A key tyrosine (Y1494) in the beta4 integrin regulates multiple signaling pathways important for tumor development and progression

Abstract

Expression of the alpha6beta4 integrin is associated with poor patient prognosis and reduced survival in a variety of human cancers. In recent years, a limited number of in vivo studies have examined the contribution of this integrin receptor to cancer progression and they have revealed that the alpha6beta4 integrin plays a multifaceted role in regulating tumor development and progression. In the current study, we investigated the mechanism by which one tyrosine residue in the beta4 subunit cytoplasmic domain, Y1494, contributes to the tumor-promoting functions of the alpha6beta4 integrin in vivo. We show that Y1494 participates in the stimulation of diverse signaling pathways that promote alpha6beta4-dependent tumor growth and invasion. Mutation of Y1494 inhibits the ability of the alpha6beta4 integrin to support anchorage-independent growth in vitro and tumor development and angiogenesis in vivo, a result that mimics the loss of total expression of the beta4 subunit. Our results support the hypothesis that Y1494 regulates alpha6beta4-dependent anchorage-independent growth through activation of the extracellular signal-regulated kinase 1/2 signaling pathway, and invasion through the combined activation of phosphatidylinositol 3-kinase and Src. Collectively, our results identify Y1494 as a major regulatory site for signaling from the alpha6beta4 integrin to promote tumor development and progression.

Figure 1

Y1494 in the β4 integrin subunit is required for α6β4 integrin-dependent anchorage independent growth and invasion of tumor cells. (A) Representative bright-field images captured at 4X magnification of Mock, wt-β4 and Y1494F-β4 transfected MDA-MB-435 cell lines grown in 0.3% agar with complete growth medium for 2 weeks. The graph shows the mean (± SEM) of three representative experiments performed in triplicate. 50 fields per well were counted for each assay. *p < 0.01 (B) Cells were recovered from 3D Matrigel after 10 days by dispase treatment and apoptosis was measured by flow cytometry after staining with Annexin V-PE and 7-AAD. The data shown represent the mean percentage (± SEM) of Annexin V-PE+, 7-AAD-cells from three independent experiments performed in duplicate. p > 0.05. (C) Representative fluorescence images captured at 10X magnification of Mock, wt-β4 and Y1494F-β4 transfected MDA-MB-435 cells that were stained with calcien dye after 10 days of growth in 3D Matrigel cultures to detect viable cells. (D) Representative images captured at 4X magnification of MDA-MB-231 cells and Mock, wt-β4 and Y1494F-β4 transfected MDA-MB-435 cells grown for 10 days in 3D Matrigel culture. (inset magnification = 20X).

Figure 2

Y1494 in the β4 integrin subunit is required for α6β4 integrin-dependent activation of ERK1/2. (A) wt-β4 and Y1494F-β4 transfected MDA-MB-435 cells were maintained in suspension or allowed to adhere to laminin-1 coated plates for 30 minutes. Aliquots of cell extracts that contained equivalent amounts of total protein were resolved by SDS-PAGE (10%) and then immunoblotted with the indicated antibodies. (B) wt-β4 and Y1494F-β4 transfected MDA-MB-435 cells were maintained in suspension or incubated with either α6- or β4-specific antibodies and allowed to adhere to anti-mouse IgG-coated plates for 30 minutes. (C) Mock, wt-β4 and Y1494F-β4 transfected MDA-MB-435 cells were recovered from 3D-Matrigel after 10 days by dispase treatment. Aliquots of cell lysates containing equivalent amounts of total protein were immunoblotted with antibodies specific for phospho-ERK1/2, phospho-Ser217/221 of MEK1/2 or tubulin. The immunoblots for the phospho-specific antibodies were stripped and reprobed for total expression levels. Densitometry measurements shown in (C) are the respective ratio of phospho-protein to total protein levels. CS, cells maintained in suspension; LC, cells adherent to laminin-1; α6, cells clustered with a α6-specific antibody (2B7); β4, cells clustered with a β4-specific antibody (UM-A9).

