Search

Purpose: Renal and hematological toxicity are side effects and dose-limiting factors of Peptide Receptor Radionuclide Therapy (PRRT). We aimed to assess the changes in renal and hematological function and associations with survival in neuroendocrine tumor (NET) patients treated with PRRT., Methods: A retrospective cohort of 448 NET patients treated with either 177 Lu-DOTATATE or 90 Y-DOTATOC were followed for changes of renal and hematological function. Renal function was assessed by monitoring changes in serum creatinine, blood urea nitrogen and estimated glomerular filtration rate. Hematological function was determined by examining changes in white blood cell counts (WBC), platelet counts, and hemoglobin levels over time. Piecewise linear mixed effect models were applied to model the longitudinal repeated measurements of renal and hematological function. Overall survival (OS) and progression-free survival (PFS) were modelled using Cox proportional hazard regressions., Results: Of the 448 PRRT treated patients, 335 received 177 Lu-DOTATATE (74.78%) and 113 were treated with 90 Y-DOTATOC (25.22%). Comparing patients treated with 177 Lu-DOTATATE to those treated with 90 Y-DOTATOC, renal function did not differ significantly prior to, during or after PRRT. Compared with patients treated with 90 Y-DOTATOC, significantly decreased indicators of hematological function were observed in those treated with 177 Lu-DOTATATE prior to and during PRRT treatment (WBC: estimate, -0.10, 95% CI, -0.15 to -0.05; P < 0.001; platelet count: estimate, -2.53, 95% CI, -3.83 to -1.24; P < 0.001), and no significant recovery was observed in hematological function post PRRT. Individuals who received 177 Lu-DOTATATE tended to have a longer PFS (hazard ratio, 0.47, 95%CI: 0.28-0.79, P = 0.004) compared with 90 Y-DOTATOC, but there was no difference in OS., Conclusion: There was no significant renal, but minor hematological toxicity, in patients treated with 177 Lu-DOTATATE compared with 90 Y-DOTATOC. Compared to 90 Y-DOTATOC, 177 Lu-DOTATATE appears to enhance PFS, but not OS. Treatment with 177 Lu-DOTATATE may necessitate follow-up for hematological toxicity irrespective of other therapies prior to PRRT., (© 2024. The Author(s).)

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, Ras-driven sarcomas characterized by loss of the NF1 tumor suppressor gene and hyperactivation of MEK and CDK4/6 kinases. MPNSTs lack effective therapies. We recently demonstrated remarkable efficacy of dual CDK4/6-MEK inhibition in mice with de novo MPNSTs, which was heightened by combined targeting of the immune checkpoint protein, PD-L1. The triple combination therapy targeting CDK4/6, MEK, and PD-L1 led to extended MPNST regression and improved survival, although most tumors eventually acquired drug resistance. Here, we consider the immune activation phenotype caused by CDK4/6-MEK inhibition in MPNSTs that uniquely involved intratumoral plasma cell accumulation. We discuss how PD-L1 and FOXM1, a tumor-promoting transcription factor, are functionally linked and may be key mediators of resistance to CDK4/6-MEK targeted therapies. Finally, the role of FOXM1 in suppressing anti-tumor immunity and potentially thwarting immune-based therapies is considered. We suggest that future therapeutic strategies targeting the oncogenic network of CDK4/6, MEK, PD-L1, and FOXM1 represent exciting future treatment options for MPNST patients.

The Iowa Neuroendocrine Tumor (NET) Clinic was founded and developed by two remarkable physicians, Thomas and Sue O'Dorisio. Tom was an Endocrinologist and close friend and colleague of Aaron Vinik. Both men were pioneers in studies of gastrointestinal hormones and the management of patients with NETs. Sue was a Pediatric Oncologist and research scientist with great expertise in new drug development and clinical trials. She and Tom were leaders in bringing somatostatin analogs and somatostatin-conjugated radioligands to the clinic for the therapy and diagnosis of NETs. All three physicians received lifetime achievement awards for their contributions to the field of NETs. This is the story of how the Iowa NET Clinic developed over the years to become a model for the multidisciplinary mantagement of patients with NETs, culminating in its designation as a European Neuroendocrine Tumor Society NET Center of Excellence, and the receipt of a Specialized Project of Research Excellence (SPORE) grant for the study of NETs from the National Institutes of Health., (Copyright © 2024 AACE. Published by Elsevier Inc. All rights reserved.)

