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, 3 (22), 3613-3625

TAZ Functions as a Tumor Suppressor in Multiple Myeloma by Downregulating MYC

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TAZ Functions as a Tumor Suppressor in Multiple Myeloma by Downregulating MYC

Stacy Grieve et al. Blood Adv.

Abstract

Multiple myeloma (MM) is an incurable blood cancer that is often characterized by amplification and overexpression of the MYC oncogene. Despite efforts, direct targeting of MYC is not yet possible; therefore, alternative strategies to inhibit MYC activity are necessary. TAZ is a transcriptional coactivator downstream of the Hippo-signaling pathway that functions as an oncogene in many solid tumors. However, its role in hematological malignancies is largely unexplored. Here, we show that, in contrast to solid tumors, expression of TAZ is lower in hematological malignancies, and that high expression of TAZ correlates with better patient outcomes. We further show that TAZ is hypermethylated in MM patient samples and in a panel of MM cell lines. Genetic overexpression of TAZ or pharmacological upregulation of TAZ by treatment with the demethylating agent decitabine induces apoptosis. Importantly, TAZ-induced apoptosis is independent of canonical Hippo components LATS1 or the TEA-domain family of transcription factors. Instead, RNA-sequencing analysis revealed that overexpression of TAZ represses a MYC transcriptional program and we show that increased TAZ expression correlates with decreased MYC expression in both cell-line models and patient samples. Furthermore, promoter derepression of TAZ expression sensitizes MM cell lines through a reciprocal reduction in MYC expression using additional therapeutics such as bortezomib, trichostatin A, and panobinostat. Our findings uncover an unexpected role for TAZ in MM tumorigenesis and provide a compelling rationale for exploring the therapeutic potential of upregulating TAZ expression to restore sensitivity to specific therapeutics in MM.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

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Figure 1.
Figure 1.
Decreased TAZ expression in MM cell lines and patient samples predicts worse outcomes. (A) Expression data comparing normal plasma cells (NPCs) from healthy subjects (n = 22), MGUS (n = 44), SMM (n = 12), and MM (n = 351), combining data from GSE5900 and GSE2658 using probe set 202134_s_at. Data are presented using box-and-whisker plots according to the Tukey method. The Student t test was used to evaluate significance: **P ≤ .01 and ***P ≤ .001. (B) Overall survival curve relative to TAZ expression in individuals affected by MM, obtained from http://www.canevolve.org/AnalysisResults/AnalysisResults.html and based on GSE2658 (log-rank test). Optimal cutoff determined using Cutoff Finder (http://molpath.charite.de/cutoff). (C) Immunoblot analysis of HMCLs for protein expression of TAZ, YAP, LATS1, and MST1 relative to A549 cells. (D) mRNA expression of TAZ, YAP, LATS1, and MST1 relative to A549 cells in HMCLs. A.U., arbitrary unit; HR, hazard ratio; ns, not significant.
Figure 2.
Figure 2.
TAZ is hypermethylated in MM. (A) Methylation β values of normal plasma and B cells (n = 9) and plasma cells from MGUS, SMM (n = 4), or MM (n = 161) are shown as mean ± standard error of the mean (SEM) using data from GSE21304. β values range from 0 to 1.0, equivalent to 0% to 100% methylation. (B) Prediction of CpG islands in TAZ promoter using MethPrimer (www.urogene.org/methprimer/) with regions highlighted for MSP. (C) MSP of 3 regions within the TAZ promoter reveals hypermethylation in KMM1, KMS11, JJN3, and U266 cell lines. Universally methylated (Meth) and unmethylated (UnMeth) DNA served as MSP controls. GC, guanine and cytosine nucleotides; M, methylated; TSS, transcription start site; U, unmethylated.
Figure 3.
Figure 3.
Overexpression of TAZ induces cell death. (A) Proliferation assay of KMM1 cells transduced with empty vector (WPI), wild-type TAZ (TAZ), or TAZ mutants unable to bind TEAD (TAZ-F52A/F53A) or LATS1/2 (TAZ-WWm). Data are mean ± standard deviation (SD) of triplicates. Single-factor analysis of variance (ANOVA) of doubling times indicated significance between all TAZ-expressing lines and either KMM1 or WPI cell lines. (B) Cell-viability assay of cell lines described in panel A after 3, 6, or 9 days posttransduction. Data are mean ± SD of triplicates. *P ≤ .05 of TAZ, #P ≤ .05 of TAZ-F52A/F53A, or $P ≤ .05 of TAZ-WWm compared with KMM1 or WPI cell lines, respectively, using a Student t test. (C) Immunoblot of cell lines described in panel A for markers of apoptosis including cleaved caspase 3 and the 3 isoforms of BIM (BIMEL, BIML, BIMS). β-actin was used as a loading control.
Figure 4.
Figure 4.
Transcriptomic analysis reveals TAZ as negative regulator of MYC. GSEA reveals enrichment of apoptotic genes (A) and downregulation of MYC targets (B-C) using the MSigDB Hallmark collection in TAZ-overexpressing KMM1 cells relative to WPI-expressing cells. (D) Table of gene sets enriched in TAZ-expressing KMM1 cells, highlighting the number of genes in each set (n), the normalized enrichment score (NES), and test of statistical significance (false discovery rate [FDR] q value). (E) Expression of TAZ and MYC are anticorrelated in MM patient samples combining data from GSE5900 and GSE2658 using probe sets 202134_s_at (TAZ) and 202431_s_at (MYC). (F) Validation of TAZ targets at the mRNA levels using qPCR. Data are mean ± SD of triplicate samples. **P ≤ .01 using the Student t test. (G) Validation of TAZ targets at the protein level. β-actin was used as a loading control.
Figure 5.
Figure 5.
Demethylating agent DAC upregulates TAZ and represses MYC transcriptional program. KMM1 (A), KMS11 (B), JJN3 (C), and U266 (D) were treated with increasing concentrations of DAC and evaluated for TAZ and MYC expression at the mRNA and/or protein levels. KMM1 (E) or JJN3 (F) cells were treated with increasing concentrations of DAC and evaluated for deregulation of MYC targets at the mRNA level. Data are mean ± SD of triplicate samples.
Figure 6.
Figure 6.
Upregulation of TAZ sensitizes MM cells to chemotherapeutics. KMM1 (A) and JJN3 (B) cells were treated with increasing concentrations of DAC for 48 hours followed by exposure to BTZ for 72 hours before cell viability was assessed. CI values were calculated using CompuSyn software. Immunoblot analysis of TAZ and MYC expression in KMM1 (C) and JJN3 (D) cells treated with DAC for 48 hours followed by BTZ exposure for 24 hours. KMM1 (E) and JJN3 (F) cells were treated with increasing concentration of DAC for 48 hours followed by exposure to TSA for 72 hours before cell viability was assessed and CI values calculated. Immunoblot analysis of TAZ and MYC expression in KMM1 (G) and JJN3 (H) cells treated with DAC for 48 hours followed by TSA exposure for 24 hours. Densitometry was performed and relative expression, normalized to β-actin, is shown below the lanes indicating expression compared with untreated cells. ns, nonspecific.

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