Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Largen: a molecular regulator of mammalian cell size control.

Little is known about how mammalian cells maintain cell size homeostasis. We conducted a novel genetic screen to identify cell-size-controlling genes and isolated Largen, the product of a gene (PRR16) that increased cell size upon overexpression in human cells. In vitro evidence indicated that Largen preferentially stimulates the translation of specific subsets of mRNAs, including those encoding proteins affecting mitochondrial functions. The involvement of Largen in mitochondrial respiration was consistent with the increased mitochondrial mass and greater ATP production in Largen-overexpressing cells. Furthermore, Largen overexpression led to increased cell size in vivo, as revealed by analyses of conditional Largen transgenic mice. Our results establish Largen as an important link between mRNA translation, mitochondrial functions, and the control of mammalian cell size.

Objective Supervised Machine Learning-Based Classification and Inference of Biological Neuronal Networks.

The classification of biological neuron types and networks poses challenges to the full understanding of the human brain's organisation and functioning. In this paper, we develop a novel objective classification model of biological neuronal morphology and electrical types and their networks, based on the attributes of neuronal communication using supervised machine learning solutions. This presents advantages compared to the existing approaches in neuroinformatics since the data related to mutual information or delay between neurons obtained from spike trains are more abundant than conventional morphological data. We constructed two open-access computational platforms of various neuronal circuits from the Blue Brain Project realistic models, named Neurpy and Neurgen. Then, we investigated how we could perform network tomography with cortical neuronal circuits for the morphological, topological and electrical classification of neurons. We extracted the simulated data of 10,000 network topology combinations with five layers, 25 morphological type (m-type) cells, and 14 electrical type (e-type) cells. We applied the data to several different classifiers (including Support Vector Machine (SVM), Decision Trees, Random Forest, and Artificial Neural Networks). We achieved accuracies of up to 70%, and the inference of biological network structures using network tomography reached up to 65% of accuracy. Objective classification of biological networks can be achieved with cascaded machine learning methods using neuron communication data. SVM methods seem to perform better amongst used techniques. Our research not only contributes to existing classification efforts but sets the road-map for future usage of brain-machine interfaces towards an in vivo objective classification of neurons as a sensing mechanism of the brain's structure.

Embedding a Panoramic Representation of Infrared Light in the Adult Rat Somatosensory Cortex through a Sensory Neuroprosthesis.

Can the adult brain assimilate a novel, topographically organized, sensory modality into its perceptual repertoire? To test this, we implemented a microstimulation-based neuroprosthesis that rats used to discriminate among infrared (IR) light sources. This system continuously relayed information from four IR sensors that were distributed to provide a panoramic view of IR sources, into primary somatosensory cortex (S1). Rats learned to discriminate the location of IR sources in <4 d. Animals in which IR information was delivered in spatial register with whisker topography learned the task more quickly. Further, in animals that had learned to use the prosthesis, altering the topographic mapping from IR sensor to stimulating electrode had immediate deleterious effects on discrimination performance. Multielectrode recordings revealed that S1 neurons had multimodal (tactile/IR) receptive fields, with clear preferences for those stimuli most likely to be delivered during the task. Neuronal populations predicted, with high accuracy, which stimulation pattern was present in small (75 ms) time windows. Surprisingly, when identical microstimulation patterns were delivered during an unrelated task, cortical activity in S1 was strongly suppressed. Overall, these results show that the adult mammalian neocortex can readily absorb completely new information sources into its representational repertoire, and use this information in the production of adaptive behaviors.

Overcoming intratumoural heterogeneity for reproducible molecular risk stratification: a case study in advanced kidney cancer.

BACKGROUND: Metastatic clear cell renal cell cancer (mccRCC) portends a poor prognosis and urgently requires better clinical tools for prognostication as well as for prediction of response to treatment. Considerable investment in molecular risk stratification has sought to overcome the performance ceiling encountered by methods restricted to traditional clinical parameters. However, replication of results has proven challenging, and intratumoural heterogeneity (ITH) may confound attempts at tissue-based stratification. METHODS: We investigated the influence of confounding ITH on the performance of a novel molecular prognostic model, enabled by pathologist-guided multiregion sampling (n = 183) of geographically separated mccRCC cohorts from the SuMR trial (development, n = 22) and the SCOTRRCC study (validation, n = 22). Tumour protein levels quantified by reverse phase protein array (RPPA) were investigated alongside clinical variables. Regularised wrapper selection identified features for Cox multivariate analysis with overall survival as the primary endpoint. RESULTS: The optimal subset of variables in the final stratification model consisted of N-cadherin, EPCAM, Age, mTOR (NEAT). Risk groups from NEAT had a markedly different prognosis in the validation cohort (log-rank p = 7.62 × 10-7; hazard ratio (HR) 37.9, 95% confidence interval 4.1-353.8) and 2-year survival rates (accuracy = 82%, Matthews correlation coefficient = 0.62). Comparisons with established clinico-pathological scores suggest favourable performance for NEAT (Net reclassification improvement 7.1% vs International Metastatic Database Consortium score, 25.4% vs Memorial Sloan Kettering Cancer Center score). Limitations include the relatively small cohorts and associated wide confidence intervals on predictive performance. Our multiregion sampling approach enabled investigation of NEAT validation when limiting the number of samples analysed per tumour, which significantly degraded performance. Indeed, sample selection could change risk group assignment for 64% of patients, and prognostication with one sample per patient performed only slightly better than random expectation (median logHR = 0.109). Low grade tissue was associated with 3.5-fold greater variation in predicted risk than high grade (p = 0.044). CONCLUSIONS: This case study in mccRCC quantitatively demonstrates the critical importance of tumour sampling for the success of molecular biomarker studies research where ITH is a factor. The NEAT model shows promise for mccRCC prognostication and warrants follow-up in larger cohorts. Our work evidences actionable parameters to guide sample collection (tumour coverage, size, grade) to inform the development of reproducible molecular risk stratification methods.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress.

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

Antitumour activity of the novel flavonoid Oncamex in preclinical breast cancer models.

BACKGROUND: The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents. METHODS: We studied the effect of a second-generation flavonoid analogue Oncamex in a panel of seven breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy and immunohistochemistry of xenograft tissue to investigate its mechanism of action. RESULTS: Proliferation assays showed that Oncamex treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in the expression of genes controlling cell cycle and apoptosis. Fluorescence microscopy showed the compound's mitochondrial targeting and reactive oxygen species-modulating properties, inducing superoxide production at concentrations associated with antiproliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no signs of untoward effects on the animals. CONCLUSIONS: Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent.

Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo.

Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 µmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.

Identifying molecular features that distinguish fluvastatin-sensitive breast tumor cells.

Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatin triggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERα)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genes encoding regulators of mevalonate production, non-sterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation.

Novel flavonoids as anti-cancer agents: mechanisms of action and promise for their potential application in breast cancer.

Flavonoids are a large group of ubiquitous polyphenolic secondary metabolites in plants with a wide range of properties, including a widely reported anti-cancer effect. The present review focuses on the different known mechanisms partaking in said anti-tumour effects, with particular emphasis on breast cancer. Their structure and reactivity allows flavonoids to work as antioxidant agents and phyto-oestrogens, modulating oestrogen signalling and metabolism to induce an overall anti-proliferative response. Other effects include the ability of flavonoids to modulate the CYP1 (cytochrome P450 1) and ABC (ATP-binding cassette) protein families, involved in carcinogenesis and drug delivery respectively. They can also induce apoptosis and cell cycle arrest and regulate other signalling pathways involved in the development and progression of cancer. In conclusion, there is accumulating evidence on the versatility of flavonoids and the numerous activities contributing to their anti-tumour effect. The complex, yet effective, mechanism of action of flavonoids, together with their interesting pharmacological properties, is the basis for their potential application in breast and other cancers. This rationale has led to the current interest in the application of flavonoids, including clinical trials currently underway and the development of novel flavonoids with improved properties, which hold great promise for tackling breast cancer.

Distinct mechanisms of non-autonomous UPRER mediated by GABAergic, glutamatergic, and octopaminergic neurons.

The capacity to deal with stress declines during the aging process, and preservation of cellular stress responses is critical to healthy aging. The unfolded protein response of the endoplasmic reticulum (UPRER) is one such conserved mechanism, which is critical for the maintenance of several major functions of the ER during stress, including protein folding and lipid metabolism. Hyperactivation of the UPRER by overexpression of the major transcription factor, xbp-1s, solely in neurons drives lifespan extension as neurons send a neurotransmitter-based signal to other tissue to activate UPRER in a non-autonomous fashion. Previous work identified serotonergic and dopaminergic neurons in this signaling paradigm. To further expand our understanding of the neural circuitry that underlies the non-autonomous signaling of ER stress, we activated UPRER solely in glutamatergic, octopaminergic, and GABAergic neurons in C. elegans and paired whole-body transcriptomic analysis with functional assays. We found that UPRER-induced signals from glutamatergic neurons increased expression of canonical protein homeostasis pathways and octopaminergic neurons promoted pathogen response pathways, while minor, but statistically significant changes were observed in lipid metabolism-related genes with GABAergic UPRER activation. These findings provide further evidence for the distinct role neuronal subtypes play in driving the diverse response to ER stress.

A metabolic atlas of mouse aging.

Humans are living longer, but this is accompanied by an increased incidence of age-related chronic diseases. Many of these diseases are influenced by age-associated metabolic dysregulation, but how metabolism changes in multiple organs during aging in males and females is not known. Answering this could reveal new mechanisms of aging and age-targeted therapeutics. In this study, we describe how metabolism changes in 12 organs in male and female mice at 5 different ages. Organs show distinct patterns of metabolic aging that are affected by sex differently. Hydroxyproline shows the most consistent change across the dataset, decreasing with age in 11 out of 12 organs investigated. We also developed a metabolic aging clock that predicts biological age and identified alpha-ketoglutarate, previously shown to extend lifespan in mice, as a key predictor of age. Our results reveal fundamental insights into the aging process and identify new therapeutic targets to maintain organ health.

Experimental characterization of next-generation expanded-bed adsorbents for capture of a recombinant protein expressed in high-cell-density yeast fermentation.

Expanded-bed adsorption (EBA) can be particularly useful in protein recovery from high-cell-density fermentation broth where conventional methods for harvest and clarification, such as continuous centrifugation and depth filtration, demand long processing times and are associated with high costs. In this work, the use of next-generation high-particle-density EBA adsorbents, including two mixed-mode resins, for the direct capture of a recombinant protein expressed in yeast at high cell densities is evaluated. Using classical experimental approaches and under different conditions (pH, salt, etc.), Langmuir isotherm parameters for these resins are obtained along with pore diffusivity values. Additional batch adsorption studies with Fastline® MabDirect, the resin that demonstrated the highest static binding capacity for the recombinant protein of interest under the conditions evaluated in this study, indicate competitive binding of nontarget proteins and approximately a 30% reduction in equilibrium binding capacity to 50 mg/mL settled bed in the presence of a 5%-10% cell concentration. Packed-bed (PB) dynamic binding capacities for the MabDirect resin (25-40 mg/mL PB) were significantly higher than for the Fastline® HSA resin and for the MabDirect MM resin in expanded-bed mode (5-10 mg/mL settled bed). Bed expansion behavior for the mMabDirect MM resin along with process yield and eluate purity are identified as a function of linear velocity and cell density, demonstrating process feasibility for pilot scale use.

The aging tumor metabolic microenvironment.

Despite the higher incidence of cancer with increasing age, few preclinical or clinical studies incorporate age. This, coupled with an aging world population, requires that we improve our understanding of how aging affects cancer development, progression, and treatment. One key area will be how the tumor microenvironment (TME) changes with age. Metabolite levels are an essential component of the TME, and they are affected by the metabolic requirements of the cells present and systemic metabolite availability. These factors are affected by aging, causing different TME metabolic states between young and older adults. In this review, we will summarize what is known about how aging impacts the TME metabolic state, and suggest how we can improve our understanding of it.

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent.

INTRODUCTION: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. METHODS: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. RESULTS: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. CONCLUSION: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Susceptibility to simvastatin-induced toxicity is partly determined by mitochondrial respiration and phosphorylation state of Akt.

Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10µM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.

Ureido-substituted sulfamates show potent carbonic anhydrase IX inhibitory and antiproliferative activities against breast cancer cell lines.

A series of 50 sulfamates were obtained by reacting 4-aminophenol with isocyanates followed by sulfamoylation. Most of the new compounds were nanomolar inhibitors of the tumor-associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII, whereas they inhibited less cytosolic offtarget isoforms CA I and II. Some of these sulfamates showed significant antiproliferative activity in several breast cancer cell lines, such as SKBR3, MCF10A, ZR75/1, MDA-MB-361 and MCF7, constituting interesting anticancer leads.

Effect of short- and long-term treatment with valproate on carnitine homeostasis in humans.

AIMS: The aim of this study was to identify the mechanisms of hypocarnitinemia in patients treated with valproate. METHODS: Plasma concentrations and urinary excretion of carnitine, acetylcarnitine, propionylcarnitine, valproylcarnitine, and butyrobetaine were determined in a patient starting valproate treatment and in 10 patients on long-term valproate treatment. Transport of carnitine and valproylcarnitine by the proximal tubular carnitine transporter OCTN2 was assessed in vitro. RESULTS: In the patient starting valproate, the plasma carnitine and acetylcarnitine levels dropped for 1-3 weeks and had recovered after 3-5 weeks, whereas the plasma levels of propionyl and valproylcarnitine increased steadily over 5 weeks. The renal excretion and excretion fractions (EFs) of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine decreased substantially after starting valproate. Compared with controls, patients on long-term valproate treatment had similar plasma levels of carnitine, acetylcarnitine, and propionylcarnitine, whereas valproylcarnitine was found only in patients. Urinary excretion and renal clearance of carnitine, acetylcarnitine, propionylcarnitine, and butyrobetaine were decreased in valproate-treated compared with that in control patients, reaching statistical significance for carnitine. The EFs of carnitine, acetylcarnitine, and propionylcarnitine were <5% of the filtered load in controls and were lower in valproate-treated patients. In contrast, the EF for valproylcarnitine approached 100%, resulting from a low affinity of valproylcarnitine for the carnitine transporter OCTN2 and competition with concomitantly filtered carnitine. CONCLUSIONS: The initial drop in plasma carnitine levels of valproate-treated patients is most likely due to impaired carnitine biosynthesis, whereas the recovery of the plasma carnitine levels is explainable by an increased renal expression of OCTN2. Renally excreted valproylcarnitine does not affect renal handling of carnitine in vivo.

Methionine restriction forces Epstein-Barr virus out of latency.

EBV gene expression is repressed during viral latency to prevent an immune response, but it is not known how metabolism contributes to this silencing. In this issue of Cell Metabolism, Guo et al. describe how methionine restriction reactivates the expression of EBV genes, offering new therapeutic approaches against EBV-driven diseases.

Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα, and cell-cycle signaling in a breast cancer model.

Aim: A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation. Methods: The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17ß-estradiol (E2)-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9). Results: In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis. Conclusions: Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.