Abstract P2-06-03: Leukemia inhibitory factor receptor as a tumor suppressor: A study on migration and invasion of breast cancer cells upon LIFR stimulation
Authors: Dempsey NG , Miller P , Lippman M 
Published: May 2016

BACKGROUND: Tumorigenesis is the result of a step-wise process during which a mutation activates an oncogene or inactivates a tumor suppressor gene. Identification of these genes is critical in order to develop effective therapies for breast cancer patients. Our group previously discovered the Leukemia Inhibitory Factor Receptor (LIFR) as a novel tumor suppressor gene via an in vivo RNAi screen in HMLE cells. HMLE is a partially transformed non-tumorigenic cell line; these cells can become tumorigenic with a single mutation, such as the Ras mutation that creates the HMLER line. HMLEs were transduced using an shRNA library targeting the entire human genome, and stably transfected cells were xenografted into NOD/SCID mice. Genomic DNA from resultant primary tumors were analyzed for the shRNA sequences that, when integrated, made HMLEs tumorigenic. LIFR emerged from this screen as a novel candidate tumor suppressor gene in breast cancer. Here we report on the decreased migration and invasion of breast cancer cells activated by LIFR stimulation.

METHODS: HMLER cells were plated at 500,000 cells per well of a six-well plate. Twenty-four hours later, HMLERs were treated with 100, 25, 12.5, 5, 2.5, or 0 ng/ml recombinant hLIF. Protein lysates were analyzed for phospho-STAT3 induction upon LIF stimulation. Based on the results, we selected 25 ng/ml as the appropriate hLIF concentration to maximally stimulate LIFR in the migration assay described here. HMLERs were serum starved for 8 hours. DMEM with 10% fetal bovine serum was added to the bottom of the migration assay plate as a chemoattractant. The cells were suspended in DMEM with 0.1% bovine serum albumin and either treated with 25 ng/ml LIF or no LIF. Thereafter, 25,000 cells were added to either a Corning Biocoat Matrigel Invasion Chamber or a control insert lacking a migration matrix. The migration assay plate was incubated at 37°C and the cells were allowed to migrate for 20 hours. Migrated cells were enumerated under the light microscope and a migration percentage was calculated.

RESULTS: In the first portion of the study, we found that low concentrations of LIF (2.5 ng/ml) resulted in p-STAT3 induction in HMLERs, but that p-STAT3 was maximally induced with 25 ng/ml of LIF. In the invasion and migration assay, HMLER cells that had not been treated with LIF displayed an aggressively invasive and migratory phenotype with 61.1% migration in matrigel compared to control inserts without the migration matrix. When HMLERs were treated with 25 ng/ml LIF, the cells displayed decreased invasion and migration with only 50.0% of cells migrating. Based on these results, LIFR stimulation inhibits the invasion and migration of breast cancer cells.

CONCLUSIONS: As a tumor suppressor gene, LIFR is vital to the normal functioning of a non-cancerous cell, and its loss can produce a tumorigenic and metastatic phenotype. Treatment with LIF converts aggressively metastatic breast cancer cells to a less invasive phenotype. Through a deeper understanding of LIFR's tumor suppressor effects, we can harness the anti-tumorigenic and anti-metastatic properties of LIFR stimulation and develop targeted therapies to prevent growth and metastasis of breast cancer.

Citation Format: Dempsey NG, Miller P, Lippman M. Leukemia inhibitory factor receptor as a tumor suppressor: A study on migration and invasion of breast cancer cells upon LIFR stimulation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-03.