RESUMO
Several emerging therapies kill cancer cells primarily by inducing necrosis. As necrosis activates immune cells, potentially, uncovering the molecular drivers of anticancer therapy-induced necrosis could reveal approaches for enhancing immunotherapy efficacy. To identify necrosis-associated genes, we performed a genome-wide CRISPR-Cas9 screen with negative selection against necrosis-inducing preclinical agents BHPI and conducted follow-on experiments with ErSO. The screen identified transient receptor potential melastatin member 4 (TRPM4), a calcium-activated, ATP-inhibited, sodium-selective plasma membrane channel. Cancer cells selected for resistance to BHPI and ErSO exhibited robust TRPM4 downregulation, and TRPM4 reexpression restored sensitivity to ErSO. Notably, TRPM4 knockout (TKO) abolished ErSO-induced regression of breast tumors in mice. Supporting a broad role for TRPM4 in necrosis, knockout of TRPM4 reversed cell death induced by four additional diverse necrosis-inducing cancer therapies. ErSO induced anticipatory unfolded protein response (a-UPR) hyperactivation, long-term necrotic cell death, and release of damage-associated molecular patterns that activated macrophages and increased monocyte migration, all of which was abolished by TKO. Furthermore, loss of TRPM4 suppressed the ErSO-induced increase in cell volume and depletion of ATP. These data suggest that ErSO triggers initial activation of the a-UPR but that it is TRPM4-mediated sodium influx and cell swelling, resulting in osmotic stress, which sustains and propagates lethal a-UPR hyperactivation. Thus, TRPM4 plays a pivotal role in sustaining lethal a-UPR hyperactivation that mediates the anticancer activity of diverse necrosis-inducing therapies. SIGNIFICANCE: A genome-wide CRISPR screen reveals a pivotal role for TRPM4 in cell death and immune activation following treatment with diverse necrosis-inducing anticancer therapies, which could facilitate development of necrosis-based cancer immunotherapies.
Assuntos
Trifosfato de Adenosina , Canais de Cátion TRPM , Camundongos , Animais , Necrose/metabolismo , Morte Celular , Membrana Celular/metabolismo , Trifosfato de Adenosina/metabolismo , Sódio/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Estrogen receptor-positive (ER+) metastatic tumors contribute to nearly 70% of breast cancer-related deaths. Most patients with ER+ metastatic breast cancer (MBC) undergo treatment with the estrogen receptor antagonist fulvestrant as standard of care. Yet, among such patients, metastasis in liver is associated with reduced overall survival compared with other metastasis sites. The factors underlying the reduced responsiveness of liver metastases to ER-targeting agents remain unknown, impeding the development of more effective treatment approaches to improve outcomes for patients with ER+ liver metastases. We therefore evaluated site-specific changes in MBC cells and determined the mechanisms through which the liver metastatic niche specifically influences ER+ tumor metabolism and drug resistance. We characterized ER activity of MBC cells both in vitro, using a novel system of tissue-specific extracellular matrix hydrogels representing the stroma of ER+ tumor metastatic sites (liver, lung, and bone), and in vivo, in liver and lung metastasis mouse models. ER+ metastatic liver tumors and MBC cells grown in liver hydrogels displayed upregulated expression of glucose metabolism enzymes in response to fulvestrant. Furthermore, differential ERα activity, but not expression, was detected in liver hydrogels. In vivo, increased glucose metabolism led to increased glycogen deposition in liver metastatic tumors, while a fasting-mimicking diet increased efficacy of fulvestrant treatment to reduce the metastatic burden. Our findings identify a novel mechanism of endocrine resistance driven by the liver tumor microenvironment. IMPLICATIONS: These results may guide the development of dietary strategies to circumvent drug resistance in liver metastasis, with potential applicability in other metastatic diseases.
