Hypoxia suppression of Bim and Bmf blocks anoikis and luminal clearing during mammary morphogenesis

Abstract

Proper adhesion to extracellular matrix is critical for epithelial cell survival. Detachment from matrix signals results in apoptosis, referred to as anoikis. Selective apoptosis of cells that become detached from matrix is associated with the formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro. Because early breast cancer lesions such as carcinoma in situ, characterized by ducts exhibiting lumens filled with cells, are often associated with hypoxic markers, we sought to examine the role of hypoxia in anoikis and lumen formation in mammary epithelial cells. Here, we show that hypoxic conditions inhibit anoikis and block expression of proapoptotic BH3-only family members Bim and Bmf in epithelial cells. Hypoxia-mediated anoikis protection is associated with increased activation of the epidermal growth factor receptor-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (Erk) kinase pathway and requires the hypoxia-activated transcription factor. Consistent with these data, hypoxic conditions inhibit luminal clearing during morphogenesis in human mammary epithelial acini when grown in three-dimensional cultures and are associated with decreased expression of Bim and Bmf as well as Erk activation. We show that hypoxia regulates specific cell survival pathways that disrupt tissue architecture related to clearing of luminal space during mammary morphogenesis and suggest that hypoxia-mediated anoikis resistance may contribute to cancer progression.

Figure 1.

Hypoxia blocks anoikis and Bim and Bmf expression. (A) Attached MCF-10A cells were incubated at 20% (normoxia) or 1% O₂ (hypoxia) for 6 h and then lysed and subjected to immunoblot analysis to determine level of HIF-1α induction. (B) Attached MCF-10A cells were cultured under normoxic or hypoxic conditions for 24 h and then lysed for total mRNA. Equal amounts of total mRNA were analyzed by quantitative RT-PCR to determine relative levels of the hypoxic target ADM. (C) Attached or suspended MCF-10A cells were incubated under normoxic or hypoxic conditions or treated with vehicle or DFOM for 36 h and assessed by fluorescence-activated cell sorting analysis to determine level of cell death by Annexin V/PI labeling. Histogram represents data from three independent experiments. Error bars indicate SE. (D) Attached or suspended MCF-10A cells were incubated under normoxic or hypoxic conditions for 48 h, and equal amounts of protein lysate were subjected to immunoblot analysis and analyzed for cleaved caspase-3, Bim, and Bmf protein levels. Actin was used as a loading control. (E) Attached (0 h in suspension) or suspended MCF-10A cells were treated with vehicle or DFOM and then lysed at indicated times and assessed for levels of cleaved caspase-3, Bim, and Bmf by using immunoblot analysis. Actin was used as a loading control (*p < 0.05, **p < 0.01).

Figure 2.

Mek–Erk signaling is maintained in suspended cells under hypoxia and is required for hypoxic inhibition of anoikis. (A) MCF-10A cells were cultured in suspension for the indicated times under normoxic or hypoxic conditions and then lysed for protein. Immunoblot analysis was used to determine levels of phospho- and total Erk, Mek, and Akt as well as level of Bim protein. Actin was used as a loading control. (B) Attached or suspended MCF-10A cells were pretreated with dimethyl sulfoxide (DMSO) or indicated inhibitor compounds then incubated at 20% or 1% O₂ for 48 h. Whole cell lysates were immunoblotted for indicated protein levels. Actin was used as a loading control. (C) Suspended MCF-10A cells were pretreated with DMSO or indicated inhibitors, grown in suspension for 36 h, and then subjected to Annexin V/PI staining and fluorescence-activated cell sorting analysis to determine level of cell death. Histogram represents data from three independent experiments. Error bars indicate SE (***p < 0.005).

Figure 3.

HIF-1α is required for hypoxia-mediated inhibition of Bim, Bmf, and anoikis. MCF-10A cells were transfected with control or HIF-1α–targeting siRNA oligonucleotides. (A) Attached cells were placed under hypoxic conditions for 6 h, and then cells were lysed and immunoblot analysis was used to assess HIF-1α knockdown. (B) After transfection, cells were placed in attached or suspended culture under normoxic or hypoxic conditions for 48 h. Cells were lysed and levels of Bim, Bmf, and cleaved caspase-3 were determined using immunoblot analysis. Actin was used as a loading control. (C) After transfection, cells were cultured in suspension for 48 h under normoxic or hypoxic conditions. Cells were then analyzed for level of cell death using Annexin V/PI staining and fluorescence-activated cell sorting analysis. Histogram represents data from three independent experiments Error bars indicate SE (*p < 0.05).

Figure 4.

Hypoxia disrupts acinar organization, blocks luminal clearance and Bim and Bmf expression in 3D culture. MCF-10A cells were placed in a morphogenesis assay under standard tissue culture conditions (20% O₂, 5% CO₂) until day 6, and cells were then incubated under normoxic or hypoxic conditions for the duration of the experiment. (A) At day 10, structures were fixed and stained with antibodies directed against cleaved caspase-3, actin, β-catenin, integrin α5, integrin α6, and E-cadherin. For all images, blue indicates nuclear stain DAPI. (B) At days 8, 10, and 12, structures were fixed and stained with the nuclear stain DAPI, and the percentage of filled acini in the population was determined for each condition. Histogram represents percentage of filled acini under normoxic and hypoxic conditions for three independent experiments. Error bars indicate SE. (C) At days 6, 8, and 10, structures were lysed and subjected to immunoblot analysis and analyzed for Bim, Bmf, and phospho- and total Erk protein levels. Actin was used as a loading control. (D) At day 8, structures were lysed for total RNA. Equal amounts of total RNA were subjected to quantitative RT-PCR analysis to determine relative level of Bim and Bmf expression. Histogram represents data from at least three independent experiments. Error bars represent SE (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001).

Figure 5.

Erk activation in hypoxic acini is required for inhibition of expression of Bim and Bmf as well as luminal apoptosis. MCF-10A cells were placed in a morphogenesis assay under standard tissue culture conditions (20% O₂, 5% CO₂) until day 6, and then cells were incubated under normoxic or hypoxic conditions after pretreatment with indicated inhibitor compounds until day 8. (A) Structures were lysed and immunoblot analysis was used to determine the level of indicated phospho- and total proteins. Actin was used as a loading control. (B) Structures were fixed and stained with an antibody against cleaved caspase-3 as well as the nuclear stain DAPI. (C) Percentage of caspase-positive acini was determined in each population. Caspase-positive acini were defined as any structure with two or more caspase-3–positive cells present in the luminal space. Histogram represents data from three individual experiments as described in B. Error bars represent SE (*p < 0.05).