Tristetraprolin impairs myc-induced lymphoma and abolishes the malignant state

Abstract

Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.

Figure 1. Myc Alters the Expression of Hundreds of Genes Containing AREs

(A and B) Gene expression profiling of arrays showing genes from the ARED Organism database whose expression is specifically altered in B220+ B cells from premalignant Eμ-Myc (n=5) and wild type (n=4) mice (A) and in Eμ-Myc lymphomas (n=13) compared to premalignant Eμ-Myc B cells (B). All probe sets shown have a >2.0-fold change and are significantly altered by unpaired t-test analysis (p<0.05). (C) Gene expression profiling comparing ARED genes differentially expressed between 44 human BL and 129 human non-BL samples from GSE4475. All probe sets shown have >2.0-fold change and are significantly altered by unpaired t-test analysis (p<0.05). See also Figure S1 and Tables S1–S3.

Figure 2. Myc Alters the Expression of AUBPs

(A–B and E) Gene expression profiling of arrays showing differentially expressed AUBP genes in B220+ B cells from premalignant Eμ-Myc versus wild type littermates (A), Eμ-Myc lymphomas compared to premalignant Eμ-Myc B cells (B) and human BL and non-BL samples (E) from GSE4475. All probe sets shown have >2.0-fold change and are significantly altered by unpaired t-test analysis (p<0.05). (C and F) qRT-PCR analysis of total RNA isolated from B220+ B cells of wild type and premalignant Eμ-Myc mice and lymphomas that arose in independent Eμ-Myc transgenics (C), and of RNA of CD19+ B cells from a healthy individual compared to human BL samples (F). The relative expression was determined for TTP (ZFP36), TIS11B (ZFP36L1), HUR, AUF1, AUF2 and NCL (Nucleolin). Individual samples are indicated by circles and the mean for each group is represented by a line. Results were normalized to the expression of Ubiquitin (Ub). (D) Immunoblots comparing the levels of TTP, Actin, and Tubulin in B220+ B cells of wild type and premalignant Eμ-Myc littermates. Student’s t-test (* p<0.05, ** p<0.01, *** p<0.001) See also Figure S2.

Figure 3. Myc Directly Represses TTP and TIS11B Transcription

(A) qRT-PCR analysis of total RNA isolated from primary wild type B220+ B cells grown ex vivo in the presence of IL7. IL7 was removed at −18 hr and re-introduced at 0 hr and cells were collected at the indicated intervals. The relative expression was determined for c-Myc, TTP and Tis11b. Results were normalized to Ubiquitin (Ub) expression. (B) Expression of c-Myc, TTP, and Tis11b throughout mouse B cell (GSE15907) and T cell (GSE30631) development. (C) Total RNA was isolated from P493-6 B cells treated with Tet for 72 hr to repress c-Myc expression. Cells were either washed to remove Tet (black lines with circles), allowing Myc activation, or were treated with β-E2 (red lines with squares), allowing proliferation via ER-EBNA2 activation. Cells were collected at the indicated intervals. qRT-PCR analysis was performed to determine the relative expression of c-Myc, TTP, TIS11B, HUR, AUF1, AUF2, and NCL, and results were normalized to Ubiquitin (Ub) expression. (D–E) ChIP analyses establish that Myc binds to the initiator element (Inr) found in the promoters of TTP and TIS11B and that this is associated with reduced binding of RNA Pol II. Chromatin was immunoprecipitated with an α-c-Myc antibody from P493-6 cells with repressed c-Myc (+Tet) or following 8 hr of c-Myc activation (−Tet) (D), or with an α-RNA Pol II antibody from P493-6 cells when c-Myc was repressed by Tet (0 hr) or following c-Myc activation by removal of Tet (24 hr) (E). Bound chromatin was evaluated by qRT-PCR and compared to the total amount of chromatin. Results show the mean with error bars indicating ± SEM. See also Figure S3.

Figure 4. Generation of Eμ-TTP and Eμ-Tis11b Transgenic Mice

(A) Schematic of Eμ-TTP and Eμ-Tis11b transgenes. The complete coding region for mouse TTP or Tis11b, was cloned into the pEμSR plasmid downstream of the SRα promoter. The transgenes also include the mouse Igh 5’ enhancer (Eμ), the rabbit globin poly A and an SV40 tag region. (B) qRT-PCR analysis of TTP or Tis11b expression in total RNA isolated from B220+ B cells from wild type and Eμ-TTP littermates from two different founders (Line 1 and Line 2) or from Eμ-Tis11b littermates. Results were normalized to Ubiquitin (Ub) expression. (C) Immunoblots analyses of TTP and Actin levels in bone marrow and splenic B220+ B cells of wild type and Eμ-TTP littermates. Results show the mean with error bars indicating ± SEM. See also Figure S4.

