RESUMO
There is an urgent need to provide immediate and effective options for the treatment of prostate cancer (PCa) to prevent progression to lethal castration-resistant PCa (CRPC). The mevalonate (MVA) pathway is dysregulated in PCa, and statin drugs commonly prescribed for hypercholesterolemia, effectively target this pathway. Statins exhibit anti-PCa activity, however the resulting intracellular depletion of cholesterol triggers a feedback loop that restores MVA pathway activity, thus diminishing statin efficacy and contributing to resistance. To identify drugs that block this feedback response and enhance the pro-apoptotic activity of statins, we performed a high-content image-based screen of a 1508 drug library, enriched for FDA-approved compounds. Two of the validated hits, Galeterone (GAL) and Quinestrol, share the cholesterol-related tetracyclic structure, which is also evident in the FDA-approved CRPC drug Abiraterone (ABI). Molecular modeling revealed that GAL, Quinestrol and ABI not only share structural similarity with 25-hydroxy-cholesterol (25HC) but were also predicted to bind similarly to a known protein-binding site of 25HC. This suggested GAL, Quinestrol and ABI are sterol-mimetics and thereby inhibit the statin-induced feedback response. Cell-based assays demonstrated that these agents inhibit nuclear translocation of sterol-regulatory element binding protein 2 (SREBP2) and the transcription of MVA genes. Sensitivity was independent of androgen status and the Fluva-GAL combination significantly impeded CRPC tumor xenograft growth. By identifying cholesterol-mimetic drugs that inhibit SREBP2 activation upon statin treatment, we provide a potent "one-two punch" against CRPC progression and pave the way for innovative therapeutic strategies to combat additional diseases whose etiology is associated with SREBP2 dysregulation.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Neoplasias da Próstata , Proteína de Ligação a Elemento Regulador de Esterol 2 , Masculino , Humanos , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Animais , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Linhagem Celular Tumoral , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos , Esteróis/farmacologia , Sinergismo Farmacológico , Camundongos Nus , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Morte Celular/efeitos dos fármacosRESUMO
Statins, a family of FDA-approved cholesterol-lowering drugs that inhibit the rate-limiting enzyme of the mevalonate metabolic pathway, have demonstrated anticancer activity. Evidence shows that dipyridamole potentiates statin-induced cancer cell death by blocking a restorative feedback loop triggered by statin treatment. Leveraging this knowledge, we develop an integrative pharmacogenomics pipeline to identify compounds similar to dipyridamole at the level of drug structure, cell sensitivity and molecular perturbation. To overcome the complex polypharmacology of dipyridamole, we focus our pharmacogenomics pipeline on mevalonate pathway genes, which we name mevalonate drug-network fusion (MVA-DNF). We validate top-ranked compounds, nelfinavir and honokiol, and identify that low expression of the canonical epithelial cell marker, E-cadherin, is associated with statin-compound synergy. Analysis of remaining prioritized hits led to the validation of additional compounds, clotrimazole and vemurafenib. Thus, our computational pharmacogenomic approach identifies actionable compounds with pathway-specific activities.
Assuntos
Neoplasias da Mama , Inibidores de Hidroximetilglutaril-CoA Redutases , Humanos , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Ácido Mevalônico/metabolismo , Farmacogenética , Vemurafenib/uso terapêutico , Nelfinavir/uso terapêutico , Clotrimazol/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Caderinas , Colesterol , DipiridamolRESUMO
Aberrant N-glycan Golgi remodeling and metabolism are associated with epithelial-mesenchymal transition (EMT) and metastasis in patients with breast cancer. Despite this association, the N-glycosylation pathway has not been successfully targeted in cancer. Here, we show that inhibition of the mevalonate pathway with fluvastatin, a clinically approved drug, reduces both N-glycosylation and N-glycan-branching, essential components of the EMT program and tumor metastasis. This indicates novel cross-talk between N-glycosylation at the endoplasmic reticulum (ER) and N-glycan remodeling at the Golgi. Consistent with this cooperative model between the two spatially separated levels of protein N-glycosylation, fluvastatin-induced tumor cell death was enhanced by loss of Golgi-associated N-acetylglucosaminyltransferases MGAT1 or MGAT5. In a mouse model of postsurgical metastatic breast cancer, adjuvant fluvastatin treatment reduced metastatic burden and improved overall survival. Collectively, these data support the immediate repurposing of fluvastatin as an adjuvant therapeutic to combat metastatic recurrence in breast cancer by targeting protein N-glycosylation at both the ER and Golgi. SIGNIFICANCE: These findings show that metastatic breast cancer cells depend on the fluvastatin-sensitive mevalonate pathway to support protein N-glycosylation, warranting immediate clinical testing of fluvastatin as an adjuvant therapy for breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Fluvastatina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Ácido Mevalônico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Retículo Endoplasmático/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Feminino , Glicosilação , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Claudins are essential for tight junction formation and paracellular transport, and they affect key cellular events including proliferation and migration. The properties of tight junctions are dynamically modulated by a variety of inputs. We previously showed that the inflammatory cytokine tumor necrosis factor-α (TNFα), a major pathogenic factor in kidney disease, alters epithelial permeability by affecting the expression of claudin-1, -2, and -4 in kidney tubular cells. Here, we explored the effect of TNFα on claudin-3 (Cldn-3), a ubiquitous barrier-forming protein. We found that TNFα elevated Cldn-3 protein expression in tubular epithelial cells (LLC-PK1 and IMCD3) as early as 3 h post treatment. Bafilomycin A and bortezomib, inhibitors of lysosomal and proteasomes, respectively, reduced Cldn-3 degradation. However, TNFα caused a strong upregulation of Cldn-3 in the presence of bafilomycin, suggesting an effect independent from lysosomes. Blocking protein synthesis using cycloheximide prevented Cldn-3 upregulation by TNFα, verifying the contribution of de novo Cldn-3 synthesis. Indeed, TNFα elevated Cldn-3 mRNA levels at early time points. Using pharmacological inhibitors and siRNA-mediated silencing, we determined that the effect of TNFα on Cldn-3 was mediated by extracellular signal regulated kinase (ERK)-dependent activation of NF-κB and PKA-induced activation of CREB1. These two pathways were turned on by TNFα in parallel and both were required for the upregulation of Cldn-3. Because Cldn-3 was suggested to modulate cell migration and epithelial-mesenchymal transition (EMT), and TNFα was shown to affect these processes, Cldn-3 upregulation may modulate regeneration of the tubules following injury.
Assuntos
Claudina-3/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Claudina-3/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Células LLC-PK1 , Masculino , Camundongos , Transdução de Sinais , Suínos , Regulação para CimaRESUMO
Multiple myeloma (MM) is a plasma cell malignancy that is often driven by chromosomal translocations. In particular, patients with t(4;14)-positive disease have worse prognosis compared to other MM subtypes. Herein, we demonstrated that t(4;14)-positive cells are highly dependent on the mevalonate (MVA) pathway for survival. Moreover, we showed that this metabolic vulnerability is immediately actionable, as inhibiting the MVA pathway with a statin preferentially induced apoptosis in t(4;14)-positive cells. In response to statin treatment, t(4;14)-positive cells activated the integrated stress response (ISR), which was augmented by co-treatment with bortezomib, a proteasome inhibitor. We identified that t(4;14)-positive cells depend on the MVA pathway for the synthesis of geranylgeranyl pyrophosphate (GGPP), as exogenous GGPP fully rescued statin-induced ISR activation and apoptosis. Inhibiting protein geranylgeranylation similarly induced the ISR in t(4;14)-positive cells, suggesting that this subtype of MM depends on GGPP, at least in part, for protein geranylgeranylation. Notably, fluvastatin treatment synergized with bortezomib to induce apoptosis in t(4;14)-positive cells and potentiated the anti-tumor activity of bortezomib in vivo. Our data implicate the t(4;14) translocation as a biomarker of statin sensitivity and warrant further clinical evaluation of a statin in combination with bortezomib for the treatment of t(4;14)-positive disease.
Assuntos
Bortezomib/farmacologia , Fluvastatina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Ácido Mevalônico/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Fosfatos de Poli-Isoprenil/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 4 , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Translocação Genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Statins are widely prescribed cholesterol-lowering drugs that inhibit HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate metabolic pathway. Multiple lines of evidence indicate that certain cancers depend on the mevalonate pathway for growth and survival, and, therefore, are vulnerable to statin therapy. However, these immediately available, well-tolerated, and inexpensive drugs have yet to be successfully repurposed and integrated into cancer patient care. In this review, we highlight recent advances and outline important considerations for advancing statins to clinical trials in oncology.
Assuntos
Antineoplásicos/uso terapêutico , Reposicionamento de Medicamentos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Neoplasias/tratamento farmacológico , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Ácido Mevalônico/metabolismo , Medicina de PrecisãoRESUMO
BACKGROUND: Statins inhibit HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Epidemiological and pre-clinical evidence support an association between statin use and delayed prostate cancer (PCa) progression. Here, we evaluated the effects of neoadjuvant fluvastatin treatment on markers of cell proliferation and apoptosis in men with localized PCa. METHODS: Thirty-three men were treated daily with 80 mg fluvastatin for 4-12 weeks in a single-arm window-of-opportunity study between diagnosis of localized PCa and radical prostatectomy (RP) (ClinicalTrials.gov: NCT01992042). Percent Ki67 and cleaved Caspase-3 (CC3)-positive cells in tumor tissues were evaluated in 23 patients by immunohistochemistry before and after treatment. Serum and intraprostatic fluvastatin concentrations were quantified by liquid chromatography-mass spectrometry. RESULTS: Baseline characteristics included a median prostate-specific antigen (PSA) level of 6.48 ng/mL (IQR: 4.21-10.33). The median duration of fluvastatin treatment was 49 days (range: 27-102). Median serum low-density lipoprotein levels decreased by 35% after treatment, indicating patient compliance. Median PSA decreased by 12%, but this was not statistically significant in our small cohort. The mean fluvastatin concentration measured in the serum was 0.2 µM (range: 0.0-1.1 µM), and in prostatic tissue was 8.5 nM (range: 0.0-77.0 nM). At these concentrations, fluvastatin induced PCa cell death in vitro in a dose- and time-dependent manner. In patients, fluvastatin treatment did not significantly alter intratumoral Ki67 positivity; however, a median 2.7-fold increase in CC3 positivity (95% CI: 1.9-5.0, p = 0.007) was observed in post-fluvastatin RP tissues compared with matched pre-treatment biopsy controls. In a subset analysis, this increase in CC3 was more pronounced in men on fluvastatin for >50 days. CONCLUSIONS: Fluvastatin prior to RP achieves measurable drug concentrations in prostatic tissue and is associated with promising effects on tumor cell apoptosis. These data warrant further investigation into the anti-neoplastic effects of statins in prostate tissue.
Assuntos
Fluvastatina/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Idoso , Apoptose , Biomarcadores Tumorais/metabolismo , Caspase 3/metabolismo , Progressão da Doença , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Projetos Piloto , Cuidados Pré-Operatórios , Prostatectomia/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgiaRESUMO
Claudin-2 (Cldn-2) is a channel-forming tight junction (TJ) protein in the proximal tubules that mediates paracellular Na+ transport and has also emerged as a regulator of proliferation and migration. Expression of Cldn-2 is altered by numerous stimuli, but the underlying mechanisms remain incompletely understood. Here we show that Cldn-2 protein and mRNA expression were low in subconfluent tubular cells and increased during junction maturation. Cldn-1 or occludin did not exhibit similar confluence-dependence. Conversely, disruption of TJs by Ca2+ removal or silencing of zonula occludens-1 (ZO-1) or ZO-2 induced a large drop in Cldn-2 abundance. Immunofluorescent staining revealed a more uneven Cldn-2 staining in nascent, Cldn-1-positive TJs. Subconfluence and ZO-1 silencing augmented Cldn-2 degradation and reduced Cldn-2 promoter activity, suggesting that insertion into the TJs slows Cldn-2 turnover. Indeed, blocking endocytosis or lysosomal degradation increased Cldn-2 abundance. Cell confluence increased expression of the junctional adapters ZO-1 and -2, and the small GTPase Rac, and elevated Rac activity and p21-activated kinase (Pak) phosphorylation, suggesting that they might mediate confluence-dependent Cldn-2 regulation. Indeed, Rac silencing or Pak inhibition strongly reduced Cldn-2 protein abundance, which was likely the combined effect on turnover, as these interventions reduced Cldn-2 promoter activity and augmented Cldn-2 degradation. Taken together, our data suggest that TJ integrity and maturity, ZO-1 expression/TJ localization, and Rac/Pak control Cldn-2 degradation and synthesis. A feedback mechanism connecting Cldn-2 expression with junction remodeling, e.g., during wound healing, epithelial-mesenchymal transition, or tumor metastasis formation, may have important downstream effects on permeability, proliferation, and migration.