[The Efficacy of topo â -pS10 Expression in Gastric Cancer as a Predictive Biomarker for Irinotecan Use].

BACKGROUND: Though irinotecan is commonly used for treating advanced gastric cancer, there is no predictive biomarker to date. We have studied the resistant mechanism for irinotecan and found that phosphorylation of serine 10 residue of topoisomerase Ⅰ(topo Ⅰ)is an important step for irinotecan resistance. We have developed an immunohistochemical staining-based biomarker; topo Ⅰ-pS10, for predicting irinotecan efficacy. PURPOSE: The purpose of this study is to test the accuracy of topo Ⅰ-pS10 immunohistochemical staining in gastric cancer clinical samples. METHODS: In this study we performed 2 sets of tests. In the training set, we stained 79 gastric cancer clinical samples which efficacy of irinotecan was measured by succinate dehydrogenase inhibition(SDI)test. In the validation set, we used 27 gastric cancer clinical samples which irinotecan was used and the efficacy was known. RESULTS: Training set: From the ROC curve the cut-off point was set at 35% positive nuclei. Sixty three cases were positive with topo Ⅰ-pS10 in the nuclei. With the result of irinotecan SDI, the sensitivity was 76.6% and the positive predictive value was 92.5%. This result showed that topo Ⅰ-pS10 positive case does not respond to irinotecan. Validation set: In this set, the sensitivity was 82.4% and the positive predictive value was 82.4%. CONCLUSION: topo Ⅰ-pS10 staining can be used as a predictive biomarker for irinotecan for gastric cancer patients.

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro.

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.

ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response.

The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or γ-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro.

The delivery of the diphtheria toxin catalytic domain (DTA) from acidified endosomes into the cytoplasm of eukaryotic cells requires protein-protein interactions between the toxin and a cytosolic translocation factor (CTF) complex. A conserved peptide motif, T1, within the DT transmembrane helix 1 mediates these interactions. Because the T1 motif is also present in the N-terminal segments of lethal factor (LF) and edema factor (EF) in anthrax toxin, we asked whether LF entry into the cell might also be facilitated by target cell cytosolic proteins. In this study, we have used LFnDTA and its associated ADP-ribosyltransferase activity (DTA) to determine the requirements for LF translocation from the lumen of endosomal vesicles to the external medium in vitro. Although low-level release of LFnDTA from enriched endosomal vesicles occurs in the absence of added factors, translocation was enhanced by the addition of cytosolic proteins and ATP to the reaction mixture. We show by GST-LFn pull-down assays that LFn specifically interacts with at least zeta-COP and beta-COP of the COPI coatomer complex. Immunodepletion of COPI coatomer complex and associated proteins from cytosolic extracts blocks in vitro LFnDTA translocation. Translocation may be reconstituted by the addition of partially purified bovine COPI to the translocation assay mixture. Taken together, these data suggest that the delivery of LF to the cytosol requires either COPI coatomer complex or a COPI subcomplex for translocation from the endosomal lumen. This facilitated delivery appears to use a mechanism that is analogous to that of DT entry.

Multicohort Retrospective Validation of a Predictive Biomarker for Topoisomerase I Inhibitors.

PURPOSE: The camptothecin (CPT) analogs topotecan and irinotecan specifically target topoisomerase I (topoI) and are used to treat colorectal, gastric, and pancreatic cancer. Response rate for this class of drug varies from 10% to 30%, and there is no predictive biomarker for patient stratification by response. On the basis of our understanding of CPT drug resistance mechanisms, we developed an immunohistochemistry-based predictive test, P-topoI-Dx, to stratify the patient population into those who did and did not experience a response. PATIENTS AND METHODS: The retrospective validation studies included a training set (n = 79) and a validation cohort (n = 27) of gastric cancer (GC) patients, and 8 cohorts of colorectal cancer (CRC) patient tissue (n = 176). Progression-free survival for 6 months was considered a positive response to CPT-based therapy. Formalin-fixed, paraffin-embedded slides were immunohistochemically stained with anti-phospho-specific topoI-Serine10 (topoI-pS10), quantitated, and analyzed statistically. RESULTS: We determined a threshold of 35% positive staining to offer optimal test characteristics in GC. The GC (n = 79) training set demonstrated 76.6% (95% confidence interval, 64-86) sensitivity; 68.8% (41-88) specificity; positive predictive value (PPV) 92.5% (81-98); and negative predictive value (NPV) 42.3% (24-62). The GC validation set (n = 27) demonstrated 82.4% (56-95) sensitivity and 70.0% (35-92) specificity. Estimated PPV and NPV were 82.4% (56-95) and 70.0% (35-92) respectively. In the CRC validation set (n = 176), the 40% threshold demonstrated 87.5% (78-94) sensitivity; 70.0% (59-79) specificity; PPV 70.7% (61-79); and NPV 87.0 % (77-93). CONCLUSION: The analysis of retrospective data from patients (n = 282) provides clinical validity to our P-topoI-Dx immunohistochemical test to identify patients with disease that is most likely to respond to topoI inhibitors.

