Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attribut... more Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long‐term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA‐seq and mass spectrometry (MS)‐based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer‐related pathways and some less established that showed higher expression in short‐term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusua...
Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with... more Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with rising incidence. Yet, molecular mechanisms supporting the formation of these lesions from CRC are unknown. We aimed to explore drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of insulin receptor substrate 2 (IRS2) gene amplification was noted in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. We subsequently constructed an in ...
1108 In recent years it has become clear that angiogenesis, new capillary blood vessel growth fro... more 1108 In recent years it has become clear that angiogenesis, new capillary blood vessel growth from pre-existing vasculature, is crucial for tumor progression and metastases. Consequently, the microvascular endothelial cell, recruited by a tumor, has become an important target in cancer therapy. Akt, a serine/threonine protein kinase, has been implicated in the vascular endothelial growth factor (VEGF) signaling pathway of many tumor types promoting VEGF secretion. Akt is chronically activated not only in tumor cells, but also in VEGF-stimulated tumor endothelial cells, and induces angiogenesis via its downstream effectors. For that reason, Akt has become an attractive drug target for cancer therapy. Several Akt inhibitors are today in clinical trials but they all suffer from lack of selectivity and specificity. In order to overcome those limitations, we propose to inhibit the overexpressed Akt1 in tumors and their surrounding endothelial cells by using RNA interference. Still, this...
Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM). Yet,... more Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM). Yet, molecular mechanisms supporting it are unknown. We aimed to explore drivers enabling adaptation of CRC cells to the brain and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of IRS2 gene amplification was observed in 13% of BM, compared to only 3% of primary tumors or other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BM. We constructed an in-vitro system mimicking the brain microenvironment using cultured human astrocytes or their conditioned media. Under these...
Brain metastases (BMs) from colorectal cancer (CRC) are currently the fourths leading cause of BM... more Brain metastases (BMs) from colorectal cancer (CRC) are currently the fourths leading cause of BMs, and their incidence is on the rise. Yet, mechanisms mediating the formation of BMs from CRC are unknown. We aimed to explore genomic drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence was noted in the insulin receptor substrate 2 (IRS2) gene, which was significantly amplified in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. Next, ...
Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, i... more Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, ...
Glioblastoma (GB) is the most lethal type of primary tumor in the central nervous system. Current... more Glioblastoma (GB) is the most lethal type of primary tumor in the central nervous system. Current treatments include surgical resection followed by chemotherapy and radiotherapy. With this therapeutic regimen, the median survival is less than two years. However, these treatments do not much improve the overall survival of GB patients. GBs are highly angiogenic and invasive tumors and often acquire resistance to therapy. The invasive nature of the disease limits the ability to achieve complete resection of the tumor and the majority of GB patients will experience disease relapse. Moreover, GB is highly heterogeneous, harboring different mutations and presenting different phenotypes. As the brain is considered to be an immune‐privileged tissue, GB is defined as a cold tumor for which current immunotherapies have not yet been demonstrated to improve survival. On top of these challenges, the blood brain barrier (BBB) restricts the uptake of drugs by the brain, thus limiting the therapeutic options. Therefore, enormous efforts are being dedicated to the development of novel nanomedicines, which will be able to cross the BBB and specifically target the cancer cells. Here, the current achievements in drug delivery and novel therapeutic approaches for GB therapy are discussed.
SummaryGlioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by... more SummaryGlioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by most patients, and a median survival time of less than two years. To examine survival-associated patterns, we assembled a cohort of 87 GBM patients whose survival ranges from less than 3 months and up to 10 years, most of which are not bearing isocitrate-dehyderogenase (IDH)-1 mutation and did not undergo prior treatment. We integrated high-resolution mass-spectrometry proteomics and RNA-sequencing to examine the yet unresolved proteomic contribution to poor patient outcome, and compared it to the more established transcriptomic contribution and to published single-cell RNA-sequencing data. Discovering both layer-specific and shared processes, we found that immune, metabolic and developmental processes distinguish short and long survival periods. Additionally, we observed a significant discrepancy in tumor classification between expression layers. Overall, our integrative findings estab...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018
Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite cur... more Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of...
Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal t... more Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal therapeutic outcome, partly due to acquirement of resistance and insufficient blood-brain barrier (BBB) penetration of chemotherapeutics. To circumvent these limitations, we conjugated the chemotherapy paclitaxel (PTX) to a dendritic polyglycerol sulfate (dPGS) nanocarrier. dPGS is able to cross the BBB, bind to P/L-selectins and accumulate selectively in intracranial tumors. We show that dPGS has dual targeting properties, as we found that P-selectin is not only expressed on tumor endothelium but also on glioblastoma cells. We delivered dPGS-PTX in combination with a peptidomimetic of the anti-angiogenic protein thrombospondin-1 (TSP-1 PM). This combination resulted in a remarkable synergistic anticancer effect on intracranial human and murine glioblastoma via induction of Fas and Fas-L, with no side effects compared to free PTX or temozolomide. This study shows that our unique therapeut...
The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment m... more The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment might rely on simultaneous targeting of multiple genes, which can be achieved by RNA interference-based therapeutic strategies. Here we show a potent combination of microRNA and siRNA delivered by an efficient nanocarrier to PDAC tumors. Using proteomic-microRNA profiles and survival data of PDAC patients from TCGA, we found a novel signature for prolonged survival. Accordingly, we used a microRNA-mimic to increase miR-34a together with siRNA to silence PLK1 oncogene. For in vivo dual-targeting of this combination, we developed a biodegradable amphiphilic polyglutamate amine polymeric nanocarrier (APA). APA-miRNA-siRNA polyplexes systemically administered to orthotopically inoculated PDAC-bearing mice showed no toxicity and accumulated at the tumor, resulting in an enhanced antitumor effect due to inhibition of MYC oncogene, a common target of both miR-34a and PLK1. Taken together, our fi...
Glioblastoma multiforme is a very heterogeneous tumor, highly infiltrative, angiogenic and resist... more Glioblastoma multiforme is a very heterogeneous tumor, highly infiltrative, angiogenic and resistant to standard therapeutic intervention. Experimental models currently used in research are mostly based on cancer cell lines, which are grown in culture for many generations and have lost many essential biological characteristics. In consequence, the xenograft tumor models derived from those cells do not maintain genomic and phenotypic characteristics present in the original tumor. It is questionable whether these artificial preclinical models can serve as reliable platforms to select the lead candidate for a novel therapeutic approach. We utilized miRNA expression patterns to evaluate the clinical relevance of some of the currently available experimental models of GBM, searching for similarities and differences in miRNA expression levels between freshly isolated tumors, patient-derived primary cells and GBM cell lines grown in culture. The study included 22 formalin-fixed paraffin-emb...
The presence of dormant, microscopic cancerous lesions possesses a major obstacle for the treatme... more The presence of dormant, microscopic cancerous lesions possesses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93 and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients' tumor samples compared to normal bone making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge, hence, we synthesized an aminated polyglycerol dendritic nanocarrier, PG-NH2, and designed PG-NH2-microRNA polyplexes to target cancer. Reconstitut...
There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor prog... more There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor progression, the point at which a dormant tumor acquires the ability to grow and metastasize. Although the tumor dormancy phenomenon has important implications for early detection and treatment of cancer, its biology and genetic characteristics are poorly understood. MicroRNAs (miRs), small non-coding RNA molecules which regulate gene expression at the post-transcriptional level, are emerging as key regulators in physiological and pathological processes, including tumorigenesis. We hypothesized that specific miRs may also play a key role in the regulation of tumor dormancy and the “angiogenic switch”. We therefore established a pair of cell lines that generate dormant avascular or fast-growing angiogenic osteosarcomas in SCID mice (Saos-2-D and Saos-2-E respectively). Although these cells share similar growth rate in vitro, when inoculated into mice, Saos-2-E cells generate vascularized and ...
