A global analysis of the reconstitution of PTEN function by translational readthrough of PTEN pathogenic premature termination codons.

The PTEN tumor suppressor gene is mutated with high incidence in tumors and in the germline of patients with cancer predisposition or with macrocephaly associated with autism. PTEN nonsense mutations generating premature termination codons (PTC) and producing nonfunctional truncated PTEN proteins are frequent in association with human disease. However, there are no studies addressing the restoration of full-length PTEN proteins from the PTC-mutated PTEN gene by translational readthrough. Here, we have performed a global translational and functional readthrough analysis of the complete collection of PTEN PTC somatic or hereditary mutations found in tumors or in the germline of patients (disease-associated PTEN PTCome), and we set standards for the analysis of the potential of readthrough functional reconstitution in disease-relevant genes. Our analysis indicates that prevalent pathogenic PTEN PTC mutations are susceptible to PTEN functional restoration in response to readthrough-inducing compounds. Comprehensive readthrough analyses of disease-associated PTComes will be valuable tools for the implementation of readthrough-based precision interventions in specific groups of patients.

Modeling human disease in yeast: recreating the PI3K-PTEN-Akt signaling pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a model organism that has been thoroughly exploited to understand the universal mechanisms that govern signaling pathways. Due to its ease of manipulation, humanized yeast models that successfully reproduce the function of human genes permit the development of highly efficient genetic approaches for molecular studies. Of special interest are those pathways related to human disease that are conserved from yeast to mammals. However, it is also possible to engineer yeast cells to implement functions that are naturally absent in fungi. Along the years, we have reconstructed several aspects of the mammalian phosphatidylinositol 3-kinase (PI3K) pathway in S. cerevisiae. Here, we briefly review the use of S. cerevisiae as a tool to study human oncogenes and tumor suppressors, and we present an overview of the models applied to the study of the PI3K oncoproteins, the tumor suppressor PTEN, and the Akt protein kinase. We discuss the application of these models to study the basic functional properties of these signaling proteins, the functional assessment of their clinically relevant variants, and the design of feasible platforms for drug discovery.

Induction of Translational Readthrough on Protein Tyrosine Phosphatases Targeted by Premature Termination Codon Mutations in Human Disease.

Nonsense mutations generating premature termination codons (PTCs) in various genes are frequently associated with somatic cancer and hereditary human diseases since PTCs commonly generate truncated proteins with defective or altered function. Induced translational readthrough during protein biosynthesis facilitates the incorporation of an amino acid at the position of a PTC, allowing the synthesis of a complete protein. This may evade the pathological effect of the PTC mutation and provide new therapeutic opportunities. Several protein tyrosine phosphatases (PTPs) genes are targeted by PTC in human disease, the tumor suppressor PTEN being the more prominent paradigm. Here, using PTEN and laforin as examples, two PTPs from the dual-specificity phosphatase subfamily, we describe methodologies to analyze in silico the distribution and frequency of pathogenic PTC in PTP genes. We also summarize laboratory protocols and technical notes to study the induced translational readthrough reconstitution of the synthesis of PTP targeted by PTC in association with disease in cellular models.

Time-dependent cross talk between spinal serotonin 5-HT2A receptor and mGluR1 subserves spinal hyperexcitability and neuropathic pain after nerve injury.

