TIS21/BTG2 inhibits breast cancer growth and progression by differential regulation of mTORc1 and mTORc2–AKT1–NFAT1–PHLPP2 signaling axis
It has been reported that PI3K/AKT pathway is altered in various cancers and AKT isoforms specifically regulate cell growth and metastasis of cancer cells; AKT1, but not AKT2, reduces invasion of cancer cells but maintains cancer growth. We propose here a novel mechanism of the tumor suppresser, TIS21/BTG2, that inhibits both growth and invasion of triple negative breast cancer cells via AKT1 activation by differential regulation of mTORc1 and mTORc2 activity.
Transduction of adenovirus carrying TIS21/BTG2 gene and transfection of short interfering RNAs were employed to regulate TIS21/BTG2 gene expression in various cell lines. Treatment of mTOR inhibitors and mTOR kinase assays can evaluate the role of mTORc in the regulation of AKT phosphorylation at S473 residue by TIS21/BTG2 in breast cancer cells. Open data and immunohistochemical analysis were performed to confirm the role of TIS21/BTG2 expression in various human breast cancer tissues.
We observed that TIS21/BTG2 inhibited mTORc1 activity by reducing Raptor-mTOR interaction along with upregulation of tsc1 expression, which lead to significant reduction of p70S6K activation as opposed to AKT1S473, but not AKT2, phosphorylation via downregulating PHLPP2 (AKT1-specific phosphatase) in breast cancers. TIS21/BTG2-induced pAKTS473 required Rictor-bound mTOR kinase, indicating activation of mTORc2 by TIS21/BTG2 gene. Additionally, the TIS21/BTG2-induced pAKTS473 could reduce expression of NFAT1 (nuclear factor of activated T cells) and its target genes, which regulate cancer microenvironment.
TIS21/BTG2 significantly lost in the infiltrating ductal carcinoma, but it can inhibit cancer growth via the TIS21/BTG2–tsc1/2–mTORc1–p70S6K axis and downregulate cancer progression via the TIS21/BTG2–mTORc2–AKT1–NFAT1–PHLPP2 pathway.
KeywordsTIS21/BTG2 mTORc1, mTORc2 AKT1 PHLPP2 NFAT1 Triple negative breast cancer
B-cell translocation gene 2
TPA-inducible sequences 21
Mammalian target of rapamycin
Rapamycin insensitive companion of mTOR
Ribosomal protein S6 kinase 1
Tuberous sclerosis complex
Phosphoinositide-dependent kinase 1
Mouse embryonic fibroblasts
Nuclear factor of activated T cells
PH domain and leucine rich repeat protein phosphatase 2
This work was supported by the grants from the National R&D Program for Cancer Control (131280) by Ministry for Health and Welfare and by the National Research Foundation (no. 2016R1A2B4006466) of the Korean government MSIP. We deeply appreciate the following scientists for their kind sharing of the materials; recombinant DNAs of GST-S6K1 and GST-AKT1 was a kind gift from professors Sungho Ryu (Pohang University of Science and Technology, South Korea) and Sungkwan An (Konkuk University, South Korea). tsc1−/− and tsc2−/− mouse embryonic fibroblasts were kindly shared by professor Mee-Sup Yoon (Gachon University, South Korea). Torin1 and PP242 were donated by professor Jin Won Cho (Yonsei University, South Korea), AKT inhibitor IV was kindly shared by professor Hong-Duk Youn (Seoul National University College of Medicine, South Korea). We also deeply appreciate professor DY Kang for his statistical analysis.
This study was funded by National R&D Program for Cancer Control (131280) by Ministry for Health and Welfare and by the National Research Foundation (no. 2016R1A2B4006466).
Compliance with ethical standards
Conflict of interest
The authors declare there is no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Written informed consent was obtained from all individual participants included in the study.
- Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, Szallasi Z (2010) An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat 123:725–731. https://doi.org/10.1007/s10549-009-0674-9 CrossRefPubMedGoogle Scholar
- Kim BC, Ryu MS, Oh SP, Lim IK (2008) TIS21/(BTG2) negatively regulates estradiol-stimulated expansion of hematopoietic stem cells by derepressing Akt phosphorylation and inhibiting mTOR signal transduction. Stem Cells 26:2339–2348. https://doi.org/10.1634/stemcells.2008-0327 CrossRefPubMedGoogle Scholar
- Ma CX et al (2016) A phase I study of the AKT inhibitor MK-2206 in combination with hormonal therapy in postmenopausal women with estrogen receptor-positive metastatic breast cancer. Clin Cancer Res 22:2650–2658. https://doi.org/10.1158/1078-0432.CCR-15-2160 CrossRefPubMedPubMedCentralGoogle Scholar
- Maroulakou IG, Oemler W, Naber SP, Tsichlis PN (2007) Akt1 ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice. Cancer Res 67:167–177. https://doi.org/10.1158/0008-5472.CAN-06-3782 CrossRefPubMedGoogle Scholar
- Powell E et al (2016) p53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer. Breast Cancer Res 18:13. https://doi.org/10.1186/s13058-016-0673-9 CrossRefPubMedPubMedCentralGoogle Scholar
- Sundaramoorthy S, Ryu MS, Lim IK (2013) B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NFkappaB pathways which enhances transcription of MnSOD by accelerating IkappaBalpha degradation in normal and cancer cells. Cell Commun Signal 11:69. https://doi.org/10.1186/1478-811X-11-69 CrossRefPubMedPubMedCentralGoogle Scholar
- Yoeli-Lerner M, Chin YR, Hansen CK, Toker A (2009) Akt/protein kinase b and glycogen synthase kinase-3beta signaling pathway regulates cell migration through the NFAT1 transcription factor. Mol Cancer Res 7:425–432. https://doi.org/10.1158/1541-7786.MCR-08-0342 CrossRefPubMedPubMedCentralGoogle Scholar