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Supplementary Tables 1-2, Figures 1-8 from PTEN Is a Major Tumor Suppressor in Pancreatic Ductal Adenocarcinoma and Regulates an NF-κB–Cytokine Network

Pathways of human epidermal growth factor (EGF) receptors are activated upon ligand-dependent or -independent homo- or heterodimerization and their subsequent transphosphorylation. Overexpression of these receptors positively correlates with transphosphorylation rates and increased tumor growth rates. MEDI4276, an anti-human epidermal growth factor receptor 2 (HER2) biparatopic antibody-drug conjugate, has two paratopes within each antibody arm. One, 39S, is aiming at the HER2 site involved in receptor dimerization and the second, single chain fragment (scFv), mimicking trastuzumab. Here we present the cocrystal structure of the 39S Fab-HER2 complex and, along with biophysical and functional assays, determine the corresponding epitope of MEDI4276 and its underlying mechanism of action. Our results reveal that MEDI4276's uniqueness is based first on the ability of its 39S paratope to block HER2 homo- or heterodimerization and second on its ability to cluster the receptors on the surface of receptor-overexpressing cells.

The use of selenocysteines and various cross-linkers to induce helicity in a bioactive peptide is described. The higher reactivity of selenocysteine, relative to cysteine, facilitates rapid cross-linking within unprotected linear peptides under mild aqueous conditions. Alkylating agents of variable topology and electrophilicity were used to link pairs of selenocysteines within a p53 peptide. Facile selenoether formation enables diverse tailoring of the helical peptide structure.

This study aimed to identify the microRNAs associated with sclerotic status of subchondral bone in the pathogenesis of osteoarthritis (OA). Total RNA was extracted from non-sclerotic and sclerotic OA subchondral bone from patients undergoing knee replacement surgeries. miRCURY™ LNA miRNA chip and qRT-PCR were used to profile and validate differential microRNA expression. In addition, we further confirmed profiles of altered miRNAs in an OA rat meniscectomy animal model and their putative targets of the miRNAs were predicted using ingenuity (IPA) software. Finally, five short-listed miRNAs were reactivated by transient in vitro overexpression (miRNA mimics) in subchondral bone osteoblasts and their phenotypes were assessed. Functional screening identified 30 differentiated miRNAs in sclerotic subchondral bone compared to non-sclerotic bone of OA patients. Data integration resulted in confirmation of the eight miRNAs, with aberrant expression in independent human OA bone sample set. In silico analysis (IPA) identified 732 mRNA transcripts as putative targets of the eight altered miRNAs, of which twenty genes were validated to be differentially expressed in sclerotic compared to non-sclerotic bone samples. Out of eight dysregulated miRNA's, five of them showed consistent time-dependent downregulation in a rat OA model. Furthermore, synthetic miR-199a-3p, miR-199a-5p, miR-590-5p, and miR-211-5p mimics rescued the abnormal osteoarthritic subchondral bone osteoblast gene expression and mineralization. We have identified four novel miRNAs that play important roles in subchondral bone pathogenesis in OA. Additional studies are required to develop these miRNAs into therapeutic modalities for OA.

Current HER2-targeted drugs are ineffective in killing cancer cells expressing relatively low levels of HER2. Therefore, more than 60% of breast cancer patients are ineligible for HER2-targeted therapies because of lack of HER2 overexpression and the vast majority of eligible patients who initially respond to the treatment will eventually relapse. MedImmune is developing a novel HER2-targeting antibody-drug conjugate (ADC) to address this unmet medical need. We show that a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 is able to induce receptor clustering on the tumor cell surface, which in turn facilitates internalization and promotes lysosomal trafficking and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic antibody can deliver a greater quantity of cytotoxin into the targeted cancer cells. As a result, it demonstrated superior antitumor activity over Kadcyla® (T-DM1) in HER2-overexpressing (HER2-positive) tumor models. It also induced complete tumor regression in a HER2-positive tumor model that had developed acquired resistance to T-DM1 through chronic exposure. Moreover, to explore the potential clinical applications in treating the HER2 non-overexpressing (HER2-negative) patients the biparatopic ADC was evaluated across 17 primary tumor models derived from HER2-negative breast cancer patients among which 13 were triple-negative. Other criteria were also considered in the selection of these 17 models, including the degree of heterogeneity in HER2 expression, ER/PR status and histopathologic subclass, to maximize the diversity of tumor subtypes in the study. The biparatopic ADC demonstrated potent antitumor activity regardless of the histopathologic subclass and ER/PR status of the tumor. At the dose of 1 mg/kg, 41% of the tumor models (7 out of 17) showed tumor regression and 6% (1 out of 17) showed tumor stasis. At the dose of 3 mg/kg, 71% of the models (12 out of 17) showed tumor regression and 12% (2 out of 17) showed tumor stasis. Overall, our findings underscore the potential use of this novel HER2-targeting ADC to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies, and thus warrant investigation in the clinic. Citation Format: Li JY, Perry SR, Muniz-Medina V, Wetzel LK, Rebelatto MC, Bezabeh BZ, Fleming RL, Dimasi N, Gao C, Wu H, Jenkins DW, Osbourn JK, Coats SR. A biparatopic HER2-targeting antibody-drug conjugate demonstrates potent antitumor activity in primary tumor models that are refractory to or ineligible for HER2-targeted therapies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD5-08.

