RESUMEN
Molecular diagnostics is a rapidly growing branch of the clinical laboratory and has accelerated the advance of personalized medicine in the fields of pharmacogenomics, pharmacogenetics, and nutrigenomics. The versatility of molecular biology allows it to be effective in several medical fields that include reproduction, immunogenetics, and virology. Implementation of molecular and sequencing technology in reproductive medicine can add another layer of understanding to better define the causes behind infertility and recurrent reproductive loss. In the following, we examine current molecular methods for probing factors behind reproductive pregnancy loss including reverse transcription polymerase chain reaction and next generation sequencing (NGS). We review several current and potential genetic (DNA) and transcriptional (RNA)-based parameters in women with infertility that can be significant in diagnosis and treatment. These molecular factors can be inferred either from genomic DNA or RNA locally within the endometrium. Furthermore, we consider infection-based abnormalities such as human herpesvirus-6 and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Finally, we present future directions as well as data demonstrating the potential role of human endogenous retroviruses in pregnancy loss. We hope these discussions will assist the clinician in delineating some of the intricate molecular factors that can contribute to infertility and recurrent reproductive failures.
Asunto(s)
Aborto Espontáneo , COVID-19 , Regulación de la Expresión Génica , Herpesvirus Humano 6 , Infertilidad Femenina , Infecciones por Roseolovirus , SARS-CoV-2 , Aborto Espontáneo/genética , Aborto Espontáneo/metabolismo , Aborto Espontáneo/virología , COVID-19/genética , COVID-19/metabolismo , Endometrio/metabolismo , Endometrio/virología , Femenino , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , Humanos , Infertilidad Femenina/genética , Infertilidad Femenina/metabolismo , Infecciones por Roseolovirus/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. In vitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Inmunidad , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Caspasa 3/metabolismo , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo Condicionados/farmacología , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Desnudos , Clasificación del Tumor , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores Toll-Like/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V-ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer-associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF-κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl-xL and Bcl-2A1) and decreased expression of proapoptotic proteins (Bax, Apaf-1, caspase-3, caspase-6, and caspase-7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF-α and IL-8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer-associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/genética , Neoplasias/metabolismo , Neutrófilos/metabolismo , Microambiente Tumoral , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-8/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Neoplasias/genética , Neutrófilos/patología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Interleukin (IL)-22 is recognized as a tumor-supporting cytokine and is implicated in the proliferation of multiple epithelial cancers. In breast cancer, the current knowledge of IL-22 function is based on cell line models and little is known about how IL-22 affects the tumor initiation, proliferation, invasion, and metastasis in the in vivo system. Here, we investigated the tumor stage-specific function of IL-22 in disease development by evaluating the stage-by-stage progression of breast cancer in an IL-22 knockout spontaneous breast cancer mouse model. We found that among all the stages, IL-22 is specifically upregulated in tumor microenvironment (TME) during the malignant transformation stage of breast tumor progression. The deletion of IL-22 gene leads to the arrest of malignant transition stage, and reduced invasion and tumor burden. Administration of recombinant IL-22 in the TME does not influence in vivo tumor initiation and proliferation but only promotes malignant transformation of cancer cells. Mechanistically, deletion of IL-22 gene causes downregulation of epithelial-to-mesenchymal transition (EMT)-associated transcription factors in breast tumors, suggesting EMT as the mechanism of regulation of malignancy by IL-22. Clinically, in human breast tumor tissues, increased number of IL-22+ cells in the TME is associated with an aggressive phenotype of breast cancer. For the first time, this study provides an insight into the tumor stage-specific function of IL-22 in breast tumorigenesis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Interleucinas/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Microambiente Tumoral/genética , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Interleucinas/administración & dosificación , Interleucinas/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Noqueados , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Estadificación de Neoplasias , Proteínas Recombinantes , Análisis de Matrices Tisulares , Regulación hacia Arriba , Interleucina-22RESUMEN
Ovarian cancer (OVCA) patients often develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase 'a2' isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible factor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. The apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. These cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2-cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy flux. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V-ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer.
