Prerna Malaney

Pathological Unfoldomics of Uncontrolled Chaos: Intrinsically Disordered Proteins and Human Diseases

Many biologically important proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro as a whole or in part.1–5 These intrinsically disordered proteins (IDPs), or intrinsically disordered protein regions (IDPRs) of hybrid proteins possessing both structured and disordered domains, do not have unique well-defined 3D structures, existing instead as collapsed or extended dynamically mobile conformational ensembles. Therefore, natural proteins can be found in one of three major protein forms: functional and folded, nonfunctional and misfolded, or functional and intrinsically disordered. Although IDPs and IDPRs are highly dynamic, their structures can be described reasonably well by a rather limited number of lower-energy conformations.6,7 The structural plasticity and conformational adaptability of IDPs/IDPRs and their intrinsic lack of rigid structure leads to a number of exceptional functional advantages, providing them with unique capabilities to act in functional modes not achievable by ordered proteins.5 As a result, intrinsic disorder is a common feature of proteins involved in signaling, regulation, and recognition, and IDPs/IDPRs play diverse roles in modulation and control of their binding partners’ functions and in promoting the assembly of supramolecular complexes. The biological actions of IDPs/IDPRs, which frequently serve as major regulators of their binding partners, are controlled by extensive posttranslational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, and sumoylation,5 and by alternative splicing.8 In fact, many IDPs/IDPRs are known to contain multiple functional elements that contribute to their ability to be involved in interaction with, regulation of, and control by multiple structurally unrelated partners.9 Given the existence of multiple functions in a single disordered protein, and given that each functional element is typically relatively short, alternative splicing could readily generate sets of protein isoforms with highly diverse regulatory elements.8 The complexity of the disorder-based interactomes is further increased by the capacity of a single IDPR to bind to multiple partners, gaining very different structures in the bound state.10 IDPs can form highly stable complexes or be involved in signaling interactions where they undergo constant “bound–unbound” transitions, thus acting as dynamic and sensitive “on–off” switches. The ability of these proteins to return to highly flexible conformations after the completion of a particular function, and their predisposition to adopt different conformations depending on their environment, are unique physiological properties of IDPs that allow them to exert different functions in different cellular contexts according to a specific conformational state.5 Although the field of protein disorder has started from careful analysis of a very limited number of biologically active proteins without unique structures (which, for a long time, were taken as rare exceptions from the general “one sequence–one unique structure–one unique function” paradigm),1–4 applications of various disorder predictors to different proteomes revealed that IDPs are highly abundant in nature,11–16 and the overall amount of disorder in proteins increases from bacteria to archaea to eukaryota, with over half of all eukaryotic proteins predicted to contain extended IDPRs.11,12,15–17 One explanation for this trend is a change in the cellular requirements for certain protein functions, particularly cellular signaling. In support of this hypothesis, an analysis of a eukaryotic signal protein database indicated that the majority of known signal transduction proteins were predicted to contain significant regions of disorder.18 A detailed study focused on the intricate mechanisms of IDP regulation inside the cell was recently conducted by Gsponer et al.19 These authors grouped all the Saccharomyces cerevisiae proteins into three classes according to their predicted disorder propensities and evaluated the correlations between intrinsic disorder and the various regulation steps of protein synthesis and degradation.19 Although the transcriptional rates of mRNAs encoding IDPs and ordered proteins were comparable, IDP-encoding transcripts were generally less abundant than transcripts encoding ordered proteins because of increased decay rates of IDP mRNAs.19 Also, IDPs were found to be less abundant than ordered proteins because of lower rates of protein synthesis and shorter protein half-lives.19 Curiously, IDPs were shown to be substrates of twice as many kinases as ordered proteins. Furthermore, the vast majority of kinases whose substrates were IDPs were either regulated in a cell-cycle-dependent manner or activated upon exposure to specific stimuli or stress.19 Similar regulation trends were also found in proteomes of Schizosaccharomyces pombe and Homo sapiens,19 suggesting that both unicellular and multicellular organisms use evolutionarily conserved mechanisms to regulate the availability of their IDPs. This tight regulation is directly related to the major roles of IDPs/IDPRs in signaling, where it is crucial for a given protein to be available in appropriate amounts and not to be present longer than needed.19 It was also pointed out5 that although the abundance of many IDPs may be closely regulated, some disordered proteins could be present in cells in large amounts or/and for long periods of time, either due to specific PTMs or via interactions with other factors. These events could promote changes in cellular localization of IDPs or protect them from degradation.3,20–23 Taken together, these data highlight that the chaos seemingly associated with highly flexible and promiscuous IDPs/IDPRs is under tight control.24

Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional Versatility

IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTENs protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.

