Inducible overexpression of c-Jun in MCF7 cells causes resistance to vinblastine via inhibition of drug-induced apoptosis and senescence at a step subsequent to mitotic arrest

Abstract

c-Jun is a major component of the AP-1 transcription factor and plays a key role in regulation of diverse biological processes including proliferation and apoptosis. Treatment of a wide variety of cells with the microtubule inhibitor vinblastine leads to a robust increase in c-Jun expression, JNK-mediated c-Jun phosphorylation, and activation of AP-1-dependent transcription. However, the role of c-Jun induction in the response of cells to vinblastine remains obscure. In this study we used MCF7 breast cancer cell lines that express the dominant-negative form of c-Jun, TAM-67, as well as cells that overexpress c-Jun, under the control of an inducible promoter. Vinblastine induced c-Jun protein expression, c-Jun phosphorylation, and AP-1 activation in MCF7 cells, and these parameters were strongly inhibited by inducible TAM-67 expression and strongly enhanced by inducible c-Jun expression. Vinblastine-induced cell death was not affected by TAM-67 expression whereas cells were protected by c-Jun overexpression. Further investigation revealed that apoptotic and senescent cells were observed after vinblastine treatment and that both outcomes were strongly inhibited by c-Jun overexpression. Although c-Jun expression inhibited cell death, it did not affect the ability of vinblastine to induce mitotic arrest. These results indicate that c-Jun expression plays a protective role in the cellular response to vinblastine and operates post-mitotic block to inhibit drug-induced apoptosis and senescence.

Introduction

Microtubule inhibitors, especially vinca alkaloids and paclitaxel and its derivatives, are widely used in cancer chemotherapy. The primary mechanism of action of these drugs is to bind to tubulin or microtubules and disrupt mitotic spindle dynamics leading to M-phase arrest [1]. In general, mitotic arrest leads to cell death by apoptosis. However, if apoptosis is suppressed or mitotic checkpoint signaling perturbed, other outcomes, such as aneuploidy, mitotic catastrophe, or senescence, can result [2], [3], [4], [5]. There is a great deal of interest in unraveling the molecular links between mitotic arrest and the eventual outcome, whether it is the immediate or eventual death of the cell, or the survival of an impaired cell. Information in this area is critical as it contributes to our knowledge of basic cellular processes and is essential for a better understanding of the mechanism of action of drugs that interfere with microtubule dynamics.

Our previous studies in KB-3 cells have shown that vinblastine activates the c-Jun NH₂-terminal protein kinase (JNK) signal transduction pathway, leading to phosphorylation and activation of c-Jun, and in turn activation of AP-1-dependent transcription [6], [7], [8]. Through an auto-amplification loop involving AP-1 sites in the c-Jun promoter, these events result in a robust increase in c-Jun protein expression. Indeed, we have observed that vinblastine causes highly increased c-Jun expression in all cell lines examined to date, suggesting it is a common and perhaps universal response to microtubule inhibition.¹ While it is established that c-Jun plays diverse roles in numerous cellular functions, including cell proliferation, differentiation, and death, its precise role in cellular responses to microtubule inhibitors is controversial. Theoretically, increased c-Jun expression could represent a protective mechanism in aid of cell survival; a destructive mechanism actively involved in the cell death process; or may simply be a bystander event with no direct role in the response to mitotic block.

Stable expression of a c-Jun dominant-negative, TAM-67, in KB-3 cells caused a modest resistance to vinblastine and a delay in caspase activation induced by the drug [6]. However, these properties were context-dependent, in that the TAM-67-expressing cells were resistant only when cells were exposed to drug for short intervals, and relative to control cells were not resistant upon continuous drug treatment.² More recent studies indicated that the kinetics and extent of apoptosis are similar for both wild-type and c-Jun knockout fibroblasts [9]. In contrast, fibroblasts expressing high levels of human c-Jun were found to be highly resistant to vinblastine [9]. However, cell systems stably expressing dominant-negative forms of a multi-functional transcription factor or with specific genetic lesions do not necessarily represent the best models because of possible cellular adaptation and compensatory mechanisms.

In order to avoid these potential drawbacks we sought to determine the effect of c-Jun inhibition or c-Jun overexpression on vinblastine sensitivity in a more controlled and defined system. For this purpose we utilized MCF7 cells that have been developed to express either the c-Jun dominant-negative TAM-67 or human c-Jun under the control of an inducible promoter [10]. Using the Tet-off system, TAM-67 or c-Jun can be expressed in a manner that is dependent upon the removal of doxycycline from the medium. In this study, we exploited this powerful system to investigate the role of c-Jun and its inhibition on the sensitivity of breast cancer cells to vinblastine. We show that MCF7 cells are highly similar to KB-3 cells in that they exhibit a robust increase in c-Jun expression, c-Jun phosphorylation, and AP-1 activation in response to vinblastine treatment. While inducible TAM-67 expression blocks these events as predicted, there is no change in the sensitivity of the cells to vinblastine. In contrast, inducible overexpression of c-Jun renders the cells markedly resistant to vinblastine, through a mechanism that operates post-mitotic block to prevent drug-induced apoptosis and senescence.