Emerging evidence implicates serotonergic descending facilitatory pathways from the brainstem to the spinal cord in the maintenance of pathologic pain. Upregulation of the serotonin receptor 2A (5-HT(2A)R) in dorsal horn neurons promotes spinal hyperexcitation and impairs spinal µ-opioid mechanisms during neuropathic pain. We investigated the involvement of spinal glutamate receptors, including metabotropic receptors (mGluRs) and NMDA, in 5-HT(2A)R-induced hyperexcitability after spinal nerve ligation (SNL) in rat. High-affinity 5-HT(2A)R agonist (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2) enhanced C-fiber-evoked dorsal horn potentials after SNL, which was prevented by mGluR1 antagonist AIDA [(RS)-1-aminoindan-1,5-dicarboxylic acid] but not by group II mGluR antagonist LY 341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid] or NMDA antagonist d-AP5 [D-(-)-2-amino-5-phosphonopentanoic acid]. 5-HT(2A)R and mGluR1 were found to be coexpressed in postsynaptic densities in dorsal horn neurons. In the absence of SNL, pharmacological stimulation of 5-HT(2A)R with TCB-2 both induced rapid bilateral upregulation of mGluR1 expression in cytoplasmic and synaptic fractions of spinal cord homogenates, which was attenuated by PKC inhibitor chelerythrine, and enhanced evoked potentials during costimulation of mGluR1 with 3,5-DHPG [(RS)-3,5-dihydroxyphenylglycine]. SNL was followed by bilateral upregulation of mGluR1 in 5-HT(2A)R-containing postsynaptic densities. Upregulation of mGluR1 in synaptic compartments was partially prevented by chronic administration of selective 5-HT(2A)R antagonist M100907 [(R)-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipidinemethanol], confirming 5-HT(2A)R-mediated control of mGluR1 upregulation triggered by SNL. Changes in thermal and mechanical pain thresholds following SNL were increasingly reversed over the days after injury by chronic 5-HT(2A)R blockade. These results emphasize a role for 5-HT(2A)R in hyperexcitation and pain after nerve injury and support mGluR1 upregulation as a novel feedforward activation mechanism contributing to 5-HT(2A)R-mediated facilitation.

Functional analysis of PTEN variants of unknown significance from PHTS patients unveils complex patterns of PTEN biological activity in disease.

Heterozygous germline mutations in PTEN gene predispose to hamartomas and tumors in different tissues, as well as to neurodevelopmental disorders, and define at genetic level the PTEN Hamartoma Tumor Syndrome (PHTS). The major physiologic role of PTEN protein is the dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), counteracting the pro-oncogenic function of phosphatidylinositol 3-kinase (PI3K), and PTEN mutations in PHTS patients frequently abrogate PTEN PIP3 catalytic activity. PTEN also displays non-canonical PIP3-independent functions, but their involvement in PHTS pathogeny is less understood. We have previously identified and described, at clinical and genetic level, novel PTEN variants of unknown functional significance in PHTS patients. Here, we have performed an extensive functional characterization of these PTEN variants (c.77 C > T, p.(Thr26Ile), T26I; c.284 C > G, p.(Pro95Arg), P95R; c.529 T > A, p.(Tyr177Asn), Y177N; c.781 C > G, p.(Gln261Glu), Q261E; c.829 A > G, p.(Thr277Ala), T277A; and c.929 A > G, p.(Asp310Gly), D310G), including cell expression levels and protein stability, PIP3-phosphatase activity, and subcellular localization. In addition, caspase-3 cleavage analysis in cells has been assessed using a C2-domain caspase-3 cleavage-specific anti-PTEN antibody. We have found complex patterns of functional activity on PTEN variants, ranging from loss of PIP3-phosphatase activity, diminished protein expression and stability, and altered nuclear/cytoplasmic localization, to intact functional properties, when compared with PTEN wild type. Furthermore, we have found that PTEN cleavage at the C2-domain by the pro-apoptotic protease caspase-3 is diminished in specific PTEN PHTS variants. Our findings illustrate the multifaceted molecular features of pathogenic PTEN protein variants, which could account for the complexity in the genotype/phenotype manifestations of PHTS patients.

The expression pattern of pyruvate dehydrogenase kinases predicts prognosis and correlates with immune exhaustion in clear cell renal cell carcinoma.

