ABSTRACT
In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.
Subject(s)
Fatty Acids , Triple Negative Breast Neoplasms , Tumor Microenvironment , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Humans , Female , Fatty Acids/metabolism , Mice , Cell Line, Tumor , Animals , Biomechanical Phenomena , Disease Models, Animal , Neoplasm MetastasisABSTRACT
BACKGROUND: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. METHODS: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. RESULTS: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. CONCLUSIONS: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.
Subject(s)
Biological Specimen Banks , Cryopreservation , Genomics , Neoplasms/diagnosis , Single-Cell Analysis , Biomarkers, Tumor , Cryopreservation/methods , Cryopreservation/standards , Gene Expression Profiling/methods , Gene Expression Regulation , Genomics/methods , High-Throughput Nucleotide Sequencing , Humans , Immunophenotyping , Neoplasms/etiology , Organ Specificity/genetics , Sequence Analysis, RNA/methods , Signal Transduction , Single-Cell Analysis/methodsABSTRACT
The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1-Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1-Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.
Subject(s)
Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 1/metabolism , Kinesins/metabolism , Triple Negative Breast Neoplasms/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/genetics , Aurora Kinase A/metabolism , Benzamides/pharmacology , Cell Cycle/genetics , Cell Line, Tumor , Cell Self Renewal/drug effects , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Kinesins/antagonists & inhibitors , Kinesins/genetics , Mice , Neoplastic Stem Cells/drug effects , Paclitaxel/pharmacology , Quinazolines/pharmacology , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolismABSTRACT
Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.
ABSTRACT
The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.
Subject(s)
Triple Negative Breast Neoplasms/immunology , Tumor Escape , Extracellular Matrix/immunology , Extracellular Matrix/pathology , Female , Humans , RNA-Seq , Stromal Cells/immunology , Stromal Cells/pathology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/pathology , Triple Negative Breast Neoplasms/pathologyABSTRACT
Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.
Subject(s)
Cancer-Associated Fibroblasts/pathology , Drug Resistance, Neoplasm/genetics , Heparan Sulfate Proteoglycans/metabolism , Pancreatic Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic/genetics , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness/pathology , Pancreas/pathology , Pancreatic Neoplasms/genetics , Signal Transduction/physiology , Tumor Suppressor Protein p53/geneticsABSTRACT
The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hedgehog ligand produced by neoplastic cells reprograms cancer-associated fibroblasts (CAFs) to provide a supportive niche for the acquisition of a chemo-resistant, cancer stem cell (CSC) phenotype via FGF5 expression and production of fibrillar collagen. Stromal treatment of patient-derived xenografts with smoothened inhibitors (SMOi) downregulates CSC markers expression and sensitizes tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. These studies identify Hedgehog signaling to CAFs as a novel mediator of CSC plasticity and an exciting new therapeutic target in TNBC.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Neoplasm/drug effects , Neoplastic Stem Cells/drug effects , Triple Negative Breast Neoplasms/drug therapy , Adult , Aged , Anilides/administration & dosage , Animals , Biphenyl Compounds/administration & dosage , Cell Line, Tumor , Docetaxel/administration & dosage , Female , Humans , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Middle Aged , Neoplastic Stem Cells/metabolism , Pyridines/administration & dosage , Treatment Outcome , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.
