Fan et al. Cell Death and Disease (2020) 11:635 https://doi.org/10.1038/s41419-020-02878-z Cell Death & Disease ARTICLE Open Access Triggering a switch from basal- to luminal-like breast cancer subtype by the small-molecule diptoindonesin G via induction of GABARAPL1 Minmin Fan1,JingweiChen1,JianGao 1,WenwenXue1,YixuanWang1, Wuhao Li1, Lin Zhou1,XinLi1, Chengfei Jiang2,YangSun1,XuefengWu1, Xudong Wu1,HuimingGe1,YanShen1 and Qiang Xu 1 Abstract Breast cancer is a heterogeneous disease that includes different molecular subtypes. The basal-like subtype has a poor prognosis and a high recurrence rate, whereas the luminal-like subtype confers a more favorable patient prognosis partially due to anti-hormone therapy responsiveness. Here, we demonstrate that diptoindonesin G (Dip G), a natural product, exhibits robust differentiation-inducing activity in basal-like breast cancer cell lines and animal models. Specifically, Dip G treatment caused a partial transcriptome shift from basal to luminal gene expression signatures and prompted sensitization of basal-like breast tumors to tamoxifen therapy. Dip G upregulated the expression of both GABARAPL1 (GABAA receptor-associated protein-like 1) and ERβ. We revealed a previously unappreciated role of GABARAPL1 as a regulator in the specification of breast cancer subtypes that is dependent on ERβ levels. Our findings shed light on new therapeutic opportunities for basal-like breast cancer via a phenotype switch and indicate that Dip G may serve as a leading compound for the therapy of basal-like breast cancer. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction targets and the poor disease prognosis have fostered a Breast cancer is a heterogeneous disease comprised of major effort to develop new treatment approaches for different molecular subtypes, which can be identified patients with basal-like breast cancer. through gene or biomarker expression analyses and are The distinguishing features of luminal and basal-like – predictive of prognosis1 3. Luminal-like breast cancer, breast cancer cells have been explained by a cell-of-origin which includes luminal A and B subtypes, is characterized hypothesis, i.e., originating from luminal progenitors and by the expression of estrogen receptor (ER) and/or pro- breast epithelial stem cells, respectively6. However, recent gesterone (PR) and confers a more favorable prognosis studies have proposed luminal progenitors as the com- partially due to anti-hormone therapy responsiveness4. mon origin of both luminal and basal-like breast can- – Conversely, basal-like breast cancer, such as ER- PR- cers7 9. A tumorigenic subpopulation of luminal HER2- triple negative breast cancer, has the highest progenitors could dedifferentiate to acquire a basal-like recurrence rate and the worst overall survival rate among phenotype. Indeed, basal-like cancer cells show a high – all the breast cancer subtypes5. The paucity of therapeutic degree of heterogeneity and plasticity10 13. Nuclear extracts of basal-like cancer cells are reported to be suf- ficient to induce a luminal-to-basal phenotype switch11. Correspondence: Yan Shen ([email protected])or Moreover, loss of luminal-defining factors, such as fork- Qiang Xu ([email protected]) 1State Key Laboratory of Pharmaceutical Biotechnology, School of Life head box transcription factor (FOXA1) or GATA-binding Sciences, Nanjing University, 22 Han Kou Road, Nanjing 210093, China protein 3 (GATA3), causes a shift from luminal to basal 2 – Department of Pathology, Nanjing Medical University, 140 Hanzhong Road, gene expression signatures12 14. A recent study demon- Nanjing 210029, China These authors contributed equally: Minmin Fan, Jingwei Chen strated that intervention of platelet-derived growth factor Edited by S. Tait © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Fan et al. Cell Death and Disease (2020) 11:635 Page 2 of 13 (PDGF)-CC activity in mouse models results in the con- antiproliferative activity of Dip G in MDA-MB-231 cells version of basal-like breast cancers into a luminal state (Fig. S2). These results help to rule out the possibility that that confers sensitivity to endocrine therapy10. Therefore, Dip G inhibits the growth of these basal-like cancer cells pharmacologic strategies for triggering the switch from via induction of caspase-dependent apoptosis, necroptosis basal-like cancer to a luminal subtype would benefit the or autophagic cell death. clinical therapy of basal-like breast cancer patients. Given the strong correlation between cell growth arrest GABARAPL1 (GABAA receptor-associated protein-like and the progression of cancer cell differentiation, we 