DIFFERENTIAL SERUM LEVEL OF SPECIFIC HAPTOGLOBIN ISOFORMS IN SMALL CELL LUNG CANCER.

Lung cancer is the leading cause of cancer death for both men and women in the United States, and similar trends are seen world wide. The lack of early diagnosis is one of the primary reasons for the high mortality rate. A number of biomarkers have been evaluated in lung cancer patients, however, their specificity and early stage diagnostic values are limited. Using traditional protein chemistry and proteomics tool we have demonstrated higher serum haptoglobin levels in small cell lung cancer (SCLC). Similar findings have been reported for other cancers including ovarian cancer and glioblastoma. Haptoglobin is an acute phase protein with at least six possible phenotypes. The six phenotypes, in combination with two post translational modifications, glycosylation and deamidation, lead to large numbers of possible haptoglobin isoforms. Recent studies indicate a possible correlation between specific haptoglobin glycosylation and particular disease conditions. In our current study, we have fractionated control and SCLC patient serum by 2-D gel electrophoresis to identify differentially expressed haptoglobin isoforms in SCLC serum samples.

CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer.

Irinotecan specifically targets topoisomerase I (topoI), and is used to treat various solid tumors, but only 13-32% of patients respond to the therapy. Now, it is understood that the rapid rate of topoI degradation in response to irinotecan causes irinotecan resistance. We have published that the deregulated DNA-PKcs kinase cascade ensures rapid degradation of topoI and is at the core of the drug resistance mechanism of topoI inhibitors, including irinotecan. We also identified CTD small phosphatase 1 (CTDSP1) (a nuclear phosphatase) as a primary upstream regulator of DNA-PKcs in response to topoI inhibitors. Previous reports showed that rabeprazole, a proton pump inhibitor (PPI) inhibits CTDSP1 activity. The purpose of this study was to confirm the effects of rabeprazole on CTDSP1 activity and its impact on irinotecan-based therapy in colon cancer. Using differentially expressing CTDSP1 cells, we demonstrated that CTDSP1 contributes to the irinotecan sensitivity by preventing topoI degradation. Retrospective analysis of patients receiving irinotecan with or without rabeprazole has shown the effects of CTDSP1 on irinotecan response. These results indicate that CTDSP1 promotes sensitivity to irinotecan and rabeprazole prevents this effect, resulting in drug resistance. To ensure the best chance at effective treatment, rabeprazole may not be a suitable PPI for cancer patients treated with irinotecan.

Developing a Phosphospecific IHC Assay as a Predictive Biomarker for Topoisomerase I Inhibitors.

Phosphorylation is the most extensively studied posttranslational modification of proteins. There are approximately 500 kinases known in the human genome. The kinase-activated pathways regulate almost every aspect of cell function and a deregulated kinase cascade leads to impaired cellular function. Impaired regulation of several kinase cascades, including the epidermal growth factor receptor (EGFR) pathway, leading to tumor pathogenesis, is well documented. Thus, a phosphospecific test with prognostic or predictive value was expected in oncology. However, no phosphospecific IHC test is used in oncology clinics. Human topoisomerase I (topoI) inhibitors, camptothecin and its analogues (CPT), are used extensively to treat various solid tumors. Depending on tumor type, the response rate is only 13-32%. We have demonstrated that the deregulated kinase cascade is at the core of CPT resistance. DNA-PKcs, a kinase central to the DNA-double-strand break (DSB) response pathway, phosphorylates topoI at serine 10 (topoI-pS10), and cells with higher basal levels of topoI-pS10 degrade topoI rapidly and are resistant to this class of drug. The higher basal level of topoI phosphorylation is due to continual activation of DNA-PKcs, and one potential mechanism of this pathway activation is failure of upstream effector phosphatases such as phosphatase and tensin homolog (PTEN). Based on this understanding, we have developed an IHC-based test (P-topoIDx) that can stratify the responder and non-responder patient population.

