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Choline kinase alpha (ChoKα) is a therapeutic target being developed for a variety of diseases, from cancer to rheumatoid arthritis and from parasites to bacterial infections. Nevertheless, the therapeutic potential of this drug target seems not exhausted and may end up as a possible solution for a larger variety of conditions. Here we present our working model for how ChoKα could play a role in obesity and for how drugs being developed as therapeutics for other diseases using ChoKα as a target, could be repurposed as prophylactic treatments for obesity. We also present preliminary observations in support of our model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lacal, Ibrahim and Zimmerman.)

Background: Primary resistance to anti-EGFR therapies affects 40% of metastatic colorectal cancer patients harbouring wild-type RAS/RAF. YAP1 activation is associated with this resistance, prompting an investigation into AURKA's role in mediating YAP1 phosphorylation at Ser397, as observed in breast cancer., Methods: We used transcriptomic analysis along with in vitro and in vivo models of RAS/RAF wild-type CRC to study YAP1 Ser397 phosphorylation as a potential biomarker for cetuximab resistance. We assessed cetuximab efficacy using CCK8 proliferation assays and cell cycle analysis. Additionally, we examined the effects of AURKA inhibition with alisertib and created a dominant-negative YAP1 Ser397 mutant to assess its impact on cancer stem cell features., Results: The RAS/RAF wild-type CRC models exhibiting primary resistance to cetuximab prominently displayed elevated YAP1 phosphorylation at Ser397 primarily mediated by AURKA. AURKA-induced YAP1 phosphorylation was identified as a key trigger for cancer stem cell reprogramming. Consequently, we found that AURKA inhibition had the capacity to effectively restore cetuximab sensitivity and concurrently suppress the cancer stem cell phenotype., Conclusions: AURKA inhibition holds promise as a therapeutic approach to overcome cetuximab resistance in RAS/RAF wild-type colorectal cancer, offering a potential means to counter the development of cancer stem cell phenotypes associated with cetuximab resistance., (© 2024. The Author(s).)

Lung cancer is one of the main causes of death in developed countries, and non-small cell lung cancer (NSCLC) is the most frequent type (80% of patients). In advanced NSCLC, platinum-based chemotherapy is the frontline palliative treatment, but less than 5% of patients achieve prolonged survival. Immunotherapy has recently been proposed as the standard of care (SoC) as either monotherapy or in combination with chemotherapy for advanced NSCLC. The levels of expression of PD-L1 are the only predictive biomarkers for patient assessment. Although around 30% of patients receiving immunotherapy achieve 5-year survival, a significant number does not benefit from this novel therapeutic approach. Therefore, there is a need for novel strategies to improve clinical outcomes. The expression level of choline kinase α (ChoKα) is increased in a large number of human tumors, including NSCLC tumors, and constitutes an independent prognostic factor for early-stage NSCLC patients. Thus, ChoKα has been postulated as a new target drug in cancer therapy. The combination of cisplatin with novel targeted drugs such as choline kinase inhibitors may improve both the survival rates and the quality of life of NSCLC patients and may serve as the basis for the development of new therapeutic approaches. To that aim, we developed several in vitro and in vivo approaches to assess the antitumor activity of a novel combination regimen using cisplatin and ChoKα inhibitors. Our results suggest that a proper combination of specific inhibitors of the NSCLC prognostic factor ChoKα and platinum-based conventional chemotherapy might constitute a new, efficient treatment approach for NSCLC patients. This novel approach may help reduce the toxicity profile associated with cisplatin since, despite the advances in NSCLC management in recent years, the overall 5-year survival rate is still poor.

Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the phosphorylation of choline to form phosphorylcholine ( P Cho) in the presence of ATP and magnesium. ChoK is required for the synthesis of key membrane phospholipids and is involved in malignant transformation in a large variety of human tumours. Active compounds against ChoK have been identified and proposed as antitumor agents. The ChoK inhibitory and antiproliferative activities of symmetrical bispyridinium and bisquinolinium compounds have been defined using quantitative structure-activity relationships (QSARs) and structural parameters. The design strategy followed in the development of the most active molecules is presented. The selective anticancer activity of these structures is also described. One promising anticancer compound has even entered clinical trials. Recently, ChoKα inhibitors have also been proposed as a novel therapeutic approach against parasites, rheumatoid arthritis, inflammatory processes, and pathogenic bacteria. The evidence for ChoKα as a novel drug target for approaches in precision medicine is discussed.

Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 μM, respectively, and were shown to be 2-4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 μM and 10 μM, respectively, and the minimum lethal concentration (MLC) was 1.6 μM and 20 μM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae.

