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Supplementary Figures 1-3 from Coordination of Centrosome Homeostasis and DNA Repair Is Intact in MCF-7 and Disrupted in MDA-MB 231 Breast Cancer Cells

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.

Current clinical tests for prostate cancer (PCa), such as the PSA test, are not fully capable of discerning patients that are highly likely to develop metastatic prostate cancer (MPCa). Hence, more accurate prediction tools are needed to provide treatment strategies that are focused on the different risk groups. Cancer/testis antigens (CTAs) are expressed during embryonic development and present aberrant expression in cancer making them ideal tumor specific biomarkers. Here, the potential use of a panel of CTAs as a biomarker for PCa detection as well as metastasis prediction is explored. We initially identified eight CTAs (CEP55, NUF2, PAGE4, PBK, RQCD1, SPAG4, SSX2andTTK) that are differentially expressed in MPCa when compared to local disease and used this panel to compare the gene and protein expression profiles in paired PCa and normal adjacent prostate tissue. We identified differential expression of all eight CTAs at the protein level when comparing 80 paired samples of PCa and the adjacent non-cancer tissue. Using multiple logistic regression we also show that a panel of these CTAs present high accuracy to discriminate normal from tumor samples. In summary, this study provides evidence that a panel of CTAs, differentially expressed in aggressive PCa, is a potential biomarker for diagnosis and prognosis to be used in combination with the current clinically available tools and is also a potential target for immunotherapy development.

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.

// Mohit Kumar Jolly 1,2 , Satyendra C. Tripathi 8,* , Dongya Jia 1,5,* , Steven M. Mooney 7 , Muge Celiktas 8 , Samir M. Hanash 8,10 , Sendurai A. Mani 9,11 , Kenneth J. Pienta 12 , Eshel Ben-Jacob 1,5,6,** and Herbert Levine 1,2,3,4 1 Center for Theoretical Biological Physics, Rice University, Houston, TX, USA 2 Department of Bioengineering, Rice University, Houston, TX, USA 3 Department of Physics and Astronomy, Rice University, Houston, TX, USA 4 Department of Biosciences, Rice University, Houston, TX, USA 5 Graduate Program in Systems, Synthetic and Physical Biology, Rice University, Houston, TX, USA 6 School of Physics and Astronomy and The Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv, Israel 7 Department of Biology, University of Waterloo, Waterloo, ON, Canada 8 Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, TX, USA 9 Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 10 Red and Charline McCombs Institute for the Early Detection and Treatment of Cancer, University of Texas MD Anderson Cancer Center, Houston, TX, USA 11 Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, TX, USA 12 The James Brady Urological Institute, and Departments of Urology, Oncology, Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA * These authors have contributed equally to this work ** Deceased on June 5, 2015 Correspondence to: Herbert Levine, email: // Keywords : partial EMT, epithelial-mesenchymal transition, cancer stem cells, multistability, cell-fate decisions Received : January 08, 2016 Accepted : March 07, 2016 Published : March 17, 2016 Abstract 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.

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.

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.

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.

// Takumi Shiraishi 1 , James E. Verdone 1 , Jessie Huang 5 , Ulf D. Kahlert 6 , James R. Hernandez 1 , Gonzalo Torga 1 , Jelani C. Zarif 1 , Tamir Epstein 7 , Robert Gatenby 7 , Annemarie McCartney 8 , Jennifer H. Elisseeff 8 , Steven M. Mooney 1 , Steven S. An 4,5,9 and Kenneth J. Pienta 1,2,3,4 1 Department of Urology, The James Buchanan Brady Urological Institute, Johns Hopkins University, Baltimore, MD, USA 2 Department of Oncology, Johns Hopkins University, Baltimore, MD, USA 3 Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA 4 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA 5 Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA 6 Department of Pathology, Division of Neuropathology, The Johns Hopkins Hospital, Baltimore, MD, USA 7 Department of Integrative Mathematical Oncology and Department of Diagnostic Imaging, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA 8 The Wilmer Eye Institute and Department of Biomedical Engineering, Baltimore, MD, USA 9 In Vivo Cellular and Molecular Imaging Center, The Johns Hopkins School of Medicine, Baltimore, MD, USA Correspondence: Kenneth J. Pienta, email: // Steven S. An, email: // Keywords : cytoskeleton, motility, cancer metabolism, glycolysis, metastasis Received : September 23, 2014 Accepted : November 15, 2014 Published : November 16, 2014 Abstract 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.

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.

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 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. 115: 1478–1485, 2014. © 2014 Wiley Periodicals, Inc.

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.

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.

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.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.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.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.

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.