Figure 3

Constitutively active MEK rescues Y1494F-β4 integrin-dependent anchorage independent growth in soft agar but not invasion in 3D Matrigel. Y1494F-β4 cells were co-transfected with either pMCL-MKK1-R4F (constitutively active MEK-DD) or empty vector and pLPCX vector for puromycin selection. (A) Aliquots of cell lysates containing equivalent amounts of total protein were immunoblotted with antibodies specific for MEK1/2, phospho-ERK1/2, or tubulin. Densitometry measurements shown below each panel are the respective ratio of MEK1/2 to tubulin and phospho-ERK1/2 to total ERK1/2 levels. (B) Representative bright-field images captured at 4X magnification of empty vector or MEK-DD-transfected Y1494F-β4 cells grown in 0.3% agar with complete growth medium for 2 weeks. Quantitation of the soft agar assays is shown in the graph on the right. The data represent the mean (± SEM) of three representative experiments performed in triplicate. 50 fields per well were counted for each assay. *p < 0.01 (C) Representative bright-field images at 10X magnification of empty vector or MEK-DD-transfected Y1494F-β4 cells grown in 3D Matrigel for 10 days. (D) Cells were recovered from the 3D Matrigel cultures by dispase treatment and aliquots of cell lysates containing equivalent protein were lysed and immunoblotted with antibodies specific for MEK1/2, phospho-ERK1/2, or tubulin. Densitometry measurements shown below each panel are the respective ratio of MEK1/2 to tubulin and phospho-ERK1/2 to total ERK1/2 levels.

Figure 4

Src and PI3K activation are required for optimal promotion of invasion by the α6β4 integrin. (A) wt-β4 and Y1494F-β4 transfected MDA-MB-435 cells were maintained in suspension or incubated with α6-specific antibodies and allowed to adhere to anti-mouse IgG-coated plates for 30 minutes. Aliquots of cell lysates containing equivalent amounts of total protein were immunoblotted with antibodies specific for phospho-SrcY418 or actin. The phospho-Src immunoblot was stripped and reprobed for total Src expression levels. CS, cells maintained in suspension; α6, cells clustered with a α6-specific antibody (2B7). (B) Representative bright field images at 10X magnification of wt-β4 cells, wt-β4 cells in the presence of 50 μM LY294002 (PI3K inhibitor) or 50μM PP2 (Src inhibitor), and Y1494F-β4 cells grown for 10 days in 3D Matrigel culture. (C) Cells were recovered from Matrigel after 10 days by dispase treatment and apoptosis was measured by flow cytometry after staining with Annexin V-PE and 7-AAD. The data shown represent the mean percentage (±SEM) of Annexin V-PE+, 7-AAD- cells from three independent experiments performed in duplicate. p < 0.01. (D) Wt-β4 cells were assayed for their invasive potential in the presence of 50 μM LY294002, 50μM PP2 or both using a 2D Matrigel Transwell assay. Five independent fields/well were counted. The data shown represent the mean (±SEM) of two independent experiments performed in triplicate. **p < 0.01.

Figure 5

Combined activation of PI3K and Src rescues Y1494F-β4-dependent invasion. (A) Y1494F-β4 cells were stably transfected with either Src Y527F, myr-p110α PI3K, or empty vector. Aliquots of cell lysates containing equivalent amounts of total protein were immunoblotted with antibodies specific for phospho-SrcY418, phospho-SrcY527, phospho-Akt-S473, or actin. The phospho-immunoblots were stripped and reprobed for total expression levels. Densitometry measurements shown below each panel are the respective ratio of phospho- to total expression levels. (B) Representative bright-field images captured at 4X magnification of Y1494F-β4 cells transfected with Src Y527F or myr-p110 PI3K grown in 0.3% agar with complete growth medium for 2 weeks. (C) Y1494F-β4 cells were transfected transiently with empty vector, MEK-DD, SrcY527F, Myr-p110 or SrcY527F and Myr-p110 together and assayed for their invasive potential using a 2D Matrigel Transwell assay. Five independent fields/well were counted. The data shown represent the mean (±SEM) of three independent experiments performed in triplicate. *p < 0.01. (D) Aliquots of tumor extracts from Mock, wt-β4 and Y1494F-β4 derived tumors (n = 3) containing equivalent amounts of total protein were immunoblotted with antibodies specific for β4, phospho-MEK1/2, phospho-ERK1/2, phosphoY418Src, phosphoS473 of Akt and actin. The immunoblots for the phospho-specific antibodies were stripped and reprobed for total expression levels.