RESUMEN
When delivering cells on a scaffold to treat a bone defect, the cell seeding technique determines the number and distribution of cells within a scaffold, however the optimal technique has not been established. This study investigated if human adipose-derived stem cells (ASCs) transduced with a lentiviral vector to overexpress bone morphogenetic protein 2 (BMP-2) and loaded on a scaffold using dynamic orbital shaker could reduce the total cell dose required to heal a critical sized bone defect when compared with static seeding. Human ASCs were loaded onto a collagen/biphasic ceramic scaffold using static loading and dynamic orbital shaker techniques, compared with our labs standard loading technique, and implanted into femoral defects of nude rats. Both a low dose and standard dose of transduced cells were evaluated. Outcomes investigated included BMP-2 production, radiographic healing, micro-computerized tomography, histologic assessment, and biomechanical torsional testing. BMP-2 production was higher in the orbital shaker cohort compared with the static seeding cohort. No statistically significant differences were noted in radiographic, histomorphometric, and biomechanical outcomes between the low-dose static and dynamic seeding groups, however the standard-dose static seeding cohort had superior biomechanical properties. The standard-dose 5 million cell dose standard loading cohort had superior maximum torque and torsional stiffness on biomechanical testing. The use of orbital shaker technique was labor intensive and did not provide equivalent biomechanical results with the use of fewer cells.
Asunto(s)
Proteína Morfogenética Ósea 2 , Regeneración Ósea , Terapia Genética , Ratas Desnudas , Andamios del Tejido , Animales , Humanos , Proteína Morfogenética Ósea 2/genética , Andamios del Tejido/química , Ratas , Terapia Genética/métodos , Células Madre/citología , Células Madre/metabolismo , Tejido Adiposo/citología , Masculino , Fémur/lesiones , Fémur/diagnóstico por imagen , Fémur/patología , Microtomografía por Rayos X , Fenómenos BiomecánicosRESUMEN
Ex-vivo gene therapy has been shown to be an effective method for treating bone defects in pre-clinical models. As gene therapy is explored as a potential treatment option in humans, an assessment of the safety profile becomes an important next step. The purpose of this study was to evaluate the biodistribution of viral particles at the defect site and various internal organs in a rat femoral defect model after implantation of human ASCs transduced with lentivirus (LV) with two-step transcriptional activation (TSTA) of bone morphogenetic protein-2 (LV-TSTA-BMP-2). Animals were sacrificed at 4-, 14-, 56-, and 84-days post implantation. The defects were treated with either a standard dose (SD) of 5 million cells or a high dose (HD) of 15 million cells to simulate a supratherapeutic dose. Treatment groups included (1) SD LV-TSTA-BMP-2 (2) HD LV-TSTA-BMP-2, (3) SD LV-TSTA-GFP (4) HD LV-TSTA-GFP and (5) SD nontransduced cells. The viral load at the defect site and ten organs was assessed at each timepoint. Histology of all organs, ipsilateral tibia, and femur were evaluated at each timepoint. There were nearly undetectable levels of LV-TSTA-BMP-2 transduced cells at the defect site at 84-days and no pathologic changes in any organ at all timepoints. In conclusion, human ASCs transduced with a lentiviral vector were both safe and effective in treating critical size bone defects in a pre-clinical model. These results suggest that regional gene therapy using lentiviral vector to treat bone defects has the potential to be a safe and effective treatment in humans.
