TBX15 overexpression abrogates breast cancer DOX resistance by suppressing glycolysis and autophagy.
To identify the mechanisms of DOX resistance in breast cancer cells, using bioinformatics methods we found potential 21 transcription factors (TFs) (ZEB1, TEAD1, NFAT5, MEOX2, TWST1, MAFF, RFX2, MEF2C, EGR2, EBF1, KLF14, JUN, MEIS2, NR3C2, BACH2, BHLHE41, NR3C1, TBX15, MSX1, LHX6, ETV5) that were downregulated in both breast cancer tissues and DOX-resistant breast cancer cells in the JASPER database (Fig. 1A). To test whether expression levels of these TFs were downregulated in a large and diverse breast cancer cohort, we analyzed their expression levels of these factors in the cancer tissues and in adjacent normal tissues. Among them, TBX15 levels were downregulated in breast cancer tissues, compared to normal adjacent tissues (Fig. 1B), and acted as a positive marker for the Relapse-Free survival prognosis of breast cancer (Fig. 1C). We induced T47D DOX-resistant breast cancer cells (T47D/ADR) by long exposure of DOX treatment in the cells, then detected its TBX15 expression level. Figure 1D showed that TBX15 protein levels were decreased in T47D/ADR cells. To further investigate the effects of TBX15 expression on DOX sensitivity, we utilized two DOX-resistant breast cancer cell lines, MCF7/ADR, and T47D/ADR, and treated these cells with varying concentrations of DOX. Following that, the cell viability assays and drug sensitivity analysis showed that TBX15 overexpression caused a significant reduction in cell growth and IC50 values; siRNAs targeting TBX15 (siTBX15) increased cell growth and IC50 values (Fig. 1E-I).
Activation of autophagy and glycolysis were reported to be important in drug resistance in various cancer cells [24,25). Additionally, TBX15 expression was associated with glycolysis in some tissues [26]. In order to determine mechanisms by which TBX15 may effects DOX resistance, we transfected TBX15 plasmid or TBX15 siRNAs into DOX-resistant cells, and assessed the expression levels of markers of autophagy or glycolysis. TBX15 overexpression decreased glycolysis by glucose and lactate assays, whereas siTBX15 increased glycolysis (Fig. 1J-K). Furthermore, TBX15 overexpression increased p62 protein expression and suppressed recruitment and conversion of LC3-I to LC3-II. In contrast, siTBX15 decreased p62 protein expression and promoted LC3-I conversion (Fig. 1L). At the same time, siTBX15 transfection promoted autophagosome formation after DOX treatment (Fig. 1M). These data indicate that TBX15 overexpression abrogates DOX resistance by inhibiting autophagy and glycolysis in breast cancer cells.
TBX15 forced expression promotes the DOX sensibility by inducing miR-152 expression.
We found that levels of TBX15 and miR-152 strongly correlated in TCGA database using Spearmen’s correlation analysis (Fig. 2A). Forced expression of TBX15 increased miR-152 expression levels in MCF7/ADR and T47D/ADR cells, while knockdown of TBX15 decreased miR-152 expression (Fig. 2BC). These data showed TBX15 upregulated miR-152 expression levels. To verify whether TBX15 promoted miR-152 expression through transcriptional regulation, we first used the JASPER database and identified three binding sites of TBX15 in the miR-152 promoter region. Subsequently, we constructed reporter plasmids and performed luciferase promoter reporter assays to determine whether TBX15 upregulated miR-152 expression by binding to miR-152 promoter region. These data showed that overexpression of TBX15 induced luciferase activities at all three binding sites we predicted (Fig. 2D).
To test whether miR-152 suppresses DOX resistance in breast cancer tissue, we analyzed miR-152 expression in the cancer tissues and adjacent tissues in TCGA database. Breast cancer tissues had lower expression levels of miR-152 when compared to the adjacent tissue group (Fig. S1A). Furthermore, we subdivided the TCGA dataset into miR-152 high and miR-152 low group according to its expression levels and sent them to gene set enrichment analysis (GSEA) which showed that lower expression levels of miR-152 were enriched in KANG DOXORUBINCIN UP dataset (NES = 1.56, FDR q = 0.01). These data indicated the strong correlation between miR-152 attenuation and DOX resistance in breast cancer (Fig. S1B).
We investigated the mechanisms by which miR-152 may affect DOX resistance. The cells overexpressing miR-152 were more sensitive to DOX treatment, as indicated by a lower IC50 value and higher apoptosis rate. Correspondingly, the cells in which miR-152 was inhibited displayed an increased resistance to DOX treatment (Fig. 2E-F). On the contrary, miR-152 inhibitor reversed TBX15-suppressed DOX resistance in breast cancer cells (Fig. 2G).
