The extracellular-regulated protein kinase 5 (ERK5) enhances metastatic burden in triple-negative breast cancer through focal adhesion protein kinase (FAK)-mediated regulation of cell adhesion

Abstract

There is overwhelming clinical evidence that the extracellular-regulated protein kinase 5 (ERK5) is significantly dysregulated in human breast cancer. However, there is no definite understanding of the requirement of ERK5 in tumor growth and metastasis due to very limited characterization of the pathway in disease models. In this study, we report that a high level of ERK5 is a predictive marker of metastatic breast cancer. Mechanistically, our in vitro data revealed that ERK5 was critical for maintaining the invasive capability of triple-negative breast cancer (TNBC) cells through focal adhesion protein kinase (FAK) activation. Specifically, we found that phosphorylation of FAK at Tyr397 was controlled by a kinase-independent function of ERK5. Accordingly, silencing ERK5 in mammary tumor grafts impaired FAK phosphorylation at Tyr397 and suppressed TNBC cell metastasis to the lung without preventing tumor growth. Collectively, these results establish a functional relationship between ERK5 and FAK signaling in promoting malignancy. Thus, targeting the oncogenic ERK5-FAK axis represents a promising therapeutic strategy for breast cancer exhibiting aggressive clinical behavior.

Fig. 1. ERK5 expression strongly associates with metastatic occurrence in breast cancer.

The relationship between the level of ERK5 expression and distant metastasis-free survival of breast cancer (BC) patients was assessed by Kaplan–Meier plotter. Independent cohorts of patients with early (grade 1) or late (grades 2 and 3) stage disease status were analyzed. Samples were divided into two groups with high (red) and low (black) expression of ERK5. Hazard ratios (HR) and logrank P values are shown.

Fig. 2. ERK5 is overexpressed in TNBC and its overexpression strongly correlates with poor prognosis.

A The level of Erk5 transcript was analyzed in 416 luminal/HER2+ tumors and 98 TNBC from TCGA dataset. Black lines in each group indicate median with interquartile range. B Kaplan–Meier analysis of distant metastasis-free survival of TNBC patients. Samples were divided into two groups with high (red) and low (black) expression of ERK5. Hazard ratio (HR) and logrank P values are shown. C A biopsy of human invasive ductal carcinoma and adjacent normal breast tissue (patient #481) was stained with H&E or with a specific antibody to ERK5 (brown). Scale bars: (2.5x) 500 μm, (10x) 100 μm, (40x) 20 μm.

Fig. 3. ERK5 silencing suppresses breast cancer cell invasion.

A, B Immunoblot analysis confirmed that shERK5(UTR) and shERK5(CDS) effectively downregulated ERK5 expression in MDA-MB-231 cells. Ectopic expression of ERK5 [+ F-ERK5(WT)] at a level similar to that of the endogenous protein was achieved by incubating shERK5(UTR)-expressing cells carrying an inducible F-ERK5(WT) construct with 10 ng/ml doxycycline (+) for 24 h. C ERK5 knockdown suppressed MDA-MB-231 cell invasion in FBS-free medium through Matrigel. The bar graph indicates the mean ± SD (N = 3). Representative pictures of the filters are shown. Scale bar: 200 μm. D Quantitative time course analysis of MDA-MB-231 cell adhesion on Matrigel by IncuCyte® live-cell imaging system. Representative pictures of cells taken 4 h after seeding in Matrigel-coated plates are shown. The data correspond to the mean ± SD (N = 3). E Lysates were obtained from cells cultured in FBS-free Matrigel-coated plates for 12 h and analyzed by immunoblot. The images are representative of three biological repeats. F Alternatively, transcript levels were measured by quantitative real-time PCR. Results are presented as fold change ± SD. The graph is representative of three independent biological repeats performed in duplicate.

Fig. 4. ERK5 co-localizes with p-FAK in focal adhesions.

iRFP720⁺ MDA-MB-231 cells carrying shERK5(UTR) were incubated for 24 h with 2 μg/ml doxycycline to induce F-ERK5(WT) expression, prior to being seeded on glass bottom dish coated with Matrigel and cultured for 6 h in FBS-containing medium. Ectopically expressed F-ERK5 and endogenous FAK were visualized by confocal microscopy using specific antibodies to the FLAG epitope (M2, green) and to p-FAK(Y397) (red). Nuclei were detected with DAPI (blue). Images at different Z positions are shown. Scale bars: 10 μm. The inset shows a higher magnification of focal adhesions where F-ERK5 and p-FAK(Y397) co-localized. Violet arrows highlight yellow staining indicative of F-ERK5/p-FAK(Y397) co-localization.

Fig. 5. ERK5 interacts with FAK signaling.

iRFP720⁺ MDA-MB-231 cells carrying shERK5(UTR) were incubated for 24 h with 2 μg/ml doxycycline to induce F-ERK5(WT) (A) or F-ERK5(D200A) (D) expression. Cells were subsequently seeded on Matrigel-coated glass bottom plate for 6 h prior to being fixed and incubated with antibodies to the FLAG epitope (M2) and p-FAK(Y397). Negative controls included incubation with each primary antibody separately and no primary antibodies. In situ detection of ERK5-FAK complexes was performed with oligonucleotide-labeled secondary antibodies according to the Duolink^® PLA fluorescence protocol (Sigma). Images were acquired by fluorescence microscopy. Scale bars: 10 μm. B, C Lysates were obtained from cells cultured in FBS-free Matrigel-coated plates for the indicated times. In panel C, iRFP720⁺ cells expressing shERK5(UTR) were incubated with 10 ng/ml doxycycline for 24 h to induce F-ERK5(WT) or F-ERK5(D200A) expression, prior to being seeded. MDA-MB-231 cells expressing shScr control or transduced with an empty pCHD plasmid (−) were utilized as controls. Similar results were obtained in two independent experiments.

Fig. 6. Induced ERK5 silencing accelerates tumor growth and suppresses FAK phosphorylation in mammary tumor grafts.

MDA-MB-231 cells carrying shERK5i were orthotopically transplanted into the mammary fad pad of CD1 nude mice. A Representative bioluminescence images of mammary tumors just before (day 20) and after mice were fed with doxycycline for 12 and 22 days. B Tumors were measured on average twice a week for the duration of the experiment. The data presented as fold increase in the volume of tumor size after the introduction of the dox diet at day 20 correspond to the mean ± SD. After 4 weeks, animals in the dox− and dox+ cohorts exhibiting large tumors or a change in normal behavior were humanely culled. Total N number of animals analyzed per condition over the duration of the experiment is indicated. C Tumors were collected 4 weeks after exposure to doxycycline and analyzed by immunohistochemistry with a specific antibody to ERK5 or to p-FAK(Y397). Scale bars: (40x) 20 μm.

Fig. 7. Induced ERK5 silencing suppresses breast cancer lung metastasis.

Control (dox−) and doxycycline-fed (dox+) mice bearing mammary tumor grafts were paired according to similar tumor size. A, B Breast metastasis was detected post mortem by quantification of live (DAPI⁻) breast cancer cells (tdT⁺) in the lung. A representative flow cytometry analysis is shown in B. The graphical analysis of the data demonstrated that ERK5 knockdown significantly reduced metastatic burden. Ratio paired t test was applied for p value calculation, p ≤ 0.01 (**). Raw numbers utilized for the graph are presented in the table. C Breast metastasis to the spleen and bone were detected by bioluminescence imaging in one control (dox−) animal of a pair sacrificed at 4 weeks after dox exposure.