Cellular Growth Rate Research Articles

Analytic derivation of bacterial growth laws from a simple model of intracellular chemical dynamics.

Experiments have found that the growth rate and certain other macroscopic properties of bacterial cells in steady-state cultures depend upon the medium in a surprisingly simple manner; these dependencies are referred to as ‘growth laws’. Here we construct a dynamical model of interacting intracellular populations to understand some of the growth laws. The model has only three population variables: an amino acid pool, a pool of enzymes that transport an external nutrient and produce the amino acids, and ribosomes that catalyze their own and the enzymes’ production from the amino acids. We assume that the cell allocates its resources between the enzyme sector and the ribosomal sector to maximize its growth rate. We show that the empirical growth laws follow from this assumption and derive analytic expressions for the phenomenological parameters in terms of the more basic model parameters. Interestingly, the maximization of the growth rate of the cell as a whole implies that the cell allocates resources to the enzyme and ribosomal sectors in inverse proportion to their respective ‘efficiencies’. The work introduces a mathematical scheme in which the cellular growth rate can be explicitly determined and shows that two large parameters, the number of amino acid residues per enzyme and per ribosome, are useful for making approximations.

A Microfluidic Platform for High-Throughput Screening of Small Mutant Libraries.

The screening and isolation of target microorganisms from mutated recombinant libraries are crucial for the advancement of synthetic biology and metabolic engineering. However, conventional screening tools present several limitations in throughput, cost, and labor. Herein, we describe a novel microfluidic high-throughput screening (HTS) platform with several advantages. The platform utilizes a fluid array to compartmentalize bacterial cells in well-ordered separated microwells and allows long-term cell culture with high throughput. The platform enables the extraction of selected target cells from the fluid array for additional culture and postanalysis by using a capillary-driven sample relocation method. To confirm the feasibility of the platform, we demonstrated two different types of HTS methods based on the levels of reporter gene expression and cellular growth rate difference. For the reporter gene-based HTS, a spike recovery approach was taken to demonstrate that target cells are successfully screened out from a mixture containing nontarget cells by repeating the culture and extraction processes. Additionally, the same platform allowed us to screen and sort target cells according to their cellular growth rate difference, which seems hard in conventional screening methods. Hence, the platform could be used for various microbiological assays, including the detection of cell-excreted metabolites, microbial biosensors, and other HTS systems.

Slowdown of growth controls cellulardifferentiation.

How can changes in growth rate affect the regulatory networks behavior and the outcomes of cellular differentiation? We address this question by focusing on starvation response in sporulating Bacillus subtilis. We show that the activity of sporulation master regulator Spo0A increases with decreasing cellular growth rate. Using a mathematical model of the phosphorelay—the network controlling Spo0A—we predict that this increase in Spo0A activity can be explained by the phosphorelay protein accumulation and lengthening of the period between chromosomal replication events caused by growth slowdown. As a result, only cells growing slower than a certain rate reach threshold Spo0A activity necessary for sporulation. This growth threshold model accurately predicts cell fates and explains the distribution of sporulation deferral times. We confirm our predictions experimentally and show that the concentration rather than activity of phosphorelay proteins is affected by the growth slowdown. We conclude that sensing the growth rates enables cells to indirectly detect starvation without the need for evaluating specific stress signals.

Impact of pnpR, a LysR-type regulator-encoding gene, on the cellular processes of Pseudomonas putida DLL-E4.

LysR-type transcriptional regulators (LTTRs) regulate various cellular processes in bacteria. pnpR is an LTTR-encoding gene involved in the regulation of hydroquinone (HQ) degradation, and its effects on the cellular processes of Pseudomonas putida DLL-E4 were investigated at the physiological, biochemical and molecular levels. Reverse transcription polymerase chain reaction revealed that pnpR positively regulated its own expression and that of the pnpC1C2DECX1X2 operon; additionally, pnpR partially regulated the expression of pnpA when P. putida was grown on para-nitrophenol (PNP) or HQ. Strains DLL-E4 and DLL-ΔpnpR exhibited similar cellular morphologies and growth rates. Transcriptome analysis revealed that pnpR regulated the expression of genes in addition to those involved in PNP degradation. A total of 20 genes were upregulated and 19 genes were downregulated by at least 2-fold in strain DLL-ΔpnpR relative to strain DLL-E4. Bioinformatic analysis revealed putative PnpR-binding sites located in the upstream regions of genes involved in PNP degradation, carbon catabolite repression and other cellular processes. The utilization of L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, γ-aminobutyric acid, D,L-lactic acid, D-saccharic acid, succinic acid and L-alaninamide was increased at least 1.3-fold in strain DLL-ΔpnpR as shown by BIOLOG assays, indicating that pnpR plays a potential negative regulation role in the utilization of carbon sources.

Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes.

Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H2O2 probes, roGFP2-Tsa2ΔCR and roGFP2-Tsa2ΔCPΔCR, that afford such sensitivity. These probes are ∼50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H2O2 homeostasis. The roGFP2-Tsa2ΔCR probe revealed real-time interplay between basal H2O2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H2O2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H2O2 levels and cellular growth rate.

A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate.

Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms.

Upregulation of the growth arrest-specific-2 in recurrent colorectal cancers, and its susceptibility to chemotherapy in a model cell system

Colorectal cancer (CRC) is one of the most common life-threatening malignances worldwide. CRC relapse markedly decreases the 5-year survival of patients following surgery. Aberrant expression of genes involved in pathways regulating the cell cycle, cell proliferation, or cell death are frequently reported in CRC tumorigenesis. We hypothesized that genes involved in CRC relapse might serve as prognostic indicators. We first evaluated the significance of gene sequences in the feces of patients with CRC relapse by consulting a public database. Tumorigenesis of target tissues was tested through tumor cell growth, cell cycle regulation, and chemotherapeutic efficacy. We found a highly significant correlation between CRC relapse and growth arrest-specific 2 (GAS2) gene expression. Based on cell models, the overexpressed GAS2 was associated with cellular growth rate, cell cycle regulation, and with chemotherapeutic sensitivity. Cell division was impaired by treating cells with 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469), even when the cells were overexpressing GAS2. Thus, downregulation of GAS2 expression might control CRC relapse after curative resection. GAS2 could serve as a noninvasive marker from the feces of patients with prediagnosed CRC. Our findings suggest that GAS2 could have potential clinical applications for predicting early CRC relapse after radical resection, and that XK469 might impair tumor cell division by reducing GAS2 expression or blocking its cellular translocation. This will help in selecting the best therapeutic option, 5-fluorouracil in combination with XK469, for patients overexpressing GAS2 in CRC cells. Thus, GAS2 might act as a prognostic biomolecule and potential therapeutic target in patients with CRC relapse.

Interactions between growth-dependent changes in cell size, nutrient supply and cellular elemental stoichiometry of marine Synechococcus.

The factors that control elemental ratios within phytoplankton, like carbon:nitrogen:phosphorus (C:N:P), are key to biogeochemical cycles. Previous studies have identified relationships between nutrient-limited growth and elemental ratios in large eukaryotes, but little is known about these interactions in small marine phytoplankton like the globally important Cyanobacteria. To improve our understanding of these interactions in picophytoplankton, we asked how cellular elemental stoichiometry varies as a function of steady-state, N- and P-limited growth in laboratory chemostat cultures of Synechococcus WH8102. By combining empirical data and theoretical modeling, we identified a previously unrecognized factor (growth-dependent variability in cell size) that controls the relationship between nutrient-limited growth and cellular elemental stoichiometry. To predict the cellular elemental stoichiometry of phytoplankton, previous theoretical models rely on the traditional Droop model, which purports that the acquisition of a single limiting nutrient suffices to explain the relationship between a cellular nutrient quota and growth rate. Our study, however, indicates that growth-dependent changes in cell size have an important role in regulating cell nutrient quotas. This key ingredient, along with nutrient-uptake protein regulation, enables our model to predict the cellular elemental stoichiometry of Synechococcus across a range of nutrient-limited conditions. Our analysis also adds to the growth rate hypothesis, suggesting that P-rich biomolecules other than nucleic acids are important drivers of stoichiometric variability in Synechococcus. Lastly, by comparing our data with field observations, our study has important ecological relevance as it provides a framework for understanding and predicting elemental ratios in ocean regions where small phytoplankton like Synechococcus dominates.

Global Rebalancing of Cellular Resources by Pleiotropic Point Mutations Illustrates a Multi-scale Mechanism of Adaptive Evolution

Pleiotropic regulatory mutations affect diverse cellular processes, posing a challenge to our understanding ofgenotype-phenotype relationships across multiple biological scales. Adaptive laboratory evolution (ALE) allows for such mutations to be found and characterized in the context of clear selection pressures. Here, several ALE-selected single-mutation variants in RNA polymerase (RNAP) of Escherichia coli are detailed using an integrated multi-scale experimental and computational approach. While these mutations increase cellular growth rates in steady environments, theyreduce tolerance to stress and environmental fluctuations. We detail structural changes in the RNAP that rewire the transcriptional machinery to rebalance proteome and energy allocation toward growth and away from several hedging and stress functions. We find that while these mutations occur in diverse locations in the RNAP, they share a common adaptive mechanism. In turn, these findings highlight the resource allocation trade-offs organisms face and suggest how the structure of the regulatory network enhances evolvability.

Algal biomass and diesel emulsions: An alternative approach for utilizing the energy content of microalgal biomass in diesel engines

The use of algal biomass for the production of sustainable biofuels has attracted significant interest due to the fast reproduction rates and high lipid content of many microalgal species. However, existing methods of extracting algal cellular lipids are complex and expensive, with regards to both energy input and economic costs. This work explores an alternative method of utilizing the energy content of microalgae through the preparation of wet algal biomass slurry/fossil diesel emulsions containing up to 6.6% wt/wt algae biomass, using a specific surfactant combination, for direct injection diesel engine combustion of microalgae without prior biomass drying or lipid extraction.A high lipid containing green microalgae, Chlorella sorokiniana, was used to produce algal biomass for the study. The preparation of wet algal slurry/diesel emulsions from algae grown under standard conditions, and also those under conditions intended to increase cellular lipid content or growth rates was investigated, and in all cases a surfactant pack of Span80, CTAB and butanol was found to produce a stable emulsion. A correlation between the engine work produced during combustion of the emulsions in a modern direct injection compression ignition and the lower heating value of the wet slurry emulsions was found, with no evidence of individual algae cells persisting to the engine exhaust. Engine exhaust emissions of nitrogen oxides (NOx) and particulate matter were lower for all of the wet algal slurry/diesel emulsions relative to a reference fossil diesel tested under similar conditions, while in the case of the emulsion prepared from algal biomass to which a flocculating agent had been added, emissions of carbon monoxide (CO) were found to increase significantly.

Mitochondrial permeabilization without caspase activation mediates the increase of basal apoptosis in cells lacking Nrf2

Nuclear factor E2-related factor-2 (Nrf2) is a cap‘n’collar/basic leucine zipper (b-ZIP) transcription factor which acts as sensor of oxidative and electrophilic stress. Low levels of Nrf2 predispose cells to chemical carcinogenesis but a dark side of Nrf2 function also exists because its unrestrained activation may allow the survival of potentially dangerous damaged cells. Since Nrf2 inhibition may be of therapeutic interest in cancer, and a decrease of Nrf2 activity may be related with degenerative changes associated with aging, it is important to investigate how the lack of Nrf2 function activates molecular mechanisms mediating cell death. Murine Embryonic Fibroblasts (MEFs) bearing a Nrf2 deletion (Nrf2KO) displayed diminished cellular growth rate and shortened lifespan compared with wild-type MEFs. Basal rates of DNA fragmentation and histone H2A.X phosphorylation were higher in Nrf2KO MEFs, although steady-state levels of reactive oxygen species were not significantly increased. Enhanced rates of apoptotic DNA fragmentation were confirmed in liver and lung tissues from Nrf2KO mice. Apoptosis in Nrf2KO MEFs was associated with a decrease of Bcl-2 but not Bax levels, and with the release of the mitochondrial pro-apoptotic factors cytochrome c and AIF. Procaspase-9 and Apaf-1 were also increased in Nrf2KO MEFs but caspase-3 was not activated. Inhibition of XIAP increased death in Nrf2KO but not in wild-type MEFs. Mitochondrial ultrastructure was also altered in Nrf2KO MEFs. Our results support that Nrf2 deletion produces mitochondrial dysfunction associated with mitochondrial permeabilization, increasing basal apoptosis through a caspase-independent and AIF-dependent pathway.

