Secretion modification region-derived peptide blocks exosome release and mediates cell cycle arrest in breast cancer cells

Abstract

PurposeDiscovery and development of a novel anticancer PEG-SMR-Clu peptide to prevent breast cancer metastasis. How breast cancer cells and primary mammary epithelial cells interact and communicate with each other to promote tumorigenesis and how to prevent tumor metastasis has long been a concern of researchers. Cancer cells secrete exosomes containing proteins and RNA. These factors can influence tumor development by directly targeting cancer cells and tumor stroma. In this study, we determined the effects of a peptide as an inhibitor of exosome secretion on breast tumors. We developed a peptide derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein that was modified with PEG on the N-terminus and with a Clusterin (Clu)-binding peptide on the C-terminus. Attachment of PEG to the SMR peptide, termed PEGylation, offers improved water solubility and stability as well as reduced clearance through the kidneys, leading to a longer circulation time. The 12-mer Clu-binding peptide plays multiple roles in tumor development and metastasis. The Clu peptide can be detected by antibody in vivo, thus it has the potential to be used to monitor tumor status and treatment efficacy in animal studies and eventually in cancer patients.ResultsPEG-SMRwt-Clu and PEG-SMRwt peptides inhibited the growth of both of MCF-7 (estrogen responsive, ER+) and MDA-MD-231 (estrogen non-responsive, ER-) human breast cancer cells in a dose and time-dependent manner, without inducing cytotoxic effects. The SMRwt peptide, combined with paclitaxel, induced G2/M phase cell cycle arrest on MCF-7 and MDA-MB-231 cells but did not promote apoptosis. PEG-SMRwt-Clu peptide treatment blocked exosome release from both MCF-7 and MDA-MB-231 cells. This effect was blocked by knockdown of the chaperone protein mortalin by either antibody or siRNA.Materials and methodsMCF-7 and MDA-MB-231 breast tumor cells were treated with PEG-SMR-Clu peptide alone and in combination with paclitaxel and cisplatin. Cell proliferation and viabilty were determined via cell cycle analysis using Cellometer imaging cytometry, Annexin V and MTT assays. The effects of the PEG-SMR-Clu peptide on tumor exosome release were determined by testing isolated exosome fractions, for (i) expression of CD63 and Alix proteins by Western blotting, (ii) NanoSight nanoparticle tracking analysis (NTA 10) to measure exosomes size and concentration, and (iii) measurement of acetylcholinesterase (AchE) for exosome specific enzyme activity.ConclusionsPEG-SMRwt-CLU peptides inhibited the growth of human breast cancer cells and blocked tumor exosome release in vitro. The peptide alone did not cause increased cytotoxicity or apoptosis induction, but did cause cell cycle G2/M phase arrest in both estrogen responsive and non-responsive breast cancer cells. These data suggest a potential therapeutic value of SMR to prevent breast cancer metastasis and as an adjuvant for the chemotherapeutic treatment of human breast cancer.

Cloning and Tissue Expression of Clusterin in Bufo gargarizans

“…According to previous reports, the tolerance to chemotherapy and radiotherapy is closely related with CLU expression in several cancers, and CLU up-regulation has been confirmed in many cancers including prostate, kidney, bladder, breast, head and neck, colon, cervical, pancreatic, lung, melanoma lymphoma [16,18] . Therefore, CLU has been considered as a biomarker for tumour malignancy, and it is believed that improvement of tumour cell sensitivity to chemotherapy and radiotherapy is promising way to control cancer by reducing CLU expression [14,[19][20][21] .…”

Exosomes in cancer: small vesicular transporters for cancer progression and metastasis, biomarkers in cancer therapeutics

“…While the SMR peptide and chemotherapeutic drugs were compound to remedy cancer cells, PEG-SMRwt-Clu synergically enhanced the anti-proliferative efficacies of drugs, considerably promoted the tumor cell growth suppression efficacy of drugs and inhibited exosomes secretion in breast cancer cell lines MCF-7 and MDA-MB-231 cells. Therefore the considerable usage of PEG-SMRwt-Clu peptide is pivotal process for the prevention and therapy of human breast tumor cells ( Huang et al, 2017 ).…”

Perspective: bidirectional exosomal transport between cancer stem cells and their fibroblast-rich microenvironment during metastasis formation

“…Inhibition of exosomal communication between cancer cells and the stromal microenvironment may have therapeutic potential. 92 , 93 GW4869 is a neutral sphingomyelinase inhibitor that blocks ceramide-mediated inward budding of MVBs and exosome release; furthermore, it is widely used in breast cancer cell and CAF experiments. 38 , 92 , 94 In combination with chemotherapeutic agents, it significantly reduced the survival of carcinoma cells in co-cultures and mouse experiments.…”