1. Targeting Sema3D in pancreatic cancer: a novel therapeutic strategy
Adrian G. Murphy1, 2, Agnieszka A. Rucki 1,2, 3 , Jennifer Kleponis 1,2 , Elizabeth M. Jaffee 1,2, 3, 4 , Lei Zheng 1,2,3,4 , Kelly Foley 1, 2, 3
1. The Sidney Kimmel Comprehensive Cancer Center, 2. Department of Oncology, 3. Graduate Program in Cellular and Molecular Medicine, 4. The Skip Viragh Center for Pancreatic Cancer
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Results
Introduction
Pancreatic ductal adenocarcinoma (PDA) is known for its chemotherapy resistance and dismal survival rates. Little is known about the mechanisms of metastasis in PDA. We identified Annexin A2 (AnxA2), an essential mediator of metastasis using sera from patients with prolonged survival after GVAX treatment. AnxA2 regulates the secretion of semaphorin3D (Sema3D) promoting invasion and metastasis of PDA cells.1 Sema3D and other axon guidance genes have been shown to be the cellular pathways most frequently altered in PDA.
KPC mice (KRAS/p53 mutations) spontaneously develop PDA and were crossed with AnxA2 knockouts to create KPCA-/- mice. Cell lines created from primary tumors of these mice have different gene expression profiles including Sema3D. When Sema3D is knocked down in KPC cells, metastases from PDA are only seen with KPC cells but not KPCA-/- cells indicating its importance in the development of metastasis in PDA (Fig. 1).
Sema3D expression has also been shown to affect prognosis in PDA. Immunohistochemistry studies show that 15/20 patients with disease-free survival < 1 year have abundant Sema3D expression (Figure 3).
Ref. 1. Foley et al. Manuscript submitted. C i ii E
Figure 1: AnxA2 is crucial for metastasis in a murine model of PDA
A. Shows representative images of primary PDA tumor from 6 month old KPC mouse (i) with metastases seen grossly in the liver (ii). B. shows representative image of primary PDA tumor in 6 month old KPCA-/- mouse (i) showing the absence of liver metastases (ii). C. shows representative images of H&E stain of primary PDA tumors obtained from KPC and KPCA-/- mice. D. shows representative images of H&E stains taken from the liver of KPC an KPCA-/- mice indicating metastatic disease in KPC mice and normal liver parenchyma in KPCA-/- mice with no invasion of the primary PDA tumor. E. Mice received orthotopic injections of KPC and KPCA (AnxA2 -/-) cells using the hemispleen method. Mice injected with KPCA cells survived significantly longer (p=0.0003) than those injected with KPC cells.
Figure 4: Sema3D is secreted from PDA cells in an AnxA2-dependent manner
A. Shows Sema3D secretion from KPC and KPCA-/- cells showing higher secretion from KPC cells compared to KPCA-/- cells. Sema3D secretion is blocked by anti-AnxA2 antibody (dose dependent). B. Co-immunoprecipitation and western blot analysis show that AnxA2 and Sema3D directly interact in KPC cells. AnxA2 antibody used to co-immunoprecipitate AnxA2 and Sema3D proteins (IgG control). C. shows schematic of regulation of Sema3D secretion by AnxA2 allowing Sema3D to bind to PlxnD1 receptor leading in invasion and metastasis of PDA cell.
Objective
Figure 2: Sema3D is differentially expressed in KPC vs. KPCA-/- cell lines
A. Shows a plot which shows 6 genes involves in cell movement (shown on top: Sdc2, Sema3D, Gfra2, Flt1, Tgfbr3, Plxnd1) and cell morphology/remodeling (shown on bottom: MBP, Dsg2, Dsc2, Cfb, Mmp15, Flnb) that have the largest fold differences in gene expression between KPC and KPCA-/- cells. B. shows the qRT-PCR validation results of the microarray data based on gene expression in primary PDA tumors in KPC and KPCA-/- mice.
The objectives of this research are to elucidate the role of Sema3D in the development of pancreatic cancer and to develop an assay to discover drugs which reduce Sema3D secretion in PDA. B
Materials and Methods
KPC mice (which spontaneously develop PDA) were generated with KRAS/p53 mutations. KPCA-/- mice were created by crossing AnxA2 knockouts with KPC mice. KPC and KPCA-/- mice were observed
Cell lines were developed from primary tumors of these mice and gene expression profiling performed comparing KPC and KPCA-/- cells. RNA was extracted from KPC and KPCA-/- cells and microarray gene expression analysis was performed using Affymetrix MoEx Mouse Exon 1.0 ST array. Pathway analysis was performed using Spotfire ™ and Ingenuity ™. qRT-PCR used to validate the findings of 6 genes with the highest fold change differences.
Immunohistochemistry (IHC) was used to analyze Sema3D and PlxnD1 expression in resected human PDA tissue.
Sema3D secretion was measured from KPC and KPCA-/- cells by ELISA (Cusabio). Anti-AnxA2 antibodies were used to see the effect of AnxA2 inhibition on Sema3D secretion. Co-immunoprecipitation was performed using Pierce Crosslink IP kit using antibodies for AnxA2 (BD Biosciences) and Sema3D (Abcam).
KxPxCx and KPCA cells were seeded in 96 well plates at the following densities (per well): 5 x 103, 1 x 104, 2.5 x104 , 5 x 104, 7.5 x 105 and 1 x 105 for the following time points: 1, 2, 4, 6, 16 and 24 hours. Sema3D secretion was measured from supernatants using ELISA (Cusabio, China).
Figure 5: Optimization of KxPxCx Sema3D secretion assay for 96 well plate format
KxPxCx cells were seeded in 96 well plates at different cell numbers (2.5 x 103 – 1 x 105/well) and Sema3D secretion measured in supernatants at varying time points (range: hours). A. shows the time points at which Sema3D was highest (16 hours) with equivalent concentrations secreted from 2.5 and 5 x 104 cells. B. shows the time course of Sema3D secretion from 2.5 x 104 cells showing 16 hours to be the optimal point for measuring the concentration of Sema3D measurement (473.1 pg/ml A B C
Figure 3: Sema3D expression has a pivotal role in metastasis in human PDA
A and C show representative images of a tumor with low Sema3D (A) and PlxnD1 (C) expression from a patient with prolonged disease-free survival (> 2 years). B and D show representative images of a patient with a short disease-free survival (< 1 year) with high expression of Sema3D (B) and PlxnD1 (D).
Conclusion
Sema3D secretion, which is regulated by AnxA2, is crucial for PDA metastasis. We are establishing a platform to detect drugs which have the capacity to reduce Sema3D secretion, thereby detecting additional drugs designed to target PDA. We have optimized this cell-based assay which can be used in a moderately high throughput manner. We are in the process of establishing a stably transfected KxPxCx cell line which secretes GFP-tagged Sema3D which will allow ready detection of Sema3D secretion.
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