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Lung adenocarcinoma (LUAD) is the major subtype in lung cancer, and cigarette smoking is essentially linked to its pathogenesis. We show that downregulation of Filamin A interacting protein 1-like (FILIP1L) is a driver of LUAD progression. Cigarette smoking causes its downregulation 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-sequencing analysis of these tumors, reduction of FILIP1L is associated with upregulated 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 LUAD, and warrant further efforts to evaluate pharmacologic regimens that either directly or indirectly restore FILIP1L-mediated gene regulation for the treatment of these neoplasms. Significance: This study identifies FILIP1L as a tumor suppressor in LUADs and demonstrates that downregulation of FILIP1L is a clinically relevant event in the pathogenesis and clinical course of these neoplasms.

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Lung adenocarcinoma (LUAD) is the major subtype in lung cancer, and cigarette smoking is essentially linked to its pathogenesis. We show that downregulation of Filamin A interacting protein 1-like (FILIP1L) is a driver of LUAD progression. Cigarette smoking causes its downregulation 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-sequencing analysis of these tumors, reduction of FILIP1L is associated with upregulated 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 LUAD, and warrant further efforts to evaluate pharmacologic regimens that either directly or indirectly restore FILIP1L-mediated gene regulation for the treatment of these neoplasms.Significance:This study identifies FILIP1L as a tumor suppressor in LUADs and demonstrates that downregulation of FILIP1L is a clinically relevant event in the pathogenesis and clinical course of these neoplasms.

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 upregulation of PFDN1 is a driver of the unique neoplastic characteristics in aggressive aneuploid MAC.Significance:This study identifies FILIP1L as a tumor suppressor in mucinous colon cancer and demonstrates that FILIP1L loss results in aberrant stabilization of a centrosome-associated chaperone protein to drive aneuploidy and disease progression.

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Supplementary Figure 2 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

Supplementary Figure 6 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

Supplementary Figure 3 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

Supplementary Legends 1-6 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

Inhibiting angiogenesis has become a major therapeutic strategy for cancer treatment. To identify common intracellular mediators, we previously analyzed gene expression profiles of endothelial cells after treatment with angiogenesis inhibitors. Filamin A interacting protein 1-like (FILIP1L; previously known as down-regulated in ovarian cancer 1) was identified as one of the genes up-regulated in endothelial cells in response to these inhibitors. However, the expression and function of FILIP1L protein is uncharacterized. Here, we provide the first description of the expression and specific subcellular localization of FILIP1L protein in human tissue. Overexpression of FILIP1L resulted in inhibition of cell proliferation and migration and increased apoptosis. In addition, overexpression of FILIP1L truncation mutants showed differential antiproliferative activity. A COOH terminal truncation mutant (FILIP1LΔC103) was more potent than wild-type FILIP1L in mediating this activity. Targeted expression of FILIP1LΔC103 in tumor vasculature inhibited tumor growth in vivo. Overall, these findings suggest that the novel protein FILIP1L may be an important mediator of the effects of angiogenesis inhibitors and that FILIP1L has the potential to be an antivascular reagent for cancer therapy. [Cancer Res 2008;68(18):7332–41]

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. [Cancer Res 2009;69(5):1858–66]

Supplementary Data from Recapitulation of Pancreatic Neuroendocrine Tumors in Human Multiple Endocrine Neoplasia Type I Syndrome via Pdx1-Directed Inactivation of Men1

Supplementary Figure 4 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

Supplementary Figure 1 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

Supplementary Figure 5 from Functional Characterization of Filamin A Interacting Protein 1–Like, a Novel Candidate for Antivascular Cancer Therapy

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/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet β-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.

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 upregulation of PFDN1 is a driver of the unique neoplastic characteristics in aggressive aneuploid MAC. Significance: This study identifies FILIP1L as a tumor suppressor in mucinous colon cancer and demonstrates that FILIP1L loss results in aberrant stabilization of a centrosome-associated chaperone protein to drive aneuploidy and disease progression.

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-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Finally, we demonstrated that upregulation of Dnmt1 resulted in hyperplasia through β-cell proliferation. We conclude that the upregulation of Dnmt1 promotes islet β-cell proliferation and targeting Dnmt1 may be a promising therapy for patients suffering from pancreatic neuroendocrine tumors.

The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.

// Mijung Kwon 1, * , Jae-Hoon Kim 3, 4, * , Yevangelina Rybak 1 , Alex Luna 1 , Chel Hun Choi 5, 6 , Joon-Yong Chung 6 , Stephen M. Hewitt 6 , Asha Adem 1 , Elizabeth Tubridy 1 , Juan Lin 2 , Steven K. Libutti 1 1 Department of Surgery, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2 Division of Biostatistics, Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA 3 Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul 135-720, Korea 4 Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul 135-720, Korea 5 Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea 6 Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA * These authors contributed equally to this work Correspondence to: Steven K. Libutti, email: slibutti@montefiore.org Mijung Kwon, email: mijung.kwon@einstein.yu.edu Keywords: ovarian cancer, prognosis, metastasis, chemoresistance, WNT Received: July 20, 2016 Accepted: October 17, 2016 Published: October 20, 2016 ABSTRACT 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 cancer cells and this phenotype was rescued by simultaneous knockdown of FILIP1L and SLUG, an EMT activator. We also demonstrated that FILIP1L regulates β-catenin degradation. FILIP1L co-localizes with phospho-β-catenin and increases phospho-β-catenin at the centrosomes, destined for proteosomal degradation. Finally, we showed that FILIP1L regulates EMT. Overall, these findings suggest that FILIP1L promotes β-catenin degradation and suppresses EMT, thereby inhibiting metastases and chemoresistance. Our study provides the first clinical relevance of FILIP1L in human cancer, and suggests that FILIP1L may be a novel prognostic marker for chemotherapy in ovarian cancer patients. Further, the modulation of FILIP1L expression may have the potential to be a target for cancer therapy.

