RESUMEN
Mutational testing for Gastrointestinal Stromal Tumor (GIST) patients remains underutilized. In this retrospective analysis, the target population (n = 1556) reported: 904 had molecular testing ("Tested") vs. 652 without testing ("Untested"). Overall survival (OS) was 14.7 vs. 12.7 years (p < 0.00001), in metastatic patients 1st line OS was 8.9 vs. 5.9 years in the Tested vs. Untested group (n = 416 vs. n = 254), respectively. From 1st - 3rd-line, no difference has been (self-)reported for progression-free survival (PFS). Dropout to/for further lines of treatment was 15% for patients with a Tested mutation vs. 47% in Untested patients.
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Antineoplásicos , Neoplasias Gastrointestinales , Tumores del Estroma Gastrointestinal , Humanos , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Mesilato de Imatinib , Estudios Retrospectivos , Supervivencia sin Enfermedad , Técnicas de Diagnóstico Molecular , Sistema de Registros , Antineoplásicos/uso terapéutico , Neoplasias Gastrointestinales/diagnóstico , Neoplasias Gastrointestinales/genéticaRESUMEN
INTRODUCTION: Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the "Warburg Effect." It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. RESULTS: To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. CONCLUSIONS: Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.
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Neoplasias , Protones , Glucosa/metabolismo , Glucólisis/fisiología , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Neoplasias/metabolismoRESUMEN
Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here, we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype, is a cellular adaption to extracellular acidity. LD marker PLIN2 is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing G-protein-coupled receptor (GPCR) OGR1, which is expressed highly in breast tumors. OGR1 depletion inhibits acid-induced lipid accumulation, while activation by a synthetic agonist triggers LD formation. Inhibition of OGR1 downstream signaling abrogates the lipogenic phenotype, which can be rescued with OGR1 ectopic expression. OGR1-depleted cells show growth inhibition under acidic growth conditions in vitro and tumor formation in vivo. Isotope tracing shows that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1-depleted cells are defective in endoplasmic reticulum stress response and autophagy and hence fail to accumulate LDs affecting survival under acidic stress.
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Lipogénesis , Neoplasias , Ácidos , Autofagia , Humanos , Lípidos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Metabolism is a compartmentalized process, and it is apparent in studying cancer that tumors, like normal tissues, demonstrate metabolic cooperation between different cell types. Metabolic profiling of cells in 2D culture systems often fails to reflect the metabolism occurring within tissues in vivo due to lack of other cell types and 3D interaction. We designed a tooling and methodology to metabolically profile and compare 2D cultures with cancer cell spheroids, and microtissue slices from tumors, and normal organs. We observed differences in the basal metabolism of 2D and 3D cell cultures in response to metabolic inhibitors, and chemotherapeutics. The metabolic profiles of microtissues derived from normal organs (heart, kidney) were relatively consistent when comparing microtissues derived from the same organ. Treatment of heart and kidney microtissues with cardio- or nephro-toxins had early and marked effects on tissue metabolism. In contrast, microtissues derived from different regions of the same tumors exhibited significant metabolic heterogeneity, which correlated to histology. Hence, metabolic profiling of complex microtissues is necessary to understand the effects of metabolic co-operation and how this interaction, not only can be targeted for treatment, but this method can be used as a reproducible, early and sensitive measure of drug toxicity.
