The p73 tumor suppressor is targeted by Pirh2 RING finger E3 ubiquitin ligase for the proteasome-dependent degradation

The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ΔN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ΔNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.     

Mammary epithelial cell polarity is regulated differentially by p73 isoforms via epithelial-to-mesenchymal transition

p73 is expressed as TA and ΔN isoforms, both of which are implicated in tumor suppression and/or promotion. To address how p73 possesses these opposing functions, we developed three-dimensional culture of MCF10A cells, which undergo cell morphogenesis to form polarized spheroids with hollow lumen similar to normal mammary acini in vivo. Here, we showed that upon knockdown of p73, particularly TAp73 but not ΔNp73, MCF10A cells formed irregular and near-normal acini without hollow lumen in three-dimensional culture. We also found that upon knockdown of p73 or TAp73, but not ΔNp73, MCF10A cells underwent epithelial-to-mesenchymal transition (EMT) via down-regulation of E-cadherin coupled with up-regulation of β-catenin and laminin V. In addition, we found that Snail-1, Slug, and Twist, all of which are known to act as EMT inducers by repressing E-cadherin expression, were increased markedly upon knockdown of p73 and TAp73 but little if any by ΔNp73. Furthermore, we showed that knockdown of p73 or TAp73 in MCF10A cells led to a marked increase in cell proliferation and migration. Together, our data suggest that TAp73 is necessary for maintaining normal cell polarity by suppressing EMT.     

Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.     

Reactivation of TAp73 tumor suppressor by protoporphyrin IX,a metabolite of aminolevulinic acid,induces apoptosis in <Emphasis Type="Italic">TP</Emphasis>53-deficient cancer cells

Background

The p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated ΔN isoform that inhibits TAp73 and p53 and thus, acts as an oncogene.

Results

By employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch. Finally, PpIX showed potent antitumor effect and inhibited the growth of xenograft human tumors in mice.

Conclusion

Our findings may in future contribute to the successful repurposing of PpIX into clinical practice.

     

Conservation of the Notch1 signaling pathway in gastrointestinal carcinoid cells

Gastrointestinal (GI) carcinoid cells secrete multiple neuroendocrine (NE) markers and hormones including 5-hydroxytryptamine and chromogranin A. We were interested in determining whether activation of the Notch1 signal transduction pathway in carcinoid cells could modulate production of NE markers and hormones. Human pancreatic carcinoid cells (BON cells) were stably transduced with an estrogen-inducible Notch1 construct, creating BON-NIER cells. In the present study, we found that Notch1 is not detectable in human GI carcinoid tumor cells. The induction of Notch1 in human BON carcinoid cells led to high levels of functional Notch1, as measured by CBF-1 binding studies, resulting in activation of the Notch1 pathway. Similar to its developmental role in the GI tract, Notch1 pathway activation led to an increase in hairy enhancer of split 1 (HES-1) protein and a concomitant silencing of human Notch1/HES-1/achaete-scute homolog 1. Furthermore, Notch1 activation led to a significant reduction in NE markers. Most interestingly, activation of the Notch1 pathway caused a significant reduction in 5-hydroxytryptamine, an important bioactive hormone in carcinoid syndrome. In addition, persistent activation of the Notch1 pathway in BON cells led to a notable reduction in cellular proliferation. These results demonstrate that the Notch1 pathway, which plays a critical role in the differentiation of enteroendocrine cells, is highly conserved in the gut. Therefore, manipulation of the Notch1 signaling pathway may be useful for expanding the targets for therapeutic and palliative treatment of patients with carcinoid tumors.