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 over-expressed 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, immunoblot and immunohistochemistry techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, ß-galactosidase and Apo-BrdU techniques were used to assess proliferation, migration, clonogenicity, senescence and apoptosis in MPM cells following SP1 knockdown, p53 over-expression, or mithramycin treatment. Murine subcutaneous and intraperitoneal (IP) xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblot and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 over-expression, 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. IP 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 mesothelioma patients.

Genomics and Functional Genomics of Malignant Pleural Mesothelioma

“…Functional consequences of deregulations in ZEB1, ERβ, SP1, and WT1 zinc-finger transcription factors are well studied in breast cancer, lung cancer, pancreatic cancer, and prostate cancer [ 169 , 170 , 171 , 172 ]. Recently, the upregulation of these genes has been shown in MPM, when compared to matched normal counterparts [ 173 , 174 , 175 , 176 ]. Horio et al showed that siRNA-mediated knockdown of ZEB1 (zinc finger e-box binding homeobox 1) led to the suppression of proliferation, and anchorage-dependent and anchorage-independent clonal growth of MPM cells.…”

Mithramycin-loaded mPEG-PLGA nanoparticles exert potent antitumor efficacy against pancreatic carcinoma

“…The transcription regulation of MIT depends on a non-covalent interaction with GC-rich DNA regions, especially the site of union of Sp1 transcription factor. 11 MIT was shown to have antiangiogenic effect in …”

“…8,9 It was also shown that MIT inhibited DNA methyltransferase (DNMT), histone deacetylase (HDAC), and antiapoptotic protein XIAP. [10][11][12] Preclinical studies demonstrated that MIT can inhibit the generation and progression of several cancers, including carcinomas of the lung, breast, pancreas, stomach, and prostate, as well as sarcoma and glioblastoma. 8,[13][14][15][16] Meanwhile, its prominent in vitro and in vivo activities linked to specific transcription regulation have triggered the clinical trials of MIT in lung cancer, esophagus cancer, and Ewing sarcoma, sponsored by the National Cancer Institute (NCT01610570, NCT01624090, and NCT02859415, www.ClinicalTrials.gov).…”

Targeting the epigenome in malignant pleural mesothelioma

“…qRT-PCR, immunoblot and IHC experiments demonstrated markedly higher SP1 expression levels in MPM lines and primary MPMs relative to cultured normal mesothelia or normal pleura. Over-expression or knock-down of SP1 significantly increased or decreased EZH2 expression, respectively; furthermore, knock-down of SP1 diminished proliferation of MPM cells (102).…”