Inhibition of Transforming Growth Factor b Signaling in MCF-7 Cells Results in Resistance to Tumor Necrosis Factor a : A Role for Bcl-21

Transforming growth factor b (TGF-b) is a multifunctional cytokine capable of regulating diverse cellular processes. In this study we investigated the effect of autocrine TGF-b signaling on tumor necrosis factor (TNF) a-induced cell death. We abrogated the TGF-b autocrine loop by overexpression of a truncated TGF-b type II receptor in MCF-7 breast carcinoma cells and found that this generated resistance to TNF-ainduced cytotoxicity. To elucidate the molecular basis of the influence of TGF-b on TNF-a-induced cytotoxicity, we evaluated the expression levels or activities of proteins involved in TNF-a signal transduction or the regulation of apoptosis in general in TGF-b-responsive and TGF-b-nonresponsive MCF-7 cells. We observed no significant difference in the expression of TNF-a receptors or the TNF receptorassociated death domain protein. In addition, downstream activation of nuclear factor kB by TNF-a was not altered in cells that had lost TGF-b responsiveness. Analysis of members of the Bcl-2 family of apoptosis-regulatory proteins revealed that Bcl-XL and Bax expression levels were not changed by disruption of TGF-b signaling. In contrast, the TGF-bnonresponsive cells expressed much higher levels of Bcl-2 protein and mRNA than did cells with an intact TGF-b autocrine loop. Furthermore, restoration of a TGF-b signal to MCF-7 cells that had spontaneously acquired resistance to TGF-b caused a reduction in Bcl-2 protein expression. Taken together, our data indicate that loss of autocrine TGF-b signaling results in enhanced resistance to TNF-a-mediated cell death and that this is likely to be mediated by derepression of Bcl-2 expression. Introduction TGF-b is a multifunctional peptide growth factor that plays a pivotal role in many physiological and pathological processes. The principle effects of TGF-b include regulation of cell proliferation, cell migration, cellular differentiation, immune cell behavior, and the synthesis and degradation of the extracellular matrix (see Refs. 1 and 2 for a general review of TGF-b). TGF-b elicits its effects by binding and activating specific receptors expressed on the surface of cells. The TbR-I and TbR-II are transmembrane serine/threonine kinases directly involved in signal propagation. Because both ligand and receptors are expressed in many different tissues and cell types, TGF-b contributes to a wide range of biological processes via autocrine and paracrine pathways. The full measure of the potential complexity of the effects of TGF-b can come into play during the initiation and progression of malignancy. Autocrine effects of TGF-b have been implicated in some models of carcinogenesis and local invasion, whereas paracrine actions of TGF-b may facilitate metastatic spread and tumor cell escape from immune surveillance (3, 4). Indeed, analysis of the production of TGF-b by tumor cells, as well as that of the functional status of the TGF-b signal transduction pathway in tumors, has generated important insights into tumor cell biology. Among the least understood aspects of TGF-b biology is its role in regulating apoptosis and cell death. Many groups have documented an increase in TGF-b mRNA and/or protein as an early event in the apoptotic cascade. Examples of increased TGF-b production by cells undergoing apoptosis include removal of hormone from hormonally dependent epithelia (5–7), antimetabolite-induced apoptosis of hormone-independent tumor cells (8), and treatment of human mammary carcinomas with pharmacological doses of antiestrogens (9, 10). What remains unclear, however, is whether the observed rise in TGF-b is an integral component of the apoptotic cascade or whether it is simply a consequence of the cytotoxic insult. An investigation by Danforth and Sgagias (11) attempted to elucidate the role, if any, of TGF-b as a determinant of cell death induced by TNF-a. TNF-a is an inflammatory cytokine capable of binding and activating cell surface receptors (TNF-RI and TNF-RII). TNF-RI is a member of a family of death-inducing receptors that includes Fas/CD95 and death receptor 3. Binding of TNF-a to TNF-RI is responsible for the generation of many of the known cellular responses of Received 8/15/00; revised 12/19/00; accepted 12/20/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants DAMD17-J-4287 and DAMD17-J-4130 from the Department of Defense Breast Cancer Research Program. 2 Supported by National Cancer Institute Training Grant CA09658. 3 To whom requests for reprints should be addressed, at Dartmouth Medical School, Kellogg Box 0128, Hanover, NH 03755. Phone: (603) 650-1550; Fax: (603) 650-1129; E-mail: Bradley.Arrick@dartmouth.edu. 4 The abbreviations used are: TGF-b, transforming growth factor b; TbR-I, TGF-b type I receptor; TbR-II, TGF-b type II receptor; TNF, tumor necrosis factor; TNF-R, tumor necrosis factor a receptor; NF-kB, nuclear factor kB; TBS, Tris-buffered saline; TRADD, TNF receptor-associated death domain protein; TBST, TBS and 0.05% Tween 20; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt. 109 Vol. 12, 109–117, February 2001 Cell Growth & Differentiation