Abstract
Background
Targeting cancer stem cells is critical for suppressing cancer progression and recurrence. Finding novel markers or related pathways could help eradicate or diagnose cancer in clinic.
Methods
By constructing STARD13-correlated ceRNA 3′UTR stable overexpression or knockdown breast cancer cells, we aimed to explore the effects of STARD13-correlated ceRNA network on breast cancer stemness in vitro and in vivo. Further RNA-sequencing was used to analyze transcriptome change in combination with functional studies on candidate signaling. Clinical samples obtained from The Cancer Genome Atlas data were used to validate the correlation between STARD13 and related pathways. Finally, in vitro and in vivo experiments were used to examine the effects of STARD13-correlated ceRNA network on chemotherapy sensitivity/resistance.
Results
Here, we revealed that this ceRNA network inhibited stemness of breast cancer. Mechanistically, we found that activation of STARD13-correlated ceRNA network was negatively correlated with YAP/TAZ activity in breast cancer. Specifically, this ceRNA network attenuated YAP/TAZ nuclear accumulation and transcriptional activity via collectively modulating Hippo and Rho-GTPase/F-actin signaling. Finally, we demonstrated that YAP/TAZ transcriptional activity regulated by this ceRNA network was involved in chemoresistance.
Conclusions
Our results uncover a novel mechanism of YAP/TAZ activation in breast cancer and propose the possibility to drive STARD13-correlated ceRNA network to inhibit breast cancer stem cell traits.
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Background
Breast cancer is the most common cancer in women worldwide, and its incidence is increasing yearly [1]. At present, chemotherapy and surgery are the main methods for breast cancer treatment; especially, chemotherapy is the only option for triple negative breast cancer [2]. However, tumor relapse and chemoresistance constitute a major detriment to patients’ treatment and survival, and the mechanisms underpinning these phenomena remain elusive, which is an urgent need to solve. Since tumor relapse and chemoresistance are a complex network that integrates multiple growth control signals through an expanding set of core elements, a single factor could not make a good assessment of chemotherapy or tumor recurrence. In fact, we have only a scattered understanding of the molecular mechanisms that are responsible for tumor relapse and chemoresistance.
Cancer stem cells (CSCs) are proposed to drive tumor relapse and chemoresistance [49].
In this work, we showed that STARD13-correlated ceRNA network could regulate CSC traits of breast cancer cells through two independent pathways, which collaboratively led to the nucleus-cytoplasm translocation of YAP/TAZ. Despite the more details that need to be elucidated, we proposed that verteporfin, an inhibitor of YAP-TEAD binding, might target breast CSCs and could be used as a combinative treatment with doxorubicin in breast cancer therapy. However, we must admit that other first-line drugs in breast cancer treatment were not examined in this work, which could be performed in our future work.
Conclusions
Our work reveals an unpredicted layer of YAP regulation and put the activation of STARD13-correlated ceRNA network as a potential novel therapeutic strategy to specifically target breast CSCs.
Abbreviations
- ceRNA:
-
Competing endogenous RNAs
- CSCs:
-
Cancer stem cells
- ECM:
-
Extracellular matrix
- EMT:
-
Epithelial-to-mesenchymal transition
- FACS:
-
Fluorescence-activated cell sorting
- FBS:
-
Fetal bovine serum
- GEO:
-
Gene Expression Omnibus
- IHC:
-
Immunohistochemistry
- LATS:
-
Large tumor suppressor
- MDR:
-
Multidrug resistance
- qRT-PCR:
-
Quantitative real-time PCR
- RIP:
-
RNA immunoprecipitation
- STR:
-
Short tandem repeat
- TCGA:
-
The Cancer Genome Atlas
- UTR:
-
Untranslated region
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Funding
This work was supported by the National Nature Science Foundation of China, No. 81702957; China Postdoctoral Science Foundation, No. 2017M620230; Postdoctoral Research Funding Scheme of Jiangsu Province (2017), No.1701197B; and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.
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Contributions
LZ and TX designed the research. LZ and CX analyzed the data. LZ, CX, XL, QG, LG, and HN performed the research. LZ, CX, and XL wrote the paper. YX contributed new reagents or analytic tools. All authors read and approved the final manuscript.
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All animal experiments were performed with the approval of Ethics Committee for Animal Experimentation of China Pharmaceutical University.