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

Most protein-protein interactions occur inside cells. Peptides can inhibit protein-protein interactions but tend not to enter cells. We systematically compare cell permeability for 8-12 residue model peptides with helix-inducing lactam/hydrocarbon linkers between amino acid sidechains. Cell uptake increases when hydrophobic residues and lactam linkers (i, i + 4) form a contiguous hydrophobic surface patch. Uptake increases further when both hydrophobic and positively charged (but not neutral or negative) residues are clustered into like surface patches. Amphipathicity alone is however insufficient for cell uptake of acyclic sequences. Changing the linker from lactam to hydrocarbon further increases uptake, but also promotes cell lysis. Helicity, positive charge and amphipathicity together promote cell permeability. Most known bioactive helical peptides do not optimally cluster residues for amphipathicity and so are likely unoptimised for cell uptake.

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that control a diverse range of biological processes during development and in adult tissues. We recently reported that somatic FGFR2 mutations are associated with shorter survival in endometrial cancer. However, little is known about how these FGFR2 mutations contribute to endometrial cancer metastasis. Here, we report that expression of the activating mutations FGFR2

The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37-25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT48-57) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.

Initiation of pancreatic ductal adenocarcinoma (PDAC) is driven by oncogenic KRAS mutation, and disease progression is associated with frequent loss of tumor suppressors. In this study, human PDAC genome analyses revealed frequent deletion of the PTEN gene as well as loss of expression in primary tumor specimens. A potential role for PTEN as a haploinsufficient tumor suppressor is further supported by mouse genetic studies. The mouse PDAC driven by oncogenic Kras mutation and Pten deficiency also sustains spontaneous extinction of Ink4a expression and shows prometastatic capacity. Unbiased transcriptomic analyses established that combined oncogenic Kras and Pten loss promotes marked NF-κB activation and its cytokine network, with accompanying robust stromal activation and immune cell infiltration with known tumor-promoting properties. Thus, PTEN/phosphoinositide 3-kinase (PI3K) pathway alteration is a common event in PDAC development and functions in part to strongly activate the NF-κB network, which may serve to shape the PDAC tumor microenvironment. Significance: Detailed molecular genetics studies established that PTEN operates as a haploinsufficient tumor suppressor to promote metastatic PDAC development. The strong activation of the NF-κB–cytokine program in Pten-deficient tumors provides additional avenues for targeted therapies in tumors with altered PI3K regulation. Cancer Discovery; 1(2); 158–69. ©2011 AACR. Read the Commentary on this article by Chiao and Ling, p. 103 This article is highlighted in the In This Issue feature, p. 91

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

SummaryA hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state, which contributes to its plasticity and therapeutic resistance. Here, integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/β-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells.

Glioblastoma (GBM) is a highly lethal primary brain cancer with hallmark features of diffuse invasion, intense apoptosis resistance and florid necrosis, robust angiogenesis, and an immature profile with developmental plasticity. In the course of assessing the developmental consequences of central nervous system (CNS)-specific deletion of p53 and Pten, we observed a penetrant acute-onset malignant glioma phenotype with striking clinical, pathological, and molecular resemblance to primary GBM in humans. This primary, as opposed to secondary, GBM presentation in the mouse prompted genetic analysis of human primary GBM samples that revealed combined p53 and Pten mutations as the most common tumor suppressor defects in primary GBM. On the mechanistic level, the "multiforme" histopathological presentation and immature differentiation marker profile of the murine tumors motivated transcriptomic promoter-binding element and functional studies of neural stem cells (NSCs), which revealed that dual, but not singular, inactivation of p53 and Pten promotes cellular c-Myc activation. This increased c-Myc activity is associated not only with impaired differentiation, enhanced self-renewal capacity of NSCs, and tumor-initiating cells (TICs), but also with maintenance of TIC tumorigenic potential. Together, these murine studies have provided a highly faithful model of primary GBM, revealed a common tumor suppressor mutational pattern in human disease, and established c-Myc as a key component of p53 and Pten cooperative actions in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential.