Both nosocomial pathogens, such as Streptococcus pneumoniae and Haemophilus influenzae and food-borne pathogens, such as Bacillus cereus and Clostridium perfringens are known to be detrimental to human and animal health. The effectiveness of currently used treatments for these pathogens becomes limited as resistant strains emerge. Therefore, new methods for eliminating bacterial pathogens must be developed continuously. This includes establishing novel targets to which drug discovery efforts could be focused. A promising method for discovering new drug targets in prokaryotes is to take advantage of the information available regarding the enzymatic pathways that have been established as drug targets in eukaryotic systems and explore the analogous pathways found in bacterial systems. This is an efficient strategy because the same inhibitors developed at considerable expense to block these pathways in eukaryotic systems could also be employed in prokaryotes. Drugs that are used to prevent diseases involving eukaryotic cells could be repurposed as antibiotics and antimicrobials for the control of bacteria pathogens. This strategy could be pursued whenever the primary and tertiary structures of a target are are conserved between eukaryotic and prokaryotes. A possible novel target fitting these parameters is choline kinase (ChoK), whose active site sequences are conserved (Figure 1) and whose tertiary structure (Figure 2) is maintained. Here, we describe why ChoK is a putative drug target by describing its role in the growth and pathogenesis of Gram-positive bacteria S. pneumoniae and the Gram-negative bacteria H. influenzae . Using S. pneumoniae as a model, we also present promising preliminary information that repurposing of drugs known to inhibit the human isoform of ChoK (hChoK), is a promising strategy for blocking the growth of S. pneumoniae cells and inhibiting the activity of the S. pneumoniae isoform of ChoK (sChok), with downstream physiological effects on the cell wall., (Copyright © 2019 Zimmerman, Lacal and Ibrahim.)

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1β-dependent inflammation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CDC42 is an oncogenic Rho GTPase overexpressed in colorectal cancer (CRC). Although CDC42 has been shown to regulate gene transcription, the specific molecular mechanisms regulating the oncogenic ability of CDC42 remain unknown. Here, we have characterized the transcriptional networks governed by CDC42 in the CRC SW620 cell line using gene expression analysis. Our results establish that several cancer-related signaling pathways, including cell migration and cell proliferation, are regulated by CDC42. This transcriptional signature was validated in two large cohorts of CRC patients and its clinical relevance was also studied. We demonstrate that three CDC42-regulated genes offered a better prognostic value when combined with CDC42 compared to CDC42 alone. In particular, the concordant overexpression of CDC42 and silencing of the putative tumor suppressor gene CACNA2D2 dramatically improved the prognostic value. The CACNA2D2/CDC42 prognostic classifier was further validated in a third CRC cohort as well as in vitro and in vivo CRC models. Altogether, we show that CDC42 has an active oncogenic role in CRC via the transcriptional regulation of multiple cancer-related pathways and that CDC42-mediated silencing of CACNA2D2 is clinically relevant. Our results further support the use of CDC42 specific inhibitors for the treatment of the most aggressive types of CRC.

Lung cancer is the leading cause of cancer death. Beyond first line treatment, few therapeutic options are available, particularly for squamous cell carcinoma (SCC). Here, we have explored the phospholipidomes of 30 human SCCs and found that they almost invariably (in 96.7% of cases) contain phospholipids with longer acyl chains compared to matched normal tissues. This trait was confirmed using in situ 2D-imaging MS on tissue sections and by phospholipidomics of tumor and normal lung tissue of the L-IkkαKA/KA mouse model of lung SCC. In both human and mouse, the increase in acyl chain length in cancer tissue was accompanied by significant changes in the expression of acyl chain elongases (ELOVLs). Functional screening of differentially expressed ELOVLs by selective gene knockdown in SCC cell lines followed by phospholipidomics revealed ELOVL6 as the main elongation enzyme responsible for acyl chain elongation in cancer cells. Interestingly, inhibition of ELOVL6 drastically reduced colony formation of multiple SCC cell lines in vitro and significantly attenuated their growth as xenografts in vivo in mouse models. These findings identify acyl chain elongation as one of the most common traits of lung SCC discovered so far and pinpoint ELOVL6 as a novel potential target for cancer intervention.

