Transcriptional Regulation of p21/CIP1 Cell Cycle Inhibitor by PDEF Controls Cell Proliferation and Mammary Tumor Progression

Abstract

The Ets family of transcription factors control a myriad of cellular processes and contribute to the underlying genetic loss of cellular homeostasis resulting in cancer. PDEF (prostate-derived Ets factor) has been under investigation for its role in tumor development and progression. However, the role of PDEF in cancer development has been controversial. Some reports link PDEF to tumor promoter, and others show tumor-suppressing functions in various systems under different conditions. So far, there has been no conclusive evidence from in vivo experiments to prove the role of PDEF. We have used both in vitro and in vivo systems to provide a conclusive role of PDEF in the progression process. PDEF-expressing cells block the cell growth rate, and this retardation was reversible when PDEF expression was silenced with PDEF-specific small interfering RNA. When these PDEF-expressing cells were orthotopically implanted into the mouse mammary gland, tumor incidence and growth rate were significantly retarded. Cell cycle analysis revealed that PDEF expression partially blocked cell cycle progression at G 1 /S without an effect on apoptosis. PDEF overexpression resulted in an increase in p21/CIP1 at both the mRNA and protein levels, resulting in decreased Cdk2 activity. Promoter deletion analysis, electrophoresis mobility shift assays, and chromatin immunoprecipitation studies identified the functional Ets DNA binding site at ؊2118 bp of the p21/CIP1 gene promoter. This site is capable of binding and responding to PDEF. Furthermore, we silenced p21/CIP1 expression in PDEFoverexpressing cells by small interfering RNA. p21-silenced PDEF cells exhibited significantly increased cell growth in vitro and in vivo, demonstrating the p21 regulation by PDEF as a key player. These experiments identified PDEF as a new transcription factor that directly regulates p21/CIP1 expression under non-stressed conditions. This study conclusively proves that PDEF is a breast tumor suppressor for the first time using both in vitro and in vivo systems. PDEF can be further developed as a target for designing therapeutic intervention of breast cancer.

PDEF downregulates stathmin expression in prostate cancer

“…The data obtained from functional assays further confirm the findings and provide new insights into the role of PDEF in regulating the prostate cancer process associated with STMN. PDEF has been studied in our laboratory as a tumor suppressor (16) and with the use of iTRAQ labeling and mass spectrometric analysis techniques we were able to shed light on a possible role of PDEF in prostate cancer cells.…”

“…Our previous in vitro and in vivo data prove that PDEF is a tumor suppressor in breast cancer cells. We showed that PDEF-expressing cells block the cell growth rate, and this retardation was reversed when PDEF expression was silenced with PDEF-specific small interfering RNA (16). Upon STMN expression PDEF overexpressing cells show increased growth potential in an MTT proliferation assay in both PC3 (Fig.…”

“…Early reports showed that PDEF was a tumor promoter (13,14). However, recent studies support its role as a tumor suppressor based on differing model systems and varying experimental conditions (14,(15)(16)(17)(18).…”

Tumor-suppressive Maspin Functions as a Reactive Oxygen Species Scavenger

“…To determine the effect of overexpression of maspin and cysteine mutations on the tumorigenic properties of TM40D cells, we assessed the anchorage-independent growth and in vitro proliferation of WT and mutant maspin TM40D cells using soft agar colony formation and MTT assays, respectively. Anchorage-independent growth is one of the hallmarks of transformation, and the soft agar assay is a commonly used in vitro assay for detecting malignant transformation of cells (28,37). The abilities of the cells to form colonies on soft agar are shown in Fig.…”

JAZ (Znf346), a SIRT1-interacting Protein, Protects Neurons by Stimulating p21 (WAF/CIP1) Protein Expression

“…The cells were lysed 36 h after transfection, and assays were performed (19,20) using the Promega Dual-Luciferase reporter assay system (catalog no. E1910) according to the manufacturer's instructions.…”