Disrupting glutamine metabolic pathways to sensitize gemcitabine-resistant pancreatic cancer.

Pancreatic cancer is a lethal disease with poor prognosis. Gemcitabine has been the first line systemic treatment for pancreatic cancer. However, the rapid development of drug resistance has been a major hurdle in gemcitabine therapy leading to unsatisfactory patient outcomes. With the recent renewed understanding of glutamine metabolism involvement in drug resistance and immuno-response, we investigated the anti-tumor effect of a glutamine analog (6-diazo-5-oxo-L-norleucine) as an adjuvant treatment to sensitize chemoresistant pancreatic cancer cells. We demonstrate that disruption of glutamine metabolic pathways improves the efficacy of gemcitabine treatment. Such a disruption induces a cascade of events which impacts glycan biosynthesis through Hexosamine Biosynthesis Pathway (HBP), as well as cellular redox homeostasis, resulting in global changes in protein glycosylation, expression and functional effects. The proteome alterations induced in the resistant cancer cells and the secreted exosomes are intricately associated with the reduction in cell proliferation and the enhancement of cancer cell chemosensitivity. Proteins associated with EGFR signaling, including downstream AKT-mTOR pathways, MAPK pathway, as well as redox enzymes were downregulated in response to disruption of glutamine metabolic pathways.

Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors.

Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.

Peroxiredoxin 1 stimulates endothelial cell expression of VEGF via TLR4 dependent activation of HIF-1α.

Chronic inflammation leads to the formation of a pro-tumorigenic microenvironment that can promote tumor development, growth and differentiation through augmentation of tumor angiogenesis. Prostate cancer (CaP) risk and prognosis are adversely correlated with a number of inflammatory and angiogenic mediators, including Toll-like receptors (TLRs), NF-κB and vascular endothelial growth factor (VEGF). Peroxiredoxin 1 (Prx1) was recently identified as an endogenous ligand for TLR4 that is secreted from CaP cells and promotes inflammation. Inhibition of Prx1 by CaP cells resulted in reduced expression of VEGF, diminished tumor vasculature and retarded tumor growth. The mechanism by which Prx1 regulates VEGF expression in normoxic conditions was investigated in the current study. Our results show that incubation of mouse vascular endothelial cells with recombinant Prx1 caused increases in VEGF expression that was dependent upon TLR4 and required hypoxia inducible factor-1 (HIF-1) interaction with the VEGF promoter. The induction of VEGF was also dependent upon NF-κB; however, NF-κB interaction with the VEGF promoter was not required for Prx1 induction of VEGF suggesting that NF-κB was acting indirectly to induce VEGF expression. The results presented here show that Prx1 stimulation increased NF-κB interaction with the HIF-1α promoter, leading to enhanced promoter activity and increases in HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. Prx1 induced HIF-1 also promoted NF-κB activity, suggesting the presence of a positive feedback loop that has the potential to perpetuate Prx1 induction of angiogenesis. Strikingly, inhibition of Prx1 expression in CaP was accompanied with reduced expression of HIF-1α. The combined findings of the current study and our previous study suggest that Prx1 interaction with TLR4 promotes CaP growth potentially through chronic activation of tumor angiogenesis.

The effect of photodynamic therapy on tumor cell expression of major histocompatibility complex (MHC) class I and MHC class I-related molecules.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) is FDA-approved anti-cancer modality for elimination of early disease and palliation in advanced disease. PDT efficacy depends in part on elicitation of a tumor-specific immune response that is dependent on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytolytic potential of CTLs and NK cells is mediated by the ability of these cells to recognize major histocompatibility complex (MHC) class I and MHC class I-related molecules. The MHC class I-related molecules MICA and MICB are induced by oxidative stress and have been reported to activate NK cells and co-stimulate CD8(+) T cells. The purpose of this study was to examine the effect of PDT on tumor cell expression of MHC classes I and II-related molecules in vivo and in vitro. STUDY DESIGN/MATERIALS AND METHODS: Human colon carcinoma Colo205 cells and murine CT26 tumors were treated with 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH)-PDT at various doses. MHC classes I and I-related molecule expression following treatment of Colo205 cells was temporally examined by flow cytometry using antibodies specific for components of MHC class I molecules and by quantitative PCR using specific primers. Expression of MHC class I-related molecules following HPPH-based PDT (HPPH-PDT) of murine tumors was monitored using a chimeric NKG2D receptor. RESULTS: In vitro HPPH-PDT significantly induces MICA in Colo205 cells, but had no effect on MHC class I molecule expression. PDT also induced expression of NKG2D ligands (NKG2DL) following in vivo HPPH-PDT of a murine tumor. Induction of MICA corresponded to increased NK killing of PDT-treated tumor cells. CONCLUSIONS: PDT induction of MICA on human tumor cells and increased expression of NKG2DL by murine tumors following PDT may play a role in PDT induction of anti-tumor immunity. This conclusion is supported by our results demonstrating that tumor cells have increased sensitivity to NK cell lysis following PDT.

