RESUMEN
The metastasis of tumor cells into vital organs is a major cause of death from diverse types of malignancies [...].
RESUMEN
Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFß and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.
Asunto(s)
Neoplasias de la Mama , Femenino , Humanos , Animales , Ratones , Neoplasias de la Mama/patología , Transducción de Señal , Metástasis de la NeoplasiaRESUMEN
Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in â¼39% of human breast cancers, and â¼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.
Asunto(s)
Neoplasias de la Mama , Carcinoma Intraductal no Infiltrante , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/patología , Epigénesis Genética , Recurrencia Local de Neoplasia/genética , Carcinoma Intraductal no Infiltrante/genética , Transformación Celular Neoplásica/genéticaRESUMEN
The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single-cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation-related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at a single-cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte, and mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.
Asunto(s)
Hígado , Análisis de la Célula Individual , Núcleo Celular/genética , Humanos , Análisis de Secuencia de ARN , Transcriptoma/genéticaRESUMEN
Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor ß (TGF-ß) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions in vitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.
Asunto(s)
Células Endoteliales , Células Madre Pluripotentes , Animales , Hepatocitos , Humanos , Hígado , Ratones , Factor de Crecimiento Transformador betaRESUMEN
Pineoblastoma is a rare pediatric cancer induced by germline mutations in the tumor suppressors RB1 or DICER1. Presence of leptomeningeal metastases is indicative of poor prognosis. Here we report that inactivation of Rb plus p53 via a WAP-Cre transgene, commonly used to target the mammary gland during pregnancy, induces metastatic pineoblastoma resembling the human disease with 100% penetrance. A stabilizing mutation rather than deletion of p53 accelerates metastatic dissemination. Deletion of Dicer1 plus p53 via WAP-Cre also predisposes to pineoblastoma, albeit with lower penetrance. In silico analysis predicts tricyclic antidepressants such as nortriptyline as potential therapeutics for both pineoblastoma models. Nortriptyline disrupts the lysosome, leading to accumulation of non-functional autophagosome, cathepsin B release and pineoblastoma cell death. Nortriptyline further synergizes with the antineoplastic drug gemcitabine to effectively suppress pineoblastoma in our preclinical models, offering new modality for this lethal childhood malignancy.
Asunto(s)
Mutación de Línea Germinal/genética , Lisosomas/metabolismo , Pinealoma/tratamiento farmacológico , Pinealoma/genética , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Autofagia/efectos de los fármacos , Análisis por Conglomerados , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Integrasas/metabolismo , Estimación de Kaplan-Meier , Lisosomas/efectos de los fármacos , Ratones , Metástasis de la Neoplasia , Nortriptilina/farmacología , Nortriptilina/uso terapéutico , Pinealoma/patología , Pinealoma/ultraestructura , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment.
Asunto(s)
Hígado/citología , Hígado/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Monocitos/citología , Monocitos/metabolismo , Análisis de Secuencia de ARNRESUMEN
The dual phosphatase CDC25 has recently been identified as a target for diverse triple-negative breast cancers including RB1/PTEN/P53-deficient tumors. Moreover, CDC25 inhibitors effectively synergize with PI3K inhibitors to suppress tumor growth. We discuss these findings and the challenges that lie ahead in bringing CDC25 inhibitors to the clinic.
