IGF2 Overexpression Research Articles

Therapeutic Targets for Adrenocortical Carcinoma in the Genomics Era

Adrenocortical carcinoma (ACC) is a rare and often fatal cancer, affecting ~1 person per million per year worldwide. Approximately 75% of patients with ACC eventually develop metastases and progress on the few available standard-of-care medical therapies, highlighting an incredible need for an improved understanding of the molecular biology of this disease. Although it has long been known that ACC is characterized by certain histological and genetic features (e.g., high mitotic activity, chromosomal instability, and overexpression of IGF2), only in the last two decades of genomics has the molecular landscape of ACC been more thoroughly characterized. In this review, we describe the findings of historical genetics and recent genomics studies on ACC and discuss how underlying concepts emerging from these studies contribute to the current model of critical pathways for adrenocortical carcinogenesis. Integrative synthesis across these studies reveals that ACC consists of three distinct molecular subtypes with divergent clinical outcomes and implicates differential regulation of Wnt signaling, cell cycle, DNA methylation, immune biology, and steroidogenesis in ACC biology. These cellular programs are pharmacologically targetable and may enable the development of therapeutic strategies to improve outcomes for patients facing this devastating disease.

The role of insulin-like growth factor system in the adrenocortical tumors.

The different presentation of adrenocortical tumors in benign adenoma (ACA) or adrenocortical carcinoma (ACC) is related to the variability at the molecular level. The insulin-like growth factor (IGF) system is one of the most frequently altered pathways in ACC. In this review we will critically analyze the evidence regarding the pathogenic role of the IGF system in adrenal tumorigenesis, focusing on ACC. We will also examine the preclinical and clinical studies which investigated the targeting of the IGF system as a therapeutic approach in ACC. The IGF system plays a crucial role in the embryogenesis of adrenal glands. No significant alterations of the IGF system were observed in ACA. In ACC, the IGF2 overexpression is one of the most frequent molecular change presented in more than 85% of cases. However, IGF2 seems to be only a tumor progression factor which requires additional hits to trigger adrenal tumorigenesis. Also, the IGF1 receptor (IGF1R) appears to be higher expressed in ACC. Many IGF1R target-drugs have been developed to inhibit the activation of the IGF system. Preclinical studies using antibody or tyrosine kinase which target the IGF1R, or the dual-targeting of IGF1R and insulin receptor (IR) reduced ACC cells proliferation both in vitro and in vivo in mouse xenograft model. However, these promising results were not confirmed in clinical trials. Nowadays, predictive markers for the response of target-IGF therapy are missing and further studies which investigate new molecular markers and evaluate the entire IGF receptors, including the IR, are urgently needed.

The role of epithelial growth factors and insulin growth factors in the adrenal neoplasms.

Human fetal and adult adrenal gland express both insulin growth factor-1 (IGF-1) and IGF-2, their receptors (IGF-Rs) and a variety of specific IGF binding proteins suggesting their potential role in the regulation of adrenal growth and function. IGF-2 overexpression is essential for the growth of monoclonal lesions, such as large benign adenomas (ACA) and adrenocortical carcinomas (ACC) and has been found to contribute to tumorigenesis in Beckwith-Wiedemann syndrome. IGF-2 is the most highly expressed gene observed in more than 85% of ACCs. However, no significant differences in clinical, biological and transcriptomic traits were found between tumors with high and low expression of IGF-2. On the contrary, the expression of IGF-1R, mediating the IGF-2 effects in vivo, was more discriminant between malignant (overexpression) and benign tumors. Data on the role of epithelial growth factor (EGF) and its receptor (EGF-R) in adrenocortical tumorigenesis are controversial. Several studies have shown EGF-R overexpression in ACCs but not in benign ACAs, suggesting that EGF-R could potentially be used as a marker for the differential diagnosis of ACAs and ACCs. Although, in vitro and animal studies provide promising results in the therapeutic role of IGF and EGF pathway inhibitors, the available data in humans are still not encouraging. Herein, we aim to present recent data on the role of IGF and EGF pathways in adrenal development and tumorigenesis and their potential implication in the treatment of the ACC, a rare malignancy with very poor prognosis.

