Tang et al. Oncogenesis (2020) 9:47 https://doi.org/10.1038/s41389-020-0232-1 Oncogenesis ARTICLE Open Access SOX9 interacts with FOXC1 to activate MYC and regulate CDK7 inhibitor sensitivity in triple-negative breast cancer Lin Tang1,JuanJin1,2,KunXu3,XinWang1,JinhaiTang 4 and Xiaoxiang Guan1,3 Abstract Triple-negative breast cancer (TNBC) is particularly sensitive to cyclin-dependent kinase 7 inhibitor, THZ1, compared to hormone receptor (HR)+ breast cancer, but our data found that different TNBC cell lines had a wide range of IC50 values of THZ1, suggesting a possible heterogeneity in sensitivity to THZ1 in TNBC. To seek potential biomarkers of THZ1 sensitivity, we re-analyzed the mRNAs profile in breast cancer cells treated with THZ1 from the previous study and demonstrated that elevated expression of SOX9 was significantly associated with the sensitivity of THZ1 in TNBC. We also verified that SOX9 expression promoted cell proliferation, migration, stemness, and predicted poor prognosis. Moreover, based on the tissue array of 278 patients and over 900 samples from TCGA data, we found that SOX9 expression was significantly higher in TNBC than HR+ breast cancers. Furthermore, ChIP-sequencing indicated that SOX9 binding to enhancer near transcription factor FOXC1, was remarkably inhibited by THZ1. And we also demonstrated that SOX9 and FOXC1 interacted with each other, which might co-operate and co-regulate the MYC signaling pathway in TNBC. Mechanistically, SOX9 may sensitize TNBC cells to THZ1, in a FOXC1-related manner, suggesting that SOX9 could be as a predictive factor of THZ1. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction cluster of particular genes, which are overexpressed in Triple-negative breast cancer (TNBC) is a particularly TNBC but not in hormone receptor (HR)+ breast cancer aggressive subtype of breast cancer, which is characterized cells containing putative oncogenes, promoting TNBC by high genetic complexity and lacks the distinct driver tumorigenicity and the disease development3. Moreover, oncogenes1. Because of the tumor heterogeneity of an excessive number of genes in this cluster are associated TNBC, the development of targeted therapies has been with super-enhancers (SEs)4,5. It is commonly accepted limited for a long time2. Surprisingly, a recent research that enhancers are regions of non-coding DNA, which identifies that TNBC is selectively sensitive to THZ1, a mediate the transcription of adjacent genes, serving as a newly developed CDK7 inhibitor3. Recent studies have cis-regulatory element6. SEs are a subset of the enhancers found that besides the regulation of cell cycle, CDK7 is with a pronounced effect on recruiting transcription fac- also essential in promoting transcription processes. CDK7 tors, cofactors, and chromatin regulators to drive the inhibitor can strongly diminish the transcription of a abundant expression of some significant oncogenes, such as MYC, CCND2, and EGFR, in various tumors. CDK7 inhibitor is expected to control TNBC cell proliferation by Correspondence: Jinhai Tang ([email protected])or inhibiting the super-enhancer-associated oncogenes3. Xiaoxiang Guan ([email protected]) 1Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing Although TNBC have showed an extreme sensitivity to University, Nanjing 210002, China THZ1 compared to HR+ breast cancer, different 2 Department of Oncology, Fudan University Shanghai Cancer Center, Shanghai responses to the CDK7 inhibitor still exist among differ- 200032, China Full list of author information is available at the end of the article ent TNBC cell lines shown by us and Wang and These authors contributed equally: Lin Tang, Juan Jin, Kun Xu © 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/. Oncogenesis Tang et al. Oncogenesis (2020) 9:47 Page 2 of 12 coworkers3. Therefore, to identify the factors that predict time-dependent manner (Fig. 1d), we focused further on against the THZ1 response in TNBC may help with a SOX9 that has not been evaluated deeply in TNBC. better selection of TNBC patients for the treatment. Moreover, in a panel of TNBC cell lines with varying Previous studies found that THZ1 elicited selective effects levels of SOX9 expression, SOX9 expression levels were mainly by hampering highly active transcription and negative with IC50 values, and SOX9-amplified cells had a