RESUMEN
CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Fosfatasas cdc25/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
A switch from catabolic to anabolic metabolism, a major hallmark of cancer, enables rapid cell duplication, and is driven by multiple oncogenic alterations, including PIK3CA mutation, MYC amplification, and TP53 loss. However, tumor growth requires active mitochondrial function and oxidative phosphorylation (OXPHOS). Recently, loss of the retinoblastoma (RB1) tumor suppressor in breast cancer was shown to induce mitochondrial protein translation (MPT) and OXPHOS. Here, we discuss how increased OXPHOS can enhance anabolic metabolism and cell proliferation, as well as cancer stemness and metastasis. Mitochondrial STAT3, FER/FER-T, and CHCHD2 are also implicated in OXPHOS. We propose that RB1 loss represents a prototypic oncogenic alteration that promotes OXPHOS, that aggressive tumors acquire lethal combinations of oncogenes and tumor suppressors that stimulate anabolism versus OXPHOS, and that targeting both metabolic pathways would be therapeutic.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Ubiquitina-Proteína Ligasas/genética , Anabolizantes/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Glucólisis/genética , Humanos , Mitocondrias/genética , Metástasis de la Neoplasia , Células Madre Neoplásicas/patología , Fosforilación Oxidativa , Proteínas de Unión a Retinoblastoma/deficiencia , Ubiquitina-Proteína Ligasas/deficienciaRESUMEN
The tumor suppressor PTEN is frequently inactivated in breast and other cancers; yet, germ-line mutations in this gene induce nonmalignant hamartomas, indicating dependency on additional cooperating events. Here we show that most tumors derived from conditional deletion of mouse pten in mammary epithelium are highly differentiated and lack transplantable tumor-initiating cells (TICs) capable of seeding new tumors following orthotopic injection of FACS-sorted or tumorsphere cells. A rare group of poorly differentiated tumors did harbor transplantable TICs. These transplantable tumors exhibited distinct molecular classification, signaling pathways, chromosomal aberrations, and mutational landscape, as well as reduced expression of microRNA-143/145 (miR-143/145). Stable knockdown of miR-143/145 conferred tumorigenic potential upon poorly transplantable pten-deficient tumor cells through a mechanism involving induction of RAS signaling, leading to increased sensitivity to MEK inhibition. In humans, miR-145 deficiency significantly correlated with elevated RAS-pathway activity in basal-like breast cancer, and patients with combined PTEN/miR-145 loss or PTEN-loss/high RAS-pathway activity exhibited poor clinical outcome. These results underscore a selective pressure for combined PTEN loss together with RAS-pathway activation, either through miR-145 loss or other mechanisms, in basal-like breast cancer, and a need to identify and prioritize these tumors for aggressive therapy.
RESUMEN
Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low-like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration-approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Mitocondriales/genética , Biosíntesis de Proteínas , Proteínas de Unión a Retinoblastoma/deficiencia , Neoplasias de la Mama Triple Negativas/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Línea Celular Tumoral , Femenino , Amplificación de Genes , Humanos , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Mapas de Interacción de Proteínas , Proteínas de Unión a Retinoblastoma/genética , Activación Transcripcional , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC), an aggressive disease comprising several subtypes including basal-like and claudin-low, involves frequent deletions or point mutations in TP53, as well as loss of PTEN. We previously showed that combined deletion of both tumor suppressors in the mouse mammary epithelium invariably induced claudin-low-like TNBC. The effect of p53 mutation plus Pten deletion on mammary tumorigenesis and whether this combination can induce basal-like TNBC in the mouse are unknown. METHODS: WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) composite mice were generated in which Pten is deleted and a p53-R270H mutation in the DNA-binding domain is induced upon expression of Cre-recombinase in pregnancy-identified alveolar progenitors. Tumors were characterized by histology, marker analysis, transcriptional profiling [GEO-GSE75989], bioinformatics, high-throughput (HTP) FDA drug screen as well as orthotopic injection to quantify tumor-initiating cells (TICs) and tail vein injection to identify lung metastasis. RESULTS: Combined Pten deletion plus induction of p53-R270H mutation accelerated formation of four distinct mammary tumors including poorly differentiated adenocarcinoma (PDA) and spindle/mesenchymal-like lesions. Transplantation assays revealed highest frequency of TICs in PDA and spindle tumors compared with other subtypes. Hierarchical clustering demonstrated that the PDA and spindle tumors grouped closely with human as well as mouse models of basal and claudin-low subtypes, respectively. HTP screens of primary Pten(∆):p53(∆) vs. Pten(∆):p53(R270H) spindle tumor cells with 1120 FDA-approved drugs identified 8-azaguanine as most potent for both tumor types, but found no allele-specific inhibitor. A gene set enrichment analysis revealed increased expression of a metastasis pathway in Pten(∆):p53(R270H) vs. Pten(∆):p53(∆) spindle tumors. Accordingly, following tail vein injection, both Pten(∆):p53(R270H) spindle and PDA tumor cells induced lung metastases and morbidity significantly faster than Pten(∆):p53(∆) double-deletion cells, and this was associated with the ability of Pten(∆):p53(R270H) tumor cells to upregulate E-cadherin expression in lung metastases. CONCLUSIONS: Our results demonstrate that WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice represent a tractable model to study basal-like breast cancer because unlike p53 deletion, p53(R270H) mutation in the mouse does not skew tumors toward the claudin-low subtype. The WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice develop basal-like breast cancer that is enriched in TICs, can readily form lung metastasis, and provides a preclinical model to study both basal-like and claudin-low TNBC in immune-competent mice.