Loss of imprinting mutations define both distinct and overlapping roles for misexpression of IGF2 and of H19 lncRNA.

Imprinted genes occur in discrete clusters that are coordinately regulated by shared DNA elements called Imprinting Control Regions. H19 and Igf2 are linked imprinted genes that play critical roles in development. Loss of imprinting (LOI) at the IGF2/H19 locus on the maternal chromosome is associated with the developmental disorder Beckwith Wiedemann Syndrome (BWS) and with several cancers. Here we use comprehensive genetic and genomic analyses to follow muscle development in a mouse model of BWS to dissect the separate and shared roles for misexpression of Igf2 and H19 in the disease phenotype. We show that LOI results in defects in muscle differentiation and hypertrophy and identify primary downstream targets: Igf2 overexpression results in over-activation of MAPK signaling while loss of H19 lncRNA prevents normal down regulation of p53 activity and therefore results in reduced AKT/mTOR signaling. Moreover, we demonstrate instances where H19 and Igf2 misexpression work separately, cooperatively, and antagonistically to establish the developmental phenotype. This study thus identifies new biochemical roles for the H19 lncRNA and underscores that LOI phenotypes are multigenic so that complex interactions will contribute to disease outcomes.

The IGF1R/INSR Inhibitor BI 885578 Selectively Inhibits Growth of IGF2-Overexpressing Colorectal Cancer Tumors and Potentiates the Efficacy of Anti-VEGF Therapy.

Clinical studies of pharmacologic agents targeting the insulin-like growth factor (IGF) pathway in unselected cancer patients have so far demonstrated modest efficacy outcomes, with objective responses being rare. As such, the identification of selection biomarkers for enrichment of potential responders represents a high priority for future trials of these agents. Several reports have described high IGF2 expression in a subset of colorectal cancers, with focal IGF2 amplification being responsible for some of these cases. We defined a novel cut-off value for IGF2 overexpression based on differential expression between colorectal tumors and normal tissue samples. Analysis of two independent colorectal cancer datasets revealed IGF2 to be overexpressed at a frequency of 13% to 22%. An in vitro screen of 34 colorectal cancer cell lines revealed IGF2 expression to significantly correlate with sensitivity to the IGF1R/INSR inhibitor BI 885578. Furthermore, autocrine IGF2 constitutively activated IGF1R and Akt phosphorylation, which was inhibited by BI 885578 treatment. BI 885578 significantly delayed the growth of IGF2-high colorectal cancer xenograft tumors in mice, while combination with a VEGF-A antibody increased efficacy and induced tumor regression. Besides colorectal cancer, IGF2 overexpression was detected in more than 10% of bladder carcinoma, hepatocellular carcinoma and non-small cell lung cancer patient samples. Meanwhile, IGF2-high non-colorectal cancer cells lines displayed constitutive IGF1R phosphorylation and were sensitive to BI 885578. Our findings suggest that IGF2 may represent an attractive patient selection biomarker for IGF pathway inhibitors and that combination with VEGF-targeting agents may further improve clinical outcomes. Mol Cancer Ther; 16(10); 2223-33. ©2017 AACR.