RESUMEN
The interaction of recruited immune effector cells and cancer cells within tumor microenvironment (TME) shapes the fate of cancer progression and metastasis. Many cancers including breast cancer, express a specific vacuolar ATPase (a2V) on their cell surface which acidifies the extracellular milieu helping cancer cell proliferation and metastasis. To understand the role of immune cell-associated-a2V during breast tumor pathogenesis, we knocked-out a2V (KO) from the hematopoietic stem cells (HSC) and generated breast tumors in mice. The a2V-KO mice developed faster growing, larger, and metastatic breast tumors compared to control mice. Further investigation of the TME revealed a significant reduction in the presence of CD4+ and CD8+ T cells in the a2V-KO tumors. Targeted RNA-Seq of the cells of the TME demonstrated that pro-inflammatory cytokines, death receptors, death receptor ligands, and cytotoxic effectors were significantly down-regulated within the a2V-KO TME. Interestingly, analysis of immune cells in the blood, spleen, and thymus of the non-tumor bearing a2V-KO mice revealed a significant decrease in CD4+ and CD8+ T cell populations. For the first time, this study demonstrates that inhibition of V-ATPase expression in HSC leads to a decrease in CD4+ and CD8+ T cell populations and thus promotes breast tumor growth and metastasis.
RESUMEN
The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.
Asunto(s)
Transducción de Señal , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Biomarcadores , Susceptibilidad a Enfermedades , Endosomas/metabolismo , Espacio Extracelular/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Lisosomas/metabolismo , Transducción de Señal/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Vacuolas/metabolismoRESUMEN
Preterm birth is widespread and causes 35% of all neonatal deaths. Infants who survive face potential long-term complications. A major contributing factor of preterm birth is infection. We investigated the role of interleukin 22 (IL22) as a potential clinically relevant cytokine during gestational infection. IL22 is an effector molecule secreted by immune cells. While the expression of IL22 was reported in normal nonpregnant endometrium and early pregnancy decidua, little is known about uterine IL22 expression during mid or late gestational stages of pregnancy. Since IL22 has been shown to be an essential mediator in epithelial regeneration and wound repair, we investigated the potential role of IL22 during defense against an inflammatory response at the maternal-fetal interface. We used a well-established model to study infection and infection-associated inflammation during preterm birth in the mouse. We have shown that IL22 is upregulated to respond to an intrauterine lipopolysaccharide administration and plays an important role in controlling the risk of inflammation-induced preterm birth. This paper proposes IL22 as a treatment method to combat infection and prevent preterm birth in susceptible patients.
Asunto(s)
Interleucinas/metabolismo , Lipopolisacáridos/farmacología , Trabajo de Parto Prematuro/metabolismo , Trabajo de Parto Prematuro/prevención & control , Regulación hacia Arriba/fisiología , Útero/metabolismo , Animales , Caspasas/metabolismo , Proteína Ligando Fas/metabolismo , Femenino , Interleucinas/genética , Ratones , Trabajo de Parto Prematuro/inducido químicamente , Embarazo , Regulación hacia Arriba/efectos de los fármacos , Útero/efectos de los fármacos , Interleucina-22RESUMEN
Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides initial congenial environment for primary tumor formation, is partly understood. Here, we report a novel role of epithelial cell-associated vacuolar ATPase 'a2' isoform (a2V) in regulating breast tissue ECM stiffness to control metastasis. Using a mammary gland-specific a2V-knockout model, we show that in the absence of a2V, breast tumors exhibit atypically soft tumor phenotype, less tumor rigidity, and necrotic tumor microenvironment. These tumors contain a decreased number of cancer cells at primary tumor site, but showed extensive metastases compared to control. Nanomechanical evaluation of normal breast tissues revealed a decrease in stiffness and collagen content in ECM of a2V-deleted breast tissues. Mechanistically, inhibition of a2V expression caused dispersed Golgi morphology with relocation of glycosyltransferase enzymes to early endosomes in mammary epithelial cells. This resulted in defective glycosylation of ECM proteins and production of compromised ECM that further influenced tumor metastasis. Clinically, in patients with cancer, low a2V expression levels in normal breast tissue correlated with lymph node metastasis. Thus, using a new knockout mouse model, we have identified a2V expression in epithelial cells as a key requirement for proper ECM formation in breast tissue and its expression levels can significantly modulate breast tumor dissemination. Evaluation of a2V expression in normal breast tissues can help in identifying patients with high risk of developing metastases.