The microenvironment is central to many aspects of cancer pathobiology and has been proposed to play a role in the development of cancer cell resistant to therapy. To examine the response to microenvironmental conditions, two paclitaxel resistant prostate cancer cell lines (stable and reversible) and one reversible heat resistant cell line were studied. In comparison to their parental cell lines, both paclitaxel resistant cell lines (stable and reversible) were more sensitive to microenvironmental heat, potentially yielding a synergistic therapeutic opportunity. In the two phenotypic cells repopulated after acute heat or paclitaxel treatments, there was an inverse correlation between paclitaxel and heat resistance: resistance to paclitaxel imparted sensitivity to heat; resistance to heat imparted sensitivity to paclitaxel. These studies indicate that as cancer cells evolve resistance to single microenvironmental stress they may be more sensitive to others, perhaps allowing us to design new approaches for prostate cancer therapy.

When cells encounter substantial DNA damage, critical cell cycle events are halted while DNA repair mechanisms are activated to restore genome integrity. Genomic integrity also depends on proper assembly and function of the bipolar mitotic spindle, which is required for equal chromosome segregation. Failure to execute either of these processes leads to genomic instability, aging, and cancer. Here, we show that following DNA damage in the breast cancer cell line MCF-7, the centrosome protein centrin2 moves from the cytoplasm and accumulates in the nucleus in a xeroderma pigmentosum complementation group C protein (XPC)–dependent manner, reducing the available cytoplasmic pool of this key centriole protein and preventing centrosome amplification. MDA-MB 231 cells do not express XPC and fail to move centrin into the nucleus following DNA damage. Reintroduction of XPC expression in MDA-MB 231 cells rescues nuclear centrin2 sequestration and reestablishes control against centrosome amplification, regardless of mutant p53 status. Importantly, the capacity to repair DNA damage was also dependent on the availability of centrin2 in the nucleus. These observations show that centrin and XPC cooperate in a reciprocal mechanism to coordinate centrosome homeostasis and DNA repair and suggest that this process may provide a tractable target to develop treatments to slow progression of cancer and aging. Cancer Res; 70(8); 3320–8. ©2010 AACR.

Mitochondria generate adenosine triphosphate (ATP) but also dangerous reactive oxygen species (ROS). One-electron reduction of dioxygen in the early stages of the electron transport chain yields a superoxide radical that is detoxified by mitochondrial superoxide dismutase to give hydrogen peroxide. The hydroxyl radical is derived from decomposition of hydrogen peroxide via the Fenton reaction, catalyzed by Fe2+ ions. Mitochondria require a constant supply of Fe2+ for heme and iron-sulfur cluster biosyntheses and therefore are particularly susceptible to ROS attack. Two main antioxidant defenses are known in mitochondria: enzymes that catalytically remove ROS, e.g. superoxide dismutase and glutathione peroxidase, and low molecular weight agents that scavenge ROS, including coenzyme Q, glutathione, and vitamins E and C. An effective defensive system, however, should also involve means to control the availability of pro-oxidants such as Fe2+ ions. There is increasing evidence that this function may be carried out by the mitochondrial protein frataxin. Frataxin deficiency is the primary cause of Friedreich's ataxia (FRDA), an autosomal recessive degenerative disease. Frataxin is a highly conserved mitochondrial protein that plays a critical role in iron homeostasis. Respiratory deficits, abnormal cellular iron distribution and increased oxidative damage are associated with frataxin defects in yeast and mouse models of FRDA. The mechanism by which frataxin regulates iron metabolism is unknown. The yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of oxygen and assembles stepwise into a 48-subunit multimer (alpha48) that sequesters2000 atoms of iron in a ferrihydrite mineral core. Assembly of mYfhlp is driven by two sequential iron oxidation reactions: a fast ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (alpha --alpha3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding alpha48. Depending on the ionic environment, stepwise assembly is associated with the sequestration ofor = 50-75 Fe(II)/subunit. This Fe(II) is initially loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. However, as iron oxidation and mineralization proceed, Fe(III) becomes progressively inaccessible and a stable iron-protein complex is produced. In conclusion, by coupling iron oxidation with stepwise assembly, frataxin can successively function as an iron chaperon or an iron store. Reduced iron availability and solubility and increased oxidative damage may therefore explain the pathogenesis of FRDA.

// Dongya Jia 1, 2, * , Mohit Kumar Jolly 1, 3, * , Marcelo Boareto 1, 7 , Princy Parsana 8 , Steven M. Mooney 9, 10 , Kenneth J. Pienta 9, 10, 11, 12 , Herbert Levine 1, 3, 4 , Eshel Ben-Jacob 1, 5, 6 1 Center for Theoretical Biological Physics, Rice University, Houston, TX 77005-1827, USA 2 Graduate Program in Systems, Synthetic and Physical Biology, Rice University, Houston, TX 77005-1827, USA 3 Department of Bioengineering, Rice University, Houston, TX 77005-1827, USA 4 Department of Physics and Astronomy, Rice University, Houston, TX 77005-1827, USA 5 Department of Biosciences, Rice University, Houston, TX 77005-1827, USA 6 School of Physics and Astronomy and The Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv 69978, Israel 7 Institute of Physics, University of Sao Paulo, Sao Paulo 05508, Brazil 8 Department of Computer Science, Johns Hopkins University, Baltimore, MD 21287, USA 9 The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 10 Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 11 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 12 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA * These authors have contributed equally to this work Correspondence to: Kenneth J. Pienta, e-mail: kpienta1@jhmi.edu Herbert Levine, e-mail: herbert.levine@rice.edu Eshel Ben-Jacob, e-mail: eshel@rice.edu Keywords: EMT, metastasis, OVOL, partial EMT, cancer systems biology Received: March 04, 2015 Accepted: April 10, 2015 Published: April 22, 2015 ABSTRACT 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.