Highlights of This Issue

Using liquid chromatography/mass spectrometry (LC/MS)-based metabolomics and the MEK inhibitor RO4987655, 21 plasma metabolite markers of MEK inhibition in two sensitive BRAF mutant, human melanoma xenograft models were identified. Of these, it was found that on-treatment changes in the concentrations of 15 metabolites significantly predicted objective responses in patients with advanced melanoma treated with RO4987655. In patients with progressive disease, it was demonstrated that propionyl carnitine and amino acid levels increased; while in those patients that responded, phosphatidylcholines and sphingomyelin concentrations decreased. In addition, the pre-treatment levels of seven plasma metabolites (phosphatidylcholines and sphingomyelins) had significant prognostic value.Given the lack of meaningful clinical efficacy of pharmacological agents targeting the IGF pathway when applied in unselected cancer patients, the identification of selection biomarkers is of crucial importance. Sanderson and colleagues report that autocrine IGF2 overexpression hyperactivates the IGF pathway in colorectal cancer (CRC) and other cancers and predicts for sensitivity to the IGF1R/INSR inhibitor BI 885578, either as monotherapy or in combination with VEGF-pathway inhibition. Coupled with the identification of substantial subsets of IGF2-overexpressing patients in diverse tumor types, these findings support the assessment of IGF2 expression for patient selection in future trials of IGF pathway inhibitors.Molecularly targeted drugs have yielded significant therapeutic advances in oncogene-driven non-small cell lung cancer (NSCLC), but most patients eventually develop acquired resistance. Here, Shien and colleagues have identified that the IL6 family cytokine oncostatin-M (OSM) induced a switch to the epithelial-to-mesenchymal phenotype and protected cells from targeted drug-induced apoptosis in OSM receptors (OSMRs)/JAK1/STAT3-dependent manner, and co-targeting inhibition of the oncogenic pathway and JAK1 revered resistance to targeted drugs. These results suggest that the OSMRs/JAK1/STAT3 axis contributes to resistance to targeted drugs in oncogene-driven NSCLC cells, implying that this pathway could be a therapeutic target.OPCML, a GPI anchored tumor-suppressor protein is a negative regulator of receptor tyrosine kinases (RTK), is inactivated by somatic methylation in many cancer types. Here, Zanini and colleagues report that restoration of OPCML in deficient cells leads to sensitization to anti-HER RTK inhibitors. OPCML interacts with HER2 and not EGFR, thereby disrupting formation of HER2-EGFR heterodimers leading to better response to both lapatinib and erlotinib in HER2-expressing cancer cell lines. High OPCML expression is associated with better clinical response to lapatinib therapy in breast cancer patients. These results suggest that OPCML/RTK inhibitor co-therapy could be a valuable sensitizing approach to RTK inhibitors.

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR.

Abstract 1742: Cancer cell-selective transcriptome analysis reveals new colorectal cancer molecular subtypes with improved biological resolution and superior predictive and prognostic performance

Abstract Molecular classification of colorectal cancer (CRC) based on gene expression profiling of tumor samples is known to be heavily affected by transcripts of stromal origin. As a consequence, current CRC transcriptional subtypes reflect an admixture of cancer cell-intrinsic traits and tumor microenvironment features. Whether selective analysis of the cancer cell transcriptome could improve CRC subtyping, remains an open issue. In patient-derived xenografts (PDXs), human transcripts only originate from cancer cells, because stromal transcripts are of mouse origin. We therefore assessed cancer-cell intrinsic transcriptional features of CRC by generating human-specific expression profiles of 515 PDXs from 244 CRC patients, and performing unsupervised class discovery. We identified five “CRC intrinsic subtypes” (CRIS A-E) only partially overlapping with the current ones, and robustly enriched for distinct molecular, functional and phenotypic traits: (i) CRIS-A: mucinous, glycolytic, CIMP, and enriched for microsatellite instability or mutations in KRAS; (ii) CRIS-B: marked TGF-β pathway activity, epithelial-mesenchymal transition and poor prognosis; (iii) CRIS-C: MSS, elevated EGFR signaling and sensitivity to EGFR-targeted treatments; (iv) CRIS-D: MSS, WNT activation, and IGF2 overexpression and amplification; (v) CRIS-E: MSS, Paneth cell-like phenotype and higher frequency of TP53 mutation. CRIS subtypes successfully categorized independent sets of primary and metastatic CRCs and cell lines, for a total of over 3000 samples profiled by microarrays and RNAseq. The new subtypes displayed unprecedented predictive and prognostic performances, indipendent from known markers including stromal signatures, whose integration with CRIS further enhanced prognostic significance. Citation Format: Claudio Isella, Sara E. Bellomo, Francesco Brundu, Francesco Galimi, Elisa Ficarra, Luigi Marchionni, Livio Trusolino, Andrea Bertotti, Enzo Medico. Cancer cell-selective transcriptome analysis reveals new colorectal cancer molecular subtypes with improved biological resolution and superior predictive and prognostic performance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1742. doi:10.1158/1538-7445.AM2017-1742

Abstract 1040: LIN28B-mediated let-7 independent activation of AKT promotes neuroblastoma pathogenesis