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Metabolómica , Neoplasias/metabolismo , Esferoides Celulares/metabolismo , Células A549 , Animales , Técnicas de Cultivo de Célula/métodos , Células HCT116 , Humanos , Ratones , Neoplasias/patología , Esferoides Celulares/patologíaRESUMEN
Genomic instability and high mutation rates cause cancer to acquire numerous mutations and chromosomal alterations during its somatic evolution; most are termed passengers because they do not confer cancer phenotypes. Evolutionary simulations and cancer genomic studies suggest that mildly deleterious passengers accumulate and can collectively slow cancer progression. Clinical data also suggest an association between passenger load and response to therapeutics, yet no causal link between the effects of passengers and cancer progression has been established. To assess this, we introduced increasing passenger loads into human cell lines and immunocompromised mouse models. We found that passengers dramatically reduced proliferative fitness (â¼3% per Mb), slowed tumor growth, and reduced metastatic progression. We developed new genomic measures of damaging passenger load that can accurately predict the fitness costs of passengers in cell lines and in human breast cancers. We conclude that genomic instability and an elevated load of DNA alterations in cancer is a double-edged sword: it accelerates the accumulation of adaptive drivers, but incurs a harmful passenger load that can outweigh driver benefit. The effects of passenger alterations on cancer fitness were unrelated to enhanced immunity, as our tests were performed either in cell culture or in immunocompromised animals. Our findings refute traditional paradigms of passengers as neutral events, suggesting that passenger load reduces the fitness of cancer cells and slows or prevents progression of both primary and metastatic disease. The antitumor effects of chemotherapies can in part be due to the induction of genomic instability and increased passenger load. Cancer Res; 77(18); 4763-72. ©2017 AACR.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Mama/patología , Transformación Celular Neoplásica/patología , Neoplasias Pulmonares/secundario , Mutación , Animales , Mama/metabolismo , Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones SCIDRESUMEN
Ongoing intratumoral evolution is apparent in molecular variations among cancer cells from different regions of the same tumor, but genetic data alone provide little insight into environmental selection forces and cellular phenotypic adaptations that govern the underlying Darwinian dynamics. In three spontaneous murine cancers (prostate cancers in TRAMP and PTEN mice, pancreatic cancer in KPC mice), we identified two subpopulations with distinct niche construction adaptive strategies that remained stable in culture: (i) invasive cells that produce an acidic environment via upregulated aerobic glycolysis; and (ii) noninvasive cells that were angiogenic and metabolically near-normal. Darwinian interactions of these subpopulations were investigated in TRAMP prostate cancers. Computer simulations demonstrated invasive, acid-producing (C2) cells maintain a fitness advantage over noninvasive, angiogenic (C3) cells by promoting invasion and reducing efficacy of immune response. Immunohistochemical analysis of untreated tumors confirmed that C2 cells were invariably more abundant than C3 cells. However, the C2 adaptive strategy phenotype incurred a significant cost due to inefficient energy production (i.e., aerobic glycolysis) and depletion of resources for adaptations to an acidic environment. Mathematical model simulations predicted that small perturbations of the microenvironmental extracellular pH (pHe) could invert the cost/benefit ratio of the C2 strategy and select for C3 cells. In vivo, 200 mmol/L NaHCO3 added to the drinking water of 4-week-old TRAMP mice increased the intraprostatic pHe by 0.2 units and promoted proliferation of noninvasive C3 cells, which remained confined within the ducts so that primary cancer did not develop. A 0.2 pHe increase in established tumors increased the fraction of C3 cells and signficantly diminished growth of primary and metastatic tumors. In an experimental tumor construct, MCF7 and MDA-MB-231 breast cancer cells were coinjected into the mammary fat pad of SCID mice. C2-like MDA-MB-231 cells dominated in untreated animals, but C3-like MCF7 cells were selected and tumor growth slowed when intratumoral pHe was increased. Overall, our data support the use of mathematical modeling of intratumoral Darwinian interactions of environmental selection forces and cancer cell adaptive strategies. These models allow the tumor to be steered into a less invasive pathway through the application of small but selective biological force. Cancer Res; 77(9); 2242-54. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/genética , Evolución Molecular , Neoplasias Pancreáticas/genética , Neoplasias de la Próstata/genética , Selección Genética/genética , Animales , Neoplasias de la Mama/patología , Linaje de la Célula/genética , Proliferación Celular/genética , Simulación por Computador , Femenino , Humanos , Células MCF-7 , Masculino , Ratones , Modelos Teóricos , Fosfohidrolasa PTEN/genética , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/patología , Miembro 25 de Receptores de Factores de Necrosis Tumoral/genética , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TH-302 is a hypoxia-activated prodrug known to activate selectively under the hypoxic conditions commonly found in solid tumors. It is currently being evaluated in clinical trials, including two trials in Pancreatic Ductal Adenocarcinomas (PDAC). The current study was undertaken to evaluate imaging biomarkers for prediction and response monitoring of TH-302 efficacy in xenograft models of PDAC. Dynamic contrast-enhanced (DCE) and diffusion weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor vasculature and cellularity, respectively. Three human PDAC xenografts with known differential responses to TH-302 were imaged prior to, and at 24 h and 48 hours following a single dose of TH-302 or vehicle to determine if imaging changes presaged changes in tumor volumes. DW-MRI was performed at five b-values to generate apparent diffusion coefficient of water (ADC) maps. For DCE-MRI, a standard clinically available contrast reagent, Gd-DTPA, was used to determine blood flow into the tumor region of interest. TH-302 induced a dramatic decrease in the DCE transfer constant (Ktrans) within 48 hours after treatment in the sensitive tumors, Hs766t and Mia PaCa-2, whereas TH-302 had no effect on the perfusion behavior of resistant SU.86.86 tumors. Tumor cellularity, estimated from ADC, was significantly increased 24 and 48 hours after treatment in Hs766t, but was not observed in the Mia PaCa-2 and SU.86.86 groups. Notably, growth inhibition of Hs766t was observed immediately (day 3) following initiation of treatment, but was not observed in MiaPaCa-2 tumors until 8 days after initiation of treatment. Based on these preclinical findings, DCE-MRI measures of vascular perfusion dynamics and ADC measures of cell density are suggested as potential TH-302 response biomarkers in clinical trials.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Hipoxia , Imagen por Resonancia Magnética , Nitroimidazoles , Neoplasias Pancreáticas , Mostazas de Fosforamida , Profármacos , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , Nitroimidazoles/farmacocinética , Nitroimidazoles/farmacología , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Mostazas de Fosforamida/farmacocinética , Mostazas de Fosforamida/farmacología , Profármacos/farmacocinética , Profármacos/farmacología , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC.
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Adenocarcinoma/genética , Adenoma/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenoma/metabolismo , Adenoma/patología , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Detección Precoz del Cáncer , Perfilación de la Expresión Génica , Células HT29 , Humanos , Inmunohistoquímica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genéticaRESUMEN
Cancer immunotherapies, such as immune checkpoint blockade or adoptive T-cell transfer, can lead to durable responses in the clinic, but response rates remain low due to undefined suppression mechanisms. Solid tumors are characterized by a highly acidic microenvironment that might blunt the effectiveness of antitumor immunity. In this study, we directly investigated the effects of tumor acidity on the efficacy of immunotherapy. An acidic pH environment blocked T-cell activation and limited glycolysis in vitro. IFNγ release blocked by acidic pH did not occur at the level of steady-state mRNA, implying that the effect of acidity was posttranslational. Acidification did not affect cytoplasmic pH, suggesting that signals transduced by external acidity were likely mediated by specific acid-sensing receptors, four of which are expressed by T cells. Notably, neutralizing tumor acidity with bicarbonate monotherapy impaired the growth of some cancer types in mice where it was associated with increased T-cell infiltration. Furthermore, combining bicarbonate therapy with anti-CTLA-4, anti-PD1, or adoptive T-cell transfer improved antitumor responses in multiple models, including cures in some subjects. Overall, our findings show how raising intratumoral pH through oral buffers therapy can improve responses to immunotherapy, with the potential for immediate clinical translation.
Asunto(s)
Antineoplásicos/inmunología , Microambiente Tumoral/inmunología , Animales , Anticuerpos/inmunología , Bicarbonatos/farmacología , Antígeno CTLA-4/inmunología , Línea Celular Tumoral , Femenino , Concentración de Iones de Hidrógeno , Inmunoterapia/métodos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Early cancers are avascular and hence, profoundly acidic. Pre-malignant cells must adapt to acidosis to thrive in this hostile microenvironment. Here, we investigate MCF-7 cells that are adapted to grow in acidic conditions using SILAC proteomics and we reveal a significant upregulation of lysosomal proteins. Prominent among these is LAMP2 that functions to protect lysosomal membranes from acid proteolysis. LAMP2 upregulation by acidosis is confirmed both in vitro and in vivo. Furthermore, we show that the depletion of LAMP2 is sufficient to increase acidosis-mediated toxicity. In breast cancer patient samples, there is a high correlation of LAMP2 mRNA and protein expression with progression. We also observe that LAMP2 is located at the plasma membrane in clinical samples and this redistribution is acid-induced in vitro. Our findings suggest a potential adaptive mechanism, wherein cells chronically exposed to an acidic environment translocate lysosomal proteins to their surface, thus protecting the plasmalemma from acid-induced hydrolysis.
