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IGF2 knockdown in two colorectal cancer cell lines decreases survival, adhesion and modulates survival-associated genes

  • Original Article
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Tumor Biology

Abstract

Increased expression of insulin-like growth factor 2 (IGF2) is found in tumors of colorectal cancer (CRC) patients exhibiting a gained region on chromosome 11q15 and is implicated in poor patient survival. This study analyzes in vitro phenotypic- and gene expression changes associated with IGF2 shRNA-mediated knockdown. Initially, doxycycline inducible IGF2 knockdown cell lines were generated in the CRC cell lines SW480 and LS174T. The cells were analyzed for changes in proliferation, cell cycle, apoptosis, adhesion, and invasion. Expression profiling analysis was performed, and, for a subset of the identified genes, expression was validated by qRT-PCR and Western blot. IGF2 knockdown inhibited cell proliferation in both cell lines induced G1 cell cycle blockade and decreased adhesion to several extracellular matrix proteins. Knockdown of IGF2 did not alter invasiveness in SW480 cells, while a slight increase in apoptosis was seen only in the LS174T cell line. Knockdown of IGF2 in SW480 deregulated 58 genes, several of which were associated with proliferation and cell-cell/cell-ECM contacts. A subset of these genes, including CDK2, YAP1, and BIRC5 (Survivin), are members of a common network. This study supports the concept of direct autocrine/paracrine tumor cell activation through IGF2 and a shows role of IGF2 in CRC proliferation, adhesion and, to a limited extent, apoptosis.

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Acknowledgments

We thank the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Whole-Genome Expression Beadchips and related services. We also wish to thank the German Research Foundation who supported this project (Peter Lichter, LI 406/12-2; Martin Schneider, SCHN 947/2) in the framework of the Clinical Research Group KFO-227.

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Authors and Affiliations

  1. Division of Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany

    Michael A. Rogers, Verena Kalter & Peter Lichter

  2. Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany

    Moritz Strowitzki & Martin Schneider

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Electronic supplementary material

Supplementary Table 1

Primers used for qRT-PCR analysis. (PDF 29 kb)

Supplementary Table 2

Antibodies used for Western blot analysis. (PDF 29 kb)

Supplementary Table 3

Microarray data for significantly deregulated genes at day 3 and 5. P. adjust, adjusted p-value; FC, fold change. (XLSX 22 kb)

Supplementary Table 4

Patient data. (DOCX 14 kb)

Supplementary Fig 1

Analysis of apoptosis in the IGF2–1 shRNA cell lines. a. Annexin-V 7-AAD measurements in the SW480 cell line (day 4). b. AnnexinV/7-AAD measurements in the LS174T cell line (day 4). c. Activated caspase-3 measurements in the LS174T cell line (day 4). *, p-values. (EPS 113 kb)

Supplementary Fig 2

a. Prediction of phenotype based on genes associated with specific terms in the Ingenuity Disease and Function database. Increased/decreased relate to the term named in the specific column (i.e. increased proliferation). Note Z-scores. b. Network analysis of a subset of the microarray data. Green, downregulated vs controls; Red, upregulated vs controls. Solid arrows, activates the affected gene; hatched arrows, inhibits the affected gene. (EPS 16932 kb)

Supplementary Fig 3

Evaluation of YAP1 overexpression in SW480 cells and in the IGF2–1 shRNA (tet-on) permanent cell line. a, b. SW480 cells. a. qRT-PCR analysis of YAP1 3 days after transfection with the YAP1 overexpression vector. b. Western blot analysis of YAP1 (similar experiment as a). c-e. IGF2–1 shRNA permanent cell line and controls. c-d. Analysis of IGF2 and YAP1 mRNA expression at day 3 after transfection with YAP1. e. Analysis of cell proliferation at day 6 after transfection with YAP1. (EPS 18855 kb)

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Rogers, M.A., Kalter, V., Strowitzki, M. et al. IGF2 knockdown in two colorectal cancer cell lines decreases survival, adhesion and modulates survival-associated genes. Tumor Biol. 37, 12485–12495 (2016). https://doi.org/10.1007/s13277-016-5115-x

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