Abstract LIN28 is well known as a RNA-binding protein and a suppressor of microRNA biogenesis, by selectively blocking the processing of let-7 precursors. It plays diverse functions in cellular reprogramming, development, metabolism and tumorigenesis. Many of these functions are executed through its ability to inhibit let-7 maturation. However, little is known about its function independent of let-7. Here we made zebrafish transgenic lines expressing high levels of either wild-type or mutant LIN28B (does not block the maturation of let-7) in the peripheral sympathetic nervous system driven by the dopamine beta-hydroxylase promoter. We bred these lines with a transgenic zebrafish line overexpressing the MYCN oncogene in these cells. Either wild-type or mutant LIN28B overexpression accelerated the onset and increased the penetrance of MYCN-induced neuroblastoma, despite the fact that only wild-type LIN28B blocked let-7 maturation compared to MYCN-only tumors. Mechanistically, both wild-type and mutant LIN28B overexpression enhanced MYCN-induced hyperplasia by increasing cell proliferation in the sympathoadrenal lineage. Further studies revealed that overexpression of either wild-type or mutant LIN28B resulted in hyperphosphorylation of AKT on both Ser473 and Thr308 in zebrafish tumors and also serum-deprived human neuroblastoma cell lines. Both wild-type and mutant LIN28B interacted with IGF2BP1, a known interactor that increases stability and translation of a subset of RNAs. We found IGF2 RNA levels to be increased in response to either form of LIN28B. Coincident with IGF2 overexpression, IGF1R was phosphorylated, providing an explanation for PI3K-AKT activation in LIN28B overexpressing cells. Finally, overexpression of a constitutively active, myristoylated murine Akt2 (myr-mAkt2) alone in zebrafish induced ganglioneuroma in the interregnal gland (the zebrafish equivalent of the human adrenal medulla), without a requirement for MCYN overexpression. Thus, our studies indicate that overexpression of LIN28B leads to AKT activation mediated through its interaction with IGF2BP1 and subsequent upregulation of IGF2. This pathway provides a mechanism underlying enhanced transformation in LIN28B overexpressing neuroblastomas, which is independent of the inhibitory activity of LIN28B on let-7 maturation. Citation Format: Ting Tao, John T. Powers, Hui Shi, Pavlos Missios, Antonio R. Perez-Atayde, Shizhen Zhu, George Q. Daley, Thomas A. Look. LIN28B-mediated let-7 independent activation of AKT promotes neuroblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1040. doi:10.1158/1538-7445.AM2017-1040

Stromal-derived IGF2 promotes colon cancer progression via paracrine and autocrine mechanisms.

The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-β (TGFβ) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFβ/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFβ to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFβ was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.

HMGA1P7-pseudogene regulates H19 and Igf2 expression by a competitive endogenous RNA mechanism.

Recent studies have revealed that pseudogene transcripts can function as competing endogenous RNAs, and thereby can also contribute to cancer when dysregulated. We have recently identified two pseudogenes, HMGA1P6 and HMGA1P7 for the HMGA1 gene whose overexpression has a critical role in cancer progression. These pseudogenes work as competitive endogenous RNA decoys for HMGA1 and other cancer related genes suggesting their role in carcinogenesis. Looking for new HMGA1 pseudogene ceRNAs, we performed RNA sequencing technology on mouse embryonic fibroblasts deriving from transgenic mice overexpressing HMGA1P7. Here, we report that HMGA1P7 mRNA sustains the H19 and Igf2 overexpression by acting as miRNA decoy. Lastly, the expression of HMGA1P7 was significantly correlated with H19 and IGF2 levels in human breast cancer thereby suggesting a role for HMGA1P7 deregulation in this neoplasia.

