Regulation of rDNA Transcription by Proto-Oncogene PELP1

Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive.Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA.Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions

A Phase I Trial of the MET/ALK/ROS1 Inhibitor Crizotinib Combined with the VEGF Inhibitor Pazopanib in Patients with Advanced Solid Malignancies

Background: Crizotinib inhibits ALK, MET and ROS1 tyrosine kinases but the development of resistance to monotherapy is an issue. The anti-angiogenic properties of pazopanib could overcome crizotinib drug resistance. Additionally, the anti-angiogenic properties of crizotinib could augment the clinical efficacy of pazopanib. Methods: We evaluated the safety and responses in patients with advanced solid tumors treated with crizotinib and pazopanib. Results: Eighty-two patients (median age 53 years, range 18-78 years) were enrolled. The median number of prior systemic therapies was 3 (range, 0-8). We were able to dose escalate to dose level 8 (crizotinib 250 mg twice daily and pazopanib 800 mg daily) with no MTD identified. Grade 3 or 4 toxicities were seen in 32% of patients with the highest prevalence being fatigue (n=9, 11%), diarrhea (n=6, 7%), vomiting (n=3, 4%), anemia (n=2, 2%) and ALT increased (n=2, 2%). Of the 82 patients, 61 (74%) had measurable disease by RECISTv1.1 and reached first restaging (6 weeks). Partial response (PR) was observed in 6/61 (10%) patients, and stable disease (SD) lasting ≥6 months was observed in 10/61 patients (16%) (total = 16/61 (26%) of patients with SD ≥6 months/PR). Conclusion: Dose level 6 (crizotinib 200 mg twice daily and pazopanib 600 mg daily) was the most tolerable dosing of the combination and can be used in future studies. We also observed moderate clinical activity in patients with advanced solid tumors that had received numerous prior therapies

PELP1 nucleolar localization depends on active rDNA transcription.

<p>HeLa cells were treated with or without actinomycin D (Pol I transcription inhibitor, 4 µg/ml for 12 h) [<b>A</b>] and with or without α-amanitin (Pol II transcription inhibitor 80 µg/ml for 12 h) [<b>B</b>]. The localization of green (PELP1), red (nucleolin) and yellow (colocalization of PELP1 with nucleolin) was analyzed by using confocal microscopy. (<b>C</b>) HeLa or Cos1 cells were transfected with pHrD Luciferase reporter vector along with control or PELP1-expressing vectors. After 6 h, cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. *<i>p-value </i><0.05.</p

PELP1 localizes to the nucleolus.

<p>(<b>A</b>) MCF7 and ZR-75 cells were serum starved for 2 days and stimulated with 10% serum for 12 h. PELP1 (green) localization was visualized by using confocal microscopy. (B) HeLa cells were grown in 10% serum and the localization of PELP1 (green), the nucleoli marker nucleolin (red) and colocalization (yellow) was analyzed by using confocal microscopy. (<b>C</b>) PELP1 localization was analyzed by using a control peptide or PELP1 epitope-specific peptide adsorbed antibody. (<b>D</b>) HeLa cells were transfected with either control or PELP1-specific siRNA and the localization of PELP1 (green) was analyzed by using confocal microscopy. (<b>E</b>) HeLa cells were fractionated into cytoplasm, nucleoplasm and nucleoli, and the presence of PELP1 in these fractions was analyzed by immunoblotting. Paxillin, lamin and nucleolin were used as cytoplamic, nuclear and nucleoli markers respectively.</p

PELP1 associates with the rDNA promoter and regulates rDNA transcription.

<p>(<b>A</b>) Schematic representation of the rDNA gene and primer pairs used in the ChIP assay. (<b>B</b>) ChIP assay was done using antibodies specific for PELP1 or isotype rabbit IgG control in ZR-75 cells. DNA recovered from ChIP or input controls was subjected to conventional PCR using the indicated primers spanning the promoter and coding regions. (<b>C</b>) Total RNA was isolated from ZR-75 overexpressing PELP1 and HeLa, ZR-75 cells expressing control or PELP1 siRNA analyzed for the status of pre-rRNA transcription by RT-PCR.</p

CDKs regulate PELP1 nucleolar localization.

<p>(<b>A</b>) ZR-75 cells at sub confluence were fixed and the localization of ^phos991PELP1was analyzed by using confocal microscopy. (<b>B</b>) Localization of GST-tagged PELP1 or PELP1 S991AMT or PELP1 S991EMT were visualized by using confocal microscopy with the antibody against the GST epitope in ZR-75 cells. (<b>C</b>) PELP1 was visualized in ZR-75 cells after serum starved for two days and released with 10% serum in absence or presence of roscovitine (10 µM) by using confocal microscopy. (<b>D</b>) 293T cells were transfected with pHrD luciferase reporter vector along with PELP1 WT or PELP1 S991AMT or PELP1 S991EMT expressing vectors. Cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. *<i>p-value </i><0.05.</p

PELP1 nucleolar localization depends on cell cycle progression.

<p>(<b>A</b>) HeLa cells were arrested in G1-S boundary by a double thymidine block and cells were released to progress into the S and G2 phases of the cell cycle. PELP1 localization was visualized by using confocal microscopy. (<b>B</b>) ZR-75 cells were synchronized to G2/M phase using nocodazole (15 µM) for 18 h and released to progress into the cell cycle by addition of 10% serum. PELP1 localization was monitored with confocal microscopy by colocalizing with nucleolin. (<b>C</b>) ZR-75 cells were synchronized to G1 phase by serum starvation and released to progress into the cell cycle by addition of 10% serum. PELP1 localization was monitored by using confocal microscopy in different time intervals by colocalizing with nucleolin.</p