Clinicopathological features of pancreatic ductal adenocarcinoma patients.

<p>Clinicopathological features of pancreatic ductal adenocarcinoma patients.</p

Whole Genome Sequencing of Newly Established Pancreatic Cancer Lines Identifies Novel Somatic Mutation (<i>c</i>.<i>2587G>A</i>) in Axon Guidance Receptor Plexin A1 as Enhancer of Proliferation and Invasion

<div><p>The genetic profile of human pancreatic cancers harbors considerable heterogeneity, which suggests a possible explanation for the pronounced inefficacy of single therapies in this disease. This observation has led to a belief that custom therapies based on individual tumor profiles are necessary to more effectively treat pancreatic cancer. It has recently been discovered that axon guidance genes are affected by somatic structural variants in up to 25% of human pancreatic cancers. Thus far, however, some of these mutations have only been correlated to survival probability and no function has been assigned to these observed axon guidance gene mutations in pancreatic cancer. In this study we established three novel pancreatic cancer cell lines and performed whole genome sequencing to discover novel mutations in axon guidance genes that may contribute to the cancer phenotype of these cells. We discovered, among other novel somatic variants in axon guidance pathway genes, a novel mutation in the PLXNA1 receptor (c.2587G>A) in newly established cell line SB.06 that mediates oncogenic cues of increased invasion and proliferation in SB.06 cells and increased invasion in 293T cells upon stimulation with the receptor’s natural ligand semaphorin 3A compared to wild type PLXNA1 cells. Mutant PLXNA1 signaling was associated with increased Rho-GTPase and p42/p44 MAPK signaling activity and cytoskeletal expansion, but not changes in E-cadherin, vimentin, or metalloproteinase 9 expression levels. Pharmacologic inhibition of the Rho-GTPase family member CDC42 selectively abrogated PLXNA1 c.2587G>A-mediated increased invasion. These findings provide <i>in-vitro</i> confirmation that somatic mutations in axon guidance genes can provide oncogenic gain-of-function signals and may contribute to pancreatic cancer progression.</p></div

Mutant PLXNA1 mediates ligand-induced invasion of SB.06 cells.

<p>A) Invasion of SB.06 cells treated with scrambled siRNA and Sem3A(150 ng/mL), B) PLXNA1 siRNA knockdown and Sem3A, and C) intact SB.06 cells with no treatment. D) Quantification of SB.06 invasion compared with equivalently treated SB.07 cells. * p<0.05 via 2-tailed Student’s t-test.</p

Invasion of various pancreas cancer cell lines in response to semaphorin stimulation.

<p>SEMA3A stimulation (150ng/mL) increases invasion of SB.06 cells, but not of pancreas cell lines not harboring a PLXNA1 mutation.</p

Growth characteristics of established cell lines.

<p>Phase contrast microscopy of cell lines A) SB.07, B) SB.12, and C) SB.06. Formation of a ‘swirl’-like growth pattern (D) in cell line SB.06 at subconfluence and with E-cadherin/DAPI immunofluorescent staining (E). (F) Growth curves for established cell lines SB.06 (red), SB.07 (green), and SB.12 (blue).</p

Summary of genomic aberrations in established cell lines.

<p>A) SB.06 and B) SB.07 during WGS displayed as Circos plots of both genomes. The outer circle shows copy number variations (red, gains; green, losses); translocations are depicted as links in the interior of the plot.</p

Gene expression changes measured by Rho pathway qPCR array in 293T cells transfected with PLXNA1 plasmid.

<p>Gene expression changes measured by Rho pathway qPCR array in 293T cells transfected with PLXNA1 plasmid.</p

Chromosomal analysis of established cell lines.

<p>Karyograms of cell lines A) SB.06, B) SB.07, C) SB.12, and FISH validation of shown chromosomal translocations in cell lines D) SB.06, E) SB.07.</p

Immune phenotyping of parent tumor samples matches established cell lines.

<p>A) Immunohistochemical staining of parent tumor samples SB.06, SB.07 and SB.12 for CEA and CA-19-9; B) Immunofluorescence DAPI/Alexa-Fluor488 for E-cadherin, CEA, and CA19-9 of cell lines SB.06, SB.07, SB.12.</p

Proliferation of SB.06 cells harboring mutant PLXNA1 is reduced following RNAi silencing of PLXNA1.

<p>Cell growth of SB.06 and SB.07 cell lines following siRNA knockdown of PLXNA1 and 48 hours of treatment with 150 ng/mL of Sem3A, as measured by Cell-Titer Glo assay. *p<0.05 via 2-tailed Student’s t-test.</p