Asha Adem

Lung adenocarcinoma (LUAD) is the major subtype in lung cancer, and cigarette smoking is essentially linked to its pathogenesis. We show that down-regulation of Filamin A interacting protein 1-like (FILIP1L) is a driver of LUAD progression. Cigarette smoking causes its down-regulation by promoter methylation in LUAD. Loss of FILIP1L increases xenograft growth, and, in lung-specific knockout mice, induces lung adenoma formation and mucin secretion. In syngeneic allograft tumors, reduction of FILIP1L and subsequent increase in its binding partner, prefoldin 1 (PFDN1) increases mucin secretion, proliferation, inflammation and fibrosis. Importantly, from the RNA-Seq analysis of these tumors, reduction of FILIP1L is associated with up-regulated Wnt/β-catenin signaling, which has been implicated in proliferation of cancer cells as well as inflammation and fibrosis within the tumor microenvironment. Overall, these findings suggest that down-regulation of FILIP1L is clinically relevant in L...

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75 % of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in a-cells or an insulin promoter in b-...

Deoxycytidine (aza-TdCyd) for the treatment of MEN1 tumors in a preclinical study Ziqiang Yuan1, Juliet Gardiner1, Zoya Gauhar2, Svetlana Bagdasarov1, Asha Adem1, Daniel Slegowski, Michael Difilippantonio3, Steven K Libutti1. 1Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey; 2Princeton University, Princeton, New Jersey; 3Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.

Aneuploid mucinous colorectal adenocarcinoma (MAC) is an aggressive subtype of colorectal cancer with poor prognosis. The tumorigenic mechanisms in aneuploid MAC are currently unknown. Here we show that downregulation of Filamin A–interacting protein 1-like (FILIP1L) is a driver of MAC. Loss of FILIP1L increased xenograft growth, and, in colon-specific knockout mice, induced colonic epithelial hyperplasia and mucin secretion. The molecular chaperone prefoldin 1 (PFDN1) was identified as a novel binding partner of FILIP1L at the centrosomes throughout mitosis. FILIP1L was required for proper centrosomal localization of PFDN1 and regulated proteasome-dependent degradation of PFDN1. Importantly, increased PFDN1, caused by downregulation of FILIP1L, drove multinucleation and cytokinesis defects in vitro and in vivo, which were confirmed by time-lapse imaging and 3D cultures of normal epithelial cells. Overall, these findings suggest that downregulation of FILIP1L and subsequent upregula...

The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1α overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor β-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia-inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1...

e13017Background: PCCs are rare neuroendocrine tumors and their pathogenesis is not well understood. Epigenetic changes are seen in many human tumors, and these changes may play a role in the development of PCCs. Using a high-throughput global DNA methylation approach, HELP-tagging, we sought to determine the differential methylation pattern of human VHL-PCCs, SDHD-PCCs, SDHB-PCCs and sporadic PCCs compared to normal adrenal tissue. Methods: Normal adrenal glands (n = 4), VHL-PCCs (n = 5), SDHD-PCCs (n = 6), SDHB-PCCs (n = 6), and sporadic-PCCs (n = 4) were analyzed. We generated HELP-tagging libraries with HpaII- or Msp- digested DNA from all samples. Massively-parallel sequencing with an Illumina HiSeq 2000 sequencer was performed. Sequences were analyzed and aligned using Illumina pipeline and customized HELP-tagging software. Differential DNA methylation profiles were compared. Differentially methylated loci were defined as those with a methylation change of > 20% and P value < 0.05. These loci were t...

Filamin A interacting protein 1-like (FILIP1L) is an inhibitor of the canonical WNT pathway. WNT/β-catenin signaling and its downstream pathway, epithelial-to-mesenchymal transition (EMT), play a key role in ovarian cancer metastasis and chemoresistance. To study the clinical implications of FILIP1L in regulating the WNT/β-catenin pathway, the expression of FILIP1L, β-catenin, SNAIL and SLUG was analyzed by immunohistochemistry on tissue microarrays of 369 ovarian samples ranging from normal to metastatic. In addition, the results were validated in mouse model and in vitro cell culture. In the present study, we demonstrated that FILIP1L expression was inversely correlated with poor prognosis, stage and chemoresistance in ovarian cancer. Notably, low FILIP1L expression was independent negative prognostic factor with respect to overall and disease-free survival. FILIP1L inhibited peritoneal metastases in orthotopic mouse model. FILIP1L knockdown induced chemoresistance in ovarian canc...