Asunto(s)
Neoplasias Mamarias Animales/genética , Neoplasias Basocelulares/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Mama Triple Negativas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Cadherinas/genética , Claudinas/genética , Epitelio/metabolismo , Epitelio/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Neoplasias Mamarias Animales/patología , Ratones , Neoplasias Basocelulares/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosfohidrolasa PTEN/metabolismo , Embarazo , Eliminación de Secuencia/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PTEN loss and PIK3CA activation both promote the accumulation of phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). While these proteins also have distinct biochemical functions, beyond the regulation of PIP3, little is known about the consequences of these differences in vivo. Here, we directly compared cancer signalling in mammary tumors from MMTV-Cre:Ptenf/f and MMTV-Cre:Pik3ca(LSL-H1047R) mice. Using unsupervised hierarchical clustering we found that whereas MMTV-Cre:Pik3ca(LSL-H1047R)-derived tumors fall into two separate groups, designated squamous-likeEx and class14(Ex), MMTV-Cre:Ptenf/f tumors cluster as one group together with PIK3CA(H1047R) class14(Ex), exhibiting a 'luminal' expression profile. Gene Set Enrichment Analysis (GSEA) of Pten(Δ) and PIK3CA(H1047R) class14(Ex) tumors revealed very similar profiles of signalling pathways as well as some interesting differences. Analysis of 18 signalling signatures revealed that PI3K signalling is significantly induced whereas EGFR signalling is significantly reduced in Pten(∆) versus PIK3CA(H1047R) tumors. Thus, Pten(∆) and PIK3CA(H1047R) tumors exhibit discernable differences that may impact tumorigenesis and response to therapy.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Mamarias Experimentales/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Fosfatidilinositol 3-Quinasa Clase I , Receptores ErbB/metabolismo , Neoplasias Mamarias Experimentales/clasificación , Neoplasias Mamarias Experimentales/patología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) encodes a tumor suppressor gene implicated in the growth of various tumor types including breast cancer. We previously demonstrated that recombinant adenovirus-mediated mda-7/IL-24 expression in the mammary glands of carcinogen-treated (methylnitrosourea, MNU) rats suppressed mammary tumor development. Since most MNU-induced tumors in rats contain activating mutations in Ha-ras, which arenot frequently detected in humans, we presently examined the effect of MDA-7/IL-24 on Her2/Neu-induced mammary tumors, in which the RAS pathway is induced. We generated tet-inducible MDA-7/IL-24 transgenic mice and crossed them with Her2/Neu transgenic mice. Triple compound transgenic mice treated with doxycycline exhibited a strong inhibition of tumor development, demonstrating tumor suppressor activity by MDA-7/IL-24 in immune-competent mice. MDA-7/IL-24 induction also inhibited growth of tumors generated following injection of Her2/Neu tumor cells isolated from triple compound transgenic mice that had not been treated with doxycycline, into the mammary fat pads of isogenic FVB mice. Despite initial growth suppression, tumors in triple compound transgenic mice lost mda-7/IL-24 expression and grew, albeit after longer latency, indicating that continuous presence of this cytokine within tumor microenvironment is crucial to sustain tumor inhibitory activity. Mechanistically, MDA-7/IL-24 exerted its tumor suppression effect on HER2+ breast cancer cells, at least in part, through PERP, a member of PMP-22 family with growth arrest and apoptosis-inducing capacity. Overall, our results establish mda-7/IL-24 as a suppressor of mammary tumor development and provide a rationale for using this cytokine in the prevention/treatment of human breast cancer.
