Correlation between p53 and Mdm2 expression with histopathological parameters in cattle squamous cell carcinomas

Background and Aim: Squamous cell carcinoma (SCC) is the most common form of carcinoma in cattle. Histopathological grading systems have been utilized over several decades for estimating the malignancy of cattle SCCs. This study aimed to detect p53 and Mdm2 expression in different SCC cases in cattle and correlate their expression with the SCC histopathological grading. Materials and Methods: Cattle SCC cases were collected at the Veterinary Teaching Hospital in Nineveh. The SCC grading system categorized the cases histologically based on their differentiation grade into three groups: Well, moderately, and poorly differentiated. The SCC cases were subsequently verified for p53 and Mdm2 immunoexpression. Results: Fourteen of 16 examined cattle SCC samples tested positive for p53 expression. Moreover, 15 out of the 16 SCC samples tested positive for Mdm2 expression. The increased immunoreactivity of both p53 and Mdm2 was associated with a poor histological grading of the cattle SCC. There is a positive correlation between the nuclear expression of p53 and Mdm2, and the degree of differentiation and the number of mitotic figures in the examined cattle SCC samples. Conclusion: Our results demonstrate an increased p53 and Mdm2 expression in cattle SCC cases characterized by poor histopathological grading, thus suggesting an essential role of these molecules in the development of moderately and poorly differentiated SCC in cattle.

Differential Regulation of Myocardial E3 Ligases and Deubiquitinases in Ischemic Heart Failure

The pathological changes of ubiquitination and deubiquitination following myocardial infarction (MI) and chronic heart failure (CHF) have been sparsely examined. We investigated the expression of muscle-specific E3 ubiquitin ligases and deubiquitinases in MI and CHF. Therefore, mice were assigned to coronary artery ligation for 3 days or 10 weeks as well as for sham operation (each n = 10). Expression of E3 ligases (MAFBX, MURF1, CHIP, ITCH, MDM2) and deubiquitinases (A20, CYLD, UCH-L1, USP14, USP19) was determined. After MI and in CHF, the mRNA expression of MURF1, CHIP and MDM2 (all p < 0.05) was decreased. Protein expression analyses revealed that ITCH expression decreased in CHF (p = 0.01), whereas MDM2 expression increased in MI (p = 0.02) and decreased in CHF (p = 0.02). Except for USP19 mRNA expression that decreased at 3 days and 10 weeks (both p < 0.01), the expression of other deubiquitinases remained unaffected after MI and CHF. The expression of myocardial E3 ligases is differentially regulated following MI, raising the question of whether an upstream regulation exists that is activated by MI for tissue protection or whether the downregulation of E3 ligases enables myocardial hypertrophy following MI.

Adipose MDM2 regulates systemic insulin sensitivity

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

Expression profile of tumour suppressor protein p53 and its regulator MDM2 in a cohort of breast cancer patients in a Tertiary Hospital in Ghana

Background Inactivation or mutation of the tumour suppressor gene p53 or its regulator mouse double minute 2 (MDM2) is the commonest event in breast cancer. These altered genes usually express abnormally high levels of their proteins in many carcinomas. The phenotypic expression of p53 and MDM2 in breast cancer cases in our setting is not known. This study investigated the expression of the tumour suppressor protein p53 and its regulator MDM2, using immunohistochemistry in a Ghana breast cancer cohort. Method A 9-year retrospective cross-sectional study on archived tissue blocks–formalin fixed paraffin embedded tissue (FFPE) was carried out. Demographic data were abstracted. Based on complete clinical data and availability of FFPE archived blocks 203 cases were selected for tissue micro array (TMA) construction. The TMA sections were subjected to immunohistochemistry (IHC) (ER, PR, HER2, p53, and MDM2). Expression of p53 and MDM2 were related to grade and molecular subtypes. Results The age ranged from 17 to 92 years (mean = 49.34 ± 13.74). Most of the cases were high grade; grade II (34.9%) and grade III (55.7%). Fifty-four percent of the cases were triple negative. Invasive ductal carcinoma no special type was the commonest histotype (87.1%). Thirty-six percent (36%) of the cases expressed p53. Significant associations were found between p53 overexpression and histological grade (p = 0.034), triple negative (p = 0.0333) and luminal B (p<0.01) tumors. Most cases (93.1%) were negative for MDM2 expression. Significant association was found between MDM2 and HER2 over-expression as well as Ki-67. There was no significant positive correlation between MDM2 and p53 co-expression (p>0.05). Conclusion The elevated level of p53 expression in the aggressive breast cancer phenotypes (high histological grade and triple negative) in our cohort suggest that P53 elevation may be a poor prognostic marker in our setting. High expression of MDM2 in our cohort with high Ki67; also in cases with Her2/neu overexpression known with predictable poor prognosis in the absence of target therapy suggest MDM2 may be associated with aggressive biological behaviour in our breast cancer cases. The non-significant association of p53 and MDM2 expression in the same cases as also documented by previous studies suggest independent genetic pathway in tumourigenesis.