Mitochondrial function depends on a continuous supply of iron to the iron-sulfur cluster (ISC) and heme biosynthetic pathways as well as on the ability to prevent iron-catalyzed oxidative damage. The mitochondrial protein frataxin plays a key role in these processes by a novel mechanism that remains to be fully elucidated. Recombinant yeast and human frataxin are able to self-associate in large molecular assemblies that bind and store iron as a ferrihydrite mineral. Moreover, either single monomers or polymers of human frataxin have been shown to serve as donors of Fe(II) to ISC scaffold proteins, oxidatively inactivated [3Fe-4S](+) aconitase, and ferrochelatase. These results suggest that frataxin can use different molecular forms to accomplish its functions. Here, stable monomeric and assembled forms of human frataxin purified from Escherichia coli have provided a tool for testing this hypothesis at the biochemical level. We show that human frataxin can enhance the availability of Fe(II) in monomeric or assembled form. However, the monomer is unable to prevent iron-catalyzed radical reactions and the formation of insoluble ferric iron oxides. In contrast, the assembled protein has ferroxidase activity and detoxifies redox-active iron by sequestering it in a protein-protected compartment.

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. Electronic supplementary material The online version of this article (doi:10.1007/s12032-015-0593-z) contains supplementary material, which is available to authorized users.

Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --alpha(3)). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios ofor = 0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions. The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction. However, only a fraction of the expected total H(2)O(2) is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2). This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer.

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.

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.

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.

formin. Moreover, we also found that metformin inhibited expression of TWIST-1, a transcriptional activator of N-cadherin. Similar findings were observed by confocal microscopy. PC3 prostate cancer cells with stable over-expression of TWIST-1 became resistant to metforminmediated inhibition. CONCLUSIONS: We demonstrate that Metformin’s anti-cancer therapeutic effect may be mediated through repression of the TWIST/ N-cadherin signaling pathway.

Chemotherapy Increases Aggressiveness of Prostate Cancer via Epithelial Mesenchymal Transition Although chemotherapy has been used as a major therapeutic weapon against advanced prostate cancer, overcoming drug resistance is still a major challenge. Cancer has been known to diminish therapeutic effect by several molecular mechanisms– increasing efflux by recruiting cell membrane pumps and limiting drug diffusion by constructing leaky vasculature. Moreover, recent studies suggest that long-term treatment with chemotherapy may have detrimental effects for patients presenting with solid tumors. Specifically, prostate cancer epithelial cells develop resistance to chemotherapy and may convert into mesenchymal cells. This switching process, known as Epithelial to Mesenchymal Transition (EMT), is a critical precursor in prostate cancer invasion and metastasis.

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is typically restricted to the normal testis but is aberrantly expressed in prostate cancer (PCa). Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the fetal prostate and is also a stress-response protein that is up-regulated in response to cellular stress. PAGE4 interacts with c-Jun and plays an important role in the development and pathology of the prostate gland. HIPK1, a component of the stress-response pathway, and CLK2, a dual specificity kinase are kinases that phosphorylate PAGE4 at predominantly at T51 and at multiple S/T residues, respectively. While phosphorylation at T51 is critical in potentiating c-Jun, the functional significance of the multisite phosphorylation by CLK2 is not known. The aim of this study was to determine the expression levels of HIPK1 and CLK2 in PCa cell lines, and in a set of 80 paired cases of PCa and benign adjacent tissue. Protein levels of HIPK1 and CLK2 were evaluated in cell lines and tissue samples by Western Blotting (WB) and immunohistochemistry (IHC), respectively. WB was performed with whole cell lysates from BPH1, DU145, LNCAP and PC3 cells using a fluorescence approach. IHC was performed with TMAs containing paired tumor and benign samples and after scanning (Aperio ScanScope), data were quantified using the ImageScope software. WB showed that HIPK1 is expressed in all cell lines selected with no significant changes in expression levels, while CLK2 was expressed only in BPH1 and LNCAP, suggesting this protein is expressed in cells displaying a less aggressive phenotype. Protein expression analysis by IHC in PCa and benign samples showed that CLK2 is more commonly expressed in PCa than HIPK1 when compared to non-cancer cases. CLK2 expression was positive in 81.7% of the cancer cases and HIPK1 was expressed in 72.2%. When compared to benign cases these differences are significant with p Citation Format: Luciane T. Kagohara, Steven Mooney, Yihong Chen, John Orban, Prakash Kulkarni, Robert W. Veltri. Phosphorylation of the intrinsically disordered cancer/testis antigen PAGE4 by HIPK1 and CLK2 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4970.

