Larry A. Donehower

Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway.

Modulation of tumor suppressor activities may provide new opportunities for cancer therapy. Here we show that disruption of the gene Ppm1d encoding Wip1 phosphatase activated the p53 and p16 (also called Ink4a)–p19 (also called ARF) pathways through p38 MAPK signaling and suppressed in vitro transformation of mouse embryo fibroblasts (MEFs) by oncogenes. Disruption of the gene Cdkn2a (encoding p16 and p19), but not of Trp53 (encoding p53), reconstituted cell transformation in Ppm1d-null MEFs. In vivo, deletion of Ppm1d in mice bearing mouse mammary tumor virus (MMTV) promoter–driven oncogenes Erbb2 (also called c-neu) or Hras1 impaired mammary carcinogenesis, whereas reduced expression of p16 and p19 by methylation-induced silencing or inactivation of p38 MAPK correlated with tumor appearance. We conclude that inactivation or depletion of the Wip1 phosphatase with resultant p38 MAPK activation suppresses tumor appearance by modulating the Cdkn2a tumor-suppressor locus.

Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas.

Loss or mutation of p53 is thought to be an early event in the malignant transformation of many human astrocytic tumors. To better understand the role of p53 in their growth and transformation, we developed a model employing cultured neonatal astrocytes derived from mice deficient in one (p53 +/-) or both (p53 -/-) p53 alleles, comparing them with wild-type (p53 +/+) cells. Studies of in vitro and in vivo growth and transformation were performed, and flow cytometry and karyotyping were used to correlate changes in growth with genomic instability. Early-passage (EP) p53 -/- astrocytes achieved higher saturation densities and had more rapid growth than EP p53 +/- and +/+ cells. The EP p53 -/- cells were not transformed, as they were unable to grow in serum-free medium or in nude mice. With continued passaging, p53 -/- cells exhibited a multistep progression to a transformed phenotype. Late-passage p53 -/- cells achieved saturation densities 50 times higher than those of p53 +/+ cells and formed large, well-vascularized tumors in nude mice. p53 +/- astrocytes exhibited early loss of the remaining wild-type p53 allele and then evolved in a manner phenotypically similar to p53 -/- astrocytes. In marked contrast, astrocytes retaining both wild-type p53 alleles never exhibited a transformed phenotype and usually senesced after 7 to 10 passages. Dramatic alterations in ploidy and karyotype occurred and were restricted to cells deficient in wild-type p53 following repeated passaging. The results of these studies suggest that loss of wild-type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes.

Absence of p53: No Effect in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Familial amyotrophic lateral sclerosis (ALS) has been linked in some families to dominantly inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (Cu-Zn SOD1). Transgenic mice expressing a mutant human Cu-Zn SOD1 (G93A) develop a dominantly inherited adult-onset paralytic disorder that replicates many of the clinical and pathological features of familial ALS. Increased p53 immunoreactivity has been reported in the motor cortex and spinal ventral horns of postmortem tissue from ALS patients. The nuclear phosphoprotein p53 is an important regulator of cellular proliferation, and increasing evidence supports the role of p53 in regulating cellular apoptosis. To assess the role of p53-mediated apoptosis in amyotrophic lateral sclerosis, mice deficient in both p53 alleles (p53-/-) were crossed with transgenic mice expressing the G93A mutant (G93A+), creating novel transgenic knockout mice. The animals (p53 +/+G93A+, p53+/-G93A+, p53-/-G93A+) were examined at regular intervals for cage activity, upper and lower extremity strength, and mortality. At 120 days from birth mice from each genotype were sacrificed, and L2-L3 anterior horn motor neurons were counted. There was no significant difference in time to onset of behavioral decline, mortality, or motor neuron degeneration between the different genotypes. Despite evidence that p53 plays an important role after acute neuronal injury, the current study suggests that p53 is not significantly involved in cell death in the G93A+ transgenic mouse model of familial ALS.

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

Allelic phasing of a mouse chromosome 11 deficiency influences p53 tumorigenicity

Most tumour suppressor genes (TSGs) have been found through linkage studies in cancer predisposed families where the mutations have a high penetrance, for example, the breast cancer genes BRCA1 and BRCA2. Loss of heterozygosity (LOH) analyses of sporadic breast tumours indicate that there are many other putative TSGs yet to be identified. One such locus is proximal to BRCA1 on human chromosome 17q21. In an attempt to isolate this putative TSG, we have assessed a portion of the orthologous region on mouse chromosome 11 for its tumorigenic potential using segmental haploidy in combination with a p53 mutation. Two populations of animals were studied, with the deleted region being either on the same (cis) or on the homologous chromosome (trans) to a targeted mutant p53 allele. The deficiency elevated the tumour susceptibility of p53 heterozygous mice and modified the tumour spectrum, but only when the deficiency was in trans with the p53 mutation. Even though the genotype of these mice is identical, allelic phasing affects both the tumour spectrum and progression.

