Dataset Information

Proteomic profiling of human cancer pseudopodia for the identification of anti-metastatic drug candidates

ABSTRACT: Cancer metastasis causes approximately 90% of all cancer-related death. Independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis progresses by cancer cell migration and invasion throughout the body. This migration of cancer cells requires the formation of pseudopodia in the direction of movement, but detailed understanding of the process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human cancer pseudopodia to examine this mechanisms essential to metastasis formation, and identify potential candidates for pharmacological interference with the process. We demonstrate that Prohibitins (PHBs) are significantly enriched in the pseudopodia fraction derived from cancer cells, and knockdown of PHBs, as well as their chemical inhibition through Rocaglamide (Roc-A), efficiently reduces cancer cell migration.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Johannes Graumann

LAB HEAD: Johannes Graumann

PROVIDER: PXD004658 | Pride | 2018-04-12

REPOSITORIES: Pride

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Proteomic profiling of human cancer pseudopodia for the identification of anti-metastatic drug candidates.

Choi Sunkyu S Bhagwat Aditya M AM Al Mismar Rasha R Goswami Neha N Ben Hamidane Hisham H Sun Lu L Graumann Johannes J

Scientific reports 20180411 1

Cancer metastasis causes approximately 90% of all cancer-related death and independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis implies cancer cell migration and invasion throughout the body. Migration requires the formation of pseudopodia in the direction of movement, but a detailed understanding of this process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human ...[more]

PMID: 29643415

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Project description:Cell motility and invasion initiate metastasis. However, only a subpopulation of cancer cells within a tumor will ultimately become invasive. Due to this stochastic and transient nature, in an experimental setting, migrating and invading cells need to be isolated from the general population in order to study the gene expression profiles linked to these processes. This report describes microarray analysis on RNA derived from migrated or invaded subpopulations of triple negative breast cancer cells in a Transwell set-up, at two different time points during motility and invasion, pre-determined as M-bM-^@M-^\earlyM-bM-^@M-^] and M-bM-^@M-^\lateM-bM-^@M-^] in real-time kinetic assessments. Invasion- and migration-related gene expression signatures were generated through comparison with non-invasive cells, remaining at the upper side of the Transwell membranes. Late-phase signatures of both invasion and migration indicated poor prognosis in a series of breast cancer data sets. Furthermore, evaluation of the genes constituting the prognostic invasion-related gene signature revealed KruM-LM-^Hppel-like factor 9 (KLF9) as a putative suppressor of invasive growth in breast cancer. Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the M-bM-^@M-^\earlyM-bM-^@M-^] invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue. Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro. In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects. After selection of two time points (M-bM-^@M-^\earlyM-bM-^@M-^] and M-bM-^@M-^\lateM-bM-^@M-^]), RNA from invasive and migratory MDA-MB-231 cells was isolated from Transwell membranes and hybridized onto an Illumina HumanHT-12 v4 Expression beadchip. Gene expressions of migrated/invaded subpopulations vs non-motile cells were compared.

Project description:Metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed both in vitro and in vivo migration of human PC-3 prostate cancer cells . We isolated 6 subpopulation of cells: TEM4-18 were isolated from in vitro transendothelial migration of PC-3 cells; GS672.Ug, GS683.LALN and JD1203.Lu are single passaged in vivo cell lines from TEM4-18; GS689.Li and GS694.LAd are twice passaged in vivo cell lines from PC-3 cells. All the subpopulations crossed an endothelial barrier more efficiently and more aggressive in a murine metastatic colonization model than parental PC-3 cells. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. These cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition, including frank loss of E-cadherin expression and upregulation of the E-cadherin repressor ZEB1. We used microarray to detail the global programme of gene expression underlying cancer metastasis. We wanted to compare the expression profiles of cells that had undergone transendothelial migration in vitro or metastasis in vivo. TEM4-18 was generated from the parental PC-3 that was plated onto a confluent human microvascular endothelial cell line from the lung and allowed to migrate across this monolayer. GS672.Ug were isolated from urogenital after intravenous injection of PC-3 cells. GS683.LALN and JD1203.Lu were isolated from lung after intravenous injection of TEM-418 cells. JD549.Ki were isolated from kidney after intravenous injection PC-3 cells. GS689.Li and GS694.LAd were isolated from liver and left adrenal after intravenous injection of JD549.Ki. All the cell lines are analyzed once except duplicate for TEM4-18 in this experiment for a total of 7 samples in this microarray. All other five sublines were then compared to TEM4-18 cells for changes in gene expression.

Dataset Information

Proteomic profiling of human cancer pseudopodia for the identification of anti-metastatic drug candidates

Publications

Proteomic profiling of human cancer pseudopodia for the identification of anti-metastatic drug candidates.

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