Asunto(s)
Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Interleucinas/fisiología , Neoplasias Mamarias Animales/patología , Virus del Tumor Mamario del Ratón/genética , Receptor ErbB-2/fisiología , Animales , Apoptosis , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Mamarias Animales/genética , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Breast cancer is associated with alterations in a number of growth factor and hormone-regulated signaling pathways. Mouse models of metastatic breast cancer typically feature mutated oncoproteins that activate PI3K, Stat3, and Ras signaling, but the individual and combined roles of these pathways in breast cancer progression are poorly understood. In this study, we examined the relationship between oncogenic pathway activation and breast cancer subtype by analyzing mouse mammary tumor formation in which each pathway was activated singly or pairwise. All three oncogenes showed cooperation during primary tumor formation, but efficient dissemination was only dependent on Ras. In addition, transcriptional profiling demonstrated that Ras induced adenocarcinomas with molecular characteristics related to human basal-like and HER2(+) tumors. In contrast, Ras combined with PIK3CA(H1047R), an oncogenic mutant linked to ERα(+)/luminal breast cancer in humans, induced metastatic luminal B-like tumors. Consistent with these data, elevated Ras signaling was associated with basal-like and HER2(+) subtype tumors in humans and showed a statistically significant negative association with estrogen receptor (ER) signaling across all breast cancer. Despite this, there are luminal tumors with elevated Ras signaling. Importantly, when considered as a continuous variable, Ras pathway activation was strongly linked to reduced survival of patients with ERα(+) disease independent of PI3K or Stat3 activation. Therefore, our studies suggest that Ras activation is a key determinant for dissemination and poor prognosis of ERα(+)/luminal breast cancer in humans, and hormone therapy supplemented with Ras-targeting agents may be beneficial for treating this aggressive subtype.
Asunto(s)
Neoplasias de la Mama/patología , Invasividad Neoplásica/patología , Transducción de Señal/fisiología , Proteínas ras/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Supervivencia sin Enfermedad , Femenino , Inmunohistoquímica , Estimación de Kaplan-Meier , Ratones , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant tumors. Twenty-four genes that were significantly and differentially expressed between WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients. Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling.
Asunto(s)
Quinasa del Factor 2 de Elongación/metabolismo , Eliminación de Gen , Fosfohidrolasa PTEN/genética , Neoplasias de la Mama Triple Negativas/enzimología , Proteína p53 Supresora de Tumor/genética , Animales , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/genética , Inhibidores Enzimáticos/farmacología , Epitelio/enzimología , Epitelio/metabolismo , Femenino , Humanos , Glándulas Mamarias Humanas/enzimología , Glándulas Mamarias Humanas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
HER2(+) breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2(+) breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that "immortalize" the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu(+) tumorsphere versus monolayer cells with a lentivirus short hairpin RNA kinome library. We identified kinases such as the mitogen-activated protein kinase and the TGFßR protein family, previously implicated in HER2(+) breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IκB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2(+) breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKε, suppressed growth of both mouse and human HER2(+) breast cancer cells. TBK1/IKKε inhibition promoted cellular senescence by suppressing p65-NF-κB and inducing p16(Ink4a). In addition, TBK1/IKKε inhibition cooperated with lapatinib, a HER2/EGFR1-targeted drug, to accelerate apoptosis and kill HER2(+) breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2(+) breast cancer may benefit from anti-TBK1/IKKε plus anti-HER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2(+) breast cancer.
Asunto(s)
Neoplasias de la Mama/terapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Receptor ErbB-2/análisis , Animales , Apoptosis/efectos de los fármacos , Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Genes p53 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Lapatinib , Ratones , Ratones SCID , Mutación , Quinazolinas/farmacología , Factor de Transcripción ReIA/antagonistas & inhibidoresRESUMEN
HER2(+) breast cancer (BC) is a highly aggressive subtype, affecting ~20% of BC patients. Current treatments include adjuvant or neoadjuvant chemotherapy plus anti-HER2 agents such as trastuzumab, a monoclonal antibody directed against HER2. Despite improvement in disease free survival, most patients eventually succumb to metastatic disease, which is largely incurable. Consequently, there is an urgent need to identify novel drugs that can efficiently kill HER2(+) BC and/or potentiate the effect of existing anti-HER2 therapies. We performed a lenti-viral shRNA kinome screen on non-adherent mouse Her2/Neu tumorspheres and identified TBK1, a non-canonical IκB kinase (IKK), as the most potent target [1]. TBK1 knock-down, or treatment with TBK1-II, a drug that efficiently inhibits TBK1 and its close relative IKKε (IKBKE), suppressed growth of human HER2(+) BC cells and induced cellular senescence. Senescence was associated with inhibition of phosphorylated/active p65-NFkB and induction of the cell cycle inhibitor, p16(ink4a). In addition, TBK1-II cooperated with lapatinib, a EGFR/HER2 inhibitor, to accelerate apoptosis in vitro and suppress tumor growth in a xenograft model of HER2(+) BC. Thus, TBK1/IKKε inhibitors may improve treatment of HER2(+) BC in cooperation with anti-HER2 therapy.
RESUMEN
Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1(met), fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of â¼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA(+)/CD24(-/low)/CD44(+) cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.