RESUMO
Breast cancer stands as the most prevalent malignancy afflicting women. Despite significant advancements in its diagnosis and treatment, breast cancer metastasis continues to be a leading cause of mortality among women. To metastasize, cancer cells face numerous challenges: breaking away from the primary tumor, surviving in the circulation, establishing in a distant location, evading immune detection and, finally, thriving to initiate a new tumor. Each of these sequential steps requires cancer cells to adapt to a myriad of stressors and develop survival mechanisms. In addition, most patients with breast cancer undergo surgical removal of their primary tumor and have various therapeutic interventions designed to eradicate cancer cells. Despite this plethora of attacks and stresses, certain cancer cells not only manage to persist but also proliferate robustly, giving rise to substantial tumors that frequently culminate in the patient's demise. To enhance patient outcomes, there is an imperative need for a deeper understanding of the molecular and cellular mechanisms that empower cancer cells to not only survive but also expand. Herein, we delve into the intrinsic stresses that cancer cells encounter throughout the metastatic journey and the additional stresses induced by therapeutic interventions. We focus on elucidating the remarkable strategies adopted by cancer cells, such as cell-cell clustering and intricate cell-cell communication mechanisms, to ensure their survival.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Comunicação CelularRESUMO
Metastasis is responsible for the majority of cancer-related fatalities. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163intCD206intPDL1intHLA-DR+ or PDL1highARG1high). Our in-depth spatial analysis demonstrates that metastasis-initiating cancer cells consistently colocalizes with distinct cell populations within the TME, suggesting a role for these cell-cell interactions in promoting metastasis.
RESUMO
Inter-patient and intra-tumoral heterogeneity complicate the identification of predictive biomarkers and effective treatments for basal triple negative breast cancer (b-TNBC). Invasion is the initiating event in metastasis and can occur by both collective and single-cell mechanisms. We cultured primary organoids from a b-TNBC genetically engineered mouse model in 3D collagen gels to characterize their invasive behavior. We observed that organoids from the same tumor presented different phenotypes that we classified as non-invasive, collective and disseminative. To identify molecular regulators driving these invasive phenotypes, we developed a workflow to isolate individual organoids from the collagen gels based on invasive morphology and perform RNA sequencing. We next tested the requirement of differentially regulated genes for invasion using shRNA knock-down. Strikingly, KRAS was required for both collective and disseminative phenotypes. We then performed a drug screen targeting signaling nodes upstream and downstream of KRAS. We found that inhibition of EGFR, MAPK/ERK, or PI3K/AKT signaling reduced invasion. Of these, ERK inhibition was striking for its ability to potently inhibit collective invasion and dissemination. We conclude that different cancer cells in the same b-TNBC tumor can express different metastatic molecular programs and identified KRAS and ERK as essential regulators of collective and single cell dissemination.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Proto-Oncogênicas p21(ras) , Linhagem Celular Tumoral , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Movimento Celular/genéticaRESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype associated with early metastatic recurrence and worse patient outcomes. TNBC tumors express molecular markers of the epithelial-mesenchymal transition (EMT), but its requirement during spontaneous TNBC metastasis in vivo remains incompletely understood. We demonstrated that spontaneous TNBC tumors from a genetically engineered mouse model (GEMM), multiple patient-derived xenografts, and archival patient samples exhibited large populations in vivo of hybrid E/M cells that lead invasion ex vivo while expressing both epithelial and mesenchymal characteristics. The mesenchymal marker vimentin promoted invasion and repressed metastatic outgrowth. We next tested the requirement for five EMT transcription factors and observed distinct patterns of utilization during invasion and colony formation. These differences suggested a sequential activation of multiple EMT molecular programs during the metastatic cascade. Consistent with this model, our longitudinal single-cell RNA analysis detected three different EMT-related molecular patterns. We observed cancer cells progressing from epithelial to hybrid E/M and strongly mesenchymal patterns during invasion and from epithelial to a hybrid E/M pattern during colony formation. We next investigated the relative epithelial versus mesenchymal state of cancer cells in both GEMM and patient metastases. In both contexts, we observed heterogeneity between and within metastases in the same individual. We observed a complex spectrum of epithelial, hybrid E/M, and mesenchymal cell states within metastases, suggesting that there are multiple successful molecular strategies for distant organ colonization. Together, our results demonstrate an important and complex role for EMT programs during TNBC metastasis.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Humanos , Camundongos , Metástase Neoplásica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , VimentinaRESUMO
Mechanical signals from the tumor microenvironment modulate cell mechanics and influence cell metabolism to promote cancer aggressiveness. Cells withstand external forces by adjusting the stiffness of their cytoskeleton. Microtubules (MTs) act as compression-bearing elements. Yet how cancer cells regulate MT dynamic in response to the locally constrained environment has remained unclear. Using breast cancer as a model of a disease in which mechanical signaling promotes disease progression, we show that matrix stiffening rewires glutamine metabolism to promote MT glutamylation and force MT stabilization, thereby promoting cell invasion. Pharmacologic inhibition of glutamine metabolism decreased MT glutamylation and affected their mechanical stabilization. Similarly, decreased MT glutamylation by overexpressing tubulin mutants lacking glutamylation site(s) decreased MT stability, thereby hampering cancer aggressiveness in vitro and in vivo. Together, our results decipher part of the enigmatic tubulin code that coordinates the fine-tunable properties of MT and link cell metabolism to MT dynamics and cancer aggressiveness.
Assuntos
Ácido Glutâmico/metabolismo , Mecanotransdução Celular/fisiologia , Microtúbulos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Células Cultivadas , Metabolismo Energético/fisiologia , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
Cancer invasion and metastasis are challenging to study in vivo since they occur deep inside the body over extended time periods. Organotypic 3D culture of fresh tumor tissue enables convenient real-time imaging, genetic and microenvironmental manipulation and molecular analysis. Here, we provide detailed protocols to isolate and culture heterogenous organoids from murine and human primary and metastatic site tumors. The time required to isolate organoids can vary based on the tissue and organ type but typically takes <7 h. We describe a suite of assays that model specific aspects of metastasis, including proliferation, survival, invasion, dissemination and colony formation. We also specify comprehensive protocols for downstream applications of organotypic cultures that will allow users to (i) test the role of specific genes in regulating various cellular processes, (ii) distinguish the contributions of several microenvironmental factors and (iii) test the effects of novel therapeutics.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Técnicas de Cultura de Tecidos/métodos , Animais , Humanos , Camundongos , Metástase NeoplásicaRESUMO
Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.
Assuntos
Ácido Aspártico/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Carcinoma/metabolismo , Ácido Glutâmico/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fibroblastos Associados a Câncer/patologia , Linhagem Celular , Matriz Extracelular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg Cancer Res; 78(18); 5229-42. ©2018 AACR.
Assuntos
Sinalização do Cálcio , Carcinoma de Células Escamosas/patologia , Matriz Extracelular/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Actomiosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Diltiazem/farmacologia , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Invasividade Neoplásica , Esferoides Celulares , Verapamil/farmacologiaRESUMO
Acto-myosin contractility in carcinoma-associated fibroblasts leads to assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock-down of LIF-dependent genes in fibroblasts, we identified the glycoprotein ICAM-1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that the membrane-bound ICAM-1 isoform is necessary and sufficient to promote inflammation-dependent extracellular matrix contraction, which favors cancer cell invasion. Indeed, ICAM-1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM-1 expression by establishing a positive feedback signaling. Thus, targeting stromal ICAM-1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination.
Assuntos
Actomiosina/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Actomiosina/genética , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Neoplasias de Cabeça e Pescoço/genética , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente TumoralRESUMO
Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.