Subject(s)
Drug Resistance, Neoplasm , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Animals , Benzamides , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Therapy, Combination , Female , Fluorescence Resonance Energy Transfer , Humans , Hypoxia , Intravital Microscopy/methods , Mice , Mice, Inbred BALB C , Morpholines/pharmacology , Morpholines/therapeutic use , Nanoparticles/chemistry , Nitroimidazoles/pharmacology , Nitroimidazoles/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphoramide Mustards/pharmacology , Phosphoramide Mustards/therapeutic use , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines , Signal Transduction/drug effects , Transplantation, Heterologous , Tumor MicroenvironmentABSTRACT
N-linked glycosylation (NLG) is a co-translational modification that is essential for the folding, stability, and trafficking of transmembrane (TM) and secretory glycoproteins. Efficient NLG requires the stepwise synthesis and en bloc transfer of a 14-sugar carbohydrate known as a lipid-linked oligosaccharide (LLO). The genetics of LLO biosynthesis have been established in yeast and Chinese hamster systems, but human models of LLO biosynthesis are lacking. In this study we report that Kato III human gastric cancer cells represent a model of deficient LLO synthesis, possessing a homozygous deletion of the LLO biosynthesis factor, MPDU1. Kato III cells lacking MPDU1 have all the hallmarks of a glycosylation-deficient cell line, including altered sensitivity to lectins and the formation of truncated LLOs. Analysis of transcription using an expression microarray and protein levels using a proteome antibody array reveal changes in the expression of several membrane proteins, including the metalloprotease ADAM-15 and the cell adhesion molecule CEACAM1. Surprisingly, the restoration of MPDU1 expression in Kato III cells demonstrated a clear phenotype of increased cell-cell adhesion, a finding that was confirmed in vivo through analysis of tumor xenografts. These experiments also confirmed that protein levels of CEACAM-1, which functions in cell adhesion, is dependent on LLO biosynthesis in vivo. Kato III cells and the MPDU1-rescued Kato IIIM cells therefore provide a novel model to examine the consequences of defective LLO biosynthesis both in vitro and in vivo.
Subject(s)
Antigens, CD/biosynthesis , Cell Adhesion Molecules/biosynthesis , Gene Expression Regulation , Glycolipids/biosynthesis , Antigens, CD/genetics , Cell Adhesion/genetics , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Glycolipids/genetics , Glycosylation , HumansABSTRACT
Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.
Subject(s)
Cell Culture Techniques/methods , Collagen/chemistry , Animals , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Coculture Techniques , Collagen/isolation & purification , Extracellular Matrix/metabolism , Gefitinib/pharmacology , Humans , Macropodidae/metabolism , Mice , Microscopy, Atomic Force , Rats , Tendons/metabolismABSTRACT
Basal-like breast cancer (BLBC) is a heterogeneous disease with poor prognosis; however, its cellular origins and aetiology are poorly understood. In this study, we show that inhibitor of differentiation 4 (ID4) is a key regulator of mammary stem cell self-renewal and marks a subset of BLBC with a putative mammary basal cell of origin. Using an ID4GFP knock-in reporter mouse and single-cell transcriptomics, we show that ID4 marks a stem cell-enriched subset of the mammary basal cell population. ID4 maintains the mammary stem cell pool by suppressing key factors required for luminal differentiation. Furthermore, ID4 is specifically expressed by a subset of human BLBC that possess a very poor prognosis and a transcriptional signature similar to a mammary stem cell. These studies identify ID4 as a mammary stem cell regulator, deconvolute the heterogeneity of BLBC and link a subset of mammary stem cells to the aetiology of BLBC.
Subject(s)
Breast Neoplasms/genetics , Inhibitor of Differentiation Proteins/genetics , Mammary Glands, Animal/cytology , RNA, Messenger/metabolism , Stem Cells/metabolism , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Gene Knock-In Techniques , Humans , Inhibitor of Differentiation Proteins/metabolism , Mammary Glands, Animal/metabolism , Mice , Neoplasm Transplantation , Phenotype , Real-Time Polymerase Chain ReactionABSTRACT
Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.
Subject(s)
Glioma/metabolism , Mannose-6-Phosphate Isomerase/metabolism , Radiation Tolerance , Receptors, Fibroblast Growth Factor/metabolism , Signal Transduction , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement/genetics , Cell Proliferation , Gene Knockdown Techniques , Glioma/genetics , Glioma/radiotherapy , Humans , Mannose-6-Phosphate Isomerase/genetics , Protein Multimerization , Protein Transport , RNA, Small Interfering , Radiation Tolerance/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 2/chemistry , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Receptors, Fibroblast Growth Factor/chemistry , Receptors, Fibroblast Growth Factor/geneticsABSTRACT
Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2ß1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2ß1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2ß1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2ß1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.
