PDX-1 Is a Therapeutic Target for Pancreatic Cancer, Insulinoma and Islet Neoplasia Using a Novel RNA Interference Platform

<div><p>Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Immunohistochemistry of human pancreatic and islet neoplasia specimens revealed marked PDX-1 overexpression, suggesting PDX-1 as a “drugable” target within these diseases. To do so, a novel RNA interference effector platform, bifunctional shRNA^PDX-1, was developed and studied in mouse and human cell lines as well as in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Systemic delivery of bi-shRNA^humanPDX-1 lipoplexes resulted in marked reduction of tumor volume and improved survival in a human pancreatic cancer xenograft mouse model. bi-shRNA^mousePDX-1 lipoplexes prevented death from hyperinsulinemia and hypoglycemia in an insulinoma mouse model. shRNA^mousePDX-1 lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of islet neoplasia. PDX-1 was overexpressed in pancreatic neuroendocrine tumors and nesidioblastosis. These data demonstrate that PDX-1 RNAi therapy controls hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia, therefore, PDX-1 is a potential therapeutic target for these pancreatic diseases.</p> </div

Knockdown of PDX-1 expression inhibits cell proliferation in β TC-6 cells.

<p>MTS assay showed the time course of cell proliferation for β TC-6 (a) cells transfected with bi-shRNA^mousePDX-1 and bi-shRNA^humanPDX-1, respectively. A comparison of the percentages of cell proliferation inhibition between bi-shRNA and shRNA transfection at different doses is shown in β TC-6 cells (b). Cell proliferation determined by BrdU also is shown (c). Western blot analysis of 20 ug lysate from β TC-6 cells that were transfected with shRNAi^mousePDX-1 after 48 h was performed with anti-PDX-1, cyclin E, Cdk2, Cdk4, p53 and p27 (d).</p

bi-shRNA^mousePDX-1 knockdown of mPDX-1 expression in β TC-6 cells <i>in vitro</i> inhibits insulin expression and secretion.

<p>Comparison of efficacy of bi-shRNA^mousePDX-1, shRNA^mousePDX-1 or empty vector in inhibition of PDX-1 expression in β TC-6 cells is shown using western blot (a) and cell immunostaining (top panel, b as indicated by arrow). Insulin expression and glucose stimulated secretion in response to knockdown of PDX-1 is shown by cell immunostaining (bottom panel, b as indicated by arrow) (×200) and ELISA assay (d), respectively. Each experiment was repeated five times. PDX-1 and insulin expression in immunostained β TC-6 cells are quantified (c). PDX-1 expression affected the RIP-directed reporter expression (RIP-mCherry) in βTC-6 cells (e and f). The cells expressing mCherry (red) and GFP (green) were visualized and photographed using fluorescence microscopy (e and f). (×200).</p

Knockdown of PDX-1 expression inhibits cell proliferation in PANC-1 cells.

<p>MTS assay showed the time course of cell proliferation for PANC-1 (a) cells transfected with bi-shRNA^mousePDX-1 and bi-shRNA^humanPDX-1, respectively. A comparison of cell proliferation inhibition percentages between bi-shRNA and shRNA transfection at different doses is shown in PANC-1 cells (b).</p

Systemic bi-shRNA^mousePDX-1 lipoplexes prevent death from severe hyperinsulinemia and hypoglycemia in an insulinoma SCID mouse model.

<p>Glucose levels A and C corresponding to insulin levels B and D were acquired from the β TC-6 mice treated with bi-shRNAi^mousePDX-1 or shRNAi^mousePDX-1, respectively. In each figure a-d, the dash line represents control group data and the dash-dot line represents treatment group data. Islet cells expressing PDX-1, insulin, PP, PCNA, p27, cyclin E and CdK4 were shown by IHC and islet cell apoptosis was shown by TUNEL assay, as indicated by arrows (×200) (e). Mouse survival after three treatment cycles of either empty vector or bi-shRNAi^mousePDX-1 and empty vector or shRNAi^mousePDX-1 was evaluated and compared using Kaplan-Meier in SPSS (f).</p

Real Time <i>RT-PCR</i> detection of <i>TUSC2</i> gene expression in patients receiving DC-<i>TUSC2</i>.

1<p>Specimens not available.</p

Systemic shRNA^mousePDX-1 lipoplexes reverse hyperinsulinemia and hypoglycemia and alter glucose tolerance in somatostatin receptor subtypes 1 and 5 (SSTR1/5^−/−) knockout mice.

<p>Glucose levels (a) and corresponding insulin levels (b) were measured as described in the method section. Immunostaining for pancreatic slides with PDX-1, PCNA and TUNEL assay were performed. Red staining in top panel of (c) indicates PDX-1 expression (arrow); green staining at middle panel (c) indicates PCNA expression (arrow). Apoptotic tumor cells from SSTR1/5^−/− mice are stained brown and are shown in the bottom panel of (c) (arrow) (×200). IPGTT assay showed glucose levels and insulin levels (d) on day 7 and glucose levels and insulin levels (e) on day 150 after shRNA^mousePDX-1 therapy. Data are presented as means ± S.E.M. and P<0.05 indicate significance.</p

shRNA^humanPDX-1 therapies reduce tumor volume and prolong survival in a xenograft SCID mouse model of pancreatic cancer.

<p>The tumor volume was evaluated and compared at 90 and 120 days following the initial treatment between treatment and control groups (a). The survival rates of mice receiving treatment and of those receiving the empty vector control were analyzed using Kaplan-Meier in SPSS (b). Immunostaining for tumor slides with PDX-1, PCNA, Cyclin E, and P53 as well as TUNEL assay were performed and analyzed (c). The positive for each marker is indicated by arrow (×200).</p

PDX-1 expression in islet neoplasia, β TC-6 tumor and islet of SSTR<i>1/5</i>^−/−.

<p>Immunostaining with anti-PDX-1 antibody demonstrates overexpression of PDX-1 in human islet neoplasia specimens (26 pancreatic neuroendocrine tumors (a), 10 nesidioblastosis specimens (b), βTC-6 tumors (c) and 6 pancreata specimens from SSTR1/5^−/− mice (e, f). PDX-1 is overexpressed in both islets (e) and acinar cells (f) of SSTR1/5^−/− mice as compared to that of wild type mice where only islet cells expressed PDX-1 (d). PDX-1 positive is indicated by arrow (×200).</p

DC-<i>TUSC2</i> metabolic tumor response in a metastatic lung cancer patient.

<p>The patient is a 54 year old female with a large cell neuroendocrine carcinoma. She had received six prior chemotherapy regimens. Prior to entry in the protocol, two hepatic metastases were progressing on gemcitabine. The patient also had a metastasis in the head of the pancreas and a peripancreatic lymph node (arrows). A. Pretreatment PET scan. The dose of Fluorodeoxyglucose(18F) was 8.8mCi B. Post-treatment PET scan performed 20 days following the fourth dose of DC-<i>TUSC2</i>. The dose of Fluorodeoxyglucose(18F) was 9.0mCi. All scans were performed within a 60 to 90 minute window after injection.</p