Asunto(s)
Proteína Morfogenética Ósea 2 , Lentivirus , Ratas , Humanos , Animales , Distribución Tisular , Lentivirus/genética , Lentivirus/metabolismo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Terapia Genética/métodos , Células Madre/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It disproportionately affects BRCA mutation carriers and young women, especially African American (AA) women. Chemoresistant TNBC is a heterogeneous and molecularly unstable disease that challenges our ability to apply personalized therapies. With the approval of immune checkpoint blockade (ICB) for TNBC, the addition of pembrolizumab to systemic chemotherapy has become standard of care (SOC) in neoadjuvant systemic therapy (NST) for high-risk early-stage TNBC. Pembrolizumab plus chemotherapy significantly increased the pathologic complete response (pCR) and improved event-free survival in TNBC. However, clinical uncertainties remain because similarly treated TNBC partial responders with comparable tumor responses to neoadjuvant therapy often experience disparate clinical outcomes. Current methods fall short in accurately predicting which high-risk patients will develop chemo-resistance and tumor relapse. Therefore, novel treatment strategies and innovative new research initiatives are needed. We propose that the EGFR-K-RAS-SIAH pathway activation is a major tumor driver in chemoresistant TNBC. Persistent high expression of SIAH in residual tumors following NACT/NST reflects that the EGFR/K-RAS pathway remains activated (ON), indicating an ineffective response to treatment. These chemoresistant tumor clones persist in expressing SIAH (SIAHHigh/ON) and are linked to early tumor relapse and poorer prognosis. Conversely, the loss of SIAH expression (SIAHLow/OFF) in residual tumors post-NACT/NST reflects EGFR/K-RAS pathway inactivation (OFF), indicating effective therapy and chemo-sensitive tumor cells. SIAHLow/OFF signal is linked to tumor remission and better prognosis post-NACT/NST. Therefore, SIAH is well-positioned to become a novel tumor-specific, therapy-responsive, and prognostic biomarker. Potentially, this new biomarker (SIAHHigh/ON) could be used to quantify therapy response, predict chemo-resistance, and identify those patients at the highest risk for tumor relapse and poor survival in TNBC.
RESUMEN
BACKGROUND: Previous reports suggest that intact SMAD4 expression is associated with a locally aggressive pancreas cancer phenotype. The objectives of this work were to determine the frequency of intact SMAD4 and its association with patterns of recurrence in patients with upfront resected pancreas cancer receiving adjuvant therapy. METHODS: A tissue microarray was constructed using resected specimens from patients who underwent upfront surgery and adjuvant gemcitabine with no neoadjuvant treatment for pancreas cancer. SMAD4 expression was determined by immunohistochemical staining. Associations of SMAD4 expression and clinicopathologic parameters with clinical outcomes were evaluated using Cox proportional hazard models. RESULTS: One hundred twenty-seven patients were included with a median follow up of 5.7 years. Most patients had stage ≥ pT3 tumors (75%) and pN1 (68%). All patients received adjuvant gemcitabine, and 79% of patients received adjuvant chemoradiotherapy. Ten (8%) patients had intact SMAD4 expression. Grade was the only clinicopathologic parameter statistically associated with SMAD4 expression (P=0.05). Median overall survival was 2.1 years. On univariate analysis, SMAD4 expression was associated with increased locoregional recurrence (hazard ratio 7.0, P<0.01, 95% confidence interval: 2.8-18.0) but not distant recurrence (P=0.06) or overall survival (P=0.73). On multivariable analysis, SMAD4 expression (hazard ratio 9.6, P<0.01, 95% confidence interval: 3.7-24.8) and adjuvant chemoradiotherapy (hazard ratio 0.3, P=0.01, 95% confidence interval: 0.1-0.8) were associated with higher and lower locoregional recurrence, respectively. CONCLUSIONS: In patients with upfront resected pancreas cancer, SMAD4 expression was associated with an increased risk of locoregional recurrence. Prospective evaluation of the frequency of SMAD4 expression and validation of its predictive utility is warranted.