IGF2 Is Up-regulated by Epigenetic Mechanisms in Hepatocellular Carcinomas and Is an Actionable Oncogene Product in Experimental Models

Effective treatments are urgently needed for hepatocellular carcinoma (HCC), which is usually diagnosed at advanced stages. Signaling via the insulin-like growth factor (IGF) pathway is aberrantly activated in HCC by IGF2 overexpression. We aimed to elucidate the mechanism of IGF2 overexpression and its oncogenic activities and evaluate the anti-tumor effects of reducing IGF2 signaling. We obtained 228 HCC samples from patients who underwent liver resection, 168 paired non-tumor adjacent cirrhotic liver samples, and 10 non-tumor liver tissues from patients undergoing resection for hepatic hemangioma. We analyzed gene expression, microRNA, and DNA methylation profiles for all samples, focusing on genes in the IGF signaling pathway. IGF2 was expressed in SNU449 and PLC5 HCC cells and knocked down with small hairpin RNAs in Hep3B and Huh7 cell lines. We analyzed these cells for proliferation, apoptosis, migration, and colony formation. We performed studies in mice engineered to express Myc and Akt1 in liver, which develop liver tumors, with or without hepatic expression of Igf2. Mice with xenograft tumors grown from HCC cells were given a monoclonal antibody against IGF1 and IGF2 (xentuzumab), along with sorafenib; tumor growth was measured and tissues were analyzed by immunohistochemistry and immunoblots. Levels of IGF2 messenger RNA and protein were increased >20-fold in 15% of human HCC tissues compared with non-tumor liver tissues. Methylation at the fetal promoters of IGF2 was reduced in the HCC samples and cell lines that overexpressed IGF2, compared with those that did not overexpress this gene, and non-tumor tissues. Tumors that overexpressed IGF2 had gene expression patterns significantly associated with hepatic progenitor cell features, stellate cell activation, NOTCH signaling, and an aggressive phenotype (P < .0001). In mice engineered to express Myc and Akt1 in liver, co-expression of Igf2 accelerated formation of liver tumors, compared to mice with livers expressing only Myc and Akt1, and shortened survival times (P= .02). The antibody xentuzumab blocked phosphorylation of IGF1 receptor in HCC cell lines and reduced their proliferation and colony formation. In mice with xenograft tumors, injection of xentuzumab, with or without sorafenib, slowed tumor growth and increased survival times compared to vehicle or sorafenib alone. Xentuzumab inhibited phosphorylation of IGF1 receptor and AKT and reduced decreased tumor vascularization compared with vehicle. A large proportion of HCC samples were found to overexpress IGF2, via demethylation of its fetal promoter. Overexpression of IGF2 accelerates formation of liver tumors in mice with hepatic expression of MYC and AKT1, via activation of IGF1 receptor signaling. An antibody against IGF1 and IGF2 slows growth of xenograft tumors and increases survival of these mice.