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor (GPCR) involved in inflammation and metabolism. It is activated through cleavage of its N-terminus by proteases. The new N-terminus functions as a tethered ligand that folds back and intramolecularly activates PAR2, initiating multiple downstream signaling pathways. The only compounds reported to date to inhibit PAR2 activation are of moderate potency. Three structural models for PAR2 have been constructed based on sequence homology with known crystal structures for bovine rhodopsin, human ORL-1 (also called nociceptin/orphanin FQ receptor), and human PAR1. The three PAR2 model structures were compared and used to predict potential interactions with ligands. Virtual screening for ligands using the Chembridge database, and either ORL-1 or PAR1 derived PAR2 models led to identification of eight new small molecule PAR2 antagonists (IC50 10-100 μM). Notably, the most potent compound 1 (IC50 11 μM) was derived from the less homologous template protein, human ORL-1. The results suggest that virtual screening against multiple homology models of the same GPCR can produce structurally diverse antagonists and that this may be desirable even when some models have less sequence homology with the target protein.

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potent cytotoxicity of small molecule drugs and have shown to provide therapeutic options for various cancers. We report herein the discovery of a HER2-targeting ADC MEDI4276 that showed potent cell killing activity in vitro in cancer cell lines that express the HER2 receptor. The observed in vitro activity translated into in vivo tumor growth inhibition in various xenograft mouse models. MEDI4276 is a homogeneous molecule with precise control of drug loading following site specific conjugation of a cytotoxic drug. The drug in MEDI4276 is MMETA, a fully synthetic analog of the tubulysin family that showed pM potency in a panel of cancer cell lines. MMETA was conjugated to the antibody via engineered cysteines with a maleimide-bearing mc-Lys protease cleavable linker. The antibody in MEDI4276 is a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 that leads to antibody-receptor clustering following binding and thus promoting internalization, lysosomal trafficking and degradation. The combination of enhanced internalization and potent cytotoxic drug allows for this ADC to kill tumor cell populations with a broader range of HER2 expression. Preclinical studies showed that MEDI4276 induced tumor regression in HER2-positive tumor models that had developed acquired resistance to T-DM1 and in a number of models with lower HER2 expression that are refractory to T-DM1 treatment. Overall, our findings underscore the potential application of MEDI4276 to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies. MEDI4276 is currently being investigated in a Phase I clinical trial. Citation Format: John Li, Dorin Toader, Samuel R. Perry, Vanessa Muniz-Medina, Leslie Wetzel, Marlon C. Rebelatto, Mary Jane Masson Hinrichs, Ryan Fleming, Binyam Bezabeh, Pamela Thompson, Nazzareno Dimasi, Brandon Lam, Xian-Qing Yu, Changshou Gao, Rakesh Dixit, Steven Coats, Jane Osbourn, Herren Wu. MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies. [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 2970.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context-dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven PDAC mouse model establishes that advanced PDAC remain strictly dependent on KrasG12D expression. Transcriptome and metabolomic analysis indicate that KrasG12D serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that KrasG12D drives glycolysis intermediates into the non-oxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC. Citation Format: Haoqiang Ying, Hailei Zhang, Jonathan L. Coloff, Haiyan Yan, Wei Wang, Shujuan Chen, Andrea Viale, Hongwu Zheng, Ji-hye Paik, Carol Lim, Alexander R. Guimaraes, Alec C. Kimmelman, Eric S. Martin, Jeffery Chang, Aram Hezel, Samuel R. Perry, Jian Hu, Boyi Gan, Yonghong Xiao, John M. Asara, Ralph Weissleder, Y. Alan Wang, Costas A. Lyssiotis, Lynda Chin, Lewis C. Cantley, Ronald A. DePinho, Sujun Hua, Gerald C. Chu, Eliot Fletcher-Sananikone, Jason W. Locasale, Jaekyoung Son. Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A101.

