Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells

Abstract

Purpose: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms.

Experimental design: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, β-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters.

Results: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression.

Conclusions: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma.

Figure 1

SP1 is overexpressed in malignant pleural mesothelioma cells. (*, P < 0.05; **, P < 0.01; ***, P < 0.001.) A). qRT-PCR (left, upper panel) and immunoblot (left, lower panel) showing that SP1 mRNA and protein expression levels are higher in cultured MPM cells relative to normal mesothelia; qRT-PCR (middle, upper panel) showing that SP1 mRNA is overexpressed in primary MPM compared to normal pleura; TMA analysis (middle, lower panel) of SP1 expression in normal mesothelial tissues (NMT, n=22) and malignant mesothelial tissues (MMT, n=59). SP1 staining was scored from 0-100 based on percentage of positive cells. The majority of the MMT had significantly increased SP1 staining compared to NMT. Correlation of increased intra-tumoral SP1 expression with overall survival in 39 patients with locally advanced MPM undergoing potentially curative resections (right panel). B). Confirmation of stable knockdown of SP1 by qRT-PCR and immunoblot analysis in MES1 and MES7 cells (left and right panels, respectively). C) and D). Time-dependent inhibition of cell proliferation, migration (after 72 hours) and clonogenicity (after 21 days) of SP1 knockdown in MES1 and MES7 cells, respectively. Both shRNA sequences significantly inhibit soft agar clonogenicity of MPM cells.

Figure 2

Effects of MM in vitro and in vivo in MPM cells. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). A). Effects of MM on proliferation and soft agar clonogenicity of MES1 and MES7 cells. B). Effects of IP MM on volume and mass of established subcutaneous MES1 xenografts (left and middle panels, respectively), and body weight in tumor bearing mice (right panel). C). Effects of IP administration of MM (1mg/kg IP) in nude mice with MES1 induced carcinomatosis. Numbers of mice with residual tumors following MM treatment vs saline injections (left panel). Representative results depicting carcinomatosis in control mice compared to no residual tumor or minimal residual tumors in mice following IP MM (right panel). D). Heatmap (left panel) and ingenuity pathway analysis (right panel) of genes modulated in MES1 and MES7 xenografts in mice receiving MM (1mg kg).

Figure 3

Validation of microarray data. (*, P < 0.05; **, P < 0.01; ***, P < 0.001.) A). qRT-PCR showing the effects of MM on ROR1, HDAC4, p53, p21, PMAIP1 and PRDM1 in MES1 and MES7 cells harvested immediately after 24 hour exposure, as well as corresponding xenografts harvested 3 days following the last IP saline or MM injection. B) Immunoblot analysis (left panel) showing the dose-dependent effects of MM for 24 hours on SP1, ROR1, HDAC4, EZH2, p53, p21, PMAIP1, PRDM1 protein levels in cultured MPM cells; representative immunoblot analysis (right panel) demonstrating the effects of MM on SP1, ROR1, HDAC4, EZH2, p53, p21, PMAIP1, PRDM1 protein levels in subcutaneous MPM xenografts from MM-treated and control mice. Three tumors (T1, T2 and T3) were selected from saline control vs MM 1mg/kg. Tumors were harvested 3 days following the last IP saline or MM injection. C). Quantitative ChIP analysis of various gene promoters in MES1 cells cultured in normal media with or without MM (100nM, 24 hours).

Figure 4

Effects of SP1 knockdown with or without overexpression of p53 in MPM cells. (*, P < 0.05; **, P < 0.01; ***, P < 0.001.) A). Left panel: SP1 depletion and p53 over-expression significantly inhibits in-vitro proliferation (left panel), and decreases volume and mass of MES7 xenografts (middle and right panels, respectively). B). Venn diagram (left panel), and corresponding heatmap (middle panel) corresponding to genes regulated by MM and overexpression of p53 in SP1-depleted MES1 and MES7 cells. Ingenuity pathway analysis of genes commonly regulated by in MES1 and/or MES7 cells by MM and SP1KD/p53OEX (right panel). C). qRT-PCR and immunoblot analysis (left and right panels, respectively) demonstrating the effects of SP1 knockdown or overexpression of p53 on genes targeted by MM in cultured MES1 and MES7 cells.

Figure 5

Effects of MM on cell cycle progression, senescence and apoptosis in MPM cells. (*, P < 0.05; **, P < 0.01; ***, P < 0.001.) A). Propidium iodide staining demonstrating that 24 hour MM treatment induces dose dependent G0/G1 arrest in MPM cells. B). ß-galactosidase staining assays demonstrating that 24 hour MM treatment induces dose dependent senescence in MES1 and MES7 cells. C) and D). Apo-BrdU analysis demonstrating minimal apoptosis in MPM cells immediately following 24 hour MM exposure (C), but significant dose-dependent apoptosis 48 hours following drug treatment (D).