Multiple endocrine neoplasia type 1 (MEN1) syndrome results from mutations in the MEN1 gene and causes tumor formation via largely unknown mechanisms. Using a novel genome-wide methylation analysis, we studied tissues from MEN1-parathyroid tumors, Men1 knockout (KO) mice, and Men1 null mouse embryonic fibroblast (MEF) cell lines. We demonstrated that inactivation of menin (the protein product of MEN1) increases activity of DNA (cytosine-5)-methyltransferase 1 (DNMT1) by activating retinoblastoma-binding protein 5 (Rbbp5). The increased activity of DNMT1 mediates global DNA hypermethylation, which results in aberrant activation of the Wnt/β-catenin signaling pathway through inactivation of Sox regulatory genes. Our study provides important insights into the role of menin in DNA methylation and its impact on the pathogenesis of MEN1 tumor development.

Background: PNETs are rare highly vascular tumors that are increasing in incidence. Management of advanced tumors, both functional and nonfunctional, can be challenging, as systemic treatment options are limited. CA4P is a systemically delivered vascular disrupting agent that has shown clinical activity for the treatment of other advanced endocrine cancers. We evaluated the efficacy of systemic administration of CA4P for the treatment of functional insulinomas in a transgenic mouse model of PNETs. Methods: Twelve month-old mice with homozygous deletion of the Men1 gene in the pancreas and confirmation of a functional insulinoma by presence of elevated serum insulin were divided into two groups (4 mice per group). The treatment group received CA4P, 100 mg/kg, IP on Monday, Wednesday and Friday for four weeks. The control group received PBS by the same route, frequency and for the same duration. Serum insulin was measured by enzyme-linked immunosorbent assay (ELISA) pre and at intervals during treatment. After four weeks, mice were euthanized and whole pancreata were dissected, formalin-fixed with paraffin embedding, and sectioned (5 μm) on a rotating microtome. Hematoxylin and eosin staining was performed and the tumor cross-sectional area in the sections with maximal tumor diameter was determined in three sections for each mouse in a blinded fashion. Tumor size was assessed by measuring the length and width of the tumor in two dimensions and then calculating the maximum tumor area for each mouse and for each group. Statistical analysis was performed with ANOVA and Student9s t-test. Results: Treatment was well tolerated in all mice without treatment related death or weight loss. At baseline, mean serum insulin was elevated, but equivalent in both groups (CA4P group 3.695 ± 0.669 µg/L, versus PBS group 3.485 ± 0.666 µg/L, p=0.672). Treatment with CA4P resulted in a significant and sustained decrease in circulating insulin, with maximum effect seen by day 17 (CA4P group 0.213 ± 0.075 µg/L, versus PBS group 4.578 ± 0.161 µg/L, p Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A4. Citation Format: Ziqiang Yuan, Nikolas Zaphiros, Asha Adem, Norvilia Etienne, Jai Balkissoon, Dai Chaplin, Steven K. Libutti. Combretastatin A-4 Phosphate (CA4P) is effective for the treatment of functional pancreatic neuroendocrine tumors (PNETs) in a transgenic mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A4.