Abstract B17: Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer

Abstract Although EGFR targeted therapy has improved the survival outcome of patients with metastatic colorectal cancer, lack of response or emergence of secondary resistance frequently occurs as result of genetic alterations that hyperactivate the RAS pathway or by activation of compensatory pathways by signals from the tumor microenvironmet that can also induce a stem-like phenotype. From patient-derived xenografts (“xenopatients”) of colorectal cancer liver metastases, we generated primary sphere cultures (“xenospheres”) that display the properties of cancer stem-like cells. Indeed, these cells can long-term self-propagate in vitro, and form phenocopies of original patient tumors in vivo (“spheropatients”), maintaining the same response to anti-EGFR therapy. We obtained a panel of xenospheres derived from more than 40 different xenopatients, all genetically characterized for the presence of mutations in genes that are predictive for response to EGFR targeted therapy. The panel included xenospheres harboring mutations in RAS genes (Rasmut), or recently identified rare predictors of resistance: ERBB2 amplification/mutation, MET amplification, EGFR mutation in the extracellular domain, and IGF2 overexpression. Moreover, we conducted an extensive exome-sequencing analysis of 10 xenospheres, for which the exome data of the matched xenopatients were available, thus allowing a complete and robust comparison. Xenospheres displayed a good genetic stability and a remarkable correspondence with xenopatients, in both the mutational and gene expression profiles. While RASmut xenospheres displayed self-sustained proliferative ability, those lacking mutations affecting the RAS pathway (RASwt) were strongly dependent of exogenous growth factors. We thus assessed the response of all RASwt xenospheres to ligands of the EGF family (TGFA, EREG, AREG, HB-EGF, NRG1), or bFGF, or HGF, testing both proliferation and response to EGFR inhibition (cetuximab). We found that TGFA and HB-EGF induced proliferation and resistance to cetuximab with almost the same potency of EGF, while EREG and AREG had a weak mitogenic activity and did not protect against cetuximab treatment, accordingly with observations in patients and xenopatients. The different proliferative activity (and protection against cetuximab) can be at least in part explained by the effect on receptor stabilization and exposure to the cell membrane exerted by different ligands. Interestingly, we found that, among all EGF family ligands, NRG1 (binding ERBB3/4) had the most potent mitogenic activity and protection against cetuximab treatment. NRG1 was able to replace EGF in sustaining xenosphere propagation, without altering the global gene expression profile, implying that it could sustain cancer-stem like cell properties similarly to EGF. To study the role of NRG1 in vivo we generated both an autocrine and a paracrine mode, by inducing its expression infecting either RASwt xenospheres or a cell line of murine fibroblasts. NRG1-expressing RASwt xenospheres became growth factor independent, resistant to cetuximab but sensitive to lapatinib (a dual EGFR/ERBB2 small molecule). Similar results were obtained using the conditioned medium of NRG1-expressing murine fibroblasts. By injecting parental and modified RASwt xenospheres into immunocompromised mice, we found that NRG1-expressing xenospheres had a strongly increased tumor onset, and that these tumors were resistant to cetuximab but sensitive to lapatinib treatment, suggesting its possible use in the clinical practice of RASwt patients. We conclude that xenospheres are a robust patient-derived in vitro model of colorectal cancer-stem like cells, amenable to study molecular mechanisms of response or resistance to targeted therapies. Citation Format: Paolo Luraghi, Viola Bigatto, Gigliola Reato, Elia Cipriano, Francesco Sassi, Claudio Isella, Andrea Bertotti, Livio Trusolino, Paolo Maria Comoglio, Carla Boccaccio. Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B17.

Abstract 3820: Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma

Abstract Background and aims Hepatocellular carcinoma (HCC) is a major health problem. Most patients are diagnosed at advanced stages when the only approved therapy is the multi-kinase inhibitor sorafenib. Consequently, there is a great need for the development of new effective treatments. IGF signaling pathway is aberrantly activated in HCC; however, its contribution to HCC pathogenesis is still unclear. Since IGF2 is overexpressed in HCC, we aimed to elucidate the oncogenic potential and mechanism of dis-regulation of this protein and determine the antitumoral efficacy of molecular abrogation of this ligand by targeted therapies. Methods Transcriptomic profiling, miRNAs expression, RNA- and whole exome- sequencing and methylation were analyzed in 228 HCCs with a focus on IGF-pathway. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out in IGF2-overexpressing tumors. Stable HCC cell lines with knock-down and ectopic overexpression of IGF2 were generated. A chemically-induced mouse model of HCC, and two genetically-engineered mosaic mouse models (GEMM) overexpressing IGF2 specifically in the liver were generated to assess IGF2 oncogenicity in hepatocarcinogenesis. The therapeutic potential of a monoclonal-antibody against IGF-ligands (IGF1/2-mAb) alone or in combination with sorafenib was tested in a xenograft model of HCC. Results Here, IGF2-overexpression occurred in 15% of HCC patients as a result of the epigenetic reactivation of IGF2-fetal promoters, mainly through loss of promoters methylation (53% of cases) and deregulation of miR-216b, miR-483-5p and miR-let7-d (35% of cases). Re-expression of IGF2 was associated with a progenitor cell-like, poorly differentiated and aggressive subtype of HCC, and poor prognosis (p&amp;lt;0.0001). In a GEMM model, IGF2-overexpression accelerated HCC progression and reduced survival (p = 0.02). Conversely, IGF2-blockage using an IGF1/2-monoclonal antibody (mAb) or specific shRNAs against IGF2 reduced viability and proliferation in vitro (p&amp;lt;0.05), and inhibited IGF-Insulin pathway activation without disturbing insulin metabolism. IGF1/2-mAb delayed tumor growth and increased survival in vivo compared to placebo or sorafenib (p&amp;lt;0.0001), through antiproliferative and antiangiogenic mechanisms. Conclusions IGF2 is the first validated epidriver in HCC and has a key role in the hepatocarcinogenic process. These results provide the rationale for testing IGF1/2-mAb in a selected subset of HCC patients. Citation Format: Iris Martinez-Quetglas, Roser Pinyol, Daniel Dauch, Sara Torrecilla, Victoria Tovar, Agrin Moeini, Clara Alsinet, Anna Portela, Augusto Villanueva, Manel Esteller, Lars Zender, Josep M Llovet. Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3820.

