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Background Colorectal cancer (CRC) remains largely incurable when diagnosed at the metastatic stage. Despite some advances in precision medicine for this disease in recent years, new molecular targets, as well as prognostic/predictive markers, are highly needed. Neuroligin 1 (NLGN1) is a transmembrane protein that interacts at the synapse with the tumor suppressor adenomatous polyposis Coli (APC), which is heavily involved in the pathogenesis of CRC and is a key player in the WNT/β-catenin pathway. Methods After performing expression studies of NLGN1 on human CRC samples, in this paper we used in vitro and in vivo approaches to study CRC cells extravasation and metastasis formation capabilities. At the molecular level, the functional link between APC and NLGN1 in the cancer context was studied. Results Here we show that NLGN1 is expressed in human colorectal tumors, including clusters of aggressive migrating (budding) single tumor cells and vascular emboli. We found that NLGN1 promotes CRC cells crossing of an endothelial monolayer (i.e. Trans-Endothelial Migration or TEM) in vitro, as well as cell extravasation/lung invasion and differential organ metastatization in two mouse models. Mechanistically, NLGN1 promotes APC localization to the cell membrane and co-immunoprecipitates with some isoforms of this protein stimulates β-catenin translocation to the nucleus, upregulates mesenchymal markers and WNT target genes and induces an “EMT phenotype” in CRC cell lines Conclusions In conclusion, we have uncovered a novel modulator of CRC aggressiveness which impacts on a critical pathogenetic pathway of this disease, and may represent a novel therapeutic target, with the added benefit of carrying over substantial knowledge from the neurobiology field.

Purpose: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers. Experimental Design: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment. Results: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance. Conclusions: Subtype classification systems represent canonical “termini a quo” (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Background Patient-derived xenografts (PDX) mice models play an important role in preclinical trials and personalized medicine. Sharing data on the models is highly valuable for numerous reasons – ethical, economical, research cross validation etc. The EurOPDX Consortium was established 8 years ago to share such information and avoid duplicating efforts in developing new PDX mice models and unify approaches to support preclinical research. EurOPDX Data Portal is the unified data sharing platform adopted by the Consortium. Main body In this paper we describe the main features of the EurOPDX Data Portal (https://dataportal.europdx.eu/), its architecture and possible utilization by researchers who look for PDX mice models for their research. The Portal offers a catalogue of European models accessible on a cooperative basis. The models are searchable by metadata, and a detailed view provides molecular profiles (gene expression, mutation, copy number alteration) and treatment studies. The Portal displays the data in multiple tools (PDX Finder, cBioPortal, and GenomeCruzer in future), which are populated from a common database displaying strictly mutually consistent views. (Short) Conclusion EurOPDX Data Portal is an entry point to the EurOPDX Research Infrastructure offering PDX mice models for collaborative research, (meta)data describing their features and deep molecular data analysis according to users’ interests.

Purpose: Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need. Experimental Design: We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response. Results: Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice. Conclusions: These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, “maintenance” treatment with PARP inhibitors warrants further clinical investigation.

Patient-derived xenografts (PDXs) are obtained by transplanting fragments of a patient's tumour into immunodeficient mice. Growth and propagation of PDXs allows correlating therapeutic response in vivo with extensive, multi-dimensional molecular annotation, leading to identification of predictive biomarkers. PDXs are increasingly recognised as clinically relevant models of cancer for several reasons, of which the main is the possibility of studying the behaviour of cancer cells in a natural microenvironment, where they interact with stromal components accrued from the mouse host. PDXs maintain close similarities with the tumour of origin, in terms of tissue architecture, molecular features and response to treatments. Indeed, preclinical trials in PDXs have been shown to match and also anticipate data obtained in patients. Exploration of more complex processes like metastatic evolution and antitumour immune responses is actively pursued with PDXs, as new generations of host models emerge on the horizon.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

Semaphorins are a family of molecular signals that guide cell migration and are implicated in the regulation of cancer cells. In particular, transmembrane semaphorins are postulated to act as both ligands ("forward" mode) and signaling receptors ("reverse" mode); however, reverse semaphorin signaling in cancer is relatively less understood. Here, we identified a previously unknown function of transmembrane semaphorin 4C (Sema4C), acting in reverse mode, to elicit nonconventional TGF-β/BMP receptor activation and selective SMAD1/5 phosphorylation. Sema4C coimmunoprecipitated with TGFBRII and BMPR1, supporting its role as modifier of this pathway. Sema4C reverse signaling led to the increased abundance of ID1/3 transcriptional factors and to extensive reprogramming of gene expression, which suppressed the typical features of the epithelial-mesenchymal transition in invasive carcinoma cells. This phenotype was nevertheless coupled with burgeoning metastatic behavior in vivo, consistent with evidence that Sema4C expression correlates with metastatic progression in human breast cancers. Thus, Sema4C reverse signaling promoted SMAD1/5- and ID1/3-dependent gene expression reprogramming and phenotypic plasticity in invasive cancer cells.

