Krithika Rajagopalan

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. J. Cell. Biochem. 112: 3256–3267, 2011.

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. J. Cell. Biochem. 114: 1286–1293, 2013.

Intrinsically disordered proteins and conformational noise: Implications in cancer

Intrinsically disordered proteins, IDPs, are proteins that lack a rigid 3D structure under physiological conditions, at least in vitro. Despite the lack of structure, IDPs play important roles in biological processes and transition from disorder to order upon binding to their targets. With multiple conformational states and rapid conformational dynamics, they engage in myriad and often “promiscuous” interactions. These stochastic interactions between IDPs and their partners, defined here as conformational noise, is an inherent characteristic of IDP interactions. The collective effect of conformational noise is an ensemble of protein network configurations, from which the most suitable can be explored in response to perturbations, conferring protein networks with remarkable flexibility and resilience. Moreover, the ubiquitous presence of IDPs as transcriptional factors and, more generally, as hubs in protein networks, is indicative of their role in propagation of transcriptional (genetic) noise. As effectors of transcriptional and conformational noise, IDPs rewire protein networks and unmask latent interactions in response to perturbations. Thus, noise-driven activation of latent pathways could underlie state-switching events such as cellular transformation in cancer. To test this hypothesis, we created a model of a protein network with the topological characteristics of a cancer protein network and tested its response to a perturbation in presence of IDP hubs and conformational noise. Because numerous IDPs are found to be epigenetic modifiers and chromatin remodelers, we hypothesize that they could further channel noise into stable, heritable genotypic changes.

Creatine kinase brain overexpression protects colorectal cells from various metabolic and non‐metabolic stresses

Creatine kinase brain (CKB) is one of three cytosolic isoforms of creatine kinase that is predominantly expressed in the brain. The enzyme is overexpressed in a wide variety of cancers, with the exception of colon cancer, where it is downregulated. The significance of this downregulation remains poorly understood. Here, we demonstrate that overexpression of CKB‐C283S, a dominant‐negative construct that lacks the kinase function but retains its ability to dimerize, causes remarkable changes in cell shape, adhesion, and invasion. Furthermore, it results in increased expression of stromal cell markers such as PAGE4 and SNAIL, suggesting an epithelial‐to‐mesenchymal transition (EMT) in these cells. In cells transfected with a CKB‐expressing construct, CKB localizes not only to the cytosol but also to the nucleus, indicating a structural or kinase role unrelated to ATP storage. Furthermore, overexpression of CFP‐tagged wild‐type (WT) CKB in Caco‐2 colon cancer cells dramatically increased the number of cells in G2/M but had little effect on cell proliferation. Taken together, these data demonstrate that the downregulation of CKB may play an important role in colon cancer progression by promoting EMT. J. Cell. Biochem. 112: 1066–1075, 2011.

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

Background: PAGE4, an intrinsically disordered protein up-regulated in prostate cancer, binds to c-Jun and potentiates its transactivation. Results: The effects of phosphorylation on PAGE4 conformation, dynamics, and c-Jun binding were determined by NMR. Conclusion: Phosphorylation induces a more compact conformational ensemble, restricting access to the c-Jun binding site. Significance: This study may help to explain how phosphorylation of PAGE4 alters its binding to c-Jun. 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.

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 [RajagopalanK. et al. (2014) Biochim, Biophys. Acta1842, 154−16324263171], 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.

Protein Folding and the Order/Disorder Paradox

Most proteins encoded by the nuclear genome are synthesized in the cytoplasm and fold into precise 3D structures. During synthesis, the nascent polypeptide begins to fold as it traverses the large subunit of the ribosome and is assisted by molecular chaperones in attaining its precise folded/highly ordered state efficiently and in a biologically relevant timescale. Proteins that are misfolded are culled, re‐routed, and marked by mechanisms such as ubiquitinylation for degradation ensuring strict quality control (QC). In addition to the highly ordered “globular” proteins, emerging evidence indicates that a large fraction of the proteome also comprises the so‐called “Intrinsically Disordered Proteins” (IDPs). IDPs are proteins that lack rigid 3D structures and instead, exist as dynamic ensembles. The dynamic structures in the IDPs have many similarities with “normal” globular proteins such as the native (ordered), and non‐native (molten globule, pre‐molten globule, and coil‐like) states seen during folding of “normal” globular proteins. However, unlike the case of the nascent globular proteins, IDPs evade being detected as “misfolded” and degraded by the cells QC system. We refer to this paradox as the order/disorder paradox and postulate that the IDPs capitalize on their intrinsic promiscuity and ability to undergo disorder‐to‐order transitions upon binding to biological targets (coupled folding and binding) to escape the cells surveillance machinery. Understanding the mechanism by which the IDPs evade the quality check has wide implications from protein folding to disease biology since the aggregation of misfolded proteins underlies several debilitating illnesses such as many neurodegenerative diseases and cancer. J. Cell. Biochem. 112: 1949–1952, 2011.

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

BackgroundThe 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.ResultsIn 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.ConclusionsThis 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.

Cancer/testis antigens and obligate participation in multiple hallmarks of cancer: an update

The Cancer/Testis Antigens (CTAs) are a group of so-called tumor antigens that exhibit provocative expression patterns in most types of cancer. However, because most CTAs appear to be intrinsically disordered, they are recalcitrant to classical structural studies. Thus, the functions of most, if not all, CTAs have remained elusive and their potential as pharmacological targets in cancer has remained largely unexplored. In this viewpoint, we suggest that perhaps, an integrative approach applying dynamical systems theory and a system-level perspective rather a merely reductionist view, may shine new light on these enigmatic molecules.