To examine the mechanisms of miR-152 on these processes, we transfected miR-152 mimics or a miR-152 inhibitor into MCF7/ADR cells and assessed its effects on autophagy or glycolysis. miR-152 mimics decreased glycolysis in glucose and lactate assays, whereas miR-152 inhibitor increased glycolysis (Fig. 2H). Furthermore, miR-152 mimics increased p62 protein expression and suppressed recruitment and conversion of LC3- I to LC3- II. In contrast, miR-152 inhibitor decreased p62 protein expression and promoted LC3-I conversion (Fig. 2I). These data indicate that TBX15/miR-152 pathway abrogates DOX resistance by inhibiting autophagy and glycolysis in breast cancer cells. TBX15 attenuation may result in miR-152 downregulation in DOX-resistant breast cancer cells, suggesting a potential new therapeutic target for breast cancer diagnosis and treatment in the future.
KIF2C, a direct target of miR-152, is upregulated in breast cancer which induces glycolysis and autophagy.
KIF2C is upregulated in some cancer tissues and is associated with resistance to chemotherapy [15–17]. By bioinformatic assays, we found KIF2C is one of potential target of miR-152. We analyzed the correlation between expression levels of miR-152 and its potential targets, and found that KIF2C was one of most significantly positive-associated gene with miR-152 expression levels, and miR-152 levels were negatively correlated with KIF2C expression in breast cancer in the GEO database (Fig. S2A,B). DOX-resistant cells transduced with TBX15 or miR-152 mimics have lower KIF2C expression levels following DOX treatment, whereas transfection with siTBX15 or miR-152 inhibitor increased KIF2C expression levels in both MCF7/ADR and T47D/ADR cells (Fig. 3A). By using bioinformatic analysis of miR-152 seed-matching site in the KIF2C gene, we found that KIF2C was a potential direct target of miR-152. To confirm this, luciferase reporter constructs were made that contained the putative binding site of the KIF2C 3’-UTR region or three nucleotide substitutions in its 3’-UTR region (mutant). Overexpression of miR-152 inhibited wild-type KIF2C reporter activity, but not that mutant region (Fig. 3B), demonstrating that miR-152 can specifically target the 3’-UTR regions of KIF2C by binding to its putative sequences.
To identify whether KIF2C promotes DOX resistance in breast cancer tissues, we obtained TCGA mRNAs expression data from Breast Cancer, and divided the samples into cancer tissues and adjacent tissue groups. We found that KIF2C expression levels were significantly higher in breast cancer tissues than those in the adjacent groups (Fig. 3C). We then divided the cancer samples into groups based on high or low expression levels of KIF2C, and assessed overall patient outcomes within these two groups. Higher expression of KIF2C indicated poor prognosis in breast cancer patients (Fig. S2C). In addition, we also observed the positive correlation between KIF2C expression and DOX resistance signature using the GSEA (NES = 1.65, FDR q = 0.01) which suggesting high expression levels of KIF2C may be responsible for DOX resistance (Fig. 3C).
Additionally, we determined the IC50 value and performed cell apoptosis assays to detect DOX sensitivity in KIF2C-upregulated or down-regulated MCF7/ADR cells. Cells overexpressing KIF2C were more sensitive to DOX treatment, while those transfected with siKIF2C displayed an increased resistance to DOX (Fig. 3D). Similarly, apoptosis assays showed KIF2C overexpression suppressed cell apoptosis after DOX treatment, while siKIF2C displayed an increased sensitivity to DOX (Fig. 3E). To identify the mechanism of KIF2C-mediated DOX resistance, overexpressed or inhibited expression of KIF2C in MCF7/ADR cells were used to assess autophagy and glycolysis. Overexpression of KIF2C increased glycolysis in glucose and lactate assays, whereas siKIF2C reduced glycolysis (Fig. 3F). Overexpression of KIF2C decreased p62 protein expression and promoted conversion of LC3-I, whereas siKIF2C increased p62 protein expression and suppressed LC3-I conversion (Fig. 3G). Collectively, these data suggest that KIF2C promotes DOX resistance by inducing autophagy and glycolysis.
To determine role of KIF2C in TBX15/miR-152-mediated drug sensitivity, we tested the effects of KIF2C on the DOX sensitivity by transfecting MCF7/ADR cells overexpressing TBX15/miR-152 with KIF2C cDNA (without the 3’-UTR region). Overexpression of KIF2C suppressed miR-152-induced DOX sensitivity, as determined by IC50 values and apoptosis assays (Fig. 4A-C). The reduction of glucose consumption, lactate production, ECAR and OCR assays induced by miR-152 overexpression was abrogated in these cells (Fig. 4D-E). Furtherly, KIF2C overexpression reversed miR-152-suppressed autophagy (Fig. 4F). Similarly, KIF2C suppressed TBX15-induced DOX sensitivity by increasing autophagy and glycolysis (Fig. 4G-I). Taken together, these data suggest that TBX15/miR-152 pathway promotes DOX sensitivity through the targeting of KIF2C by miR-152.
KIF2C increased PKM2 expression by promoting its protein stability.