Choline kinase α (CHKα) plays a crucial role in the regulation of membrane phospholipid synthesis and has oncogenic properties in vitro. We have analyzed the expression of CHKα in cell lines derived from pancreatic ductal adenocarcinoma (PDAC) and have found increased CHKα expression, associated with differentiation. CHKα protein expression was directly correlated with sensitivity to MN58b, a CHKα inhibitor that reduced cell growth through the induction of apoptosis. Accordingly, CHKα knockdown led to reduced drug sensitivity. In addition, we found that gemcitabine-resistant PDAC cells displayed enhanced sensitivity to CHKα inhibition and, in vitro, MN58b had additive or synergistic effects with gemcitabine, 5-fluorouracil, and oxaliplatin, three active drugs in the treatment of PDAC. Using tissue microarrays, CHKα was found to be overexpressed in 90% of pancreatic tumors. While cytoplasmic CHKα did not relate to survival, nuclear CHKα distribution was observed in 43% of samples and was associated with longer survival, especially among patients with well/moderately differentiated tumors. To identify the mechanisms involved in resistance to CHKα inhibitors, we cultured IMIM-PC-2 cells with increasingly higher concentrations of MN58b and isolated a subline with a 30-fold higher IC50. RNA-Seq analysis identified upregulation of ABCB1 and ABCB4 multidrug resistance transporters, and functional studies confirmed that their upregulation is the main mechanism involved in resistance. Overall, our findings support the notion that CHKα inhibition merits further attention as a therapeutic option in patients with PDAC and that expression levels may predict response., (©2016 American Association for Cancer Research.)

Objectives: Little is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage., Methods: Choline metabolic profile of FLS cells was determined by (1)H magnetic resonance spectroscopy ((1)HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC₅₀=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration., Results: The enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease., Conclusions: These data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Choline kinase alpha (ChoKα) is a critical enzyme in the synthesis of phosphatidylcholine, a major structural component of eukaryotic cell membranes. ChoKα is overexpressed in a large variety of tumor cells and has been proposed as a target for personalized medicine, both in cancer therapy and rheumatoid arthritis., Materials and Methods: Triterpene quinone methides (TPQ) bioactive compounds isolated from plants of the Celastraceae family and a set of their semisynthetic derivatives were tested against the recombinant human ChoKα. Those found active as potent enzymatic inhibitors were tested in vitro for antiproliferative activity against HT29 colorectal adenocarcinoma cells, and one of the active compounds was tested for in vivo antitumoral activity in mice xenographs of HT29 cells., Results: Among 59 natural and semisynthetic TPQs tested in an ex vivo system, 14 were highly active as inhibitors of the enzyme ChoKα with IC50 <10 μM. Nine of these were potent antiproliferative agents (IC50 <10 μM) against tumor cells. At least one compound was identified as a new antitumoral drug based on its in vivo activity against xenographs of human HT-29 colon adenocarcinoma cells., Conclusions: The identification of a new family of natural and semisynthetic compounds with potent inhibitory activity against ChoKα and both in vitro antiproliferative and in vivo antitumoral activity supports further research on these inhibitors as potential anticancer agents. Their likely role as antiproliferative drugs deserves further studies in models of rheumatoid arthritis.

Choline kinase α (CHKA; here designated as ChoKα) is the first enzyme in the CDP-choline pathway, implicated in phospholipids metabolism. It is overexpressed in several human tumors such as breast, lung, bladder, colorectal, prostate, ovary, and liver. The overexpression of ChoKα has oncogenic potential and synergizes with other known oncogenes. It has been proposed as a novel cancer drug target with a distinct mechanism of action. We have generated a set of ChoKα inhibitors with potent in vitro antiproliferative and in vivo antitumoral activity against human xenografts in mice, showing high efficacy with low toxicity profiles. Among these inhibitors, RSM-932A has been chosen for further clinical development due to its potent antiproliferative activity in vitro against a large variety of tumor-derived cell lines, a potent in vivo anticancer activity, and lack of toxicity at the effective doses. Here, we provide the preclinical evidence to support the use of RSM-932A as a good candidate to be tested in clinical trials as the "first in humans" drug targeting ChoKα., (©2014 American Association for Cancer Research.)

Aim: The relevance of the cytidine diphosphate-choline and Rho GTPases pathways in the pathogenesis of cancer has been previously demonstrated. We investigate by a case-control association study if genetics variants in these pathways are associated with risk of developing lung cancer., Methods: Thirty-seven tag SNPs were evaluated as risk factor of NSCLC in 897 cases and 904 controls., Results: Six SNPs were nominally associated with lung cancer risk, which were not significant after the Bonferroni correction for multiple comparisons. No association was observed with the remaining 31 analyzed SNPs, neither it was found significant in haplotype frequencies., Conclusions: Although the implication of the two pathways investigated in our study in carcinogenesis is well established, our null results suggest that common genetic variants in CDP-choline and Rho GTPases-related genes are not risk factors for lung cancer.