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Membrana Celular/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Neoplasias/metabolismo , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Femenino , Humanos , Concentración de Iones de Hidrógeno , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Análisis por Matrices de Proteínas , ProteómicaRESUMEN
The dynamic cancer ecosystem, with its rich temporal and spatial diversity in environmental conditions and heritable cell phenotypes, is remarkably robust to therapeutic perturbations. Even when response to therapy is clinically complete, adaptive tumor strategies almost inevitably emerge and the tumor returns. Although evolution of resistance remains the proximate cause of death in most cancer patients, a recent analysis found that evolutionary terms were included in less than 1% of articles on the cancer treatment outcomes, and this has not changed in 30 years. Here, we review treatment methods that attempt to understand and exploit intratumoral evolution to prolong response to therapy. In general, we find that treating metastatic (i.e., noncurable) cancers using the traditional strategy aimed at killing the maximum number of tumor cells is evolutionarily unsound because, by eliminating all treatment-sensitive cells, it enables rapid proliferation of resistant populations-a well-known evolutionary phenomenon termed "competitive release." Alternative strategies, such as adaptive therapy, "ersatzdroges," and double-bind treatments, shift focus from eliminating tumor cells to evolution-based methods that suppress growth of resistant populations to maintain long-term control.
Asunto(s)
Evolución Molecular , Neoplasias/patología , Neoplasias/terapia , Animales , HumanosRESUMEN
BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.
RESUMEN
Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic, both of which are associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are specifically activated in hypoxic environments to release cytotoxic or cytostatic effectors. TH-302 is a HAP that is currently being evaluated in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the "steal" phenomenon, in which atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. We show that MIA PaCa-2 tumors exhibit a "steal" effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MIA PaCa-2 tumor volume growth after 18 days of treatment. These studies support a combination mechanism of action for TH-302 with a vasodilator that transiently increases tumor hypoxia.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/patología , Antineoplásicos/farmacología , Nitroimidazoles/farmacología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/patología , Mostazas de Fosforamida/farmacología , Profármacos/farmacología , Animales , Antineoplásicos/metabolismo , Circulación Sanguínea/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Hidralazina/farmacología , Concentración de Iones de Hidrógeno , Ratones , Nitroimidazoles/metabolismo , Mostazas de Fosforamida/metabolismo , Profármacos/metabolismo , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (â¼ 550 mm(3)), significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.
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Antineoplásicos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nitroimidazoles/farmacología , Consumo de Oxígeno/efectos de los fármacos , Mostazas de Fosforamida/farmacología , Profármacos/farmacología , Ácido Pirúvico/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Oxígeno/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Many studies have shown that the acidity of solid tumors contributes to local invasion and metastasis. Oral pH buffers can specifically neutralize the acidic pH of tumors and reduce the incidence of local invasion and metastatic formation in multiple murine models. However, this effect is not universal as we have previously observed that metastasis is not inhibited by buffers in some tumor models, regardless of buffer used. B16-F10 (murine melanoma), LL/2 (murine lung) and HCT116 (human colon) tumors are resistant to treatment with lysine buffer therapy, whereas metastasis is potently inhibited by lysine buffers in MDA-MB-231 (human breast) and PC3M (human prostate) tumors. In the current work, we confirmed that sensitive cells utilized a pH-dependent mechanism for successful metastasis supported by a highly glycolytic phenotype that acidifies the local tumor microenvironment resulting in morphological changes. In contrast, buffer-resistant cell lines exhibited a pH-independent metastatic mechanism involving constitutive secretion of matrix degrading proteases without elevated glycolysis. These results have identified two distinct mechanisms of experimental metastasis, one of which is pH-dependent (buffer therapy sensitive cells) and one which is pH-independent (buffer therapy resistant cells). Further characterization of these models has potential for therapeutic benefit.
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Tampones (Química) , Concentración de Iones de Hidrógeno , Neoplasias/patología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Lisina/química , Lisina/farmacología , Melanoma Experimental , Ratones , Metástasis de la Neoplasia , Neoplasias/mortalidad , Neoplasias/terapiaRESUMEN
One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8) affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D) cultures. For these studies, we used 1) 3D reconstituted basement membrane overlay cultures of human carcinomas, 2) live cell imaging assays to assess proteolysis, and 3) in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.
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Neoplasias de la Mama/metabolismo , Catepsina B/metabolismo , Neoplasias del Colon/metabolismo , Animales , Línea Celular Tumoral , Colágeno Tipo IV/metabolismo , Precursores Enzimáticos/metabolismo , Líquido Extracelular/metabolismo , Femenino , Xenoinjertos , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones SCID , Trasplante de Neoplasias , ProteolisisRESUMEN
The pH of solid tumors is acidic due to increased fermentative metabolism and poor perfusion. It has been hypothesized that acid pH promotes local invasive growth and metastasis. The hypothesis that acid mediates invasion proposes that H(+) diffuses from the proximal tumor microenvironment into adjacent normal tissues where it causes tissue remodeling that permits local invasion. In the current work, tumor invasion and peritumoral pH were monitored over time using intravital microscopy. In every case, the peritumoral pH was acidic and heterogeneous and the regions of highest tumor invasion corresponded to areas of lowest pH. Tumor invasion did not occur into regions with normal or near-normal extracellular pH. Immunohistochemical analyses revealed that cells in the invasive edges expressed the glucose transporter-1 and the sodium-hydrogen exchanger-1, both of which were associated with peritumoral acidosis. In support of the functional importance of our findings, oral administration of sodium bicarbonate was sufficient to increase peritumoral pH and inhibit tumor growth and local invasion in a preclinical model, supporting the acid-mediated invasion hypothesis. Cancer Res; 73(5); 1524-35. ©2012 AACR.
Asunto(s)
Concentración de Iones de Hidrógeno , Invasividad Neoplásica , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral , Animales , Medicamentos Herbarios Chinos , Humanos , Ratones , Ratones SCID , Modelos Biológicos , Bicarbonato de Sodio/farmacologíaRESUMEN
Cells maintain intracellular pH (pHi) within a narrow range (7.1-7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the pHi, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs). In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including cofilin and talin regulated actin (de-)polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate, and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H(+)-sensors, suggesting a mechanistic disconnect between intra- and extracellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.
RESUMEN
The observation of aerobic glycolysis by tumor cells in 1924 by Otto Warburg, and subsequent innovation of imaging glucose uptake by tumors in patients with PET-CT, has incited a renewed interest in the altered metabolism of tumors. As tumors grow in situ, a fraction of it is further away from their blood supply, leading to decreased oxygen concentrations (hypoxia), which induces the hypoxia response pathways of HIF1α, mTOR, and UPR. In normal tissues, these responses mitigate hypoxic stress and induce neoangiogenesis. In tumors, these pathways are dysregulated and lead to decreased perfusion and exacerbation of hypoxia as a result of immature and chaotic blood vessels. Hypoxia selects for a glycolytic phenotype and resultant acidification of the tumor microenvironment, facilitated by upregulation of proton transporters. Acidification selects for enhanced metastatic potential and reduced drug efficacy through ion trapping. In this review, we provide a comprehensive summary of preclinical and clinical drugs under development for targeting aerobic glycolysis, acidosis, hypoxia and hypoxia response pathways. Hypoxia and acidosis can be manipulated, providing further therapeutic benefit for cancers that feature these common phenotypes.
Asunto(s)
Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Microambiente Tumoral , Animales , Hipoxia de la Célula , Diagnóstico por Imagen , Glucosa/metabolismo , Humanos , Neoplasias/patologíaRESUMEN
The microenvironment of solid tumors tends to be more acidic (6.5-7.0) than surrounding normal (7.2-7.4) tissue. Chaotic vasculature, oxygen limitation and major metabolic changes all contribute to the acidic microenvironment. We have previously proposed that low extracellular pH (pHe) plays a critical role in the development and progression of solid tumors. While extracellular acidosis is toxic to most normal cells, cancer cells can adapt and survive under this harsh condition. In this study, we focused on identifying survival strategies employed by cancer cells when challenged with an acidic pHe (6.6-6.7) either acutely or for many generations. While acutely acidic cells did not grow, those acclimated over many generations grew at the same rate as control cells. We observed that these cells induce autophagy in response to acidosis both acutely and chronically, and that this adaptation appears to be necessary for survival. Inhibition of autophagy in low pH cultured cells results in cell death. Histological analysis of tumor xenografts reveals a strong correlation of LC3 protein expression in regions projected to be acidic. Furthermore, in vivo buffering experiments using sodium bicarbonate, previously shown to raise extracellular tumor pH, decreases LC3 protein expression in tumor xenografts. These data imply that autophagy can be induced by extracellular acidosis and appears to be chronically employed as a survival adaptation to acidic microenvironments.