RESUMEN
Triple-negative breast cancer (TNBC), characterized by the absence or low expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2), is the most aggressive subtype of breast cancer. TNBC accounts for about 15% of breast cancer cases in the U.S., and is known for high relapse rates and poor overall survival (OS). Chemo-resistant TNBC is a genetically diverse, highly heterogeneous, and rapidly evolving disease that challenges our ability to individualize treatment for incomplete responders and relapsed patients. Currently, the frontline standard chemotherapy, composed of anthracyclines, alkylating agents, and taxanes, is commonly used to treat high-risk and locally advanced TNBC. Several FDA-approved drugs that target programmed cell death protein-1 (Keytruda) and programmed death ligand-1 (Tecentriq), poly ADP-ribose polymerase (PARP), and/or antibody drug conjugates (Trodelvy) have shown promise in improving clinical outcomes for a subset of TNBC. These inhibitors that target key genetic mutations and specific molecular signaling pathways that drive malignant tumor growth have been used as single agents and/or in combination with standard chemotherapy regimens. Here, we review the current TNBC treatment options, unmet clinical needs, and actionable drug targets, including epidermal growth factor (EGFR), vascular endothelial growth factor (VEGF), androgen receptor (AR), estrogen receptor beta (ERß), phosphoinositide-3 kinase (PI3K), mammalian target of rapamycin (mTOR), and protein kinase B (PKB or AKT) activation in TNBC. Supported by strong evidence in developmental, evolutionary, and cancer biology, we propose that the K-RAS/SIAH pathway activation is a major tumor driver, and SIAH is a new drug target, a therapy-responsive prognostic biomarker, and a major tumor vulnerability in TNBC. Since persistent K-RAS/SIAH/EGFR pathway activation endows TNBC tumor cells with chemo-resistance, aggressive dissemination, and early relapse, we hope to design an anti-SIAH-centered anti-K-RAS/EGFR targeted therapy as a novel therapeutic strategy to control and eradicate incurable TNBC in the future.
RESUMEN
BACKGROUND: The treatment of complex bone loss scenarios remains challenging. This study evaluates the efficacy of ex vivo regional gene therapy using transduced human adipose-derived stem cells (ASCs) overexpressing bone morphogenetic protein-2 (BMP-2) to treat critical-sized bone defects. METHODS: Critical-sized femoral defects created surgically in immunocompromised rats were treated with ASCs transduced with a lentivirus encoding BMP-2 (Group 1, n = 14), or green fluorescent protein (Group 2, n = 5), nontransduced ASCs (Group 3, n = 5), or rhBMP-2 (Group 4, n = 14). At 12 weeks, femurs were evaluated for quantity and quality of bone formation with plain radiographs, micro-computed tomography, histology/histomorphometry, and biomechanical strength testing. RESULTS: Thirteen of 14 samples in Group 1 and all 14 samples in Group 4 showed radiographic healing, while no samples in either Groups 2 or 3 healed. Groups 1 and 4 had significantly higher radiographic scores (p < 0.001), bone volume fraction (BV/TV) (p < 0.001), and bone area fraction (BA/TA) than Groups 2 and 3 (p < 0.001). Radiographic scores, BV/TV, and BA/TA were not significantly different between Groups 1 and 4. No difference with regards to mean torque, rotation at failure, torsional stiffness, and energy to failure was seen between Groups 1 and 4. CONCLUSIONS: Human ASCs modified to overexpress BMP-2 resulted in abundant bone formation, with the quality of bone comparable to that of rhBMP-2. This strategy represents a promising approach in the treatment of large bone defects in the clinical setting. CLINICAL RELEVANCE: Large bone defects may require sustained protein production to induce an appropriate osteoinductive response. Ex vivo regional gene therapy using a lentiviral vector has the potential to be part of a comprehensive tissue engineering strategy for treating osseous defects.
Asunto(s)
Proteína Morfogenética Ósea 2 , Lentivirus , Tejido Adiposo , Animales , Proteína Morfogenética Ósea 2/genética , Regeneración Ósea , Terapia Genética , Humanos , Lentivirus/genética , Osteogénesis , Ratas , Células Madre , Microtomografía por Rayos XRESUMEN
Chemo-resistant breast cancer is a major barrier to curative treatment for a significant number of women with breast cancer. Neoadjuvant chemotherapy (NACT) is standard first- line treatment for most women diagnosed with high-risk TNBC, HER2+, and locally advanced ER+ breast cancer. Current clinical prognostic tools evaluate four clinicopathological factors: Tumor size, LN status, pathological stage, and tumor molecular subtype. However, many similarly treated patients with identical residual cancer burden (RCB) following NACT experience distinctly different tumor relapse rates, clinical outcomes and survival. This problem is particularly apparent for incomplete responders with a high-risk RCB classification following NACT. Therefore, there is a pressing need to identify new prognostic and predictive biomarkers, and develop novel curative therapies to augment current standard of care (SOC) treatment regimens to save more lives. Here, we will discuss these unmet needs and clinical challenges that stand in the way of precision medicine and personalized cancer therapy.