The Rho GTPase Rac1 is a multifunctional protein working through different effector pathways. The emerging physiological significance of glycanlectin recognition gives reason to testing the possibility for an influence of modulation of Rac1 expression on these molecular aspects. Using human colon adenocarcinoma (SW620) cells genetically engineered for its up- and down-regulation (Rac1+ and Rac1- cells) along with wild-type and mock-transfected control cells, the questions are addressed whether the presence of adhesion/growth-regulatory galectins and distinct aspects of cell surface glycosylation are affected. Proceeding from RT-PCR data to Western blotting after two-dimensional gel electrophoresis and flow cytofluorimetry with non-crossreactive antibodies against six members of this lectin family (i.e. galectins-1, -3, -4, -7, -8 and -9), a reduced extent of the presence of galectins-1, -7 and -9 was revealed in the case of Rac1 cells. Application of these six galectins as probes to determination of cell reactivity for human lectins yielded relative increases in surface labelling of Rac1- cells with galectins-1, -3 and -7. Examining distinct aspects of cell surface glycosylation with a panel of 14 plant/fungal lectins disclosed a decrease in α2,6-sialylation of N-glycans and an increase in PNA-reactive sites (i.e. non-sialylated core 1 O-glycans), two alterations known to favour reactivity for galectins-1 and -3. Thus, manipulation of Rac1 expression selectively affects the expression pattern within the galectin network at the level of proteins and distinct aspects of cell surface glycosylation.

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum (p.f.-ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f.-ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f.-ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f.-ChoK as an accessible drug target against the parasite.

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

Background: Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy., Methodology/principal Findings: ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action., Conclusion/significance: Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Three main molecular mechanisms are considered to contribute expanding the repertoire and diversity of proteins present in living organisms: first, at DNA level (gene polymorphisms and single nucleotide polymorphisms); second, at messenger RNA (pre-mRNA and mRNA) level including alternative splicing (also termed differential splicing or cis-splicing); finally, at the protein level mainly driven through PTM and specific proteolytic cleavages. Chimeric mRNAs constitute an alternative source of protein diversity, which can be generated either by chromosomal translocations or by trans-splicing events. The occurrence of chimeric mRNAs and proteins is a frequent event in cells from the immune system and cancer cells, mainly as a consequence of gene rearrangements. Recent reports support that chimeric proteins may also be expressed at low levels under normal physiological circumstances, thus, representing a novel source of protein diversity. Notably, recent publications demonstrate that chimeric protein products can be successfully identified through bottom-up proteomic analyses. Several questions remain unsolved, such as the physiological role and impact of such chimeric proteins or the potential occurrence of chimeric proteins in higher eukaryotic organisms different from humans. The occurrence of chimeric proteins certainly seems to be another unforeseen source of complexity for the proteome. It may be a process to take in mind not only when performing bottom-up proteomic analyses in cancer studies but also in general bottom-up proteomics experiments., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Management of locally advanced rectal cancer (RC) consists of neoadjuvant chemoradiotherapy (CRT) with fluoropyrimidines, followed by total mesorectal excision. We sought to evaluate the expression of selected genes, some of which were derived from a previous undirected SAGE (serial analysis of gene expression)-based approach, before and after CRT, to identify mechanisms of resistance., Methods: This retrospective cohort study included 129 consecutive patients. Quantitative polymerase chain reaction of 53 candidate genes was performed on the biopsy specimen before treatment and on the surgical specimen after CRT. A paired-samples t test was performed to determine genes that were significantly changed after CRT. The result was correlated with patients' disease-free survival., Results: Twenty-two genes were significantly upregulated, and two were significantly downregulated. Several of the upregulated genes have roles in cell cycle control; these include CCNB1IP1, RCC1, EEF2, CDKN1, TFF3, and BCL2. The upregulation of TFF3 was associated with worse disease-free survival on multivariate analyses (hazard ratio, 2.64; P=.027). Patients whose surgical specimens immunohistochemically showed secretion of TFF3 into the lumen of the tumoral microglands had a higher risk of relapse (hazard ratio, 2.51; P=.014). In vitro experiments showed that DLD-1 cells stably transfected with TFF3 were significantly less sensitive to 5-fluorouracil and showed upregulation of genes involved in the transcriptional machinery and in resistance to apoptosis., Conclusion: Upregulation of TFF3 after CRT for RC is associated with a higher risk of relapse. The physiological role of TFF3 in restoring the mucosa during CRT could be interfering with treatment efficacy. Our results could reveal not only a novel RC prognostic marker but also a therapeutic target., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have analyzed the response of primary cultures derived from tumor specimens of non small cell lung cancer (NSCLC) patients to choline kinase α (ChoKα) inhibitors. ChoKα inhibitors have been demonstrated to increase ceramides levels specifically in tumor cells, and this increase has been suggested as the mechanism that explain its proapoptotic effect in cancer cells. Here, we have investigated the molecular mechanism associated to the intrinsic resistance, and found that other enzyme involved in lipid metabolism, acid ceramidase (ASAH1), is specifically upregulated in resistant tumors. NSCLC cells with acquired resistance to ChoKα inhibitors also display increased levels of ASAH1. Accordingly, ASAH1 inhibition synergistically sensitizes lung cancer cells to the antiproliferative effect of ChoKα inhibitors. Thus, the determination of the levels of ASAH1 predicts sensitivity to targeted therapy based on ChoKα specific inhibition and represents a model for combinatorial treatments of ChoKα inhibitors and ASAH1 inhibitors. Considering that ChoKα inhibitors have been recently approved to enter Phase I clinical trials by the Food and Drug Administration (FDA), these findings are anticipating critical information to improve the clinical outcome of this family of novel anticancer drugs under development.

The objective of the ONCODEATH consortium [EU Research Consortium "ONCODEATH" (2006-2010)] was to achieve sensitization of solid tumor cells to death receptor related therapies using rational mechanism-based drug combinations of targeted therapies. In this collaborative effort, during a period of 42 mo, cell and animal model systems of defined oncogenes were generated. Exploitation of generated knowledge and tools enabled the consortium to achieve the following research objectives: (1) elucidation of tumor components which confer sensitivity or resistance to TRAIL-induced cell death; (2) providing detailed knowledge on how small molecule Hsp90, Aurora, Choline kinase, BRAF inhibitors, DNA damaging agents, HDAC and DNMT inhibitors affect the intrinsic apoptotic amplification and execution machineries; (3) optimization of combined action of TRAIL with these therapeutics for optimum effects with minimum concentrations and toxicity in vivo. These findings provide mechanistic basis for a pharmacogenomic approach, which could be exploited further therapeutically, in order to reach novel personalized therapies for cancer patients.

Recent technological advances, combined with the development of bioinformatic tools, allow us to better address biological questions combining -omic approaches (i.e., genomics, metabolomics and proteomics). This novel comprehensive perspective addresses the identification, characterisation and quantitation of the whole repertoire of genes, proteins and metabolites occurring in living organisms. Here we provide an overview of recent significant advances and technologies used in genomics, metabolomics and proteomics. We also underline the importance and limits of mass accuracy in mass spectrometry-based -omics and briefly describe emerging types of fragmentation used in mass spectrometry. The range of instruments and techniques used to address the study of each -omic approach, which provide vast amounts of information (usually termed "high-throughput" technologies in the literature) is briefly discussed, including names, links and descriptions of the main databases, data repositories and resources used. Integration of multiple -omic results and procedures seems necessary. Therefore, an emerging challenge is the integration of the huge amount of data generated and the standardisation of the procedures and methods used. Functional data integration will lead to answers to unsolved questions, hopefully, applicable to clinical practice and management of patients.

In the last decade, several reports have focused on the identification and characterization of proteins present in urine. In an effort to build a list of proteins of interest as biomarkers, we reviewed the largest urine proteomes and built two updated lists of proteins of interest (available as supplementary tables). The first table includes a consensus list of 443 proteins found in urine by independent laboratories and reported on the top three largest urine proteomes currently published. This consensus list of proteins could serve as biomarkers to diagnose, monitor and manage a number of diseases. Here, we focus on a reduced list of 35 proteins with potential interest as cancer biomarkers in urine following two criteria: first, proteins previously detected in urine using bottom-up proteomic experiments, and second, those suggested as cancer protein biomarkers in human plasma. In an effort to standardize the information presented and its use in future studies, here we include the updated International Protein Index (v. 3.80) and primary Swiss-Prot accession numbers, official gene symbols and recommended full names. The main variables that influence urine proteomic experiments are also discussed.

Recent reviews pinpointed the enormous diversity of proteins found in living organisms, especially in higher eukaryotes. Protein diversity is driven through three main processes: first, at deoxyribonucleic acid (DNA) level (i.e. gene polymorphisms), second, at precursor messenger ribonucleic acid (pre-mRNA) or messenger ribonucleic acid (mRNA) level (i.e. alternative splicing, also termed as differential splicing) and, finally, at the protein level (i.e. PTM). Current proteomic technologies allow the identification, characterization and quantitation of up to several thousands of proteins in a single experiment. Nevertheless, the identification and characterization of protein species using these technologies are still hampered. Here, we review the use of the terms "protein species" and "protein isoform." We evidence that the appropriate selection of the database used for searches can impede or facilitate the identification of protein species. We also describe examples where protein identification search engines systematically fail in the attribution of protein species. We briefly review the characterization of protein species using proteomic technologies including gel-based, gel-free, bottom-up and top-down analysis and discuss their limitations. As an example, we discuss the theoretical characterization of the two human choline kinase species, α-1 and α-2, sharing the same catalytic activity but generated by alternative splicing on CHKA gene., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have summarized here the importance of ChoKα1 in human carcinogenesis. ChoKα1 displays its oncogenic activity through activation of specific signaling pathways that influence on cell proliferation and survival. It is overexpressed in a large number of human tumors with an incidence of 40-60% of all tumors investigated. Currently, there is an active effort in the development of strategies to knockdown the activity of ChoKα through specific siRNA or small molecules inhibitors. Results from genetic silencing or from treatment with MN58b, a well characterized ChoKα inhibitor showing antiproliferative and antitumoral effect in mice xenografts, provide strong support to this concept, indicating that the design of new antitumoral drugs must be selective against this isoform. However, affecting the other two known isoforms of ChoK may have also therapeutic consequences since the physiologically active form of ChoK may be constituted by homo or heterodimers. Furthermore, alteration of the ChoKβ activity might lead to a change in the lipid content of the cells of particular tissues such as skeletal muscle as described in the ChoKβ null mice (Sher et al., 2006). Finally, the identification of the ChoKα1 isoform as an excellent novel tool for the diagnosis and prognosis of cancer patients may have clinical consequences of immediate usefulness. On one hand, the use of specific monoclonal antibodies against ChoKα1 as a tool for diagnosis in paraffin embedded samples from patient biopsies, through standard immunohistochemistry techniques, can now be achieved (Gallego-Ortega et al., 2006). On the other hand, it has been recently described the prognostic value of determination of ChoKα1 expression levels in non-small cell lung cancer using real time quantitative PCR technology (Ramírez de Molina et al., 2007). Therefore, further research should be supported on the utility of ChoK isoforms as a promising area to improve cancer diagnosis and treatment.

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors., (Copyright 2010 AACR.)

Background: The serine/threonine kinase PKB/Akt plays essential role in various cellular processes including cell growth and proliferation, metabolism and cell survival. The importance of the Akt pathway is highlighted by the mutation of various components of the pathway such as the PTEN and PI3-kinase (P110alpha) in human cancers. In this paper, we employed an RNA interference library targeting all human kinases to screen for kinases involved in the regulation of Akt activation, in particular serine 473 phosphorylation. Here, we transfected the MDA-MB 468 breast cell line with the human kinome siRNA library and measured Akt activation using an antibody specific for phosphoserine 473 of Akt., Results: The screen revealed that phosphorylation of Akt(ser473) can be regulated by more than 90 kinases. Interestingly, phosphorylation of Akt(ser473), but not thr308, can be severely reduced by inhibition of Choline kinase activity via siRNA or small molecule inhibitors. We show here that the regulation of Akt phosphorylation by Choline kinase is PI3K-independent. In addition, xenograft tumors treated with Choline kinase inhibitors demonstrated a statistically significant decrease in Akt(ser473) phosphorylation. Importantly, the reduction in phosphorylation correlates with regression of these xenograft tumors in the mouse model., Conclusion: High Choline kinase expression and activity has previously been implicated in tumor development and metastasis. The mechanism by which Choline kinase is involved in tumor formation is still not fully resolved. From our data, we proposed that Choline kinase plays a key role in regulating Akt(ser473) phosphorylation, thereby promoting cell survival and proliferation.

Background: The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKalpha and ChoKbeta isoforms, the first one with two different variants of splicing. Recently ChoKalpha has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKbeta in carcinogenesis has been reported., Methodology/principal Findings: Here we compare the in vitro and in vivo properties of ChoKalpha1 and ChoKbeta in lipid metabolism, and their potential role in carcinogenesis. Both ChoKalpha1 and ChoKbeta showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKbeta display an ethanolamine kinase role, ChoKalpha1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKalpha1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKbeta overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKalpha1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKbeta mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKalpha1 than ChoKbeta., Conclusion/significance: This study represents the first evidence of the distinct metabolic role of ChoKalpha and ChoKbeta isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.

Bladder cancer is one of the most common causes of death in industrialized countries. New tumor markers and therapeutic approaches are still needed to improve the management of bladder cancer patients. Choline kinase-alpha (ChoKalpha) is a metabolic enzyme that has a role in cell proliferation and transformation. Inhibitors of ChoKalpha show antitumoral activity and are expected to be introduced soon in clinical trials. This study aims to assess whether ChoKalpha plays a role in the aggressiveness of bladder tumors and constitutes a new approach for bladder cancer treatment. We show here that ChoKalpha is constitutively altered in human bladder tumor cells. Furthermore, in vivo murine models, including an orthotopic model to mimic as much as possible the physiological conditions, revealed that increased levels of ChoKalpha potentiate both tumor formation (P< or =0.0001) and aggressiveness of the disease on different end points (P=0.011). Accordingly, increased levels of ChoKalpha significantly reduce survival of mice with bladder cancer (P=0.05). Finally, treatment with a ChoKalpha-specific inhibitor resulted in a significant inhibition of tumor growth (P=0.02) and in a relevant increase in survival (P=0.03).

Background: TWIST1 is a basic helix-loop-helix (bHLH) transcription factor that has been involved in tumor progression and metastasis in several cancer types, although no evidence has been provided yet on its implication in colorectal carcinogenesis., Methods: We examined the expression pattern of TWIST1 messenger RNA (mRNA) in 54 colorectal cancer biopsies compared with each respective adjacent normal mucosa by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) methodology., Results: TWIST1 mRNA was found significantly overexpressed in colorectal cancer samples compared to nontumorous colon mucosa (P < 0.0001). Receiver operating characteristic (ROC) curve analysis demonstrated that TWIST1 mRNA levels are significantly increased in patients with nodal invasion and, interestingly, a significant correlation with patient sex was also found., Conclusions: Evidence for upregulation of TWIST1 mRNA in colorectal cancer is provided, suggesting its implication in the onset of malignant progression of this disease. In addition, significant higher levels of TWIST1 mRNA were found in men than in women, suggesting a possible transcriptional regulation of TWIST1 by sexual hormones. The use of TWIST1 as a new prognostic marker of advanced malignancy, and as a potential therapeutic target in colorectal cancer, is proposed.

Choline Kinase (ChoK) comprises a family of cytosolic enzymes involved in the synthesis of phosphatidylcholine (PC), the most abundant phospholipid in eukaryotic cell membranes. One of the ChoK isoforms, Choline Kinase alpha (ChoKalpha), is found over expressed in human tumours. Chemical inhibitors able to interfere with ChoK activity have proven to be effective antitumoral drugs in vitro and in vivo. To validate the use of selective ChoKalpha inhibitors in cancer therapy, we have developed a genetic strategy to interfere specifically with ChoKalpha activity based on the generation of a shRNA against the alpha isoform of ChoK. Here we demonstrate that specific inhibition of ChoKalpha by shRNA has antitumor activity. The specific depletion of ChoKalpha induces apoptosis in several tumor-derived cell lines from breast, bladder, lung and cervix carcinoma tumors, while the viability of normal primary cells is not affected. Furthermore, this selective antiproliferative effect is achieved both under in vitro and in vivo conditions, as demonstrated by an inducible ChoKalpha suppression system in human tumour xenografts. These results demonstrate that ChoKalpha inhibition is a useful antitumoral strategy per se, and provides definitive and non-ambiguous evidence that ChoKalpha can be used as an efficient and selective drug target for cancer therapy.

Cdc42, a member of Rho GTPases family, is involved in the regulation of several cellular functions, such as rearrangement of actin cytoskeleton, membrane trafficking, cell-cycle progression, and transcriptional regulation. Aberrant expression or activity of Cdc42 has been reported in several tumours. Here, the specific role of Cdc42 in development and progression of colorectal cancer was analyzed through microarrays technology. A comparative analysis of Cdc42 overexpressing cells versus cells with decreased Cdc42 levels through siRNA revealed that Cdc42 overexpression down-regulated the potential tumour suppressor gene ID4. Results were validated by quantitative RT-PCR and the methylation status of the specific promoter, analyzed. Methylation-specific PCR and bisulfite sequencing PCR analysis revealed that Cdc42 induced the methylation of the CpG island of the ID4 promoter. Colorectal adenocarcinoma samples were compared with the corresponding adjacent normal tissue of the same patient in order to determine specific gene expression levels. The downregulation of ID4 by Cdc42 was also found of relevance in colorectal adenocarcinoma biopsies. Cdc42 was found to be overexpressed with high incidence (60%) in colorectal cancer samples, and this expression was associated with silencing of ID4 with statistical significance (p<0.05). Cdc42 may have a role in the development of colon cancer. Furthermore, inhibition of Cdc42 activity may have a direct impact in the management of colorectal cancer.

Colorectal adenocarcinoma is the second cause of cancer mortality in developed countries. Rac1 is a member of the family of Rho GTPases that regulates many intracellular signaling pathways, including those involved in tumorigenesis, invasion, and metastasis. We have investigated the role of Rac1 in colorectal tumor progression by genetic modification of the human colorectal adenocarcinoma cell line SW620 to either overexpress Rac1 or lack Rac1 expression. Tumor behavior was studied by orthotopic injection of stably modified cell lines into the cecal wall of athymic nude mice, a model that replicates the histopathological appearance and clinical behavior of human colorectal adenocarcinoma in humans. While overexpression of Rac1 resulted in an accelerated tumorigenic process, inducing a faster mortality rate, inhibition of Rac1 completely suppressed tumor formation. These results suggest that Rac1 plays a major role in colorectal adenocarcinoma progression. Finally, interference with Rac1 function may provide an important tool to block the malignant phenotype of colorectal adenocarcinoma cells.

