OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition

Abstract

Background: Hormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT).

Methods: The relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells.

Results: ER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don't detach at significant rates.

Conclusion: OSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.

Keywords: Oncostatin M; breast tumor metastasis; cluster of differentiation 44; epithelial to mesenchymal transition.

Figure 1

OSM induces EMT and a BCSC phenotype in human breast cancer cells (A) MCF-7 luc human breast cancer cells were treated with OSM (25 ng/mL) for 72 hrs. OSM induces an increased mesenchymal phenotype and a decrease in E-cadherin expression, as measured by immunofluorescence using antibodies for E-cadherin and actin (63× magnification). (B) MCF-7 cells were treated with OSM (25 ng/mL) for 1 hr, and qPCR was performed with primers for the EMT transcription factors Slug and Snail (*P<0.05, unpaired t-test). (C) MCF-7 cells were treated with OSM (25 ng/mL) for 24, 48 and 72 hrs, and cell lysates were assessed by Western blot analysis for E-cadherin and α-catenin (D and E). ER+ MCF-7 and T47D cells were treated with OSM (25 ng/mL) for 72 hrs before flow cytometry was performed. In T47D cells, OSM induces the CD44⁺/CD24⁻ BCSC population 3-fold, while a small non-significant increase was seen in MCF-7 cells. **P<0.01, *P<0.05, unpaired t-test.

Figure 2

OSM induces CD44s and CD44 isoforms (above 85 kDa) and cleaved products (below 50 kDa) in ER+ cells, but not ER- cells (A) tumor cells were treated with OSM for 24, 48, and 72 hrs and whole cell lysates were collected followed by immunoblotting for CD44 using a CD44 antibody kindly provided by Dr. Mellor CD44 expression in MCF-7 (ER+), and (B) quantification of (A). (C) CD44 expression in T47D (ER+), and (D) quantification of (C). (E) CD44 expression in MDA-MB-231 (ER-), and (F). quantification of (E). All OSM treatment time points were compared to the corresponding non-treatment time points after normalizing to ß-actin. Unpaired t-tests were performed for each time point. **P<0.01, *P<0.05.

Figure 3

Reduced CD44 expression does not abrogate OSM-induced EMT in ER+ cells (A) Both MCF-7 luc-shCD44 and MDA-MB-231 luc-shCD44 cells show knockdown of CD44 protein expression compared to non-targeting control (shNTC) cells by Western blot analysis, and quantification in (B). MCF7 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) for one hour, and qPCR analysis for Slug and Snail was performed. Knockdown of CD44 expression did not decrease OSM-induced Slug (C), or Snail (D) in these cells (n.s. = no significance, *p<0.05, **P<0.005, ***P<0.001, One way Anova). (E) MCF7 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) for 72 hrs, and E-cadherin was observed via immunofluorescence (20x magnification, scale bar-20 nm). (F) MCF7 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) for 72 hrs to observe EMT morphology by phase contrast images (20x magnification, scale bar-100 µm).

Figure 4

Reduced CD44 expression decreases OSM-induced tumor cell detachment in ER+ cells (A) ER+ MCF7 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) over a 5-day period, detached cells were counted on days 1, 3, and 5, and data is represented as fold-change in graphical representation (*P<0.05, **P<0.005, ***P<0.001, one-way Anova). (B) ER- MDA-MB-231 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) over a 5-day period, detached cells were counted on days 1, 3, and 5, and data is represented as fold change calculated by comparing non-OSM treated cells to OSM treated cells and differences between OSM treated shNTC and shCD44 as shown in graphical representation. ***P<0.001, one-way Anova.

Figure 5

Reduced CD44 expression decreases OSM-induced tumor cell migration in ER- cells greater than ER+ cells (A) ER+ MCF-7 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) for 3 days, wound healing was analyzed using ImageJ software, and data was interpreted as fold-change. (B) ER- MDA-MB-231 luc-shNTC and shCD44 cells were treated with OSM (25 ng/mL) for 3 days, wound healing was analyzed using Image J software, and data was interpreted as fold-change. ***P<0.001, one-way Anova.

Figure 6

OSM signaling induces an EMT and upregulates CD44 and cleavage of CD44 in breast tumor cells: Once bound to its receptor, OSM induces cells to undergo EMT and subsequent detachment and migration. OSM signaling increases expression of the transmembrane protein CD44 and also cleavage via MMPs. OSM-induced CD44 is involved in migration and detachment as well as docking, which is the step immediately before extravasation during the metastatic cascade.