RESUMEN
Umbilical cord blood (UCB) has been increasingly explored as an alternative source of stem cells for use in regenerative medicine due to several advantages over other stem-cell sources, including the need for less stringent human leukocyte antigen matching. Combined with an osteoinductive signal, UCB-derived mesenchymal stem cells (MSCs) could revolutionize the treatment of challenging bone defects. This study aimed to develop an ex vivo regional gene-therapy strategy using BMP-2-transduced allogeneic UCB-MSCs to promote bone repair. To this end, human UCB-MSCs were transduced with a lentiviral vector carrying the cDNA for BMP-2 (LV-BMP-2). In vitro assays to determine the UCB-MSC osteogenic potential and BMP-2 production were followed by in vivo implantation of LV-BMP-2-transduced UCB-MSCs in a mouse hind-limb muscle pouch. Non-transduced and LV-GFP-transduced UCB-MSCs were used as controls. Transduction with LV-BMP-2 was associated with abundant BMP-2 production and induction of osteogenic differentiation in vitro. Implantation of BMP-2-transduced UCB-MSCs led to robust heterotopic bone formation 4 weeks postoperatively, as seen on radiographs and histology. These results, along with the fact that UCB-MSCs can be easily collected with no donor-site morbidity and low immunogenicity, suggest that UCB might be a preferable allogeneic source of MSCs to develop an ex vivo gene-therapy approach to treat difficult bone-repair scenarios.
Asunto(s)
Proteína Morfogenética Ósea 2/genética , Sangre Fetal/citología , Terapia Genética , Vectores Genéticos/genética , Lentivirus/genética , Células Madre Mesenquimatosas/metabolismo , Animales , Biomarcadores , Regeneración Ósea , Diferenciación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Trasplante de Células Madre Mesenquimatosas , Ratones , Fenotipo , Transducción Genética , TransgenesRESUMEN
Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.
Asunto(s)
Modelos Animales de Enfermedad , Neurofibromatosis 1 , Neurofibromina 1/metabolismo , Porcinos , Animales , Manchas Café con Leche , Exones/genética , Fibroblastos/metabolismo , Proteínas Activadoras de GTPasa/genética , Ganglios Espinales/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Canales Iónicos , Aprendizaje , Masculino , Memoria , Mutación , Neurofibroma , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/fisiología , Transducción de SeñalRESUMEN
Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.
RESUMEN
Ex vivo regional gene therapy strategies using animal mesenchymal stem cells genetically modified to overexpress osteoinductive growth factors have been successfully used in a variety of animal models to induce both heterotopic and orthotopic bone formation. However, in order to adapt regional gene therapy for clinical applications, it is essential to assess the osteogenic capacity of transduced human cells and choose the cell type that demonstrates the best clinical potential. Bone-marrow stem cells (BMSC) and adipose-derived stem cells (ASC) were selected in this study for in vitro evaluation, before and after transduction with a lentiviral two-step transcriptional amplification system (TSTA) overexpressing bone morphogenetic protein 2 (BMP-2; LV-TSTA-BMP-2) or green fluorescent protein (GFP; LV-TSTA-GFP). Cell growth, transduction efficiency, BMP-2 production, and osteogenic capacity were assessed. The study demonstrated that BMSC were characterized by a slower cell growth compared to ASC. Fluorescence-activated cell sorting analysis of GFP-transduced cells confirmed successful transduction with the vector and revealed an overall higher but not statistically significant transduction efficiency in ASC versus BMSC (90.2 ± 4.06% vs. 80.4 ± 8.51%, respectively; p = 0.146). Enzyme-linked immunosorbent assay confirmed abundant BMP-2 production by both cell types transduced with LV-TSTA-BMP-2, with BMP-2 production being significantly higher in ASC versus BMSC (239.5 ± 116.55 ng vs. 70.86 ± 24.7 ng; p = 0.001). Quantitative analysis of extracellular deposition of calcium (Alizarin red) and alkaline phosphatase activity showed that BMP-2-transduced cells had a higher osteogenic differentiation capacity compared to non-transduced cells. When comparing the two cell types, ASC/LV-TSTA-BMP-2 demonstrated a significantly higher mineralization potential compared to BMSC/LV-TSTA-BMP-2 7 days post transduction (p = 0.014). In conclusion, this study demonstrates that transduction with LV-TSTA-BMP-2 can significantly enhance the osteogenic potential of both human BMSC and ASC. BMP-2-treated ASC exhibited higher BMP-2 production and greater osteogenic differentiation capacity compared to BMP-2-treated BMSC. These results, along with the fact that liposuction is an easy procedure with lower donor-site morbidity compared to BM aspiration, indicate that adipose tissue might be a preferable source of MSCs to develop a regional gene therapy approach to treat difficult bone-repair scenarios.
