PML represses lung cancer metastasis by suppressing the nuclear EGFR-mediated transcriptional activation of MMP2

Abstract

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-β (IFNβ) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNβ induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.

Keywords: ATRS; ATRS, AT-rich sequence; EGFR, EGF receptor; IFNβ, interferon-β; INM, inner nuclear membrane; Interferon-β; LAC, lung adenocarcinoma; MMP2; MMP2, matrix metalloprotease-2; PML; PML, promyelocytic leukemia protein; STAT3; lung adenocarcinoma; nEGFR, nuclear EGFR; nuclear EGFR.

Figure 1.

PML suppressed lung cancer cell invasion when EGFR was activated. (A) Left, Luciferase-expressing A549-shControl or shPML cells were injected via the tail vein into immunocompromised NOD/SCID mice (n = 5 for each group). The mice were monitored for colonization of the injected cells in the lungs as described in the Materials and Methods. Representative images from week 6 post-injection are shown. Right, The mean total photon flux measured by IVIS at week 6 is shown. The data are presented as the mean ± SEM. (B) The cumulative survival rate of the same mice described in Figure 1A was monitored weekly until 13^th week after cell injection. (C) Left, H1975-shControl and H1975-shPML cell invasion was assayed as described in the Materials and Methods. Right, A549-shControl and A549-shPML cells were cultured overnight in low-serum starvation conditions (0.5% FBS). These cells were then subjected to the cell invasion assay in the low serum-medium with 50 ng/ml EGF. (D) A549-Control and A549-PML cells were cultured overnight in low-serum starvation conditions (0.5% FBS). These cells were then subjected to the cell invasion assay in the low serum-medium with 50 ng/ml EGF. (E) CL1-5-Control and CL1-5-PML cell invasion was assayed as described in (D).

Figure 2.

PML repressed nEGFR-induced MMP2 expression. (A) RT-qPCR was performed to determine the relative mRNA expression of PML, MMP2 and MMP9 in H1975-shControl, H1975-shPML and H1975-shPML-2 cells as described in the Materials and Methods. (B) H1975 shPML cells with or without overexpression of shRNA-resistant PML (rPML) were analyzed by RT-qPCR to determine the relative mRNA expression of PML and MMP2. (C) 293T cells transfected with pMMP2 Luc and 100 ng of pCMV-EGFR NLS or pCMV-EGFR were analyzed using the luciferase reporter gene assay as described in the Materials and Methods. (D) A549 cells were transfected with pMMP2 Luc and increasing amounts of the pHA-PML expression plasmid. Twenty-four hours after transfection, the cells were treated with or without 50 ng/ml EGF for 18 h and then subjected to a luciferase reporter gene assay. (E) 293T cells were transfected with pMMP2 Luc, 100 ng of pCMV-EGFR NLS or empty vector and increasing amounts of PML expression plasmid as indicated. The cells were subjected to a luciferase reporter gene assay 48 h after transfection. (F) 293T cells were transfected in 6-well plate with 1 μg of pCMV-EGFR NLS or empty vector, and increasing amounts of pHA-PML as indicated. Forty-eight hours after transfection, the cells were analyzed by RT-qPCR to detect MMP2 mRNA expression.

Figure 3.

Identification of an ATRS in the MMP2 promoter. (A) Left, A list of the MMP2 promoter luciferase reporter constructs. The promoter region is indicated relative to the transcription start site. The arrows indicate the 2 primer pairs used for PCR amplification in the ChIP assay. The putative ATRS in the middle region of the MMP2 promoter is indicated. Right, 293T cells transfected with the 3 pMMP2 Luc constructs with or without pCMV-NLS-EGFR, were subjected to a luciferase reporter gene assay. (B) H1975 cells were analyzed by ChIP using preimmune IgG or anti-EGFR antibody as described in the Materials and Methods. The relative amount of immunoprecipitated DNA from the middle or distal regions of the MMP2 promoter was quantified by RT-qPCR with specific primers. (C) Upper, The sequences of the wild-type and mutant pMMP2 Luc near the ATRS are provided. The wild-type and mutated ATRS sequences are underlined with the mutated nucleotides in boldface. Lower, 293T cells transfected with wild-type or mutant ATRS pMMP2 Luc with or without pCMV-NLS-EGFR were subjected to a luciferase reporter gene assay. (D) 293T cells transfected with pMMP2 Luc with a wild-type or a mutant ATRS and pCMV-NLS-EGFR were subjected to ChIP with an anti-EGFR antibody. The relative level of immunoprecipitated wild-type or mutant MMP2 ATRS were Figure 3 (See previous page). quantified by RT-qPCR with specific primers. (E) 293T cells transfected with pMMP2 Luc with a wild-type or a mutated ATRS and increasing amounts of pCMV-NLS-EGFR were subjected to ChIP with an anti-H3K9Ac antibody. The relative amount of ATRS derived from the immunoprecipitated MMP2 promoter was quantified by RT-qPCR with specific primers.

