Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level.

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.

The GRHL2/ZEB Feedback Loop-A Key Axis in the Regulation of EMT in Breast Cancer.

More than 90% of cancer-related deaths are caused by metastasis. Epithelial-to-Mesenchymal Transition (EMT) causes tumor cell dissemination while the reverse process, Mesenchymal-to-Epithelial Transition (MET) allows cancer cells to grow and establish a potentially deadly metastatic lesion. Recent evidence indicates that in addition to E and M, cells can adopt a stable hybrid Epithelial/Mesenchymal (E/M) state where they can move collectively leading to clusters of Circulating Tumor Cells-the "bad actors" of metastasis. EMT is postulated to occur in all four major histological breast cancer subtypes. Here, we identify a set of genes strongly correlated with CDH1 in 877 cancer cell lines, and differentially expressed genes in cell lines overexpressing ZEB1, SNAIL, and TWIST. GRHL2 and ESRP1 appear in both these sets and also correlate with CDH1 at the protein level in 40 breast cancer specimens. Next, we find that GRHL2 and CD24 expression coincide with an epithelial character in human mammary epithelial cells. Further, we show that high GRHL2 expression is highly correlated with worse relapse-free survival in all four subtypes of breast cancer. Finally, we integrate CD24, GRHL2, and ESRP1 into a mathematical model of EMT regulation to validate the role of these players in EMT. Our data analysis and modeling results highlight the relationships among multiple crucial EMT/MET drivers including ZEB1, GRHL2, CD24, and ESRP1, particularly in basal-like breast cancers, which are most similar to triple-negative breast cancer (TNBC) and are considered the most dangerous subtype. J. Cell. Biochem. 118: 2559-2570, 2017. © 2017 Wiley Periodicals, Inc.

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

Phenotypic plasticity in prostate cancer: role of intrinsically disordered proteins.

A striking characteristic of cancer cells is their remarkable phenotypic plasticity, which is the ability to switch states or phenotypes in response to environmental fluctuations. Phenotypic changes such as a partial or complete epithelial to mesenchymal transition (EMT) that play important roles in their survival and proliferation, and development of resistance to therapeutic treatments, are widely believed to arise due to somatic mutations in the genome. However, there is a growing concern that such a deterministic view is not entirely consistent with multiple lines of evidence, which indicate that stochasticity may also play an important role in driving phenotypic plasticity. Here, we discuss how stochasticity in protein interaction networks (PINs) may play a key role in determining phenotypic plasticity in prostate cancer (PCa). Specifically, we point out that the key players driving transitions among different phenotypes (epithelial, mesenchymal, and hybrid epithelial/mesenchymal), including ZEB1, SNAI1, OVOL1, and OVOL2, are intrinsically disordered proteins (IDPs) and discuss how plasticity at the molecular level may contribute to stochasticity in phenotypic switching by rewiring PINs. We conclude by suggesting that targeting IDPs implicated in EMT in PCa may be a new strategy to gain additional insights and develop novel treatments for this disease, which is the most common form of cancer in adult men.

Stability of the hybrid epithelial/mesenchymal phenotype.

Epithelial-to-Mesenchymal Transition (EMT) and its reverse - Mesenchymal to Epithelial Transition (MET) - are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During EMT, epithelial cells lose cell-cell adhesion and gain migratory and invasive traits either partially or completely, leading to a hybrid epithelial/mesenchymal (hybrid E/M) or a mesenchymal phenotype respectively. Mesenchymal cells move individually, but hybrid E/M cells migrate collectively as observed during gastrulation, wound healing, and the formation of tumor clusters detected as Circulating Tumor Cells (CTCs). Typically, the hybrid E/M phenotype has largely been tacitly assumed to be transient and 'metastable'. Here, we identify certain 'phenotypic stability factors' (PSFs) such as GRHL2 that couple to the core EMT decision-making circuit (miR-200/ZEB) and stabilize hybrid E/M phenotype. Further, we show that H1975 lung cancer cells can display a stable hybrid E/M phenotype and migrate collectively, a behavior that is impaired by knockdown of GRHL2 and another previously identified PSF - OVOL. In addition, our computational model predicts that GRHL2 can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of these PSFs may be predictive of poor patient outcome. Finally, based on these specific examples, we deduce certain network motifs that can stabilize the hybrid E/M phenotype. Our results suggest that partial EMT, i.e. a hybrid E/M phenotype, need not be 'metastable', and strengthen the emerging notion that partial EMT, but not necessarily a complete EMT, is associated with aggressive tumor progression.