Asunto(s)
Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/genética , Terapia Genética , Osteogénesis/genética , Tejido Adiposo/citología , Tejido Adiposo/trasplante , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 2/farmacología , Regulación del Desarrollo de la Expresión Génica , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Transducción GenéticaRESUMEN
BACKGROUND: The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. RESULTS: Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. CONCLUSION: Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.
Asunto(s)
Proteínas Nucleares/clasificación , Filogenia , Ubiquitina-Proteína Ligasas/clasificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Secuencia de Consenso , Evolución Molecular , Regulación Neoplásica de la Expresión Génica , Humanos , Invertebrados/enzimología , Familia de Multigenes , Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Vertebrados/metabolismoRESUMEN
Dysregulation of the MAPK pathway correlates with progression of pancreatic ductal adenocarcinoma (PDAC) progression. IQ motif containing GTPase-activating protein 1 (IQGAP1) is a MAPK scaffold that directly regulates the activation of RAF, MEK, and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in gluconeogenesis, is transcriptionally downregulated in various cancers, including PDAC. Here, we demonstrate that FBP1 acts as a negative modulator of the IQGAP1-MAPK signaling axis in PDAC cells. FBP1 binding to the WW domain of IQGAP1 impeded IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity. Conversely, decreased FBP1 expression induced pERK1/2 levels in PDAC cell lines and correlated with increased pERK1/2 levels in patient specimens. Treatment with gemcitabine caused undesirable activation of ERK1/2 in PDAC cells, but cotreatment with the FBP1-derived small peptide inhibitor FBP1 E4 overcame gemcitabine-induced ERK activation, thereby increasing the anticancer efficacy of gemcitabine in PDAC. These findings identify a primary mechanism of resistance of PDAC to standard therapy and suggest that the FBP1-IQGAP1-ERK1/2 signaling axis can be targeted for effective treatment of PDAC. Cancer Res; 77(16); 4328-41. ©2017 AACR.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Desoxicitidina/análogos & derivados , Fructosa-Bifosfatasa/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores , Línea Celular Tumoral , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Fructosa-Bifosfatasa/genética , Humanos , Neoplasias Pancreáticas/metabolismo , Transducción de Señal , Transfección , Proteínas Activadoras de ras GTPasa/metabolismo , GemcitabinaRESUMEN
BACKGROUND: Metastatic breast cancer exhibits diverse and rapidly evolving intra- and inter-tumor heterogeneity. Patients with similar clinical presentations often display distinct tumor responses to standard of care (SOC) therapies. Genome landscape studies indicate that EGFR/HER2/RAS "pathway" activation is highly prevalent in malignant breast cancers. The identification of therapy-responsive and prognostic biomarkers is paramount important to stratify patients and guide therapies in clinical oncology and personalized medicine. METHODS: In this study, we analyzed matched pairs of tumor specimens collected from 182 patients who received neoadjuvant systemic therapies (NST). Statistical analyses were conducted to determine whether EGFR/HER2/RAS pathway biomarkers and clinicopathological predictors, alone and in combination, are prognostic in breast cancer. FINDINGS: SIAH and EGFR outperform ER, PR, HER2 and Ki67 as two logical, sensitive and prognostic biomarkers in metastatic breast cancer. We found that increased SIAH and EGFR expression correlated with advanced pathological stage and aggressive molecular subtypes. Both SIAH expression post-NST and NST-induced changes in EGFR expression in invasive mammary tumors are associated with tumor regression and increased survival, whereas ER, PR, and HER2 were not. These results suggest that SIAH and EGFR are two prognostic biomarkers in breast cancer with lymph node metastases. INTERPRETATION: The discovery of incorporating tumor heterogeneity-independent and growth-sensitive RAS pathway biomarkers, SIAH and EGFR, whose altered expression can be used to estimate therapeutic efficacy, detect emergence of resistant clones, forecast tumor regression, differentiate among partial responders, and predict patient survival in the neoadjuvant setting, has a clear clinical implication in personalizing breast cancer therapy. FUNDING: This work was supported by the Dorothy G. Hoefer Foundation for Breast Cancer Research (A.H. Tang); Center for Innovative Technology (CIT)-Commonwealth Research Commercialization Fund (CRCF) (MF14S-009-LS to A.H. Tang), and National Cancer Institute (CA140550 to A.H. Tang).
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Receptores ErbB/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas ras/metabolismo , Biomarcadores de Tumor , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Progresión de la Enfermedad , Receptores ErbB/genética , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Modelos Biológicos , Terapia Neoadyuvante , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteínas Nucleares/genética , Pronóstico , Modelos de Riesgos Proporcionales , Resultado del Tratamiento , Ubiquitina-Proteína Ligasas/genética , Proteínas ras/genéticaRESUMEN
Recombinant human BMP-2 (rhBMP-2) is a potent osteoinductive agent, but has been associated not only with bone formation, but also osteoclastogenesis and bone resorption. Osteoprotegerin (OPG) is a RANKL inhibitor that blocks differentiation and function of osteoclasts. We hypothesized that the combination of local BMP-2 (recombinant protein or a product of gene therapy) plus systemic OPG-Fc is more effective than BMP-2 alone in promoting bone repair. To test this hypothesis we used a mouse critical-sized femoral defect model. Col2.3eGFP (osteoblastic marker) male mice were treated with rhBMP-2 (group I), rhBMP-2 and systemic OPG (group II), rhBMP-2 and delayed administration of OPG (group III), mouse BM cells transduced with a lentiviral vector containing the BMP-2 gene (LV-BMP-2; group IV), LV-BMP-2 and systemic OPG (group V), a carrier alone (group VI) and administration of OPG alone (group VII). All bone defects treated with BMP-2 (alone or combined with OPG) healed, whereas minimal bone formation was noted in animals treated with the carrier alone or OPG alone. MicroCT analysis showed that bone volume (BV) in rhBMP-2+OPG and LV-BMP-2+OPG groups was significantly higher compared to rhBMP-2 alone (p<0.01) and LV-BMP-2 alone (p<0.001). Similar results were observed in histomorphometry, with rhBMP-2 alone defects exhibiting significantly lower bone area (B.Ar) compared to rhBMP-2+OPG defects (p<0.005) and LV-BMP-2 defects having a significantly lower B.Ar compared to all BMP-2+OPG treated groups (p≤0.01). TRAP staining demonstrated a major osteoclast response in the groups that did not receive OPG (rhBMP-2, LV-BMP-2 and sponge alone) beginning as early as 7days post-operatively. In conclusion, we demonstrated that locally delivered BMP-2 (recombinant protein or gene therapy) in combination with systemically administered OPG improved bone healing compared to BMP-2 alone in a mouse critical-sized bone defect. These data indicate that osteoclasts can diminish healing responses to BMP-2 and that RANKL inhibition may thus accentuate BMP-2 efficacy.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Fémur/patología , Osteoprotegerina/farmacología , Ligando RANK/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Animales , Recuento de Células , Quimioterapia Combinada , Fémur/diagnóstico por imagen , Fémur/efectos de los fármacos , Fémur/cirugía , Secciones por Congelación , Humanos , Isoenzimas/metabolismo , Lentivirus/genética , Masculino , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteogénesis , Ligando RANK/metabolismo , Proteínas Recombinantes/farmacología , Fosfatasa Ácida Tartratorresistente , Transducción Genética , Microtomografía por Rayos XRESUMEN
Metastatic breast cancer is a highly heterogeneous, rapidly evolving and devastating disease that challenges our ability to find curative therapies. RAS pathway activation is an understudied research area in breast cancer. EGFR/RAS pathway activation is prevalent in breast cancer with poor prognosis. The prognostic RAS pathway biomarkers can be used to identify resistant tumour clones, stratify patients and guide therapies.
RESUMEN
BACKGROUND: Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab. METHODS: A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 µg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. RESULTS: At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001). CONCLUSIONS: In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora. CLINICAL RELEVANCE: Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Proteína Morfogenética Ósea 2/uso terapéutico , Proteínas Morfogenéticas Óseas/uso terapéutico , Fracturas del Fémur/tratamiento farmacológico , Fijación Interna de Fracturas , Factor de Crecimiento Transformador beta/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Quimioterapia Adyuvante , Esquema de Medicación , Quimioterapia Combinada , Fracturas del Fémur/diagnóstico por imagen , Fracturas del Fémur/patología , Fracturas del Fémur/cirugía , Curación de Fractura , Marcadores Genéticos , Humanos , Inyecciones Subcutáneas , Masculino , Radiografía , Distribución Aleatoria , Ratas , Proteínas Recombinantes/uso terapéuticoRESUMEN
Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.
Asunto(s)
Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Humanos , Ratones , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Esclerosis Tuberosa/fisiopatologíaRESUMEN
The mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a therapeutic target for many types of cancers. NMDA receptors regulate mTOR signalling activity; their inappropriate expression on several human cancer cell lines represents a potential therapeutic avenue to control dysregulated growth, division and invasiveness. Targeting these receptors with selective ligands (e.g., glycineB site ligands) may be a less toxic and more tolerable approach than administering compounds acting at the mTORC1 complex itself, such as rapamycin and its derivatives. Thus, testing glycineB site ligands in relevant in vitro and in vivo paradigms with established human cancer cells that express NMDA receptors on their surface could provide proofs of concept/principle that would encourage exploration of these and other "non-toxic" strategies. Interestingly, in some cancer models that express NMDA receptors on their surface, NMDA receptor antagonists, such as MK-801 (dizocilpine), were shown to possess anti-proliferative and anti-invasive effects, which conflict with hypotheses about promoting NMDA receptor activation as a cancer chemotherapeutic strategy. Whether NMDA receptor activation or antagonism is associated with anti-proliferative and anti-invasive effects may reflect differences between cancer cell lines in terms of the proteins associated with the NMDA receptors on their cell surfaces, which, in turn, could lead to different "downstream" effects on cascades of intracellular phosphorylations. Irrespective of whether activation or antagonism is associated with anti-proliferative and anti-invasive effects for specific types of cancer, data are emerging that support exploration of targeting NMDA receptors expressed on the surface of cancer cells as a therapeutic strategy.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Antineoplásicos/efectos adversos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ligandos , Terapia Molecular Dirigida , Neoplasias/patología , Fosforilación/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Overactivation of the mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of syndromic forms of autism spectrum disorders (ASDs), such as tuberous sclerosis complex, neurofibromatosis 1, and fragile X syndrome. Administration of mTORC1 (mTOR complex 1) inhibitors (e.g. rapamycin) in syndromic mouse models of ASDs improved behavior, cognition, and neuropathology. However, since only a minority of ASDs are due to the effects of single genes (â¼10%), there is a need to explore inhibition of mTOR activity in mouse models that may be more relevant to the majority of nonsyndromic presentations, such as the genetically inbred BTBR T(+)Itpr3(tf)/J (BTBR) mouse model of ASDs. BTBR mice have social impairment and exhibit increased stereotypic behavior. In prior work, d-cycloserine, a partial glycineB site agonist that targets the N-methyl-d-aspartate (NMDA) receptor, was shown to improve sociability in both Balb/c and BTBR mouse models of ASDs. Importantly, NMDA receptor activation regulates mTOR signaling activity. The current study investigated the ability of rapamycin (10mg/kg, i.p.×four days), an mTORC1 inhibitor, to improve sociability and stereotypic behavior in BTBR mice. Using a standard paradigm to assess mouse social behavior, rapamycin improved several measures of sociability in the BTBR mouse, suggesting that mTOR overactivation represents a therapeutic target that mediates or contributes to impaired sociability in the BTBR mouse model of ASDs. Interestingly, there was no effect of rapamycin on stereotypic behaviors in this mouse model.