AAVP displaying octreotide for ligand-directed therapeutic transgene delivery in neuroendocrine tumors of the pancreas.

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.

In vivo proton MR spectroscopy of pancreatic neuroendocrine tumors in a multiple endocrine neoplasia type 1 conditional knockout mouse model.

PURPOSE: 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. METHODS: 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). RESULTS: 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. CONCLUSION: These results indicate the potential use of tCho levels estimated via in vivo (1)H-MRS in differentiating malignant pancreatic tumors from benign tumors.

Filamin A interacting protein 1-like inhibits WNT signaling and MMP expression to suppress cancer cell invasion and metastasis.

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 ß-catenin-directed transcriptional activity, suggesting inhibition of the canonical WNT pathway. Nuclear ß-catenin, an indicator of an active canonical WNT pathway, was reduced in FILIP1L-expressing tumors. Overall, these findings suggest that FILIP1L reduces ß-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.

Smoking-associated Downregulation of FILIP1L Enhances Lung Adenocarcinoma Progression Through Mucin Production, Inflammation, and Fibrosis.

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/ß-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.

FILIP1L Loss Is a Driver of Aggressive Mucinous Colorectal Adenocarcinoma and Mediates Cytokinesis Defects through PFDN1.

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.

B7 immune-checkpoints as targets for the treatment of neuroendocrine tumors.

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 ß-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 ß-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.

Tissue-specific induced DNA methyltransferase 1 (Dnmt1) in endocrine pancreas by RCAS-TVA-based somatic gene transfer system promotes ß-cell proliferation.

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 ß-cells of the pancreas in a RIP-TVA mouse model. In the present study, we mediated Dnmt1 expression in islet ß-cells in RIP-TVA mice by utilizing the RCAS-TVA system to test if the upregulation of Dnmt1 can promote ß-cell proliferation. In vitro, we demonstrated that upregulation of Dnmt1 increased ß-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 ß-cell proliferation. We conclude that the upregulation of Dnmt1 promotes islet ß-cell proliferation and targeting Dnmt1 may be a promising therapy for patients suffering from pancreatic neuroendocrine tumors.

Reduced expression of FILIP1L, a novel WNT pathway inhibitor, is associated with poor survival, progression and chemoresistance in ovarian cancer.

Filamin A interacting protein 1-like (FILIP1L) is an inhibitor of the canonical WNT pathway. WNT/ß-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/ß-catenin pathway, the expression of FILIP1L, ß-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 ß-catenin degradation. FILIP1L co-localizes with phospho-ß-catenin and increases phospho-ß-catenin at the centrosomes, destined for proteosomal degradation. Finally, we showed that FILIP1L regulates EMT. Overall, these findings suggest that FILIP1L promotes ß-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.

Loss of MEN1 activates DNMT1 implicating DNA hypermethylation as a driver of MEN1 tumorigenesis.

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/ß-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.

Down-regulation of Filamin A interacting protein 1-like Is associated with promoter methylation and an invasive phenotype in breast, colon, lung and pancreatic cancers [corrected].

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.