Chemotherapy of HER2- and MDM2-Enriched Breast Cancer Subtypes Induces Homologous Recombination DNA Repair and Chemoresistance

Analyzing the TCGA breast cancer database, we discovered that patients with the HER2 cancer subtype and overexpression of MDM2 exhibited decreased post-treatment survival. Inhibition of MDM2 expression in the SKBR3 cell line (HER2 subtype) diminished the survival of cancer cells treated with doxorubicin, etoposide, and camptothecin. Moreover, we demonstrated that inhibition of MDM2 expression diminished DNA repair by homologous recombination (HR) and sensitized SKBR3 cells to a PARP inhibitor, olaparib. In H1299 (TP53−/−) cells treated with neocarzinostatin (NCS), overexpression of MDM2 WT or E3-dead MDM2 C478S variant stimulated the NCS-dependent phosphorylation of ATM, NBN, and BRCA1, proteins involved in HR DNA repair. However, overexpression of chaperone-dead MDM2 K454A variant diminished phosphorylation of these proteins as well as the HR DNA repair. Moreover, we demonstrated that, upon NCS treatment, MDM2 K454A interacted with NBN more efficiently than MDM2 WT and that MDM2 WT was degraded more efficiently than MDM2 K454A. Using a proliferation assay, we showed that overexpression of MDM2 WT, but not MDM2 K454A, led to acquisition of resistance to NCS. The presented results indicate that, following chemotherapy, MDM2 WT was released from MDM2-NBN complex and efficiently degraded, hence allowing extensive HR DNA repair leading to the acquisition of chemoresistance by cancer cells.

MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α–MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α–MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.

Feasibility of Developing Radiotracers for MDM2: Synthesis and Preliminary Evaluation of an 18F-Labeled Analogue of the MDM2 Inhibitor SP-141

Murine double minute 2 (MDM2), a negative regulator of the p53 tumor suppressor protein, is overexpressed in several human cancers. Herein we investigate the feasibility of developing 18F-labeled compounds based on the small molecule inhibitor SP-141 for imaging tumor MDM2 expression levels with positron emission tomography (PET). Three nonradioactive fluorinated SP-141 analogues, 1–3, were synthesized, and their binding to the MDM2 protein was analyzed by surface plasmon resonance (SPR). One of these, a fluoroethoxy analogue, was labeled with fluorine-18 (18F) using 18F-fluorethyl bromide to provide [18F]1 and evaluated in vitro and in vivo. SPR analysis confirmed the binding of the fluorinated analogues to MDM2 at 1.25–20 µM concentrations. Cell uptake studies revealed high uptake (67.5–71.4 %/mg protein) and specificity of [18F]1 in MCF7 and HepG2 cells. The uptake of [18F]1 in these cells could be modulated using 100 µM SP-141, potentially reflecting changes in MDM2 expression because of p53 activation by SP-141. [18F]1 exhibited stable uptake and retention in HepG2 tumor xenografts (~3 %ID/g) in vivo, but poor clearance from blood and other normal tissues, yielding low tumor-to-background ratios (< 2) at 2 h post injection. Our results suggest that [18F]1 has suboptimal characteristics for in vivo evaluation as a PET tracer for MDM2, but warrant radiolabeling and assessment of the other fluorinated analogues synthesized in this work, 2 and 3, and potentially other molecular scaffolds for developing MDM2 targeted radiotracers.

High Glucose Treatment Limits Drosha Protein Expression and Alters AngiomiR Maturation in Microvascular Primary Endothelial Cells via an Mdm2-dependent Mechanism

Diabetes promotes an angiostatic phenotype in the microvascular endothelium of skeletal muscle and skin. Angiogenesis-related microRNAs (angiomiRs) regulate angiogenesis through the translational repression of pro- and anti-angiogenic genes. The maturation of micro-RNA (miRs), including angiomiRs, requires the action of DROSHA and DICER proteins. While hyperglycemia modifies the expression of angiomiRs, it is unknown whether high glucose conditions alter the maturation process of angiomiRs in dermal and skeletal muscle microvascular endothelial cells (MECs). Compared to 5 mM of glucose, high glucose condition (30 mM, 6–24 h) decreased DROSHA protein expression, without changing DROSHA mRNA, DICER mRNA, or DICER protein in primary dermal MECs. Despite DROSHA decreasing, high glucose enhanced the maturation and expression of one angiomiR, miR-15a, and downregulated an miR-15a target: Vascular Endothelial Growth Factor-A (VEGF-A). The high glucose condition increased Murine Double Minute-2 (MDM2) expression and MDM2-binding to DROSHA. Inhibition of MDM2 prevented the effects evoked by high glucose on DROSHA protein and miR-15a maturation in dermal MECs. In db/db mice, blood glucose was negatively correlated with the expression of skeletal muscle DROSHA protein, and high glucose decreased DROSHA protein in skeletal muscle MECs. Altogether, our results suggest that high glucose reduces DROSHA protein and enhances the maturation of the angiostatic miR-15a through a mechanism that requires MDM2 activity.