Jacob A. Eitel

TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer

The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%-80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-beta1. Whether TGF-beta1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-beta1-activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-beta1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-beta1-mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-beta1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.

PTEN and p53 are required for hypoxia induced expression of maspin in glioblastoma cells.

In response to genotoxic stress, p53 induces the tumor suppressors maspin and PTEN. Here we demonstrate that in response to limited oxygen conditions PTEN and p53 work in tandem to induce maspin in glioblastoma cells. In response to hypoxia a portion of PTEN migrates to the nucleus and complexes with p53, while cytoplasmic PTEN prevents Mdm2 nuclear localization by attenuating Akt signaling. Subcellular distribution of PTEN in the cytoplasm or nucleus protects p53 from inac-tivation and degradation. The presence of nuclear PTEN and p53 coordinates the induction of maspin and p21 (both p53 gene targets) in response to hypoxia. Altering the expression of PTEN and/or p53 attenuated maspin gene induction under hypoxic conditions. Furthermore, implanting U87 (PTEN null) and PTEN reconstituted U87 cells (U87PTEN) in mice we observed by immuno-histochemistry and western blot that Maspin was only detectable in cells with PTEN. The integra-tion of PTEN and p53 into a common pathway for the induction of another tumor suppressor, Maspin, constitutes a tumor suppressor network of PTEN/p53/Mapsin that is operational under limited oxygen conditions.

Whole-Body PET/CT Evaluation of Tumor Perfusion Using Generator-Based 62Cu-Ethylglyoxal Bis(Thiosemicarbazonato)Copper(II): Validation by Direct Comparison to 15O-Water in Metastatic Renal Cell Carcinoma

This study was undertaken to demonstrate the feasibility of whole-body 62Cu-ethylglyoxal bis(thiosemicarbazonato)copper(II) (62Cu-ETS) PET/CT tumor perfusion imaging in patients with metastatic renal carcinoma and to validate 62Cu-ETS as a quantitative marker of tumor perfusion by direct comparison with 15O-water perfusion imaging. Methods: PET/CT imaging of 10 subjects with stage IV renal cell cancer was performed after intravenous administration of 15O-water (10-min dynamic list-mode study) with the heart and at least 1 tumor in the PET field of view, followed 10 min later by intravenous 62Cu-ETS (6-min list-mode study). Whole-body 62Cu imaging was then performed from 6 to 20 min at 2–3 min/bed position. Blood flow (K1) was quantified with both agents for normal and malignant tissues in the 21.7-cm dynamic field of view. The required arterial input functions were derived from the left atrium and, in the case of 62Cu-ETS, corrected for partial decomposition of the agent by blood with data from an in vitro analysis using a sample of each patient’s blood. This imaging protocol was repeated at an interval of 3–4 wk after initiation of a standard clinical treatment course of the antiangiogenic agent sunitinib. Results: All subjects received the scheduled 62Cu-ETS doses for the dynamic and subsequent whole-body PET/CT scans, but technical issues resulted in no baseline 15O-water data for 2 subjects. Direct comparisons of the perfusion estimates for normal tissues and tumor metastases were made in 18 paired baseline and treatment studies (10 subjects; 8 baseline studies, 10 repeated studies during treatment). There was an excellent correlation between the blood flow estimates made with 62Cu-ETS and 15O-water for normal tissues (muscle, thyroid, myocardium) and malignant lesions (pulmonary nodules, bone lesions); the regression line was y = 0.85x + 0.15, R2 = 0.83, for the 88 regions analyzed. Conclusion: 62Cu-ETS provided high-quality whole-body PET/CT images, and 62Cu-ETS measures of blood flow were highly and linearly correlated with 15O-water–derived K1 values (mL−1⋅min−1⋅g). This tracer is suitable for use as a PET tracer of tumor perfusion in patients with metastatic renal cell carcinoma.

Estimation of radiation dosimetry for 68Ga-HBED-CC (PSMA-11) in patients with suspected recurrence of prostate cancer

INTRODUCTION This study was performed to estimate the human radiation dosimetry for [68Ga]Ga-HBED-CC (PSMA-11) (68Ga PSMA-11). METHODS Under an RDRC-approved research protocol, we evaluated the biodistribution and pharmacokinetics of 68Ga PSMA-11 with serial PET imaging following intravenous administration to nine prostate cancer patients in whom clinical [11C]acetate PET/CT exams had been independently performed under Expanded Access IND 118,204. List-mode imaging was performed over the initial 0-10min post-injection with the pelvis in the field-of-view. Whole-body images were acquired, pelvis-to-head, at 15, 60, and 90-min post-injection. Additional images of the pelvis were acquired at 40-min and 115-min, and voided urine collected from each subject at 48-min and 120-min post-injection. Radiation dosimetry estimates were calculated from these data using the OLINDA software package. RESULTS Renal uptake was high and relatively invariant, ranging from 11% to 14% of the injected dose between 15 and 90-min post-injection. Radioactivity collected in the voided urine accounted for 14% of the injected dose over a period of 120-min. Lymph nodes and skeletal metastases suspicious for prostate cancer recurrence were detected in a greater number of patients using 68Ga PSMA-11 than using 11C-acetate. CONCLUSION Kidneys are the critical organ following 68Ga PSMA-11 administration, receiving an estimated dose of 0.413mGy/MBq. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE This study confirms that the kidneys will be the critical organ following intravenous administration of 68Ga PSMA-11, and provided data consistent with the expectation that 68Ga PSMA-11 will be superior to [11C]acetate for defining sites of recurrence in prostate cancer patients presenting with biochemical relapse.

Therapeutic considerations for Mdm2: not just a one trick pony

Background: The mdm2 proto-oncogene is elevated in numerous late stage cancers. The Mdm2 protein manifests its oncogenic properties in part through inactivation of the tumor suppressor protein p53. Recent efforts in anticancer drug design have focused on the identification of small molecules that disrupt the Mdm2–p53 interaction, in hope of re-engaging the p53 pathway. Objective: In addition to binding p53, Mdm2 complexes with numerous proteins involved in DNA repair, translation, metabolic activities, tumor growth and apoptosis. Further biochemical analysis is required to understand how Mdm2 integrates into all of these cellular processes. Post-translational modifications to Mdm2 can alter its ability to associate with numerous proteins. Changes in protein structure may also affect the ability of small molecule inhibitors to effectively antagonize Mdm2. Conclusion: The complexity of Mdm2 modification has been largely neglected during the development of previous Mdm2 inhibitors. Future high-throughput or in silico screening efforts will need to recognize the importance of post-translational modifications to Mdm2. Furthermore, the identification of molecules that target other domains in Mdm2 may provide a tool to prevent other pivotal p53-independent functions of Mdm2. These aims provide a useful roadmap for the discovery of new Mdm2-binding compounds with therapeutic potency that may exceed its predecessors.

Serdemetan antagonizes the Mdm2-HIF1α axis leading to decreased levels of glycolytic enzymes.

Serdemetan (JNJ-26854165), an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1), were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2.