Targeting the human MUC1-C oncoprotein with an antibody-drug conjugate.

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed on the surface of diverse human carcinomas and is an attractive target for the development of mAb-based therapeutics. However, attempts at targeting the shed MUC1 N-terminal subunit have been unsuccessful. We report here the generation of mAb 3D1 against the nonshed oncogenic MUC1 C-terminal (MUC1-C) subunit. We show that mAb 3D1 binds with low nM affinity to the MUC1-C extracellular domain at the restricted α3 helix. mAb 3D1 reactivity is selective for MUC1-C-expressing human cancer cell lines and primary cancer cells. Internalization of mAb 3D1 into cancer cells further supported the conjugation of mAb 3D1 to monomethyl auristatin E (MMAE). The mAb 3D1-MMAE antibody-drug conjugate (ADC) (a) kills MUC1-C-positive cells in vitro, (b) is nontoxic in MUC1-transgenic (MUC1.Tg) mice, and (c) is active against human HCC827 lung tumor xenografts. Humanized mAb (humAb) 3D1 conjugated to MMAE also exhibited antitumor activity in (a) MUC1.Tg mice harboring syngeneic MC-38/MUC1 tumors, (b) nude mice bearing human ZR-75-1 breast tumors, and (c) NCG mice engrafted with a patient-derived triple-negative breast cancer. These findings and the absence of associated toxicities support clinical development of humAb 3D1-MMAE ADCs as a therapeutic for the many cancers with MUC1-C overexpression.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.

Induction of heat shock proteins by heregulin beta1 leads to protection from apoptosis and anchorage-independent growth.

Elevation of heat shock protein (HSP) levels is widespread in cancer and predicts a poor prognosis and resistance to therapy. We show that HSP elevation in tumor cells can be induced by the highly malignant factor heregulin beta1 (HRGbeta1), which induces HSP expression through heat shock transcription factor 1 (HSF1). Inactivation of the hsf1 gene prevents HSP induction by HRGbeta1. HSP expression is induced through a cascade response initiated by HRGbeta1 binding to c-erbB receptors on the cell surface and which leads to the inhibition of intracellular HSF1 antagonist glycogen synthase kinase 3. HSF1 activated by this pathway plays a key role in the protection of cells from apoptosis and the mediation of anchorage independent growth by HRGbeta1, indicating a role for HSF1 in this tumorigenic pathway.

Titin determines the Frank-Starling relation in early diastole.

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target.

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.

Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) frequently presents as metastatic disease. It would be useful to detect serum tumor biomarkers at an earlier stage in order to improve the overall survival. MATERIALS AND METHODS: Serum samples from SCLC patients (6 limited disease, 7 extensive disease, 4 relapsed disease, 4 no evidence of disease post-treatment) and 5 normal controls were used to identify tumor biomarkers utilizing proteomics. Serum hepatocyte growth factor was also studied using standard ELISA. RESULTS: Utilizing MALDI-TOF-Mass Spectrometry (MS) based protein identification techniques, a SCLC specific overexpressed protein was identified to be haptoglobin alpha-subunit, with its serum level correlating with the disease stage. The mean level of alpha-haptoglobin was increased in SCLC serum as compared to the normal controls. Serum HGF was also studied as potential tumor biomarker and was found to correlate with the disease status. Either serum alpha-haptoglobin relative level (above 1.9 U), or HGF level (above 500 pg/ml) was associated with a trend towards worse survival. CONCLUSION: Our current findings suggest that serum levels of alpha-haptoglobin and HGF may serve as useful serum tumor biomarkers in SCLC. It would now also be useful to determine if these serum biomarkers are altered in response to therapy for this disease.

Circulating tumor cells and serum tumor biomarkers in small cell lung cancer.

