Mutant p53 Cooperates with ETS and Selectively Up-regulates Human MDR1 Not MRP1

Abstract

The most frequently expressed drug resistance genes, MDR1 and MRP1, occur in human tumors with mutant p53. However, it was unknown if mutant p53 transcriptionally regulated both MDR1 and MRP1. We demonstrated that mutant p53 did not activate either the MRP1 promoter or the endogenous gene. In contrast, mutant p53 strongly up-regulated the MDR1 promoter and expression of the endogenous MDR1 gene. Notably, cells that expressed either a transcriptionally inactive mutant p53 or the empty vector showed no endogenous MDR1 up-regulation. Transcriptional activation of the MDR1 promoter by mutant p53 required an Ets binding site, and mutant p53 and Ets-1 synergistically activated MDR1 transcription. Biochemical analysis revealed that Ets-1 interacted exclusively with mutant p53s in vivo but not with wild-type p53. These findings are the first to demonstrate the induction of endogenous MDR1 by mutant p53 and provide insight into the mechanism.The emergence of drug resistance poses a major obstacle to the success of cancer chemotherapy. Tumor cells acquire drug resistance via many routes including alterations in transport, drug targets, metabolism, and/or genes regulating cell survival. The most common alterations in drug transport are increased expression of MDR1 1 (the gene product is P-glycoprotein (1, 2)) and the multidrug resistance-associated protein (MRP1) (3, 4). Both are energy-dependent anticancer drug efflux pumps and play critical roles in the response to chemotherapeutic drugs (e.g. vinca alkaloids, taxanes, and epipodophyllotoxins). Further, both MDR1 and MRP1 are expressed in colon tumors that frequently express mutant (MT) forms of p53 (5, 6) and are intractable to chemotherapy. Notably, we have shown directly that MDR1 in colon tumors is normally repressed by wild-type (wt) p53 (7). In an analogous fashion Wang and Beck (9) as well as Sullivan et al. (8) have shown that wt p53 represses MRP1. Many clinical studies show that MT p53 expression is associated with increased MDR1 and/or MRP1 expression (5, 10, 11). These findings are fully consistent with a loss of p53 repression leading to MDR1 or MRP1 up-regulation. However, it is just as likely that these genes could be up-regulated by the "gain-offunction" activity of MT p53s (12-14).p53 deletion or mutation is one of the most frequent alterations in human malignancy and is clearly a critical step in the progression of colorectal cancer (15). Close to 90% of the p53 mutations in human tumors results in a disruption of the DNA binding domain. This not only disrupts transrepression and sequence-specific transactivation but also confers a gain-offunction activity that was first demonstrated for many MT p53s as acquiring the ability to induce tumors (13). This property was associated with the capability of these MT p53s to stimulate the expression of an alternate set of endogenous genes (13, 14, 16) that could potentially promote tumor progression and impair therapeutic response. However, although c-myc has unequivocally been demonstrated to be an endogenous t...

Transcription regulation by mutant p53

“…For example, transcriptional regulation of the MDR-1 promoter by wtp53 and by mutp53 is mediated by different promoter regions (Sampath et al, 2001) and similar observations were demonstrated for CD95 (Zalcenstein et al, 2003), suggesting that the assembly of functionally distinct complexes may be determined by different mechanisms. Either way, it seems that the functional consequence of mutp53 presence in the complex is tumor promotion while the presence of wtp53 results in tumor suppression.…”

“…Although wtp53 inhibits Sp1-dependent activation, presumably by interfering with DNA binding by Sp1 (Bargonetti et al, 1997), mutp53 proteins cooperate with Sp1 and amplify its activating effects on transcription. Similarly, the physical association between wtp53 and Ets-1 is inhibitory to Ets-1 activity (Pastorcic and Das, 2000) whereas the mutp53/Ets-1 interaction potentiates Ets-1-mediated transcription (Sampath et al, 2001). A similar picture emerges with regard to NF-Y.…”

“…One such example is Sp1 (Gualberto and Baldwin, 1995;Chicas et al, 2000); the interaction of mutp53 with Sp1 was shown to elicit cooperative effects and amplify the activating effects of Sp1 on transcription. Similarly, activation of the MDR-1 promoter by mutp53 was shown to require its association with Translation regulation by mutant p53 L Weisz et al transcription factors of the Ets family (Sampath et al, 2001). The notion that mutp53 is tethered to specific chromatin regions via interactions with sequence-specific transcription factors is reinforced by the work of Di- Agostino et al (2006) demonstrating a functionally significant interaction of mutp53 with NF-Y, an important transcription factor involved in cell-cycle progression, which binds specifically to CCAAT box elements in the DNA.…”

“…It appears most likely that functional interactions of the wtp53 N-terminal domain with the basal transcriptional machinery are also retained in mutp53 and are required for full transcriptional potency. Indeed, several of the proteins reported to interact with mutp53 and to affect its function are known to interact also with wtp53; these include Sp1 (Bargonetti et al, 1997;Pastorcic and Das, 2000;Kim and Deppert, 2003), Ets-1 (Sampath et al, 2001), and NF-Y (Di Agostino et al, 2006). Interestingly, the interactions of these proteins with wtp53 often result in opposite transcriptional outcomes than those observed with mutp53.…”

Interactions of mutant p53 with DNA: guilt by association

“…Therefore, the lack of sequence homology among putative mutp53-REs can be explained at least in part by the fact that the specificity of the association of mutp53 with DNA will be determined by the DNA binding specificity of other factors interacting with mutp53 proteins. Indeed, several binding partners of mutp53 proteins are sequence-specific DNA binding transcription factors recognizing distinct sequence motifs in DNA (Chicas et al, 2000;Sampath et al, 2001;Scian et al, 2004;Di Agostino et al, 2006). Notably, the association of mutp53 proteins with certain promoters seems to be predominantly determined by the DNA binding properties of the interacting partner (Chicas et al, 2000;Di Agostino et al, 2006).…”