HER2 drives Mucin-like 1 to control proliferation in breast cancer cells

Abstract

Mucin-like 1 (MUCL1) was first identified as a breast-specific gene over a decade ago. Based on its highly restricted mRNA expression in breast tissue and continued expression during breast tumorigenesis and progression, MUCL1 is an attractive tumor-associated antigen and a potential therapeutic target. However, very little is known about the cellular location, biological functions and regulation of the MUCL1 protein, which will have a major impact on its druggability. Here we describe our efforts to fully characterize the cellular localization of MUCL1, investigate its regulation by key breast cancer oncogenes such as human epidermal growth factor receptor 2 (HER2) and discover its functional roles in breast cancer. Although some mucins are membrane bound, our data indicate that MUCL1 is secreted by some breast cancer cells, whereas others only express high levels of intracellular MUCL1. MUCL1 expression is highest in HER2-amplified breast tumors and inhibiting HER2 activity in tumor cells resulted in a decreased MUCL1 expression. In-depth investigation demonstrated that phosphoinositide3-kinase/Akt pathway, but not Ras/MEK pathway, controls MUCL1 expression downstream of HER2. Phenotypic assays revealed a strong dependence of HER2-positive cells on MUCL1 for cell proliferation. We further identified the mechanism by which MUCL1 regulates cell growth. Knockdown of MUCL1 induced a G1/S phase arrest concomitant with decreased cyclin D and increased p21 and p27 levels. Finally, we investigated the impact of MUCL1 loss on kinase signaling pathways in breast cancer cells through phospho-kinase array profiling. MUCL1 silencing abrogated phospho-focal adhesion kinase (FAK), Jun NH2-terminal kinase (JNK) and c-Jun signals, but not extracellular signal-regulated kinase or Akt pathway activities, thereby pointing to FAK/JNK pathway as the downstream effector of MUCL1 signaling. We are the first to identify an important role for MUCL1 in the proliferation of breast cancer cells, probably mediated via the FAK/JNK signaling pathway. Taken together, these data suggest a potential utility for therapeutic targeting of this protein in breast cancer.

Figures

Figure 1

A schematic of the MUCL1 amino acid sequence is presented. A hydrophobic signal peptide is present at residues 1–20 and a triple serine- and threonine-rich tandem repeat is present at residues 46–69. The antibody used for the current studies was generated against amino acids 19–53.

Figure 2

MUCL1 is highly expressed in normal breast tissue and breast cancer.(a)MUCL1 expression is highest in mammary gland in a cDNA array from Origene. (b)MUCL1 expression examined across a panel of human cancer samples shows breast cancer having the highest expression level. In addition, the normal tissue samples exhibiting the highest expression were all from breast samples and are highlighted in the box. Analyses were done using the Oncomine Power Tools database (powertools.oncomine.com).

Figure 3

MUCL1 RNA and protein expression was examined in a panel of breast and lung cancer cell lines as described in Table 1. (a) KPL4 cells were transiently transfected with either NT siRNA or MUCL1 siRNA for 48 h. Cell lysates were probed using a rabbit polyclonal anti-MUCL1 antibody. β-Actin was used as a loading control. (b) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, cell lysates and culture supernatant were immunoblotted using anti-DDK and anti-MUCL1 antibodies. (c) MDA-MB-361 cells were transiently transfected with either NT siRNA or MUCL1 siRNA for 48 h. Cell media was changed and conditioned media was collected 48 h later and assessed by MUCL1 enzyme-linked immunosorbent assay (ELISA). *P<0.01 (n=3). Cell lysates were probed using an anti-MUCL1 antibody. (d) RNA was extracted from cells and assessed forMUCL1 expression by reverse transcription PCR. RNA expression is shown as fold expression in each cell line divided by the median of MUCL1 expression across the panel of cell lines ±s.d. (n=3 technical replicates). For assessing protein levels, cells were grown for 48 h in serum-free media. Culture supernatant was collected and the secreted MUCL1 was measured by ELISA and normalized to the cell number. Intracellular MUCL1 levels were examined in whole-cell lysates by western blotting for comparison. The experiments were repeated twice with similar results.

