Abstract
BackgroundMolecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here we investigated the pathophysiologic role of Mcl-1 in Her2-positive BC cells under hypoxic conditions.MethodsRNA interference and a novel small molecule inhibitor, EU-5346, were used to examine the role of Mcl-1 in Her2-positive BC cell lines and primary BC cells (sensitive or intrinsically resistant to Her2 inhibitors) under hypoxic conditions (using a hypoxic incubation chamber). Mechanisms-of-action were investigated by RT-PCR, mitochondrial isolation, as well as immunoprecipitation/blotting analysis, and microscopy. The specificity against Mcl-1 of the novel small molecule inhibitor EU5346 was verified in Mcl-1Δ/nullversus Mcl-1wt/wt Murine Embryonic Fibroblasts (MEFs). Proliferation, survival, and spheroid formation were assessed in response to Mcl-1 and Her2 inhibition.ResultsWe demonstrate for a strong correlation between high Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line.ConclusionOur data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0686-4) contains supplementary material, which is available to authorized users.
Highlights
Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown
Our data demonstrate a critical role of myeloid cell leukemia-1 (Mcl-1) in human epidermal growth factor receptor 2 (Her2)-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC
Mcl-1 protein levels of BC exposed to hypoxia were transiently increased 2.41-fold (standard deviation (SD) ±0.44), 2.57-fold (SD ±0.34), and 3.31-fold (SD ±0.31) in Her2-positive BT474, SKBR3, and HCC-1954 BC cells, respectively
Summary
Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. Luminal-A BC, the most common subtype, is ERpositive and is characterized by the absence of Her expression, a low rate of proliferation (Ki67), and histologically low-grade tumors. Inherent and acquired resistance to these agents remains a significant barrier to further reduce mortality in this BC patient subtype, highlighting the urgent need for novel therapies [11]. These drugs do not penetrate the blood–brain barrier as as they reach the rest of the body, with lapatinib in combination with capecitabine and T-DM1 monotherapy being a possible exception [12]
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