Phosphorylation-induced Conformational Ensemble Switching in an Intrinsically Disordered Cancer/Testis Antigen.

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells (Zeng, Y., He, Y., Yang, F., Mooney, S. M., Getzenberg, R. H., Orban, J., and Kulkarni, P. (2011) The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J. Biol. Chem. 286, 13985-13994). Phosphorylation of PAGE4 at Thr-51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at Thr-51. The population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on Thr-51. This central region therefore becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Although flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at Thr-51, most likely because of masking the effects of the more compact phosphorylated state. Therefore, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble, with large functional consequences. The changes in the structural ensemble induced by posttranslational modifications are similar conceptually to the conformational switching events seen in some marginally stable ("metamorphic") folded proteins in response to mutation or environmental triggers.

OVOL guides the epithelial-hybrid-mesenchymal transition.

Metastasis involves multiple cycles of Epithelial-to-Mesenchymal Transition (EMT) and its reverse-MET. Cells can also undergo partial transitions to attain a hybrid epithelial/mesenchymal (E/M) phenotype that has maximum cellular plasticity and allows migration of Circulating Tumor Cells (CTCs) as a cluster. Hence, deciphering the molecular players helping to maintain the hybrid E/M phenotype may inform anti-metastasis strategies. Here, we devised a mechanism-based mathematical model to couple the transcription factor OVOL with the core EMT regulatory network miR-200/ZEB that acts as a three-way switch between the E, E/M and M phenotypes. We show that OVOL can modulate cellular plasticity in multiple ways - restricting EMT, driving MET, expanding the existence of the hybrid E/M phenotype and turning both EMT and MET into two-step processes. Our theoretical framework explains the differences between the observed effects of OVOL in breast and prostate cancer, and provides a platform for investigating additional signals during metastasis.

Alternative CD44 splicing identifies epithelial prostate cancer cells from the mesenchymal counterparts.

An epithelial to mesenchymal transition (EMT) has been shown to be a necessary precursor to prostate cancer metastasis. Additionally, the differential expression and splicing of mRNAs has been identified as a key means to distinguish epithelial from mesenchymal cells by qPCR, western blotting and immunohistochemistry. However, few markers exist to differentiate between these cells by flow cytometry. We previously developed two cell lines, PC3-Epi (epithelial) and PC3-EMT (mesenchymal). RNAseq was used to determine the differential expression of membrane proteins on PC3-Epi/EMT. We used western blotting, qPCR and flow cytometry to validate the RNAseq results. CD44 was one of six membrane proteins found to be differentially spliced between epithelial and mesenchymal PC3 cells. Although total CD44 was positive in all PC3-Epi/EMT cells, PC3-Epi cells had a higher level of CD44v6 (CD44 variant exon 6). CD44v6 was able to differentiate epithelial from mesenchymal prostate cancer cells using either flow cytometry, western blotting or qPCR.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells.

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

The presence of androgen receptor elements regulates ZEB1 expression in the absence of androgen receptor.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that plays a central role in the epithelial to mesenchymal transition (EMT) of cancer cell lines. Studies on its regulation have mostly focused on the negative 3'UTR binding of miR200c. Interestingly, it has been previously reported that androgen receptor (AR) regulates ZEB1 expression in breast and prostate cancers. In order to validate this, various ZEB1 promoter deletions were cloned into a luciferase reporter system to elucidate the contribution of two putative androgen response elements (AREs). The in vivo contribution of AR was also assessed in cell lines after R1881 treatment using qPCR with prostate specific antigen (PSA) as the positive control. We discovered that AR upregulates the levels of expression of ZEB1 10-fold on a luciferase promoter that only contains the distal ARE. However, when the proximal ARE is included, no additional activation is apparent with AR or its hormone independent variant, AR-V7. Furthermore, we demonstrate here that a promoter construct containing both AREs activates transcription of ZEB1 even in the AR-null cell lines DU145 and PC3. Incubation of the AR-positive cell line, LNCaP with R1881, failed to substantially increase the expression levels of ZEB1. Despite the presence of AREs in the promoter region, it appears that ZEB1 expression can be induced even without AR. In addition, the region around the distal ARE is a potent repressor in AR-null cell lines.

