Transcriptional regulation of biosynthetic genes of the plant MEP-derived pathway to boost the metabolic flux towards bioactive diterpenes

2013 - 2014This project was aimed at enhancing the synthesis of tri-cyclic bioactive abietane diterpenes (e.g. aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol), synthesized in the roots of Salvia sclarea and other Salvia species, with known anti-inflammatory and antitumoral activities. There is a great demand of novel molecules to treat melanoma, the most aggressive form of skin cancer, since advanced stages are inevitably resistant to conventional therapeutic agents.We have recently shown that aethiopinone is cytotoxic against the human melanoma A357 cell line at a concentration not toxic to normal cells. In addition, by using the web server IdTarget a number of putative proteins overexpressed in melanoma were identified as potential cellular target of aethiopinone. Despite this interesting evidence, this compound can not be easily synthesized by chemical means, and it is only produced in the roots of Salvia species in minute amounts (less than 0.5% DW) wich are not sufficient to yield reliable amounts for a deeper understanding of their molecular targets and potential future commercialization. In order to produce sufficient quantity of this interesting class of compounds, we targeted the plastidial terpenoid MEP-dependent pathway, from which they derive, by two different metabolic engineering strategies in S. sclarea hairy roots. The first approach was based on the coordinated activation of MEP-pathway biosynthetic genes by elicitation or by overexpression of transcription factors. An enhanced content (about a 20-fold increase) of abietane diterpenes in S. sclarea hairy roots was induced by elicitation with Methyl-Jasmonate (MJ), due to the increased expression levels of the several MEP-pathway biosynthetic genes, indicating a possible coordinate gene regulation by transcription factors. Four transcription factors (WRKYs and Myc2) of A. thaliana were selected on the basis of the presence of MJRE-box in their promoter region. Overexpression of AtWRKY and AtMyc2 genes in S. sclarea hairy roots positively regulated transcription of several genes of the terpenoid MEP-pathway. High-level induced-expression of genes acting up-stream [1-Deoxy-D-Xylulose-5-Phosphate Synthase (DXS) and 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase (DXR)] or downstream [geranylgeranyl-diphosphate synthase (GGPPS) and copalyl-diphosphate synthase (CPPS)] of this pathway, correlated with high-level of abietane-type diterpenes (3-5 fold increase). To our knowledge, this is the first evidence of TFs activating this specific diterpene pathway. One drawback of this strategy was the impaired growth, at varying level, of transgenic S. sclarea hairy roots. However, it was possible to select the best performing over-expressing hairy root lines in which high final biomass was coupled to high content of abietane diterpenes. The second strategy was aimed at blocking the Ent-copalyl-diphosphate synthase (Ent-CPPS), the first enzyme acting at the lateral competing route from GGPP to gibberellins. Either chemical inhibition of the enzymatic activity of Ent-CPPS with CCC (chlorocholine chloride), a known plant growth retardant, or RNAi-mediated silencing of this gene in S. sclarea hairy roots enhanced significantly (>4-fold) the total abietane diterpenes content, without causing any growth impairment compared to control hairy roots. Overall, these complementary approaches were successful in increasing the content of aethiopinone and other tricyclic abietane diterpenes (from a 3-fold up to a 5-fold increase compared to the content in the control line) in engineered S. sclarea hairy roots and might be extended to different plant species synthesizing other bioactive specialized terpenes. Moreover, the combination of these two approaches are expected to further enhance the accumulation of abietane diterpenes, as for chemical elicitation (with MJ, coronatine etc) coupled with metabolic engineering approaches, currently in progress in our laboratory, are also expected to increase the efficiency of the synthesis of this interesting class of compounds. Finally, the promising results presented in this study pave the way to a rational design of a hairy root-based production platform to yield reliable amounts of tricyclic abietane diterpenes towards a deeper understanding of their molecular targets and the potential future exploitation as novel plant-derived anti-tumor molecules. [edited by author]XIII n.s

Boosting the synthesis of pharmaceutically active abietane diterpenes in S. sclarea hairy roots by engineering the GGPPS and CPPS genes

Abietane diterpenoids (ADs), synthesized in the roots of different Salvia species, such as aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol, have a variety of known biological activities. We have shown that aethiopinone has promising cytotoxic activity against several human tumor cell lines, including the breast adenocarcinoma MCF7, HeLa, epithelial carcinoma, prostate adenocarcinoma PC3, and human melanoma A375. The low content of these compounds in natural sources, and the limited possibility to synthesize them chemically at low cost, prompted us to optimize the production of abietane diterpenoids by targeting genes of the methylerythritol phosphate (MEP) pathway, from which they are derived. Here, we report our current and ongoing efforts to boost the metabolic flux towards this interesting class of compounds in Salvia sclarea hairy roots (HRs). Silencing the gene encoding the ent-copalyl-diphosphate synthase gene (entCPPS), acting at the lateral geranylgeranyl pyrophosphate (GGPP) competitive gibberellin route, enhanced the content of aethiopinone and other ADs in S. sclarea HRs, indicating indirectly that the GGPP pool is a metabolic constraint to the accumulation of ADs. This was confirmed by overexpressing the GGPPS gene (geranyl-geranyl diphosphate synthase) which triggered also a significant 8-fold increase of abietane diterpene content above the basal constitutive level, with a major boosting effect on aethiopinone accumulation in S. sclarea HRs. A significant accumulation of aethiopinone and other AD compounds was also achieved by overexpressing the CPPS gene (copalyl diphosphate synthase) pointing to this biosynthetic step as another potential metabolic target for optimizing the biosynthesis of this class of compounds. However, by co-expressing of GGPPS and CPPS genes, albeit significant, the increase of abietane diterpenoids was less effective than that obtained by overexpressing the two genes individually. Taken together, the results presented here add novel and instrumental knowledge to a rational design of a hairy root-based platform to yield reliable amounts of aethiopinone and other ADs for a deeper understanding of their molecular pharmacological targets and potential future commercialization

Oral-Facial-Digital syndrome Type I cells exhibit impaired DNA repair; unanticipated consequences of defective OFD1 outside of the cilia network

Defects in OFD1 underlie the clinically complex ciliopathy, Oral-Facial-Digital syndrome Type I (OFD Type I). Our understanding of the molecular, cellular and clinical consequences of impaired OFD1 originate from its characterised roles at the centrosome/basal body/cilia network. Nonetheless, the first described OFD1 interactors were components of the TIP60 histone acetyltransferase complex. We find that OFD1 can also localise to chromatin and its reduced expression is associated with mislocalization of TIP60 in patient-derived cell lines. TIP60 plays important roles in controlling DNA repair. OFD Type I cells exhibit reduced histone acetylation and altered chromatin dynamics in response to DNA double strand breaks (DSBs). Furthermore, reduced OFD1 impaired DSB repair via homologous recombination repair (HRR). OFD1 loss also adversely impacted upon the DSB-induced G2-M checkpoint, inducing a hypersensitive and prolonged arrest. Our findings show that OFD Type I patient cells have pronounced defects in the DSB-induced histone modification, chromatin remodelling and DSB-repair via HRR; effectively phenocopying loss of TIP60. These data extend our knowledge of the molecular and cellular consequences of impaired OFD1, demonstrating that loss of OFD1 can negatively impact upon important nuclear events; chromatin plasticity and DNA repair

Identification of uPAR Variants Acting as ceRNAs in Leukaemia Cells

The 3′untranslated region (3′UTR) of the urokinase (uPA) receptor (uPAR) mRNA can act as a competitive endogenous RNA (ceRNA) in acute myeloid leukaemia (AML) cells, promoting the expression of pro-tumoral targets, including uPAR. Here, we identified three variants of uPAR mRNA containing the 3′UTR, in KG1 and U937 leukaemia cells expressing low and high uPAR levels, respectively. Identified variants lack exon 5 (uPAR Δ5) or exon 6 (uPAR Δ6) or part of exon 6, exon 7 and part of 3′UTR (uPAR Δ6/7). uPAR Δ5 and uPAR Δ6 transcript levels were higher in U937 cells compared to KG1 cells. Both uPAR variants were expressed also in AML blasts, at higher levels as compared to CD34 hematopoietic cells from healthy donors. The presence of the 3′UTR conferred high instability to the uPAR Δ5 variant transcript, preventing its translation in protein. Overexpression of the uPAR Δ5-3′UTR variant regulated the expression of some pro-tumoral factors previously reported to be regulated by the 3′UTR of uPAR and increased KG1 cell adhesion, migration and proliferation. These results demonstrate the expression of uPAR mRNA variants containing the 3′UTR in AML cells and the ceRNA activity and the biological effects of the uPAR Δ5-3′UTR variant

Posttranscriptional Regulation of the Plasminogen Activation System by Non-Coding RNA in Cancer

Various species of non-coding RNAs (ncRNAs) may act as functional molecules regulating diverse biological processes. In cancer cell biology, ncRNAs include RNAs that regulate the expression of oncogenes and tumor suppressor genes through various mechanisms. The urokinase (uPA)-mediated plasminogen activation system (PAS) includes uPA, its inhibitors PAI-1 and PAI-2 and its specific cellular receptor uPAR; their increased expression represents a negative prognostic factor in several cancers. Here, we will briefly describe the main uPA-mediated PAS components and ncRNA species; then, we will review more recent evidence of the roles that ncRNAs may play in regulating the expression and functions of uPA-mediated PAS components in cancer

Posttranscriptional Regulation of the Plasminogen Activation System by Non-Coding RNA in Cancer

Various species of non-coding RNAs (ncRNAs) may act as functional molecules regulating diverse biological processes. In cancer cell biology, ncRNAs include RNAs that regulate the expression of oncogenes and tumor suppressor genes through various mechanisms. The urokinase (uPA)-mediated plasminogen activation system (PAS) includes uPA, its inhibitors PAI-1 and PAI-2 and its specific cellular receptor uPAR; their increased expression represents a negative prognostic factor in several cancers. Here, we will briefly describe the main uPA-mediated PAS components and ncRNA species; then, we will review more recent evidence of the roles that ncRNAs may play in regulating the expression and functions of uPA-mediated PAS components in cancer

Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots

Diterpenoids are important compounds for plant survival and have beneficial properties for humans. Bioactive abietanic diterpenes are synthesized in roots of Salvia sclarea (e.g. aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol), but at a very low level (about 1 % of root dry weight). To enhance the biosynthesis of this interesting class of compounds, heterologous AtDXS (d-xylulose 5-phosphate synthase) or AtDXR (1-deoxy-d-xylulose 5 phosphate reductoisomerase) genes, encoding the up-stream enzymes of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP)-dependent terpenoid pathway, were ectopically expressed in S. sclarea hairy roots. Quantitative targeted metabolic analysis (HPLC–DAD) revealed that three independent root lines, expressing different levels of DXS or DXR transcripts and proteins, synthesized a significant higher content of abietanic diterpenes, compared to the control hairy root line transformed with the empty vector. The increase was gene-dependent, since the overexpression of the AtDXR triggered a 4.4-fold increase in aethiopinone, an abietane quinone-type tricyclic diterpene. In addition, aethiopinone was proved to be cytotoxic to different solid tumor cell lines, with the highest effect on human melanoma A375 cell line (IC50 11.4 µM). Overall these results show that it is possible to boost the metabolic flow towards the synthesis of abietanic diterpenes in S. sclarea hairy roots by overexpressing genes involved in the first steps of the MEP-pathway and provide new insights for the large-scale production of this class of compounds, with potential application in cancer treatment

Grapefruit-Derived Micro and Nanovesicles Show Distinct Metabolome Profiles and Anticancer Activities in the A375 Human Melanoma Cell Line

Fruit juice is one of the most easily accessible resources for the isolation of plant-derived vesicles. Here we found that micro- and nano-sized vesicles (MVs and NVs) from four Citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, specifically inhibit the proliferation of lung, skin and breast cancer cells, with no substantial effect on the growth of non-cancer cells. Cellular and molecular analyses demonstrate that grapefruit-derived vesicles cause cell cycle arrest at G2/M checkpoint associated with a reduced cyclins B1 and B2 expression levels and the upregulation of cell cycle inhibitor p21. Further data suggest the inhibition of Akt and ERK signalling, reduced intercellular cell adhesion molecule-1 and cathepsins expressions, and the presence of cleaved PARP-1, all associated with the observed changes at the cellular level. Gas chromatography-mass spectrometry-based metabolomics reveals distinct metabolite profiles for the juice and vesicle fractions. NVs exhibit a high relative amount of amino acids and organic acids whereas MVs and fruit juice are characterized by a high percentage of sugars and sugar derivatives. Grapefruit-derived NVs are in particular rich in alpha-hydroxy acids and leucine/isoleucine, myo-inositol and doconexent, while quininic acid was detected in MVs. Our findings reveal the metabolite signatures of grapefruit-derived vesicles and substantiate their potential use in new anticancer strategies

Functional Characterization of the OFD1 Protein Reveals a Nuclear Localization and Physical Interaction with Subunits of a Chromatin Remodeling Complex

Oral-facial-digital (OFD) type I syndrome is an X-linked dominant disease (MIM311200) characterized by malformations of oral cavity, face, and digits and by cystic kidneys. We previously identified OFD1, the gene responsible for this disorder, which encodes for a centrosomal protein with an unknown function. We now report that OFD1 localizes both to the primary cilium and to the nucleus. Moreover, we demonstrate that the OFD1 protein is able to self-associate and that this interaction is mediated by its coiled-coil rich region. Interestingly, we identify an OFD1-interacting protein RuvBl1, a protein belonging to the AAA+-family of ATPases, which has been recently associated to cystic kidney in zebrafish and to ciliary assembly and function in Chlamydomonas reinhardtii. We also provide experimental evidence that OFD1, together with RuvBl1, is able to coimmunoprecipitate with subunits of the human TIP60 histone acetyltransferase (HAT) multisubunit complex. On the basis of these results, we hypothesize that OFD1 may be part of a multi-protein complex and could play different biological functions in the centrosome-primary cilium organelles as well as in the nuclear compartment

Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors

Plant abietane diterpenoids (e.g. aethiopinone, 1- oxoaethiopinone, salvipisone and ferruginol), synthesized in the roots of several Salvia spp, have antibacterial, antifungal, sedative and anti-proliferative properties. Recently we have reported that content of these compounds in S. sclarea hairy roots is strongly depending on transcriptional regulation of genes belonging to the plastidial MEP-dependent terpenoid pathway, from which they mostly derive. To boost the synthesis of this interesting class of compounds, heterologous AtWRKY18, AtWRKY40, and AtMYC2 TFs were overexpressed in S. sclarea hairy roots and proved to regulate in a coordinated manner the expression of several genes encoding enzymes of the MEP-dependent pathway, especially DXS, DXR, GGPPS and CPPS. The content of total abietane diterpenes was enhanced in all overexpressing lines, although in a variable manner due to a negative pleiotropic effect on HR growth. Interestingly, in the best performing HR lines overexpressing the AtWRKY40 TF induced a significant 4-fold increase in the final yield of aethiopinone, for which we have reported an interesting anti-proliferative activity against resistant melanoma cells. The present results are also informative and instrumental to enhance the synthesis of abietane diterpenes derived from the plastidial MEP-derived terpenoid pathway in other Salvia species