preferentially induced selective inactivation of super- high sensitivity to THZ1, indicating that SOX9 would be a enhancer-associated oncogenes4,5,7,8. If some oncogenic predictive factor for the THZ1 sensitivity (Fig. 1e, f). Next, transcription factors are associated with SEs, malignant we silenced SOX9 in MDA-468 and BT549 but over- potency of the transcription factors will be amplified, expressed SOX9 in MDA-157 that had a relatively low which can trigger numerous and deregulate transcription baseline expression level of SOX9. We found that SOX9 processes in tumors. So, we focused on the super- knockdown increased the IC50 values in MDA-468 and enhancer-associated transcription factors and demon- BT549, while ectopic expression of SOX9 in MDA-157 strated that SOX9, which is a super-enhancer-associated sensitized cells to THZ1 (Fig. 1g). Together, these results transcription factor in TNBC cells, predicted the sensi- showed that the SOX9 expression level was positively tivity of TNBC to THZ1. Most of the published studies associated with the sensitivity to THZ1 in TNBC cells. reported that SOX9 as an oncogenic protein was over- expressed in various solid tumors, inducing cancer cell SOX9 facilitated malignant properties in TNBC cells – growth, proliferation, migration, and invasion9 14. More- Although numerous studies have investigated the roles over, co-expression of exogenous Slug and Sox9 increased of SOX9 in cancers of the prostate and colorectum10,12,14, the tumorigenic and metastatic abilities of breast cancer few investigated molecular functions of SOX9 in breast cells and was associated with poor outcomes in breast cancer. As shown in Supplementary Fig. S1, the cell cancer patients11. In the present study, we demonstrated proliferation, colony formation and migration ability of that high SOX9 expression was associated with malignant TNBC cells treated with THZ1 were significantly phenotype in both TNBC cells and TNBC patients. decreased, compared to the control group. To adequately However, the exact mechanisms underlying SOX9 func- address the mechanisms underlying the functional asso- tions during tumor formation and development largely ciation between SOX9 and CDK7 inhibitor, we investi- remain unclear until now. Here, we are the first to gated the biological functions of SOX9 in TNBC cells. demonstrate the roles of SOX9 in TNBC and the possible Colony formation assay showed that SOX9 silence dis- mechanisms of its predictive role in the THZ1 sensitivity. tinctly inhibited cell proliferation, while SOX9 over- expression promoted cell proliferation (Fig. 2a). We also Results investigated the effect of SOX9 on cell migration in TNBC SOX9 predicted sensitivity to THZ1 in TNBC cells cells and showed that SOX9 inhibition weakened the We demonstrated that THZ1 treatment indeed showed migration potential of MDA-468 and BT549. In contrast, strong cytotoxicity in TNBC cell lines, consistent with a the forced overexpression of SOX9 promoted cell previously published study3, but we also observed that migration in MDA-157 (Fig. 2b, c). some TNBC cell lines, such as BT-20 and MDA-157, were Because several studies have identified SOX9 as an not sensitive to THZ1 (Fig. 1a). Notably, the most important stem cell marker, we tested the effect of SOX9 11 insensitive cell lines were with IC50 values averaging on tumorsphere formation . SOX9 knockdown sup- almost ten times higher than those of sensitive ones pressed tumor-initiating ability with less tumorsphere (Fig. 1a), which hint that there are probably some factors formation compared to the control group. However, a responsible for the THZ1 efficacy in TNBC cells. We statistic difference was not observed between MDA-157 analyzed the gene expression values of super-enhancer- and MDA-157 with SOX9 overexpression (Fig. 2d). Pre- associated transcription factors based on the microarray vious studies showed that epithelial-mesenchymal tran- data of two TNBC cell lines and two HR+ breast cancer sition (EMT) progresses supported the stemness of cancer cell lines treated with THZ1 from the previously pub- cells and regulated cell migration ability15. As shown in lished study3 (Fig. 1b). The ideal predictive factor is Fig. 2e, knockdown of SOX9 significantly increased the identified as elevated in TNBC cells compared to HR+ expression levels of the epithelial marker