Reciprocal regulation of the Cadherin-11/Stat3 axis by caveolin-1 in mouse fibroblasts and lung carcinoma cells.

Caveolin-1 (Cav1) is an integral plasma membrane protein and a complex regulator of signal transduction. The Signal Transducer and Activator of Transcription-3 (Stat3) is activated by a number of receptor and non-receptor tyrosine kinases and is positively implicated in cancer. Despite extensive efforts, the relationship between Cav1 and Stat3 has been a matter of controversy. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 cell to cell adhesion molecules, as occurs with confluence of cultured cells, triggers a dramatic increase in the levels of tyr705 phosphorylated i.e. activated Stat3, by a mechanism requiring the cRac1 small GTPase. Since confluence was not taken into account in previous studies, we revisited the question of the relationship between Cav1 and Stat3-ptyr705 in non-transformed mouse fibroblasts and in human lung carcinoma cells, by examining their effect at different cell densities. Our results unequivocally demonstrate that Cav1 downregulates cadherin-11, by a mechanism which requires the Cav1 scaffolding domain. This cadherin-11 downregulation, in turn, leads to a reduction in cRac1 and Stat3 activity levels. Furthermore, in a feedback loop possibly through p53 upregulation, Stat3 downregulation increases Cav1 levels. Our data reveal the presence of a potent, negative regulatory loop between Cav1 and cadherin-11/Stat3, leading to Stat3 inhibition and apoptosis.

Structure-activity relationship study of ProxyPhos chemosensors for the detection of proximal phosphorylation and other phosphate species.

Chemosensors for the detection of phosphate-containing biological species are in high need. Detection of proximally phosphorylated sites of PPi and those found in peptides and proteins has been demonstrated using chemosensors containing pyrene, as a fluorescent reporter, and a Zn2+-chelate, as a phosphate-binding group. Using these sensors, detection of proximal phosphate groups is afforded by binding of at least two of the sensor molecules to the adjacent phosphates, via the Zn2+ centres, leading to excimer formation between the pyrene groups and the corresponding shift in emission from 376 to 476 nm. Although several reports of this chemosensor class have been made, no detailed studies of selectivity of these sensors among major phosphate targets have been reported. In this study, a library of this class of chemosensors, termed ProxyPhos, which contained various linkers and Zn2+-chelating groups (i.e. DPA, cyclen and cyclam), was prepared and the effects of structural variation on the sensing efficiency and selectivity were evaluated among proximally phosphorylated peptides, proteins, nucleotides, Pi and PPi. As a result of this study, we have identified ProxyPhos library members that are most suitable for the detection of proximally phosphorylated peptides, PPi, UTP, and a DpYD peptide motif, and have generally provided a foundation for the selection of ProxyPhos chemosensors for further development of specific biologically relevant assays. The broad utility of ProxyPhos is further supported by the demonstrated lack of these sensors' cytotoxicity, ability to rapidly permeate into live and fixed cells and compatibility with gel staining methods.

Characterization and application studies of ProxyPhos, a chemosensor for the detection of proximally phosphorylated peptides and proteins in aqueous solutions.

Proximal phosphorylation on proteins appears to have functional significance and has been associated with several diseases, including Alzheimer's and cancer. While much remains to be learned about the role of proximal phosphorylation in biological systems, no simple and/or affordable technique is available for its detection. To this end, we have previously developed a ProxyPhos chemosensor, which detects proximally phosphorylated peptides and proteins over mono- and non-phosphorylated motifs in aqueous solutions. In this follow-up work, we performed extensive characterization of peptide and protein ProxyPhos assay conditions to achieve enhanced detection, and further explored the selectivity of ProxyPhos, and its potential off-targets. As a result of characterization studies, selective sensing of proximally phosphorylated over mono-phosphorylated peptides and proteins was achieved. Moreover, studies demonstrated that ProxyPhos was compatible with the detection of all commonly phosphorylated residues (i.e. tyrosine, serine and threonine residues). Under optimized conditions, ProxyPhos efficiently discriminated between peptides derived from the activated (proximally phosphorylated, disease-relevant) and inactive (mono-phosphorylated) forms of JAK2, SYK and MAPK1 kinases. In addition, ProxyPhos can be used to probe phosphatase activity on peptides and proteins via detecting changes in proximal phosphorylation, demonstrating immediate utility of this chemosensing system.

CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

Electrochemical detection of the Fc-STAT3 phosphorylation and STAT3-Fc-STAT3 dimerization and inhibition.

Signal transducer and activator of transcription 3 (STAT3) protein is involved in regulatory functions in cell proliferation, differentiation and survival, and is linked to cancer phenotype and tumorigenesis. Towards developing new methodologies for screening STAT3 interactions, the electrochemical method based on the use of redox active protein was proposed. The electrochemical signal, due to the redox (ferrocene)-labeled STAT3 protein immobilized on a gold surface, was modulated due to protein dimerization with the unlabeled STAT3 molecule. The dramatic decrease in current density from 2.7 µA cm(-2) to 0.5 µA cm(-2) was observed following the STAT3-ferrocene-STAT3 dimerization. The electrochemical approach was further extended for screening the potential dimerization inhibitors. Previously published potent salicylic acid derivatives were the most promising candidates for inhibition of STAT3 dimerization in this assay. We expect that other SH2-containing proteins may be monitored by the proposed electrochemical method.