Awais Mughal - Academia.edu (original) (raw)
Papers by Awais Mughal
Molecular Cancer, 2015
Background: Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal p... more Background: Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nα-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets. Methods: Microarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation. Results: While decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1α and altered expression of several other genes involved in the hypoxia response. Furthermore, NAT12/NAA30 knockdown correlated with expressional dysregulation of genes involved in the p53 pathway, ribosomal assembly and cell proliferation. Western blot analysis revealed reduction of HIF1α, phospho-MTOR(Ser2448) and higher levels of p53 and GFAP in these cultures. Conclusion: NAT12/NAA30 plays an important role in growth and survival of GICs possibly by regulating hypoxia response (HIF1α), levels of p-MTOR (Ser2448) and the p53 pathway.
Molecular Cancer Therapeutics, 2011
Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most maligna... more Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most malignant human cancers (median survival 14 months from diagnosis despite combined surgery/irradiation/chemotherapy). Evidence from our and other groups indicates that glioblastoma stem-like cells (GSCs) contained within these tumors are critically important in GBM initiation, progression, and resistance to therapy. To identify new targets for glioblastoma treatment, we have systematically compared gene expression in nine primary GBM cell cultures to that in five primary neural stem cell cultures from the adult human brain (ahNSCs), using microarrays. Rigorous bioinformatic filtering identified 20 genes whose RNA expression levels were very high in primary GSC cultures but were undetectable in ahNSCs. The identified genes are involved in cell-cycle/division, epigenetic regulation, signaling and down-regulation of tumor-suppressors. Several of the candidate genes are implicated in cancer (breast, ovary and colon), while others have no known associations to cancer or have unknown functions.
Molecular Cancer Therapeutics, 2011
ABSTRACT Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the mos... more ABSTRACT Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most malignant human cancers (median survival 14 months from diagnosis despite combined surgery/irradiation/chemotherapy). Evidence from our and other groups indicates that glioblastoma stem-like cells (GSCs) contained within these tumors are critically important in GBM initiation, progression, and resistance to therapy. To identify new targets for glioblastoma treatment, we have systematically compared gene expression in nine primary GBM cell cultures to that in five primary neural stem cell cultures from the adult human brain (ahNSCs), using microarrays. Rigorous bioinformatic filtering identified 20 genes whose RNA expression levels were very high in primary GSC cultures but were undetectable in ahNSCs. The identified genes are involved in cell-cycle/division, epigenetic regulation, signaling and down-regulation of tumor-suppressors. Several of the candidate genes are implicated in cancer (breast, ovary and colon), while others have no known associations to cancer or have unknown functions. A total of nine new GBM primary cultures were further analyzed by real-time polymerase chain reaction (qPCR), Western blot and Immunohistochemistry on tissue sections and cell cultures. qPCR confirmed the increased expression of 17 of the candidate genes. These results were in good accordance with the information provided by public data bases (Rembrant, TCGA). Western blot performed on 17 candidates showed increased protein levels in 9 cases thus demarcating these as best candidates for molecular targeting. The rest of the proteins featured aberrant isoforms, probably due to alternate splicing, modifications, cleavage or degradation. Further bioinformatics analysis identified a subset of 5 candidates that are particularly highly up-regulated in GBM when compared to low-grade gliomas. To explore the functional importance of the potential target genes we established lentiviral-based shRNA delivery and started to test the potential of gene knock-downs (KDs) to inhibit growth of tumor cells. Our preliminary results look very promising. Three different lentiviral vectors, designed to silence a candidate gene encoding a tumor-suppressor binding protein kinase, were tested in three different primary GBM cultures and achieved a KD efficiency of 60-70%. We used proliferation essays to describe the growth speed of GBMs. To estimate relative numbers of sphere-forming cells (widely referred to as cancer stem cells) we performed sphere-forming assays (SFAs). The KD of the protein kinase gene resulted both in dramatic reduction of growth speed (50-70%) and in the number of sphere-forming cells (50-70%). In addition, the volume of the spheres was reduced to 50%. Cell cycle analysis revealed that there was doubling of the number of KD cells arrested in G2/M phase compared to non-silencing controls. There was also an increase (20-65%) in the proportion of apoptotic cells in 2 out of 3 GBM KD cultures.