Glioblastoma multiforme (GBM) is an aggressive primary neoplasm of the brain that exhibit notable... more Glioblastoma multiforme (GBM) is an aggressive primary neoplasm of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed deregulated molecular networks as potential targets for therapeutic development. MicroRNAs (miRNAs) are noncoding RNA molecules which act as post-transcriptional regulators of specific messenger RNA transcripts. miRNAs play major roles in normal developmental processes, and their deregulation significantly contributes to various aspects of carcinogenesis. Nevertheless, in vivo delivery of small interfering RNA (siRNA) and miRNA remains a crucial challenge for their therapeutic success. siRNAs and miRNAs on their own are not taken-up by most mammalian cells in a way that preserves their activity. In order to circumvent these limitations, we developed a cationic carrier system, which can strongly improve its stability, intracellular trafficking and silencing efficacy. Polyglycerol (PG)-Amine, ...
Publisher Summary The chapter presents a system that enables to study properties of protein phosp... more Publisher Summary The chapter presents a system that enables to study properties of protein phosphatase 2Cα (PP2Cα) expression in 293 cells.PP2Cα (also referred as PPM1A) is the most characterized member of the PP2C group. It is a monomeric enzyme of about 42 kDa that shows broad substrate specificity. The catalytic domain, of 300 amino acid residues, in the N terminus, consists of 6 α-helices and 11 β-sheets, and is common to all enzymes that belong to the PP2C family. The C-terminal domain includes a sequence of 80 amino acids that forms three α-helices. This domain determines the substrate specificity of the enzyme and displays no similarity to the other paralogs except for the closely related PP2C β. Growing list of substrates, specifically dephosphorylated by PP2C α in eukaryotic cells has been suggested. In human embryo kidney (HEK) 293 cells over expression of PP2C α and PP2C β were highly toxic to the cells and it is not possible to obtain stable cell lines overexpressing PP2C α. The chapter discusses several methods including establishment of inducible PP2C α cells; plating efficiency; antibodies; cell extracts and western blot analysis; XTT assay; and malachite-green assay of protein phosphatase activity.
Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attribut... more Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long‐term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA‐seq and mass spectrometry (MS)‐based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer‐related pathways and some less established that showed higher expression in short‐term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusua...
Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with... more Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with rising incidence. Yet, molecular mechanisms supporting the formation of these lesions from CRC are unknown. We aimed to explore drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of insulin receptor substrate 2 (IRS2) gene amplification was noted in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. We subsequently constructed an in ...
1108 In recent years it has become clear that angiogenesis, new capillary blood vessel growth fro... more 1108 In recent years it has become clear that angiogenesis, new capillary blood vessel growth from pre-existing vasculature, is crucial for tumor progression and metastases. Consequently, the microvascular endothelial cell, recruited by a tumor, has become an important target in cancer therapy. Akt, a serine/threonine protein kinase, has been implicated in the vascular endothelial growth factor (VEGF) signaling pathway of many tumor types promoting VEGF secretion. Akt is chronically activated not only in tumor cells, but also in VEGF-stimulated tumor endothelial cells, and induces angiogenesis via its downstream effectors. For that reason, Akt has become an attractive drug target for cancer therapy. Several Akt inhibitors are today in clinical trials but they all suffer from lack of selectivity and specificity. In order to overcome those limitations, we propose to inhibit the overexpressed Akt1 in tumors and their surrounding endothelial cells by using RNA interference. Still, this...
Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM). Yet,... more Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM). Yet, molecular mechanisms supporting it are unknown. We aimed to explore drivers enabling adaptation of CRC cells to the brain and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of IRS2 gene amplification was observed in 13% of BM, compared to only 3% of primary tumors or other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BM. We constructed an in-vitro system mimicking the brain microenvironment using cultured human astrocytes or their conditioned media. Under these...
Brain metastases (BMs) from colorectal cancer (CRC) are currently the fourths leading cause of BM... more Brain metastases (BMs) from colorectal cancer (CRC) are currently the fourths leading cause of BMs, and their incidence is on the rise. Yet, mechanisms mediating the formation of BMs from CRC are unknown. We aimed to explore genomic drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence was noted in the insulin receptor substrate 2 (IRS2) gene, which was significantly amplified in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. Next, ...
Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, i... more Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, ...
Glioblastoma (GB) is the most lethal type of primary tumor in the central nervous system. Current... more Glioblastoma (GB) is the most lethal type of primary tumor in the central nervous system. Current treatments include surgical resection followed by chemotherapy and radiotherapy. With this therapeutic regimen, the median survival is less than two years. However, these treatments do not much improve the overall survival of GB patients. GBs are highly angiogenic and invasive tumors and often acquire resistance to therapy. The invasive nature of the disease limits the ability to achieve complete resection of the tumor and the majority of GB patients will experience disease relapse. Moreover, GB is highly heterogeneous, harboring different mutations and presenting different phenotypes. As the brain is considered to be an immune‐privileged tissue, GB is defined as a cold tumor for which current immunotherapies have not yet been demonstrated to improve survival. On top of these challenges, the blood brain barrier (BBB) restricts the uptake of drugs by the brain, thus limiting the therapeutic options. Therefore, enormous efforts are being dedicated to the development of novel nanomedicines, which will be able to cross the BBB and specifically target the cancer cells. Here, the current achievements in drug delivery and novel therapeutic approaches for GB therapy are discussed.
SummaryGlioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by... more SummaryGlioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by most patients, and a median survival time of less than two years. To examine survival-associated patterns, we assembled a cohort of 87 GBM patients whose survival ranges from less than 3 months and up to 10 years, most of which are not bearing isocitrate-dehyderogenase (IDH)-1 mutation and did not undergo prior treatment. We integrated high-resolution mass-spectrometry proteomics and RNA-sequencing to examine the yet unresolved proteomic contribution to poor patient outcome, and compared it to the more established transcriptomic contribution and to published single-cell RNA-sequencing data. Discovering both layer-specific and shared processes, we found that immune, metabolic and developmental processes distinguish short and long survival periods. Additionally, we observed a significant discrepancy in tumor classification between expression layers. Overall, our integrative findings estab...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018
Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite cur... more Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of...
Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal t... more Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal therapeutic outcome, partly due to acquirement of resistance and insufficient blood-brain barrier (BBB) penetration of chemotherapeutics. To circumvent these limitations, we conjugated the chemotherapy paclitaxel (PTX) to a dendritic polyglycerol sulfate (dPGS) nanocarrier. dPGS is able to cross the BBB, bind to P/L-selectins and accumulate selectively in intracranial tumors. We show that dPGS has dual targeting properties, as we found that P-selectin is not only expressed on tumor endothelium but also on glioblastoma cells. We delivered dPGS-PTX in combination with a peptidomimetic of the anti-angiogenic protein thrombospondin-1 (TSP-1 PM). This combination resulted in a remarkable synergistic anticancer effect on intracranial human and murine glioblastoma via induction of Fas and Fas-L, with no side effects compared to free PTX or temozolomide. This study shows that our unique therapeut...
The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment m... more The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment might rely on simultaneous targeting of multiple genes, which can be achieved by RNA interference-based therapeutic strategies. Here we show a potent combination of microRNA and siRNA delivered by an efficient nanocarrier to PDAC tumors. Using proteomic-microRNA profiles and survival data of PDAC patients from TCGA, we found a novel signature for prolonged survival. Accordingly, we used a microRNA-mimic to increase miR-34a together with siRNA to silence PLK1 oncogene. For in vivo dual-targeting of this combination, we developed a biodegradable amphiphilic polyglutamate amine polymeric nanocarrier (APA). APA-miRNA-siRNA polyplexes systemically administered to orthotopically inoculated PDAC-bearing mice showed no toxicity and accumulated at the tumor, resulting in an enhanced antitumor effect due to inhibition of MYC oncogene, a common target of both miR-34a and PLK1. Taken together, our fi...
