Samuel R. Perry

Telomere dysfunction induces metabolic and mitochondrial compromise

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.

p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation.

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.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

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.

Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.

To 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.

PLAGL2 Regulates Wnt Signaling to Impede Differentiation in Neural Stem Cells and Gliomas

A 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/beta-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.

Pten Is a Major Tumor Suppressor in Pancreatic Ductal Adenocarcinoma and Regulates an NF-κB-Cytokine Network

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.

Imaging guided trials of the angiogenesis inhibitor sunitinib in mouse models predict efficacy in pancreatic neuroendocrine but not ductal carcinoma

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.

Erratum: Telomere dysfunction induces metabolic and mitochondrial compromise (Nature (2011) 470 (359-365))

This corrects the article DOI: 10.1038/nature09787

Abstract A101: Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism.

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.

Abstract A202: A role of AXL in pancreatic ductal adenocarcinoma.

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.