1), a member of the GABARAP family, is highly evolu- assessed cellular morphology and the expression of tionarily conserved15. Its coding gene was first identified cell–cell adhesion molecules in Dip G-treated basal-like as an early estrogen-induced gene16. Apart from its cancer cells. Both MDA-MB-231 and SUM1315 cells function in autophagy, GABARAPL1 plays a potential role displayed a distinct appearance from the original spindle- during tumor progression17,18. GABARAPL1 expression is shaped morphology after a 72-h treatment with 7.5-μMof robustly downregulated in breast tumors and those Dip G (Fig. 1a). E-cadherin expression was increased, patients with high GABARAPL1 levels have a low risk of whereas the expression of vimentin and N-cadherin was metastasis19. GABARAPL1 overexpression inhibits cell decreased by Dip G at both the mRNA and protein levels proliferation, colony formation and invasion in breast in MDA-MB-231 cells (Fig. 1b, c). Using Oil red O cancers in vitro19,20. Decreased GABARAPL1 expression staining to analyze neutral fat contents, we observed a has also been observed in patients with acute myeloid higher intensity of Oil red O in the cells treated with Dip leukemia (AML)21. Interestingly, its expression can be G, which is similar to the result with the cell differentia- induced during all-trans retinoic acid (ATRA)-induced tion inducer NaB (Fig. 1d). Notably, Dip G downregulated neutrophil differentiation, implying a novel function in the expression of the basal-like genes CD44 and cytoker- cell differentiation. Our previous study revealed that the atin (CK) 5, accompanied by upregulation of the luminal- oligostilbenoid natural product diptoindonesin G (Dip G) like genes FOXA1 and GATA3 expression in a time- induced myeloid differentiation of AML cells to an dependent manner (Fig. 1e). The wound-healing assay equivalent or higher extent than ATRA22. Herein, we also showed that Dip G-treated MDA-MB-231 cells were found that Dip G could induce cell differentiation in less migratory than the untreated control (Fig. 1f), indi- basal-like breast cancer cells. Gene profiling analyses cative of the functional property of luminal-like breast + showed that Dip G treatment caused a partial tran- cancer cells. Furthermore, considering that CD44 − scriptome shift from basal to luminal gene expression CD24 subpopulation enriched in basal-like breast cancer signatures. Such a phenotype switch conferred sensitivity cells has the stem/progenitor cell properties25, we eval- to tamoxifen therapy in basal-like breast tumors. In uated the effects of Dip G on the stemness. Real-time RT- addition, our data identified GABARAPL1, whose PCR analysis showed reduced levels of mRNA transcripts expression was increased by Dip G in an ERβ-dependent of stemness-associated genes, such as EpCAM, ALDH1, manner, as a functional regulator of the molecular sub- BMI1, and NANOG, in Dip G-treated MDA-MB-231 cells type of breast cancer. These findings shed light on new (Fig. 1g). Accordingly, Dip G reduced the percentage of + − therapeutic opportunities via a phenotype switch for CD44 CD24 cells and downregulated the canonical basal-like breast cancer. Wnt/β-catenin signaling pathway known to regulate the self-renewal and stemness of cancer cells, as evidenced by Results the increase in the levels of p-β-catenin (Ser33/37/Thr41) Dip G induces luminal differentiation in basal-like cancer and p-β-catenin (Thr41/Ser45) and the decrease in the cells levels of p-β-catenin (Ser552), p-β-catenin (Ser675) and c- A previous study reported that Dip G, a resveratrol Myc in both MDA-MB-231 and SUM1315 cells (Fig. S3). aneuploid either naturally isolated from the stem bark of tropical plants such as Hopea chinensis or totally syn- Dip G induces a partial transcriptome shift from basal to thesized, has robust antiproliferative activity in cancer luminal gene expression signatures – cells22 24. In line with these findings, a dose-and time- Next, the gene expression profile of MDA-MB-231 dependent inhibition of cell growth by Dip G was (classified as basal B1) cells treated with Dip G was ana- observed in the basal-like cancer cell lines MDA-MB-231, lyzed using HiSeq deep sequencing. After 24 h of treat- SUM1315, and MDA-MB-468 using the trypan blue dye ment, Dip G significantly downregulated 61 of 77 (79.2%) exclusion, soft agar colony formation, and bromodeox- of previously defined basal B cell line classifier genes, yuridine assays (Fig. S1). Pretreatment with various inhi- while concomitantly upregulating 9 of 25 (36%) of luminal bitors, including a pan-caspase inhibitor (z-VAD-FMK), a classifier genes (Table 1)1.