Prostate cancer (PCa) is one of the most heritable cancers. However, germline genetic variations that are consistently and highly associated with PCa have remained elusive. Recently, a variety of non-synonymous SNPs in HOXB13 have been identified in prostate cancer patients from distinct ethnic populations. Among the HOXB13 variants, HOXB13 G84E has been consistently shown to associate strongly with increased PCa risk in men of European descent. HOXB13 is a prostate-specific transcription factor that plays a role in prostate development. HOXB13 has also been implicated in various aspects of PCa biology. However, to this date, the exact role of HOXB13 in normal prostate physiology and in PCa biology remains obscure. Our lab previously reported that neither HOXB13 WT nor G84E alone can transform prostate cells and that the G84E variant does not behave differently from HOXB13 WT in interactions with cofactors (AR, MEIS2) and protein half-life. To further delineate the function of HOXB13 in the prostate and to identify G84E-induced alterations that subject G84E carriers to PCa susceptibility, we performed RNA-seq of the LAPC4 prostate cancer cell line overexpressing a control vector, HOXB13 WT or G84E. Comparisons of the RNA-seq datasets were made using Ingenuity Pathway Analysis (IPA) by selecting genes whose expression was altered by 2 standard deviations or higher. Among pathways altered by HOXB13 WT or G84E compared to the vector control dataset, the VDR/RXR activation pathway was negatively regulated by both WT and G84E. When the G84E dataset was compared to the WT dataset, the IPA analysis revealed that G84E can up-regulate the e-NOS signaling pathway. Interestingly, the top contributor to the difference imparted by HOXB13 G84E compared to WT was identified to be a set of genes regulated by HOXB13 itself. Overall, this preliminary analysis suggests that HOXB13 G84E may be a gain-of-function mutation whereby potential tumor-promoting functions of HOXB13 are over-activated. Finally, to further identify targets directly regulated by HOXB13 WT and G84E, we combined the RNA-seq datasets with HOXB13 overexpression ChIP-seq datasets we previously reported and a HOXB13 knockdown dataset in LAPC4 (Norris et al. 2009). We focused initially on genes that were shown to be up- or down-regulated in the same direction in the RNA-seq and HOXB13 knockdown datasets. Those genes were screened for nearby HOXB13 binding sites as annotated in our ChIP-seq analyses, followed by validation with droplet digital PCR. Among the genes that were down-regulated by HOXB13 WT and G84E include INPP4B, TNFSF10, IGFBP3, KLF5 and SOX9, which all have tumor suppressing functions in PCa. Among HOXB13-upregulated genes was BCHE, which is also implicated in the suppression of PCa initiation and progression. These results provide us with potential areas of further investigation in which HOXB13 G84E may differentially regulate transcription of these genes. Citation Format: Dorhyun Johng, Michael C. Haffner, Steven M. Mooney, David M. Esopi, Charles M. Ewing, Shuangling Chen, William B. Isaacs. Global analyses of HOXB13-regulated transcription reveal a potential link between HOXB13 G84E and prostate cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2027.

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.

INTRODUCTION AND OBJECTIVES: Prostate-Associated Gene 4 (PAGE4) is a member of the Cancer/Testis Antigens (CTAs) that is up-regulated in prostate cancer (PCa) and symptomatic but not asymptomatic benign prostatic hyperplasia (BPH). However, its functional role and biological significance in PCa remain elusive. The goal of this study is to investigate the PAGE4 expression pattern in PCa and its potential relevance to PCa development and progression. METHODS: PAGE4 mRNA expression was determined by real-time RT-PCR in the PCa tissue samples obtained from 90 organconfined tumors and 21 metastatic sites. The relationship between PAGE4 mRNA level and PSA recurrence was evaluated. The mRNA level of PAGE4 in isolated cell types was analyzed in samples obtained from 8 cancer containing prostates and 5 that did not have cancer by laser capture microdissection. Immunohistochemical staining was used to detect PAGE4 protein expression in PCa. Expression of PAGE4 was up-regulated by pCMV-PAGE4-GFP transfection and cell viability was determined using the WST-1 assay. Cell cycle distribution, apoptosis, DNA damage, and cell migration were evaluated. RESULTS: PAGE4 mRNA is higher in the adjacent normal region (pathologically normal area from diseased prostate) than in cancer itself. Moreover, the lower mRNA level of PAGE4 in cancer tissues is correlated with higher tumor recurrence and lower PSA-free survival in patients with organ-confined PCa. Immunohistochemistry revealed that PAGE4 was highly expressed in the epithelial cells associated with infiltrating inflammatory cells, namely proliferative inflammatory atrophy, which is recognized as a precursor of PCa. Furthermore, in PCa tissues, the mRNA level of PAGE4 is positively associated with that of Glutathione S-transferase P1, which is a stress response gene protecting PCa development. In PCa cells, PAGE4 is induced by various stress factors including treatment with the proinflammatory cytokine TNF. Over-expression of PAGE4 favored cell survival under stress stimulation, such as glucose-deprivation and chemical drug treatment, and also decreased PCa cell migration. CONCLUSIONS: These data suggest that PAGE4 appears to represent an atypical CTA that is up-regulated in the diseased prostate and may be associated with less aggressive PCa.

The presence of arachidonate 12-lipoxygenase (12-LOX) potentiates prostate cancer (PCa) progression and therefore may be a good therapeutic target and/or a potential diagnostic predictor for PCa. In this study, we examined the expression of 12-LOX in PCa stem cells (PCa SCs) to test if it can serve as a unique marker and therapeutic target for PCa SCs. To this end, we isolated the cancer stem cell-like side population (SP) cells from the human PCa cell line DU-145 by a flow cytometry-based SP technique. The isolated DU-145 SP cells comprised a small population of the DU-145 cells. The SP cells had an up-regulation of ATP-binding cassette sub-family G member 2 (ABCG2) which enables these cells to efflux vital dyes and chemotherapeutic drugs. Furthermore, we detected a strong up-regulation of 12-LOX in these DU-145 SP cells compared to the parental DU-145 cells by RT-PCR and Western blot approaches. We also detected 12-LOX overexpression in PCa SCs in human PCa tissue samples by paraffin-section based immunofluorescent 4-channel confocal microscopy. However, no 12-LOX was detected in normal prostate epithelial SCs in normal prostate tissue samples. These multiple lines of evidence support the possibility that 12-LOX may serve as a unique marker and therapeutic target for PCa SCs.

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.

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.

Here, we demonstrate that p68 (DDX5) and p72 (DDX17), two homologous RNA helicases and transcriptional cofactors, are substrates for the acetyltransferase p300 in vitro and in vivo. Mutation of acetylation sites affected the binding of p68/p72 to histone deacetylases, but not to p300 or estrogen receptor. Acetylation additionally increased the stability of p68 and p72 RNA helicase and stimulated their ability to coactivate the estrogen receptor, thereby potentially contributing to its aberrant activation in breast tumors. Also, acetylation of p72, but not of p68 RNA helicase, enhanced p53-dependent activation of the MDM2 promoter, pointing at another mechanism of how p72 acetylation may facilitate carcinogenesis by boosting the negative p53-MDM2 feedback loop. Furthermore, blocking p72 acetylation caused cell cycle arrest and apoptosis, revealing an essential role for p72 acetylation. In conclusion, our report has identified for the first time that acetylation modulates RNA helicases and provides multiple mechanisms how acetylation of p68 and p72 may affect normal and tumor cells.

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.

The levels of ceramides, sphingosine and their related metabolites have been reported to play an important role in cell fate by determining the balance between apoptosis and cell survival signaling through what has been called the sphingolipid rheostat. One of sphingosine metabolic product, sphingosine-1-phosphate, has been widely proven to be related with tumor growth, resistance to apoptosis, angiogenesis, metastasis and invasiveness in several tumors. In prostate cancer, sphingosine-1-phosphate levels correlate with risk of progression to castration-resistant prostate cancer (CRPC), PSA levels, Gleason score, positive surgical margins, lymph node extension, metastatic extension and mortality. Hypoxia-inducible factors (HIFs) act as pleiotropic transcription factors which serve as major components for cancer progression, upregulation of HIFs are essential for metastasis and tumor growth once hypoxia has ensued. Several relations between HIFs and ceramides/sphingosine metabolism have been reported recently, suggesting that both mechanisms might be somehow interconnected. Despite decades of research inhibitors to HIF have been few and far between. The DNA-intercalating agent doxorubicin was identified one such inhibitor by a mechanism not yet understood. The major mechanism of doxorubicin-mediated apoptosis has been established to be induced by the transcription factor, p53. The p53 cell cycle arrest is usually through p21 which has been shown to have enhanced expression in response to sphingosine kinase 2, one of the enzymes that mediate the conversion of sphingosine to sphingosine-1-phosphate. Several studies associating chemotherapy with different drugs that attempt to modify sphingosine and ceramides metabolism have been published with some remarkable results, although never complete responses. We hypothesize that the incomplete responses may be due to an incomplete blockade of the conversion of ceramides and sphingosine towards their phosphorylated metabolites, sphingosine-1-phosphate and ceramide-1-phospate respectively. Previous work has focused on single agents to enhance chemotherapy. In this work we are interested in studying the combination of different modifiers in the metabolism of ceramides/sphingosine with doxorubicin in order to achieve a total metabolic blockade in an effort to elicit a complete clinical response for prostate cancer. Citation Format: Gonzalo Torga, Steven Mooney, Jelani C. Zarif, Kenneth J. Pienta. Induction of apoptosis in prostate cancer cells by altering cell metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2939. doi:10.1158/1538-7445.AM2015-2939

The concept of the cancer stem cell (CSC) is neither new nor without controversy. It is important to study these cells since they are often hypothesized to be androgen insensitive and resistant to chemotherapeutics. Therefore, finding reliable markers for identifying and targeting these cells has become increasingly attractive. The hyaluronic acid receptor, CD44, is associated with a wide variety of cell types, including CSCs. CD44 has many different isoforms which can be categorized as belonging to one of two groups; one expressing different combinations of 10 variant extracellular exons (CD44v), and the other only expressing standard exons found in all isoforms, but none of the 10 variant exons (CD44s). CD44s is typically associated with the mesenchymal stem cell niche, while the various CD44v forms are related to hematopoietic stem cells and increased cell adhesion. However, both CD44s and CD44v have been linked to the CSC niche and cancer progression. Similar to CD44, the aldehyde dehydrogenases (ALDH) superfamily expression is also associated with CSCs and cancer progression. Previously, our laboratory demonstrated that epithelial cancer cells undergo an EMT upon exposure to M2 macrophages. In order to obtain a purely epithelial PCa population, our laboratory isolated a single cell clone of PC3 that had high E-cadherin expression, denoted PC3-Epi. PC3-Epi cells were then incubated with M2 macrophages, which caused a stable EMT to occur after only a few days in culture, denoted PC3-EMT. Our finding demonstrate that these PC3 clones exhibit numerous CSC characteristics, such as high ALDH activity, CD44 expression and cell plasticity. FACS analysis of CD44 isoform expression show distinct CSC subpopulations within these PC3-Epi and PC3-EMT cell types. These subpopulations showed the ability to recapitulate the presorted parental population after being returned to culture. This data suggests not only that the PC3 cell line displays numerous CSC characteristics, but also that distinct CSC subpopulations may exist. Note: This abstract was not presented at the meeting. Citation Format: James R. Hernandez, Steven M. Mooney, Gonzalo Torga, James E. Verdone, Kenneth J. Pienta. CD44 isoform expression illuminates multiple CSC subpopulations in PC3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1394. doi:10.1158/1538-7445.AM2015-1394

Our previous work has demonstrated that exposure of M2 - Tumor Associated Macrophages M2-TAMs) to epithelial cancer cells can induce an Epithelial to Mesenchymal Transition (EMT). The resulting mesenchymal cancer cells exhibit a more aggressive phenotype, which places patients at a higher risk of relapse and death. Although most patients are cured by radical prostatectomy or radiation, it has been demonstrated that the majority of patients have disseminated tumor cells in their bone marrow at the time of primary treatment and approximately 10-15% of these men will eventually suffer a relapse after a period of dormancy. Further characterization of these disseminated cells both at the time of primary treatment and over time, is needed. It is unclear if these cells, especially those destined to eventually proliferate, are “mesenchymal”, “epithelial”, or “cancer stem” cells (CSCs). In this work, we investigate the CSC molecular and gene expression profile across multiple cancer cell lines and cell phenotypes. In part, this was done by utilizing various culturing conditions, such as 3D growth in stem cell media in order to generate sphere-like cultures known as tumorspheres. Flow cytometric analysis was performed based on ALDH, CD44 variant and CD133 expression. This was followed by gene expression analysis and characterization of the resulting tumorspheres and standard 2D cultures. The results demonstrate that there may be a level of plasticity that allows these cancer cells to alter gene expression and shift towards a CSC state and/or that there is a common CSC progenitor that exists within the population which can give rise to both epithelial and mesenchymal cell types. Therefore, furthering our understanding of the dynamic relationship between CSCs, EMT, and the converse mesenchymal to epithelial transition, is critical in developing novel therapeutics and prognostic tools in the field of cancer medicine. Citation Format: James R. Hernandez, Steven M. Mooney, Takumi Shiraishi, James E. Verdone, Calvin A. Harberg, Donald Vindivich, Kenneth J. Pienta. Profiling of cancer cell lines demonstrates a dynamic relationship between epithelial, mesenchymal and cancer stem states. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1919. doi:10.1158/1538-7445.AM2014-1919

Background: Epithelial-mesenchymal transition (EMT) is associated with metastasis in human prostate cancer (PCa). As metastatic tumors are the major cause of death in most cancer patients, identifying instances of EMT in patients may serve as a good prognostic indicator of progressive disease. Abnormal morphologic deformation of the cell nucleus is strongly associated with an aggressive cancer phenotype and computer-aided quantitative nuclear morphometry (QNM) has been used to quantify this deformation. In the present study, we go further to investigate the association of nuclear morphologic features between an epithelial PCa phenotype and an EMT-induced mesenchymal PCa phenotype across a panel of extracellular matrix (ECM) substrates on which cells are grown in vitro. Methods: PC3 epithelial cells (PC3-Epi) are established human PCa cultures from bone metastatic lesions. M2 macrophages induced PC3-Epi cells to undergo EMT and become mesenchymal PC3 cells (PC3- EMT). PC3-Epi and PC3-EMT cells were cultured independently on glass tissue-culture slides and also on a panel of ECM component substrates, including collagen 1, poly-D-Lysine, and various molecular weight hyaluronic acid polymers. Slides were stained with Feulgen or hematoxylin and eosin (H&E). The images were digitized with a slide-scanning microscope. Computer aided image processing software measured a total of 41 non-redundant features. This included nuclear size, shape, and texture features. Features were statistically analyzed using both supervised and unsupervised machine learning algorithms. Results: Statistical classification between PC3-Epi and PC3-EMT was performed on a panel of ECM substrates. The PC3-Epi and PC3-EMT morphological features were characterized by the type of ECM substrate on which cells were cultured. Chromatin texture and shape features, such as Feret diameter, were among the most significant in contributing to separation of the PC3-Epi and PC3-EMT. In contrast, the morphological features could not be separated when cells were grown on glass surfaces. Conclusions: The PC3-Epi and PC3-EMT cells were statistically separated more effectively with extracellular matrix substrates than with glass. Furthermore, our findings suggest that ECM components affect chromatin texture, a function of chromatin organization, and nuclear morphology. Our results also indicate quantitative nuclear morphometric analysis may be a viable tool for demonstrating instances of EMT in clinical samples, potentially adding quantitative prognostic value to this technique. Nuclear morphometric factors, such as those used here, have also found applications to distinguish aggressive human prostate cancers in fixed biopsy samples (See review: Veltri, R.W., et al. “Nuclear morphometry, nucleomics and prostate cancer progression”. Asian Journal of Andrology (2012) 14, 375-384.) Citation Format: James E. Verdone, Robert W. Veltri, Steven M. Mooney, James R. Hernandez, Calvin A. Harberg, Donald S. Coffey, Kenneth J. Pienta. Extracellular matrix components direct chromatin texture and nuclear morphological changes in epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1139. doi:10.1158/1538-7445.AM2014-1139

In collaboration with Dr. K. Cooney's lab at the Univ of Michigan, our group recently discovered that a recurrent glycine-to-glutamic acid germline mutation (G84E) in HOXB13 is associated with an increased prostate cancer (PCa) risk. HOXB13 is an androgen receptor (AR)-independent prostate-specific transcription factor shown to play a critical role in the development of the mouse prostate. However, the exact function of HOXB13 in normal human prostate biology and the mechanism by which HOXB13 G84E predisposes carriers to PCa remain unknown. The purpose of this study is to test our hypothesis that HOXB13 G84E, by itself or together with other oncogenes, may reprogram normal prostate cells to be prone to transformation, possibly due to altered molecular characteristics that have downstream effects on transcriptional targets. To this end, using patient-derived normal prostate epithelial cells immortalized with hTERT in the presence or absence of transduced AR (957e/hTERT and 957e/hTERT/AR), we generated cell lines stably expressing HOXB13 WT or G84E to examine neoplastic phenotypes and differentiation. In the presence of AR, HOXB13 WT and G84E caused a change in cell morphology to a fibroblast-like appearance and suppressed proliferation without any significant differences between WT and G84E. In the absence of AR, the morphological and growth suppressive effects of HOXB13 were ablated, which indicates a cooperative relationship between HOXB13 and AR. In addition, we analyzed several molecular aspects of HOXB13 WT and G84E such as protein-interactions, subcellular localization and half-life which could hypothetically contribute to carcinogenesis when altered. Whether the HOXB13-MEIS and HOXB13-AR interactions are affected by G84E was studied for the following reasons. Most HOXB13 mutations identified in PCa lie in the MEIS-interacting domains (MID) of HOXB13 and the biological significance of HOX-MEIS complexes has been shown in leukemic transformation. HOXB13 mediates the transcription of a subset of AR-target genes and the HOXB13-AR interaction has been documented. Immunoprecipitation analysis in 957e/hTERT/AR demonstrated that HOXB13 and MEIS2 interact through the MIDs and G84E did not alter this interaction. Additionally, G84E did not disturb the HOXB13-AR interaction. Finally, HOXB13 G84E localized properly to the nucleus and no significant differences were found in the half-lives of HOXB13 WT and G84E, at least in the absence of AR. Taken together, our study suggests that HOXB13 G84E alone or with AR cannot initiate PCa in our cell line model with a caveat that our model does not accurately recapitulate human PCa since patients harbor the G84E mutation throughout development. Moreover, the lack of molecular differences between WT and G84E calls for further investigation of potential areas of G84E-induced alterations, such as gene-protein and protein-protein interactions on a global scale. Citation Format: Dorhyun Johng, Charles M. Ewing, Steven M. Mooney, Shuangling Chen, William B. Isaacs. HOXB13: Investigating mechanisms of G84E mutation associated with prostate carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2948. doi:10.1158/1538-7445.AM2014-2948

Introduction and Objectives: The Mechanisms contributing to androgen-independent progression in prostate cancer (PCa) are not fully elucidated. PCa lesions are heterogeneous and thus, it is important to understand whether among the heterogeneous collectives of cell types, androgen-independent cells exist prior to hormonal manipulation. The identification of these cells may predict patient prognosis as well as provide a better understanding of treatment resistance. Methods: LNCaP cells (10,000 cells in 10cm dish) were grown for 40 days in normal medium. Fifty colonies were selected and 22 subclones were established. To compare their androgen-dependence, six of these clones were grown in normal and androgen-depleted medium and their cell proliferation rates were examined. Between an androgen-dependent clone (LNCaP-ADc) and an androgen-independent clone (LNCaP-AIc) gene copy number and gene expression were compared using Human SNP array 6.0 and Human Genome U133 Plus 2.0, respectively. One of the genes differentially expressed between these clones was knocked down by siRNA in LNCaP cells and the influence on cell proliferation was evaluated. The expression levels of this gene in PCa tisssue samples were examined by real-time PCR. Results: Cell proliferation was significantly suppressed by androgen depletion in LNCaP-ADc, but not in LNCaP-AIc. Gene copy numbers were different at 1q (ADcAIc), indicating that these clones contain genetic differences. The most differentially expressed gene was SPRY1 (ADc>AIc, ratio=5.699). Knocking down of SPRY1enhanced proliferation and PSA expression of LNCaP cells. SPRY1 expression in PCa tissues were negatively associated with PSA recurrence after surgical treatment (p=0.0076). Conclusions: A random population of LNCaP cells comprises a genetically heterogeneous group of cells that have different levels of androgen dependence. Included among them are those that appear to be able to be resistant to androgen depletion. Among the changes associated with this independence, SPRY1 appears to be differentially expressed in such clones and may represent a novel biomarker for differentiating aggressive cells in PCa. Citation Format: Naoki Terada, Takumi Shiraishi, Yu Zeng, Koh-Meng Aw-Yong, Steven M. Mooney, Jun Luo, Prakash Kulkarni, Robert H. Getzenberg. The identification and characterization of androgen-independent cells prior to hormonal manipulation in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C44.

Introduction: It has long been thought that the stem cell-like features of cancer cells contribute to their evolutionary capability to escape therapeutic strategies, such as chemotherapy, radiotherapy, or hormone deprivation in prostate cancer (PCa). At the same time, temperature is perhaps one of the most important elements of the environment. One of the most clinically relevant examples of the effect of temperature on stem-like cells is cryptorchidism, in which germ cells maintained at body temperature that is several degrees higher than in the scrotum do not function to form sperm. In contrast, these pluripotential cells have a high likelihood to develop cancer if they are not rescued by relocating them to the scrotum and therefore a lower environmental temperature, in very early childhood. RNA binding motif 3 (RBM3) belongs to a cold-inducible RNA binding protein family and has been shown to be highly expressed in normal testis tissue but is decreased in cryptorchid mouse. We previously reported that RBM3 was decreased by heat treatment in PCa cell lines. The goal of this study is to investigate the effect of RBM3 over-expression on stem cell-like features of PC-3 cells. Methods: RBM3 mRNA expression was detected by RT-PCR in fetal and adult human tissues, as well as in PCa samples. The normal prostate cell line, PrEc, was sorted with the stem cell surface marker CD133 using flow cytometry. PC-3 cells were transfected with pCMV6-RBM3-GFP vector. The soft agar clonogenic assay was performed with RBM3- or control vector-transfected cells. The expression of stem cell-related genes and miRNAs was determined using PCR array. Results: The RBM3 mRNA level is decreased in fetal tissues and CD133 (+) PrEc cells in comparison to adult tissues and CD133 (−) cells, respectively. Metastatic PCa expresses lower RBM3 mRNA than primary tumors. Moreover, RBM3 expression was decreased upon treatment of chemotherapy and maintained at a low level in the surviving clones after intensive chemo- or heat treatment in both PC-3 and DU-145 cells. PC-3 cells that over-express RBM3 demonstrated less heterogenic morphology than those with a control vector. Importantly, RBM3 over-expression greatly attenuates the anchorage-independent clonogenic ability of PC-3 cells. Additionally, a panel of genes and miRNAs that are involved in neural cell differentiation and self-renew are altered upon RBM3 expression. Conclusion: Taken together, these data suggest that lowering RBM3 may play an important role in maintaining the stem cell-like features of cancer cells during development and progression under environmental stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 495. doi:10.1158/1538-7445.AM2011-495

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.