Assuntos
Neoplasias da Mama , Neoplasias Hepáticas , Animais , Neoplasias da Mama/patologia , Dieta , Feminino , Fulvestranto/efeitos adversos , Glucose , Humanos , Hidrogéis/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Receptores de Estrogênio/metabolismo , Microambiente TumoralRESUMO
Approximately 75% of breast cancers are estrogen receptor alpha-positive (ERα+), and targeting ERα directly with ERα antagonists/degraders or indirectly with aromatase inhibitors is a successful therapeutic strategy. However, such treatments are rarely curative and development of resistance is universal. We recently reported ErSO, a compound that induces ERα-dependent cancer cell death through a mechanism distinct from clinically approved ERα drugs, via hyperactivation of the anticipatory unfolded protein response. ErSO has remarkable tumor-eradicative activity in multiple ERα+ tumor models. While ErSO has promise as a new drug, it has effects on ERα-negative (ERα-) cells in certain contexts. Herein, we construct modified versions of ErSO and identify variants with enhanced differential activity between ERα+ and ERα- cells. We report ErSO-DFP, a compound that maintains antitumor efficacy, has enhanced selectivity for ERα+ cancer cells, and is well tolerated in rodents. ErSO-DFP and related compounds represent an intriguing new class for the treatment of ERα+ cancers.
Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Receptores de Estrogênio/metabolismo , Resposta a Proteínas não DobradasRESUMO
Metastatic estrogen receptor α (ERα)-positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14 to 21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did not undergo complete regression and regrew remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression.
Assuntos
Neoplasias da Mama , Recidiva Local de Neoplasia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Cães , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Camundongos , Ratos , Resposta a Proteínas não DobradasRESUMO
The majority of breast cancer specific deaths in women with estrogen receptor positive (ER+) tumors occur due to metastases that are resistant to therapy. There is a critical need for novel therapeutic approaches to achieve tumor regression and/or maintain therapy responsiveness in metastatic ER+ tumors. The objective of this study was to elucidate the role of metabolic pathways that undermine therapy efficacy in ER+ breast cancers. Our previous studies identified Exportin 1 (XPO1), a nuclear export protein, as an important player in endocrine resistance progression and showed that combining selinexor (SEL), an FDA-approved XPO1 antagonist, synergized with endocrine agents and provided sustained tumor regression. In the current study, using a combination of transcriptomics, metabolomics and metabolic flux experiments, we identified certain mitochondrial pathways to be upregulated during endocrine resistance. When endocrine resistant cells were treated with single agents in media conditions that mimic a nutrient deprived tumor microenvironment, their glutamine dependence for continuation of mitochondrial respiration increased. The effect of glutamine was dependent on conversion of the glutamine to glutamate, and generation of NAD+. PGC1α, a key regulator of metabolism, was the main driver of the rewired metabolic phenotype. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and our findings reveal a critical role that ERα-XPO1 crosstalk plays in reducing cancer recurrences. Combining SEL with current therapies used in clinical management of ER+ metastatic breast cancer shows promise for treating and keeping these cancers responsive to therapies in already metastasized patients.
RESUMO
Breast cancer remains one of the leading causes of cancer mortality in the US. Elevated cholesterol is a major risk factor for breast cancer onset and recurrence, while cholesterol-lowering drugs, such as statins, are associated with a good prognosis. Previous work in murine models showed that cholesterol increases breast cancer metastasis, and the pro-metastatic effects of cholesterol were due to its primary metabolite, 27-hydroxycholesterol (27HC). In our prior work, myeloid cells were found to be required for the pro-metastatic effects of 27HC, but their precise contribution remains unclear. Here we report that 27HC impairs T cell expansion and cytotoxic function through its actions on myeloid cells, including macrophages, in a Liver X receptor (LXR) dependent manner. Many oxysterols and LXR ligands had similar effects on T cell expansion. Moreover, their ability to induce the LXR target gene ABCA1 was associated with their effectiveness in impairing T cell expansion. Induction of T cell apoptosis was likely one mediator of this impairment. Interestingly, the enzyme responsible for the synthesis of 27HC, CYP27A1, is highly expressed in myeloid cells, suggesting that 27HC may have important autocrine or paracrine functions in these cells, a hypothesis supported by our finding that breast cancer metastasis was reduced in mice with a myeloid specific knockout of CYP27A1. Importantly, pharmacologic inhibition of CYP27A1 reduced metastatic growth and improved the efficacy of checkpoint inhibitor, anti-PD-L1. Taken together, our work suggests that targeting the CYP27A1 axis in myeloid cells may present therapeutic benefits and improve the response rate to immune therapies in breast cancer.
Assuntos
Neoplasias da Mama/patologia , Colestanotriol 26-Mono-Oxigenase/genética , Hidroxicolesteróis/efeitos adversos , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Colestanotriol 26-Mono-Oxigenase/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Receptores X do Fígado/metabolismo , Camundongos , Células Mieloides/efeitos dos fármacos , Transplante de Neoplasias , Linfócitos T/efeitos dos fármacosRESUMO
Accumulation of unfolded protein, or other stresses, activates the classical reactive unfolded protein response (UPR). In the recently characterized anticipatory UPR, receptor-bound estrogen, progesterone and other mitogenic hormones rapidly elicit phosphorylation of phospholipase C γ (PLCγ), activating the anticipatory UPR. How estrogen and progesterone activating their receptors couples to PLCγ phosphorylation and anticipatory UPR activation was unknown. We show that the oncogene c-Src is a rate-limiting regulator whose tyrosine kinase activity links estrogen and progesterone activating their receptors to anticipatory UPR activation. Supporting Src coupling estrogen and progesterone to anticipatory UPR activation, we identified extranuclear complexes of estrogen receptor α (ERα):Src:PLCγ and progesterone receptor:Src:PLCγ. Moreover, Src inhibition protected cancer cells against cell death. To probe Src's role, we used the preclinical ERα biomodulator, BHPI, which kills cancer cells by inducing lethal anticipatory UPR hyperactivation. Notably, Src inhibition blocked BHPI-mediated anticipatory UPR activation and the resulting rapid increase in intracellular calcium. After unbiased long-term selection for BHPI-resistant human breast cancer cells, 4/11 BHPI-resistant T47D clones, and nearly all MCF-7 clones, exhibited reduced levels of normally growth-stimulating Src. Notably, Src overexpression by virus transduction restored sensitivity to BHPI. Furthermore, in wild type cells, several-fold knockdown of Src, but not of ERα, strongly blocked BHPI-mediated UPR activation and subsequent HMGB1 release and necrotic cell death. Thus, Src plays a previously undescribed pivotal role in activation of the tumor-protective anticipatory UPR, thereby increasing the resilience of breast cancer cells. This is a new role for Src and the anticipatory UPR in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Estrogênio/metabolismo , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Humanos , Complexos Multiproteicos/metabolismo , Fosfolipase C gama/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The oncofetal mRNA-binding protein, IMP1 or insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1), promotes the overexpression of several oncogenic proteins by binding to and stabilizing their mRNAs. IMP1 is frequently overexpressed in melanoma and is associated with a poor prognosis, but the full spectrum of IMP1 target transcripts remains unknown. Here, we report the identification of protein kinase C-α (PKCα), as a novel molecular target of IMP1. Overexpression of IMP1 resulted in increased levels of PKCα, while RNAi knockdown of IMP1 resulted in decreased PKCα mRNA stability, PKCα protein levels, and MAPK/ERK activation. In addition to IMP1 acting as a positive regulator of PKCα mRNA, we also report the identification of miR-340 as a negative regulator of PKCα mRNA. In melanoma cancer cells, inhibition of miR-340 led to increased PKCα protein levels. PKCα plays important roles in numerous signaling pathways including the MAPK/ERK signaling pathway. PKCα activates RAF1, which in turn activates MEK1, and activates downstream transcriptional targets of MAPK through activation of JNK signaling. Together, these pathways provide a way to activate MAPK signaling downstream of BRAF and MEK1 inhibitors, which are commonly used to treat melanoma. Analysis of 117 melanoma tumors samples showed that overexpression of PKCα is associated with poorer overall survival. In patients harboring BRAF or NRAS mutations, PKCα overexpression is associated with an 11-fold increased risk of death. Thus, PKCα mRNA is a novel target of IMP1, which is commonly overexpressed in melanoma and is linked to poorer overall survival.
Assuntos
Melanoma/genética , Proteína Quinase C-alfa/genética , Proteínas de Ligação a RNA/genética , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases , Melanoma/mortalidade , Proteínas de Membrana/genética , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas B-raf/genética , Interferência de RNA , Risco , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Resultado do Tratamento , Regulação para CimaRESUMO
Approximately 30% of metastatic breast cancers harbor estrogen receptor α (ERα) mutations associated with resistance to endocrine therapy and reduced survival. Consistent with their constitutive proliferation, T47D and MCF7 cells in which wild-type ERα is replaced by the most common mutations, ERαY537S and ERαD538G, exhibit partially estrogen-independent gene expression. A novel invasion/dissociation/rebinding assay demonstrated that the mutant cells have a higher tendency to dissociate from invasion sites and rebind to a second site. Compared to ERαD538G breast tumors, ERαY537S tumors exhibited a dramatic increase in lung metastasis. Transcriptome analysis showed that the ERαY537S and ERαD538G mutations each elicit a unique gene expression profile. Gene set enrichment analysis showed Myc target pathways are highly induced in mutant cells. Moreover, chromatin immunoprecipitation showed constitutive, fulvestrant-resistant, recruitment of ERα mutants to the Myc enhancer region, resulting in estrogen-independent Myc overexpression in mutant cells and tumors. Knockdown and virus transduction showed Myc is necessary and sufficient for ligand-independent proliferation of the mutant cells but had no effect on metastasis-related phenotypes. Thus, Myc plays a key role in aggressive proliferation-related phenotypes exhibited by breast cancer cells expressing ERα mutations.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Fulvestranto/farmacologia , Mutação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Células MCF-7 , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cells react to a variety of stresses, including accumulation of unfolded or misfolded protein, by activating the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). The UPR is highly conserved and plays a key role in the maintenance of protein folding quality control and homeostasis. In contrast to the classical reactive mode of UPR activation, recent studies describe a hormone-activated anticipatory UPR. In this pathway, mitogenic hormones, such as estrogen (E2), epidermal growth factor, and vascular endothelial growth factor rapidly activate the UPR in anticipation of a future need for increased protein folding capacity upon cell proliferation. Here, we focus on this recently unveiled pathway of E2-estrogen receptor α (ERα) action. Notably, rapid activation of the anticipatory UPR pathway is essential for subsequent activation of the E2-ERα regulated transcription program. Moreover, activation of the UPR at diagnosis is a powerful prognostic marker in ERα positive breast cancer. Furthermore, in cells containing ERα mutations that confer estrogen independence and are common in metastatic breast cancer, the UPR is constitutively activated and linked to antiestrogen resistance. Lethal ERα-dependent hyperactivation of the anticipatory UPR represents a promising therapeutic approach exploited by a new class of small molecule ERα biomodulator.
RESUMO
The endoplasmic reticulum stress sensor, the unfolded protein response (UPR), regulates intracellular protein homeostasis. While transient activation of the reactive UPR by unfolded protein is protective, prolonged and sustained activation of the reactive UPR triggers CHOP-mediated apoptosis. In the recently characterized, evolutionarily conserved anticipatory UPR, mitogenic hormones and other effectors pre-activate the UPR; how strong and sustained activation of the anticipatory UPR induces cell death was unknown. To characterize this cell death pathway, we used BHPI, a small molecule that activates the anticipatory UPR through estrogen receptor α (ERα) and induces death of ERα+ cancer cells. We show that sustained activation of the anticipatory UPR by BHPI kills cells by inducing depletion of intracellular ATP, resulting in classical necrosis phenotypes, including plasma membrane disruption and leakage of intracellular contents. Unlike reactive UPR activation, BHPI-induced hyperactivation of the anticipatory UPR does not induce apoptosis or sustained autophagy. BHPI does not induce CHOP protein or PARP cleavage, and two pan-caspase inhibitors, or Bcl2 overexpression, have no effect on BHPI-induced cell death. Moreover, BHPI does not increase expression of autophagy markers, or work through recently identified programmed-necrosis pathways, such as necroptosis. Opening of endoplasmic reticulum IP3R calcium channels stimulates cell swelling, cPLA2 activation, and arachidonic acid release. Notably, cPLA2 activation requires ATP depletion. Importantly, blocking rapid cell swelling or production of arachidonic acid does not prevent necrotic cell death. Rapid cell death is upstream of PERK activation and protein synthesis inhibition, and results from strong and sustained activation of early steps in the anticipatory UPR. Supporting a central role for ATP depletion, reversing ATP depletion blocks rapid cell death, and the onset of necrotic cell death is correlated with ATP depletion. Necrotic cell death initiated by strong and sustained activation of the anticipatory UPR is a newly discovered role of the UPR.
Assuntos
Apoptose , Resposta a Proteínas não Dobradas , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácido Araquidônico/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Necrose , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismoRESUMO
Ovarian cancers often recur and tumors acquire resistance to chemotherapy due to overexpression of the ATP-dependent efflux pump, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1). Nontoxic small molecule inhibitors targeting MDR1 have remained largely elusive. Instead, in a novel application of our recently described estrogen receptor α (ERα) biomodulator, BHPI, we targeted MDR1's substrate, ATP. BHPI depletes intracellular ATP and nearly blocks MDR1-mediated drug efflux in ovarian cancer cells by inducing toxic hyperactivation of the endoplasmic reticulum stress sensor, the unfolded protein response (UPR). BHPI increased sensitivity of MDR1 overexpressing multidrug resistant OVCAR-3 ovarian cancer cells to killing by paclitaxel by >1,000 fold. BHPI also restored doxorubicin sensitivity in OVCAR-3 cells and in MDR1 overexpressing breast cancer cells. In an orthotopic OVCAR-3 xenograft model, paclitaxel was ineffective and the paclitaxel-treated group was uniquely prone to form large secondary tumors in adjacent tissue. BHPI alone strongly reduced tumor growth. Notably, tumors were undetectable in mice treated with BHPI plus paclitaxel. Compared to control ovarian tumors, after the combination therapy, levels of the plasma ovarian cancer biomarker CA125 were at least several hundred folds lower; moreover, CA125 levels progressively declined to undetectable. Targeting MDR1 through UPR-dependent ATP depletion represents a promising therapeutic strategy.
RESUMO
Many estrogen receptor α (ERα)-positive breast cancers develop resistance to endocrine therapy via mutation of ERs whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ERs, we describe here our development and characterization of three chemically novel AEs that effectively suppress proliferation of breast cancer cells and tumors. Our AEs are effective against wild-type and Y537S and D538G ERs, the two most commonly occurring constitutively active ERs. The three new AEs suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared with WT ER, mutants exhibited approximately 10- to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER-containing MCF7 and T47D cells revealed that AE responses were compound, cell-type, and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER-expressing tumor xenografts in NOD/SCID-γ mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ERs to different AEs and make clear the value of having a toolkit of AEs for treatment of endocrine therapy-resistant tumors driven by different constitutively active ERs. Cancer Res; 77(20); 5602-13. ©2017 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Mutação , Animais , Neoplasias da Mama/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Moduladores de Receptor Estrogênico/química , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição Aleatória , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oncofetal mRNA-binding protein, IMP1 or insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP1), binds to and stabilizes c-Myc, ß-TrCP1, and other oncogenic mRNAs, leading to increased expression of the proteins encoded by its target mRNAs. IMP1 is frequently overexpressed in cancer and is strongly correlated with a poor prognosis and reduced survival in melanoma, ovarian, breast, colon, and lung cancer. While IMP1 is an attractive anticancer drug target, there are no small molecule inhibitors of IMP1. A fluorescence anisotropy-based assay was used to screen 160,000 small molecules for their ability to inhibit IMP1 binding to fluorescein-labeled c-Myc mRNA. The small molecule, BTYNB, was identified as a potent and selective inhibitor of IMP1 binding to c-Myc mRNA. In cells, BTYNB downregulates several mRNA transcripts regulated by IMP1. BTYNB destabilizes c-Myc mRNA, resulting in downregulation of c-Myc mRNA and protein. BTYNB downregulates ß-TrCP1 mRNA and reduces activation of nuclear transcriptional factors-kappa B (NF-κB). The oncogenic translation regulator, eEF2, emerged as a new IMP1 target mRNA, enabling BTYNB to inhibit tumor cell protein synthesis. BTYNB potently inhibited proliferation of IMP1-containing ovarian cancer and melanoma cells with no effect in IMP1-negative cells. Overexpression of IMP1 reversed BTYNB inhibition of cell proliferation. BTYNB completely blocked anchorage-independent growth of melanoma and ovarian cancer cells in colony formation assays. With its ability to target c-Myc and to inhibit proliferation of difficult-to-target melanomas and ovarian cancer cells, and with its unique mode of action, BTYNB is a promising small molecule for further therapeutic evaluation and mechanistic studies.
RESUMO
Outgrowth of metastases expressing ERα mutations Y537S and D538G is common after endocrine therapy for estrogen receptor α (ERα) positive breast cancer. The effect of replacing wild type ERα in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ERαY537S or ERαD538G replace one or both wild-type ERα genes. In 2-dimensional, and in quantitative anchorage-independent 3-dimensional cell culture, ERαY537S and ERαD538G cells exhibited estrogen-independent growth. A progestin further increased their already substantial proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/ICI 182,780 (ICI). Our recently described ERα biomodulator, BHPI, which hyperactivates the unfolded protein response (UPR), completely blocked proliferation. In ERαY537S and ERαD538G cells, estrogen-ERα target genes were constitutively active and partially antiestrogen resistant. The UPR marker sp-XBP1 was constitutively activated in ERαY537S cells and further induced by progesterone in both cell lines. UPR-regulated genes associated with tamoxifen resistance, including the oncogenic chaperone BiP/GRP78, were upregulated. ICI displayed a greater than 2 fold reduction in its ability to induce ERαY537S and ERαD538G degradation. Progestins, UPR activation and perhaps reduced ICI-stimulated ERα degradation likely contribute to antiestrogen resistance seen in ERαY537S and ERαD538G cells.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Indóis/farmacologia , Resposta a Proteínas não Dobradas , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Progestinas/farmacologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Botanical estrogen (BE) dietary supplements are consumed by women as substitutes for loss of endogenous estrogens at menopause. To examine the roles of estrogen receptor α (ERα) and aryl hydrocarbon receptor (AhR) and their crosstalk in the actions of BEs, we studied gene regulation and proliferation responses to four widely used BEs, genistein, daidzein, and S-equol from soy, and liquiritigen from licorice root in breast cancer and liver cells. BEs and estradiol (E2), acting through ERα, stimulated proliferation, ERα chromatin binding and target-gene expression. BEs but not E2, acting through AhR, bound to xenobiotic response element-containing chromatin sites and enhanced AhR target-gene expression (CYP1A1, CYP1B1). While E2 and TCDD acted quite selectively through their respective receptors, BEs acted via both receptors, with their AhR activity moderated by negative crosstalk through ERα. Both ERα and AhR should be considered as mediators of the biology and pharmacology of BEs.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Células MCF-7 , Modelos Biológicos , Dibenzodioxinas Policloradas/farmacologia , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Progesterona/metabolismoRESUMO
The endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), plays a key role in regulating intracellular protein homeostasis. The extensively studied reactive mode of UPR activation is characterized by unfolded protein, or other EnR stress, triggering UPR activation. Here we focus on the emerging anticipatory mode of UPR activation in which mitogenic steroid and peptide hormones and other effectors preactivate the UPR and anticipate a future need for increased protein folding capacity. Mild UPR activation in breast cancer can be protective and contributes to antiestrogen resistance. Hyperactivation of the anticipatory UPR pathway in cancer cells with a small molecule converts it from cytoprotective to cytotoxic, highlighting its potential as a therapeutic target in estrogen receptor-positive breast cancer.
Assuntos
Neoplasias/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasias/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/genéticaRESUMO
To identify new pathways of estrogen action and novel estrogen receptor α (ERα) biomodulators, we performed high throughput screening and used follow on assays and bioinformatics to identify small molecule ERα inhibitors with a novel mode of action. These studies led to identification of rapid extranuclear activation of the endoplasmic reticulum stress sensor, the unfolded protein response (UPR), as a new pathway of estrogen-ERα action. Moreover, increasing evidence indicates that the mechanism underlying anticipatory activation of the UPR is shared among steroid and peptide hormones and is conserved from insects to humans. It is likely that this newly unveiled extranuclear pathway is used by diverse mitogenic hormones to prepare cells for the increased protein folding load that will occur during subsequent cell proliferation. Demonstrating biological relevance, elevated expression of a UPR gene signature in ERα positive breast cancer is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen, tumor recurrence and poor survival. In addition, overexpression of epidermal growth factor receptor and HER2/neu is positively correlated with increased UPR activation in breast cancer. This review describes recent research that demonstrates the importance of anticipatory UPR activation in therapy resistant tumors and discusses a promising small molecule biomodulator that inhibits tumor growth by tuning this UPR signaling pathway.
Assuntos
Neoplasias da Mama/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Resposta a Proteínas não Dobradas/genéticaRESUMO
The onco-protein epidermal growth factor (EGF) initiates a cascade that includes activation of the ERK and AKT signaling pathways and alters gene expression. We describe a new action of EGF-EGF receptor (EGFR), rapid anticipatory activation of the endoplasmic reticulum stress sensor, the unfolded protein response (UPR). Within 2 min, EGF elicits EGFR dependent activation of phospholipase C γ (PLCγ), producing inositol triphosphate (IP3), which binds to IP3 receptor (IP3R), opening the endoplasmic reticulum IP3R Ca(2+) channels, resulting in increased intracellular Ca(2+). This calcium release leads to transient and moderate activation of the IRE1α and ATF6α arms of the UPR, resulting in induction of BiP chaperone. Knockdown or inhibition of EGFR, PLCγ or IP3R blocks the increase in intracellular Ca(2+). While blocking the increase in intracellular Ca(2+) by locking the IP3R calcium channel with 2-APB had no effect on EGF activation of the ERK or AKT signaling pathways, it abolished the rapid EGF-mediated induction and repression of gene expression. Knockdown of ATF6α or XBP1, which regulate UPR-induced chaperone production, inhibited EGF stimulated cell proliferation. Supporting biological relevance, increased levels of EGF receptor during tumor progression were correlated with increased expression of the UPR gene signature. Anticipatory activation of the UPR is a new role for EGF. Since UPR activation occurs in <2 min, it is an initial cell response when EGF binds EGFR.
Assuntos
Neoplasias da Mama/genética , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Precoces/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration.