Sam D. Molyneux

Prkar1a is an osteosarcoma tumor suppressor that defines a molecular subclass in mice.

Some cancers have been stratified into subclasses based on their unique involvement of specific signaling pathways. The mapping of human cancer genomes is revealing a vast number of somatic alterations; however, the identification of clinically relevant molecular tumor subclasses and their respective driver genes presents challenges. This information is key to developing more targeted and personalized cancer therapies. Here, we generate a new mouse model of genomically unstable osteosarcoma (OSA) that phenocopies the human disease. Integrative oncogenomics pinpointed cAMP-dependent protein kinase type I, alpha regulatory subunit (Prkar1a) gene deletions at 11qE1 as a recurrent genetic trait for a molecularly distinct subclass of mouse OSA featuring RANKL overexpression. Using mouse genetics, we established that Prkar1a is a bone tumor suppressor gene capable of directing subclass development and driving RANKL overexpression during OSA tumorigenesis. Finally, we uncovered evidence for a PRKAR1A-low subset of human OSA with distinct clinical behavior. Thus, tumor subclasses develop in mice and can potentially provide information toward the molecular stratification of human cancers.

Notch Activation by the Metalloproteinase ADAM17 Regulates Myeloproliferation and Atopic Barrier Immunity by Suppressing Epithelial Cytokine Synthesis

Epithelial cells of mucosal tissues provide a barrier against environmental stress, and keratinocytes are key decision makers for immune cell function in the skin. Currently, epithelial signaling networks that instruct barrier immunity remain uncharacterized. Here we have shown that keratinocyte-specific deletion of a disintegrin and metalloproteinase 17 (Adam17) triggers T helper 2 and/or T helper 17 (Th2 and/or Th17) cell-driven atopic dermatitis and myeloproliferative disease. In vivo and in vitro deficiency of ADAM17 dampened Notch signaling, increasing production of the Th2 cell-polarizing cytokine TSLP and myeloid growth factor G-CSF. Ligand-independent Notch activation was identified as a regulator of AP-1 transcriptional activity, with Notch antagonizing c-Fos recruitment to the promoters of Tslp and Csf3 (G-CSF). Further, skin inflammation was rescued and myeloproliferation ameliorated by delivery of active Notch to Adam17(-)(/-) epidermis. Our findings uncover an essential role of ADAM17 in the adult epidermis, demonstrating a gatekeeper function of the ADAM17-Notch-c-Fos triad in barrier immunity.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K–NRF2-regulated pathway

Significance Our establishment of a connection between the phosphatidylinositol 3-kinase (PI3K) and NRF2 pathways provides the basis for the tissue specificity of BRCA1-related cancers. Because BRCA1 is a vital component of the intracellular machinery maintaining genomic stability, BRCA1 functions as a major tumor suppressor in cells of all types. However, BRCA1-related cancers occur overwhelmingly in breasts and ovaries. Our work demonstrates that estrogen (E2) acts via the PI3K–AKT pathway to regulate NRF2 activation in BRCA1-deficient cells, resulting in the induction of antioxidant genes that protect the cell from reactive oxygen species-induced death. BRCA1-deficient cells are thus allowed to survive and may accumulate mutations, such as loss of PTEN, that perpetuate NRF2 activation. Our work supports the emerging clinical strategy of treating BRCA1-related cancers with PI3K inhibitors. Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1’s tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase–AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

