Targeted proteomics of potential biomarkers is often challenging. Hence, we developed an intermediate workflow to streamline potential urinary biomarkers of prostate cancer (PCa).
Using previously ...discovered potential PCa biomarkers; we selected proteotypic peptides for targeted validation. Preliminary in silico immunohistochemical and single reaction monitoring (SRM) verification was performed. Successful PTPs were then prevalidated using parallel reaction monitoring (PRM) and reconfirmed in 15 publicly available databases.
Stringency-based targetable potential biomarkers were shortlisted following in silico screening. PRM reveals top 12 potential biomarkers including the top ranking seven in silico verification-based biomarkers. Database reconfirmation showed differential expression between PCa and benign/normal prostatic urine samples.
The pragmatic penultimate screening step, described herein, would immensely improve targeted proteomics validation of potential disease biomarkers.
The NF-κB/IκB signaling pathway is a critical regulator of cell survival in cancer. Here, we report that combined down-regulation of growth arrest- and DNA-damage-inducible proteins (GADD)45α and γ ...expression by NF-κB is an essential step for various cancer types to escape programmed cell death. We demonstrate that inhibition of NF-κB in cancer cells results in GADD45α- and γ-dependent induction of apoptosis and inhibition of tumor growth. Inhibition of GADD45α and γ in cancer cells by small interfering RNA abrogates apoptosis induction by the inhibitor of NF-κB and blocks c-Jun N-terminal kinase activation, whereas overexpression of GADD45α and γ activates c-Jun N-terminal kinase and induces apoptosis. These results establish an unambiguous role for the GADD45 family as an essential mediator of cell survival in cancer cells with implications for cancer chemotherapy and novel drug discovery.
Cervical cancer is the third most frequent cancer in women worldwide. Adenine nucleotide signaling is modulated by the ectonucleotidases that act in sequence, forming an enzymatic cascade. ...Considering the relationship between the purinergic signaling and cancer, we studied the E-NTPDases, ecto-5′-nucleotidase, and E-NPPs in human cervical cancer cell lines and keratinocytes. We evaluated the expression profiles of these enzymes using RT-PCR and quantitative real-time PCR analysis. The activities of these enzymes were examined using ATP, ADP, AMP, and p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as substrate, in a colorimetric assay. The extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The hydrolysis of all substrates exhibited a linear pattern and these activities were cation-dependent. An interesting difference in the degradation rate was observed between cervical cancer cell lines SiHa, HeLa, and C33A and normal imortalized keratinocytes, HaCaT cells. The mRNA of ecto-5′-nucleotidase, E-NTPDases 5 and 6 were detectable in all cell lines, and the dominant gene expressed was the Entpd 5 enzyme, in SiHa cell line (HPV16 positive). In accordance with this result, a higher hydrolysis activity for UDP and GDP nucleotides was observed in the supernatant of the SiHa cells. Both normal and cancer cells presented activity and mRNAs of members of the NPP family. Considering that these enzymes exert an important catalytic activity, controlling purinergic nucleotide concentrations in tumors, the presence of ectonucleotidases in cervical cancer cells can be important to regulate the levels of extracellular adenine nucleotides, limiting their effects.
Purpose
Improvement in diagnostic accuracy of prostate cancer (PCa) progression using MS‐based methods to analyze biomarkers in our African, Caucasian, and Mixed Ancestry patients can advance early ...detection and treatment monitoring.
Experimental design
MS‐based proteomic analysis of pooled (N = 36) and individual samples (N = 45) of PCa, benign prostatic hyperplasia, normal healthy controls, and patients with other uropathies was used to identify differences in proteomics profile. Samples were analyzed for potential biomarkers and proteome coverage in African, Caucasian, and Mixed Ancestry PCa patients.
Results
A total of 1102 and 5595 protein groups and nonredundant peptides, respectively, were identified in the pooling experiments (FDR = 0.01). Twenty potential biomarkers in PCa were identified and fold differences ± 2SD were observed in 17 proteins using intensity‐based absolute quantification. Analysis of 45 individual samples yielded 1545 and 9991 protein groups and nonredundant peptides, respectively. Seventy‐three (73) proteins groups, including existing putative PCa biomarkers, were found to be potential biomarkers of PCa by label‐free quantification and demonstrated ethnic trends within our PCa cohort.
Conclusion and clinical relevance
Urinary proteomics is a promising route to PCa biomarker discovery and may serve as source of ethnic‐related biomarkers of PCa.
GADD45β (growth arrest- and DNA damage-inducible) interacts with upstream regulators of the JNK and p38 stress response kinases. Previously, we reported that the hypertrophic zone of the Gadd45β−/− ...mouse embryonic growth plate is compressed, and expression of type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13) genes is decreased. Herein, we report that GADD45β enhances activity of the proximal Col10a1 promoter, which contains evolutionarily conserved AP-1, cAMP-response element, and C/EBP half-sites, in synergism with C/EBP family members, whereas the MMP13 promoter responds to GADD45β together with AP-1, ATF, or C/EBP family members. C/EBPβ expression also predominantly co-localizes with GADD45β in the embryonic growth plate. Moreover, GADD45β enhances C/EBPβ activation via MTK1, MKK3, and MKK6, and dominant-negative p38αapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45β and C/EBPβ on the Col10a1 promoter. Importantly, GADD45β knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPβ binding to the Col10a1 promoter in vivo, indicating that GADD45β influences the transactivation function of DNA-bound C/EBPβ. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPβ is the target of the GADD45β-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45β via the MTK1/MKK3/6/p38 axis to C/EBPβ-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.