A Unified Nomenclature and Amino Acid Numbering for Human PTEN

With the discovery of an isoform based on an alternative translation start site, PTEN nomenclature needs an update. The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

PTEN proteoforms in biology and disease

Proteoforms are specific molecular forms of protein products arising from a single gene that possess different structures and different functions. Therefore, a single gene can produce a large repertoire of proteoforms by means of allelic variations (mutations, indels, SNPs), alternative splicing and other pre-translational mechanisms, post-translational modifications (PTMs), conformational dynamics, and functioning. Resulting proteoforms that have different sizes, alternative splicing patterns, sets of post-translational modifications, protein–protein interactions, and protein–ligand interactions, might dramatically increase the functionality of the encoded protein. Herein, we have interrogated the tumor suppressor PTEN for its proteoforms and find that this protein exists in multiple forms with distinct functions and sub-cellular localizations. Furthermore, the levels of each PTEN proteoform in a given cell may affect its biological function. Indeed, the paradigm of the continuum model of tumor suppression by PTEN can be better explained by the presence of a continuum of PTEN proteoforms, diversity, and levels of which are associated with pathological outcomes than simply by the different roles of mutations in the PTEN gene. Consequently, understanding the mechanisms underlying the dysregulation of PTEN proteoforms by several genomic and non-genomic mechanisms in cancer and other diseases is imperative. We have identified different PTEN proteoforms, which control various aspects of cellular function and grouped them into three categories of intrinsic, function-induced, and inducible proteoforms. A special emphasis is given to the inducible PTEN proteoforms that are produced due to alternative translational initiation. The novel finding that PTEN forms dimers with biological implications supports the notion that PTEN proteoform–proteoform interactions may play hitherto unknown roles in cellular homeostasis and in pathogenic settings, including cancer. These PTEN proteoforms with unique properties and functionalities offer potential novel therapeutic opportunities in the treatment of various cancers and other diseases.

Loss of phosphatase and tensin homolog (PTEN) induces leptin-mediated leptin gene expression: feed-forward loop operating in the lung.

Background: Leptin expression is induced in lung diseases and lung cancer, but the mechanism of leptin gene expression remains elusive. Results: Leptin mediates leptin and leptin receptor expression, setting up a feed-forward loop. Conclusion: DNA elements and intracellular signals activating leptin gene expression were identified. Significance: Mechanism of leptin/leptin receptor gene regulation will aid in targeting leptin signaling in lung pathologies. Elevated levels of systemic and pulmonary leptin are associated with diseases related to lung injury and lung cancer. However, the role of leptin in lung biology and pathology, including the mechanism of leptin gene expression in the pathogenesis of lung diseases, including lung cancer, remains elusive. Here, using conditional deletion of tumor suppressor gene Pten in the lung epithelium in vivo in transgenic mice and human PTEN-null lung epithelial cells, we identify the leptin-driven feed-forward signaling loop in the lung epithelial cells. Leptin-mediated leptin/leptin-receptor gene expression likely amplifies leptin signaling that may contribute to the pathogenesis and severity of lung diseases, resulting in poor clinical outcomes. Loss of Pten in the lung epithelial cells in vivo activated adipokine signaling and induced leptin synthesis as ascertained by genome-wide mRNA profiling and pathway analysis. Leptin gene transcription was mediated by binding of transcription factors NRF-1 and CCAAT/enhancer-binding protein δ (C/EBP) to the proximal promoter regions and STAT3 to the distal promoter regions as revealed by leptin promoter-mutation, chromatin immunoprecipitation, and gain- and loss-of-function studies in lung epithelial cells. Leptin treatment induced expression of the leptin/leptin receptor in the lung epithelial cells via activation of MEK/ERK, PI3K/AKT/mammalian target of rapamycin (mTOR), and JAK2/STAT3 signaling pathways. Expression of constitutively active MEK-1, AKT, and STAT3 proteins increased expression, and treatment with MEK, PI3K, AKT, and mTOR inhibitors decreased LEP expression, indicating that leptin via MAPK/ERK1/2, PI3K/AKT/mTOR, and JAK2/STAT3 pathways, in turn, further induces its own gene expression. Thus, targeted inhibition of the leptin-mediated feed-forward loop provides a novel rationale for pharmacotherapy of disease associated with lung injury and remodeling, including lung cancer.

PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer

ABSTRACT PTEN phosphorylation at its C-terminal (C-tail) serine/threonine cluster negatively regulates its tumor suppressor function. However, the consequence of such inhibition and its downstream effects in driving lung cancer remain unexplored. Herein, we ascertain the molecular mechanisms by which phosphorylation compromises PTEN function, contributing to lung cancer. Replacement of the serine/threonine residues with alanine generated PTEN-4A, a phosphorylation-deficient PTEN mutant, which suppressed lung cancer cell proliferation and migration. PTEN-4A preferentially localized to the nucleus where it suppressed E2F1-mediated transcription of cell cycle genes. PTEN-4A physically interacted with the transcription factor E2F1 and associated with chromatin at gene promoters with E2F1 DNA-binding sites, a likely mechanism for its transcriptional suppression function. Deletion analysis revealed that the C2 domain of PTEN was indispensable for suppression of E2F1-mediated transcription. Further, we uncovered cancer-associated C2 domain mutant proteins that had lost their ability to suppress E2F1-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with these findings, we observed increased PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples establishing phosphorylation as a bona fide inactivation mechanism for PTEN in lung cancer. Thus, use of small molecule inhibitors that hinder PTEN phosphorylation is a plausible approach to activate PTEN function in the treatment of lung cancer. AbbreviationsAKT V-Akt Murine Thymoma Viral OncogeneCA Cancer adjacentCDK1 Cyclin dependent kinase 1CENPC-C Centromere Protein CChIP Chromatin Immunoprecipitationco-IP Co-immunoprecipitationCOSMIC Catalog of Somatic Mutations In CancerCREB cAMP Responsive Element Binding ProteinC-tail Carboxy terminal tailE2F1 E2F Transcription Factor 1ECIS Electric Cell-substrate Impedance SensingEGFR Epidermal Growth Factor ReceptorGSI Gamma Secretase InhibitorHDAC1 Histone Deacetylase 1HP1 Heterochromatin protein 1KAP1/TRIM28 KRAB-Associated Protein 1/Tripartite Motif Containing 28MAF1 Repressor of RNA polymerase III transcription MAF1 homologMCM2 Minichromosome Maintenance Complex Component 2miRNA micro RNAMTF1 Metal-Regulatory Transcription Factor 1PARP Poly(ADP-Ribose) PolymerasePD-1 Programmed Cell Death 1PD-L1 Programmed Cell Death 1 Ligand 1PI3K Phosphatidylinositol-4,5-Bisphosphate 3-KinasePLK Polo-like KinasepPTEN Phosphorylated PTENPTEN Phosphatase and Tensin Homolog deleted on chromosome tenPTM Post Translational ModificationRad51 RAD51 RecombinaseRad52 RAD52 RecombinaseRPA1 Replication protein ASILAC Stable Isotope Labeling with Amino Acids in Cell CultureSRF Serum Response FactorTKI Tyrosine Kinase inhbitorsTMA Tissue MicroarrayTOP2A DNA Topoisomerase 2A

Abstract 3682: Phosphorylation mediated conformational changes defines nuclear role of phosphatase and tensin homology (PTEN) in tumor suppression