Choline kinase alpha (ChoKalpha) is an enzyme involved in the metabolism of phospholipids recently found to play a relevant role in the regulation of cell proliferation, oncogenic transformation and human carcinogenesis. In addition, this novel oncogene has been recently defined as a prognostic factor in human cancer, and as a promising target for therapy since its specific inhibitors display efficient antitumoral activity in vivo. However, the mechanism by which this enzyme is involved in the regulation of these processes is not yet understood. Using differential microarray analysis, we identify target genes that provide the basis for the understanding of the molecular mechanism for the regulation of cell proliferation and transformation mediated by over-expression of the human ChoKalpha. These results fully support a critical role of this enzyme in the regulation of the G1-->S transition at different levels, and its relevant role in human carcinogenesis. The molecular basis to understand the connection between phospholipids metabolism and cell cycle regulation through choline kinase is reported.

Background: Adequate prognostic markers to predict outcome of patients with lung cancer are still needed. The aim of this study was to assess whether choline kinase alpha (ChoKalpha) gene expression could identify patients with different prognoses. ChoKalpha is an enzyme involved in cell metabolism and proliferation and has a role in oncogene-mediated transformation in several human tumours, including lung cancer., Methods: 60 patients with non-small-cell lung cancer (NSCLC) who had undergone surgical resection in a single centre were enrolled into the study as the training group. We used real-time reverse-transcriptase PCR (RT-PCR) to measure ChoKalpha gene expression and analyse the association between ChoKalpha expression and survival in evaluable patients. Additionally, a second group of 120 patients with NSCLC from a different hospital were enrolled into the study as the validation group. We did an overall analysis of all 167 patients who had available tissue to confirm the cut-off point for future studies. The primary endpoints were lung-cancer-specific survival and relapse-free survival., Findings: Seven of the 60 patients in the training group were not evaluable due to insufficient tissue. In the 53 evaluable patients, the cut-off for those with ChoKalpha overexpression was defined by receiver operator under the curve (ROC) methodology. 4-year lung-cancer-specific survival was 54.43% (95% CI 28.24-80.61) for 25 patients with ChoKalpha expression above the ROC-defined cut-off compared with 88.27% (75.79-100) for 28 patients with concentrations of the enzyme below this cut-off (hazard ratio [HR] 3.14 [0.83-11.88], p=0.07). In the validation group, six of the 120 enrolled patients were not evaluable due to insufficient tissue. For the other 114 patients, 4-year lung-cancer-specific survival was 46.66% (32.67-59.65) for those with ChoKalpha expression above the ROC-defined cut-off compared with 67.01% (50.92-81.11) for patients with concentrations of ChoKalpha below the cut-off (HR 1.87 [1.01-3.46], p=0.04). A global analysis of all 167 patients further confirmed the association between ChoKalpha overexpression and worse clinical outcome of patients with NSCLC: 4-year lung-cancer-specific survival for ChoKalpha expression above the ROC-defined cut-off was 49.00% (36.61-60.38) compared with 70.52% (59.80-76.75) for those with concentrations of ChoKalpha below the cut-off (HR 1.98 [1.14-3.45], p=0.01). The overall analysis confirmed the cut-off for ChoKalpha expression should be 1.91-times higher than concentrations noted in healthy tissues when ChoKalpha is used as an independent predictive factor of relapse-free and lung-cancer-specific survival in patients with early-stage NSCLC., Interpretation: ChoKalpha expression is a new prognostic factor that could be used to help identify patients with early-stage NSCLC who might be at high risk of recurrence, and to identify patients with favourable prognosis who could receive less aggressive treatment options or avoid adjuvant systemic treatment. New treatments that inhibit ChoKalpha expression or activity in patients with lung cancer should be studied further.

Despite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern.

The small GTPase Rac1 is involved in the regulation of critical cellular functions, such as transcription control, cell cycle, and organization of actin cytoskeleton. Rac1 signalling modulates cancer progression since its overexpression leads to an increased tumour growth of xenografts of human colorectal tumour cells, while a drastic reduction of Rac1 expression by siRNA interferes with cancer progression (Espina et al., unpublished results). We aimed to study the molecular basis for the specific contribution of Rac1 in the progression of colorectal cancer. Comparative microarray analysis of a human colorectal carcinoma cell line genetically engineered to display different levels of Rac1 identified novel target genes for this GTPase. These results suggest that Rac1 plays a critical role in signalling transduction pathways relevant to human colorectal tumour progression, such as activation of Wnt signalling, inhibition of TGF-beta signalling, and enhancement of metastasis-inducing genes.