Functional copy-number alterations (fCNAs) are DNA copy-number changes with concordant differential gene expression. These are less likely to be bystander genetic lesions and could serve as robust and reproducible tumor biomarkers. To identify candidate fCNAs in neuroendocrine tumors (NETs), we integrated chromosomal microarray (CMA) and RNA-seq differential gene-expression data from 31 pancreatic (pNETs) and 33 small-bowel neuroendocrine tumors (sbNETs). Tumors were resected from 47 early-disease-progression (<24 months) and 17 late-disease-progression (>24 months) patients. Candidate fCNAs that accurately differentiated these groups in this discovery cohort were then replicated using fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded (FFPE) tissues in a larger validation cohort of 60 pNETs and 82 sbNETs (52 early- and 65 late-disease-progression samples). Logistic regression analysis revealed the predictive ability of these biomarkers, as well as the assay-performance metrics of sensitivity, specificity, and area under the curve. Our results indicate that copy-number changes at chromosomal loci 4p16.3, 7q31.2, 9p21.3, 17q12, 18q21.2, and 19q12 may be used as diagnostic and prognostic NET biomarkers. This involves a rapid, cost-effective approach to determine the primary tumor site for patients with metastatic liver NETs and to guide risk-stratified therapeutic decisions.

Cognitive or motor impairment is common among individuals with neurofibromatosis type 1 (NF1), an autosomal dominant tumor-predisposition disorder. As many as 70% of children with NF1 report difficulties with spatial/working memory, attention, executive function, and fine motor movements. In contrast to the utilization of various Nf1 mouse models, here we employ an NF1 +/ex42del miniswine model to evaluate the mechanisms and characteristics of these presentations, taking advantage of a large animal species more like human anatomy and physiology. The prefrontal lobe, anterior cingulate, and hippocampus from NF1 +/ex42del and wild-type miniswine were examined longitudinally, revealing abnormalities in mature oligodendrocytes and astrocytes, and microglial activation over time. Imbalances in GABA: Glutamate ratios and GAD67 expression were observed in the hippocampus and motor cortex, supporting the role of disruption in inhibitory neurotransmission in NF1 cognitive impairment and motor dysfunction. Moreover, NF1 +/ex42del miniswine demonstrated slower and shorter steps, indicative of a balance-preserving response commonly observed in NF1 patients, and progressive memory and learning impairments. Collectively, our findings affirm the effectiveness of NF1 +/ex42del miniswine as a valuable resource for assessing cognitive and motor impairments associated with NF1, investigating the involvement of specific neural circuits and glia in these processes, and evaluating potential therapeutic interventions., (© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Malignant peripheral nerve sheath tumors (MPNSTs) are deadly sarcomas, which desperately need effective therapies. Half of all MPNSTs arise in patients with neurofibromatosis type I (NF1), a common inherited disease. NF1 patients can develop benign lesions called plexiform neurofibromas (PNFs), often in adolescence, and over time, some PNFs, but not all, will transform into MPNSTs. A deeper understanding of the molecular and genetic alterations driving PNF-MPNST transformation will guide development of more targeted and effective treatments for these patients. This review focuses on an oncogenic transcription factor, FOXM1, which is a powerful oncogene in other cancers but little studied in MPNSTs. Elevated expression of FOXM1 was seen in patient MPNSTs and correlated with poor survival, but otherwise, its role in the disease is unknown. We discuss what is known about FOXM1 in MPNSTs relative to other cancers and how FOXM1 may be regulated by and/or regulate the most commonly altered players in MPNSTs, particularly in the MEK and CDK4/6 kinase pathways. We conclude by considering FOXM1, MEK, and CDK4/6 as new, clinically relevant targets for MPNST therapy.

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models., Experimental Design: Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response., Results: Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression., Conclusions: CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes., (©2023 American Association for Cancer Research.)

The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pretreatment for anticancer therapies. In a variation of this idea, this study explores the concept of adding low-dose decitabine (DAC) following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of DAC following treatment with the chemotherapy agent gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, DAC did not induce a robust antitumor T cell response in sarcoma. Furthermore, DAC synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces biphasic cell cycle arrest and apoptosis. Therapeutic efficacy was sequence dependent, with gemcitabine priming cells for treatment with DAC through inhibition of ribonucleotide reductase. This study identifies an apparently unique application of DAC to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising paradigm for cancer treatment by augmenting chemotherapy through addition of DAC to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.