Loss of function of tumor suppressor PTEN (Phosphatase and tensin homolog) causes cancer in various tissues. PTEN C-terminal phosphorylation (pPTEN) inactivates PTEN, leading to multiple malignancies with increased severity. However, little is known about the molecular mechanisms underlying such inactivation. Therefore, the objective of our work is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN C-terminal phosphorylation at a serine-threonine cluster (Ser380, Thr382, Thr383 and Ser385) conformationally inactivates PTEN, abrogating its tumor suppressor function. Replacement of these serine/threonine residues with alanine generated an artificial phosphorylation-deficient mutant of PTEN (PTEN-4A), which is constitutively active. PTEN-4A suppressed cell proliferation and migration to a greater extent as compared to PTEN-WT. PTEN-4A preferentially localized to the nucleus and suppressed the E2F-mediated transcription of cell cycle genes. The nuclear localization sequence and phosphatase activity of PTEN-4A is critical for this transcriptional suppression. Immuno-precipitation assays show that PTEN physically associates with the transcription factor E2F1, a likely mechanism for its suppressive effect on E2F1 related genes. Further, deletion analysis of PTEN-4A protein revealed that the C2 domain is indispensable for suppression of E2F-related genes. Systematic transcriptional assays identify disease-associated C2 domain mutations that lose their ability to suppress E2F-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Taken together, we reveal nuclear functions of PTEN-4A in tumor suppression that can be therapeutically leveraged for developing adjunctive cancer therapies. Small molecule inhibitors that hinder PTEN phosphorylation maybe utilized to activate PTEN nuclear function in tumors. Such adjunctive therapy has a high likelihood to reduce toxic doses of chemotherapeutic agents and targeted inhibitors, including kinase inhibitors that are being used in clinical settings. Citation Format: Prerna Malaney, Jonathan Semidey-Hurtado, Jamaal Hardee, Deepal Patel, Daniel Hennessey, Kate Stanford, Emily Palumbo, Zhi Tian, Diane Allen-Gipson, Vrushank Dave. Phosphorylation mediated conformational changes defines nuclear role of phosphatase and tensin homology (PTEN) in tumor suppression. [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 3682.

Abstract B09: Loss of PTEN cooperates with mutant KRAS initiating EMT and increased stemness in a mouse model of lung cancer

We have developed a preclinical model of lung cancer associated with hyper-activated PI3K/mTOR and KRAS. Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers; of which >50% have aberrant PI3K/AKT/mTOR signaling and ∼30% harbor oncogenic KRAS, conferring chemo/radio-resistance and poor prognosis. KRAS inhibitors are difficult to develop, and rapalogs targeting mTOR are toxic and induce compensatory activation of AKT, causing resistance to apoptosis. Hence, development of effective inhibitors of KRAS and PI3K/mTOR pathways is imperative. Further, clinical studies implicate that tumors with aberrant KRAS and PI3K/mTOR signaling are associated with epithelial-mesenchymal transition (EMT), metastasis, poor differentiation, and chemo/radio-resistance. However, molecular details of EMT mediated by KRAS and PI3K/mTOR pathway is lacking, limiting the development of drugs targeting EMT-mediators. Using a CCSP-promoter driven Cre/LoxP mediated deletion of PTEN in an oncogenic KRAS status (PTENΔΔ/KRasG12D), we have developed a preclinical mouse model of lung cancer with hyper-activation of PI3K/mTOR and KRAS pathways. Microarray based RNA profiling followed by bioinformatic analysis on PTEN-null lung epithelial cells revealed transcriptional activation of RAS/RAF/MAPK/ERK pathway associated candidate genes. On the other hand, Dox-induced PTENΔΔ/KRasG12D lung tumors showed induction of SNAIL1, SLUG, ZEB-2 and SOX2 genes. Taken together, our analysis reveals that PTEN loss predisposes lung tumors to hyper-activation of the KRAS pathway, which is further accentuated by oncogenic mutation in KRAS, initiating EMT and increased stemness. Citation Format: Prerna Malaney, Vrushank Dave. Loss of PTEN cooperates with mutant KRAS initiating EMT and increased stemness in a mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B09. doi: 10.1158/1557-3125.RASONC14-B09