Critical roles for nitric oxide and ERK in the completion of prosurvival autophagy in 4OHTAM-treated estrogen receptor-positive breast cancer cells

Highlights

• Tamoxifen induces breast cancer cell production of superoxide and nitric oxide.

• Nitric oxide is critical for formation of functional autolysosomes.

• Superoxide activates ERK, which is critical for autolysosomal integrity.

• Inhibition of ERK and nitric oxide destroys autolysosomes, leading to necrosis.

Abstract

Autophagy is a mechanism of tamoxifen (TAM) resistance in ER-positive (ER+) breast cancer cells. In this study, we showed in ER+ MCF7 cells that 4-hydroxytamoxifen (4OHTAM) induced cellular nitric oxide (NO) that negatively regulates cellular superoxide (O2−) and cytotoxicity. 4OHTAM stimulated LC3 lipidation and formation of monodansylcadaverine (MDC)-labeled autophagic vesicles dependent on O2−. Depletion of NO increased O2− and LC3 lipidation, yet reduced formation of MDC-labeled autophagic vesicles. Instead, NO-depleted cells formed remarkably large vacuoles with rims decorated by LC3. The vacuoles were not labeled by MDC or the acidic lysosome-specific fluorescence dye acridine orange (AO). The vacuoles were increased by the late stage autophagy inhibitor chloroquine, which also increased LC3 lipidation. These results suggest NO is required for proper autophagic vesicle formation or maturation at a step after LC3 lipidation. In addition, 4OHTAM induced O2−-dependent activation of ERK, inhibition of which destabilized lysosomes/autolysosomes upon 4OHTAM treatment and together with depletion of NO led to necrotic cell death. These results suggest an essential role for endogenous NO and ERK activation in the completion of pro-survival autophagy.

Introduction

Approximately 70% of breast cancers express estrogen receptor (ER), so endocrine therapy with tamoxifen (TAM) benefits patients by significantly reducing recurrence [1], [2]. Unfortunately, acquired TAM resistance is not uncommon [2], [3], emphasizing the challenge of emerging resistance [4]. TAM treatment changes different aspects of cell physiology, which is reflected in the complexity of TAM resistance mechanisms [2], [5], [6]. Besides inhibition of cell proliferation, in vitro TAM can also induce senescence and cell death [7], [8], [9], [10]. Increased autophagy with a shift in balance between cell death and survival may be critical for in vivo response to TAM [11], [12]. Inhibition of autophagy enhances TAM-induced cell death in 4OHTAM-resistant cells [11], [12], [13], consistent with its role in cell survival [14], [15]. However, excessive autophagy in ER-positive (ER+) breast cancer cells treated with anti-estrogens can lead to type-II programmed cell death, autophagic death [8], [16], and necrosis [17]. Regulation of survival and apoptosis in response to TAM is poorly understood, which makes elucidation of appropriate mechanisms an important task for anti-estrogen therapy research.

TAM induces oxidative stress through reactive oxygen species (ROS) [18], [19]. Low levels of ROS activate stress signaling pathways and promote proliferation and survival while excessive ROS may cause irreversible damage to DNA, protein, and cell membranes leading to cell death [20], [21]. Increased transcription of antioxidant genes and activation of stress signaling pathways are associated with in vivo TAM-resistance in animal models [18], [22] and human breast cancers [23] suggesting adaptation to oxidative stress occurs in acquired TAM resistance. ROS stimulates autophagy by regulation of ATG4 and stress signaling pathways [24], [25], [26] suggesting autophagy may protect against ROS [26]. Active autophagy is observed in acquired TAM-resistance [11], [12], [13], implying that oxidative stress may function in both TAM-induced death and activation of pro-survival autophagy.

Nitric oxide (NO) is an integral part of ROS [27], [28] produced by nitric oxide synthases [29]. At low levels, NO is a scavenger of superoxide (O2−) [28]. However, excess NO can aggravate oxidative stress when converted to peroxynitrite [30]. NO regulates cellular signaling and is involved in tumorigenesis and cancer progression [31], [32]. Excessive NO production in mitochondria mediates TAM-induced cell death [33]. Lower expression of eNOS is associated with worse prognosis in ER+ breast cancer [34], [35] implying that NO regulates TAM response. Exogenous NO induces autophagy [36], [37]. However, the regulatory role of endogenous NO in TAM-induced oxidative stress, autophagy and cell death remains to be elucidated.

In this study, we investigated the role of NO in 4OHTAM-induced oxidative stress, autophagy, and cell death. We showed that endogenous NO was essential for completion of autophagy and protection of ER+ MCF7 breast cancer cells from 4OHTAM-induced cytotoxicity.