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Epigenetic dysregulation is involved in the initiation and progression of many epithelial cancers. BMI1, a component of the polycomb protein family, plays a key role in these processes by controlling the histone ubiquitination and long-term repression of multiple genomic loci. BMI1 has previously been implicated in pancreatic homeostasis and the function of pancreatic cancer stem cells. However, no work has yet addressed its role in the early stages of pancreatic cancer development. Here, we show that BMI1 is required for the initiation of murine pancreatic neoplasia using a novel conditional knockout of Bmi1 in combination with a Kras(G12D)-driven pancreatic cancer mouse model. We also demonstrate that the requirement for Bmi1 in pancreatic carcinogenesis is independent of the Ink4a/Arf locus and at least partially mediated by dysregulation of reactive oxygen species. Our data provide new evidence of the importance of this epigenetic regulator in the genesis of pancreatic cancer.

C. Akay, K. A. Lindl, N. Shyam, B. Nabet, Y. Goenaga-Vazquez, J. Ruzbarsky, Y. Wang, D. L. Kolson and K. L. Jordan-Sciutto (2012) Neuropathology and Applied Neurobiology38, 175–200 Activation status of integrated stress response pathways in neurones and astrocytes of HIV-associated neurocognitive disorders (HAND) cortex Aims: Combined anti-retroviral therapy (cART) has led to a reduction in the incidence of HIV-associated dementia (HAD), a severe motor/cognitive disorder afflicting HIV(+) patients. However, the prevalence of subtler forms of neurocognitive dysfunction, which together with HAD are termed HIV-associated neurocognitive disorders (HAND), continues to escalate in the post-cART era. The microgliosis, astrogliosis, dendritic damage, and synaptic and neuronal loss observed in autopsy cases suggest an underlying neuroinflammatory process, due to the neurotoxic factors released by HIV-infected/activated macrophages/microglia in the brain, might underlie the pathogenesis of HAND in the post-cART era. These factors are known to induce the integrated stress response (ISR) in several neurodegenerative diseases; we have previously shown that BiP, an indicator of general ISR activation, is upregulated in cortical autopsy tissue from HIV-infected patients. The ISR is composed of three pathways, each with its own initiator protein: PERK, IRE1α and ATF6. Methods: To further elucidate the specific ISR pathways activated in the central nervous system of HAND patients, we examined the protein levels of several ISR proteins, including ATF6, peIF2α and ATF4, in cortical tissue from HIV-infected patients. Results: The ISR does not respond in an all-or-none fashion in HAND, but rather demonstrates a nuanced activation pattern. Specifically, our studies implicate the ATF6 pathway of the ISR as a more likely candidate than the PERK pathway for increases in BiP levels in astrocytes. Conclusion: These findings begin to characterize the nature of the ISR response in HAND and provide potential targets for therapeutic intervention in this disease.

Malignant rhabdoid tumors (MRTs) are aggressive tumors associated with mutations in the SMARCB1 gene. In experimental systems, the loss of SMARCB1 is hypothesized to alter p16(INK4A) pathways resulting in the repression of tumor suppressors. To determine whether these pathways are deregulated in human MRT, we used immunohistochemistry on tissue microarrays to evaluate p16(INK4A)/E2F1/RB and p14(ARF)/MDM2/p53 pathways in 25 atypical teratoid/rhabdoid tumors (AT/RT) and 11 non-CNS MRT. p16(INK4A) was negative or showed focal weak expression. p16(INK4A) downstream targets CDK4/cyclin D1/ppRB were variably expressed at moderate to low levels; E2F1 was negative. Unexpectedly, p14(ARF) expression was seen in many cases, which correlated positively with p53 and inversely with MDM2 immunostaining in AT/RT. TP53 mutational analysis in 19 of 25 AT/RT and in 8 of 11 non-CNS MRT cases showed point mutations in only 3 AT/RT cases, suggesting that p53 expression was driven mainly by p14(ARF). Finally, nucleophosmin, a protein that stabilizes p53, was positive in most cases and colocalized with p53. Together, these data suggest that, in MRT, there is deregulation not only of p16(INK4A) but also of the p14(ARF) pathway. These results provide insights into cell cycle deregulation in the pathogenesis of human MRT and may aid in the design and evaluation of potential therapies for these tumors.

As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV-associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary postmitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By Q-PCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, Cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19(ARF)) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19(ARF), Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Many characteristic mutations in PDAC are known, but this information has so far failed to produce the development of effective treatments, highlighting the need for deeper understanding of the processes that drive tumorigenesis. B-cell specific Moloney murine leukemia virus insertion site 1 (Bmi1), a Polycomb repressive group protein, is upregulated in PDAC and associated with poor prognosis. Our lab has shown that despite an oncogenic K-ras mutation, mice with pancreas specific loss of Bmi1 do not develop precancerous lesions, termed PanINs (Pancreatic Intraepithelial Neoplasia). This lack of PanIN development was also seen in the presence of pancreatitis, which is usually known to synergize with oncogenic K-ras to speed PanIN development. In other cancer types, Bmi1's effect on tumorigenesis was mechanistically linked to its regulation of the Ink4a/ARF genetic locus. However, we found that in PDAC, PanIN initiation was independent of Bmi1 control this locus. Further, impairment in the regulation of ROS generation was seen in vitro in pancreatic cancer cell lines lacking Bmi1. Regulating ROS generation is a vital step in the neoplastic process and has been shown in other systems to be controlled by Bmi1. Overall, in this work we have shown that expression of the Polycomb group protein Bmi1 is necessary for the initiation of pancreatic precancerous lesions, and that the mechanism of Bmi1 requirement is independent of its repression of the Ink4a/ARF genetic locus. Given the recent pre-clinical development of a Bmi1 inhibitor, this work could provide rationale for future treatment of pancreatic cancer, a truly devastating disease. Citation Format: Heather Schofield, Filip Bednar, Meredith Collins, Wei Yan, Yaqing Zhang, Nikhil Shyam, Jaime Eberle, Kenneth Olive, Nabeel Bardeesy, Daisuke Nakada, Diane Simeone, Sean Morrison, Marina Pasca di Magliano. Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-061. doi:10.1158/1538-7445.AM2015-LB-061