The interaction between Myc and Miz1 is required to antagonize TGFbeta-dependent autocrine signaling during lymphoma formation and maintenance

Abstract

The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to Miz1; whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. Here we show that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor beta-2 (TGFbeta-2) and TGFbeta-3. Upon Myc suppression, TGFbeta signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFbeta. We demonstrate that, since lymphomas express high levels of TGFbeta, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFbeta is a key factor that establishes oncogene addiction of T-cell lymphomas.

Figure 1.

MycV394D is impaired relative to wild-type Myc in its ability to induce lymphomas. (A) Time course of the experiment. (B) Doxycycline-regulated expression of Myc in animals derived from the five founder lines selected for the experiment. Double-transgenic mice were sacrificed at the time points shown in A. Immunoblots of thymic lysates were probed with antibody 9E10, which detects human Myc, and Cdk2 as loading control. For comparison, equal amounts of a HeLa cell lysate were loaded in the last lane. (C) Kaplan-Meier plot summarizing the tumor development in double-transgenic mice expressing wild-type Myc and MycV394D, as well as the single-transgenic controls; double-transgenic mice of different founder lines with the same genotype are summarized in one graph here. The number of mice analyzed is shown on the right, together with the median survival of mice for each genotype. The P-value is shown for the difference between double-transgenic mice expressing wild-type Myc and MycV394D, and was calculated using a log-rank test. All single-transgenic or nontransgenic mice are included as one control group. (D) Summary of FACS analyses documenting the percentage of cells expressing the T-cell markers CD4 and CD8 in nontransgenic mouse (control) thymus (n = 2), and in thymi (wild-type Myc: n = 7; MycV394D: n = 7) and lymphomas (wild-type Myc: n = 10; MycV394D: n = 11) arising in mice of the indicated genotypes.

Figure 2.

Molecular analysis of lymphomas arising in mice expressing wild-type Myc or MycV394D. (A) The left panels show representative sections stained with the indicated antibodies or reagents to detect the presence of Ki67, a marker of cells in the G1, S, or G2 (but not G0) phases of the cycle; of apoptotic cells (using terminal desoxynucleotide transferase and biotinylated dUTP “TUNEL”); and of senescent cells using either SA-β-Gal or H3K9triMe as markers. The right panels show a quantification of stainings of multiple lymphomas for each marker together with the P-value determined by a two-tailed Mann-Whitney test. (B) Immunofluorescence pictures of representative sections of lymphomas stained with antibodies against both Ki67 and H3K9triMe. Pictures in the bottom row show enlargements of the indicated regions documenting the presence of double-positive cells in MycV394D lymphomas. (C) Quantification of immunoblots documenting expression of p27, Cdk1, and Cdk1 phosphorylated at Tyr 15 in lymphomas expressing either wild-type Myc or MycV394D. (D) The panel on the right shows the percentage of lymphoma cells incorporating BrdU. Mice of each genotype that had developed lymphomas were injected with BrdU 3 h before they were sacrificed (wild-type Myc: n = 8; MycV349D: n = 5). The pictures show representative sections stained with α-BrdU antibodies.

Figure 3.

Elevated expression of cdk2nb and cdkn1c expression in lymphomas expressing MycV394D. (A) Immunoblots documenting the expression of the indicated proteins in lymphomas of mice of the indicated genotypes. For comparison, prelymphomagenic and control thymi are included as indicated. Furthermore, lysates of mouse embryo fibroblasts are shown; where indicated, these cells were exposed to UVB irradiation prior to harvesting to induce Atr-dependent phosphorylation of p53 at Ser18. (B) RQ-PCR analysis documenting expression of the indicated mRNAs in lymphomas expressing either wild-type Myc or MycV394D. Each dot indicates expression in one individual lymphoma. The Y-axis shows expression levels relative to the thymus of a nontransgenic mouse. P-values refer to the difference between the two different genotypes, and were calculated using a two-tailed Mann-Whitney test.

Figure 4.