Figure 4

(a) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, live cells were stained with anti-DDK or anti-MUCL1 antibodies followed by staining with a fluorescently labeled secondary antibody and analyzed by FCM. Cells expressing a known DDK-tagged membrane protein (positive control) demonstrated a significant shift in fluorescence. No shift in fluorescence was detected in HEK-293 cells transfected with DDK-tagged MUCL1 by either antibody. (b) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, DDK-tagged MUCL1 was probed for using the ThermoScientific Cell Surface Protein Isolation Kit. Cells were treated with a biotinylation reagent to label surface proteins and then collected, lysed and labeled proteins were purified using neutravidin agarose resin. The eluate contains the isolated, labeled cell surface proteins and the flow-through (FT) contains unlabeled, intracellular proteins. The experiments were repeated twice with similar results.

Figure 5

(a) Breast cancer cell lines were treated with the indicated doses of lapatinib for 72 h. Phospho-HER2 and MUCL1 levels were assessed by western blotting. β-Actin was used as a loading control. The percentage of MUCL1 relative to the vehicle control (0 μM) is shown. (b) The dose–response effect of growth inhibition following a 6-day lapatinib treatment is shown and the half maximal effective concentration (EC50) values for each cell line were calculated. The mean ±s.d. is plotted (n=5). (c) Breast cancer cells were treated with the indicated doses of the phosphoinositide3-kinase (PI3K) inhibitor GSK1059615 or the MEK1/2 inhibitor selumetinib for 48 h. Phospho-Akt, phospho-ERK1/2 and MUCL1 levels were assessed by western blotting. β-Actin was used as a loading control. The experiments were repeated twice with similar results.

Figure 6

(a) Breast cancer cell lines were transfected with MUCL1 siRNA or NT control siRNA. Cell proliferation was assessed by Cell Titer Glo Assay each day for 1 week. Mean ±s.d. is shown (n=10). *P<0.001 for difference of growth rate. MUCL1 knockdown was confirmed by western blotting. (b) BT474 and KPL4 cells were transfected with MUCL1 or NT siRNA for 96 h, methanol-fixed and stained with propidium iodide for cell cycle analysis. The percent of cells in each cell cycle phase is shown as the mean ±s.d. (n=3). (c) Western blots of cell cycle regulators 72 h post transfection shows significant decreases in cyclins D1 and D3, as well as increases in the cyclin-dependent kinase (Cdk) inhibitors p21cip1 and p27kip1. The experiments were repeated twice with similar results.

Figure 7

(a) KPL4 and SKBR3 cells were transiently transfected with MUCL1 siRNA or NT control siRNA and lysed for western blotting 48 h later. Immunoblot analysis showed a decrease in the phosphorylation of FAK, JNK and c-Jun in MUCL1 cells as compared with control cells, and phosphorylated MKK4 was decreased in KPL4 cells only. No changes were detected in activation of MKK7, Akt, ERK1/2 or HER2. β-Actin was used as a loading control. (b) Breast cancer cells were transfected with FAK siRNA or NT control siRNA. Cell proliferation was assessed by Cell Titer Glo Assay each day for 1 week. Mean ±s.d. is shown (n=10). *P<0.001 for difference of growth rate. FAK knockdown was confirmed by western blotting. (c) Proposed model depicting MUCL1-mediated FAK activation and signaling to downstream JNK. A potential interaction between MUCL1 with FAK is likely to be intracellular. Following integrin engagement, JNK activation requires association of FAK with a Src kinase and p130Cas, and the recruitment of Crk. The activation of JNK may be through MKK4 or other mediators depending on the cell type. On stimulation by JNK, c-Jun translocates to the nucleus and mediates G1/S phase transition leading to cell proliferation. The experiments were repeated twice with similar results.