Renal cancer cells constitute a paradigm of tumor cells with a glycolytic reprogramming which drives metabolic alterations favouring cell survival and transformation. We studied the expression and activity of pyruvate dehydrogenase kinases (PDK1-4), key enzymes of the energy metabolism, in renal cancer cells. We analysed the expression, subcellular distribution and clinicopathological correlations of PDK1-4 by immunohistochemistry of tumor tissue microarray samples from a cohort of 96 clear cell renal cell carcinoma (ccRCC) patients. Gene expression analysis was performed on whole tumor tissue sections of a subset of ccRCC samples. PDK2 and PDK3 protein expression in tumor cells correlated with lower patient overall survival, whereas PDK1 protein expression correlated with higher patient survival. Gene expression analysis revealed molecular association of PDK2 and PDK3 expression with PI3K signalling pathway, as well as with T cell infiltration and exhausted CD8 T cells. Inhibition of PDK by dichloroacetate in human renal cancer cell lines resulted in lower cell viability, which was accompanied by an increase in pAKT. Together, our findings suggest a differential role for PDK enzymes in ccRCC progression, and highlight PDK as actionable metabolic proteins in relation with PI3K signalling and exhausted CD8 T cells in ccRCC.

Heterogeneous Expression and Subcellular Localization of Pyruvate Dehydrogenase Complex in Prostate Cancer.

Background: Pyruvate dehydrogenase (PDH) complex converts pyruvate into acetyl-CoA by pyruvate decarboxylation, which drives energy metabolism during cell growth, including prostate cancer (PCa) cell growth. The major catalytic subunit of PDH, PDHA1, is regulated by phosphorylation/dephosphorylation by pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). There are four kinases, PDK1, PDK2, PDK3 and PDK4, which can phosphorylate and inactivate PDH; and two phosphatases, PDP1 and PDP2, that dephosphorylate and activate PDH. Methods: We have analyzed by immunohistochemistry the expression and clinicopathological correlations of PDHA1, PDP1, PDP2, PDK1, PDK2, PDK3, and PDK4, as well as of androgen receptor (AR), in a retrospective PCa cohort of patients. A total of 120 PCa samples of representative tumor areas from all patients were included in tissue microarray (TMA) blocks for analysis. In addition, we studied the subcellular localization of PDK2 and PDK3, and the effects of the PDK inhibitor dichloroacetate (DCA) in the growth, proliferation, and mitochondrial respiration of PCa cells. Results: We found heterogeneous expression of the PDH complex components in PCa tumors. PDHA1, PDP1, PDK1, PDK2, and PDK4 expression correlated positively with AR expression. A significant correlation of PDK2 immunostaining with biochemical recurrence and disease-free survival was revealed. In PCa tissue specimens, PDK2 displayed cytoplasmic and nuclear immunostaining, whereas PDK1, PDK3 and PDK4 showed mostly cytoplasmic staining. In cells, ectopically expressed PDK2 and PDK3 were mainly localized in mitochondria compartments. An increase in maximal mitochondrial respiration was observed in PCa cells upon PDK inhibition by DCA, in parallel with less proliferative capacity. Conclusion: Our findings support the notion that expression of specific PDH complex components is related with AR signaling in PCa tumors. Furthermore, PDK2 expression associated with poor PCa prognosis. This highlights a potential for PDH complex components as targets for intervention in PCa.

The role of protein tyrosine phosphatases in prostate cancer biology.

Prostate cancer (PCa) is the most frequent malignancy in the male population of Western countries. Although earlier detection and more active surveillance have improved survival, it is still a challenge how to treat advanced cases. Since androgen receptor (AR) and AR-related signaling pathways are fundamental in the growth of normal and neoplastic prostate cells, targeting androgen synthesis or AR activity constitutes the basis of the current hormonal therapies in PCa. However, resistance to these treatments develops, both by AR-dependent and -independent mechanisms. Thus, alternative therapeutic approaches should be developed to target more efficiently advanced disease. Protein tyrosine phosphatases (PTPs) are direct regulators of the protein- and residue-specific phosphotyrosine (pTyr) content of cells, and dysregulation of the cellular Tyr phosphorylation/dephosphorylation balance is a major driving event in cancer, including PCa. Here, we review the current knowledge on the role of classical PTPs in the growth, differentiation, and survival of epithelial prostate cells, and their potential as important players and therapeutic targets for modulation in PCa.

