Inhibition of GSK-3 induces differentiation and impaired glucose metabolism in renal cancer

Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases including Type II diabetes, Alzheimer's disease, bipolar disorder, inflammation and cancer. Consequently it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacological and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent anti-proliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The anti-proliferative activity is most likely caused by G0-G1 and G2-M phase arrest as evident from cell cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. Additionally, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft RCC tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells

Neuropilin-1 Modulates p53/Caspases Axis to Promote Endothelial Cell Survival

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets

Genetic status of KRAS modulates the role of Neuropilin-1 in tumorigenesis

Abstract Neuropilin-1 (NRP1), a non–tyrosine kinase receptor, is overexpressed in many cancers including pancreatic and lung cancers. Inhibition of NRP1 expression, however, has differing pro-tumor vs. anti-tumor effects, depending on the cancer types. To understand the differential role of NRP1 in tumorigenesis process, we utilized cells from two different cancer types, pancreatic and lung, each containing either wild type KRAS (KRAS wt) or mutant KRAS (KRAS mt). Inhibition of NRP1 expression by shRNA in both pancreatic and lung cancer cells containing dominant active KRAS mt caused increased cell viability and tumor growth. On the contrary, inhibition of NRP1, in the tumor cells containing KRAS wt showed decreased tumor growth. Importantly, concurrent inhibition of KRAS mt and NRP1 in the tumor cells reverses the increased viability and leads to tumor inhibition. We found that NRP1 shRNA expressing KRAS mt tumor cells caused increased cell viability by decreasing SMAD2 phosphorylation. Our findings demonstrate that the effects of NRP1 knockdown in cancer cells are dependent on the genetic status of KRAS

Chemically Modified Peptides Targeting the PDZ Domain of GIPC as a Therapeutic Approach for Cancer

GIPC (GAIP-interacting protein, C terminus) represents a new target class for the discovery of chemotherapeutics. While many of the current generation of anticancer agents function by directly binding to intracellular kinases or cell surface receptors, the disruption of cytosolic protein–protein interactions mediated by non-enzymatic domains is an underdeveloped avenue for inhibiting cancer growth. One such example is the PDZ domain of GIPC. Previously we developed a molecular probe, the cell-permeable octapeptide CR1023 (<i>N</i>-myristoyl-PSQSSSEA), which diminished proliferation of pancreatic cancer cells. We have expanded upon that discovery using a chemical modification approach and here report a series of cell-permeable, side chain-modified lipopeptides that target the GIPC PDZ domain <i>in vitro</i> and <i>in vivo</i>. These peptides exhibit significant activity against pancreatic and breast cancers, both in cellular and animal models. CR1166 (<i>N</i>-myristoyl-PSQSK­(ε<i>N</i>-4-bromobenzoyl)­SK­(ε<i>N</i>-4-bromobenzoyl)­A), bearing two halogenated aromatic units on alternate side chains, was found to be the most active compound, with pronounced down-regulation of EGFR/1GF-1R expression. We hypothesize that these organic acid-modified residues extend the productive reach of the peptide beyond the canonical binding pocket, which defines the limit of accessibility for the native proteinogenic sequences that the PDZ domain has evolved to recognize. Cell permeability is achieved with <i>N</i>-terminal lipidation using myristate, rather than a larger CPP (cell-penetrating peptide) sequence. This, in conjunction with optimization of targeting through side chain modification, has yielded an approach that will allow the discovery and development of next-generation cellular probes for GIPC PDZ as well as for other PDZ domains

Inhibition of endoglin-GIPC interaction inhibits pancreatic cancer cell growth

Endoglin, a 180-kDa disulfide-linked homodimeric transmembrane receptor protein mostly expressed in tumor-associated endothelial cells, is an endogenous binding partner of GAIP-interacting protein, C terminus (GIPC). Endoglin functions as a coreceptor of T&#946;RII that binds TGF&#946; and is important for vascular development and consequently has become a compelling target for antiangiogenic therapies. A few recent studies in Gastrointestinal Stromal Tumor (GIST), breast cancer and ovarian cancer, however, suggest that endoglin is upregulated in tumor cells and is associated with poor prognosis. These findings indicate a broader role of endoglin in tumor biology, beyond angiogenic effects. The goal of our current study is to evaluate the effects of targeting endoglin in pancreatic cancer both in vitro and in vivo. We analyzed the antiproliferative effect of both RNAi-based and peptide ligand-based inhibition of endoglin in pancreatic cancer cell lines, the latter yielding a GIPC PDZ domain-targeting lipopeptide with notable antiproliferative activity. We further demonstrated that endoglin inhibition induced a differentiation phenotype in the pancreatic cancer cells and sensitized them against conventional chemotherapeutic drug gemcitabine. Most importantly, we have demonstrated the antitumor effect of both RNAi-based and competitive inhibitor–based blocking of endoglin in pancreatic cancer xenograft models in vivo. To our knowledge, this is the first report exploring the effect of targeting endoglin in pancreatic cancer cells