Abstract
Anoikis resistance is an essential prerequisite for tumor metastasis, but the underlying molecular mechanisms remain unknown. Herein, we report that the oncoprotein hepatitis B X-interacting protein (HBXIP) is prominently upregulated in breast cancer cells following ECM detachment. Altering HBXIP expression can impair the anchorage-independent growth ability of tumor cells. Mechanistically, HBXIP, which binds to Kelch-like ECH-associated protein 1 (Keap1) to activate nuclear factor E2-related factor 2 (Nrf2), contains a cis-acting antioxidant response element (ARE) in the gene promoter and is a target gene of Nrf2. The HBXIP/Nrf2 axis forms a reciprocal positive feedback loop that reinforces the expression and tumor-promoting actions of each protein. In response to ECM detachment, Nrf2 reduces reactive oxygen species (ROS) accumulation, protects the mitochondrial membrane potential and increases cellular ATP, GSH and NADPH levels to maintain breast cancer cell survival. Meanwhile, the reinforcement of HBXIP induced by Nrf2 inhibits JNK1 activation by inhibiting ubiquitin-mediated degradation of Prdx1, which also plays an essential role in promoting ECM-detached cell survival. Furthermore, a strong positive correlation was identified between HBXIP expression and Prdx1 expression in clinical breast cancer tissues and TCGA Pan-Cancer Atlas clinical data of breast invasive carcinoma based on the cBioPortal cancer genomics database. Co-expression of HBXIP and Prdx1 predicts a poor prognosis for breast cancer patients. Collectively, our findings reveal a significant mechanism by which the HBXIP/Nrf2 feedback loop contributes to anoikis resistance by maintaining redox homeostasis and inhibiting JNK1 activation and support the likely therapeutic value of the HBXIP/Nrf2 axis in breast cancer patients.
Highlights
Breast cancer metastasis is a multi-step and multi-factor process, including the reintegration and degradation of the extracellular matrix (ECM), detachment from local tissue, invasion of blood or the lymphatic system, and the formation of new tumors in distant locations
Compared with hepatitis B X-interacting protein (HBXIP) KD/Nrf[2] OE cells, HBXIP OE/Nrf[2] KD had limited effects on rescuing the oxidate situation, phosphate pathway (PPP) flux, mitochondrial permeabilization and ATP production relative to HBXIP/Nrf[2] KD cells following ECM detachment (Fig. 4C and Supplementary Fig. 2A). These results demonstrate that HBXIP alone has little ability to modulate reactive oxygen species (ROS), PPP flux, and ATP production unless Nrf[2] is coexpressed, which is consistent with our previous study showing that HBXIP modulates ROS in breast cancer cells by competing with Nrf[2] for binding with KEAP1 and promoting Nrf[2] accumulation and activation[9]
Metastatic breast cancer cells must be able to resist anoikis to survive under anchorage-independent conditions when they circulate in the bloodstream
Summary
Breast cancer metastasis is a multi-step and multi-factor process, including the reintegration and degradation of the extracellular matrix (ECM), detachment from local tissue, invasion of blood or the lymphatic system, and the formation of new tumors in distant locations. Use of the antioxidant NAC to neutralize ROS (or H2O2 treatment) showed little effect on JNK1 phosphorylation or the Prdx[1] protein levels in ECM-detached HBXIP/Nrf2-deficient MDA-MB-436 cells (or HBXIP/ Nrf[2] double OE MCF-7 cells) (Fig. 5C, left panel and Fig. 5D, left panel) This pattern suggests that Prdx1-mediated JNK1 activation is involved in HBXIP/Nrf[2] feedback loop-induced anoikis resistance in addition to diminishing ROS levels in breast cancer cells. Prdx[1] knockdown increased the apoptosis rate of HBXIP OE/Nrf[2] KD and HBXIP KD/Nrf[2] OE breast cancer cells (Fig. 5I) These results indicate that the reciprocal HBXIP/Nrf[2] feedback loop prevents unconstrained ROS accumulation and promotes Prdx[1] accumulation and stabilizes the Prdx1-GSTπ-JNK1 complex to reduce JNK1 phosphorylation following ECM detachment. Simultaneous inhibition of HBXIP and Nrf[2] expression is a possible effective treatment for breast cancer
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