PKM2 is involved in multidrug resistance in some cancer cells [ 22, 27]. However, role of PKM2 in mediating chemoresistance in breast cancer remains to be studied. In our previous study, we found that PKM2 promoted cell proliferation and angiogenesis in breast cancer [12]. In order to identify whether PKM2 is involved in the TBX15/miR-152/KIF2C-mediated DOX resistance, we assessed PKM2 expression in MCF7/ADR cells when expression of TBX15/miR-152 or KIF2C was altered in the cells. TBX15 overexpression decreased PKM2 expression, whereas its siRNAs increased PKM2 levels (Fig. 5A). Conversely, overexpressing KIF2C increased PKM2 expression, whereas knockdown of KIF2C caused its expression decrease (Fig. 5B). Notably, overexpression of KIF2C reversed TBX15 or miR-152-mediated downregulation of PKM2 (Fig. 5C,D). In addition, PKM2 overexpression restored siKIF2C-inhibited autophagy (Fig. 5E). Thus, these data suggest that PKM2 is regulated in TBX15/miR-152/KIF2C pathway for regulating DOX resistance.
In order to determine the mechanism by which KIF2C regulates PKM2 expression, mRNA expression levels of PKM2 were determined in MCF7/ADR cells after the transfection of KIF2C. Overexpression of KIF2C did not change PKM2 mRNA expression levels (data not shown), suggesting a potential posttranscriptional mechanism modulated PKM2 expression. Co-IP analysis showed that KIF2C directly bound to PKM2, and that a weaker PKM2 band was co-immunoprecipitated by anti-KIF2C antibodies when TBX15 was overexpressed. In contrast, transfection of siTBX15 resulted in a stronger interaction between KIF2C and PKM2 (Fig. 6A). Similarly, KIF2C overexpression also resulted in a stronger interaction between KIF2C and PKM2; and siKIF2C decreased the binding (Fig. 6B).
To determine whether KIF2C modulates PKM2 protein stability in MCF7/ADR cells, we treated cells with CHX, which inhibits protein production. CHX treatment did not change KIF2C-mediated upregulation of PKM2 (Fig. 6C). However, knockdown of KIF2C enhanced PKM2 degradation following CHX treatment in a time dependent manner (Fig. 6D). We also treated cells with MG132, which inhibited protein degradation. It restored the downregulation of PKM2 following knockdown of KIF2C (Fig. 6E, F). Considering the ubiquitin (Ub)-editing degradation of PKM2, we examined the ubiquitylation pattern of PKM2 in MCF7/ADR cells. Ub-HA plasmid was co-transfected with KIF2C-pCMV or siKIF2C. Co-immunoprecipitation experiments revealed that overexpression of KIF2C induced weaker bands, which corresponded to PKM2 ubiquitination, whereas knockdown of KIF2C enhanced PKM2 ubiquitination (Fig. 6G). Therefore, these data suggest that KIF2C directly binds to PKM2 and prevent PKM2 degradation by ubiquitination.
There are three domains (Domain-1, Domain-2, and Domain-3) of human KIF2C protein (Fig. S2D, http://www.uniprot.org/uniprot/Q99661). To confirm which domain(s) was/were responsible for PKM2 ubiquitylation, we constructed three plasmids encoding different domains of KIF2C, respectively. The three plasmids were transfected into MCF7/ADR cells, respectively, and PKM2 expression levels were analyzed by Western blotting. Only overexpression of the plasmid encoding Domain-2 of KIF2C increased PKM2 expression and showed more binding to PKM2 in co-immunoprecipitation experiments (Fig. 6H). Ubiquitin immunoprecipitation assays also revealed that only overexpression of KIF2C Domain-2 resulted in a weak band that corresponded to PKM2 ubiquitination (Fig. 6I). These data suggest that KIF2C Domain-2 plays a major role in the binding of KIF2C and PKM2 to increase PKM2 stability.
TBX15/miR-152 overexpression abrogates DOX resistance in vivo.
In order to investigate the effects of miR-152 on DOX sensitivity in vivo, we established tumor xenografts by subcutaneously implanting MCF7/ADR cells that were stably expressing miR-152 in nude mice. Twenty-four days after subcutaneous injection of the cancer cells, the mice were treated with DOX (4 mg/kg/week) and or PBS by intraperitoneal injection. Analysis of implanted tumor xenografts revealed that overexpression of miR-152 significantly suppressed tumor growth. Furthermore, xenografts with overexpression of miR-152 were more sensitive to DOX treatment (Fig. 7A-C). These data suggested that miR-152 promotes sensitivity to DOX treatment in vivo. Using immunohistochemistry assays, we found that overexpression of miR-152 significantly suppressed KIF2C and PKM2 expression, and that this suppression was much greater following DOX treatment (Fig. 7D, E). Collectively, these data suggest that miR-152 abrogates DOX resistance by inhibiting KIF2C and PKM2 expression.
In order to elucidate the effect of TBX15 on miR-152, KIF2C and PKM2 expression in vivo, we established tumor xenografts by implanting MCF7/ADR cells stably expressing TBX15. The tumors were found 10 days after subcutaneous injection. The mice were euthanized 21 days after the cell injection, and tumors were resected. Analysis of tumor tissues revealed that overexpression of TBX15 significantly decreased tumor growth (Fig. 7F-H). Furthermore, qRT-PCR and immunohistochemistry assays showed that TBX15 overexpression not only induced miR-152 expression levels, but also decreased KIF2C and PKM2 expression levels in vivo (Fig. 7I, J).