Abstract A70: Targeting pediatric bone sarcoma stem cell with metabolic inhibitors

Our project tests whether metabolic inhibitors are able to target putative sarcoma stem cells in osteosarcoma and Ewing sarcoma. The cure rate for the two most common bone sarcomas affecting children and adolescents, osteosarcoma and Ewing sarcoma, has plateaued around 70% over the past twenty years, and the cure rate for patients with metastatic bone sarcomas has not significantly improved over 20% in several decades. The subpopulation of cells in these tumors referred to as sarcoma stem cells are thought to be responsible for resistance to current therapies, relapses and metastases. To date therapies targeting the sarcoma stem cell population have not been identified. We studied the ability of metabolic inhibitors to eliminate the sarcoma stem cell population. A panel of established and verified osteosarcoma (HOS, 143B, MNNG, SAOS2) and Ewing sarcoma (TC71, TC32, MHH, A4573, CHLA9, CHLA10) cell lines was grown in 2-dimensional culture and treated with metabolic inhibitors, including 2-deoxyglucose, dichloroacetate, oxamate, lonidamine, 3-Bromopyruvate, and metformin for 72hrs establishing dose response curves for each individual agent. Most agents showed IC50s in the millimolar range. Combinations of two metabolic inhibitors at their IC50 were then tested and evaluated for synergy and if the combination would allow for dosing at physiologically attainable concentrations. The combination of 2-deoxyglucose and metformin proved toxic to the osteosarcoma and Ewing sarcoma cell lines (p The cells were then grown in 3-dimensional cultures using Magnetic 3D Bioprinting kit(n3D Biosciences, Houston, TX) and exposed to physiologically attainable concentrations of 2-deoxyglucose (500uM) and metformin (5uM), based on Phase I studies of these individual agents in humans. The resulting 3-dimentional cell cultures were incubated for 10 days with 2-deoxyglucose and metformin and there was a significant reduction in cell proliferation as measured by trypan blue dye exclusion and reduced aldehyde dehydrogenase expression. Additionally, the functional assay of cancer stem cells, sphere-forming efficiency, was impeded in all cell lines treated with the combination of 2-deoxyglucose and metformin. Finally, the ability to establish orthotopic tumors in immune deficient mice was significantly reduced for intratibialy-injected cells pretreated with 2-deoxyglucose and metformin. In vivo testing of the combination of 2-deoxyglucose and metformin to treat established xenograft osteosarcoma and Ewing sarcomas is currently underway, and resulting tumors will be harvested and assessed for aldehyde dehydrogenase expression, sphere-forming efficiency, and further characterization of the stem cell properties and metabolic alterations. Our study will provide the preclinical basis for testing metabolic inhibitors, in particular the combination of 2-deoxyglucose and metformin, in clinical trials for pediatric bone sarcomas. Citation Format: Matteo Trucco, Nino Rainsusso, Piti Techavichit, Ronald Bernardi, Ryan Shuck, Laura Satterfield, Wendy Allen-Rhoades, Larry Donehower, David Loeb, Jason Yustein. Targeting pediatric bone sarcoma stem cell with metabolic inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A70.

Abstract 5036: Oncogenic role of Runx2 in the development of osteosarcoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnOsteosarcoma (OS) is a potentially fatal bone malignancy principally affecting adolescents or young adults, and comprises about 3-4% of all pediatric tumors. Although the overall survival rate has approached 70% in patients with localized disease, outcomes are still inferior compared to most other pediatric malignancies and significant improvements have been difficult to obtain over the past 20 years. Current treatment for OS includes a combination of neoadjuvant chemotherapy and surgery, with patients being monitored for recurrence following treatment. The etiology of OS is not well defined; however, most OS tumors arise sporadically, and exhibit high genome instability and chromosomal abnormalities. One gene often found abnormally expressed in osteosarcoma is the transcription factor Runx2. Runx2 is the master regulatory protein of bone development, and activates the transcription of the genes necessary for osteoblast differentiation. Due to this obvious importance in normal bone development, we have hypothesized that Runx2 also has a pronounced role in osteosarcoma development and progression. Evidence to support this hypothesis include: (1) a report that Runx2 was the most significantly gained gene in copy number, resulting in a 9-fold over-expression in human osteosarcoma tumors; (2) patients with tumors expressing higher amounts of Runx2 have an overall poorer survival rate. Dysregulation of Runx2 results in constitutively high protein levels, however, the consequence of over-expression of Runx2 in osteosarcoma remains unclear. Our studies have focused on investigating the biological and functional role of Runx2 in OS. We have shown that the transcriptional activity of Runx2 is increased in osteosarcoma cells when compared to normal osteoblasts. Additionally, we have shown that the knockdown of Runx2 results in reduced OS cell and tumor growth in vitro and in vivo, respectively. Our functional data is corroborated by gene expression analysis, which shows Runx2 regulates multiple cellular pathways involved in cell cycle regulation, cell death, and proliferation. Moreover, our gene expression analysis reveals that Runx2 regulates pathways involved in drug metabolism, and we have shown that knockdown of Runx2 sensitizes cells to treatment with chemotherapeutics. We have provided a mechanism for the dox-induced apoptosis in our Runx2 knockdown, and have shown that c-Myc plays a role in the activation of caspase-3 after dox treatment, and inhibition of c-Myc results in a significant decrease in apoptosis in shRunx2 cells when compared to control cells. Thus, we have shown a novel role chemoprotective role for Runx2 in OS, and suggest that inhibitors of Runx2 are an encouraging therapeutic prospect for patients with OS.nnCitation Format: Alison Roos, Jason T. Yustein, Larry Donehower. Oncogenic role of Runx2 in the development of osteosarcoma. [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 5036. doi:10.1158/1538-7445.AM2013-5036nnNote: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.