Subject(s)
Bone Neoplasms/metabolism , G(M1) Ganglioside/metabolism , Integrin alpha2/metabolism , Prostatic Neoplasms/metabolism , Sialic Acids/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Neoplasms/secondary , Cell Line, Tumor , G(M1) Ganglioside/genetics , Humans , Integrin alpha2/genetics , Male , Neoplasm Metastasis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Sialic Acids/geneticsABSTRACT
Despite the progress achieved in breast cancer screening and therapeutic innovations, the basal-like subtype of breast cancer (BLBC) still represents a particular clinical challenge. In order to make an impact on survival in this type of aggressive breast cancer, new targeted therapeutic agents are urgently needed. Aberrant activation of the Hedgehog (Hh) signalling pathway has been unambiguously tied to cancer development and progression in a variety of solid malignancies, and the recent approval of vismodegib, an orally bioavailable small-molecule inhibitor of Smoothened, validates Hh signalling as a valuable therapeutic target. A number of recent publications have highlighted a role for Hh signalling in breast cancer models and clinical specimens. Interestingly, Hh ligand overexpression is associated with the BLBC phenotype and a poor outcome in terms of metastasis and breast cancer-related death. In this review, we provide a comprehensive overview of the canonical Hh signalling pathway in mammals, highlight its roles in mammary gland development and breast carcinogenesis and discuss its potential therapeutic value in BLBC.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Transformation, Neoplastic/drug effects , Hedgehog Proteins/metabolism , Molecular Targeted Therapy , Signal Transduction/drug effects , Breast Neoplasms/pathology , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Female , HumansABSTRACT
Outcomes have improved significantly for many women diagnosed with breast cancer. For the heterogeneous group of tumours lacking expression of the oestrogen, progesterone and HER2 receptors, 'triple negative' breast cancers (TNBC), the prognosis overall has remained quite poor. When TNBC recurs, there is often little response to chemotherapy, and there are a few treatment options in this setting. Thus, there is an urgent clinical need to identify new therapeutic targets in order to improve the outlook for these patients. This review highlights the most promising therapeutic targets identified through new sequencing technologies, as well as through studies of apoptosis. We also present mounting evidence that the developmental signalling pathways Wnt/ß-catenin, NOTCH and Hedgehog play an important role in the pathogenesis and progression of TNBC with new therapeutic approaches inhibiting these pathways in advanced preclinical studies or early clinical trials.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Humans , Molecular Targeted Therapy , Receptor, ErbB-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Signal TransductionABSTRACT
Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward glycolipid substrates. ST8Sia I (EC 2.4.99.8, SAT-II, SIAT 8a) is the key enzyme controlling the biosynthesis of b- and c-series gangliosides. ST8Sia I is expressed at early developmental stages whereas in adult human tissues, ST8Sia I transcripts are essentially detected in brain. ST8Sia I together with b- and c-series gangliosides are also over-expressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, neuroblastoma and in estrogen receptor (ER) negative breast cancer, where they play a role in cell proliferation, migration, adhesion and angiogenesis. We have stably expressed ST8Sia I in MCF-7 breast cancer cells and analyzed the glycosphingolipid composition of wild type (WT) and GD3S+ clones. As shown by mass spectrometry, MCF-7 expressed a complex pattern of neutral and sialylated glycosphingolipids from globo- and ganglio-series. WT MCF-7 cells exhibited classical monosialylated gangliosides including G(M3), G(M2), and G(M1a). In parallel, the expression of ST8Sia I in MCF-7 GD3S+ clones resulted in a dramatic change in ganglioside composition, with the expression of b- and c-series gangliosides as well as unusual tetra- and pentasialylated lactosylceramide derivatives G(Q3) (II(3)Neu5Ac(4)-Gg(2)Cer) and G(P3) (II(3)Neu5Ac(5)-Gg(2)Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gangliosides/metabolism , Gene Expression , Sialyltransferases/genetics , Sialyltransferases/metabolism , Female , Gangliosides/biosynthesis , Glycosphingolipids/metabolism , Humans , MCF-7 Cells , MethylationABSTRACT
We have recently established and characterized cellular clones deriving from MDA-MB-231 breast cancer cells that express the human G(D3) synthase (GD3S), the enzyme that controls the biosynthesis of b- and c-series gangliosides. The GD3S positive clones show a proliferative phenotype in the absence of serum or growth factors and an increased tumor growth in severe immunodeficient mice. This phenotype results from the constitutive activation of the receptor tyrosine kinase c-Met in spite of the absence of ligand and subsequent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt pathways. Here, we show by mass spectrometry analysis of total glycosphingolipids that G(D3) and G(D2) are the main gangliosides expressed by the GD3S positive clones. Moreover, G(D2) colocalized with c-Met at the plasma membrane and small interfering RNA silencing of the G(M2)/G(D2) synthase efficiently reduced the expression of G(D2) as well as c-Met phosphorylation and reversed the proliferative phenotype. Competition assays using anti-G(D2) monoclonal antibodies also inhibit proliferation and c-Met phosphorylation of GD3S positive clones in serum-free conditions. Altogether, these results demonstrate the involvement of the disialoganglioside G(D2) in MDA-MB-231 cell proliferation via the constitutive activation of c-Met. The accumulation of G(D2) in c-Met expressing cells could therefore reinforce the tumorigenicity and aggressiveness of breast cancer tumors.
