DLX5 (distal-less homeobox 5) promotes tumor cell proliferation by transcriptionally regulating MYC

Abstract

The human DLX homeobox genes, which are related to Dll (Drosophila distal-less gene), encode transcription factors that are expressed primarily in embryonic development. Recently, DLX5 was reported to act as an oncogene in lymphomas and lung cancers, although the mechanism is not known. The identification of target genes of DLX5 can facilitate our understanding of oncogenic mechanisms driven by overexpression of DLX5. The MYC oncogene is aberrantly expressed in many human cancers and regulates transcription of numerous target genes involved in tumorigenesis. Here we demonstrate by luciferase assay that the MYC promoter is specifically activated by overexpression of DLX5 and that two DLX5 binding sites in the MYC promoter are important for transcriptional activation of MYC. We also show that DLX5 binds to the MYC promoter both in vitro and in vivo and that transfection of a DLX5 expression plasmid promotes the expression of MYC in a dose-dependent manner in mammalian cells. Furthermore, overexpression of DLX5 results in increased cell proliferation by up-regulating MYC. Knockdown of DLX5 in lung cancer cells overexpressing DLX5 resulted in decreased expression of MYC and reduced cell proliferation, which was rescued by overexpression of MYC. Because DLX5 has a restricted pattern of expression in adult tissues, it may serve as a potential therapeutic target for the treatment of cancers that overexpress DLX5.

FIGURE 1.

Expression of Dlx5 and Myc in inv(6)-positive T-cell lymphomas from Lck-Myr-Akt2 transgenic mice. Real time RT-PCR analysis was performed with total RNA samples extracted from wild-type T cells (WT) or T-cell lymphomas from 10 different mice represented by numbers 1–10. Values were normalized against expression of Actin.

FIGURE 2.

DLX5 regulates MYC transcription in vitro. A, annotation of the MYC promoter. SNM and XNM, sequences inserted in SNM-Luc and XNM-Luc reporter vectors, respectively. The MYC promoter contains four putative DLX5 binding sites with the core motif of TAAT. P1 and P2 sites are major transcription start sites. B and C, luciferase activity in cells transfected with XNM-Luc or SNM-Luc, respectively, normalized by Renilla luciferase. pcDNA3.1 (Vec) in samples 1 and 3 or pcDNA3.1-DLX5 (DLX5) in samples 2 and 4 was co-transfected with luciferase reporter constructs and Renilla luciferase plasmid. Statistical analysis was performed by a two-tailed t test. D, MYC promoter reporter constructs bearing four different DLX5 binding sites were subjected to site-directed mutagenesis to substitute putative DLX5 binding sequences with the designated sequences (underlined). E, promoter activity measured using XNM-Luc or XNM-M4-Luc (Xmut-Luc), which contains a mutation in the D4 binding site of DLX5 in HeLa cells. Luciferase activity was normalized by Renilla luciferase. F, promoter activity of four different SNM-Luc mutant vectors (M1-Luc, M2-Luc, M3-Luc, and M4-Luc) containing different mutations in putative DLX5 binding sites in SNM-Luc in HeLa cells. Luciferase activity was normalized by Renilla luciferase.

FIGURE 3.

DLX5 binds to MYC promoter in vitro and in vivo. A, gel shift assays were performed using recombinant human DLX5 protein and radiolabeled oligonucleotides. DLX5 binding could be competed by excess molar concentrations (25-, 50-, or 250-fold excess) of unlabeled probes, as indicated by the triangles. Supershift was observed by adding a DLX5-specific antibody (α-DLX5) into each reaction. The arrow indicates oligonucleotides binding to DLX5. B, ChIP assay carried out using ChIP-grade antibody against DLX5 (or normal goat IgG as a negative control) with HeLa cells transiently transfected with DLX5 or NCI-H322M cells. PCR products represent a portion of the MYC promoter (MYC). The upstream control (Upstream Con) represents a portion of the upstream MYC promoter containing nucleotides −2305 to −2015. The second control (Downstream Con) represents a downstream sequence containing nucleotides 1801–2062. Nucleotide numbers are relative to the P2 transcription start site (+1) in the MYC promoter. CON, positive control using total genomic DNA as template; DLX5, PCR products amplified from the ChIP samples mediated by anti-DLX5 antibody; IgG, absence of PCR product corresponding to MYC promoter in sample mediated by IgG.

FIGURE 4.

DLX5 promotes Jurkat cell proliferation by regulating the expression of MYC. A, expression of MYC protein in Jurkat cells transfected by vector (1) or DLX5 (2). The histogram in A represents the density of the MYC bands in immunoblot. B, real time RT-PCR analysis of MYC mRNA levels in Jurkat cells transfected by vector and DLX5. C, expression of MYC in Jurkat cells transfected with increasing amounts of DLX5 plasmid. D, real time RT-PCR analysis of MYC mRNA level in Jurkat cells transfected with increasing amounts of DLX5 plasmid. E, cell numbers counted at 0, 24, and 48 h in Jurkat cells transfected with vector (Vec), DLX5, DLX5 + non-targeting siRNA (siCon), DLX5 + siRNA1 against MYC (siMYC1), DLX5 + siRNA2 against MYC (siMYC2), and MYC. F, expression of MYC, DLX5, and β-ACTIN in Jurkat cells transfected with Vec (1), DLX5 (2), DLX5 + non-targeting siRNA (3); DLX5 + siRNA1 against MYC (4), DLX5 + siRNA2 against MYC (5), and MYC (6).

FIGURE 5.

DLX5 promotes proliferation of lung cancer cells by regulating the expression of MYC. A, real time RT-PCR analyses of -fold change of DLX5 and MYC mRNA levels in NCI-H322M, NCI-H520, or NCI-H23 lung cancer cells transfected with siRNA against DLX5 (siDLX5; Invitrogen Oligo ID, HSS102810) or non-targeting siRNA (siCON) for 16 h or 24 h. The DLX5 and MYC mRNA levels were normalized against the respective DLX5 and MYC mRNA levels observed in cells transfected with control non-targeting siRNA. B, the same number of cells were transfected with buffer (Normal), non-targeting siRNA (siCON), siRNA1 against DLX5 (siDLX5-1; Invitrogen Oligo ID, HSS102808), or siRNA2 against DLX5 (siDLX5-2; Invitrogen oligo ID, HSS102810) and counted 48 h after transfection. C, cell numbers counted at 48 h in NCI-H322M cells transfected with buffer (NCI-H322M), non-targeting siRNA (siCONTROL), siRNA1 against DLX5 (siDLX5-1), siRNA2 against DLX5 (siDLX5-2), siRNA1 against DLX5 + pcDNA3-MYC (siDLX5-1 + MYC), or siRNA2 against DLX5 + pcDNA3-MYC (siDLX5-2 + MYC). D, expression of DLX5, CASPASE 3, and β-ACTIN in NCI-H322M cells transfected with buffer (Normal), non-targeting siRNA (siCON), siRNA1 against DLX5 (siDLX5-1), or siRNA2 against DLX5 (siDLX5-2). The arrow points to the bands representing the cleaved form of active CASPASE 3.