Purpose MR spectroscopy (MRS) can improve diagnosis and follow treatment in cancer. However, no study has yet reported application of in vivo 1H-MRS in malignant pancreatic lesions. This study quantitatively determined whether in vivo 1H-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. Methods Relative tCho levels in pancreatic tumors or pancreata from KO and WT mice were determined using in vivo 1H-MRS at 9.4 T. The levels of Cho-containing compounds were also quantified using in vitro 1H-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). Results tCho levels measured by in vivo 1H-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 1H-NMR. Conclusion These results indicate the potential use of tCho levels estimated via in vivo 1H-MRS in differentiating malignant pancreatic tumors from benign tumors. Magn Reson Med 74:1221–1226, 2015. © 2014 Wiley Periodicals, Inc.

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. [Cancer Res 2009;69(5):1858–66]

BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor-α (TNF-α) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF-α, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF-α targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno-associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF-α (AAVP-TNF-α). The activity of AAVP-TNF-α was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP-TNF-α infection of human melanoma cells resulted in high levels of TNF-α expression. Systemic administration of targeted AAVP-TNF-α to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF-α, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF-α specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF-α is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy. Cancer 2009. Published 2008 by the American Cancer Society.

Inhibiting angiogenesis has become a major therapeutic strategy for cancer treatment. To identify common intracellular mediators, we previously analyzed gene expression profiles of endothelial cells after treatment with angiogenesis inhibitors. Filamin A interacting protein 1-like (FILIP1L; previously known as down-regulated in ovarian cancer 1) was identified as one of the genes up-regulated in endothelial cells in response to these inhibitors. However, the expression and function of FILIP1L protein is uncharacterized. Here, we provide the first description of the expression and specific subcellular localization of FILIP1L protein in human tissue. Overexpression of FILIP1L resulted in inhibition of cell proliferation and migration and increased apoptosis. In addition, overexpression of FILIP1L truncation mutants showed differential antiproliferative activity. A COOH terminal truncation mutant (FILIP1LΔC103) was more potent than wild-type FILIP1L in mediating this activity. Targeted expression of FILIP1LΔC103 in tumor vasculature inhibited tumor growth in vivo. Overall, these findings suggest that the novel protein FILIP1L may be an important mediator of the effects of angiogenesis inhibitors and that FILIP1L has the potential to be an antivascular reagent for cancer therapy. [Cancer Res 2008;68(18):7332–41]

Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.

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.

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 present 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 down-regulated, 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 down-regulation 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.

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 devel...

In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-α (AAVP-TNF-α) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-α alone (AAVP-TNF-α plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-α plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-α production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-α and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-α and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity.

Background Pasireotide (SOM230), a long-acting somatostatin analogue (LAR), has improved agonist activity at somatostatin receptors. We tested the effect of SOM230 on insulin secretion, serum glucose concentrations, tumor growth, and survival using an MEN1 transgenic mouse model. Methods Eight 12-month-old conditional Men1 knockout mice with insulinoma were assessed. The treatment (n = 4) and control groups (n = 4) received monthly subcutaneous injections of SOM230 or PBS. Serum insulin and glucose levels were determined by enzyme-linked immunosorbent assay and enzymatic colorimetric assay, respectively. Tumor activity, growth, and apoptosis were determined by microPET/CT scan and histologic analysis. Results On day 7, there was a decrease in serum insulin levels from 1.06 ± 0.28 μg/L to 0.37 ± 0.17 μg/L (P = .0128) and a significant increase in serum glucose from 4.2 ± 0.45 mmol/L to 7.12 ± 1.06 mmol/L (P = .0075) in the treatment group but no change in the control group. Tumor size was less in the treatment group (2,098 ± 388 μm2) compared with the control group (7,067 ± 955 μm2; P = .0024). Furthermore, apoptosis was increased in the treatment group (6.9 ± 1.23%) compared with the control group (0.29 ± 0.103%; P = .002). Conclusion SOM230 demonstrates antisecretory, antiproliferative, and proapoptotic activity in our MEN1 model of insulinoma. Further studies of the effects of SOM230 in PNET patients with MEN1 mutations are warranted.

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.

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 alpha-cells or an insulin promoter in beta-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated alpha-cells or beta-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.

Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor-alpha (TNF-alpha) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF-alpha, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF-alpha targeted to tumor vasculature by systemic delivery to examine its antitumor activity.A hybrid adeno-associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF-alpha (AAVP-TNF-alpha). The activity of AAVP-TNF-alpha was analyzed in various in vitro and in vivo settings using a human melanoma tumor model.In vitro, AAVP-TNF-alpha infection of human melanoma cells resulted in high levels of TNF-alpha expression. Systemic administration of targeted AAVP-TNF-alpha to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF-alpha, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed.Targeted AAVP vectors can be used to deliver TNF-alpha specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF-alpha is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy.

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 Student's 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.