Objective: Inflammation is present in many diseases and identification of immune cell infiltration is a common assessment. CD138 (syndecan-1) is a recommended immunohistochemical marker for human plasmacytes although it is also expressed in various epithelia and tumors. Similarly, CD138 is a marker for murine plasmacytes, but its tissue immunostaining is not well-defined. Endogenous CD138 expression is an important confounding factor when evaluating plasmacyte infiltration. We studied two plasmacyte markers (CD138 and Kappa light chains) for endogenous immunostaining in five organs and one tumor from B6 mice., Results: Plasmacytes in Peyer's patches were positive for CD138 and Kappa markers without endogenous immunostaining. Endogenous CD138 immunostaining was widespread in liver, kidney, lung and a malignant peripheral nerve sheath tumor (MPNST) versus regionalized immunostaining in skin and small intestine wall. Endogenous Kappa immunostaining was absent in all tissues except for plasmacytes. Tissues with widespread endogenous CD138 immunostaining were contrasted by absence of endogenous Kappa immunostaining. Here, plasmacytes would not be distinguished by CD138, but would be obvious by Kappa immunostaining. Our study suggests that utility of immunostaining for plasmacytes by CD138 is tissue dependent in mice. Additionally, Kappa immunostaining may be a useful alternative in mouse tissues with confounding endogenous CD138 immunostaining., (© 2022. The Author(s).)

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted along with NF1 , a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of MPNSTs is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown., Methods: Using CRISPR-Cas9 targeting of Nf1 + Cdkn2a or Nf1 + Tp53 in the mouse sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Terminal tumors were evaluated by western blot, qRT-PCR, and immunohistochemistry., Results: Mice lacking Rabl6 displayed slower tumor progression and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in Rabl6 -null, Nf1 + Cdkn2a lesions whereas loss of RABL6A caused upregulation of the CDK inhibitor, p27, in all tumors. Paradoxically, both models displayed elevated Myc protein and Ki67 staining in terminal tumors lacking RABL6A. In Nf1 + p53 tumors, cellular atypia and polyploidy were evident and increased by RABL6A loss., Conclusions: These findings demonstrate that RABL6A is required for optimal progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for unwanted alterations, including increased Myc, cellular atypia, and polyploidy, that ultimately promote a hyper-proliferative tumor phenotype akin to drug-resistant lesions., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Pancreatic neuroendocrine tumors (pNETs) are unique, slow-growing malignancies whose molecular pathogenesis is incompletely understood. With rising incidence of pNETs over the last four decades, larger and more comprehensive 'omic' analyses of patient tumors have led to a clearer picture of the pNET genomic landscape and transcriptional profiles for both primary and metastatic lesions. In pNET patients with advanced disease, those insights have guided the use of targeted therapies that inhibit activated mTOR and receptor tyrosine kinase (RTK) pathways or stimulate somatostatin receptor signaling. Such treatments have significantly benefited patients, but intrinsic or acquired drug resistance in the tumors remains a major problem that leaves few to no effective treatment options for advanced cases. This demands a better understanding of essential molecular and biological events underlying pNET growth, metastasis, and drug resistance. This review examines the known molecular alterations associated with pNET pathogenesis, identifying which changes may be drivers of the disease and, as such, relevant therapeutic targets. We also highlight areas that warrant further investigation at the biological level and discuss available model systems for pNET research. The paucity of pNET models has hampered research efforts over the years, although recently developed cell line, animal, patient-derived xenograft, and patient-derived organoid models have significantly expanded the available platforms for pNET investigations. Advancements in pNET research and understanding are expected to guide improved patient treatments.