Glioblastoma multiforme is a very heterogeneous tumor, highly infiltrative, angiogenic and resist... more Glioblastoma multiforme is a very heterogeneous tumor, highly infiltrative, angiogenic and resistant to standard therapeutic intervention. Experimental models currently used in research are mostly based on cancer cell lines, which are grown in culture for many generations and have lost many essential biological characteristics. In consequence, the xenograft tumor models derived from those cells do not maintain genomic and phenotypic characteristics present in the original tumor. It is questionable whether these artificial preclinical models can serve as reliable platforms to select the lead candidate for a novel therapeutic approach. We utilized miRNA expression patterns to evaluate the clinical relevance of some of the currently available experimental models of GBM, searching for similarities and differences in miRNA expression levels between freshly isolated tumors, patient-derived primary cells and GBM cell lines grown in culture. The study included 22 formalin-fixed paraffin-emb...
The presence of dormant, microscopic cancerous lesions possesses a major obstacle for the treatme... more The presence of dormant, microscopic cancerous lesions possesses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93 and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients' tumor samples compared to normal bone making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge, hence, we synthesized an aminated polyglycerol dendritic nanocarrier, PG-NH2, and designed PG-NH2-microRNA polyplexes to target cancer. Reconstitut...
There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor prog... more There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor progression, the point at which a dormant tumor acquires the ability to grow and metastasize. Although the tumor dormancy phenomenon has important implications for early detection and treatment of cancer, its biology and genetic characteristics are poorly understood. MicroRNAs (miRs), small non-coding RNA molecules which regulate gene expression at the post-transcriptional level, are emerging as key regulators in physiological and pathological processes, including tumorigenesis. We hypothesized that specific miRs may also play a key role in the regulation of tumor dormancy and the “angiogenic switch”. We therefore established a pair of cell lines that generate dormant avascular or fast-growing angiogenic osteosarcomas in SCID mice (Saos-2-D and Saos-2-E respectively). Although these cells share similar growth rate in vitro, when inoculated into mice, Saos-2-E cells generate vascularized and ...
Glioblastoma multiforme (GBM) is an aggressive primary neoplasm of the brain that exhibit notable... more Glioblastoma multiforme (GBM) is an aggressive primary neoplasm of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed deregulated molecular networks as potential targets for therapeutic development. MicroRNAs (miRNAs) are noncoding RNA molecules which act as post-transcriptional regulators of specific messenger RNA transcripts. miRNAs play major roles in normal developmental processes, and their deregulation significantly contributes to various aspects of carcinogenesis. Nevertheless, in vivo delivery of small interfering RNA (siRNA) and miRNA remains a crucial challenge for their therapeutic success. siRNAs and miRNAs on their own are not taken-up by most mammalian cells in a way that preserves their activity. In order to circumvent these limitations, we developed a cationic carrier system, which can strongly improve its stability, intracellular trafficking and silencing efficacy. Polyglycerol (PG)-Amine, ...
Publisher Summary The chapter presents a system that enables to study properties of protein phosp... more Publisher Summary The chapter presents a system that enables to study properties of protein phosphatase 2Cα (PP2Cα) expression in 293 cells.PP2Cα (also referred as PPM1A) is the most characterized member of the PP2C group. It is a monomeric enzyme of about 42 kDa that shows broad substrate specificity. The catalytic domain, of 300 amino acid residues, in the N terminus, consists of 6 α-helices and 11 β-sheets, and is common to all enzymes that belong to the PP2C family. The C-terminal domain includes a sequence of 80 amino acids that forms three α-helices. This domain determines the substrate specificity of the enzyme and displays no similarity to the other paralogs except for the closely related PP2C β. Growing list of substrates, specifically dephosphorylated by PP2C α in eukaryotic cells has been suggested. In human embryo kidney (HEK) 293 cells over expression of PP2C α and PP2C β were highly toxic to the cells and it is not possible to obtain stable cell lines overexpressing PP2C α. The chapter discusses several methods including establishment of inducible PP2C α cells; plating efficiency; antibodies; cell extracts and western blot analysis; XTT assay; and malachite-green assay of protein phosphatase activity.
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