Figure 5. TTP Suppresses Myc-driven Lymphomagenesis

(A) Survival curves of Eμ-Myc;Eμ-TTP-1 (n=19), Eμ-Myc;Eμ-TTP-2 (n=25), and Eμ-Myc;Eμ-Tis11b (n=23) transgenic mice was compared to Eμ-Myc littermates (n=16, 30 or 22, respectively). Tick marks above survival curve indicate mice that were still alive when the analysis was completed. P-values were determined by using Mantel-Cox logrank test. (B) Immunoblots analyses of Myc and Actin levels in Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 lymphomas. (C) Splenic mass and numbers of B220+ B cells in the spleens of 5-week-old wild type, Eμ-TTP-1, Eμ-Myc, and Eμ-Myc;Eμ-TTP-1 littermates. (D–E) Analysis of TUNEL-positive (D) and BrdU+ (E) B220+ cells, separated based upon their IgM expression, from BM and spleen of 5-week-old wild type, Eμ-TTP-1, Eμ-Myc, and Eμ-Myc;Eμ-TTP-1 mice. Results show the mean with error bars indicating ± SEM. Student’s t-test (* p<0.05, ** p<0.01, *** p<0.001) See also Figure S5.

Figure 6. TTP Impairs Maintenance of Eμ-Myc Lymphoma

(A–B) Nude mice were injected with unsorted Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-TTP-I-GFP retrovirus (left panel) or with Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-Tis11b-I-GFP retrovirus (right panel). Shown is analysis of the change in the percentage of GFP+ B cells found in tumors compared to the percentage of GFP+ lymphoma cells that were injected (A) and the percentage of GFP+ B cells found in peripheral blood (B) at the time of sacrifice. Individual samples are indicated by circles and the mean for each group is represented by a line. (C) Percentage of GFP+ unsorted ex vivo Eμ-Myc lymphoma cells infected with MSCVI-GFP versus MSCV-TTP-I-GFP retrovirus (left panel) or with MSCV-I-GFP versus MSCV-Tis11b-I-GFP retrovirus (right panel) was determined at the indicated intervals. (D) Changes in cell cycle distribution of unsorted ex vivo Eμ-Myc lymphoma cells infected with MSCV-I-GFP versus MSCV-TTP-I-GFP retrovirus based upon the presence or absence of GFP within the lymphoma cells (i.e., GFP+ cells minus GFP-negative cells). Cells were labeled with BrdU, harvested and analyzed by FACS. The graph represents the average of three individually infected plates of Eμ-Myc lymphoma cells for each retrovirus. (E) Survival curves of Nude mice injected with Eμ-Myc lymphoma cells sorted for GFP+ following infection with MSCV-I-GFP or MSCV-TTP-I-GFP retrovirus. P-values were determined by using Mantel-Cox log-rank test. Results show the mean with error bars indicating ± SEM. Student’s t-test (**p<0.01, ***p<0.001, *****p<0.00001) See also Figure S6.

Figure 7. TTP-dependent Myc Targets Associated with Tumorigenesis

(A and E) Gene expression profiling of arrays showing ARED genes whose expression is specifically altered in B220+ BM B cells from premalignant Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 mice (A) or all genes whose expression is specifically altered in lymphomas from Eμ-Myc versus Eμ-Myc;Eμ-TTP-1 mice (E). Genes labeled in (A) have known roles in cancer. All probe sets shown have >2.0-fold change and are significantly altered by unpaired t-test analysis (p<0.05). (B and C) qRT-PCR analysis of total RNA isolated from B220+ BM B cells of wild type and Eμ-TTP-1 transgenics and from premalignant Eμ-Myc and Eμ-Myc;Eμ-TTP-1 mice (B) or from wild type B220+ splenic B cells and from Eμ-Myc and Eμ-Myc;Eμ-TTP-1 lymphomas (C). The relative expression of Ccnd1, Fstl1, Gabarapl1, Tes, Tns3 and Uaca is shown. Results were normalized to Ubiquitin (Ub) expression. (D) qRT-PCR (top left panel) and immunoblot (top right panel) analyses show the mRNA and protein levels of TTP-Flag, Cyclin D1 and Actin in HeLa Tet-Off/TTP-Flag cells that were grown +Dox (repressed TTP-Flag) or −Dox (induced TTP-Flag) for 48 hr. For RNP-IP analyses (bottom panel), RNPs were immunoprecipitated with α-Flag antibody from control or TTP-Flag-expressing HeLa cells and used for qPCR detection of cyclin D1 mRNA. Results show the mean with error bars indicating ± SEM. Student’s t-test (* p<0.05, ** p<0.01, *** p<0.001) See also Figure S7 and Tables S4–S7.