EZH2 is overexpressed in adrenocortical carcinoma and is associated with disease progression.

Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/β-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.

Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation.

Although insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in steatosis and steatohepatitis, a causal role of IGF2 in steatosis development remains elusive. Aim of our study was to decipher the role of IGF2 in steatosis development. Hydrodynamic gene delivery of an Igf2 plasmid used for transient Igf2 overexpression employing codon-optimized plasmid DNA resulted in a strong induction of hepatic Igf2 expression. The exogenously delivered Igf2 had no influence on endogenous Igf2 expression. The downstream kinase AKT was activated in Igf2 animals. Decreased ALT levels mirrored the cytoprotective effect of IGF2. Serum cholesterol was increased and sulfo-phospho-vanillin colorimetric assay confirmed lipid accumulation in Igf2-livers while no signs of inflammation were observed. Interestingly, hepatic cholesterol and phospholipids, determined by thin layer chromatography, and free cholesterol by filipin staining, were specifically increased. Lipid droplet (LD) size was not changed, but their number was significantly elevated. Furthermore, free cholesterol, which can be stored in LDs and has been reported to be critical for steatosis progression, was elevated in Igf2 overexpressing mice. Accordingly, Hmgcr/HmgCoAR was upregulated. To have a closer look at de novo lipid synthesis we investigated expression of the lipogenic transcription factor SREBF1 and its target genes. SREBF1 was induced and also SREBF1 target genes were slightly upregulated. Interestingly, the expression of Cpt1a, which is responsible for mitochondrial fatty acid oxidation, was induced. Hepatic IGF2 expression induces a fatty liver, characterized by increased cholesterol and phospholipids leading to accumulation of LDs. We therefore suggest a causal role for IGF2 in hepatic lipid accumulation.

Abstract PR02: Aneuploidy as a driver of liver cancer

Abstract Aneuploidy has long been associated with cancer but whether it is a cause or consequence of oncogenesis remains unknown. Hepatocellular Carcinomas have frequent aberrations in chromosomes 1 and 8, and oncogene addiction has yet to be clearly demonstrated in this tumor type. Therefore, we developed a mouse model to test the hypothesis that chromosome instability was a driver of liver cancer. To induce chromosomal instability in mouse hepatocytes, we generated a floxed allele of the spindle accessory checkpoint protein Mad2 and induced Cre-mediated deletion in hepatocytes by intercrossing with an Albumin-Cre transgenic. Loss of Mad2 in hepatocytes resulted in the development of both benign Hepatocellular Adenomas (HCA) and malignant Hepatocellular Carcinomas (HCC) by 12 mo. Backcrossing onto a p53-deficient background increased the penetrance and speed of disease with nearly all double deficient mice having HCC by 4 mo. Analysis of the tumor genome by array CGH showed recurrent gain and loss of whole chromosomes with a lack of small focal alterations. We showed whole chromosomes were under selective pressure by comparing HCCs with HCAs as well as T cell lymphomas that arose in the Mad2 floxed line crossed to an Lck-Cre transgenic. Comparing HCCs to HCAs showed that the magnitude but not the pattern of chromosomal aberrations is what distinguishes these tumor types. Further genetic analysis of HCCs and HCAs from the same mouse showed half of the tumor pairs shared a common ancestor, suggesting that in this model HCAs can progress to HCCs through continuing chromosomal instability. Comparing the genomes of HCCs and T cell lymphomas that resulted from Mad2-deficiency in these different tissues showed tissue-specific gain and loss patterns. Chromosomes 4 and 12 were specifically lost in HCCs but gained in lymphomas while chromosome 18 was gained in HCCs but lost in lymphomas. In addition, chromosome 15 was frequently gained in both tumors. Combined these findings show that gain and loss of whole chromosomes can be evolutionarily selected, resulting in tumorigenesis. Next, we tested how aneuploidy alters cell phenotype to induce cancer by performing matched array CGH and gene expression arrays on 29 HCCs. Gene dosage significantly altered expression since chromosome ploidy was significantly correlated with mRNA expression from that chromosome. We also identified a set of twelve genes that was recurrently over-expressed in over 80% of HCCs; most of this set was also over-expressed in human HCCs. Notably Igf2 in murine HCC was over-expressed by 50 fold. IGF2 over-expression in human HCCs has been linked to aneuploidy through loss of loss of Chr8p which causes global demethylation and loss of imprinting at the IGF2 locus. Thus, aneuploidy events are also able to affect the expression of specific genes. Since aneuploidy is frequently observed in human HCC, we performed a synteny analysis of human and mouse chromosomes to find regions that are gained or lost in both species. Five regions were found to be significantly gained by aneuploidy in both species. These regions were located on human chromosomes 1q and 8q, and mouse chromosomes 1 and 15, with the most frequently amplified region containing MYC (hChr8q/mChr15). In total, we show that chromosomal instability is sufficient to drive liver cancer through evolutionary selection acting on whole chromosomes, that aneuploidy alters cell phenotype through gene dosage and other mechanisms, and that the genetic and phenotypic similarities between this mouse model and human liver cancer suggest that aneuploidy is also a driver of human cancer. Citation Format: Lee A. Albacker, Floris Foijer, Ying Yue, Darin Takemoto, Stehpanie Davis, Rodney T. Bronson, Peter K. Sorger. Aneuploidy as a driver of liver cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR02.