Precise Immunodetection of PTEN Protein in Human Neoplasia.

PTEN is a major tumor-suppressor protein whose expression and biological activity are frequently diminished in sporadic or inherited cancers. PTEN gene deletion or loss-of-function mutations favor tumor cell growth and are commonly found in clinical practice. In addition, diminished PTEN protein expression is also frequently observed in tumor samples from cancer patients in the absence of PTEN gene alterations. This makes PTEN protein levels a potential biomarker parameter in clinical oncology, which can guide therapeutic decisions. The specific detection of PTEN protein can be achieved by using highly defined anti-PTEN monoclonal antibodies (mAbs), characterized with precision in terms of sensitivity for the detection technique, specificity for PTEN binding, and constraints of epitope recognition. This is especially relevant taking into consideration that PTEN is highly targeted by mutations and posttranslational modifications, and different PTEN protein isoforms exist. The precise characterization of anti-PTEN mAb reactivity is an important step in the validation of these reagents as diagnostic and prognostic tools in clinical oncology, including their routine use in analytical immunohistochemistry (IHC). Here, we review the current status on the use of well-defined anti-PTEN mAbs for PTEN immunodetection in the clinical context and discuss their potential usefulness and limitations for a more precise cancer diagnosis and patient benefit.

Expression of Human PTEN-L in a Yeast Heterologous Model Unveils Specific N-Terminal Motifs Controlling PTEN-L Subcellular Localization and Function.

The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP3 counteracts the stimulation of the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway. Recently, several extended versions of PTEN produced in the cell by alternative translation initiation have been described, among which, PTEN-L and PTEN-M represent the longest isoforms. We previously developed a humanized yeast model in which the expression of PI3K in Saccharomyces cerevisiae led to growth inhibition that could be suppressed by co-expression of PTEN. Here, we show that the expression of PTEN-L and PTEN-M in yeast results in robust counteracting of PI3K-dependent growth inhibition. N-terminally tagged GFP-PTEN-L was sharply localized at the yeast plasma membrane. Point mutations of a putative membrane-binding helix located at the PTEN-L extension or its deletion shifted localization to nuclear. Also, a shift from plasma membrane to nucleus was observed in mutants at basic amino acid clusters at the PIP2-binding motif, and at the Cα2 and CBR3 loops at the C2 domain. In contrast, C-terminally tagged PTEN-L-GFP displayed mitochondrial localization in yeast, which was shifted to plasma membrane by removing the first 22 PTEN-L residues. Our results suggest an important role of the N-terminal extension of alternative PTEN isoforms on their spatial and functional regulation.

Precise definition of PTEN C-terminal epitopes and its implications in clinical oncology.

Anti-PTEN monoclonal antibodies (mAb) are arising as important tools for immunohistochemistry (IHC) and protein quantification routine analysis in clinical oncology. Although an effort has been made to document the reliability of tumor tissue section immunostaining by anti-PTEN mAb, and to standardize their IHC use in research and in the clinical practice, the precise topological and biochemical definition of the epitope recognized by each mAb has been conventionally overlooked. In this study, six commercial anti-PTEN mAb have been validated and characterized for sensitivity and specificity by IHC and FISH, using a set of prostate and urothelial bladder tumor specimens, and by immunoblot, using PTEN positive and PTEN negative human cell lines. Immunoblot precise epitope mapping, performed using recombinant PTEN variants and mutations, revealed that all mAb recognized linear epitopes of 6-11 amino acid length at the PTEN C-terminus. Tumor-associated or disease-associated mutations at the PTEN C-terminus did not affect subcellular localization or PIP3 phosphatase activity of PTEN in cells, although resulted in specific loss of reactivity for some mAb. Furthermore, specific mimicking-phosphorylation mutations at the PTEN C-terminal region also abolished binding of specific mAb. Our study adds new evidence on the relevance of a precise epitope mapping in the validation of anti-PTEN mAb for their use in the clinics. This will be substantial to provide a more accurate diagnosis in clinical oncology based on PTEN protein expression in tumors and biological fluids.

