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.

Redox control of glutamine utilization in cancer.

Glutamine utilization promotes enhanced growth of cancer cells. We propose a new concept map of cancer metabolism in which mitochondrial NADH and NADPH, in the presence of a dysfunctional electron transfer chain, promote reductive carboxylation from glutamine. We also discuss why nicotinamide nucleotide transhydrogenase (NNT) is required in vivo for glutamine utilization by reductive carboxylation. Moreover, NADPH, generated by both the pentose phosphate pathway and the cancer-specific serine glycolytic diversion, appears to sustain glutamine utilization for amino-acid synthesis, lipid synthesis, and for ROS quenching. The fact that the supply of NAD(+) precursors reduces tumor aggressiveness suggests experimental approaches to clarify the role of the NADH-driven redox network in cancer.

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response.

Cancer cells, which use more glucose than normal cells and accumulate extracellular lactate even under normoxic conditions (Warburg effect), have been reported to undergo cell death under glucose deprivation, whereas normal cells remain viable. As it may be relevant to exploit the molecular mechanisms underlying this biological response to achieve new cancer therapies, in this paper we sought to identify them by using transcriptome and proteome analysis applied to an established glucose-addicted cellular model of transformation, namely, murine NIH-3T3 fibroblasts harboring an oncogenic K-RAS gene, compared with parental cells. Noteworthy is that the analyses performed in high- and low-glucose cultures indicate that reduction of glucose availability induces, especially in transformed cells, a significant increase in the expression of several unfolded protein response (UPR) hallmark genes. We show that this response is strictly associated with transformed cell death, given that its attenuation, by reducing protein translation or by increasing cell protein folding capacity, preserves the survival of transformed cells. Such an effect is also observed by inhibiting c-Jun NH2-terminal kinase, a pro-apoptotic signaling mediator set downstream of UPR. Strikingly, addition of N-acetyl-D-glucosamine, a specific substrate for the hexosamine biosynthesis pathway (HBP), to glucose-depleted cells completely prevents transformed cell death, stressing the important role of glucose in HBP fuelling to ensure UPR attenuation and increased cell survival. Interestingly, these results have been fully recognized in a human model of breast cancer, MDA-MB-231 cells. In conclusion, we show that glucose deprivation, leading to harmful accumulation of unfolded proteins in consequence of a reduction of protein glycosylation, induces a UPR-dependent cell death mechanism. These findings may open the way for new therapeutic strategies to specifically kill glycolytic cancer cells.

Oncogenic K-ras expression is associated with derangement of the cAMP/PKA pathway and forskolin-reversible alterations of mitochondrial dynamics and respiration.

The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs.

Remodelling of supraspinal neuroglial network in neuropathic pain is featured by a reactive gliosis of the nociceptive amygdala.

BACKGROUND: Many brain areas participate to supraspinal control of nociception. In these regions, few studies have investigated the role of glial cells in supraspinal plasticity and the effect of 7-day intrathecal nerve growth factor-like (BB14®, Blueprint Biotech, Milano, Italy) treatment. METHODS: In male Sprague-Dawley rats, we evaluated by immunohistochemistry the morphological and molecular rearrangement of neuroglial network occurring in several supraspinal brain regions involved in pain processing following spared nerve injury (SNI) of the sciatic nerve. In particular, the medial prefrontal cortex, the amygdala (Amy), the nucleus accumbens (Acb), the thalamus and the periaqueductal gray were analysed. RESULTS: Despite the modifications occurring in the dorsal horn of spinal cord following SNI, no significant changes in the Iba1 and glial fibrillary acidic protein (GFAP) expression were detected in all the analysed supraspinal regions, except for the Amy, showing a remarkable GFAP increase. Interestingly, neuropathic rats also displayed a significant increase of glial transporters (GTs) in all the supraspinal regions. Finally, the analysis of vesicular glutamate transporter 1 (vGLUT1) and vesicular gamma-aminobutyric acid (GABA) transporter (vGAT) expression revealed a significant enhancement of glutamatergic/GABAergic ratio in all selected brain regions of SNI animals, except for Acb. Both glial activation in the Amy and alteration of GTs and vGLUT/vGAT levels observed in neuropathic animals were largely reversed by BB14® treatment. CONCLUSIONS: All together, these data strengthen the role of supraspinal neuroglial network plasticity in the establishment of neuropathic pain syndrome. The hallmark is represented by the divergence between glial reaction confined to Amy and the widespread changes in the GT distribution and glutamate/GABA ratio detected in the other supraspinal region.

Integrative transcriptional analysis between human and mouse cancer cells provides a common set of transformation associated genes.