Identifying key mediators of cancer invasion and metastasis is crucial to the development of new and more effective therapies. We previously identified Filamin A interacting protein 1-like (FILIP1L) as an important mediator of cell migration and invasion. FILIP1L expression was inversely correlated with the invasive potential of ovarian cancer cell lines and ovarian cancer specimens. In addition, it was shown that intraperitoneal delivery of the FILIP1L gene resulted in inhibition of metastatic ovarian cancer spread into the peritoneum and intra-abdominal organs. Although these observations demonstrate that FILIP1L inhibits metastasis, it is not clear which step(s) of metastasis is inhibited by FILIP1L. To this end, in our present study we chose an orthotopic ovarian cancer model in mice where cancer cells metastasize to distant organs such as lungs which lung metastasis can occur through vessels, not by exfoliation and peritoneal spread. In addition, FILIP1L expression was controlled by a doxycycline-inducible expression system which enabled us to determine the direct effect of FILIP1L expression in vivo. Using this model, we observed that expression of FILIP1L in ovarian cancer cells inhibited spontaneous lung metastasis. Experimental lung metastases (established via tail vein injection of cancer cells) as well as the extravasation step of metastasis were not inhibited by FILIP1L, suggesting that FILIP1L inhibits the earlier steps of metastasis such as invasion and intravasation. FILIP1L inhibited matrix metallo¬proteinase (MMP)-dependent invasion in vivo. MMP3, -7 and -9 were transcriptionally down-regulated, and MMP9 protein expression and activity were inhibited in FILIP1L-expressing tumors. Furthermore, our studies suggest that FILIP1L regulates invasion and metastasis by inhibiting components of the WNT signaling pathway. FILIP1L expression reduced the induction of WNT target genes such as MMP3, -7 and -9, and β-catenin-directed transcriptional activity, suggesting inhibition of the canonical WNT pathway. Nuclear β-catenin, an indicator of an active canonical WNT pathway, was reduced in FILIP1L-expressing tumors. We conclude that FILIP1L reduces β-catenin levels, which leads to the transcriptional down-regulation of WNT target genes such as MMPs, resulting in inhibition of metastasis. Thus, modulation of FILIP1L expression has the potential to be a target for cancer therapy. Citation Format: Mijung Kwon, Soo Jin Lee, Yarong Wang, Yevangelina Ryvak, Alex Luna, Srilakshmi Reddy, Asha Adem, Brian Beaty, John Condeelis, Steven Libutti. Filamin A interacting protein 1-like inhibits WNT signaling and MMP expression to suppress ovarian cancer cell invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A61.

The human neuroblastoma cells SH-SY5Y were treated with the differentiation agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethyl sulfoxide (DMSO) and the muscarinic receptor subtype M1 and M2 RNA levels analyzed. After a decrease induced by both agents, the M1 level gradually returned to normal in the presence of TPA but remained minimal with DMSO. As for M2, several phases were observed with TPA, while DMSO caused a drastic increase. The data obtained with TPA were tentatively correlated with the amounts of immunoreactive PKC alpha. In conclusion, our results reveal: (a) differential regulation of M1 and M2 muscarinic receptor subtypes by either treatment; (b) opposing effects of TPA and DMSO on both subtypes.

Dementia refers to a global deterioration in all aspects of mental functioning, including memory, general intellect, emotional attributes, and distinctive features of personality. Alzheimer’s disease/ senile dementia of Alzheimer type (AD/SDAT) accounts for approximately 40% to 60% of all cases of dementia. Biochemical changes in brains of patients with AD/ SDAT include decreased activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) and a decline in the number of muscarinic and nicotinic receptors, indicating reduced activity in the cholinergic system. Reduced activity has also been reported in the dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT) systems. These biochemical changes may explain some of the symptoms seen in AD/ SDAT. Disturbances in the cholinergic system may explain memory impairments; abnormalities in the DA system may explain Parkinson-like symptoms; and disturbances in the NA and 5-HT systems may explain mood changes and emotional symptoms in AD / SDAT. The cholinergic hypothesis has formed the basis of many therapeutic strategies aimed at correcting the cognitive deficits associated with this disorder. Several attempts have been made to treat the disease by enhancing the effectiveness of the remaining functional cholinergic neurons (1). Some of the strategies that have been tried include the use of cholinoceptor agonists to activate the target neurons directly, drugs that act presynaptically to facilitate acetylcholine release, administration of biosynthetic precursors to increase acetylcholine levels available for release, and anticholinesterase therapy to prolong the effects of released acetylcholine. Although most of these therapeutic strategies for AD / SDAT have been disappointing, the most promising results in clinical drug trials have been obtained using anticholinesterases like physostigmine and tetrahydroaminoacridine (2). The use of physostigmine in AD / SDAT treatment is limited by the extremely short half-life (approximately 30 minutes) and narrow therapeutic window. Tacrine or 1,2,3,4-tetrahydroacridine (THA) is a reversible, moderately long acting, cholinesterase inhibitor that has been known for over 40 years. It has been used as a decurarizing agent and in the management of intractable pain, myasthenia gravis, overdose with tricyclic antidepressant agents, tardive dyskinesia, and in intoxications with anticholinergic drugs and poisons. THA in combination with lecithin was reported to improve cognitive function in Alzheimer patients (3, 4). The cognitive improvement seen after administration of THA could be due to its multiple mechanisms of action.