Peroxiredoxin 1 controls prostate cancer growth through Toll-like receptor 4-dependent regulation of tumor vasculature.

In recent years a number of studies have implicated chronic inflammation in prostate carcinogenesis. However, mitigating factors of inflammation in the prostate are virtually unknown. Toll-like receptor 4 (TLR4) activity is associated with inflammation and is correlated with progression risk in prostate cancer (CaP). TLR4 ligands include bacterial cell wall proteins, danger signaling proteins, and intracellular proteins such as heat shock proteins and peroxiredoxin 1 (Prx1). Here we show that Prx1 is overexpressed in human CaP specimens and that it regulates prostate tumor growth through TLR4-dependent regulation of prostate tumor vasculature. Inhibiting Prx1 expression in prostate tumor cells reduced tumor vascular formation and function. Furthermore, Prx1 inhibition reduced levels of angiogenic proteins such as VEGF within the tumor microenvironment. Lastly, Prx1-stimulated endothelial cell proliferation, migration, and differentiation in a TLR4- and VEGF-dependent manner. Taken together, these results implicate Prx1 as a tumor-derived inducer of inflammation, providing a mechanistic link between inflammation and TLR4 in prostate carcinogenesis. Our findings implicate Prx1 as a novel therapeutic target for CaP.

Peroxiredoxin 1 stimulates secretion of proinflammatory cytokines by binding to TLR4.

Peroxiredoxin 1 (Prx1) is an antioxidant and molecular chaperone that can be secreted from tumor cells. Prx1 is overexpressed in many cancers, and elevation of Prx1 is associated with poor clinical outcome. In the current study, we demonstrate that incubation of Prx1 with thioglycollate-elicited murine macrophages or immature bone marrow-derived dendritic cells resulted in TLR4-dependent secretion of TNF-alpha and IL-6 and dendritic cell maturation. Optimal secretion of cytokines in response to Prx1 was dependent upon serum and required CD14 and MD2. Binding of Prx1 to thioglycollate macrophages occurred within minutes and resulted in TLR4 endocytosis. Prx1 interaction with TLR4 was independent of its peroxidase activity and appeared to be dependent on its chaperone activity and ability to form decamers. Cytokine expression occurred via the TLR-MyD88 signaling pathway, which resulted in nuclear translocation and activation of NF-kappaB. These findings suggest that Prx1 may act as danger signal similar to other TLR4-binding chaperone molecules such as HSP72.

Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2.

Human peroxiredoxins 1 and 2, also known as Prx1 and Prx2, are more than 90% homologous in their amino acid sequences. Prx1 and Prx2 are elevated in various cancers and are shown to influence diverse cellular processes. Although their growth regulatory role has traditionally been attributed to the peroxidase activity, the physiological significance of this function is unclear because the proteins are highly susceptible to inactivation by H(2)O(2). A chaperone activity appears to emerge when their peroxidase activity is lost. Structural studies suggest that they may form a homodimer or doughnut-shaped homodecamer. However, little information is available whether human Prx1 and Prx2 are duplicative in structure and function. We noted that Prx1 contains a cysteine (Cys(83)) at the putative dimer-dimer interface, which is absent in Prx2. We studied the role of Cys(83) in regulating the peroxidase and chaperone activities of Prx1, because the redox status of Cys(83) might influence the oligomeric structure and consequently the functions of Prx1. We show that Prx1 is more efficient as a molecular chaperone, whereas Prx2 is better suited as a peroxidase enzyme. Substituting Cys(83) with Ser(83) (Prx1C83S) results in dramatic changes in the structural and functional characteristics of Prx1 in a direction similar to those of Prx2. Here we also report the first crystal structure of human Prx1 and the presence of the Cys(83)-Cys(83) bond at the dimer-dimer interface of decameric Prx1. These findings are consistent with the hypothesis that human Prx1 and Prx2 possess unique functions and regulatory mechanisms and that Cys(83) bestows a distinctive identity to Prx1.