Figure 4.

nEGFR and STAT3 cooperatively activated the MMP2 promoter. (A) The sequence near the ATRS in MMP2 promoter is illustrated. The ATRS is underlined. The wild-type and mutant STAT3 binding sites are specified in the open box. The mutated nucleotides in the STAT3 binding motif are capitalized. (B) shControl, shSTAT3-1 and shSTAT3-2 H1975 cells were subjected to RT-qPCR to analyze the MMP2 and STAT3 mRNA expression. (C) 293T cells transfected with pMMP2 Luc, pCMV-NLS-EGFR and the STAT3 shRNA-expression plasmid were subjected to a luciferase reporter gene assay. (D) 293T cells transfected with wild-type or mutant pMMP2 Luc and pCMV-NLS-EGFR were subjected to a luciferase reporter gene assay. (E) 293T cells transfected with pMMP2 Luc, pCMV-NLS-EGFR and/or a STAT3-expressing plasmid were subjected to a luciferase reporter gene assay. (F) Left, H1975 cells were subjected to ChIP with preimmune IgG or an anti-EGFR or anti-STAT3 antibody. Right, H1975 cells were subjected to ChIP with sequential immunoprecipitation with preimmune IgG or an anti-EGFR or anti-STAT3 antibody. The relative amount of the immunoprecipitated MMP2 ATRS DNA fragment was quantified by RT-qPCR with specific primers.

Figure 5.

PML repressed the coactivation of the MMP2 promoter by nEGFR and STAT3. (A) 293T cells transfected with pMMP2 Luc, pCMV-NLS-EGFR, the STAT3 expression vector and increasing amounts of the PML expression plasmid were subjected to a luciferase reporter assay. (B) PML-knockdown H1975 cells with or without shRNA-resistant PML expression were subjected to ChIP with preimmune IgG or an anti-EGFR or anti-STAT3 antibody. The relative amount of the immunoprecipitated MMP2 ATRS was quantified by RT-qPCR. (C and D) PML-knockdown H1975 cells with or without shRNA-resistant PML expression were subjected to ChIP with preimmune IgG or an anti-PML or anti-H3K9Ac antibody. The relative amount of the immunoprecipitated MMP2 ATRS was quantified by RT-qPCR. (E) Left, H1975 cells infected with control, shEGFR or shSTAT3-2 lentiviral vectors were subjected to Western blotting with indicated antibodies 2 d after infection. Right, The same stable cell lines as in the left panel were subjected to ChIP with preimmune IgG or an anti-PML antibody. The amount of immunoprecipitated MMP2 ATRS was quantified by RT-qPCR.

Figure 6.

PML upregulation was involved in the IFNβ-mediated suppression of lung cancer cell invasion. (A) After overnight serum starvation, A549 cells were treated with EGF and IFNβ for the indicated periods of time. The cells were then subjected to RT-qPCR and Western blotting for PML mRNA and protein expression, respectively. (B) After overnight serum starvation, H1975 cells were treated with IFNβ for the indicated periods of time. The cells were analyzed as in (A). (C) After overnight serum starvation, A549 cells were treated with EGF and IFNβ for the indicated periods of time. The relative MMP2 mRNA expression was quantified by RT-qPCR with specific primers. (D) Control and PML-knockdown A549 cells were treated with EGF or IFNβ for 3 d and then analyzed by RT-qPCR to determine the relative MMP2 mRNA expression. (E) A549 cells treated with EGF and/or IFNβ for 24 h were subjected to a cell invasion assay as described in the Materials and Methods.