BackgroundNeoantigens that arise as a consequence of tumour-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumour types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.MethodsWe performed Whole Exome Sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines,in vitroandvivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures and predicted neoantigens.ResultsThe majority of CRC models showed remarkably stable mutational and neoantigen profiles, however those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures, and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly-evolving CRC.ConclusionsThese results indicate that CRC carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

Purpose: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available. Experimental Design: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach. Results: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion. Conclusions: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.

Cancer cell dependence on activated oncogenes is therapeutically targeted, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs and prevented or even reversed the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, which are responsible for loss of responsiveness to oncogene-targeted therapies.

Summary The microenvironment influences cancer drug response and sustains resistance to therapies targeting receptor-tyrosine kinases. However, if and how the tumor microenvironment can be altered during treatment, contributing to resistance onset, is not known. We show that, under prolonged treatment with tyrosine kinase inhibitors (TKIs), EGFR- or MET-addicted cancer cells displayed a metabolic shift toward increased glycolysis and lactate production. We identified secreted lactate as the key molecule instructing cancer-associated fibroblasts to produce hepatocyte growth factor (HGF) in a nuclear factor κB-dependent manner. Increased HGF, activating MET-dependent signaling in cancer cells, sustained resistance to TKIs. Functional or pharmacological targeting of molecules involved in the lactate axis abrogated in vivo resistance, demonstrating the crucial role of this metabolite in the adaptive process. This adaptive resistance mechanism was observed in lung cancer patients progressed on EGFR TKIs, demonstrating the clinical relevance of our findings and opening novel scenarios in the challenge to drug resistance.

Purpose: Patient-derived xenografts (“xenopatients”) of colorectal cancer metastases have been essential to identify genetic determinants of resistance to the anti-EGFR antibody cetuximab and to explore new therapeutic strategies. From xenopatients, a genetically annotated collection of stem-like cultures (“xenospheres”) was generated and characterized for response to targeted therapies. Experimental Design: Xenospheres underwent exome-sequencing analysis, gene expression profile, and in vitro targeted treatments to assess genetic, biological, and pharmacologic correspondence with xenopatients, and to investigate nongenetic biomarkers of therapeutic resistance. The outcome of EGFR family inhibition was tested in an NRG1-expressing in vivo model. Results: Xenospheres faithfully retained the genetic make-up of their matched xenopatients over in vitro and in vivo passages. Frequent and rare genetic lesions triggering primary resistance to cetuximab through constitutive activation of the RAS signaling pathway were conserved, as well as the vulnerability to their respective targeted treatments. Xenospheres lacking such alterations (RASwt) were highly sensitive to cetuximab, but were protected by ligands activating the EGFR family, mostly NRG1. Upon reconstitution of NRG1 expression, xenospheres displayed increased tumorigenic potential in vivo and generated tumors completely resistant to cetuximab, and sensitive only to comprehensive EGFR family inhibition. Conclusions: Xenospheres are a reliable model to identify both genetic and nongenetic mechanisms of response and resistance to targeted therapies in colorectal cancer. In the absence of RAS pathway mutations, NRG1 and other EGFR ligands can play a major role in conferring primary cetuximab resistance, indicating that comprehensive inhibition of the EGFR family is required to achieve a significant therapeutic response. Clin Cancer Res; 24(4); 807–20. ©2017 AACR. See related commentary by Napolitano and Ciardiello, p. 727

Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-β pathway activity, epithelial–mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances., Stromal cells contribute to the gene expression profiles based on which colorectal cancer (CRC) molecular subtypes are classified. Here, patient-derived xenografts enable the authors to obtain cancer cell-specific transcriptomes by excluding transcripts from murine stromal cells, based on which they define CRC intrinsic subtypes (CRIS) and evaluate their prognostic and predictive potential.

Background The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing. Methods We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry. Associations between pevonedistat sensitivity and CRC molecular features were assessed by Student's t test. A 184-gene transcriptional predictor was generated in cell lines and applied to 87 metastatic CRC samples for which patient-derived xenografts (PDXs) were available. In vivo reponse to pevonedistat was assessed in PDX models (≥5 mice per group). All statistical tests were two-sided. Results Sixteen (13.1%) cell lines displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block, and increased apoptosis. Pevonedistat sensitivity did not statistically significantly correlate with microsatellite instability or mutations in KRAS or BRAF and was functionally associated with low EGFR pathway activity. While ineffective on predicted resistant PDXs, in vivo administration of pevonedistat statistically significantly impaired growth of five out of six predicted sensitive models (P < .01). In samples from CRC patients, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery (hazard ratio [HR] = 2.49, 95% confidence interval [CI] = 1.34 to 4.62, P = .003) and early progression under cetuximab treatment (HR = 3.59, 95% CI = 1.60 to 8.04, P < .001). Histological and immunohistochemical analyses revealed that the pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma. Conclusions These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective treatment for poorly differentiated, clinically aggressive CRC.

MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis.We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression.MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175).MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.

Only a fraction of patients with metastatic colorectal cancer receive clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) antibodies, which calls for the identification of novel biomarkers for better personalized medicine. We produced large xenograft cohorts from 85 patient-derived, genetically characterized metastatic colorectal cancer samples (“xenopatients”) to discover novel determinants of therapeutic response and new oncoprotein targets. Serially passaged tumors retained the morphologic and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or nonresponders on the basis of several predictive biomarkers. Genotype–response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multiarm study in HER2-amplified xenopatients revealed that the combined inhibition of HER2 and EGFR induced overt, long-lasting tumor regression. Our results suggest promising therapeutic opportunities in cetuximab-resistant patients with metastatic colorectal cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need. Significance: Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting. Cancer Discovery; 1(6); 508–23. ©2011 AACR. Read the Commentary on this article by Ciardiello and Normanno, p. 472 This article is highlighted in the In This Issue feature, p. 457

Purpose: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease. Experimental Design: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets. Results: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer. Conclusions: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation. Clin Cancer Res; 17(10); 3146–56. ©2011 AACR.

The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2alpha phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2alpha, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2alpha phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2alpha phosphorylation and the UPR.

Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.

Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.

Background: Bone metastases represent a common and severe complication in breast cancer, and the involvement of cancer stem cells (CSCs) in the promotion of bone metastasis is currently under discussion. Here, we used a human-in-mice model to study bone metastasis formation due to primary breast CSCs-like colonisation. Methods: Primary CD44+CD24− breast CSCs-like were transduced by a luciferase-lentiviral vector and injected through subcutaneous and intracardiac (IC) routes in non-obese/severe-combined immunodeficient (NOD/SCID) mice carrying subcutaneous human bone implants. The CSCs-like localisation was monitored by in vivo luciferase imaging. Bone metastatic CSCs-like were analysed through immunohistochemistry and flow cytometry, and gene expression analyses were performed by microarray techniques. Results: Breast CSCs-like colonised the human-implanted bone, resulting in bone remodelling. Bone metastatic lesions were histologically apparent by tumour cell expression of epithelial markers and vimentin. The bone-isolated CSCs-like were CD44−CD24+ and showed tumorigenic abilities after injection in secondary mice. CD44−CD24+ CSCs-like displayed a distinct bone tropism signature that was enriched in genes that discriminate bone metastases of breast cancer from metastases at other organs. Conclusion: Breast CSCs-like promote bone metastasis and display a CSCs-like bone tropism signature. This signature has clinical prognostic relevance, because it efficiently discriminates osteotropic breast cancers from tumour metastases at other sites.

KRAS mutations are present at a high frequency in human cancers. The development of therapies targeting mutated KRAS requires cellular and animal preclinical models. We exploited adeno-associated virus–mediated homologous recombination to insert the Kras G12D allele in the genome of mouse somatic cells. Heterozygous mutant cells displayed a constitutively active Kras protein, marked morphologic changes, increased proliferation and motility but were not transformed. On the contrary, mouse cells in which we overexpressed the corresponding Kras cDNA were readily transformed. The levels of Kras activation in knock-in cells were comparable with those present in human cancer cells carrying the corresponding mutation. Kras-mutated cells were compared with their wild-type counterparts by gene expression profiling, leading to the definition of a “mutated Kras-KI signature” of 345 genes. This signature was capable of classifying mouse and human cancers according to their KRAS mutational status, with an accuracy similar to or better than published Ras signatures. The isogenic cells that we have developed recapitulate the oncogenic activation of KRAS occurring in cancer and represent new models for studying Kras-mediated transformation. Our results have implications for the identification of human tumors in which the oncogenic KRAS transcriptional response is activated and suggest new strategies to build mouse models of tumor progression. [Cancer Res 2007;67(18):8468–76]

Prior data suggest pre-diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple-negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes.NOD/SCID mice were implanted with HER2+ MDA-MB-231/LN/2-4/H2N, trastuzumab-resistant HER2+ HCC1954 or a TNBC patient-derived xenograft (PDX). A daily low-dose aspirin regimen commenced until primary tumours reached ~250 mmAspirin delayed time to metastasis in MDA-MB-231/LN/2-4/H2N xenografts and decreased growth of HER2Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo-preventative agent in the HER2

Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying MET amplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naive GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other MET-addicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.

Background Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed. Results We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach. Conclusion The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning.