ABSTRACT
Tumours are subject to external environmental variability. However, in vitro tumour spheroid experiments, used to understand cancer progression and develop cancer therapies, have been routinely performed for the past fifty years in constant external environments. Furthermore, spheroids are typically grown in ambient atmospheric oxygen (normoxia), whereas most in vivo tumours exist in hypoxic environments. Therefore, there are clear discrepancies between in vitro and in vivo conditions. We explore these discrepancies by combining tools from experimental biology, mathematical modelling, and statistical uncertainty quantification. Focusing on oxygen variability to develop our framework, we reveal key biological mechanisms governing tumour spheroid growth. Growing spheroids in time-dependent conditions, we identify and quantify novel biological adaptation mechanisms, including unexpected necrotic core removal, and transient reversal of the tumour spheroid growth phases.
Subject(s)
Neoplasms , Spheroids, Cellular , Humans , Spheroids, Cellular/pathology , Oxygen , Models, Biological , Neoplasms/pathology , Models, TheoreticalABSTRACT
AIMS: Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. METHODS: Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. RESULTS: H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. CONCLUSION: Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.
Subject(s)
AMP-Activated Protein Kinases , Histones , Humans , Histones/genetics , Down-Regulation , Mixed Function Oxygenases , Proto-Oncogene ProteinsABSTRACT
Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer's disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-ß (oAß) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAß challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aß precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aß toxicity.
Subject(s)
Amyloid beta-Peptides/metabolism , Cell Cycle/physiology , Neurons/physiology , Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/pharmacology , Amyloid beta-Peptides/toxicity , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Biomarkers , Cell Survival/drug effects , Disease Models, Animal , Disease Susceptibility , Humans , Mice , Mice, TransgenicABSTRACT
Head and neck cancers (HNC) are the seventh most prevalent cancer type globally. Despite their common categorisation, HNCs are a heterogeneous group of malignancies arising in various anatomical sites within the head and neck region. These cancers exhibit different clinical and biological manifestations, and this heterogeneity also contributes to the high rates of treatment failure and mortality. To evaluate patients who will respond to a particular treatment, there is a need to develop in vitro model systems that replicate in vivo tumour status. Among the methods developed, patient-derived cancer organoids, also known as tumouroids, recapitulate in vivo tumour characteristics including tumour architecture. Tumouroids have been used for general disease modelling and genetic instability studies in pan-cancer research. However, a limited number of studies have thus far been conducted using tumouroid-based drug screening. Studies have concluded that tumouroids can play an essential role in bringing precision medicine for highly heterogenous cancer types such as HNC.
Subject(s)
Head and Neck Neoplasms , Humans , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Tumor Cells, CulturedABSTRACT
BACKGROUND: Population-wide screening for melanoma is not cost-effective, but genetic characterization could facilitate risk stratification and targeted screening. Common Melanocortin-1 receptor (MC1R) red hair colour (RHC) variants and Microphthalmia-associated transcription factor (MITF) E318K separately confer moderate melanoma susceptibility, but their interactive effects are relatively unexplored. OBJECTIVES: To evaluate whether MC1R genotypes differentially affect melanoma risk in MITF E318K+ vs. E318K- individuals. MATERIALS AND METHODS: Melanoma status (affected or unaffected) and genotype data (MC1R and MITF E318K) were collated from research cohorts (five Australian and two European). In addition, RHC genotypes from E318K+ individuals with and without melanoma were extracted from databases (The Cancer Genome Atlas and Medical Genome Research Bank, respectively). χ2 and logistic regression were used to evaluate RHC allele and genotype frequencies within E318K+/- cohorts depending on melanoma status. Replication analysis was conducted on 200 000 general-population exomes (UK Biobank). RESULTS: The cohort comprised 1165 MITF E318K- and 322 E318K+ individuals. In E318K- cases MC1R R and r alleles increased melanoma risk relative to wild type (wt), P < 0.001 for both. Similarly, each MC1R RHC genotype (R/R, R/r, R/wt, r/r and r/wt) increased melanoma risk relative to wt/wt (P < 0.001 for all). In E318K+ cases, R alleles increased melanoma risk relative to the wt allele [odds ratio (OR) 2.04 (95% confidence interval 1.67-2.49); P = 0.01], while the r allele risk was comparable with the wt allele [OR 0.78 (0.54-1.14) vs. 1.00, respectively]. E318K+ cases with the r/r genotype had a lower but not significant melanoma risk relative to wt/wt [OR 0.52 (0.20-1.38)]. Within the E318K+ cohort, R genotypes (R/R, R/r and R/wt) conferred a significantly higher risk compared with non-R genotypes (r/r, r/wt and wt/wt) (P < 0.001). UK Biobank data supported our findings that r did not increase melanoma risk in E318K+ individuals. CONCLUSIONS: RHC alleles/genotypes modify melanoma risk differently in MITF E318K- and E318K+ individuals. Specifically, although all RHC alleles increase risk relative to wt in E318K- individuals, only MC1R R increases melanoma risk in E318K+ individuals. Importantly, in the E318K+ cohort the MC1R r allele risk is comparable with wt. These findings could inform counselling and management for MITF E318K+ individuals.
Subject(s)
Melanoma , Skin Neoplasms , Humans , Alleles , Receptor, Melanocortin, Type 1/genetics , Microphthalmia-Associated Transcription Factor/genetics , Australia/epidemiology , Melanoma/genetics , Genotype , Genetic Predisposition to Disease/genetics , Skin Neoplasms/geneticsABSTRACT
Co-culture tumour spheroid experiments are routinely performed to investigate cancer progression and test anti-cancer therapies. Therefore, methods to quantitatively characterise and interpret co-culture spheroid growth are of great interest. However, co-culture spheroid growth is complex. Multiple biological processes occur on overlapping timescales and different cell types within the spheroid may have different characteristics, such as differing proliferation rates or responses to nutrient availability. At present there is no standard, widely-accepted mathematical model of such complex spatio-temporal growth processes. Typical approaches to analyse these experiments focus on the late-time temporal evolution of spheroid size and overlook early-time spheroid formation, spheroid structure and geometry. Here, using a range of ordinary differential equation-based mathematical models and parameter estimation, we interpret new co-culture experimental data. We provide new biological insights about spheroid formation, growth, and structure. As part of this analysis we connect Greenspan's seminal mathematical model to co-culture data for the first time. Furthermore, we generalise a class of compartment-based spheroid mathematical models that have previously been restricted to one population so they can be applied to multiple populations. As special cases of the general model, we explore multiple natural two population extensions to Greenspan's seminal model and reveal biological mechanisms that can describe the internal dynamics of growing co-culture spheroids and those that cannot. This mathematical and statistical modelling-based framework is well-suited to analyse spheroids grown with multiple different cell types and the new class of mathematical models provide opportunities for further mathematical and biological insights.
Subject(s)
Neoplasms , Spheroids, Cellular , Humans , Coculture Techniques , Spheroids, Cellular/pathology , Models, Biological , Mathematical Concepts , Neoplasms/pathology , Models, TheoreticalABSTRACT
Understanding synchrony in growing populations is important for applications as diverse as epidemiology and cancer treatment. Recent experiments employing fluorescent reporters in melanoma cell lines have uncovered growing subpopulations exhibiting sustained oscillations, with nearby cells appearing to synchronize their cycles. In this study, we demonstrate that the behavior observed is consistent with long-lasting transient phenomenon initiated and amplified by the finite-sample effects and demographic noise. We present a novel mathematical analysis of a multistage model of cell growth, which accurately reproduces the synchronized oscillations. As part of the analysis, we elucidate the transient and asymptotic phases of the dynamics and derive an analytical formula to quantify the effect of demographic noise in the appearance of the oscillations. The implications of these findings are broad, such as providing insight into experimental protocols that are used to study the growth of asynchronous populations and, in particular, those investigations relating to anticancer drug discovery.
Subject(s)
Cell Cycle , Cell Line , DemographyABSTRACT
Three-dimensional (3D) in vitro tumour spheroid experiments are an important tool for studying cancer progression and potential cancer drug therapies. Standard experiments involve growing and imaging spheroids to explore how different conditions lead to different rates of spheroid growth. These kinds of experiments, however, do not reveal any information about the spatial distribution of the cell cycle within the expanding spheroid. Since 2008, a new experimental technology called fluorescent ubiquitination-based cell cycle indicator (FUCCI) has enabled real-time in situ visualisation of the cell cycle progression. Observations of 3D tumour spheroids with FUCCI labelling reveal significant intratumoural structure, as the cell cycle status can vary with location. Although many mathematical models of tumour spheroid growth have been developed, none of the existing mathematical models are designed to interpret experimental observations with FUCCI labelling. In this work, we adapt the mathematical framework originally proposed by Ward and King (Math Med Biol 14:39-69, 1997. https://doi.org/10.1093/imammb/14.1.39 ) to produce a new mathematical model of FUCCI-labelled tumour spheroid growth. The mathematical model treats the spheroid as being composed of three subpopulations: (i) living cells in G1 phase that fluoresce red; (ii) living cells in S/G2/M phase that fluoresce green; and (iii) dead cells that are not fluorescent. We assume that the rates at which cells pass through different phases of the cell cycle, and the rate of cell death, depend upon the local oxygen concentration. Parameterising the new mathematical model using experimental measurements of cell cycle transition times, we show that the model can qualitatively capture important experimental observations that cannot be addressed using previous mathematical models. Further, we show that the mathematical model can be used to qualitatively mimic the action of anti-mitotic drugs applied to the spheroid. All software programs required to solve the nonlinear moving boundary problem associated with the new mathematical model are available on GitHub. at https://github.com/wang-jin-mathbio/Jin2021.
Subject(s)
Models, Biological , Neoplasms , Cell Cycle , Cell Division , Disease Progression , Humans , Neoplasms/diagnostic imaging , Neoplasms/pathology , Neoplasms/therapyABSTRACT
We present a novel mathematical model of heterogeneous cell proliferation where the total population consists of a subpopulation of slow-proliferating cells and a subpopulation of fast-proliferating cells. The model incorporates two cellular processes, asymmetric cell division and induced switching between proliferative states, which are important determinants for the heterogeneity of a cell population. As motivation for our model we provide experimental data that illustrate the induced-switching process. Our model consists of a system of two coupled delay differential equations with distributed time delays and the cell densities as functions of time. The distributed delays are bounded and allow for the choice of delay kernel. We analyse the model and prove the nonnegativity and boundedness of solutions, the existence and uniqueness of solutions, and the local stability characteristics of the equilibrium points. We find that the parameters for induced switching are bifurcation parameters and therefore determine the long-term behaviour of the model. Numerical simulations illustrate and support the theoretical findings, and demonstrate the primary importance of transient dynamics for understanding the evolution of many experimental cell populations.
Subject(s)
Cell Proliferation , Eukaryotic Cells/cytology , Models, Biological , Cell Count , Cell Division , Computer Simulation , Neoplasms/physiopathologyABSTRACT
The go-or-grow hypothesis states that adherent cells undergo reversible phenotype switching between migratory and proliferative states, with cells in the migratory state being more motile than cells in the proliferative state. Here, we examine go-or-grow in two-dimensional in vitro assays using melanoma cells with fluorescent cell-cycle indicators and cell-cycle-inhibiting drugs. We analyze the experimental data using single-cell tracking to calculate mean diffusivities and compare motility between cells in different cell-cycle phases and in cell-cycle arrest. Unequivocally, our analysis does not support the go-or-grow hypothesis. We present clear evidence that cell motility is independent of the cell-cycle phase and that nonproliferative arrested cells have the same motility as cycling cells.
Subject(s)
Cell Tracking , Pharmaceutical Preparations , Cell Cycle , Cell Division , Cell MovementABSTRACT
Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that control a diverse range of biological processes during development and in adult tissues. We recently reported that somatic FGFR2 mutations are associated with shorter survival in endometrial cancer. However, little is known about how these FGFR2 mutations contribute to endometrial cancer metastasis. Here, we report that expression of the activating mutations FGFR2N550K and FGFR2Y376C in an endometrial cancer cell model induce Golgi fragmentation, and loss of polarity and directional migration. In mutant FGFR2-expressing cells, this was associated with an inability to polarise intracellular pools of FGFR2 towards the front of migrating cells. Such polarization defects were exacerbated in three-dimensional culture, where FGFR2 mutant cells were unable to form well-organised acini, instead undergoing exogenous ligand-independent invasion. Our findings uncover collective cell polarity and invasion as common targets of disease-associated FGFR2 mutations that lead to poor outcome in endometrial cancer patients.
Subject(s)
Cell Movement/physiology , Cell Polarity/physiology , Endometrial Neoplasms/metabolism , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Biotinylation , Cell Line, Tumor , Cell Movement/genetics , Cell Polarity/genetics , Cell Proliferation/genetics , Cell Proliferation/physiology , Chemotaxis/genetics , Chemotaxis/physiology , Endometrial Neoplasms/genetics , Female , Fluorescent Antibody Technique , HEK293 Cells , Humans , Immunoblotting , Lentivirus/genetics , Mutation/genetics , Receptor, Fibroblast Growth Factor, Type 2/geneticsABSTRACT
Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.
Subject(s)
Exosomes/metabolism , Melanoma/metabolism , Melanoma/pathology , rab27 GTP-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Cell Movement/physiology , Clustered Regularly Interspaced Short Palindromic Repeats , Culture Media, Conditioned , Exosomes/genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , Melanoma/genetics , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Melanosomes/genetics , Melanosomes/metabolism , Mice , Neoplasm Invasiveness , Nevus/genetics , Nevus/metabolism , Proteomics , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Spheroids, Cellular , rab27 GTP-Binding Proteins/biosynthesis , rab27 GTP-Binding Proteins/geneticsABSTRACT
The fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI, allows the visualization of the G1 and S/G2/M cell cycle phases of individual cells. FUCCI consists of two fluorescent probes, so that cells in the G1 phase fluoresce red and cells in the S/G2/M phase fluoresce green. FUCCI reveals real-time information about cell cycle dynamics of individual cells, and can be used to explore how the cell cycle relates to the location of individual cells, local cell density, and different cellular microenvironments. In particular, FUCCI is used in experimental studies examining cell migration, such as malignant invasion and wound healing. Here we present, to our knowledge, new mathematical models that can describe cell migration and cell cycle dynamics as indicated by FUCCI. The fundamental model describes the two cell cycle phases, G1 and S/G2/M, which FUCCI directly labels. The extended model includes a third phase, early S, which FUCCI indirectly labels. We present experimental data from scratch assays using FUCCI-transduced melanoma cells, and show that the predictions of spatial and temporal patterns of cell density in the experiments can be described by the fundamental model. We obtain numerical solutions of both the fundamental and extended models, which can take the form of traveling waves. These solutions are mathematically interesting because they are a combination of moving wavefronts and moving pulses. We derive and confirm a simple analytical expression for the minimum wave speed, as well as exploring how the wave speed depends on the spatial decay rate of the initial condition.
Subject(s)
Cell Cycle , Cell Movement , Models, Biological , Cell Line, Tumor , Humans , KineticsABSTRACT
Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Melanoma/drug therapy , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Animals , Cell Line, Tumor , Drug Synergism , Heterografts , Histone Deacetylase Inhibitors/administration & dosage , Humans , Melanoma/enzymology , Mice , Mice, Inbred NOD , Mice, SCID , Protein Kinase Inhibitors/administration & dosage , Random Allocation , Signal Transduction/drug effectsABSTRACT
The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches.
Subject(s)
Cell Cycle , Melanoma/metabolism , Melanoma/therapy , Skin Neoplasms/metabolism , Skin Neoplasms/therapy , Antineoplastic Agents/pharmacology , Combined Modality Therapy , Disease Progression , Drug Delivery Systems , Humans , Imaging, Three-Dimensional , Molecular Targeted Therapy , Protein Kinase Inhibitors/therapeutic use , Spheroids, CellularABSTRACT
The nuclear transactive response DNA-binding protein 43 (TDP-43) undergoes relocalization to the cytoplasm with formation of cytoplasmic deposits in neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Pathogenic mutations in the TDP-43-encoding TARDBP gene in familial ALS as well as non-mutant human TDP-43 have been utilized to model FTD/ALS in cell culture and animals, including mice. Here, we report novel A315T mutant TDP-43 transgenic mice, iTDP-43(A315T), with controlled neuronal over-expression. Constitutive expression of human TDP-43(A315T) resulted in pronounced early-onset and progressive neurodegeneration, which was associated with compromised motor performance, spatial memory and disinhibition. Muscle atrophy resulted in reduced grip strength. Cortical degeneration presented with pronounced astrocyte activation. Using differential protein extraction from iTDP-43(A315T) brains, we found cytoplasmic localization, fragmentation, phosphorylation and ubiquitination and insolubility of TDP-43. Surprisingly, suppression of human TDP-43(A315T) expression in mice with overt neurodegeneration for only 1 week was sufficient to significantly improve motor and behavioral deficits, and reduce astrogliosis. Our data suggest that functional deficits in iTDP-43(A315T) mice are at least in part a direct and transient effect of the presence of TDP-43(A315T). Furthermore, it illustrates the compensatory capacity of compromised neurons once transgenic TDP-43 is removed, with implications for future treatments.
Subject(s)
Amyotrophic Lateral Sclerosis/physiopathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Frontotemporal Lobar Degeneration/physiopathology , Mutation , Recovery of Function/physiology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Astrocytes/pathology , Astrocytes/physiology , Brain/pathology , Brain/physiopathology , Disease Models, Animal , Doxycycline , Female , Frontotemporal Lobar Degeneration/genetics , Frontotemporal Lobar Degeneration/pathology , Gliosis/pathology , Gliosis/physiopathology , Hand Strength/physiology , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/physiology , Neurons/metabolism , Neurons/pathology , Spatial Memory/physiologyABSTRACT
Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.
Subject(s)
Amino Acid Transport System ASC/biosynthesis , Glutamine/metabolism , Large Neutral Amino Acid-Transporter 1/biosynthesis , Melanoma/pathology , Multiprotein Complexes/antagonists & inhibitors , Skin Neoplasms/pathology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Amino Acid Transport System ASC/antagonists & inhibitors , Amino Acid Transport System ASC/genetics , Amino Acids, Cyclic/pharmacology , Benzyl Compounds/pharmacology , Biological Transport , CDC2 Protein Kinase/biosynthesis , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cell Survival , Humans , Leucine/metabolism , Mechanistic Target of Rapamycin Complex 1 , Melanoma/metabolism , Minor Histocompatibility Antigens , Multiprotein Complexes/genetics , Proto-Oncogene Proteins B-raf/genetics , RNA Interference , RNA, Small Interfering/genetics , Serine/analogs & derivatives , Serine/pharmacology , Signal Transduction , Skin Neoplasms/metabolism , Spheroids, Cellular , TOR Serine-Threonine Kinases/genetics , Tumor Cells, Cultured , Ubiquitin-Conjugating Enzymes/biosynthesisABSTRACT
Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Targeting apoptosis regulators is thus considered a promising approach to sensitizing melanoma to therapy. In the previous issue of Experimental Dermatology, Plötz and Eberle discuss the role that apoptosis plays in melanoma progression and drug resistance and the utility of apoptosis-inducing BH3-mimetics as targeted therapy. There are a number of compounds in clinical development and the field seems close to translating recent findings into benefits for patients with melanoma. Thus, this viewpoint is timely and achieves a valuable summary of the current state of apoptosis-inducing therapy of melanoma.
Subject(s)
Apoptosis , Drug Resistance, Neoplasm , Melanoma/drug therapy , Melanoma/pathology , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Proliferation , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Mitochondrial Membrane Transport Proteins , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
Melanoma is responsible for most skin cancer-associated deaths globally. The progression of melanoma is influenced by a number of pathogenic processes. Understanding the VEGF/VEGFR axis, which includes VEGF-A, PlGF, VEGF-B, VEGF-C, and VEGF-D and their receptors, VEGFR-1, VEGFR-2, and VEGFR-3, is of great importance in melanoma due to its crucial role in angiogenesis. This axis generates multifactorial and complex cellular signaling, engaging the MAPK/ERK, PI3K/AKT, PKC, PLC-γ, and FAK signaling pathways. Melanoma cell growth and proliferation, migration and metastasis, survival, and acquired resistance to therapy are influenced by this axis. The VEGF/VEGFR axis was extensively examined for their potential as diagnostic/prognostic biomarkers in melanoma patients and results showed that VEGF overexpression can be associated with unfavorable prognosis, higher level of tumor invasion and poor response to therapy. MicroRNAs linking to the VEGF/VEGFR axis were identified and, in this review, divided into two categories according to their functions, some of them promote melanoma angiogenesis (promotive group) and some restrict melanoma angiogenesis (protective group). In addition, the approach of treating melanoma by targeting the VEGF/VEGFR axis has garnered significant interest among researchers. These agents can be divided into two main groups: anti-VEGF and VEGFR inhibitors. These therapeutic options may be a prominent step along with the modern targeting and immune therapies for better coverage of pathological processes leading to melanoma progression and therapy resistance.
Subject(s)
Melanoma , Humans , Melanoma/drug therapy , Melanoma/pathology , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor A/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal TransductionABSTRACT
Aberrant cell cycle progression is a hallmark of solid tumors. Therefore, cell cycle analysis is an invaluable technique to study cancer cell biology. However, cell cycle progression has been most commonly assessed by methods that are limited to temporal snapshots or that lack spatial information. In this chapter, we describe a technique that allows spatiotemporal real-time tracking of cell cycle progression of individual cells in a multicellular context. The power of this system lies in the use of 3D melanoma spheroids generated from melanoma cells engineered with the fluorescent ubiquitination-based cell cycle indicator (FUCCI). This technique, combined with mathematical modeling, allows us to gain further and more detailed insight into several relevant aspects of solid cancer cell biology, such as tumor growth, proliferation, invasion, and drug sensitivity.