Subject(s)
Breast Neoplasms/metabolism , Gangliosides/metabolism , Proto-Oncogene Proteins c-met/metabolism , Sialyltransferases/genetics , Antibodies, Monoclonal/pharmacology , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Proliferation/drug effects , Gangliosides/analysis , Humans , Mass Spectrometry , Phenotype , Phosphorylation/drug effects , Proto-Oncogene Proteins c-met/antagonists & inhibitors , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Sialyltransferases/metabolism , Tumor Cells, CulturedABSTRACT
The disialoganglioside G(D3) is overexpressed in â¼50% of invasive ductal breast carcinoma, and the G(D3) synthase gene (ST8SIA1) displays higher expression among estrogen receptor-negative breast cancer tumors, associated with a decreased overall survival of breast cancer patients. However, no relationship between ganglioside expression and breast cancer development and aggressiveness has been reported. We have previously shown that overexpression of G(D3) synthase induces the accumulation of b- and c-series gangliosides (G(D3), G(D2), and G(T3)) at the cell surface of MDA-MB-231 breast cancer cells together with the acquisition of a proliferative phenotype in the absence of serum. Here, we show that phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways are constitutively activated in G(D3) synthase-expressing cells. Analysis of phosphorylation of tyrosine kinase receptors shows a specific c-Met constitutive activation in G(D3) synthase-expressing cells, in the absence of its ligand, hepatocyte growth factor/scatter factor. In addition, inhibition of c-Met or downstream signaling pathways reverses the proliferative phenotype. We also show that G(D3) synthase expression enhances tumor growth in severe combined immunodeficient mice. Finally, a higher expression of ST8SIA1 and MET in the basal subtype of human breast tumors are observed. Altogether, our results show that G(D3) synthase expression is sufficient to enhance the tumorigenicity of MDA-MB-231 breast cancer cells through a ganglioside-dependent activation of the c-Met receptor.
Subject(s)
Breast Neoplasms/enzymology , Proto-Oncogene Proteins c-met/metabolism , Sialyltransferases/biosynthesis , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Growth Processes/physiology , Cell Line, Tumor , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gangliosides/metabolism , Humans , Mice , Mice, SCID , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Sialyltransferases/genetics , Sialyltransferases/metabolismABSTRACT
Glycosylation changes that occur in cancer often lead to the expression of tumour-associated carbohydrate antigens. In breast cancer, these antigens are usually associated with a poor prognosis and a reduced overall survival. Cellular models have shown the implication of these antigens in cell adhesion, migration, proliferation and tumour growth. The present review summarizes our current knowledge of glycosylation changes (structures, biosynthesis and occurrence) in breast cancer cell lines and primary tumours, and the consequences on disease progression and aggressiveness. The therapeutic strategies attempted to target tumour-associated carbohydrate antigens in breast cancer are also discussed.
Subject(s)
Antigens, Tumor-Associated, Carbohydrate/metabolism , Breast Neoplasms/metabolism , Female , Humans , PrognosisABSTRACT
Changes in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis(x) or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G(D3) synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness.