Derrick J. Morton

TGF-β Effects on Prostate Cancer Cell Migration and Invasion Are Mediated by PGE2 through Activation of PI3K/AKT/mTOR Pathway

TGF-β plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor-promoting activities. Correlations between cyclooxygenase (COX)-2 overexpression and enhanced production of prostaglandin (PG)E2 have been implicated in cancer progression; however, there are no studies indicating that TGF-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were detected only in prostate cancer cells. TGF-β treatment increased COX-2 protein levels and PGE2 secretion in PC3 cells. Exogenous PGE2 and PGF2α had no effects on cell proliferation in LNCaP, DU145, and PC3 cells whereas PGE2 and TGF-β induced migration and invasive behavior in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting small interfering RNA inhibited PGE2 and TGF-β-induced migration of PC3 cells. TGF-β and PGE2 induce activation of PI3K/AKT/mammalian target of rapamycin pathway as indicated by increased AKT, p70S6K, and S6 phosphorylation. Rapamycin completely blocked the effects of TGF-β and PGE2 on phosphorylation of p70S6K and S6 but not on AKT phosphorylation. PGE2 and TGF-β induced phosphorylation of AKT, which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3K inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-β on cell migration, whereas LY294002 or rapamycin completely eliminated PGE2, TGF-β, and epidermal growth factor-induced migration in PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediate TGF-β effects on cell migration and invasion through the activation of PI3K/AKT/mammalian target of rapamycin pathway.

Snail Promotes Epithelial Mesenchymal Transition in Breast Cancer Cells in Part via Activation of Nuclear ERK2

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

Inhibitor of differentiation 4 (ID4): From development to cancer.

Highly conserved Inhibitors of DNA-Binding (ID1-ID4) genes encode multi-functional proteins whose transcriptional activity is based on dominant negative inhibition of basic helix-loop-helix (bHLH) transcription factors. Initial animal models indicated a degree of compensatory overlap between ID genes such that deletion of multiple ID genes was required to generate easily recognizable phenotypes. More recently, new model systems have revealed alterations in mice harboring deletions in single ID genes suggesting complex gene and tissue specific functions for members of the ID gene family. Because ID genes are highly expressed during development and their function is associated with a primitive, proliferative cellular phenotype there has been significant interest in understanding their potential roles in neoplasia. Indeed, numerous studies indicate an oncogenic function for ID1, ID2 and ID3. In contrast, the inhibitor of differentiation 4 (ID4) presents a paradigm shift in context of well-established role of ID1, ID2 and ID3 in development and cancer. Apart from some degree of functional redundancy such as HLH dependent interactions with bHLH protein E2A, many of the functions of ID4 are distinct from ID1, ID2 and ID3: ID4 proteins a) regulate distinct developmental processes and tissue expression in the adult, b) promote stem cell survival, differentiation and/or timing of differentiation, c) epigenetic inactivation/loss of expression in several advanced stage cancers and d) increased expression in some cancers such as those arising in the breast and ovary. Thus, in spite of sharing the conserved HLH domain, ID4 defies the established model of ID protein function and expression. The underlying molecular mechanism responsible for the unique role of ID4 as compared to other ID proteins still remains largely un-explored. This review will focus on the current understanding of ID4 in context of development and cancer.

Id4 dependent acetylation restores mutant-p53 transcriptional activity

BackgroundThe mechanisms that can restore biological activity of mutant p53 are an area of high interest given that mutant p53 expression is observed in one third of prostate cancer. Here we demonstrate that Id4, an HLH transcriptional regulator and a tumor suppressor, can restore the mutant p53 transcriptional activity in prostate cancer cells.MethodsId4 was over-expressed in prostate cancer cell line DU145 harboring mutant p53 (P223L and V274F) and silenced in LNCaP cells with wild type p53. The cells were used to quantitate apoptosis, p53 localization, p53 DNA binding and transcriptional activity. Immuno-precipitation/-blot studies were performed to demonstrate interactions between Id4, p53 and CBP/p300 and acetylation of specific lysine residues within p53.ResultsEctopic expression of Id4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, the transcriptional targets of p53. Mutant p53 gained DNA binding and transcriptional activity in the presence of Id4 in DU145 cells. Conversely, loss of Id4 in LNCaP cells abrogated wild type p53 DNA binding and transactivation potential. Gain of Id4 resulted in increased acetylation of mutant p53 whereas loss of Id4 lead to decreased acetylation in DU145 and LNCaP cells respectively. Id4 dependent acetylation of p53 was in part due to a physical interaction between Id4, p53 and acetyl-transferase CBP/p300.ConclusionsTaken together, our results suggest that Id4 regulates the activity of wild type and mutant p53. Id4 promoted the assembly of a macromolecular complex involving CBP/P300 that resulted in acetylation of p53 at K373, a critical post-translational modification required for its biological activity.

The RNA exosome and RNA exosome-linked disease

The RNA exosome is an evolutionarily conserved, ribonuclease complex that is critical for both processing and degradation of a variety of RNAs. Cofactors that associate with the RNA exosome likely dictate substrate specificity for this complex. Recently, mutations in genes encoding both structural subunits of the RNA exosome and its cofactors have been linked to human disease. Mutations in the RNA exosome genes EXOSC3 and EXOSC8 cause pontocerebellar hypoplasia type 1b (PCH1b) and type 1c (PCH1c), respectively, which are similar autosomal-recessive, neurodegenerative diseases. Mutations in the RNA exosome gene EXOSC2 cause a distinct syndrome with various tissue-specific phenotypes including retinitis pigmentosa and mild intellectual disability. Mutations in genes that encode RNA exosome cofactors also cause tissue-specific diseases with complex phenotypes. How mutations in these genes give rise to distinct, tissue-specific diseases is not clear. In this review, we discuss the role of the RNA exosome complex and its cofactors in human disease, consider the amino acid changes that have been implicated in disease, and speculate on the mechanisms by which exosome gene mutations could underlie dysfunction and disease.

Intra-tumoral delivery of functional ID4 protein via PCL/maltodextrin nano-particle inhibits prostate cancer growth

ID4, a helix loop helix transcriptional regulator has emerged as a tumor suppressor in prostate cancer. Epigenetic silencing of ID4 promotes prostate cancer whereas ectopic expression in prostate cancer cell lines blocks cancer phenotype. To directly investigate the anti-tumor property, full length human recombinant ID4 encapsulated in biodegradable Polycaprolactone/Maltodextrin (PCL-MD) nano-carrier was delivered to LNCaP cells in which the native ID4 was stably silenced (LNCaP(-)ID4). The cellular uptake of ID4 resulted in increased apoptosis, decreased proliferation and colony formation. Intratumoral delivery of PCL-MD ID4 into growing LNCaP(-)ID4 tumors in SCID mice significantly reduced the tumor volume compared to the tumors treated with chemotherapeutic Docetaxel. The study supports the feasibility of using nano-carrier encapsulated ID4 protein as a therapeutic. Mechanistically, ID4 may assimilate multiple regulatory pathways for example epigenetic re-programming, integration of multiple AR co-regulators or signaling pathways resulting in tumor suppressor activity of ID4.

Relative Stability of Wild-Type and Mutant p53 Core Domain: A Molecular Dynamic Study.

The p53 protein is a stress response protein that functions primarily as a tetrameric transcription factor. A tumor suppressor p53 binds to a specific DNA sequence and transactivates target genes, leading to cell cycle apoptosis. Encoded by the human gene TP53, p53 is a stress response protein that functions primarily as a tetrameric transcription factor. This gene regulates a large number of genes in response to a variety of cellular functions, including oncogene activation and DNA damage. Mutations in p53 are common in human cancer types. Herein we mutate a wild-type p53, 1TSR with four of its mutated proteins. The energy for the wild-type and mutated proteins is calculated by using molecular dynamics simulations along with simulated annealing. Our results show significant differences in energy between hotspot mutations and the wild type. Based on the findings, we investigate the correlation between molar masses of the target residue and the relative energy with respect to the wild type. Our results indicate that the relative energy changes play a pivotal role in bioactivity, in conformity with observations in the rate of mutation in biology.

ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation

Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52

Castration‐resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen‐depleted environment of the prostate. In recent years, targeting multiple chaperones and co‐chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant‐negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(−)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR‐associated co‐chaperones in promoting constitutively active AR signaling in L(−)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52‐kDa FK506‐binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52‐dependent AR transcriptional activity was observed in L(−)ID4 cells. Moreover, pharmacological inhibition of FKBP52‐AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(−)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52‐mediated AR signaling.

Abstract 1219: ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity

The physiological mechanisms that can restore biological activity of mutant p53 is an area of high interest given that mutant p53 expression is observed in one third of prostate cancer and more than 50% of all cancers. Here we demonstrate that ID4 (inhibitor of differentiation-4) a dominant negative regulator of bHLH transcription factors can restore mutant p53 transcriptional activity in prostate cancer cells. ID4 is highly expressed in the normal prostate and decreased in prostate cancer due to promoter hypermethylation. Prostate cancer cell lines: DU145 harbors mutant p53 and also lacks ID4 expression; LNCaP cells express wild-type p53 and ID4; whereas PC3 cells are null for p53 and express low levels of ID4. p53 mutants (P223L and V274F) in DU145 cells are within the DNA binding domain and abrogate p53 transcriptional activity due to structural de-stabilization and/or DNA interactions. Ectopic expression of ID4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, transcriptional targets of p53. DNA binding, p53 luciferase reporter studies and ChIP analysis demonstrated that mutant p53 gains ID4 dependent DNA binding and transcriptional activity in part due to CBP/p300 dependent acetylation of p53 at lysine 373. Loss of ID4 in LNCaP cells also abrogated wild-type p53 DNA binding and transcriptional activity with concomitant loss of CBP/p300 requirement and decreased acetylation of p53. To further elucidate ID4 dependent restoration of biological activity of p53 we stably transfected p53-null cell line PC3, which has endogenous ID4 with mutant p53 mimicking DU145 cells. mRNA, protein levels and apoptosis assays were used to determine the effects on cell death and to determine if mutant p53 had the ability to transactivate upstream/downstream targets (MDM2, PUMA, and p21). To further validate our gene expression profile of p53 responsive targets, we performed DNA binding assays as well as p53 luciferase reporter assays to demonstrate wild-type and mutant p53 transcriptional activity in PC3 cells. Also, to elucidate the mechanism by which ID4 promotes apoptosis, we performed acetylation studies in LNCaP and DU145 xenografts (+/-) ID4 as well as p53 co-immunoprecipation studies to demonstrate interaction of both lysine residues 320 and 373. Our results indicate that transfected mutant p53 in PC3 cells were able to induce expression of downstream targets as well as effect biological processes such as apoptosis and cell cycle arrest. Also, post-translational modifications on residues lys320 and lys373 indicated activation of mutant p53. Collectively, based on our studies we demonstrate that ID4 promotes post-translational modifications of p53, which in turn regulates p53 transcriptional activity. NIH grant #R01CA128914 Citation Format: Derrick Jerone Morton, Divya Patel, Jugal Joshi, Pankaj Sharma, Ashley Knowell, Aisha Hunt, Jaideep Chaudhary. ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity. [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 1219. doi:10.1158/1538-7445.AM2015-1219