Niche inheritance: a cooperative pathway to enhance cancer cell fitness through ecosystem engineering.

Cancer cells can be described as an invasive species that is able to establish itself in a new environment. The concept of niche construction can be utilized to describe the process by which cancer cells terraform their environment, thereby engineering an ecosystem that promotes the genetic fitness of the species. Ecological dispersion theory can then be utilized to describe and model the steps and barriers involved in a successful diaspora as the cancer cells leave the original host organ and migrate to new host organs to successfully establish a new metastatic community. These ecological concepts can be further utilized to define new diagnostic and therapeutic areas for lethal cancers.

Correlation of Sprouty1 and Jagged1 with aggressive prostate cancer cells with different sensitivities to androgen deprivation.

Prostate cancer is a heterogeneous disease and thus, it is important to understand whether among the heterogeneous collection of cell types, androgen-deprivation insensitive cells exist prior to hormonal manipulation. We established several LNCaP subclones with distinct insensitivities to androgen deprivation from a parental LNCaP cell line. In the resulting clones, the sensitivity to androgen-deprivation negatively correlated with their PSA expression levels. In two of these clones, an androgen insensitive clone, LNCaP-cl1, and an androgen sensitive clone, LNCaP-cl5, the DNA copy number differed significantly, indicating that these clones contain genetically distinct cells. LNCaP-cl1 had higher PSA expression but lower invasiveness and tumor growth potential than LNCaP-cl5. The expression levels of two genes that are known to be regulated by miR-21, an androgen-regulated microRNA, Sprouty1 (SPRY1) and Jagged1 (JAG1) were significantly lower in LNCaP-cl1 than in LNCaP-cl5. Knocking down SPRY1 in LNCaP cells enhanced PSA expression and cell proliferation. JAG1 administration in LNCaP cells enhanced cell invasion and JAG1 knockdown in PC3 cells suppressed cell invasion and tumor formation. These results indicated that the expression differences in SPRY1 and JAG1 may contribute to the phenotypic differences between the LNCaP-cl1 and LNCaP-cl5 clones. In tissue samples, SPRY1 expression levels were significantly lower in prostate cancer patients with PSA recurrence after surgical treatment (P = 0.0076) and JAG1 expression levels were significantly higher in Gleason sum (GS) 8-9 disease than in GS 5-6 (P = 0.0121). In summary a random population of LNCaP cells comprises a heterogeneous group of cells with different androgen-deprivation sensitivities and potential for invasiveness.

Cancer/testis antigen PAGE4, a regulator of c-Jun transactivation, is phosphorylated by homeodomain-interacting protein kinase 1, a component of the stress-response pathway.

Prostate-associated gene 4 (PAGE4) is a cancer/testis antigen that is typically restricted to the testicular germ cells but is aberrantly expressed in cancer. Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the developing prostate and is also a stress-response protein that is upregulated in response to cellular stress. PAGE4 interacts with c-Jun, which is activated by the stress-response kinase JNK1, and plays an important role in the development and pathology of the prostate gland. Here, we have identified homeodomain-interacting protein kinase 1 (HIPK1), also a component of the stress-response pathway, as a kinase that phosphorylates PAGE4 at T51. We show that phosphorylation of PAGE4 is critical for its transcriptional activity since mutating this T residue abolishes its ability to potentiate c-Jun transactivation. In vitro single molecule FRET indicates phosphorylation results in compaction of (still) intrinsically disordered PAGE4. Interestingly, however, while our previous observations indicated that the wild-type nonphosphorylated PAGE4 protein interacted with c-Jun [Rajagopalan , K. et al. ( 2014 ) Biochim, Biophys. Acta 1842 , 154 -163], here we show that phosphorylation of PAGE4 weakens its interaction with c-Jun in vitro. These data suggest that phosphorylation induces conformational changes in natively disordered PAGE4 resulting in its decreased affinity for c-Jun to promote interaction of c-Jun with another, unidentified, partner. Alternatively, phosphorylated PAGE4 may induce transcription of a novel partner, which then potentiates c-Jun transactivation. Regardless, the present results clearly implicate PAGE4 as a component of the stress-response pathway and uncover a novel link between components of this pathway and prostatic development and disease.