We performed a time-course microarray experiment to define the transcriptional response to carboplatin in vitro, and to correlate this with clinical outcome in epithelial ovarian cancer (EOC). RNA ...was isolated from carboplatin and control-treated 36M2 ovarian cancer cells at several time points, followed by oligonucleotide microarray hybridization. Carboplatin induced changes in gene expression were assessed at the single gene as well as at the pathway level. Clinical validation was performed in publicly available microarray datasets using disease free and overall survival endpoints.
Time-course and pathway analyses identified 317 genes and 40 pathways (designated time-course and pathway signatures) deregulated following carboplatin exposure. Both types of signatures were validated in two separate platinum-treated ovarian and NSCLC cell lines using published microarray data. Expression of time-course and pathway signature genes distinguished between patients with unfavorable and favorable survival in two independent ovarian cancer datasets. Among the pathways most highly induced by carboplatin in vitro, the NRF2, NF-kB, and cytokine and inflammatory response pathways were also found to be upregulated prior to chemotherapy exposure in poor prognosis tumors.
Dynamic assessment of gene expression following carboplatin exposure in vitro can identify both genes and pathways that are correlated with clinical outcome. The functional relevance of this observation for better understanding the mechanisms of drug resistance in EOC will require further evaluation.
The epithelium-specific Ets transcription factor, SPDEF, plays a critical role in metastasis of prostate and breast cancer cells. While enhanced SPDEF expression blocks migration and invasion, ...knockdown of SPDEF expression enhances migration, invasion, and metastasis of cancer cells. SPDEF expression and activation is tightly regulated in cancer cells; however, the precise mechanism of SPDEF regulation has not been explored in detail. In this study we provide evidence that the cell cycle kinase CDK11p58, a protein involved in G2/M transition and degradation of several transcription factors, directly interacts with and phosphorylates SPDEF on serine residues, leading to subsequent ubiquitination and degradation of SPDEF through the proteasome pathway. As a consequence of CDK11p58 mediated degradation of SPDEF, this loss of SPDEF protein results in increased prostate cancer cell migration and invasion. In contrast, knockdown of CDK11p58 protein expression by interfering RNA or SPDEF overexpression inhibit migration and invasion of cancer cells. We demonstrate that CDK11p58 mediated degradation of SPDEF is attenuated by Growth Arrest and DNA damage-inducible 45 (GADD45) α and , two proteins inducing G2/M cell cycle arrest. We show that GADD45 α and γ, directly interact with CDK11p58 and thereby inhibit CDK11p58 activity, and consequentially SPDEF phosphorylation and degradation, ultimately reducing prostate cancer cell migration and invasion. Our findings provide new mechanistic insights into the complex regulation of SPDEF activity linked to cancer metastasis and characterize a previously unidentified SPDEF/CDK11p58/GADD45α/γ pathway that controls SPDEF protein stability and SPDEF-mediated effects on cancer cell migration and invasion.
The NF-kB/IkB signaling pathway is a critical regulator of cell survival, and constitutive activation of NF-kB is a crucial step for many types of cancers to escape programmed cell death. ...Furthermore, chemotherapeutic agents activate NF-kB in cancer cells, and this may partially explain the resistance of cancer cells to chemotherapy. The precise mechanism of the anti-apoptotic action of NF-kB is not known, but involves the regulation of several cell cycle regulatory and anti-apoptotic genes. We recently demonstrated that NF-kB mediated cell survival is absolutely dependent on two GADD45 family members, GADD45a and g. In line with this, inhibition of NF-kB in cancer cells results in GADD45a and g dependent induction of apoptosis, JNK activation and inhibition of tumor growth. These findings establish an unambiguous role for the GADD45 family as an essential mediator of cell survival in cancer cells with implications for cancer chemotherapy and novel drug discovery.
The growth arrest and DNA damage-inducible 45β (GADD45β) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of ...GADD45β in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45β as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45β mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45β protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45β-/- mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45β in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45β stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45β plays an essential role during chondrocyte terminal differentiation.
Prostate cancer (PCa) is one of the most common malignancies found in males. The development of PCa involves several mutations in prostate epithelial cells, usually linked to developmental changes, ...such as enhanced resistance to apoptotic death, constitutive proliferation, and, in some cases, to differentiation into an androgen deprivation-resistant phenotype, leading to the appearance of castration-resistant PCa (CRPCa), which leads to a poor prognosis in patients. In this review, we summarize recent findings concerning the main deregulations into signaling pathways that will lead to the development of PCa and/or CRPCa. Key mutations in some pathway molecules are often linked to a higher prevalence of PCa, by directly affecting the respective cascade and, in some cases, by deregulating a cross-talk node or junction along the pathways. We also discuss the possible environmental and nonenvironmental inducers for these mutations, as well as the potential therapeutic strategies targeting these signaling pathways. A better understanding of how some risk factors induce deregulation of these signaling pathways, as well as how these deregulated pathways affect the development of PCa and CRPCa, will further help in the development of new treatments and prevention strategies for this disease.
PDF
