. We separated, in somatic cell hybrids, the chromosomes 4 from each family member, to determine the phase of marker alleles on each chromosome. We excluded nonpaternity by performing DNA fingerprint analyses on all six family members, and we found no evidence for chromosomal rearrangements when we used high-resolution karyotype analysis. We found that two affected siblings, including one of the patients originally described by Robbins et al., inherited alleles from the non-HD chromosome 4 of their affected parents, throughout the 6-Mb region. We found that a third affected sibling, also studied by Robbins et al., inherited alleles from the HD chromosome 4 of the affected parent, throughout the 6-Mb region. Finally, we found that a fourth sibling, who is likely affected with HD, has both a recombination event within the 6-Mb region and an additional recombination event in a more centromeric region of the short arm of chromosome 4. Our results argue against a telomeric location for HD and suggest that the HD mutation in this family is either associated with DNA predisposed to double recombination and/or gene conversion within the 6-Mb region or is in a gene that is outside this region and that is different from that mutated in most other families with HD.

A panel of over 50 hybrid cells containing varying portions of the long arm of the human X chromosome have been obtained by chromosome-mediated gene transfer (CMGT) of human chromosomes to mouse cells deficient in HPRT. This panel is used to investigate the size and integrity of transfected human chromosome fragments and also to examine the effect of including a selectable DNA plasmid in the transfection mix. Chromosomal rearrangements are found to be generated in the chromosome transfer process, and the human X centromeric region is detected in the transfected cells at an unusually high frequency. Extensive lengths of X chromosome DNA are transferred intact, suggesting potential uses of CMGT in cloning large genes and loci for which only the chromosomal map position is known.

The human Y chromosome is composed of two different parts: a pseudoautosomal region shared with the X chromosome which is responsible for sex chromosome pairing and a Y-specific part that encodes the sex determining gene1. Previously we have shown that the pseudoautosomal gene MIC2 only rarely recombines between the sex chromosomes and, based on the elevated recombination rates in the pseudoautosomal region, we predicted that this gene would lie close to the Y-specific region2. In this report we describe a test of this prediction using long-range restriction mapping techniques. We conclude that MIC2 is less than 200 kilobases (kb) away from Y-specific sequences. During these experiments we have identified an HTF island in a position consistent with the proposed location of the human sex determining gene.

A radiation-induced hybrid cell line containing 10–20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.

We have developed an X-irradiation: cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.