The Stress-response protein prostate-associated gene 4, interacts with c-Jun and potentiates its transactivation.

The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Förster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa.

CETN1 is a cancer testis antigen with expression in prostate and pancreatic cancers.

BACKGROUND: The Cancer Testis Antigens (CTAs) are a group of genes that are highly expressed in the normal testis and several types of cancer. Due to their restricted expression in normal adult tissues, CTAs have been attractive targets for immunotherapy and biomarker development. In this work, we discovered that Centrin 1 (CETN1) which is found in the centrosome of all eukaryotes, may be a member of this group and is highly expressed in prostate and pancreatic cancer. Three members of the centrin family of calcium binding proteins (CETN) are localized to the centrosome in all eukaryotes with CDC31 being the sole yeast homolog. CETN1 is a retrogene that probably arose from a retrotransposition of CETN2, an X-linked gene. A previous mouse study shows that CETN1 is expressed solely in the testis, while CETN2 is expressed in all organs. RESULTS: In this work, we show that CETN1 is a new member of the growing group of CTAs. Through the mining of publicly available microarray data, we discovered that human CETN1 expression but not CETN2 or CETN3 is restricted to the testis. In fact, CETN1 is actually down-regulated in testicular malignancies compared to normal testis. Using q-PCR, CETN1 expression is shown to be highly up-regulated in cancer of the prostate and in pancreatic xenografts. Unexpectedly however, CETN1 expression was virtually absent in various cell lines until they were treated with the DNA demethylation agent 5'AZA-2'Deoxycytidine (AZA) but showed no increased expression upon incubation with Histone deacetylase inhibitor Trichostatin-A (TSA) alone. Additionally, like most CTAs, CETN1 appears to be an intrinsically disordered protein which implies that it may occupy a hub position in key protein interaction networks in cancer. Neither CETN1 nor CETN2 could compensate for loss of CDC31 expression in yeast which is analogous to published data for CETN3. CONCLUSIONS: This work suggests that CETN1 is a novel CTA with expression in cancer of the prostate and pancreas. In cell lines, the expression is probably regulated by promoter methylation, while the method of regulation in normal adult tissues remains unknown.

Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer.

Drug resistance is a major limitation to the successful treatment of advanced prostate cancer (PCa). Patients who have metastatic, castration-resistant PCa (mCRPC) are treated with chemotherapeutics. However, these standard therapy modalities culminate in the development of resistance. We established paclitaxel resistance in a classic, androgen-insensitive mCRPC cell line (DU145) and, using a suite of molecular and biophysical methods, characterized the structural and functional changes in vitro and in vivo that are associated with the development of drug resistance. After acquiring paclitaxel-resistance, cells exhibited an abnormal nuclear morphology with extensive chromosomal content, an increase in stiffness, and faster cytoskeletal remodeling dynamics. Compared with the parental DU145, paclitaxel-resistant (DU145-TxR) cells became highly invasive and motile in vitro, exercised greater cell traction forces, and formed larger and rapidly growing tumors in mouse xenografts. Furthermore, DU145-TxR cells showed a discrete loss of keratins but a distinct gain of ZEB1, Vimentin and Snail, suggesting an epithelial-to-mesenchymal transition. These findings demonstrate, for the first time, that paclitaxel resistance in PCa is associated with a trans-differentiation of epithelial cell machinery that enables more aggressive and invasive phenotype and portend new strategies for developing novel biomarkers and effective treatment modalities for PCa patients.

