CCT6A and triple-negative breast cancer metastasis: Activating the AKT/MDM2/P53 pathway.

Abstract

e13087 Background: Triple-negative breast cancer (TNBC) patients are prone to distant recurrence and poorer prognosis. The key molecules and underlying mechanisms of TNBC's aggressive phenotype remain to be further clarified. Methods: First, using the Kaplan-Meier Plotter ( https://kmplot.com/analysis ) and analyzing our collected clinical samples, we established the expression pattern and clinical value of the chaperonin containing TCP1 subunit 6A (CCT6A) in TNBC. We carried out the Cell Counting Kit-8 (CCK8), Transwell, and flow cytometry assay in the TNBC cell lines including MDA-MB-231 and BT549 to assess the effects of knockdown and overexpress of CCT6A. The mechanism of CCT6A in TNBC was identified by using the RNA-seq, KEGG analysis and Co-Immunoprecipitation Mass Spectrometry. Rescue assays verified the role of the AKT Serine/Threonine Kinase 1 (AKT) / Mouse double minute 2 homolog (MDM2)/P53 pathway on CCT6A. Then, the effect of CCT6A on tumor progression and metastasis in vivo was evaluated by the mouse models of tumorigenesis in situ and caudal artery lung metastasis. APG115, a pharmacological p53 activator and immune stimulatory MDM2 inhibitor, combined with anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody Ipilimumab was found to prevent TNBC metastasis. Finally, we did a profiling of the tumor immune environment in mice. Results: CCT6A was highly expressed in TNBC ( p<0.0001), and patients with high expression of CCT6A had poor prognosis ( p=0.0053, HR=2.36 [1.27-4.39]). Of note, CCT6A was significantly associated with lymph node metastasis ( p<0.0001). CCT6A promoted cell proliferation, migration, and invasion while inhibiting cell apoptosis and achieving G1/S stage arrest through gain- and loss-of-function approaches. In terms of mechanism, Co-IP and Immunofluorescence assay verified that CCT6A interacted with Keratin 10 (KRT10) which can promote tumor metastasis. Compared with the control group, RNA-seq, and KEGG analysis suggested that AKT/MDM2/P53 signaling was inhibited after knockdown of CCT6A in MDA-MB-231. Furthermore, we found that the tumor volume and the number of lung metastasis sites were significantly reduced in the APG115 combined with Ipilimumab treatment group. Flow cytometry and multiple Immunofluorescence assay showed that TGF-β level from regulatory T cells decreased, while IFN-γ amount from CD8+T cells significantly increased with the combination (both p<0.05). Conclusions: Our research demonstrated that CCT6A can enhance metastasis of triple-negative breast cancer via up-regulating the AKT/MDM2/P53 pathway. These results provide rationales for MDM2 inhibitor combined with anti-CTLA-4 antibody for preventing TNBC metastasis.