RANKL blockade prevents and treats aggressive osteosarcomas

Denosumab, an antibody targeting RANKL, is effective against osteosarcoma in mouse models. OutRANKing osteosarcoma Osteosarcoma is the most common primary bone cancer, and it can be difficult to treat, especially in patients with metastatic disease. Chen et al. developed a series of genetically engineered mouse models of osteosarcoma and used these models to dissect the role of receptor activator of nuclear factor κB ligand (RANKL) signaling in the progression of this disease. The authors also showed that denosumab, an antibody against RANKL that is already used in patients with some bone diseases, is effective in mouse models of osteosarcoma and is a viable candidate for future testing in human patients. Osteosarcoma (OS) is the most common primary bone cancer, which occurs primarily in children and adolescents, severely affecting survivors’ quality of life. Despite its chemosensitivity and treatment advances, long-term survival rates for OS patients have stagnated over the last 20 years. Thus, it is necessary to develop new molecularly targeted therapies for this metastatic bone cancer. Mutations in TP53 and RB are linked to OS predisposition and to the evolution of spontaneous OS. We established receptor activator of nuclear factor κB ligand (RANKL) as a therapeutic target for suppression and prevention of OS. Combined conditional osteoblast-specific deletions of Rb, p53, and the protein kinase A (PKA) regulatory subunit Prkar1α genes in genetically engineered mouse models (GEMMs) generate aggressive osteosarcomas, characterized by PKA, RANKL, and osteoclast hyperactivity. Whole-body Rankl deletion completely abrogates tumorigenesis. Although osteoblastic Rank deletion has little effect, osteoclastic Rank deletion delays tumorigenesis and prolongs life span. The latter is associated with inactivation of osteoclastogenesis and up-regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Further, we use these GEMMs as preclinical platforms to show that RANKL blockade with RANK-Fc arrests tumor progression and improves survival and also inhibits lung metastasis. Moreover, preemptive administration of RANK-Fc completely prevents tumorigenesis in mice highly predisposed to this aggressive cancer. Denosumab, a fully human monoclonal antibody against RANKL, is currently used to treat patients with osteoporosis or bone metastases. Our studies provide a strong rationale to consider RANKL blockade for the treatment and prevention of aggressive RANKL-overexpressing OS in humans.

Human somatic cell mutagenesis creates genetically tractable sarcomas

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis.

Cross-species genomics identifies DLG2 as a tumor suppressor in osteosarcoma

Leveraging the conserved cancer genomes across mammals has the potential to transform driver gene discovery in orphan cancers. Here, we combine cross-species genomics with validation across human–dog–mouse systems to uncover a new bone tumor suppressor gene. Comparative genomics of spontaneous human and dog osteosarcomas (OS) expose Disks Large Homolog 2 (DLG2) as a tumor suppressor candidate. DLG2 copy number loss occurs in 42% of human and 56% of canine OS. Functional validation through pertinent human and canine OS DLG2-deficient cell lines identifies a regulatory role of DLG2 in cell division, migration and tumorigenesis. Moreover, osteoblast-specific deletion of Dlg2 in a clinically relevant genetically engineered mouse model leads to acceleration of OS development, establishing DLG2 as a critical determinant of OS. This widely applicable cross-species approach serves as a platform to expedite the search of cancer drivers in rare human malignancies, offering new targets for cancer therapy.

Abstract 398: Genome-wide comparison of matched canine osteosarcoma primary tumours and metastases by array comparative genomic hybridization.

Canine osteosarcoma is a common and highly aggressive cancer that shares many characteristics with human osteosarcoma and can serve as a model of this disease. We collected snap frozen normal muscle, primary appendicular skeleton tumour, and matched terminal lung metastases from 8 canine osteosarcoma patients. DNA was isolated from these samples and gene copy number aberrations were assessed using high-resolution oligonucleotide array comparative genomic hybridization (Agilent G3 Canine 180k array). Data analysis was performed using Agilent Genomic Workbench 5.0 analysis software and the Aberration Detection Method 2 and Circular Binary Segmentation algorithms were applied to identify aberrant chromosomal intervals with the log2 ratio threshold set to 0.5. As expected, many cases had copy number aberrations that contain genes commonly altered in canine and human osteosarcoma, including MYC, CDKN2A/CDKN2B, PTEN, p53, and RB. To assess the degree of genomic similarity of the 8 primary tumours to their corresponding metastasis, we used these same samples to generate 8 random pairings of unmatched primaries and metastases, as well as 8 random pairs of primary tumours and 8 random pairs of metastases. A set of 11,000 random 10 base pair segments spanning the entire genome was generated and a correlation analysis was conducted between pairs of samples using the log2 ratios for each segment. Matched samples showed significant correlation with the square of the correlation coefficient (R2) being 0.44 (p 0.05 for each). Thus, as a group, primary tumours are genomically more similar to their corresponding metastasis than to unmatched metastases, and are also more similar than primary tumours or metastases are to each other. However, the range of similarity between matched primaries and metastases was relatively wide, with half the cases showing very high similarity and half showing moderate similarity that was close to the upper range of unmatched samples. These results have implications for therapies that rely on analysis of primary tumours to derive strategies that target metastases. Citation Format: Jonathan H W Liu, Yang Washington Shao, Sam D. Molyneux, Rama Khokha, Geoffrey A. Wood. Genome-wide comparison of matched canine osteosarcoma primary tumours and metastases by array comparative genomic hybridization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 398. doi:10.1158/1538-7445.AM2013-398