We reported that inactivation of menin (the protein product of MEN1) increases activity of Dnmt1 and mediates DNA hypermethylation in the development of multiple endocrine neoplasia type 1 (MEN1) syndrome. We have developed a RCAS-TVA-based somatic gene transfer system that enables tissue-specific delivery of Dnmt1 to individual β-cells of the pancreas in a RIP-TVA mouse model. In the present study, we mediated Dnmt1 expression in islet β-cells in RIP-TVA mice by utilizing the RCAS-TVA system to test if the upregulation of Dnmt1 can promote β-cell proliferation. In vitro, we demonstrated that upregulation of Dnmt1 increased β-cell proliferation. In vivo, our results showed that the levels of serum insulin were increased in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Furthermore, we confirmed that mRNA and protein expression of Dnmt1 as well as Dnmt1 enzyme activity were upregulated in the RIP-TVA mice with RCASBP-Dnm...

MR spectroscopy (MRS) can improve diagnosis and follow treatment in cancer. However, no study has yet reported application of in vivo (1) H-MRS in malignant pancreatic lesions. This study quantitatively determined whether in vivo (1) H-MRS on multiple endocrine neoplasia type 1 (Men1) conditional knockout (KO) mice and their wild type (WT) littermates could detect differences in total choline (tCho) levels between tumor and control pancreas. Relative tCho levels in pancreatic tumors or pancreata from KO and WT mice were determined using in vivo (1) H-MRS at 9.4 T. The levels of Cho-containing compounds were also quantified using in vitro (1) H-NMR on extracts of pancreatic tissues from KO and WT mice, respectively, and on extracts of pancreatic tissues from patients with pancreatic neuroendocrine tumors (PNETs). tCho levels measured by in vivo (1) H-MRS were significantly higher in PNETs from KO mice compared to the normal pancreas from WT mice. The elevated choline-containing compounds were also identified in pancreatic tumors from KO mice and tissues from patients with PNETs via in vitro (1) H-NMR. These results indicate the potential use of tCho levels estimated via in vivo (1) H-MRS in differentiating malignant pancreatic tumors from benign tumors. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.

Identifying key mediators of cancer cell invasion and metastasis is critical to the development of more effective cancer therapies. We previously identified Filamin A interacting protein 1-like (FILIP1L) as an important inhibitor of cell migration and invasion in ovarian cancer. FILIP1L expression was inversely correlated with the invasive potential of ovarian cancer cell lines and ovarian cancer specimens. We also demonstrated that DNA methylation in the FILIP1L promoter was a mechanism by which FILIP1L was down-regulated in ovarian cancer. In our present study, we tested this observation in other cancer histologies: breast, colon, lung and pancreatic cancers. Both mRNA and protein expression of FILIP1L were down-regulated in these cancer cells compared with their normal epithelial cells. As in ovarian cancer, DNA methylation is a mechanism by which FILIP1L is down-regulated in these cancer histologies. Methylation status of the FILIP1L promoter was inversely correlated with FILIP1L expression. Reduced methylation in the FILIP1L promoter following treatment with a DNA demethylating agent was associated with restoration of FILIP1L expression in these cancer cells. Further, FILIP1L expression was inversely correlated with the invasive potential of these cancer cells. Re-expression of FILIP1L in FILIP1L-low expressing, highly-invasive cancer cell lines resulted in inhibition of cell invasion. Correspondingly, knockdown of FILIP1L in FILIP1L-high expressing, low-invasive cancer cell lines resulted in increase of cell invasion. Overall, these findings suggest that down-regulation of FILIP1L associated with DNA methylation is related with the invasive phenotype in various cancers. Thus, modulation of FILIP1L expression has the potential to be a target for cancer therapy.

