Edward Ratovitski

ΔNp63 induces β-catenin nuclear accumulation and signaling

Abstract The P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that ΔNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with ΔNp63α and mediated its degradation. Here, we report that ΔNp63 associates with the B56α regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3β (GSK3β), leading to a dramatic inhibition of PP2A-mediated GSK3β reactivation. The inhibitory effect of ΔNp63 on GSK3β mediates a decrease in phosphorylation levels of β-catenin, which induces intranuclear accumulation of β-catenin and activates β-catenin-dependent transcription. Our results suggest that ΔNp63 isotypes act as positive regulators of the β-catenin signaling pathway, providing a basis for their oncogenic properties.

Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients. Experimental Design: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control. Results: Promoter hypermethylation of at least one of the genes studied was detected in all 31 lung primary tumors; 27 (87%) CDH1, 17 (55%) APC, 14 (45%) RASSF1A, 12 (39%) MGMT, 7 (23%) p16, 3 (10%) GSTP1, 3 (10%) RAR-β2, and 0 (0%) ARF. Methylation was detected in CDH1 (48%), APC (29%), RASSF1A (29%), MGMT (58%), p16 (14%), GSTP1 (33%), RAR-β2 (0%), and ARF (0%) of BAL samples from matched methylation-positive primary tumors, and in every case, aberrant methylation in BAL DNA was accompanied by methylation in the matched tumor samples. BAL samples from 10 controls without evidence of cancer revealed no methylation of the MGMT, GSTP1, p16, ARF, or RAR-β2 genes whereas methylation of RASSF1, CDH1, and APC was detected at low levels. Overall, 21 (68%) of 31 BAL samples from cancer patients were positive for aberrant methylation. Conclusion: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.

Kalirin Inhibition of Inducible Nitric-oxide Synthase

Nitric oxide (NO) acts as a neurotransmitter. However, excess NO produced from neuronal NO synthase (nNOS) or inducible NOS (iNOS) during inflammation of the central nervous system can be neurotoxic, disrupting neurotransmitter and hormone production and killing neurons. A screen of a hippocampal cDNA library showed that a unique region of the iNOS protein interacts with Kalirin, previously identified as an interactor with a secretory granule peptide biosynthetic enzyme. Kalirin associates with iNOS in vitroand in vivo and inhibits iNOS activity by preventing the formation of iNOS homodimers. Expression of exogenous Kalirin in pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Thus Kalirin may play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity.

p63α Mutations Lead to Aberrant Splicing of Keratinocyte Growth Factor Receptor in the Hay-Wells Syndrome

p63, a p53 family member, is required for craniofacial and limb development as well as proper skin differentiation. However, p63 mutations associated with the ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome (Hay-Wells syndrome) were found in the p63 carboxyl-terminal region with a sterile α-motif. By two-hybrid screen we identified several proteins that interact with the p63α carboxyl terminus and its sterile α-motif, including the apobec-1-binding protein-1 (ABBP1). AEC-associated mutations completely abolished the physical interaction between ABBP1 and p63α. Moreover the physical association of p63α and ABBP1 led to a specific shift of FGFR-2 alternative splicing toward the K-SAM isoform essential for epithelial differentiation. We thus propose that a p63α-ABBP1 complex differentially regulates FGFR-2 expression by supporting alternative splicing of the K-SAM isoform of FGFR-2. The inability of mutated p63α to support this splicing likely leads to the inhibition of epithelial differentiation and, in turn, accounts for the AEC phenotype.

The Effect of Tuning Cold Plasma Composition on Glioblastoma Cell Viability

Previous research in cold atmospheric plasma (CAP) and cancer cell interaction has repeatedly proven that the cold plasma induced cell death. It is postulated that the reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a major role in the CAP cancer therapy. In this paper, we seek to determine a mechanism of CAP therapy on glioblastoma cells (U87) through an understanding of the composition of the plasma, including treatment time, voltage, flow-rate and plasma-gas composition. In order to determine the threshold of plasma treatment on U87, normal human astrocytes (E6/E7) were used as the comparison cell line. Our data showed that the 30 sec plasma treatment caused 3-fold cell death in the U87 cells compared to the E6/E7 cells. All the other compositions of cold plasma were performed based on this result: plasma treatment time was maintained at 30 s per well while other plasma characteristics such as voltage, flow rate of source gas, and composition of source gas were changed one at a time to vary the intensity of the reactive species composition in the plasma jet, which may finally have various effect on cells reflected by cell viability. We defined a term “plasma dosage” to summarize the relationship of all the characteristics and cell viability.