Lung cancer accounts for approximately 30% of all cancer mortalities in the United States. Small cell lung cancer (SCLC), which is an aggressive malignancy with frequent and early metastases, accounts for about 15% of all of the lung cancer cases with a dismal 5-year survival rate of < 5% with current standard therapies. Early detection of SCLC is challenging, in part due to the lack of adequate serum tumor markers. The goal of this review is to summarize the current knowledge of circulating tumor cells and serum biomarkers in small cell lung cancer. The role of circulating tumor cells in prognostication is controversial, but may be better defined with advancing technologies of detection of such cells with higher precision, and improved clinico-pathological correlations. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC, such as CEA, chromogranin-A and neuron-specific enolase will be presented. Serum cytokines, such as vascular endothelial growth factor (VEGF), stem cell factor (SCF) and hepatocyte growth factor/scatter factor (HGF/SF) are also discussed. New findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomics and proteomics technologies are emphasized. It is our hope that validation of these new research platforms and technologies will result in improved early detection, prognostication and finally treatment of SCLC with potential novel molecularly-targeted therapeutics.

Modulation of c-Kit/SCF pathway leads to alterations in topoisomerase-I activity in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, for which cytotoxic chemotherapy appears to have reached its maximal efficacy. This neoplasm is characterized by the overexpression of several receptor tyrosine kinases (RTKs), especially c-Kit. The ligand for c-Kit is stem cell factor (SCF). In SCLC, SCF can influence c-Kit activation by autocrine or paracrine mechanisms. We have recently shown that the c-Kit/SCF pathway is operational in SCLC and can be inhibited by Glivec (STI571). Because the inhibition of topoisomerase-I (topo-I) is one approach used to treat SCLC, we determined the effects of c-Kit/SCF signaling on topo-I activity. A unique phosphorylation of c-Kit on amino acid 823 and amino acid 703 was identified with the SCF stimulation of H526 cells. We demonstrate that with SCF stimulation over 16 hours (dose response 0-100 ng/mL) in H526 SCLC cells (c-Kit positive, SCF responsive), a decrease in topo-I activity was observed, whereas in H82 SCLC cells (c-Kit negative, SCF unresponsive) there was no modulation of topo-I activity by SCF. Using STI571 (5 microM, 16 hours) to inhibit the c-Kit pathway following stimulation with SCF (100 ng/mL), an upregulation of topo-I activity was observed in H526 cells but not in H82 cells. Performing viability assays, we show that STI571 in combination with topo-I inhibition by camptothecin or SN38, the active metabolite of irinotecan, can cooperatively inhibit H526 cell viability (but not H82 cell viability) for 72 hours. We also show that STI571 does not directly inhibit topo-I activity in SCLC. The combination of STI571 with topo-I inhibition could provide a useful combination in the treatment of SCLC.

Combined MET inhibition and topoisomerase I inhibition block cell growth of small cell lung cancer.

Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)-induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC.

Protein kinase A binds and activates heat shock factor 1.

BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1), a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA). HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα) and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320), both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1) and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.

The von Hippel-Lindau protein pVHL suppresses renal tumorigenesis in part by promoting the degradation of hypoxia-inducible HIF-alpha transcription factors; additional mechanisms have been proposed. pVHL also stabilizes the plant homeodomain protein Jade-1, which is a candidate renal tumour suppressor that may correlate with renal cancer risk. Here we show that Jade-1 binds the oncoprotein beta-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type beta-catenin but not a cancer-causing form of beta-catenin. Whereas the well-established beta-catenin E3 ubiquitin ligase component beta-TrCP ubiquitylates only phosphorylated beta-catenin, Jade-1 ubiquitylates both phosphorylated and non-phosphorylated beta-catenin and therefore regulates canonical Wnt signalling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of beta-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL downregulates beta-catenin in a Jade-1-dependent manner and inhibits Wnt signalling, supporting a role for Jade-1 and Wnt signalling in renal tumorigenesis. The pVHL tumour suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

Biomarker discovery in lung cancer--promises and challenges of clinical proteomics.

Lung cancer is a devastating illness with an overall poor prognosis. To effectively address this disease, early detection and novel therapeutics are required. Early detection of lung cancer is challenging, in part because of the lack of adequate tumor biomarkers. The goal of this review is to summarize the knowledge of current biomarkers in lung cancer, with a focus on important serum biomarkers. The current knowledge on the known serum cytokines and tumor biomarkers of lung cancer will be presented. Emerging trends and new findings in the search for novel diagnostic and therapeutic tumor biomarkers using proteomics technologies and platforms are emphasized, including recent advances in mass spectrometry to facilitate tumor biomarker discovery program in lung cancer. It is our hope that validation of these new research platforms and technologies will result in improved early detection, prognostication, and finally the treatment of lung cancer with potential novel molecularly targeted therapeutics.