MUC4 overexpression augments cell migration and metastasis through EGFR family proteins in triple negative breast cancer cells

Abstract

Introduction: Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method: In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results: MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions: MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.

Figure 1. MUC4 promotes proliferation and growth of MDA-MB-231 cells.

(A) Detection of MUC4 protein expression in control (MDA-MB-231-SCR) and MUC4 knockdown (MDA-MB-231-shMUC4) cells. Immunoblot showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. Immunostained cells using human anti-MUC4 mouse monoclonal antibody (8G7) showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. (B) In proliferation analyses, when the number of cells was plotted against the incubation period (hours), control cells showed a significantly higher proliferation rate than MUC4 knockdown cells, p = 0.03. Population doubling time of control cells was less than MUC4 knockdown cells when calculated from the number of cells growing in log phase (day 2 to 6) using the formula, T_d = 0.693t/ln (N_t/N₀). (C) Cells, following synchronization and serum re-stimulation and stained with Telford reagent (containing propidium iodide) and analyzed by FACS, showed that the number of MUC4 knockdown cells in the G1 phase was higher than control cells suggesting the inhibition of cell cycle progression. (D) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-dependent conditions. After staining, colonies of >50 µm in size were counted using Quantity One software, p = 0.003. Images of anchorage–dependent growth assays were shown at the bottom. (E) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-independent conditions. Colonies were counted and plotted. Columns: mean of triplicates; bars: SD, p = 0.0001. Phase-contrast images were recorded at 10× magnification. Microscopic images of colonies found in anchorage–independent growth assays. Higher magnification of a typical colony was shown in box on bottom right corner to emphasize a (i) big colony with migratory outer cells versus (ii) smaller and compact colonies. SCR are control and shMUC4 are MUC4 knockdown cells. All data presented are the average of 3 independent experiments.

Figure 2. MUC4 up-regulates EGFR family receptors and induces downstream Erk1/2 and PKC-γ pathways.

(A) Immunoblot analyses showed reduced expression of EGFR, ErbB2, and ErbB3 in MUC4 knockdown cells compared with control cells. (B) Reduced expression (using immunoblot) of Sprouty 2 was detected in MUC4 knockdown cell when compared with control cells. (C) Immunoblot showed that reduced phosphorylation of Erk1/2 and expression of PKC-γ in MUC4 knockdown cells compared with control cells. β-actin was used as a loading control. (D) Immunoblot analyses showed reduced expression of β-catenin and its target gene product cyclin D1in MUC4 knockdown compared with control cells.

Figure 3. MUC4 enhances migratory and invasive potential.

(A) After 24 hours serum starvation, a wound was created with a plastic tip on plates containing control or MUC4 knockdown cells. Cells were incubated in complete media for 12 hours. Motility of cells was photographed under bright-field microscopy (left, 10× magnification). After 12 hours, the migration of control cells and MUC4 knockdown cells was measured (in µm²) using DatInf Measure setup wizard software (http://tucows.texasonline.net). Values were calculated and plotted (right). (B and C) Control and MUC4 knockdown cells were serum starved for 48 h and seeded on non-coated or Matrigel-coated membranes for motility (B) and invasion (C) assays, respectively, and incubated for 24 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemo-attractant. Cells that did not migrate through the Matrigel and/or pores in the membrane were removed using a cotton swab, and cells on the other side of the membrane were stained and photographed under bright-field microscopy (10× magnification). The number of cells that migrated and invaded was higher in control than the MUC4 knockdown cells. Data presented are the average number of cells/field for 10 fields. Columns: average of three independent experiments; bars: SE, p = 0.002 and p = 0.001, respectively. Representative images of control and MUC4 knockdown cells were shon in both figures. (D) Phalloidin staining showed that visualized F-actin under a laser scanning microscope is reduced in MDA-MB-231-shMUC4 cells compared with the control cells. (E) Immunoblot analysis showed reduced phosphorylation of FAK in MUC4 knockdown cells compared with the control cells. (F) Immunoblot analysis showed reduced expression of mesenchymal markers such as vimentin and vitronectin; and increased expression of CK-18 in MUC4 knockdown cells compared to the control.

Figure 4. MUC4 contributes to a altered phenotype.

(A) The control or MUC4 knockdown cells were seeded in 2% Matrigel on top of a 100% Matrigel layer, and fed with media every 3 days. After 7 days, acini-like structures were photographed under a phase-contrast microscope. The acini-like structures (examples shown in the boxes) that were regular (smooth and spherical shape) or irregular (irregular outline, multi-lobular) were counted and plotted as a percentage of the total count (p = 0.0005 for regular and p = 0.002 for irregular). A minimum of 120 structures was counted for each of control cells or MUC4 knockdown cells. Reduced irregular outline, multi-lobular and increased smooth and spherical shape colonies were found in MUC4 knock down cells when compared with control cells. (B) Structures were stained with the anti-ZO-1 antibody. 4,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Optical sections (0.7–0.9 µm) were captured using a laser scanning confocal microscope. The images presented here are the central planes of the acini. Bar = 20 µm.

Figure 5. MUC4 promotes growth of MDA-MB-231 xenografts.

(A) MUC4 knockdown and control cells (0.1 × 10⁶ cells/animal) were orthotopically implanted in mouse mammary fat pad of each mouse (right 3^rd mammary gland). Tumor volumes were calculated every week. The MUC4 knockdown cells started to grow tumors during the 5^th week, but control cells started to grow tumors during the 3^rd week, p = 0.0001. (B) Eight weeks after implantation, mice were sacrificed and tumors were excised and weighed. Stable silencing of MUC4 was found to decrease tumor growth, p = 0.0001.

Figure 6. Differential over-expression of MUC4 mucin in TNBC tissues compared with normal breast tissues.

Immunohistological analyses were performed using the anti-MUC4 mouse monoclonal antibody (2214, generated in our laboratory, against a sequence close to the N-terminus of human MUC4) on tumor microarrays (BR1503 and BR10010) containing normal breast and invasive TNBC tissues and observed under a Nikon light microscope. MUC4 expression in invasive primary (n = 35) TNBC tissues were compared with normal breast tissue (n = 6) in a set of arrays. High immune-reactivity for MUC4 was detected in invasive TNBC tissues, but not in normal breast tissues. The image presented was taken at 4× magnification, and the higher magnification images (marked with a red box) were taken at 10× magnification.

Figure 7. MUC4-associated gene expression, pathways, and interaction networks in MDA-MB-231 cells.

Array data were validated using RT-PCR using specific primers: 20 ng mRNA from control and MUC4 knockdown cells were reverse transcribed and used for RT-PCR using MUC4 specific primers and the LightCycler SYBR Green 1 Master. The β-actin specific primers were used as control. CT values were calculated and plotted to verify the up-regulated and down-regulated genes.

Figure 8. A schematic diagram showing the contribution of MUC4 in the overall aggressiveness of TNBC cells.

MUC4 maintains the sustained expression of EGFR family proteins and thereby potentiates downstream signaling events mediated through the PKC-γ and Erk1/2 signaling. MUC4 maintains sustained expression of β-catenin, which induces proliferation, tumorigenesis, migration, invasion, and metastasis.