CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer.

Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.

Ras-dependent carbon metabolism and transformation in mouse fibroblasts.

Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.

An approach to address Candida rugosa lipase regioselectivity in the acylation reactions of trytilated glucosides.

Candida rugosa lipase crude preparations (CRL) catalyse the regioselective acylation of methyl 6-O-trytil beta-d-glucopyranoside in organic solvents, using vinyl acetate as acyl donor. The ratio of the two products formed, namely methyl 2-O acetyl 6-O-trytil beta-d-glucopyranoside and methyl 3-O acetyl 6-O-trytil beta-d-glucopyranoside was found to be markedly affected by the nature of the reaction medium. In hydrophobic solvents values up to 80% of the monoacetylated product in position C-3 were obtained compared to less than 30% in solvents with low hydrophobicity. Computational studies were carried out to simulate the interactions between methyl 6-O-trytil beta-d-glucopyranoside and both CRL and the solvents, in order to rationalize the experimental results.

Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

Towards a blueprint of the cell cycle.

The understanding of the organisation of cell cycle events is of utmost importance to devise effective therapeutic strategies for cancer. In this article we gather evidences from the literature in support of a system model of the cell cycle, in which a growth-sensitive threshold controls entry into S phase and the sequential activation of cyclin-dependent kinases. The cycle is terminated by an End function, that comprises events from the onset of mitosis to cell division and that may also be modulated by the increase of cell size. This blueprint allows quantitative predictions by computer simulations of steady and transitory states. In fact, we show that the proposed control system applies to budding yeast populations during nutritional shift-up and following hyperactivation of the cAMP signalling pathway. Besides the growth-sensitive control system it is shown to apply to mammalian cells both in the exit from quiescence and in active proliferation. The putative molecular determinants that set the threshold controlling S phase entry are consistently altered in cancer cells. Finally, we discuss an input/output analysis based on the simulated behaviour derived from the blueprint as a new tool to investigate the road to cancer.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Posttranscriptional regulation of the expression of MET2 gene of Saccharomyces cerevisiae.

The first step of the specific pathway for methionine biosynthesis in the yeast Saccharomyces cerevisiae is catalyzed by the enzyme L-homoserine-O-acetyltransferase (HSTase) (EC 2.3.1.31), encoded by the MET2 gene. In order to ascertain whether there is a posttranscriptional control on the MET2 gene expression, as suggested by previous results on the expression of the cloned gene, systems for high inducible expression of MET2 gene were constructed. In these constructs the MET2 gene was cloned in yeast expression vectors under the control of an inducible yeast GAL promoter element so that the MET2 was transcribed at very high levels under induced conditions. Measurements of the specific mRNA levels showed a strong stimulation of MET2 gene transcription in yeast transformants grown on galactose as carbon source, corresponding to 50-100-fold the repressed conditions, while only a 2-fold increase of the enzymatic activity was observed. In addition, no evidence of a strong induced polypeptide of appropriate size on two dimensional gel electrophoresis was obtained. To understand the functional role of the non-coding 5' region of MET2 mRNA, we performed either a partial and a complete deletion of the 5' leader sequence, but even with these constructs an elevated mRNA level was not associated to a marked increase of the HSTase activity. These data support the idea of a posttranscriptional regulation of MET2 gene expression and show that the untranslated region of the specific mRNA is not involved in this regulatory mechanism.

Expression of cloned Saccharomyces diastaticus glucoamylase under natural and inducible promoters.

Any one of three homologous genes - STA1, STA2 and STA3 - encoding glucoamylase isozymes I, II and III respectively, allows the Saccharomyces species to utilize starch as a sole carbon source. We show in this paper that glucoamylase II production can be increased 4-fold over the level produced by STA2 strains, by using a two-step fermentation and a yeast strain transformed with a high-copy-number plasmid carrying the STA2 gene. The accumulation of anomalous STA2 mRNA species, mainly differing at their 5' ends, and saturation of step(s) in the secretory pathway appear to be among the major factors limiting glucoamylase expression in synthetic media.

Reduction of ribosome activity and synthesis of stable RNA in Neurospora crassa.

The addition of cycloheximide (0.02 micrograms/ml) to exponentially growing cultures of Neurospora crassa causes a reduction in growth rate and a decrease in the rate of protein accumulation, due to a partial inhibition of protein synthesis, while RNA accumulation is unaffected for about 1 h. Thus, an increased RNA:protein ratio is established in the presence of the inhibitor. RNA that accumulates during treatment with cycloheximide has the same characteristics as that of the control cultures and this, together with the enhancement of the relative rate of synthesis of ribosomal proteins induced by cycloheximide, seems to indicate that more mature ribosomes are present in cycloheximide-treated cultures. The endocellular level of several amino acids begins to increase significantly only 60 min after cycloheximide addition. A possible explanation of the stimulation of ribosome production induced by cycloheximide is given in terms of the existence of a feed-back mechanism controlling ribosome synthesis.

Homology-derived three-dimensional structure prediction of Candida cylindracea lipase.

We propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed.

Cloning and nucleotide sequences of two lipase genes from Candida cylindracea.

Two lipase-encoding genes (LIP1 and LIP2) have been isolated from a SacI genomic library of the yeast Candida cylindracea and their nucleotide sequences have been determined. Comparison with the sequence of a cDNA ruled out the presence of introns in the two genes. Both ORFs encode for mature proteins of 534 residues with putative signal peptides of 15 and 14 amino acids, respectively. When compared with other lipase sequences, the two C. cylindracea lipases showed homology only with the Geotrichum candidum lipase, whereas they shared a significant similarity with several esterases.

Analysis of the secondary structure of the catalytic domain of mouse Ras exchange factor CDC25Mm.

The minimal active domain (GEF domain) of the mouse Ras exchange factor CDC25Mm was purified to homogeneity from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism (CD) spectra in the far UV region indicate that this domain is highly structured with a high content of alpha-helix (42%). Near UV CD spectra evidenced good signal due to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this domain. The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the binary complex with H-ras p21, suggesting that the Trp residues, which are well conserved in the GEF domain of several Ras-exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the interaction with Ras proteins.

The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools.

The CDC25 gene is transcribed at a very low level in S. cerevisiae cells. We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter. The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose. This strain does not show any apparent alteration of growth, both in glucose and in galactose. Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium. The nucleotides were extracted from transformed cells, separated by HPLC and quantitated. The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool. The disruption of RAS genes was only partially able to 'cure' this phenotype. A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C. These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol.

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

Bombesin stimulates a high affinity GTPase activity in membranes of Swiss 3T3 fibroblasts.

Peptides of the bombesin family are mitogenic for Swiss 3T3 fibroblasts and in these cells stimulate the turnover of polyphosphoinositides. Recent studies have suggested that G protein(s) may be involved in the signal transduction pathway triggered by bombesin. In this study we have found and characterized a high affinity GTPase activity stimulated by bombesin in membranes of Swiss 3T3 fibroblasts. Our results support the involvement of a G protein in the response of Swiss 3T3 cells to bombesin.

Effect of the growth conditions on the expression of cell-surface-associated platelet-derived growth factor receptors in mouse fibroblasts.

The conditions affecting the appearance and disappearance of platelet-derived growth factor (PDGF) receptors from the pool of active cell surface-associated receptors were studied. Receptor molecules were revealed in intact Swiss 3T3 fibroblasts by their ability to bind 125I-labeled PDGF and, due to their property to become phosphorylated in tyrosine following ligand binding, by antibodies to phosphotyrosine. PDGF receptor molecules were found to be quite scarce in exponentially growing fibroblasts as compared to quiescent cells. When growing cells were either shifted to a medium containing plasma or received suramin in the culture medium, cell surface-associated PDGF receptors largely increased. This process required about 12 h. Incubation of quiescent cells in serum, but not in plasma, induced a slow decrement of ligand-activatable receptors. In the presence of PDGF the rate of receptor removal from the cell surface was very rapid and was a function of the PDGF concentration. Quiescent cells deprived of cell-surface receptors by incubation with PDGF reexpressed PDGF receptors in about 14 h.

In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies.

Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase.

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.

Evolutionary origin of nonuniversal CUGSer codon in some Candida species as inferred from a molecular phylogeny.

CUG, a universal leucine codon, has been reported to be read as serine in various yeast species belonging to the genus Candida. To gain a deeper insight into the origin of this deviation from the universal genetic code, we carried out a phylogenetic analysis based on the small-subunit ribosomal RNA genes from some Candida and other related Hemiascomycetes. Furthermore, we determined the phylogenetic relationships between the tRNA(Ser)CAG, responsible for the translation of CUG, from some Candida species and the other serine and leucine isoacceptor tRNAs in C. cylindracea. We demonstrate that the group of Candida showing the genetic code deviation is monophyletic and that this deviation could have originated more than 150 million years ago. We also describe how phylogenetic analysis can be used for genetic code predictions.