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

Schwann cells are normally quiescent, myelinating glia cells of the peripheral nervous system. Their aberrant proliferation and transformation underlie the development of benign tumors (neurofibromas) as well as deadly malignant peripheral nerve sheath tumors (MPNSTs). We discovered a new driver of MPNSTs, an oncogenic GTPase named RABL6A, that functions in part by inhibiting the RB1 tumor suppressor. RB1 is a key mediator of cellular senescence, a permanent withdrawal from the cell cycle that protects against cell immortalization and transformation. Based on the RABL6A-RB1 link in MPNSTs, we explored the hypothesis that RABL6A promotes Schwann cell proliferation and abrogates their senescence by inhibiting RB1. Using sequentially passaged normal human Schwann cells (NHSCs), we found that the induction of replicative senescence was associated with reduced expression of endogenous RABL6A. Silencing RABL6A in low passage NHSCs caused premature stress-induced senescence, which was largely rescued by co-depletion of RB1. Consistent with those findings, Rabl6 -deficient MEFs displayed impaired proliferation and accelerated senescence compared to wildtype MEFs. These results demonstrate that RABL6A is required for maintenance of proper Schwann cell proliferation and imply that aberrantly high RABL6A expression may facilitate malignant transformation.

Pancreatic neuroendocrine neoplasms (pNENs) are slow growing cancers of increasing incidence that lack effective treatments once they become metastatic. Unfortunately, nearly half of pNEN patients present with metastatic liver tumors at diagnosis and current therapies fail to improve overall survival. Pre-clinical models of pNEN metastasis are needed to advance our understanding of the mechanisms driving the metastatic process and for the development of novel, targeted therapeutic interventions. To model metastatic dissemination of tumor cells, human pNEN cell lines (BON1 and Qgp1) stably expressing firefly luciferase (luc) were generated and introduced into NSG immunodeficient mice by intracardiac (IC) or intravenous (IV) injection. The efficiency, kinetics and distribution of tumor growth was evaluated weekly by non-invasive bioluminescent imaging (BLI). Tumors formed in all animals in both the IC and IV models. Bioluminescent Qgp1.luc cells preferentially metastasized to the liver regardless of delivery route, mimicking the predominant site of pNEN metastasis in patients. By comparison, BON1.luc cells most commonly formed lung tumors following either IV or IC administration and colonized a wider variety of tissues than Qgp1.luc cells. These models provide a unique platform for testing candidate metastasis genes and anti-metastatic therapies for pNENs.

Additional prognostic and therapeutic biomarkers effective across different histological types of sarcoma are needed. Herein we evaluate expression of TAZ and YAP, the p53-MDM2 axis, and RABL6A, a novel oncoprotein with potential ties to both pathways, in sarcomas of different histological types. Immunohistochemical staining of a tissue microarray including 163 sarcomas and correlation with clinical data showed that elevated YAP and TAZ independently predict worse overall and progression-free survival, respectively. In the absence of p53 expression, combined TAZ and YAP expression adversely affect overall, progression free, and metastasis free survival more than TAZ or YAP activation alone. RABL6A independently predicted shorter time to metastasis and was positively correlated with p53, MDM2 and YAP expression, supporting a possible functional relationship between the biomarkers. Network analysis further showed that TAZ is positively correlated with MDM2 expression. The data implicate all five proteins as clinically relevant downstream players in the Hippo pathway. Finally, a novel inhibitor of MDM2 (MA242), effectively suppressed the survival of sarcoma cell lines from different histological types regardless of p53 status. These findings suggest both independent and cooperative roles for all five biomarkers across different histological types of sarcoma in predicting patient outcomes and potentially guiding future therapeutic approaches., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Desai et al.)

Precision medicine relies on a detailed molecular understanding of disease pathogenesis. Here, we consider urgently needed therapeutic options for malignant peripheral nerve sheath tumors (MPNSTs) based on emerging insights into druggable pathway alterations found to drive this deadly cancer. Recent observations demonstrate an essential role for an oncogenic GTPase, RABL6A, in promoting MPNST progression through hyperactivation of cyclin-dependent kinases (CDKs) and inactivation of the retinoblastoma (RB1) tumor suppressor. Monotherapies with CDK4/6 inhibitors have shown limited efficacy and durability in pre-clinical studies of MPNSTs and in clinical studies of other tumors. Therefore, we discuss the rationale and clinical benefits of inhibiting multiple RABL6A effectors, particularly CDK4/6 and MEK kinases, in targeted combination therapies suitable for MPNSTs and other Ras-driven malignancies., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Kohlmeyer et al.)

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs., Experimental Design: We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors., Results: RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G 1 arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in vitro in an RABL6A-dependent manner and suppressed MPNST growth in vivo . Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation., Conclusions: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs., (©2020 American Association for Cancer Research.)

Sarcomas represent one of the most challenging tumor types to treat due to their diverse nature and our incomplete understanding of their underlying biology. Recent work suggests cyclin-dependent kinase (CDK) pathway activation is a powerful driver of sarcomagenesis. CDK proteins participate in numerous cellular processes required for normal cell function, but their dysregulation is a hallmark of many pathologies including cancer. The contributions and significance of aberrant CDK activity to sarcoma development, however, is only partly understood. Here, we describe what is known about CDK-related alterations in the most common subtypes of sarcoma and highlight areas that warrant further investigation. As disruptions in CDK pathways appear in most, if not all, subtypes of sarcoma, we discuss the history and value of pharmacologically targeting CDKs to combat these tumors. The goals of this review are to (1) assess the prevalence and importance of CDK pathway alterations in sarcomas, (2) highlight the gap in knowledge for certain CDKs in these tumors, and (3) provide insight into studies focused on CDK inhibition for sarcoma treatment. Overall, growing evidence demonstrates a crucial role for activated CDKs in sarcoma development and as important targets for sarcoma therapy.

Purpose: Pancreatic neuroendocrine tumors (pNETs) are uncommon malignancies noted for their propensity to metastasize and comparatively favorable prognosis. Although both the treatment options and clinical outcomes have improved in the past decades, most patients will die of metastatic disease. New systemic therapies are needed., Experimental Design: Tissues were obtained from 43 patients with well-differentiated pNETs undergoing surgery. Gene expression was compared between primary tumors versus liver and lymph node metastases using RNA-Seq. Genes that were selectively elevated at only one metastatic site were filtered out to reduce tissue-specific effects. Ingenuity pathway analysis (IPA) and the Connectivity Map (CMap) identified drugs likely to antagonize metastasis-specific targets. The biological activity of top identified agents was tested in vitro using two pNET cell lines (BON-1 and QGP-1)., Results: A total of 902 genes were differentially expressed in pNET metastases compared with primary tumors, 626 of which remained in the common metastatic profile after filtering. Analysis with IPA and CMap revealed altered activity of factors involved in survival and proliferation, and identified drugs targeting those pathways, including inhibitors of mTOR, PI3K, MEK, TOP2A, protein kinase C, NF-kB, cyclin-dependent kinase, and histone deacetylase. Inhibitors of MEK and TOP2A were consistently the most active compounds., Conclusions: We employed a complementary bioinformatics approach to identify novel therapeutics for pNETs by analyzing gene expression in metastatic tumors. The potential utility of these drugs was confirmed by in vitro cytotoxicity assays, suggesting drugs targeting MEK and TOP2A may be highly efficacious against metastatic pNETs. This is a promising strategy for discovering more effective treatments for patients with pNETs., (©2020 American Association for Cancer Research.)

Neurofibromatosis type 1 (NF1) is a rare, autosomal dominant disease with variable clinical presentations. Large animal models are useful to help dissect molecular mechanisms, determine relevant biomarkers, and develop effective therapeutics. Here, we studied a NF1 minipig model (NF1 +/ex42del ) for the first 12 months of life to evaluate phenotype development, track disease progression, and provide a comparison to human subjects. Through systematic evaluation, we have shown that compared to littermate controls, the NF1 model develops phenotypic characteristics of human NF1: [1] café-au-lait macules, [2] axillary/inguinal freckling, [3] shortened stature, [4] tibial bone curvature, and [5] neurofibroma. At 4 months, full body computed tomography imaging detected significantly smaller long bones in NF1 +/ex42del minipigs compared to controls, indicative of shorter stature. We found quantitative evidence of tibial bowing in a subpopulation of NF1 minipigs. By 8 months, an NF1 +/ex42del boar developed a large diffuse shoulder neurofibroma, visualized on magnetic resonance imaging, which subsequently grew in size and depth as the animal aged up to 20 months. The NF1 +/ex42del minipig model progressively demonstrates signature attributes that parallel clinical manifestations seen in humans and provides a viable tool for future translational NF1 research.

Genetically modified swine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given the limited history of these model systems, there remains a great need for proven molecular reagents in swine tissue. Here, to provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers in a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumor predisposition disorder stemming from mutations in NF1, a gene that encodes the Ras-GTPase activating protein neurofibromin. Patients classically present with benign neurofibromas throughout their bodies and can also present with neurological associated symptoms such as chronic pain, cognitive impairment, and behavioral abnormalities. As validated antibodies for immunohistochemistry applications are particularly difficult to find for swine models of neurological disease, we present immunostaining validation of antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4 and Olig2), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These immunostaining protocols for CNS markers provide a useful resource to the scientific community, furthering the utility of genetically modified miniswine for translational and clinical applications., Competing Interests: Our association with Sanford Research does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors have declared that no competing interests exist. The specific roles of these authors are articulated in the ‘author contributions’ section.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder resulting from germline mutations in the NF1 gene, which encodes neurofibromin. Patients experience a variety of symptoms, but pain in the context of NF1 remains largely underrecognized. Here, we characterize nociceptive signaling and pain behaviors in a miniswine harboring a disruptive NF1 mutation (exon 42 deletion). We present the first characterization of pain-related behaviors in a pig model of NF1, identifying unchanged agitation scores, lower tactile thresholds (allodynia), and decreased response latencies to thermal laser stimulation (hyperalgesia) in NF1 (females only) pigs. Male NF1 pigs with tumors showed reduced sleep quality and increased resting, 2 health-related quality-of-life symptoms found to be comorbid in people with NF1 pain. We explore these phenotypes in relationship to suppression of the increased activity of the N-type voltage-gated calcium (CaV2.2) channel by pharmacological antagonism of phosphorylation of a regulatory protein-the collapsin response mediator protein 2 (CRMP2), a known interactor of neurofibromin, and by targeting the interface between the α subunit of CaV2.2 and the accessory β-subunits with small molecules. Our data support the use of NF1 pigs as a large animal model for studying NF1-associated pain and for understanding the pathophysiology of NF1. Our findings demonstrate the translational potential of 2 small molecules in reversing ion channel remodeling seen in NF1. Interfering with CaV2.2, a clinically validated target for pain management, might also be a promising therapeutic strategy for NF1-related pain management.

Background: This study explores whether objective, quantitative radiomic biomarkers derived from magnetic resonance (MR), positron emission tomography (PET), and computed tomography (CT) may be useful in reliably distinguishing malignant peripheral nerve sheath tumors (MPNST) from benign plexiform neurofibromas (PN)., Methods: A registration and segmentation pipeline was established using a cohort of NF1 patients with histopathological diagnosis of PN or MPNST, and medical imaging of the PN including MR and PET-CT. The corrected MR datasets were registered to the corresponding PET-CT via landmark-based registration. PET standard-uptake value (SUV) thresholds were used to guide segmentation of volumes of interest: MPNST-associated PET-hot regions (SUV≥3.5) and PN-associated PET-elevated regions (2.0

Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.

Mutations in the "guardian of the genome" TP53 predominate in solid tumors. In addition to loss of tumor suppressor activity, a specific subset of missense mutations confers additional oncogenic properties. These "gain-of-function" (GOF) mutations portend poor prognosis across cancer types regardless of treatment. Our objective in this study was to identify novel therapeutic opportunities to overcome the deleterious effects of GOF TP53 mutants. Using gynecologic cancer cell lines with known TP53 mutational status, we established that treatment with a proteasome inhibitor induced cell death in cells with two recurrent GOF TP53 mutations (R175H and R248Q), and addition of a histone deacetylase inhibitor (HDACi) enhanced this effect. By contrast, p53-null cancer cells were relatively resistant to the combination. Proteasome inhibition promoted apoptosis of cells with TP53 GOF mutations, potentially through induction of the unfolded protein response. In line with the reported hyperstabilization of GOF p53 protein, cells treated with HDACi exhibited reduced levels of p53 protein. Together, these data form the basis for future clinical studies examining therapeutic efficacy in a preselected patient population with GOF TP53 mutations.

Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1 tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.

MYST histone acetyltransferases have crucial functions in transcription, replication and DNA repair and are hence implicated in development and cancer. Here we characterise Myst2/Kat7/Hbo1 protein interactions in mouse embryonic stem cells by affinity purification coupled to mass spectrometry. This study confirms that in embryonic stem cells Myst2 is part of H3 and H4 histone acetylation complexes similar to those described in somatic cells. We identify a novel Myst2-associated protein, the tumour suppressor protein Niam (Nuclear Interactor of ARF and Mdm2). Human NIAM is involved in chromosome segregation, p53 regulation and cell proliferation in somatic cells, but its role in embryonic stem cells is unknown. We describe the first Niam embryonic stem cell interactome, which includes proteins with roles in DNA replication and repair, transcription, splicing and ribosome biogenesis. Many of Myst2 and Niam binding partners are required for correct embryonic development, implicating Myst2 and Niam in the cooperative regulation of this process and suggesting a novel role for Niam in embryonic biology. The data provides a useful resource for exploring Myst2 and Niam essential cellular functions and should contribute to deeper understanding of organism early development and survival as well as cancer. Data are available via ProteomeXchange with identifier PXD005987.

Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

Mechanisms of neuroendocrine tumor (NET) proliferation are poorly understood, and therapies that effectively control NET progression and metastatic disease are limited. We found amplification of a putative oncogene, RABL6A, in primary human pancreatic NETs (PNET) that correlated with high-level RABL6A protein expression. Consistent with those results, stable silencing of RABL6A in cultured BON-1 PNET cells revealed that it is essential for their proliferation and survival. Cells lacking RABL6A predominantly arrested in G1 phase with a moderate mitotic block. Pathway analysis of microarray data suggested activation of the p53 and retinoblastoma (Rb1) tumor-suppressor pathways in the arrested cells. Loss of p53 had no effect on the RABL6A knockdown phenotype, indicating that RABL6A functions independent of p53 in this setting. By comparison, Rb1 inactivation partially restored G1 to S phase progression in RABL6A-knockdown cells, although it was insufficient to override the mitotic arrest and cell death caused by RABL6A loss. Thus, RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET proliferation and development. This work identifies RABL6A as a novel negative regulator of Rb1 that is essential for PNET proliferation and survival. We suggest RABL6A is a new potential biomarker and target for anticancer therapy in PNET patients., (©2014 American Association for Cancer Research.)

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAM(m/m)) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM(+/m) heterozygous animals developed lesions. In the spleen, NIAM(m/m) mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

Cancer is the second deadliest disease in the United States, necessitating improvements in tumor diagnosis and treatment. Current model systems of cancer are informative, but translating promising imaging approaches and therapies to clinical practice has been challenging. In particular, the lack of a large-animal model that accurately mimics human cancer has been a major barrier to the development of effective diagnostic tools along with surgical and therapeutic interventions. Here, we developed a genetically modified porcine model of cancer in which animals express a mutation in TP53 (which encodes p53) that is orthologous to one commonly found in humans (R175H in people, R167H in pigs). TP53(R167H/R167H) mutant pigs primarily developed lymphomas and osteogenic tumors, recapitulating the tumor types observed in mice and humans expressing orthologous TP53 mutant alleles. CT and MRI imaging data effectively detected developing tumors, which were validated by histopathological evaluation after necropsy. Molecular genetic analyses confirmed that these animals expressed the R167H mutant p53, and evaluation of tumors revealed characteristic chromosomal instability. Together, these results demonstrated that TP53(R167H/R167H) pigs represent a large-animal tumor model that replicates the human condition. Our data further suggest that this model will be uniquely suited for developing clinically relevant, noninvasive imaging approaches to facilitate earlier detection, diagnosis, and treatment of human cancers.

The RAS protooncogene has a central role in regulation of cell proliferation, and point mutations leading to oncogenic activation of Ras occur in a large number of human cancers. Silencing of tumor-suppressor genes by DNA methyltransferase 1 (Dnmt1) is essential for oncogenic cellular transformation by Ras, and Dnmt1 is overexpressed in numerous human cancers. Here we provide new evidence that the pleiotropic regulator of G protein signaling (RGS) family member RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated degradation of Dmnt1 and promoting apoptosis. Employing mouse embryonic fibroblasts from wild-type and RGS6(-/-) mice, we found that oncogenic Ras induced upregulation of RGS6, which in turn blocked Ras-induced cellular transformation. RGS6 functions to suppress cellular transformation in response to oncogenic Ras by downregulating Dnmt1 protein expression leading to inhibition of Dnmt1-mediated anti-apoptotic activity. Further experiments showed that RGS6 functions as a scaffolding protein for both Dnmt1 and Tip60 and is required for Tip60-mediated acetylation of Dnmt1 and subsequent Dnmt1 ubiquitylation and degradation. The RGS domain of RGS6, known only for its GTPase-activating protein activity toward Gα subunits, was sufficient to mediate Tip60 association with RGS6. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogene-induced transformation and provides new insights into our understanding of the mechanisms underlying Ras-induced oncogenic transformation and regulation of Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target for developing new cancer treatments.