A pathogenic role for germline PTEN variants which accumulate into the nucleus.

The PTEN gene encodes a master regulator protein that exerts essential functions both in the cytoplasm and in the nucleus. PTEN is mutated in the germline of both patients with heterogeneous tumor syndromic diseases, categorized as PTEN hamartoma tumor syndrome (PHTS), and a group affected with autism spectrum disorders (ASD). Previous studies have unveiled the functional heterogeneity of PTEN variants found in both patient cohorts, making functional studies necessary to provide mechanistic insights related to their pathogenicity. Here, we have functionally characterized a PTEN missense variant [c.49C>G; p.(Gln17Glu); Q17E] associated to both PHTS and ASD patients. The PTEN Q17E variant displayed partially reduced PIP3-catalytic activity and normal stability in cells, as shown using S. cerevisiae and mammalian cell experimental models. Remarkably, PTEN Q17E accumulated in the nucleus, in a process involving the PTEN N-terminal nuclear localization sequence. The analysis of additional germline-associated PTEN N-terminal variants illustrated the existence of a PTEN N-terminal region whose targeting in disease causes PTEN nuclear accumulation, in parallel with defects in PIP3-catalytic activity in cells. Our findings highlight the frequent occurrence of PTEN gene mutations targeting PTEN N-terminus whose pathogenicity may be related, at least in part, with the retention of PTEN in the nucleus. This could be important for the implementation of precision therapies for patients with alterations in the PTEN pathway.

Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest.

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.

PTEN recruitment controls synaptic and cognitive function in Alzheimer's models.

Dyshomeostasis of amyloid-ß peptide (Aß) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aß appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer's disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aß-induced depression. Mechanistically, Aß triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aß-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aß-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aß signaling.

Selective impairment of spinal mu-opioid receptor mechanism by plasticity of serotonergic facilitation mediated by 5-HT2A and 5-HT2B receptors.

Opioid analgesia is compromised by intracellular mediators such as protein kinase C (PKC). The phosphatidylinositol hydrolysis-coupled serotonin receptor 5-HT2 is ideally suited to promote PKC activation. We test the hypothesis that 5-HT2A and 5-HT2B receptors, which have been previously shown to become pro-excitatory after spinal nerve ligation (SNL), can negatively influence the ability of opioids to depress spinal excitation evoked by noxious input. Spinal superfusion with (100 nM) mu-opioid receptor (MOR)-agonist DAMGO significantly depressed C fiber-evoked spinal field potentials. Simultaneous administration of subclinical 5-HT2AR antagonist 4F 4PP (100 nM) or 5-HT2BR antagonist SB 204741 (100 nM) significantly reduced the IC50 value for DAMGO in nerve-ligated rats (97.56 nM ± 1.51 and 1.20 nM ± 1.28 respectively, relative to 104 nM ± 1.08 at the baseline condition), but not in sham-operated rats. Both antagonists failed to alter depression induced by delta-opioid receptor (DOR)-agonist D-ala2-deltorphin II after SNL as well as in the sham condition. Western blot analysis of dorsal horn homogenates revealed bilateral upregulation of 5-HT2AR and 5-HT2BR protein band densities after SNL. As assessed from double immunofluorescence labeling for confocal laser scanning microscopy, scarce dorsal horn cell processes showed co-localization color overlay for 5-HT2AR/MOR, 5-HT2BR/MOR, 5-HT2AR/DOR, or 5-HT2BR/DOR in sham-operated rats. Intensity correlation-based analyses showed significant increases in 5-HT2AR/MOR and 5-HT2BR/MOR co-localizations after SNL. These results indicate that plasticity of spinal serotonergic neurotransmission can selectively reduce spinal MOR mechanisms via 5-HT2A and 5-HT2B receptors, including upregulation of the latter and increased expression in dorsal horn neurons containing MOR.