Preclinical trials in mice represent a critical step in the evaluation of experimental therapeutics. Genetically engineered mouse models (GEMMs) represent a promising platform for the evaluation of drugs, particularly those targeting the tumor microenvironment. We evaluated sunitinib, an angiogenesis inhibitor that targets VEGF and PDGF receptor signaling, in two GEMMs of pancreatic cancer. Sunitinib did not reduce tumor burden in pancreatic ductal adenocarcinoma (PDAC), whereas tumor burden was reduced in the pancreatic neuroendocrine tumor (PNET) model, the latter results confirming and extending previous studies. To explore the basis for the lack of pathologic response in PDAC, we used noninvasive microbubble contrast-enhanced ultrasound imaging, which revealed that sunitinib reduced blood flow both in PDAC and in PNET, concomitant with a reduction in vessel density; nevertheless, PDAC tumors continued to grow, whereas PNET were growth impaired. These results parallel the response in humans, where sunitinib recently garnered FDA and European approval in PNET, whereas two antiangiogenic drugs failed to demonstrate efficacy in PDAC clinical trials. The demonstration of on-target activity but with discordant benefit in the PDAC and PNET GEMMs illustrates the potential value of linked preclinical and clinical trials.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

SummaryClinical and genomic evidence suggests that the metastatic potential of a primary tumor may be dictated by prometastatic events that have additional oncogenic capability. To test this “deterministic” hypothesis, we adopted a comparative oncogenomics-guided function-based strategy involving: (1) comparison of global transcriptomes of two genetically engineered mouse models with contrasting metastatic potential, (2) genomic and transcriptomic profiles of human melanoma, (3) functional genetic screen for enhancers of cell invasion, and (4) evidence of expression selection in human melanoma tissues. This integrated effort identified six genes that are potently proinvasive and oncogenic. Furthermore, we show that one such gene, ACP5, confers spontaneous metastasis in vivo, engages a key pathway governing metastasis, and is prognostic in human primary melanomas.

Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.

Introduction Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function. Methods De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies. Results The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell’s attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression. Conclusion This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.

Pancreatic ductal adenocarinoma (PDAC) is the 4th leading cause of cancer-related death in the US. Due to a combination of late diagnosis with highly metastatic nature and therapeutic resistance, it is a near lethal malignancy. The identification of genes forming the molecular basis of these attributes is essential for the development of improved therapies. AXL is a receptor tyrosine kinase that is implicated in many common epithelial malignancies and has been shown to play a role in invasion, migration and innate immunity. In this study, we investigated the clinical relevance and oncogenic potential of AXL in PDAC progression to assess its potential as a therapeutic target. We performed IHC analysis on a total of 306 human primary PDAC samples and found that AXL is highly expressed in 50% of samples tested, suggestive of a role in PDAC. Subsequent screening of cell lines by immunoblot demonstrated expression and activation of AXL protein in 17 of 19 PDAC cell lines. Subsequently, we selected 4 cell lines with varying levels of AXL expression, and generated derivatives of these lines with constitutive knockdown of AXL using shRNA technology. Two shRNAs were selected based on their effect on AXL expression. We found significant impairment of proliferation, the ability to form colonies in soft agar as well as on migration and invasion in a dose-dependent manner. These results suggest that AXL may be a contributing factor to the metastatic phenotype of PDAC. Targeted inhibition of AXL expression in vitro led to 5-fold reduction in ERK and 2-fold reduction in AKT activation compared to control cells, demonstrating that these signaling pathways mediate the effects of AXL on tumor growth and invasion. In order to address the role of AXL on tumor initiation in vivo, shRNA derivatives of Capan-2 and PaTu-8988T cells were injected subcutaneously into nude mice. We found that knockdown of AXL resulted in significant reduction of tumor growth in both models. However, the most striking observation was in diminished invasion in AXL-knockdown tumors compared to control xenograft tumors. Histopathological analyzes revealed extensive invasion of tumor cells beyond the cutaneous muscle in control xenograft tumors, whereas AXL-knockdown xenograft tumors lacked such invasion but had formed a fibrous capsule which separated the tumor mass from the skin. Furthermore, these tumors exhibited reduced degradation of matrigel matrix used in injection of tumor cells, suggesting that inhibition of AXL may lead to impairment of matrix-degrading enzymes. To assess if targeting of AXL might have potential therapeutic implications, we tested the effect of AXL inhibition on maintenance and progression of existing tumors. We used the doxycycline inducible shRNA system to knockdown expression once tumors had reached 120 mm3 in size in Capan-2 and SW1990 xenograft tumors. Inducible inhibition of AXL greatly reduced tumor growth resulting in 36% and 60% reduction respectively, compared to control xenograft tumors, demonstrating the significance of continued AXL expression in tumor maintenance. Our data suggest that inhibition of AXL in patients could lead to reduced tumor growth and invasion warranting further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A202.

Nature 470, 359–365 (2011) In this Article, references 1,2,3 reporting an association between telomere dysfunction and mitochondrial impairment or TERT deficiency and mitochondrial impairment were inadvertently omitted.

SummaryTo determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-β/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.