The p53 tumor suppressor is controlled by an interactive network of factors that stimulate or inhibit its transcriptional activity. Within that network, Mdm2 functions as the major antagonist of p53 by promoting its ubiquitylation and degradation. Conversely, Tip60 activates p53 through direct association on target promoters as well as acetylation of p53 at lysine 120 (K120). This study examines the functional relationship between Mdm2 and Tip60 with a novel p53 regulator, NIAM (nuclear interactor of ARF and Mdm2). Previous work showed NIAM can suppress proliferation and activate p53 independently of ARF, indicating that other factors mediate those activities. Here, we demonstrate that NIAM is a chromatin-associated protein that binds Tip60. NIAM can promote p53 K120 acetylation, although that modification is not required for NIAM to inhibit proliferation or induce p53 transactivation of the p21 promoter. Notably, Tip60 silencing showed it contributes to but is not sufficient for NIAM-mediated p53 activation, suggesting other mechanisms are involved. Indeed, growth-inhibitory forms of NIAM also bind to Mdm2, and increased NIAM expression levels disrupt p53-Mdm2 association, inhibit p53 polyubiquitylation, and prevent Mdm2-mediated inhibition of p53 transcriptional activity. Importantly, loss of NIAM significantly impairs p53 activation. Together, these results show that NIAM activates p53 through multiple mechanisms involving Tip60 association and Mdm2 inhibition. Thus, NIAM regulates 2 critical pathways that control p53 function and are altered in human cancers, implying an important role for NIAM in tumorigenesis.

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53-/- MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early recurrence following pancreatectomy, rapid progression, and chemoresistance. Novel prognostic and predictive biomarkers are urgently needed to both stratify patients for clinical trials and select patients for adjuvant therapy regimens. This study sought to determine the biological significance of RABL6A (RAB, member RAS oncogene family-like protein 6 isoform A), a novel pancreatic protein, in PDAC. Analyses of RABL6A protein expression in PDAC specimens from 73 patients who underwent pancreatic resection showed that RABL6A levels are altered in 74% of tumors relative to adjacent benign ductal epithelium. Undetectable RABL6A expression, found in 7% (5/73) of patients, correlated with improved overall survival (range 41 to 118 months with 3/5 patients still living), while patients with RABL6A expression had a worse outcome (range 3.3 to 100 months, median survival 20.3 months) (P = 0.0134). In agreement with those findings, RABL6A expression was increased in pancreatic cancer cell lines compared to normal pancreatic epithelial cells, and its knockdown inhibited pancreatic cancer cell proliferation and induced apoptosis. Moreover, RABL6A depletion selectively sensitized cells to oxaliplatin-induced arrest and death. This work reveals that RABL6A promotes the proliferation, survival, and oxaliplatin resistance of PDAC cells, whereas its loss is associated with extended survival in patients with resected PDAC. Such data suggest RABL6A is a novel biomarker of PDAC and potential target for anticancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies., (©2011 AACR.)

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala(14) and Thr(31)) were found to destabilize the protein while two others (Val(24) and Ala(41)) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val(24) was required for p53-independent growth suppression whereas multiple residues (Val(24), Thr(31), Ala(41) and His(60)) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.

Nuclear interactor of ARF and Mdm2 (NIAM) is a newly discovered growth inhibitor that helps maintain chromosomal stability. It is functionally linked to the ARF-Mdm2-p53 tumor suppressor pathway and is predicted to be a tumor suppressor, but the lack of antibodies capable of detecting the endogenous human protein has delayed efforts to define its role in human tumorigenesis. This study reports the development, screening, and characterization of several monoclonal antibodies (MAbs) that specifically recognize endogenous human NIAM protein by Western blotting, immunoprecipitation, immunofluorescence, and immunohistochemistry. These MAbs are predicted to be important tools for evaluating the expression and physiological function of NIAM in normal versus neoplastic human cells and tissues.

Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1-6 h after infection resulting in a decrease in p53 6-24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.

The ARF tumor suppressor protects us against cancer through protein-protein interactions in partially defined p53-dependent and p53-independent pathways. We performed a two-hybrid screen using ARF as bait and present the identification of several new ARF partners that may regulate its growth inhibitory signaling. The potential physiological roles of these novel ARF binding proteins in regulating ARF signaling are discussed.

The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.