Insulin Growth Factor-2 Binding Protein 3 (IGF2BP3) Is a Glioblastoma-specific Marker That Activates Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase (PI3K/MAPK) Pathways by Modulating IGF-2

Glioblastoma is the most common and malignant form of primary astrocytoma. Upon investigation of the insulin-like growth factor (IGF) pathway, we found the IGF2BP3/IMP3 transcript and protein to be up-regulated in GBMs but not in lower grade astrocytomas (p<0.0001). IMP3 is an RNA binding protein known to bind to the 5'-untranslated region of IGF-2 mRNA, thereby activating its translation. Overexpression-and knockdown-based studies establish a role for IMP3 in promoting proliferation, anchorage-independent growth, invasion, and chemoresistance. IMP3 overexpressing B16F10 cells also showed increased tumor growth, angiogenesis, and metastasis, resulting in poor survival in a mouse model. Additionally, the infiltrating front, perivascular, and subpial regions in a majority of the GBMs stained positive for IMP3. Furthermore, two different murine glioma models were used to substantiate the above findings. In agreement with the translation activation functions of IMP3, we also found increased IGF-2 protein in the GBM tumor samples without a corresponding increase in its transcript levels. Also, in vitro IMP3 overexpression/knockdown modulated the IGF-2 protein levels without altering its transcript levels. Additionally, IGF-2 neutralization and supplementation studies established that the proproliferative effects of IMP3 were indeed mediated through IGF-2. Concordantly, PI3K and MAPK, the downstream effectors of IGF-2, are activated by IMP3 and are found to be essential for IMP3-induced cell proliferation. Thus, we have identified IMP3 as a GBM-specific proproliferative and proinvasive marker acting through IGF-2 resulting in the activation of oncogenic PI3K and MAPK pathways.

Cooperative Regulation of NOTCH1 Protein-Phosphatidylinositol 3-Kinase (PI3K) Signaling by NOD1, NOD2, and TLR2 Receptors Renders Enhanced Refractoriness to Transforming Growth Factor-beta (TGF-beta)- or Cytotoxic T-lymphocyte Antigen 4 (CTLA-4)-mediated Impairment of Human Dendritic Cell Maturation

Dendritic cells (DCs) as sentinels of the immune system are important for eliciting both primary and secondary immune responses to a plethora of microbial pathogens. Cooperative stimulation of a complex set of pattern-recognition receptors, including TLR2 and nucleotide-binding oligomerization domain (NOD)-like receptors on DCs, acts as a rate-limiting factor in determining the initiation and mounting of the robust immune response. It underscores the need for ``decoding'' these multiple receptor interactions. In this study, we demonstrate that TLR2 and NOD receptors cooperatively regulate functional maturation of human DCs. Intriguingly, synergistic stimulation of TLR2 and NOD receptors renders enhanced refractoriness to TGF-beta- or CTLA-4-mediated impairment of human DC maturation. Signaling perturbation data suggest that NOTCH1-PI3K signaling dynamics assume critical importance in TLR2- and NOD receptor-mediated surmounting of CTLA-4- and TGF-beta -suppressed maturation of human DCs. Interestingly, the NOTCH1-PI3K signaling axis holds the capacity to regulate DC functions by virtue of PKC delta-MAPK-dependent activation of NF-kappa B. This study provides mechanistic and functional insights into TLR2-and NOD receptor-mediated regulation of DC functions and unravels NOTCH1-PI3K as a signaling cohort for TLR2 and NOD receptors. These findings serve in building a conceptual foundation for the design of improved strategies for adjuvants and immunotherapies against infectious diseases.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with beta-catenin in breast cancer lymph node metastasis

Background: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of beta-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for beta-catenin and IGFBP2 expression. Results: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of beta-catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of beta-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and beta-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion: This study highlights regulation of beta-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and beta-catenin is associated with lymph node metastasis of breast tumors.

Insulin like growth factor binding protein 4 promotes GBM progression and regulates key factors involved in EMT and invasion

Insulin like growth factor binding protein 4 (IGFBP4) regulates growth and development of tissues and organs by negatively regulating IGF signaling. Among most cancers, IGFBP4 has growth inhibitory role and reported as a down-regulated gene, except for renal cell carcinoma, wherein IGFBP4 promotes tumor progression. IGFBP4 expression has been shown to be higher in increasing grades of astrocytoma. However, the functional role of IGFBP4 in gliomas has not been explored. Surgical biopsies of 20 normal brain and 198 astrocytoma samples were analyzed for IGFBP4 expression by qRT-PCR. Highest expression of IGFBP4 mRNA was seen in GBM tumors compared to control brain tissues (median log2 of 2.035, p < 0.0001). Immunohistochemical analysis of 53 tissue samples revealed predominant nuclear staining of IGFBP4, seen maximally in GBMs when compared to DA and AA tumors (median LI = 29.12 +/- A 16.943, p < 0.001). Over expression of IGFBP4 in U343 glioma cells resulted in up-regulation of molecules involved in tumor growth, EMT and invasion such as pAkt, pErk, Vimentin, and N-cadherin and down-regulation of E-cadherin. Functionally, IGFBP4 over expression in these cells resulted in increased proliferation, migration and invasion as assessed by MTT, transwell migration, and Matrigel invasion assays. These findings were confirmed upon IGFBP4 knockdown in U251 glioma cells. Our data suggest a pro-tumorigenic role for IGFBP4 in glioma.

Insulin-Like Growth Factor System in Cancer: Novel Targeted Therapies

Insulin-like growth factors (IGFs) are essential for growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis, and metastatic activities in various cancers. The IGFs actions are mediated through the IGF-1 receptor that is involved in cell transformation induced by tumour. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). We describe here the role of the IGF system in cancer, proposing new strategies targeting this system. We have attempted to expand the general viewpoint on IGF-1R, its inhibitors, potential limitations of IGF-1R, antibodies and tyrosine kinase inhibitors, and IGFBP actions. This review discusses the emerging view that blocking IGF via IGFBP is a better option than blocking IGF receptors. This can lead to the development of novel cancer therapies.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-alpha, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKC delta-MAPK pathway to suppress beta-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.