TGF-β enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity

Abstract

Inhibition of tumor growth factor (TGF)-β-mediated cell cycle exit is considered an important tumorigenic function of Myc oncoproteins. Here we found that TGF-β1 enforced G₁ cell cycle arrest and cellular senescence in human U-937 myeloid tumor cells ectopically expressing v-Myc, which contains a stabilizing mutation frequently found in lymphomas. This correlated with induced expression of the Myc antagonist Mad1, resulting in replacement of Myc for Mad1 at target promoters, reduced histone acetylation and strong repression of Myc-driven transcription. The latter was partially reversed by histone deacetylase (HDAC) inhibitors, consistent with involvement of Mad1. Importantly, knockdown of MAD1 expression prevented TGF-β1-induced senescence, underscoring that Mad1 is a crucial component of this process. Enforced Mad1 expression sensitized U-937-myc cells to TGF-β and restored phorbol ester-induced cell cycle exit, but could not alone induce G₁ arrest, suggesting that Mad1 is required but not sufficient for cellular senescence. Our results thus demonstrate that TGF-β can override Myc activity despite a stabilizing cancer mutation and induce senescence in myeloid tumor cells, at least in part by induction of Mad1. TGF-β-induced senescence, or signals mimicking this pathway, could therefore potentially be explored as a therapeutic principle for treating hematopoietic and other tumors with deregulated MYC expression.

Introduction

The MYC gene family encodes oncoproteins/transcription factors that regulate fundamental cellular processes such as cell growth and proliferation, apoptosis, metabolism, differentiation and stem cell function. The MYC genes are frequently deregulated in cancer, thereby contributing to tumor development (for reviews see [1], [2], [3], [4]). Myc contains a basic region/helix-loop-helix/leucine zipper (bHLHZip) motif that mediates heterodimerization with the obligatory partner Max and DNA binding to E-box recognition sequences in promoters of target genes [5]. Myc activates transcription from these sites by recruiting various cofactors, including histone acetyltransferase (HAT) complexes, but can also repress transcription via association with Miz-1 [1], [2]. Max also forms heterodimers with the Mad (Mxd)/Mnt-family bHLHZip proteins Mad1, Mxi1, Mad3, Mad4 and Mnt as well as Mga that all function as antagonists of Myc [6], [7]. These heterodimers also bind to E-boxes, but, in contrast to Myc, repress transcription by interacting with repressor complexes containing histone deacetylases (HDAC). Recent studies suggest that c-Myc regulates as many as 10–15% of all cellular genes [1], [2], [8]. It is unclear how large fraction of these genes is also regulated by Mad/Mnt proteins. MYC-family genes are usually expressed in immature, proliferating cells, while MAD-family genes are preferentially expressed in non-dividing, differentiated tissues [6], [7]. Switch from Myc:Max to Mad:Max predominance and thereby repression of common target promoters is considered to play an important role during differentiation and cell cycle exit [9], [10].

The transforming growth factor-β (TGF-β) family of cytokines maintains homeostasis in many organ systems by regulating a variety of processes including cell proliferation, apoptosis, differentiation, adhesion, production of extra-cellular matrix and cytokines during development and in the adult organism (for review see [11], [12], [13]). TGF-β signaling is initiated by ligand binding to TGF-β type II/I Ser/Thr kinase receptors, resulting in phosphorylation and activation of the receptor-regulated Smad transcription factors (R-Smads) Smad2 and Smad3. The R-Smads form active complexes with the Co-Smad, Smad4, and different cofactors, and translocate to the cell nucleus where they activate or repress transcription of TGF-β-responsive target genes [11], [12], [13]. TGF-β is a growth inhibitor for many cell types, and a number of cancers, including hematopoietic tumors, exhibit aberrations in components of the TGF-β signaling pathway [11], [12], [13]. For instance, expression of the Smad3 protein is frequently lost in T-acute lymphoblastic leukemia (ALL) [14], and the Smad3 cofactor AML-1 is often involved in translocations generating RUNX1–RUNX1T1 (AML–ETO) and RUNX1–EVI-1 (AML–EVI-1) fusion proteins in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), respectively. Generally, TGF-β carries out its anti-proliferative activities by both upregulating cyclin dependent kinase (CDK) inhibitors such as p15Ink4b, p21Cip1, p27Kip1 and p57Kip2 and downregulating the expression of growth stimulatory genes such as MYC and ID genes [11], [12]. Downregulation of MYC expression is an important part of the anti-proliferative response to TGF-β, since enforced Myc expression blocks TGF-β-induced cell cycle exit in mouse keratinocytes [15], and represses p15Ink4b expression and p15-mediated G₁ arrest in response to TGF-β in mink lung epithelial cells [16]. The repression of MYC expression by TGF-β occurs by direct interaction of a repressor complex consisting of Smad3, the transcription factors E2F4/5 and DP1 and the retinoblastoma family member p107 with a regulatory element in the MYC promoter [17]. Since deregulated MYC-family gene expression occurs frequently in many types of cancers, including in leukemia and lymphoma [4], this is another potential mechanism to interfere with TGF-β function.

In this report, we have utilized human U-937 myeloid tumor cells ectopically expressing a potent viral v-myc oncogene to investigate the relationship between Myc and TGF-β pathways in hematopoietic cells. v-Myc has previously been shown to efficiently override differentiation and G₁ cell cycle arrest induced by, for instance, the phorbol ester TPA, vitamin D₃ (VitD3) or retinoic acid (RA) in these cells [18], [19], [20]. Unexpectedly, our present data show that TGF-β1 forces v-Myc-transformed U-937 cells into cellular senescence correlating with potent induction of Mad1. Knockdown of MAD1 by RNA interference blocked induction of senescence by TGF-β1, suggesting that Mad1 is a critical component of TGF-β1 signaling in the reversal of transformation by Myc.