By Jamshed Arslan, Pharm D, PhD
Breast cancer is the most common cancer among women that causes the greatest number of cancer-related deaths worldwide. After radiotherapy or cytotoxic chemotherapy like paclitaxel, the surviving breast cancer stem cells (BCSCs) lead to tumor recurrence and metastasis. Resistant BCSCs undergo self-renewal and multi-lineage differentiation similar to embryonic stem cells (ESCs). The master regulators for such processes in ESCs and BCSCs are the transcription factors KLF4, NANOG, OCT4 and SOX2, however the regulation of these pluripotency factors in BCSCs is poorly understood. The role of chromatin remodeling in the genetic expression of pluripotency factors and BCSC phenotype under chemotherapy have never been studied, until now!
A team of researchers under the leadership of Dr. Gregg Semenza, a Nobel laureate at the John Hopkins University School of Medicine, Maryland, USA, discovered that paclitaxel induces the expression of S100A10 in Hypoxia Inducible Factor-1 (HIF-1)-dependent manner. S100A10 is a small calcium-binding protein that forms a complex with a calcium-dependent cytoskeletal protein Annexin A2 (ANXA2) to trigger breast cancer stemness through epigenetic activation of pluripotency factors.
Immunohistochemical staining of S100A10 was detected in immersion fixed paraffin-embedded sections of human kidney cancer tissue using 5 µg/mL Goat Anti-Human S100A10 Antigen Affinity-purified Polyclonal Antibody (AF1698) overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; CTS008) and counterstained with hematoxylin (blue).
Chemotherapy induces S100A10 in HIF-1-dependent manner
The researchers first confirmed the chemotherapy-induced expression of S100A10 at both gene and protein levels in breast cancer cells and animal models. Analysis of The Cancer Genome Atlas revealed that S100A10 mRNA levels in human breast cancer patients are significantly correlated with distinguishable HIF-regulated mRNAs (ANGPTL4, BNIP3, CA9, CXCR3, L1CAM, LDHA, PGK1, P4HA1, P4HA2 and PLOD1). Likewise, shRNA-knockdown of HIF-1-alpha with/without HIF-2-alpha abrogated paclitaxel-induced S100A10 mRNA and proteins. ChIP-qPCR and pharmacological analyses validated HIF-1-dependent transactivation of S100A10 by chemotherapy.
The next goal was to identify the key players in the S100A10-mediated activation of pluripotency factors.
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S100A10-ANXA2 complex interacts with histone chaperone SPT6 to express pluripotency factors
The team performed reciprocal co-immunoprecipitation (co-IP) assays and shRNA-mediated knockdowns in breast cancer cells to show the formation of S100A10-ANXA2 complex in chemotherapy-induced BCSC specification. Co-IP assays in a nuclear lysate of breast cancer cells with antibodies against S100A10 and SPT6 revealed that S100A10-ANXA2 complex recruits SPT6 to OCT4. Furthermore, ChIP-qPCR assays showed that chemotherapy increased the OCT4 binding to KLF4, NANOG, and SOX2 genes only in samples that had functional S100A10, ANXA2, SPT6 and HIF-1α.
At this point, the researchers hypothesized that a decrease in H3K27me3 explains how S100A10-ANXA2-SPT6 interaction regulates OCT4 binding to pluripotency factor genes. H3K27me3 is a chromatin marker of epigenetically blocked genes that is known to be negatively regulated by SPT6. ChIP-qPCR and co-IP analyses indicated that paclitaxel treatment caused histone demethylase KDM6A to occupy OCT4 binding sites and this binding was dependent on S100A10, ANXA2, and SPT6. Immunoblot and ChIP assays further showed that shRNA-mediated knockdown of S100A10, ANXA2, SPT6 or HIF-1α increased H3K27me3 marks at OCT4 binding sites of NANOG, SOX2, and KLF4 genes without affecting total H3 occupancy at these sites.
Based on these findings, the authors concluded that chemotherapy induces HIF-1-mediated S100A10-ANXA2-SPT6 interaction that decreases H3K27me3 at OCT4 binding sites of pluripotency factor genes in breast cancer.
Western blot shows Caki-1 cell lysates treated with varying concentrations of CoCl2 for either 4 hours or 6 hours to induce HIF-1 alpha expression. The membrane was probed with Mouse Monoclonal HIF-1 alpha Antibody (H1alpha67) [NB100-105] and followed by specific secondary antibody. HIF-1 alpha expression is not present in control/untreated lysates but increases with CoCl2 induction. β-actin is shown as a loading control.
Understanding chemoresistance can provide optimal clinical outcomes in breast cancer
This study sheds light on the constitutive and acquired chemoresistance that leads to poor prognosis. It helps in ascertaining the molecular pathways that initiate BCSC stemness. Recently, researchers at University of Toledo, Ohio, USA, pharmacologically and genetically targeted mRNA helicase eIF4A to successfully downregulate the pluripotency transcription factors NANOG, OCT4 and SOX2 in breast cancer cells. These findings indicate that targeting cancer plasticity provides a significant and durable clinical outcome in breast cancers.
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Jamshed Arslan, Pharm D, PhD
Dr Arslan is an Assistant Professor at Barrett Hodgson University, Pakistan,
where he uses various pedagogical methods to teach Pharm D students.
Research in focus
Lu, Haiquan, et al. (2020). Chemotherapy-induced S100A10 recruits KDM6A to facilitate OCT4-mediated breast cancer stemness. Journal of Clinical Investigation. https://doi.org/10.1172/JCI138577.
Sridharan, Sangita, et al. (2020). Targeting of the eukaryotic translation initiation factor 4A against breast cancer stemness. Frontiers in Oncology. https://doi.org/10.3389/fonc.2019.01311.