Mouse functional genomics is largely used to investigate relevant aspects of mammalian physiology and pathology. To which degree mouse models may offer accurate representations of molecular events underlining human diseases such as cancer is not yet fully established. Herein we compare gene expression signatures between a set of human cancer cell lines (NCI-60 cell collection) and a mouse cellular model of oncogenic K-ras dependent transformation in order to identify their closeness at the transcriptional level. The results of our integrative and comparative analysis show that in both species as compared to normal cells or tissues the transformation process involves the activation of a transcriptional response. Furthermore, the cellular mouse model of K-ras dependent transformation has a good degree of similarity with several human cancer cell lines and in particular with cell lines containing oncogenic Ras mutations. Moreover both species have similar genetic signatures that are associated to the same altered cellular pathways (e.g. Spliceosome and Proteasome) or to deregulation of the same genes (e.g. cyclin D1, AHSA1 and HNRNPD) detected in the comparison between cancer cells versus normal cells or tissues. In summary, we report one of the first in-depth analysis of global gene expression profiles of a K-ras dependent mouse cell model of transformation and a large collection of human cancer cells as compared to their normal counterparts. Taken together our findings show a strong correlation in the transcriptional and pathway alteration responses between the two species, therefore validating the use of the mouse model as an appropriate tool to investigate human cancer, and indicating that the comparative analysis, as described here, offers a useful approach to identify cancer-specific gene signatures.

From cancer metabolism to new biomarkers and drug targets.

Great interest is presently given to the analysis of metabolic changes that take place specifically in cancer cells. In this review we summarize the alterations in glycolysis, glutamine utilization, fatty acid synthesis and mitochondrial function that have been reported to occur in cancer cells and in human tumors. We then propose considering cancer as a system-level disease and argue how two hallmarks of cancer, enhanced cell proliferation and evasion from apoptosis, may be evaluated as system-level properties, and how this perspective is going to modify drug discovery. Given the relevance of the analysis of metabolism both for studies on the molecular basis of cancer cell phenotype and for clinical applications, the more relevant technologies for this purpose, from metabolome and metabolic flux analysis in cells by Nuclear Magnetic Resonance and Mass Spectrometry technologies to positron emission tomography on patients, are analyzed. The perspectives offered by specific changes in metabolism for a new drug discovery strategy for cancer are discussed and a survey of the industrial activity already going on in the field is reported.

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.

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.

Glucose modulation of cell size in yeast.

Saccharomyces cerevisiae cells grown in glucose have larger average size than cells grown in ethanol. Besides, yeast must reach a carbon source-modulated critical cell size in order to enter S phase at Start. This control is of outmost physiological relevance, since it allows us to coordinate cell growth with cell cycle progression and it is responsible for cell size homeostasis. The cell sizer mechanism requires the overcoming of two sequential thresholds, involving Cln3 and Far1, and Clb5,6 and Sic1, respectively. When both thresholds are non-functional, carbon source modulation of cell size at Start is completely abolished. Since inactivation of extracellular glucose sensing through deletion of either the GPR1 or the GPA2 gene causes a marked, but partial, reduction in the ability to modulate cell size and protein content at Start, it is proposed that both extracellular and intracellular glucose signalling is required for properly setting the cell sizer in glucose media.

Acquired glucose sensitivity of k-ras transformed fibroblasts.

Mutational activation of the ras gene is critical for the onset of different malignant phenotypes. We constructed a dominant negative mutant (GEF-DN) of a Ras activator protein (guanine nucleotide-exchange factor) that upon over-expression in k-ras transformed NIH 3T3 fibroblasts strongly reduces intracellular Ras*GTP, reverting these cells to wild-type phenotype for morphology, anchorage-independent growth and reduction of tumour formation in nude mice. Here we review evidence showing that the enhanced proliferation potential of NIH-ras cells requires high initial glucose concentration in the medium and sustained Ras pathway activation. The exquisite sensitivity of NIH-ras fibroblasts to a shortage in nutrient and energy supply highlights an acquired fragility of cancer cells that may be exploited for therapeutic purposes.

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.

Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells.

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

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.

Relating growth dynamics and glucoamylase excretion of individual Saccharomyces cerevisiae cells.

We have developed a novel flow cytometric procedure that allows determinations of properties of protein excretion in the growth medium on a cell-by-cell basis in Saccharomyces cerevisiae. The procedure is based on labelling of a periplasmically secreted protein with antibodies conjugated to a fluorescent marker such as fluorescein isothiocyanate (FITC). The staining conditions did not perturb cell growth after resuspension of stained cells in growth medium. Decrease in fluorescence was found to correlate with excretion of glucoamylase into the growth medium. The analysis of the staining pattern over time provides information on the behaviour of individual cells belonging to different cell-cycle phases and can be used to calculate the specific excretion rate of the overall population.

Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae.

A genetic and analytical methodology was developed based on a green fluorescent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL)(1-10)/CYC1 promoter and plasmids that are maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) were expressed in low to high concentration. Flow cytometric analysis was then applied to directly quantify Gfp((S65T)) (both wild type and mutant protein) expression at the single-cell level, and to indirectly measure the concentrations of non-fluorescent apoGfp((S65T)) and fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversion during aerobic growth showed that the time required for complete apoGfp((S65T)) conversion is limited only by the amount of apoprotein that is expressed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a very strong promoter.

Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae.

The CDK (cyclin-dependent kinase) family of enzymes is required for the G(1)-to-S-phase and G(2)-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G(1) phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317-20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G(1) stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G(1) progression.

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa.

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

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.