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Additional files
Additional file 1:
Table S1. Sequences of siRNA against specific target in this study. (DOC 34 kb)
Additional file 2:
Table S2. Sequences of primers used for qRT-PCR in this study (DOC 44 kb)
Additional file 3:
Table S3. Primary antibodies used in this study. (DOC 36 kb)
Additional file 4:
Table S4. Sequences of primers used for plasmid constructions. (DOC 41 kb)
Additional file 5:
Figure S1. The infection efficiency of lentivirus. (A) Lentiviral infection efficiency of MDA-MB-231 cells stably expressing STARD13-3′UTR, CDH5-3′UTR, HOXD1-3′UTR, and HOXD10-3′UTR was examined by qRT-PCR. (B) Lentiviral infection efficiency of MCF-7 cells stably depleted of STARD13, CDH5, HOXD1, and HOXD10 was verified by Western blot analysis. Data were presented as the mean ± SD, n = 3, ***p < 0.001 vs. Ctrl. (TIF 1373 kb)
Additional file 6:
Figure S2. MCF-7 cells depleted of STARD13-correlated ceRNAs gain CSC traits. (A) Phase contract images of mammospheres formed by MCF-7 cells with STARD13-correlated ceRNA knockdown. (B) Quantification of mammospheres formed in (A). (C) Represented FACS profile of MCF-7 cells described in (A). (D) Identification of stemness-related genes expression (ALDH1, OCT4, and Nanog) by Western blot analysis in MCF-7 cells described in (A). Data were presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01 vs. pLVX-Ctrl. (TIF 3016 kb)
Additional file 7:
Figure S3. STARD13-correlated ceRNA network inhibits CSC traits of breast cancer cells in vivo. (A, B, and C) Images (left) and weight (right) of tumors harvested when serially diluted MDA-MB-231 cells with STARD13-correlated ceRNAs-3′UTR overexpression were planted. (D and E) Images (left) and weight (right) of tumors harvested when serially diluted MCF-7 cells with STARD13 or its ceRNA knockdown were planted. (TIF 4230 kb)
Additional file 8:
Figure S4. (A) Correlation analysis between LATS1/2 and STARD13, CDH5, HOXD1, and HOXD10, based on the microarray data downloaded from the TCGA data portal. (B) The ceRNA sequence and the genomic locus. (TIF 1525 kb)
Additional file 9:
Figure S5. STARD13-correlated ceRNAs-3′UTRs regulate TAZ nuclear abundance. Confocal images of TAZ distribution in MDA-MB-231 cells with STARD13-correlated ceRNAs-3′UTR overexpression or not. Data were presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01 vs. Vector. (TIF 4364 kb)
Additional file 10:
Table S5. The number of common miRNA binding sites on STARD13 3′UTR and LATS1/2 3′UTR is predicted using Targetscan 6.2 and microRNA.org. (DOC 33 kb)
Additional file 11:
Figure S6. Target miRNAs attenuated the promotive effects of STARD13-correlated ceRNA network on Hippo signaling. Target miRNAs (miR-424, miR-374a, miR-590-3p, miR-448, and miR-15a) mimics mix was co-transfected with STARD13-correlated ceRNAs-3′UTR overexpression constructs or not, the protein level of LATS1/2 and downstream effectors (p-YAP/p-TAZ, YAP/TAZ, and CTGF) was examined. (TIF 1437 kb)
Additional file 12:
Figure S7. CDH5, HOXD1, HOXD10-3′UTRs regulate Hippo signaling and CSC traits of breast cancer cells through STARD13. (A) Western blot analysis of lysates from MCF-7 cells with its ceRNA knockdown plus STARD13 3′UTR co-transfection or not. (B) Western blot analysis of lysates from MDA-MB-231 cells with STARD13 ceRNAs-3′UTR overexpression plus STARD13 knockdown or not. (C and D) Phase contract images of mammospheres (C) formed by MDA-MB-231 cells described in (A). Representative FACS profile (D) of them with CD24− and CD44+ markers by flow cytometry analysis. (E) Represented images of p-YAP, LATS1, and LATS2 staining of tumors harvested when 1,000,000 cells were injected in Fig. 2a. (TIF 5010 kb)
Additional file 13:
Figure S8. Representative FACS profile of MDA-MB-231 cells with STARD13- or its ceRNAs-3′UTR overexpression plus LATS1 or LATS2 or LATS1/2 knockdown by lentiviral infection. (TIF 1568 kb)
Additional file 14:
Figure S9. STARD13-correlated ceRNA network regulates breast cancer EMT through LATS1/2. EMT marker (see in main text) expressions were measured in MDA-MB-231 cells with STARD13- or its ceRNAs-3′UTR overexpression plus LATS1 or LATS2 or LATS1/2 knockdown by lentiviral infection. (TIF 2846 kb)
Additional file 15:
Figure S10. STARD13-correlated ceRNA network regulate TAZ nuclear abundance by inhibiting RhoA-ROCK signaling. (A) Confocal images of TAZ distribution in MCF-7 cells with STARD13-correlated ceRNA knockdown. (B) Confocal images of TAZ distribution in MCF-7 cells with STARD13-correlated ceRNA knockdown plus ROCK inhibitor (Y-27632) treatment or not. (C) Representative FACS profile of MCF-7 cells with STARD13 knockdown plus ROCK inhibitor treatment. Data were presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01 VS. Control or Rock inhibitor. (TIF 5007 kb)
Additional file 16:
Figure S11. Depletion of STARD13-correlated ceRNAs dampens the response of breast cancer cells to doxorubicin. (A) IC50 curves of MCF-7 cells with STARD13-correlated ceRNA knockdown and were fitted with a nonlinear regression model and were presented as log (Doxorubicin) vs cell viability. (B) Western blot assay of lysates from MCF-7 cells with STARD13-correlated ceRNA knockdown. (C) Images of tumors harvested when STARD13 3′UTR stable overexpression cells were planted and followed by doxorubicin treatment or not. The weight of tumors harvested in (C) was monitored. (D) The weight of mice depicted in (C) was monitored. (E) The volume of tumors harvested in (C) was monitored. (G) Confocal images of MCF-7 cells described in (B) with doxorubicin treatment. Depletion of STARD13-correlated ceRNAs impaired the cellular retention of doxorubicin. (F) Western blot assay of lysates from MCF-7 cells with STARD13-correlated ceRNA knockdown plus si-Dicer or not. (TIF 3999 kb)
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Zheng, L., **ang, C., Li, X. et al. STARD13-correlated ceRNA network-directed inhibition on YAP/TAZ activity suppresses stemness of breast cancer via co-regulating Hippo and Rho-GTPase/F-actin signaling. J Hematol Oncol 11, 72 (2018). https://doi.org/10.1186/s13045-018-0613-5
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DOI: https://doi.org/10.1186/s13045-018-0613-5