Related Experiment Video
Updated: Jul 17, 2026
An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Published on: September 18, 2013
Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells
1Department of Oncology, Lombardi Comprehensive Cancer Center/Georgetown University, Washington, DC 20057, USA.
Abstract:
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
Insights
Cytokine scatter factor (SF) protects cancer cells from DNA damage-induced apoptosis via phosphoinositol-3-kinase/c-Akt signaling. New research identifies specific SF-inducible genes that contribute to this cytoprotective effect.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- Cytokine scatter factor (SF), also known as hepatocyte growth factor, mediates crucial cellular processes including apoptosis inhibition.
- SF plays a significant role in tumor cell survival, particularly during detachment, metastasis, and in response to therapies.
- Previous studies demonstrated SF's protective mechanism against DNA damage-induced apoptosis involves phosphoinositol-3-kinase/c-Akt signaling.
Purpose of the Study:
- To identify novel c-Akt-regulated genes involved in SF-mediated cytoprotection against DNA damage.
- To elucidate the specific genes that contribute to SF's role in promoting tumor cell survival.
Main Methods:
- Utilized DNA microarray assays (Affymetrix) to analyze gene expression in DU-145 prostate cancer cells treated with SF and/or a dominant-negative Akt mutant.
- Employed semiquantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR) for validation of microarray findings.
- Applied RNA interference (RNAi) to assess the functional contribution of identified SF-inducible/c-Akt-dependent genes to cytoprotection against adriamycin.
Main Results:
- Identified several SF-regulated genes, distinguishing between those induced in a c-Akt-dependent versus c-Akt-independent manner.
- Demonstrated that knockdown of AMPD3, MX1, and WNT4 individually resulted in a significant reduction in SF-mediated protection against adriamycin-induced apoptosis.
- Showed that a combination of three small interfering RNAs targeting these genes largely abolished SF's protective effect in both DU-145 and T47D cells.
Conclusions:
- Identified novel genes regulated by the c-Akt pathway that contribute to SF-mediated cytoprotection.
- These findings provide new molecular targets and insights into the mechanisms underlying tumor cell survival promoted by SF.
- The study highlights the complex genetic network regulated by SF/c-Akt signaling in conferring resistance to DNA-damaging agents.
More Related Videos
10:59Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment
Published on: November 23, 2017
11:32Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter
Published on: March 27, 2020