Molecular Cancer, 2015
Background: Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal p... more Background: Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nα-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets. Methods: Microarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation. Results: While decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1α and altered expression of several other genes involved in the hypoxia response. Furthermore, NAT12/NAA30 knockdown correlated with expressional dysregulation of genes involved in the p53 pathway, ribosomal assembly and cell proliferation. Western blot analysis revealed reduction of HIF1α, phospho-MTOR(Ser2448) and higher levels of p53 and GFAP in these cultures. Conclusion: NAT12/NAA30 plays an important role in growth and survival of GICs possibly by regulating hypoxia response (HIF1α), levels of p-MTOR (Ser2448) and the p53 pathway.
Molecular Cancer Therapeutics, 2011
Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most maligna... more Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most malignant human cancers (median survival 14 months from diagnosis despite combined surgery/irradiation/chemotherapy). Evidence from our and other groups indicates that glioblastoma stem-like cells (GSCs) contained within these tumors are critically important in GBM initiation, progression, and resistance to therapy. To identify new targets for glioblastoma treatment, we have systematically compared gene expression in nine primary GBM cell cultures to that in five primary neural stem cell cultures from the adult human brain (ahNSCs), using microarrays. Rigorous bioinformatic filtering identified 20 genes whose RNA expression levels were very high in primary GSC cultures but were undetectable in ahNSCs. The identified genes are involved in cell-cycle/division, epigenetic regulation, signaling and down-regulation of tumor-suppressors. Several of the candidate genes are implicated in cancer (breast, ovary and colon), while others have no known associations to cancer or have unknown functions.
Molecular Cancer Therapeutics, 2011
ABSTRACT Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the mos... more ABSTRACT Glioblastoma multiformae (GBM) is the most common primary brain tumor and one of the most malignant human cancers (median survival 14 months from diagnosis despite combined surgery/irradiation/chemotherapy). Evidence from our and other groups indicates that glioblastoma stem-like cells (GSCs) contained within these tumors are critically important in GBM initiation, progression, and resistance to therapy. To identify new targets for glioblastoma treatment, we have systematically compared gene expression in nine primary GBM cell cultures to that in five primary neural stem cell cultures from the adult human brain (ahNSCs), using microarrays. Rigorous bioinformatic filtering identified 20 genes whose RNA expression levels were very high in primary GSC cultures but were undetectable in ahNSCs. The identified genes are involved in cell-cycle/division, epigenetic regulation, signaling and down-regulation of tumor-suppressors. Several of the candidate genes are implicated in cancer (breast, ovary and colon), while others have no known associations to cancer or have unknown functions. A total of nine new GBM primary cultures were further analyzed by real-time polymerase chain reaction (qPCR), Western blot and Immunohistochemistry on tissue sections and cell cultures. qPCR confirmed the increased expression of 17 of the candidate genes. These results were in good accordance with the information provided by public data bases (Rembrant, TCGA). Western blot performed on 17 candidates showed increased protein levels in 9 cases thus demarcating these as best candidates for molecular targeting. The rest of the proteins featured aberrant isoforms, probably due to alternate splicing, modifications, cleavage or degradation. Further bioinformatics analysis identified a subset of 5 candidates that are particularly highly up-regulated in GBM when compared to low-grade gliomas. To explore the functional importance of the potential target genes we established lentiviral-based shRNA delivery and started to test the potential of gene knock-downs (KDs) to inhibit growth of tumor cells. Our preliminary results look very promising. Three different lentiviral vectors, designed to silence a candidate gene encoding a tumor-suppressor binding protein kinase, were tested in three different primary GBM cultures and achieved a KD efficiency of 60-70%. We used proliferation essays to describe the growth speed of GBMs. To estimate relative numbers of sphere-forming cells (widely referred to as cancer stem cells) we performed sphere-forming assays (SFAs). The KD of the protein kinase gene resulted both in dramatic reduction of growth speed (50-70%) and in the number of sphere-forming cells (50-70%). In addition, the volume of the spheres was reduced to 50%. Cell cycle analysis revealed that there was doubling of the number of KD cells arrested in G2/M phase compared to non-silencing controls. There was also an increase (20-65%) in the proportion of apoptotic cells in 2 out of 3 GBM KD cultures.