Asunto(s)
Matriz Extracelular/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , ATPasas de Translocación de Protón/metabolismo , Animales , Línea Celular Tumoral , Epitelio , Femenino , Glicosilación , Humanos , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , ATPasas de Translocación de Protón/genéticaRESUMEN
In ovarian cancer (OVCA), treatment failure due to chemo-resistance is a serious challenge. It is therefore critical to identify new therapies that are effective against resistant tumors and have reduced side effects. We recently identified 4-H-chromenes as tubulin depolymerizing agents that bind to colchicine site of beta-tubulin. Here, we screened a chemical library of substituted 4-H-chromenes and identified SP-6-27 to exhibit most potent anti-proliferative activity towards a panel of human cisplatin sensitive and resistant OVCA cell lines with 50% inhibitory concentration (IC50; mean ± SD) ranging from 0.10 ± 0.01 to 0.84 ± 0.20 µM. SP-6-27 exhibited minimum cytotoxicity to normal ovarian epithelia. A pronounced decrease in microtubule density as well as G2/M cell cycle arrest was observed in SP-6-27 treated cisplatin sensitive/resistant OVCA cells. The molecular mechanism of SP-6-27 induced cell death revealed modulation in cell-cycle regulation by upregulation of growth arrest and DNA damage inducible alpha transcripts (GADD45). An enhanced intrinsic apoptosis was observed in OVCA cells through upregulation of Bax, Apaf-1, caspase-6, -9, and caspase-3. In vitro wound healing assay revealed reduced OVCA cell migration upon SP-6-27 treatment. Additionally, SP-6-27 and cisplatin combinatorial treatment showed enhanced cytotoxicity in chemo-sensitive/resistant OVCA cells. Besides effect on cancer cells, SP-6-27 further restrained angiogenesis by inhibiting capillary tube formation by human umbilical vein endothelial cells (HUVEC). Together, these findings show that the chromene analog SP-6-27 is a novel chemotherapeutic agent that offers important advantages for the treatment of ovarian cancer.
RESUMEN
The vacuolar (Hâº)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I-IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a-e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 µM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.
Asunto(s)
Antineoplásicos/química , Bisbenzimidazol/análogos & derivados , Bisbenzimidazol/química , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Antineoplásicos/farmacología , Bisbenzimidazol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
While many investigators have described the biochemical and physiological similarities between tumor cells and trophoblast cells, in this discourse we will compare primarily their leucocytes, which constitute a large portion of the tumor and its microenvironment as well as the placenta and its microenvironment. There is a remarkable similarity between the cells that support placental growth and development and tumor growth and development. In many cases over half of the cells present in the tumor and the placenta are non-tumor or nontrophoblast cells, immune cells. Most of these immune cells are prevented from attacking the fetal derived placental cells and the self-derived tumor cells. Nevertheless, these leucocytes, in our opinion, are very active and support tumor and placental cell growth through the production of growth factors and angiogenic factors. These cells do this by activating the portion of the immune response which initiates and helps control tissue repair.
Asunto(s)
Placenta/inmunología , Complicaciones Neoplásicas del Embarazo/inmunología , Animales , Procesos de Crecimiento Celular/inmunología , Femenino , Humanos , Embarazo , Complicaciones Neoplásicas del Embarazo/patología , Microambiente Tumoral/inmunologíaRESUMEN
Neutrophils play significant regulatory roles within the tumor microenvironment by directly promoting tumor progression that leads to poor clinical outcomes. Identifying the tumor associated molecules that regulate neutrophil infiltration into tumors may provide new and specific therapeutic targets for cancer treatment. The a2-isoform of vacuolar ATPase (a2V) is uniquely and highly expressed on cancer cell plasma membrane. Cancer cells secrete a peptide from a2V (a2NTD) that promotes the pro-tumorigenic properties of neutrophils. This provides a2V the propensity to control neutrophil migration. Here, we report that the treatment of human neutrophils with recombinant a2NTD leads to neutrophil adherence and polarization. Moreover, a2NTD treatment activates surface adhesion receptors, as well as FAK and Src kinases that are essential regulators of the migration process in neutrophils. Functional analysis reveals that a2NTD can act as a chemo-attractant and promotes neutrophil migration. In addition, a2Neuɸ secrete high levels of IL-8 via NF-κB pathway activation. Confirmatory assays demonstrate that the promoted migration of a2Neuɸ was dependent on the autocrine secretion of IL-8 from a2Neuɸ. These findings demonstrate for the first time the direct regulatory role of cancer associated a2-isoform V-ATPase on neutrophil migration, suggesting a2V as a potential target for cancer therapy.
Asunto(s)
Neoplasias de la Mama/inmunología , Interleucina-8/metabolismo , Neutrófilos/citología , Péptidos/farmacología , ATPasas de Translocación de Protón/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neutrófilos/efectos de los fármacos , ATPasas de Translocación de Protón/química , Microambiente Tumoral , Familia-src Quinasas/metabolismoRESUMEN
Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of 'a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker ß-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-ß (transforming growth factor-ß) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-ß pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-ß are crucial for cell survival.
Asunto(s)
Glándulas Mamarias Animales/embriología , Glándulas Mamarias Animales/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Epitelio/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/metabolismo , Lactancia , Ratones Noqueados , Modelos Biológicos , Morfogénesis , ARN Interferente Pequeño/metabolismoRESUMEN
Nearly 65 years have passed since Peter Medawar posed the following question: "How does the pregnant mother contrive to nourish within itself, for many weeks or months, a fetus that is an antigenically foreign body." Now, understanding of reproductive immunology has demonstrated that the HLA antigens in the placenta are non-classical and do not induce rejection. In the placenta and in tumors, 50% or more of the cells are cells of the immune system and were once thought to be primed and ready for killing tumors or the "fetal transplant" but these cells are not potential killers but abet the growth of either the tumor or the placenta. We believe that these cells are there to create an environment, which enhances either placental or tumor growth. By examining the similarities of the placenta's and tumor's immune cells, novel mechanisms to cause tumors to be eliminated can be devised.
Asunto(s)
Intercambio Materno-Fetal/inmunología , Modelos Inmunológicos , Neoplasias/inmunología , Placenta/inmunología , Animales , Femenino , Humanos , EmbarazoRESUMEN
Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its 'a2' isoform (V-ATPase-V0a2) is a component of functional plasma-membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the 'a' subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Ovario/efectos de los fármacos , ATPasas de Translocación de Protón/genética , Carboplatino/farmacología , Línea Celular Tumoral , Aductos de ADN/genética , Daño del ADN/efectos de los fármacos , Femenino , Humanos , Neoplasias Ováricas/patología , Ovario/metabolismo , Ovario/patología , ATPasas de Translocación de Protón/análisis , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Notch signaling pathways exert effects throughout pregnancy and are activated in response to TLR ligands. To investigate the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligand Delta-like protein-1, transcriptional repressor hairy and enhancer of split-1, and Notch deregulator Numb were assessed. Preterm labor was initiated on gestation d 14.5 by 1 of 2 methods: 1) inflammation-induced preterm labor: intrauterine injection of LPS (a TLR4 agonist) and 2) hormonally induced preterm labor: subcutaneous injection of mifepristone. Delta-like protein-1, Notch1, and hairy and enhancer of split-1 were elevated significantly, and Numb was decreased in the uterus and placenta of inflammation-induced preterm labor mice but remained unchanged in hormonally induced preterm labor compared with their respective controls. F4/80(+) macrophage polarization was skewed in the uterus of inflammation-induced preterm labor toward M1-positive (CD11c(+)) and double-positive [CD11c(+) (M1) and CD206(+) (M2)] cells. This process is dependent on activation of Notch signaling, as shown by suppression of M1 and M2 macrophage-associated cytokines in decidual macrophages in response to γ-secretase inhibitor (an inhibitor of Notch receptor processing) treatment ex vivo. γ-Secretase inhibitor treatment also diminished the LPS-induced secretion of proinflammatory cytokines and chemokines in decidual and placental cells cultured ex vivo. Furthermore, treatment with recombinant Delta-like protein-1 ligand enhanced the LPS-induced proinflammatory response. Notch ligands (Jagged 1 and 2 and Delta-like protein-4) and vascular endothelial growth factor and its receptor involved in angiogenesis were reduced significantly in the uterus and placenta during inflammation-induced preterm labor. These results suggest that up-regulation of Notch-related inflammation and down-regulation of angiogenesis factors may be associated with inflammation-induced preterm labor but not with hormonally induced preterm labor.
Asunto(s)
Decidua/patología , Inflamación/patología , Lipopolisacáridos/toxicidad , Macrófagos/patología , Trabajo de Parto Prematuro/patología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Decidua/efectos de los fármacos , Decidua/metabolismo , Femenino , Inflamación/inducido químicamente , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Trabajo de Parto Prematuro/inducido químicamente , Trabajo de Parto Prematuro/metabolismo , EmbarazoRESUMEN
In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy.
Asunto(s)
Neoplasias de la Mama/enzimología , Neutrófilos/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Isoenzimas , Células MCF-7 , Neovascularización Patológica/enzimología , Neovascularización Patológica/inmunología , Neutrófilos/inmunología , Microambiente TumoralRESUMEN
Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1ß) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.
Asunto(s)
Inflamación , Receptores Notch/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Fluorescente , Trabajo de Parto Prematuro , Peptidoglicano/farmacología , Placenta/efectos de los fármacos , Placenta/metabolismo , Poli I-C , Polinucleótidos/farmacología , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Factor de Transcripción HES-1 , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer with poor prognosis for which no targeted therapies are currently available. Notch signaling has been implicated in breast cancer but the factors that control Notch in TNBC are unknown. Because the Vacuolar ATPase has been shown to be important in breast cancer invasiveness, we investigated the role of a2-subunit isoform of Vacuolar ATPase (a2V) in regulating Notch signaling in TNBC. Confocal microscopy revealed that among all the 'a' subunit isoforms, a2V was uniquely expressed on the plasma membrane of breast cancer cells. Both a2V and NOTCH1 were elevated in TNBC tumors tissues and cell lines. a2V knockdown by siRNA as well as V-ATPase inhibition by Bafilomycin A1 (Baf A1) in TNBC cell lines enhanced Notch signaling by increasing the expression of Notch1 intracellular Domain (N1ICD). V-ATPase inhibition blocked NICD degradation by disrupting autophagy and lysosomal acidification as demonstrated by accumulation of LC3B and diminished expression of LAMP1 respectively. Importantly, treatment with Baf A1 or anti-a2V, a novel-neutralizing antibody against a2V hindered cell migration of TNBC cells. Our findings indicate that a2V regulates Notch signaling through its role in endolysosomal acidification and emerges as a potential target for TNBC.