RESUMEN
(1) Background: Pathologic necrosis of soft tissue sarcomas (STS) has been used to determine treatment response, but its relationship to neoadjuvant treatments remains indeterminate. In this retrospective, single institution study, we hypothesized that neoadjuvant chemoradiation (NA-CRT) yields higher rates of pathologic complete response (pCR) than neoadjuvant radiation (NA-XRT) or chemotherapy (NA-CT) alone. (2) Methods: Patients with extremity STS between 2011-2020 who received neoadjuvant treatment were included. pCR was defined as percent necrosis of the surgical specimen greater than or equal to 90%. (3) Results: 79 patients were analyzed. 51.9% of the population were male with a mean age of 58.4 years. 49.4% identified as Non-Hispanic White. Twenty-six (32.9%) patients achieved pCR while 53 (67.1%) did not. NA-CT (OR 15.82, 95% CI = 2.58-96.9, p = 0.003 in univariate (UVA) and OR 24.7, 95% CI = 2.88-211.2, p = 0.003 in multivariate (MVA), respectively) and NA-XRT (OR 5.73, 95% CI = 1.51-21.8, p = 0.010 in UVA and OR 7.95, 95% CI = 1.87-33.7, p = 0.005 in MVA, respectively) was significantly associated with non- pCR when compared to NA-CRT. The analysis also demonstrated that grade 3 tumors, when using grade 2 as reference, also had significantly higher odds of achieving pCR (OR 0.23, 95% CI = 0.06-0.80, p = 0.022 in UVA and OR 0.16, 95% CI = 0.04-0.70, p = 0.015 in MVA, respectively). (4) Conclusion: NA-CRT yields superior pCR compared to other neoadjuvant regimens. This extends to higher grade tumors.
RESUMEN
Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alaninerich Ckinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rhoassociated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracyclineinducible system in PTENnull U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the nonphosphorylatable ED mutant exerted growthsuppressive and radiationsensitizing effects, while the pseudophosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS proteinprotein interactions using coimmunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKSassociated proteins, including importinß and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Dominios Proteicos , Tolerancia a Radiación , Animales , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Femenino , Glioblastoma/mortalidad , Glioblastoma/radioterapia , Humanos , Autoantígeno Ku/metabolismo , Ratones , Ratones Desnudos , Fosforilación , Mapeo de Interacción de Proteínas , Análisis de Supervivencia , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , beta Carioferinas/metabolismoRESUMEN
Lung cancer is the leading cause of cancer-associated mortality in the United States. Kinase hyperactivation is a known mechanism of tumorigenesis. The phosphorylation status of the plasma membrane-associated protein myristoylated alanine rich C-kinase substrate (MARCKS) effector domain (ED) was previously established as being important in the sensitivity of lung cancer to radiation. Specifically, when MARCKS ED was in a non-phosphorylated state, lung cancer cells were more susceptible to ionizing radiation and experienced prolonged double-strand DNA breaks. Additional studies demonstrated that the phosphorylation status of MARCKS ED is important for gene expression and in vivo tumor growth. The present study used a peptide mimetic of MARCKS ED as a therapeutic intervention to modulate MARCKS phosphorylation. Culturing A549, H1792 and H1975 lung cancer cell lines with the MARCKS ED peptide led to reduced levels of phosphorylated MARCKS and phosphorylated Akt serine/threonine kinase 1. Further investigation demonstrated that the peptide therapy was able to reduce lung cancer cell proliferation and increase radiation sensitivity. In addition, the MARCKS peptide therapy was able to prolong double-strand DNA breaks following ionizing radiation exposure. The results of the present study demonstrate that a peptide mimetic of MARCKS ED is able to modulate MARCKS phosphorylation, leading to an increase in sensitivity to radiation.
RESUMEN
Translocation to the nucleus of diacylglycerol kinase (DGK)- ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.
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Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Señales de Localización Nuclear/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Transporte Biológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Señales de Localización Nuclear/química , Estructura Terciaria de Proteína , Células Tumorales CultivadasRESUMEN
UNLABELLED: Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of receptor activator of NF-κB ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remain the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL, is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to TNF-related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Toward uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL, and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage. IMPLICATIONS: OPG variants were developed that lack TRAIL binding, yet retain RANKL binding and suggest new possibilities for therapeutic targeting in osteolytic malignancies.
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Neoplasias Óseas/metabolismo , Remodelación Ósea , Osteoprotegerina/metabolismo , Neoplasias de la Próstata/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Apoptosis/fisiología , Neoplasias Óseas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Masculino , Ratones , Ratones Desnudos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteólisis , Neoplasias de la Próstata/patología , Transducción de SeñalRESUMEN
Lung cancer is the leading cause of cancer related deaths. Common molecular drivers of lung cancer are mutations in receptor tyrosine kinases (RTKs) leading to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pro-growth, pro-survival signaling pathways. Myristoylated alanine rich C-kinase substrate (MARCKS) is a protein that has the ability to mitigate this signaling cascade by sequestering the target of PI3K, phosphatidylinositol (4,5)-bisphosphate (PIP2). As such, MARCKS has been implicated as a tumor suppressor, though there is some evidence that MARCKS may be tumor promoting in certain cancer types. Since the MARCKS function depends on its phosphorylation status, which impacts its subcellular location, MARCKS role in cancer may depend highly on the signaling context. Currently, the importance of MARCKS in lung cancer biology is limited. Thus, we investigated MARCKS in both clinical specimens and cell culture models. Immunohistochemistry scoring of MARCKS protein expression in a diverse lung tumor tissue array revealed that the majority of squamous cell carcinomas stained positive for MARCKS while other histologies, such as adenocarcinomas, had lower levels. To study the importance of MARCKS in lung cancer biology, we used inducible overexpression of wild-type (WT) and non-phosphorylatable (NP)-MARCKS in A549 lung cancer cells that had a low level of endogenous MARCKS. We found that NP-MARCKS expression, but not WT-MARCKS, enhanced the radiosensitivity of A549 cells in part by inhibiting DNA repair as evidenced by prolonged radiation-induced DNA double strand breaks. We confirmed the importance of MARCKS phosphorylation status by treating several lung cancer cell lines with a peptide mimetic of the phosphorylation domain, the effector domain (ED), which effectively attenuated cell growth as measured by cell index. Thus, the MARCKS ED appears to be an important target for lung cancer therapeutic development.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Proteínas de la Membrana/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Secuencia de Aminoácidos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/efectos de la radiación , Reparación del ADN/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Imitación Molecular , Datos de Secuencia Molecular , Terapia Molecular Dirigida/métodos , Mutación , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Fosforilación , Estructura Terciaria de Proteína , Tolerancia a Radiación , Radiación Ionizante , Análisis de Matrices TisularesRESUMEN
Kinase inhibitors are among the fastest growing class of anti-cancer therapies. One family of kinases that has recently gained attention as a target for treating malignant disorders is the Tec kinase family. Evidence has been published that one member of this family; the Bmx kinase, may play a role in the pathogenesis of glioblastoma, prostate, breast and lung cancer. Bmx has also shown potential as an anti-vascular therapy in combination with radiation or as a sensitizer to chemotherapeutic agents. Therefore, several companies such as Pharmacyclics, Avila Therapeutics, Merck and Co., Metaproteomics, IRM, and Moerae Matrix have developed compounds or peptides that function as Bmx kinase inhibitors. These companies have subsequently been issued patents for these inhibitors. Additionally, it has been shown that current clinical stage EGFR inhibitors can irreversibly inhibit Bmx, suggesting these compounds might be rapidly moved to clinical trials for other malignancies. This review will discuss current patents issued since 2009 that contain data specifically on inhibition of the Bmx kinase, and will also discuss the scientific literature that suggests their potential application as therapeutics in the treatment of the aforementioned malignancies.
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Antineoplásicos/uso terapéutico , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Humanos , Terapia Molecular Dirigida , Neoplasias/enzimología , Neoplasias/patología , Patentes como Asunto , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Ionizing radiation treatment is used in over half of all cancer patients, thus determining the mechanisms of response or resistance is critical for the development of novel treatment approaches. MATERIALS AND METHODS: In this report, we utilize a high-content peptide array platform that performs multiplex kinase assays with real-time kinetic readout to investigate the mechanism of radiation response in vascular endothelial cells. We applied this technology to irradiated human umbilical vein endothelial cells (HUVEC). RESULTS: We identified 49 specific tyrosine phosphopeptides that were differentially affected by irradiation over a time course of 1h. In one example, the Tropomyosin receptor kinase (Trk) family members, TrkA and TrkB, showed transient activation between 2 and 15 min following irradiation. When we targeted TrkA and TrkB using small molecule inhibitors, HUVEC were protected from radiation damage. Conversely, stimulation of TrkA using gambogic amide promoted radiation enhancement. CONCLUSIONS: Thus, we show that our approach not only can identify rapid changes in kinase activity but also identify novel targets such as TrkA. TrkA inhibition resulted in radioprotection that correlated with enhanced repair of radiation-induced damage while TrkA stimulation by gambogic amide produced radiation sensitization.
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Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Tolerancia a Radiación , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Ciclo Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Reparación del ADN/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Fosforilación , Análisis por Matrices de Proteínas , Proteínas Quinasas/metabolismo , Dosis de Radiación , Tolerancia a Radiación/efectos de los fármacos , Receptor trkA/agonistas , Xantonas/farmacologíaRESUMEN
PURPOSE: This study assessed whether myristoylated alanine-rich C-kinase substrate (MARCKS) can regulate glioblastoma multiforme (GBM) growth, radiation sensitivity, and clinical outcome. EXPERIMENTAL DESIGN: MARCKS protein levels were analyzed in five GBM explant cell lines and eight patient-derived xenograft tumors by immunoblot, and these levels were correlated to proliferation rates and intracranial growth rates, respectively. Manipulation of MARCKS protein levels was assessed by lentiviral-mediated short hairpin RNA knockdown in the U251 cell line and MARCKS overexpression in the U87 cell line. The effect of manipulation of MARCKS on proliferation, radiation sensitivity, and senescence was assessed. MARCKS gene expression was correlated with survival outcomes in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) Database and The Cancer Genome Atlas (TCGA). RESULTS: MARCKS protein expression was inversely correlated with GBM proliferation and intracranial xenograft growth rates. Genetic silencing of MARCKS promoted GBM proliferation and radiation resistance, whereas MARCKS overexpression greatly reduced GBM growth potential and induced senescence. We found MARCKS gene expression to be directly correlated with survival in both the REMBRANDT and TCGA databases. Specifically, patients with high MARCKS expressing tumors of the proneural molecular subtype had significantly increased survival rates. This effect was most pronounced in tumors with unmethylated O(6)-methylguanine DNA methyltransferase (MGMT) promoters, a traditionally poor prognostic factor. CONCLUSIONS: MARCKS levels impact GBM growth and radiation sensitivity. High MARCKS expressing GBM tumors are associated with improved survival, particularly with unmethylated MGMT promoters. These findings suggest the use of MARCKS as a novel target and biomarker for prognosis in the proneural subtype of GBM.
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Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Técnicas de Silenciamiento del Gen , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Trasplante de Neoplasias , Pronóstico , Tolerancia a Radiación , Trasplante HeterólogoRESUMEN
Glioblastoma multiforme is the most common primary malignant brain tumor and despite treatment with surgery, radiation, and chemotherapy, the median survival of patients with glioblastoma multiforme is approximately 1 year. Glioblastoma multiforme explants and cell lines have been reported to overexpress the interleukin-13 receptor alpha2 subunit (IL13Ralpha2) relative to nonneoplastic brain. Based on this finding, a recombinant cytotoxin composed of IL13 ligand and a truncated form of Pseudomonas aeruginosa exotoxin A (IL13-PE38QQR) was developed for the targeted treatment of glioblastoma multiforme tumors. In a recently completed phase III clinical trial, however, IL13-PE38QQR was found to be no more effective than an existing therapy in prolonging survival. To determine possible explanations for this result, we analyzed the relative expression levels of IL13Ralpha2 in glioblastoma multiforme and nonneoplastic brain specimens using publicly available oligonucleotide microarray databases, quantitative real-time reverse transcription PCR, and immunohistochemical staining. Increased expression of the IL13Ralpha2 gene relative to nonneoplastic brain was observed in 36 of 81 (44%) and 8 of 17 (47%) tumor specimens by microarray and quantitative real-time reverse transcription PCR analyses, respectively. Immunohistochemical staining of tumor specimens showed highly variable expression of IL13Ralpha2 protein both within and across specimens. These data indicate that prescreening of subjects may be of benefit in future trials of IL13Ralpha2 targeting therapies.