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Cancer‐associated fibroblasts (CAFs) as a predominant cell component in the tumour microenvironment (TME) play an essential role in tumour progression. Our earlier studies revealed oxidized ATM activation in breast CAFs, which is independent of DNA double‐strand breaks (DSBs). Oxidized ATM has been found to serve as a redox sensor to maintain cellular redox homeostasis. However, whether and how oxidized ATM in breast CAFs regulates breast cancer progression remains poorly understood. In this study, we found that oxidized ATM phosphorylates BNIP3 to induce autophagosome accumulation and exosome release from hypoxic breast CAFs. Inhibition of oxidized ATM kinase by KU60019 (a small‐molecule inhibitor of activated ATM) or shRNA‐mediated knockdown of endogenous ATM or BNIP3 blocks autophagy and exosome release from hypoxic CAFs. We also show that oxidized ATM phosphorylates ATP6V1G1, a core proton pump in maintaining lysosomal acidification, leading to lysosomal dysfunction and autophagosome fusion with multi‐vesicular bodies (MVB) but not lysosomes to facilitate exosome release. Furthermore, autophagy‐associated GPR64 is enriched in hypoxic CAFs‐derived exosomes, which stimulates the non‐canonical NF‐κB signalling to upregulate MMP9 and IL‐8 in recipient breast cancer cells, enabling cancer cells to acquire enhanced invasive abilities. Collectively, these results provide novel insights into the role of stromal CAFs in promoting tumour progression and reveal a new function of oxidized ATM in regulating autophagy and exosome release. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are considered the roots of cancer metastasis and recurrence (CSCs), due in part to their self‐renewal and therapy resistance properties. However, the underlying mechanisms for the regulation of CSC stemness are poorly understood. Recently, increasing evidence shows that long non‐coding RNAs (lncRNAs) are critical regulators for cancer cell function in various malignancies including breast cancer, but how lncRNAs regulate the function of breast cancer stem cells (BCSCs) remains to be determined. Herein, using lncRNA/mRNA microarray assays, a novel lncRNA (named lnc030) is identified, which is highly expressed in BCSCs in vitro and in vivo, as a pivotal regulator in maintaining BCSC stemness and promoting tumorigenesis. Mechanistically, lnc030 cooperates with poly(rC) binding protein 2(PCBP2) to stabilize squalene epoxidase (SQLE) mRNA, resulting in an increase of cholesterol synthesis. The increased cholesterol in turn actives PI3K/Akt signaling, which governs BCSC stemness. In summary, these findings demonstrate that a new, lnc030‐based mechanism for regulating cholesterol synthesis and stemness properties of BCSCs. The lnc030‐SQLE‐cholesterol synthesis pathway may serve as an effective therapeutic target for BCSC elimination and breast cancer treatment. [ABSTRACT FROM AUTHOR]

Background/Aims: The ataxia-telangiectasia mutated (ATM) protein kinase is critical for the maintenance of genomic stability and acts as tumor suppressor. Although evidence shows that a DNA damage-independent ATM (oxidized ATM) may be involved in cancer progression, the underlying mechanism is still unclear. Methods: Immunohistochemistry, immunofluorescence and western blotting were applied to detect the levels of oxidized ATM. Transwell assay was used to detect the cell migration and invasion abilities in different treatments. Quantitative phosphoproteome analysis was performed using hypoxic BT549 cells, in the presence or absence of Ku60019, a specific inhibitor of ATM kinase. The phosphorylated cortactin, the target protein of oxidized ATM, was confirmed by immunoprecipitation-western blots and in vitro kinase assay. The functions of phosphorylated cortactin were studied by specific short hairpin RNA, site-directed mutation, transwell assay, and actin polymerization assay. Results: Enhanced oxidized ATM proteins were present not only in the advanced and invasive breast tumor tissues but also malignant hypoxic breast cancer cells, in the absence of DNA damage. Loss of ATM expression or inhibiting oxidized ATM kinase activity reduced breast cancer cell migration and invasion. Using quantitative phosphoproteomics approach, 333 oxidized ATM target proteins were identified, some of these proteins govern key signaling associated with gap junction, focal adhesion, actin cytoskeleton rearrangement. Cortactin, one of the biggest changed phospho-protein, is a novel oxidized ATM-dependent target in response to hypoxia. Mechanically, we reveal that hypoxia-activated ATM can enhance the binding affinity of cortactin with Arp2/3 complex by phosphorylating cortactin at serine 113, and as a result, in favor of breast cancer cell migration and invasion. Conclusion: Oxidized ATM can phosphorylate cortactin at serine 113, playing a critical role in promoting breast tumor cell mobility and invasion via actin polymerization. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs), as parts of tumor initiation cells, play a crucial role to tumorigenesis, development and recurrence. However, the complicated mechanisms of CSCs to adapt to tumor microenvironment and its stemness maintenance remains unclear. Here, we show that oxidized ATM, a hypoxia-activated cytoplasm ATM, acts a novel function to maintain CSC stemness in triple-negative breast cancer cells (BCSCs) via regulating histone H4 acetylation. Mechanistically, oxidized ATM phosphorylates TRIM21 (a E3 ubiquitin ligase) serine 80 and serine 469. Serine 80 phosphorylation of TRIM21 is essential for the ubiquitination activity of TRIM21. TRIM21 binds with SIRT1 (one of deacetylase), resulting in ubiquitylation-mediated degradation of SIRT1. The reduced SIRT1 leads to increase of histone H4 acetylation, thus facilitating CSC-related gene expression. Clinical data verify that high level of ATM in breast tumors is positively correlated with malignant grade, and is closely related with low SIRT1, high p-TRIM21, and high CD44 expression. In conclusion, our study provides a novel mechanism by which oxidized ATM governing BCSCs stemness and reveals an important link among oxidized ATM, histone acetylation, and BCSCs maintenance. Hypoxia activates oxidative ATM, a pattern of DNA damage-independent ATM, in triple-negative breast cancer stem cells. The oxidized ATM phosphorylates TRIM21 to cause its activation. The activated TRIM21, an E3 ubiquitin ligases, results in degradation of SIRT1 via ubiquitination. The decreased SIRT1 (one of deacetylase) leads to high acetylation of histone H4ac in TNBC, which promotes CSC-related gene (e.g., Sox2, Oct4) expression and subsequent stemness maintenance in hypoxic breast cancer. [Display omitted] • Upregulation of oxidized ATM was observed in breast cancer cytoplasm under hypoxia. • Oxidized ATM promotes stemness maintenance of breast cancer stem cells. • Oxidized ATM affects histone acetylation through TRIM21/SIRT1/H4 acetylation axis. [ABSTRACT FROM AUTHOR]

Rescue chemotherapy is usually the preferred treatment for patients with advanced estrogen receptor-positive (ER+) breast cancer with endocrinotherapy resistance. However, these patients often simultaneously show a poor response to cytotoxic drugs, and thus the detailed mechanism of this resistance needs to be further investigated. Our previous research indicated that the G-protein-coupled estrogen receptor (GPER) is a novel mediator of the development of multidrug resistance, including resistance to both endocrinotherapy and chemotherapy, and ATP binding cassette subfamily G member 2 (ABCG2) has been identified as an engine that confers cancer cells with chemoresistance by expelling xenobiotics and chemotherapeutics. Here, we are the first to show that the expression levels of GPER and ABCG2 are markedly increased in tamoxifen-resistant ER + metastases compared to the corresponding primary tumors. A plasma membrane expression pattern of GPER and ABCG2 was observed in patients with metastases. Furthermore, both ER modulator tamoxifen, GPER-specific agonist G1 and pure ER antagonist ICI 182,780 significantly enhanced ABCG2 expression in tamoxifen-resistant breast cancer cells (MCF-7R) but not in tamoxifen-sensitive cells (MCF-7). The activated downstream GPER/EGFR/ERK and GPER/EGFR/AKT signaling pathways were responsible for regulating the expression and cell membrane localization of ABCG2, respectively, in MCF-7R cells. Interestingly, the above phenomenon could be alleviated by inhibitors of both the indicated signaling pathways and by knockdown of GPER in MCF-7R cells. More importantly, the tamoxifen-induced GPER/ABCG2 signaling axis was shown to play a pivotal role in the development of chemotherapy (doxorubicin) resistance both in vitro and in vivo. The clinical data further revealed that tamoxifen-resistant patients with high GPER/ABCG2 signaling activation had poor progression-free survival (PFS) when given rescue anthracycline chemotherapy. Therefore, our data provide novel insights into GPER-mediated chemoresistance and provide a rationale for the GPER/ABCG2 signaling axis being a promising target for reversing chemoresistance in patients with advanced ER + tamoxifen-resistant breast cancer. • GPER and ABCG2 expression are positively correlated in TAM-resistant breast cancer. • GPER signals regulate ABCG2 expression and localization of TAM-resistant cancer cells. • GPER/ABCG2 axis confers the resistance to chemotherapy in TAM-resistant breast cancer. • GPER/ABCG2 axis is an alternative target to reverse chemoresistance in breast cancer. [ABSTRACT FROM AUTHOR]