Cancer/testis antigens and urological malignancies.

Cancer/testis antigens (CTAs) are a group of tumour-associated antigens (TAAs) that display normal expression in the adult testis--an immune-privileged organ--but aberrant expression in several types of cancers, particularly in advanced cancers with stem cell-like characteristics. There has been an explosion in CTA-based research since CTAs were first identified in 1991 and MAGE-1 was shown to elicit an autologous cytotoxic T-lymphocyte (CTL) response in a patient with melanoma. The resulting data have not only highlighted a role for CTAs in tumorigenesis, but have also underscored the translational potential of these antigens for detecting and treating many types of cancers. Studies that have investigated the use of CTAs for the clinical management of urological malignancies indicate that these TAAs have potential roles as novel biomarkers, with increased specificity and sensitivity compared to those currently used in the clinic, and therapeutic targets for cancer immunotherapy. Increasing evidence supports the utilization of these promising tools for urological indications.

Cyr61 is regulated by cAMP-dependent protein kinase with serum levels correlating with prostate cancer aggressiveness.

BACKGROUND: Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previously, we demonstrated that Cyr61 was highly expressed in prostate cancer (PCa) but that the expression levels were associated with a lower risk of PCa recurrence. In the present study, we demonstrate that serum Cyr61 is a potential biomarker that correlates with PCa aggressiveness. Furthermore, we also explore the potential mechanism underlying the changes in Cyr61 expression during PCa progression. METHODS: Cyr61 concentrations in the medium from PCa cell lines and in serum samples obtained from PCa patients were measured by sandwich ELISA. Serum Cyr61 levels were correlated with disease characteristics and the association between Cyr61 expression changes by several types of stimulation or stress and cAMP/cAMP-dependent protein kinase (PKA) pathway were examined. RESULTS: There was a positive correlation between Cyr61 levels in cell supernatants and mRNA expression in these cell lines. Serum Cyr61 levels were significantly higher in non-organ-confined PCa patients (116.3 ± 140.2 ng/ml) than in organ-confined PCa patients (79.7 ± 56.1 ng/ml) (P = 0.031). Cyr61 expression was up-regulated in response to both lysophosphatidic acid and androgen treatments which promoted PCa cell invasion. Serum starvation and phosphoinositide-3-kinase inhibition also resulted in Cyr61 up-regulation; however, they suppressed cell proliferation. Cyr61 up-regulation was correlated with an increase in cAMP and suppressed by PKA inhibition. CONCLUSIONS: These findings suggest that Cyr61 expression in PCa is regulated by the cAMP/PKA pathway and that circulating Cyr61 levels are a potential serum-based biomarker for characterizing PCa.

A majority of the cancer/testis antigens are intrinsically disordered proteins.

The cancer/testis antigens (CTAs) are a group of heterogeneous proteins that are typically expressed in the testis but aberrantly expressed in several types of cancer. Although overexpression of CTAs is frequently associated with advanced disease and poorer prognosis, the significance of this correlation is unclear since the functions of the CTAs in the disease process remain poorly understood. Here, employing a bioinformatics approach, we show that a majority of CTAs are intrinsically disordered proteins (IDPs). IDPs are proteins that, under physiological conditions in vitro, lack rigid 3D structures either along their entire length or in localized regions. Despite the lack of structure, most IDPs can transition from disorder to order upon binding to biological targets and often promote highly promiscuous interactions. IDPs play important roles in transcriptional regulation and signaling via regulatory protein networks and are often associated with dosage sensitivity. Consistent with these observations, we find that several CTAs can bind DNA, and their forced expression appears to increase cell growth implying a potential dosage-sensitive function. Furthermore, the CTAs appear to occupy "hub" positions in protein regulatory networks that typically adopt a "scale-free" power law distribution. Taken together, our data provide a novel perspective on the CTAs implicating them in processing and transducing information in altered physiological states in a dosage-sensitive manner. Identifying the CTAs that occupy hub positions in protein regulatory networks would allow a better understanding of their functions as well as the development of novel therapeutics to treat cancer.