Identifying key mediators of cancer invasion and metastasis is crucial to the development of new and more effective therapies. We previously identified FILamin A Interacting Protein 1-Like (FILIP1L) as an important inhibitor of cell migration and invasion. FILIP1L expression was inversely correlated with the invasive potential of ovarian tumors. In our study, we established an orthotopic ovarian cancer model, wherein FILIP1L expression can be regulated in vivo. Using this model, we observed that expression of FILIP1L in ovarian cancer cells inhibited spontaneous lung metastasis. Experimental lung metastases (established via tail vein injection of cancer cells) as well as the extravasation step of metastasis were not inhibited by FILIP1L, suggesting that FILIP1L inhibits the earlier steps of metastasis such as invasion and intravasation. FILIP1L inhibited matrix metalloproteinase (MMP)-dependent invasion in vivo. MMP3, -7 and -9 were transcriptionally downregulated, and MMP9 protein expression and activity were inhibited in FILIP1L-expressing tumors. Importantly, overexpression of MMP9 compensated for the anti-invasive activity of FILIP1L. Furthermore, our studies suggest that FILIP1L regulates invasion and metastasis by inhibiting components of the WNT signaling pathway. FILIP1L expression reduced the induction of WNT target genes such as MMP3, -7 and -9, and β-catenin-directed transcriptional activity, suggesting inhibition of the canonical WNT pathway. Nuclear β-catenin, an indicator of an active canonical WNT pathway, was reduced in FILIP1L-expressing tumors. Overall, these findings suggest that FILIP1L reduces β-catenin levels, which may lead to the transcriptional downregulation of WNT target genes such as MMPs, resulting in inhibition of metastasis. Modulation of FILIP1L expression has the potential to be a target for cancer therapy.

The pancreatic alpha- and beta-cells are critical components in regulating blood glucose homeostasis via secretion of glucagon and insulin, respectively. Both cell types are typically localized in the islets of Langerhans. However, little is known about the roles of paracrine interactions that contribute to their physiological functions. The lack of suitable cell lines to study alpha- and beta-cells interactions have led us to develop an alpha-cell-specific Cre-expressing transgenic line utilizing a glucagon promoter sequence, the Glu-Cre transgenic mouse. Here, we demonstrate that the Glu-Cre could specifically and efficiently excise floxed target genes in adult islet alpha-cells. We further showed that deletion of the tumor suppressor gene, multiple endocrine neoplasia type 1 (Men1), in alpha-cells led to tumorigenesis. However, to our surprise, the lack of Men1 in alpha-cells did not result in glucagonomas but rather beta-cell insulinomas. Because deletion of the Men1 alleles was only present in alpha-cells, our data suggested that cross communication between alpha- and beta-cells contributes to tumorigenesis in the absence of Men1. Together, we believed that the new model systems described here will allow future studies to decipher cellular interactions between islet alpha- and beta-cells in a physiological context.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal syndrome caused by mutations in the MEN1 tumor suppressor gene. Whereas the protein product of MEN1, menin, is ubiquitously expressed, somatic loss of the remaining wild-type MEN1 allele results in tumors primarily in parathyroid, pituitary, and endocrine pancreas. To understand the endocrine specificity of the MEN1 syndrome, we evaluated biallelic loss of Men1 by inactivating Men1 in pancreatic progenitor cells using the Cre-lox system. Men1 deletion in progenitor cells that differentiate into exocrine and endocrine pancreas did not affect normal pancreas morphogenesis and development. However, mice having homozygous inactivation of the Men1 in pancreas developed endocrine tumors with no exocrine tumor manifestation, recapitulating phenotypes seen in the MEN1 patients. In the absence of menin, the endocrine pancreas showed increase in cell proliferation, vascularity, and abnormal vascular structures; such changes were lacking in exocrine pancreas. Further analysis revealed that these endocrine manifestations were associated with up-regulation in vascular endothelial growth factor expression in both human and mouse MEN1 pancreatic endocrine tumors. Together, these data suggest the presence of cell-specific factors for menin and a permissive endocrine environment for MEN1 tumorigenesis in endocrine pancreas. Based on our analysis, we propose that menin's ability to maintain cellular and microenvironment integrity might explain the endocrine- restrictive nature of the MEN1 syndrome.