He Zhu

AKT and 14-3-3 Regulate Notch4 Nuclear Localization

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.

Elevated Expression of Notch1 Is Associated With Metastasis of Human Malignancies

The expression level of Notch1 has been studied in many primary tumor types, but has not been widely investigated in metastatic lesions from human malignancies. Using immunohistochemistry (IHC), the expression level of Notch1 was evaluated and compared between primary and metastatic tumors in 12 different cancers. The mean IHC score of Notch1 was significantly increased in metastatic hepatocellular carcinoma (HCC; 5.4 ± 0.7) and in metastatic renal cell carcinoma (RCC; 5.0 ± 2.3) compared with primary HCC (3.1 ± 0.7, P = .035) and RCC (1.3 ± 0.6, P = .049), respectively. Similarly, the expression level of Notch1 showed an increasing trend in the metastatic malignancies in the larynx, prostate, and stomach compared with corresponding primary malignancies (P values are .055, .072, and .074, respectively). The results demonstrate elevated expression of Notch1 in some metastatic tumors, suggesting that Notch1 may play an important role in the development or maintenance of metastatic lesions, and targeting of Notch1 might be a therapeutic approach against tumor metastasis.

Abstract 410: Elevated Jagged-1 and Notch-1 expression in high grade and metastatic prostate cancers.

BACKGROUND Emerging evidence has suggested that Notch signaling pathway may be involved in the development, progression and metastasis of prostate cancer (PCa). In the present study, we investigated the expression levels of Jagged-1 and Notch-1 in human prostate tumors and their associations with PCa progression and metastasis. METHODS Immunohistochemistry (IHC) for Jagged-1 and Notch-1 was performed on tissue microarray (TMA) slides containing 286 formalin-fixed and paraffin-embedded (FFPE) tissue specimens with various prostatic pathologies, including benign changes, high grade prostatic intraepithelial neoplasia (HGPIN), low- and high-grade PCas as well as metastatic PCa. RESULTS Cytoplasmic and membranous IHC scores for Jagged-1 in both metastatic PCa and high grade PCa were significantly higher than those in low grade PCa and in benign prostatic tissues. Similarly, cytoplasmic IHC scores of Notch-1 in both metastatic PCa and high grade PCa were significantly elevated compared with those observed in low grade PCa and in benign prostatic tissues. A statistically significant correlation was identified between the expression of Jagged-1 and Notch-1 in human prostatic tissues. Furthermore, significantly more highly expressed Jagged-1 in membrane was observed in Caucasian patients with high-grade or metastatic PCa (vs. African Americans) and in PCa patients with positive surgical margins (vs. negative surgical margins). CONCLUSION Our results provide strong evidence that up-regulation of Jagged1-Notch1 signaling plays a role in PCa progression and metastasis and suggest that Jagged-1 and Notch-1 may be useful markers in distinguishing indolent and aggressive PCas.

Sound abnormally stimulates the vestibular system in canal dehiscence syndrome by generating pathological fluid-mechanical waves

Individuals suffering from Tullio phenomena experience dizziness, vertigo, and reflexive eye movements (nystagmus) when exposed to seemingly benign acoustic stimuli. The most common cause is a defect in the bone enclosing the vestibular semicircular canals of the inner ear. Surgical repair often corrects the problem, but the precise mechanisms underlying Tullio phenomenon are not known. In the present work we quantified the phenomenon in an animal model of the condition by recording fluid motion in the semicircular canals and neural activity evoked by auditory-frequency stimulation. Results demonstrate short-latency phase-locked afferent neural responses, slowly developing sustained changes in neural discharge rate, and nonlinear fluid pumping in the affected semicircular canal. Experimental data compare favorably to predictions of a nonlinear computational model. Results identify the biophysical origin of Tullio phenomenon in pathological sound-evoked fluid-mechanical waves in the inner ear. Sound energy entering the inner ear at the oval window excites fluid motion at the location of the defect, giving rise to traveling waves that subsequently excite mechano-electrical transduction in the vestibular sensory organs by vibration and nonlinear fluid pumping.

Abstract 2092: Elevated PKCα expression and its association with Notch signaling in localized and metastatic prostate cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Conventional protein Kinase C alpha (PKCα) is linked to the regulation of cell proliferation, differentiation, motility, survival, and apoptosis. Elevated PKCα expression has been reported in human early prostate cancer (PCa) and rat prostatic tumor cell lines. In this study, the expression level of PKCα was evaluated by immunohistochemistry (IHC) and correlated with the expression levels of Notch1 and Jagged1 in localized and metastatic PCa. Methods: : IHC for PKCα was performed on tissue microarray (TMA) slides containing 156 formalin-fixed and paraffin-embedded (FFPE) prostatic tissues specimens, including 39 benign prostatic changes, 97 localized prostate cancers and 20 metastatic prostate cancers. The expression level of PKCα was reported as a final IHC score calculated as staining extent score (0-3) multiplied by intensity score (0-3) with a maximal score of 9. ANOVA was employed to reveal the differences in IHC scores among pathologic categories, followed by Tukey-adjusted pairwise comparisons. Spearman correlation coefficient was evaluated to measure correlation between expression levels of PKCα, Notch1, and Jagged1. Results: IHC staining for PKCα was predominantly observed in cytoplasm (in 94% specimens). The score of PKCα cytoplasmic staining in metastatic PCa (5.48 ± 1.84, Mean ±SD) was significantly higher than those in localized PCa (3.30 ± 2.10, P = 2.95E-05) and in benign prostatic tissue (1.92 ± 1.53, P = 6.45E-09). Similarly, IHC score in localized PCa was significantly higher than that observed in benign prostatic changes (P = 0.001). Correlation analysis revealed that the expression level of cytoplasmic PKCα was significantly correlated with the expression levels of cytoplasmic Notch1 (r = 0.396, P = 5.85E-05), cytoplasmic Jagged1 (r = 0.610, P = 3.42E-11), and membranous Jagged1 (r = 0.354, P = 3.76E-04, respectively) in localized and metastatic prostate cancers. Conclusions: Our results indicate that up-regulation of PKCα plays a role not only in PCa formation and progression, but also in PCa metastasis. The evidence of significant correlation between PKCα and Jagged1-Notch1 signaling pathway supports the notion that synergy of PKCα with Jagged1-Notch1 signaling may play an important role in modulating the tumorgenesis and metastasis in human PCa. Citation Format: Xinchun Zhou, He Zhu, Xu Zhang, Jack Lewin, Lucio Miele. Elevated PKCα expression and its association with Notch signaling in localized and metastatic prostate cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2014-2092