Molecular pathways of human adrenocortical carcinoma - translating cell signalling knowledge into diagnostic and treatment options.

Adrenocortical carcinoma is associated with a low cure rate and a high recurrence rate. The prognosis is poor, and at diagnosis 30-40% of cases are already metastatic. The current therapeutic options (surgical resection, followed by adjuvant mitotane treatment +/- chemotherapy) are limited, and the results remain unsatisfactory. Key molecular events that contribute to formation of adrenocortical cancer are IGF2 overexpression, TP53-inactivating mutations, and constitutive activation of the Wnt/b-catenin signalling pathway via activating mutations of the b-catenin gene. The underlying genetic causes of inherited tumour syndromes have provided insights into molecular pathogenesis. The increased occurrence of adrenocortical tumours in Li-Fraumeni and Beckwith-Wiedemann syndromes, and Carney complex, has highlighted the roles of specific susceptibility genes: TP53, IGF2, and PRKAR1A, respectively. Further studies have confirmed that these genes are also involved in sporadic tumour cases. Crucially, transcriptome-wide studies have determined the differences between malignant and benign adrenocortical tumours, providing potential diagnostic tools. In conclusion, enhancing our understanding of the molecular events of adrenocortical tumourigenesis, especially with regard to the signalling pathways that may be disrupted, will greatly contribute to improving a range of available diagnostic, prognostic, and treatment approaches. (Endokrynol Pol 2016; 67 (4): 427-440).

Molecular genetic investigation of placental mesenchymal dysplasia

Molecular genetic investigation of placental mesenchymal dysplasia

Genotype-Epigenotype Interaction at the IGF2 DMR.

Paternally expressed Insulin-like Growth Factor II (IGF2) encodes a gene whose protein product functions as a potent growth mitogen. Overexpression of IGF2 has been implicated in a wide number of disorders and diseases. IGF2 is regulated in part by differential methylation of the two parentally derived alleles. The differentially methylated region (DMR) located upstream of the imprinted promoters of IGF2 exhibits plasticity under environmental stress and is hypomethylated in several types of cancer. Through bisulfite pyrosequencing and confirmation by nucleotide sequencing, we discovered a CpG to CpC transversion that results in hypomethylation of one of the three CpGs comprising this DMR. The presence of the polymorphism introduces a genetic rather than an environmentally-driven epigenetic source of hypomethylation that is additive to non-genetic sources.