The Myc/Miz1 complex is continuously required to suppress expression of cdk2nb and cdkn1c in T-cell lymphomas. (A) ChIP assays demonstrating the doxycycline-regulated presence of Myc and the constitutive presence of Miz1 at the start sites of the cdkn2b gene in T-cell lymphomas. Lymphoma cells either were harvested untreated or, where indicated, doxycycline was added to the medium and cells were harvested 24 h later. Relative enrichment of Myc and Miz1 at the cdkn2b transcription start site is shown compared with a −1-kb upstream control region. (B) Summary of the time course experiments of cultured lymphoma cells expressing either Myc or MycV394D in response to addition of doxycycline. Immunoblots were probed with antibodies directed against Myc(9E10), p15Ink4b, and Cdk2. Cells were grown in culture in the absence of doxycycline, or were harvested at the indicated times after addition of doxycycline. (C) Lymphoma cells shown in B were analyzed by RQ-PCR assays documenting the expression of cdkn2b, cdkn1a, and cdkn1c mRNAs under these experimental conditions. (D) Lymphoma cells expressing doxycycline-regulated wild-type Myc were infected with retroviruses that constitutively express either wild-type Myc or MycV394D. After selection, doxycycline was added to switch off expression of the doxycycline-regulated Myc allele, and cells were harvested 48 h later. Control experiments established that the doxycycline-regulated Myc is undetectable after 6 h. The panels show RQ-PCR and immunoblot analysis documenting expression of the indicated mRNAs and proteins. (E) Growth curve documenting the growth of cells described in C in the presence of doxycycline.

Figure 5.

TGFβ-dependent signal transduction is required for expression of cell cycle inhibitors and senescence in cultured lymphoma cells. (A) Activation of the TGFβ pathway during lymphomagenesis. The panel documents RQ-PCR assays of mRNAs encoding the indicated TGFβ isoforms in nontransgenic thymus (n = 2), in transgenic thymi (wild-type Myc: n = 2; MycV394D: n = 2), and in lymphoma cells expressing either wild-type Myc (n = 8) or MycV394D (n = 11). This and the subsequent panels show average expression value of the indicated numbers of samples. Expression in nontransgenic thymus was arbitrarily set to 1. (B) Immunohistochemistry using antibodies recognizing TGFβ-2 (top row) and phosphorylated Smad2 and Smad3 (bottom row). The stainings show representative examples of three tumors analyzed for each genotype. (C) RQ-PCR assays documenting the expression of selected downstream target genes of the TGFβ pathway in the same samples shown in A. The panels show expression levels relative to nontransgenic thymus. (D) TGFβ-dependent signaling is required to induce expression of cdkn2b and cdkn1a when expression of Myc is inhibited. T-cell lymphoma cells were transduced with either control viruses or retroviruses expressing the soluble TβR-II-ED. Where indicated, doxycycline was added to the medium, and cells were harvested 48 h later. The panels document RQ-PCR assays measuring the expression of the indicated mRNAs. (E) FACS analysis documenting the accumulation of both control lymphoma cells and of lymphoma cells expressing TβR-II-ED in the G1 phase of the cell cycle upon inhibition of Myc expression. (F) Addition of recombinant TGFβ1 induces p15Ink4b protein expression only in MycV394D-expressing lymphoma cells as shown by immunoblot analysis. (G) Addition of recombinant TGFβ1 induces cell cycle arrest in lymphoma cells expressing MycV394D, but not in cells expressing wild-type Myc. (H) Addition of recombinant TGFβ1 induces senescence in lymphoma cells expressing MycV394D, but not in cells expressing wild-type Myc. Senescence was analyzed using SA-β-Gal staining.

Figure 6.

TGFβ-dependent senescence occurs in vivo. (A) TGFβ-dependent signal transduction is required for induction of senescence when Myc is turned off. The panel shows the percentage of either control lymphoma cells (“GFP”) or cells expressing TβR-II-ED that stain positive for SA-β-Gal in response to addition of doxycycline. (B) TGFβ-dependent signal transduction regulates expression of CKIs in vivo. Control lymphoma cells and cells expressing TβR-II-ED were injected subcutaneously into syngeneic, immunocompetent mice (FVB/N). After palpable tumors had developed, doxycycline was added to the drinking water where indicated, and tumors were recovered at the indicated times thereafter. The panels document RQ-PCR assays analyzing expression of the indicated genes. (C) Induction of apoptosis occurs independently of TGFβ in T-cell lymphomas when expression of Myc is switched off. Mice were injected and tumors harvested as described above. The panels show representative sections of lymphoma tissues stained by TUNEL to visualize apoptotic cells. (D) TGFβ-dependent induction of senescence in T-cell lymphomas. The experiment was performed as before, and sections were frozen and stained for SA-β-Gal. (E) Parallel sections to those shown in B were stained for H3K9triMe.

Figure 7.

TGFβ-dependent senescence is required for sustained regression when expression of Myc is reversed. Mice were injected as described above, and doxycycline was added to the drinking water when palpable tumors had developed. Subsequently, mice were monitored for relapse.

Figure 8.

A model for oncogene addiction of Myc-induced T-cell lymphomas. We suggest that, since lymphoma cells express high levels of TGFβ, there is a constant pressure to elicit an autocrine program of differentiation and senescence, which is absent in normal lymphocytes.