RACK1 and Stratifin Target ΔNp63α for a Proteasome Degradation in Head and Neck Squamous Cell Carcinoma Cells upon DNA Damage

P53 family members with a transactivation domain induce cell cycle arrest and promoteapoptosis. However, ΔNp63 isotypes lacking the transactivation (TA)- domain promote cellproliferation and tumorigenesis in vitro and in vivo. Although p53, TAp63 or TAp73 are stabilizedupon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease andphosphorylation of ΔNp63α in squamous cell carcinoma cells. Further work revealed that RACK1physically associated with the p63α C-terminal domain through its WD40 domain. However,stratifin binds with phosphorylated ΔNp63α in response to cisplatin. Upon DNA damage inducedby cisplatin, stratifin mediated a nuclear export of ΔNp63α into cytoplasm and then RACK1targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase.Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and proteindegradation of ΔNp63α, respectively. Our data suggest that modification and down regulation ofΔNp63α is one of the major determinants of the cellular response to DNA damage in human headand neck cancers.

ΔNp63&alpha Overexpression Induces Downregulation of Sirt1 and an Accelerated Aging Phenotype in the Mouse

p63 is highly expressed in the skin and appears to be an early marker of keratinocyte differentiation. To examine the role of p63 in vivo, we generated transgenic mice that overexpress ΔNp63&alpha in the skin. These mice exhibited an accelerated aging phenotype in the skin characterized by striking wound healing defects, decreased skin thickness, decreased subcutaneous fat tissue, hair loss, and decreased cell proliferation. The accelerated skin aging was accompanied by a dramatic decrease in longevity of the mice. We found that aging in ΔNp63&alpha transgenic mice and other mouse models correlated with levels of Sirt1, a mammalian SIR2 orthologue thought to extend the lifespan in lower species. Moreover, increased ΔNp63&alpha expression induced cellular senescence that was rescued by Sirt1. Our data suggest that ΔNp63&alpha levels may affect aging in mammals, at least in part, through regulation of Sirt1.

An Inducible Nitric-oxide Synthase (NOS)-associated Protein Inhibits NOS Dimerization and Activity

A variety of transcriptional and post-transcriptional mechanisms regulate the expression of the inducible nitric-oxide synthase (iNOS, or NOS2). Although neurons and endothelial cells express proteins that interact with and inhibit neuronal NOS and endothelial NOS, macrophage proteins that inhibit NOS2 have not been identified. We show that murine macrophages express a 110-kDa protein that interacts with NOS2, which we call NOS-associated protein-110 kDa (NAP110). NAP110 directly interacts with the amino terminus of NOS2, and inhibits NOS catalytic activity by preventing formation of NOS2 homodimers. Expression of NAP110 may be a mechanism by which macrophages expressing NOS2 protect themselves from cytotoxic levels of nitric oxide.

Phospho-ΔNp63α-dependent regulation of autophagic signaling through transcription and micro-RNA modulation

Cisplatin was shown to induce the ataxia telangiectasia mutated (ATM)-dependent phosphorylation of tumor protein p63 isoform, (ΔNp63α), leading to a transcriptional regulation of specific genes implicated in the control of cell death of squamous cell carcinoma (SCC) cells. We previously observed that the cisplatin-induced phosphorylated (p)-ΔNp63α transcriptionally regulates the expression of specific microRNAs (miRNAs) in SCC cells. We found here that cisplatin exposure of SCC cells led to modulation of the members of the autophagic pathway, such as Atg1/Ulk1, Atg3, Atg4A, Atg5, Atg6/Becn1, Atg7, Atg9A and Atg10, by a direct p-ΔNp63α-dependent transcriptional regulation. We further found that specific miRNAs (miR-181a, miR-519a, miR-374a and miR-630), which are critical downstream targets of the p-ΔNp63α, modulated the protein levels of ATG5, ATG6/BECN1, ATG10, ATG12, ATG16L1 and UVRAG, adding another level of expression control for autophagic pathways in SCC cells upon cisplatin exposure. Our data support the notion that the cisplatin-induced p-ΔNp63α could regulate key pathways implicated in response of cancer cells to chemotherapeutics.

ΔNp63α levels correlate with clinical tumor response to cisplatin

After exposure to damaging agents, the p53 tumor suppressor is stabilized mediating cell cycle arrest and apoptosis. p53 family member, ΔNp63α promotes cell proliferation and accelerates tumor growth. We previously found that the genotoxic stress agents induced a decrease of ΔNp63α . We further observed that genotoxic stress mediated phosphorylation of ΔNp63α targeting it into proteasome degradation. Here, we found that high ΔNp63 protein levels in primary tumors accurately predicted response to platinum based chemotherapy and a favorable outcome in head and neck cancer patients. Our data suggest that degradation of ΔNp63α is part of the cellular response to DNA damage in head and neck cancers. The findings may have implications for the rational use of DNA damaging agents in human cancer.