Cellular growth of 2M-wollastonite in flux growth process relevant to preparation of high aspect ratio particles

High aspect ratio 2M-wollastonite particles were prepared using a flux growth process, in which a unidirectional temperature gradient was established in the melt. The one-dimensional heat transfer caused the crystals to display clear cellular structure. The measured cellular growth rate decreased from ∼12 to 8 μm s− 1 within 20 min of crystallisation time. However, the primary arm spacing remained nearly constant at ∼7.5 μm. B2O3 can significantly lower the melting point of the mixture, which made the handling of the molten silicate mixtures easier. The cellular/dendritic structures were maintained up to a maximum B2O3 content of 20 wt-%. The primary arm spacing increased from ∼8 μm at 8 wt-% B2O3 to ∼40 μm at 20 wt-% B2O3. The corresponding mean aspect ratio of 2M-wollastonite particles decreased from ∼12:1 (a high aspect ratio grade) at 8 wt-% B2O3 to ∼4:1 (a low aspect ratio grade) at 20 wt-% B2O3.

A biorefinery approach to microbial oil production from glycerol by Rhodotorula glutinis

The use of biodiesel-derived glycerol as a carbon source for microbial oil production is a biorefinery engineering strategy that aims to reduce the glycerol surplus and make the microbial oil process more cost-effective. In this work, glycerol was used as the sole carbon source for the cultivation of the oleaginous yeast Rhodotorula glutinis along with only yeast extract as a nutrient supply and without pH control. Shake-flask cultivations showed that the specific growth rate and glycerol consumption of Rhodotorula glutinis were higher at lower glycerol concentrations (≤40 g L−1), while higher C/N atom ratios enhanced oil content. The present study extends the knowledge on the influence of the aeration rate and oxygen supply in cellular growth rate and microbial oil production, providing a wiser use of glycerol as an attempt to further reduce process costs. Cultivations at different air flow rates were performed in a 2 L bioreactor and showed that a low aeration rate of 0.5 L min−1 gave the best glycerol and nitrogen uptake rates, resulting in the highest growth (5.3 g L−1) and oil mass fraction (33% of the dry cell weight). A further 68% increase in cellular growth (16.8 g L−1) and a 34% oil mass fraction of the dry cell weight was achieved after applying a feeding strategy targeting combined growth and oil production.

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth.

No Mutations of SF3B1, U2AF1, and SRSF2 spliceosomal Gene but Polymorphisms Associated with the Worse Prognosis in Multiple Myeloma

Abstract Recently, a striking example of the effects of acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The DNA sequences from abnormal blood cells of patients with hematologic malignancies has shown that a high proportion of these diseases are associated with somatic mutations of spliceosomal proteins. However, little is known about the aberrant splicing pathways in multiple myeloma (MM) patients. Therefore, this study investigated the presence and prognostic implication of splicing gene aberration in MM patients. Also, we planned functional experiments in cultured normal and Hela cell line to determine the effects of the common spliceosome mutations on cellular growth rate, cell count, and apoptosis. In total, 87 MM patients and 100 healthy controls were examined for somatic mutations in SF3B1, U2AF1, and SRSF2 by direct sequencing. Also, the clinical and laboratory characteristics of MM patients were reviewed. Regarding to the initial findings in recent reports, this study expressed the wild-type and the mutant (Q157P) U2AF1 in Hela cells and examined their proliferation. MM patients did not show mutation in SF3B1, U2AF1,and SRSF2 splicing genes. However, the patients displayed 39198T>C or 39198T>T/C polymorphism (70.1 %) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8 %) in exon 2 of U2AF1, and 5399C>T or 5399C>C/T polymorphism (100.0 %) in exon 1 of SRSF2 (Fig. 1). In the entire cohort, the number of patients with one polymorphism, two polymorphisms, and three polymorphisms was counted up to 24.1 %, 67.8 %, and 8.0 %, respectively. Meanwhile, in 100 normal controls, polymorphism of SF3B1 exon 18 and U2AF1 exon 2 was counted up to 82.0 % and 10.0 %, respectively. Free light chain (FLC) kappa/lambda (K/L) ratio and chromosome analysis showed significant difference according to polymorphism status (p=0.002) while other clinical characteristics did not. The patient with polymorphisms in both SF3B1 and U2AF1 gene had unfavorable overall survival (p =0.067) and disease-free survival (p=0.001), compared to patients without polymorphism. Our results show no recurrent SF3B1 and U2AF1 mutations in MM patients. Instead, polymorphisms were found in SF3B1 or U2AF1, which were significantly implicated in worse prognosis. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

ON-LINE MONITORING OF BIOMASS CONCENTRATION BASED ON A CAPACITANCE SENSOR: ASSESSING THE METHODOLOGY FOR DIFFERENT BACTERIA AND YEAST HIGH CELL DENSITY FED-BATCH CULTURES

The performance of an in-situ capacitance sensor for on-line monitoring of biomass concentration was evaluated for some of the most important microorganisms in the biotechnology industry: Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Bacillus megaterium. A total of 33 batch and fed-batch cultures were carried out in a bench-scale bioreactor and biomass formation trends were followed by dielectric measurements during the growth phase as well as the induction phase, for 5 recombinant E. coli strains. Permittivity measurements and viable cellular concentrations presented a linear correlation for all the studied conditions. In addition, the permittivity signal was further used for inference of the cellular growth rate. The estimated specific growth rates mirrored the main trends of the metabolic states of the different cells and they can be further used for setting-up control strategies in fed-batch cultures.

Abstract POSTER-BIOL-1346: Growth-suppressive effect of WRN RECQ helicase inactivation in breast and ovarian cancer cells

Abstract One important and high-yielding paradigm in the development of novel, targeted anticancer therapies is to identify genes that are not essential to normal cells but that have become critically important to particular cancer cells as they rely on these genes for survival. This concept, sometimes called addiction of cancer cells to specific genes or pathways can be exploited in the clinic because the function of the genes that cancer cells are addicted to can be disrupted using genetic or chemical manipulation, leading to selective killing of cancer cells while normal cells are minimally affected. For example, it was shown that homologous recombination-defective BRCA1-deficient cancer cells rely heavily on other repair pathways such as NHEJ, one of the pathways facilitated by PARP1. This finding has led to clinical trials designed to determine if PARP inhibitor drugs are an effective treatment for patients with BRCA1-deficient breast and ovarian cancer. We have previously discovered, via a high throughput screen, that simultaneous deficiency in BRCA1 and the WRN RECQ helicase resulted in additive growth-suppressive phenotype in human fibroblasts. WRN is a gene mutated in the Werner syndrome of premature aging, and it is a multifunctional exonuclease/helicase involved in maintenance of genomic stability. Congenital loss of WRN also predisposes to sarcomas and melanomas, though interestingly, not to the most common type of age-related cancer – carcinoma. Near-complete inactivation of WRN in human fibroblasts and common cell lines such as sarcoma-derived U2OS and HT1080, is typically non-lethal, though somewhat reduces cellular growth rate and fitness. Following our original observation, we were interested to determine whether loss of WRN is synthetically lethal with loss of BRCA1 in breast and ovarian cancer cell lines, and if loss of WRN restores sensitivity to platinum or impairs growth in platinum resistant clones derived from breast and ovarian cancer cell lines. Our results show that depletion of WRN in BRCA1-negative or positive breast or ovarian cancer cell lines dramatically suppresses their growth. Both in breast and ovarian cancer cells, at least some of this growth suppression could be attributed to the induction of RNAi-triggered cytotoxicity by WRN downregulation. In addition, WRN depletion also hypersensitized the HCC1937 breast cancer cell line to platinum, however this effect of WRN was attenuated in platinum-resistant clones derived in vitro from this line. Strikingly, non-cancerous mammary epithelial line MCF10a showed virtually no growth suppression by WRN depletion, suggesting that breast and ovarian cancer cells may be more reliant on WRN than normal cells of at least some of the relevant epithelial lineages — which is a therapeutically exploitable trait. We are currently following up on this observation using other non-cancerous epithelial cells, such as primary mammary and fallopian tube epithelial cells, as well as primary human keratinocytes. We will present these data as well as the update on reproducing WRN growth-suppressive phenotype using small molecule inhibitors of WRN. Significance: Our results highlight WRN as a potential novel target for development of breast and ovarian cancer therapies. Citation Format: Piri Welcsh, Keffy Kehrli, Paul Lazarchuk, Julia Sidorova. Growth-suppressive effect of WRN RECQ helicase inactivation in breast and ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1346.

Abstract 5484: FGFR2 amplification is predictive of sensitivity to regorafenib in gastric and colorectal cancers

Abstract Purpose Regorafenib is an orally bioavailable, small molecular multikinase inhibitor of VEGFR, PDGFR, FGFR, KIT, RET, BRAF, BRAFV600E, CRAF, and TIE2. Although regorafenib has demonstrated survival benefits in patients with metastatic colorectal and gastrointestinal stromal tumors, there is no proven biomarker predicting sensitivity to regorafenib. We investigated preclinical activity of regorafenib in gastric and colorectal cancer cell-lines to identify genetic alterations associated with sensitivity to regorafenib. Methods Fourteen gastric cancer cell lines and 18 colorectal cancer cell lines were screened for regorafenib sensitivity with cell proliferation assays. We performed targeted exome and transcriptome sequencing of the cell lines to identify genetic alterations associated with sensitivity to regorafenib. Mechanisms of regorafenib sensitivity were investigated in regorafenib sensitive cell lines with immunoblotting and flow cytometry. Results Two gastric (SNU-16, KATO-III) and 1 colorectal (NCI-H716) cancer cells were sensitive to regorafenib with GI50 values of less than 1 μM. Mutation profiles and mRNA expression levels of regorafenib target molecules in regorafenib sensitive and insensitive cell lines demonstrated that amplification of FGFR2 was the only genetic alteration associated with in vitro sensitivity to regorafenib. FGFR2 amplified gastric (SNU-16, KATO-III) and colorectal (NCI-H716) cancer cell lines revealed activated signaling through FGFR2 pathway, and knock-down of FGFR2 with siRNA significantly repressed cellular growth rate and decreased sensitivity to regorafenib. Regorafenib effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules in dose-dependent manner selectively in FGFR2 amplified cells. In cell cycle analysis, regorafenib induced G1/S arrest (SNU-16, KATO-III) and apoptosis (NCI-H716), however no significant changes were seen in cell lines without FGFR2 amplification. Conclusions Signaling through FGFR2 pathway plays a key role in cellular growth and survival in FGFR2 amplified cancers. Regorafenib effectively abrogates activated FGFR2 signaling in FGFR2 amplified gastric and colorectal cancer cells, and therefore should be considered for integration into treatment in patients with FGFR2 amplified gastric and colorectal cancers. Citation Format: Yongjun Cha, Hwang-Phill Kim, Sae-won Han, Jiyeon Yun, Sang-Hyun Song, Tae-You Kim. FGFR2 amplification is predictive of sensitivity to regorafenib in gastric and colorectal cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2015-5484

The roles of IP3 receptor in energy metabolic pathways and reactive oxygen species homeostasis revealed by metabolomic and biochemical studies

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are calcium channels modulating important calcium-mediated processes. Recent studies implicate IP3R in cell metabolism, but specific evidence is missing regarding IP3R's effects on actual metabolic pathways and key energy metabolites. Here, we applied metabolomics and molecular biology to compare DT40 cell lines devoid of IP3R (KO) and its wild-type (WT) counterpart. NMR and LC–MS metabolomic data showed that the KO cell line has a very different basic energy metabolism from the WT cell line, showing enhanced Warburg effect. In particular, the KO cells exhibited significant perturbation in energy charge, reduced glutathione and NADPH ratios with slower cellular growth rate. Subsequent flow cytometry results showed that the KO cell line has a higher level of general reactive oxygen species (ROS) but has a lower level of peroxynitrites. This ROS disturbance could be explained by observing lower expression of superoxide dismutase 2 (SOD2) and unchanged expression of catalase. The higher ROS seems to be involved in the slower growth rate of the KO cells, with an ROS scavenger increasing their growth rate. However, the KO and WT cell lines did not show any noticeable differences in AMPK and phosphorylated AMPK levels, suggesting possible saturation of AMPK-mediated metabolic regulatory circuit in both cells. Overall, our study reveals IP3R's roles in ROS homeostasis and metabolic pathways as well as the effects of its KO on cellular phenotypes.

A combined systems and structural modeling approach repositions antibiotics for Mycoplasma genitalium

Bacteria are increasingly resistant to existing antibiotics, which target a narrow range of pathways. New methods are needed to identify targets, including repositioning targets among distantly related species. We developed a novel combination of systems and structural modeling and bioinformatics to reposition known antibiotics and targets to new species. We applied this approach to Mycoplasma genitalium, a common cause of urethritis. First, we used quantitative metabolic modeling to identify enzymes whose expression affects the cellular growth rate. Second, we searched the literature for inhibitors of homologs of the most fragile enzymes. Next, we used sequence alignment to assess that the binding site is shared by M. genitalium, but not by humans. Lastly, we used molecular docking to verify that the reported inhibitors preferentially interact with M. genitalium proteins over their human homologs. Thymidylate kinase was the top predicted target and piperidinylthymines were the top compounds. Further work is needed to experimentally validate piperidinylthymines. In summary, combined systems and structural modeling is a powerful tool for drug repositioning.