Euphemia Leung

Gene transfer of antisense hypoxia inducible factor-1 α enhances the therapeutic efficacy of cancer immunotherapy

Solid tumors meet their demands for nascent blood vessels and increased glycolysis, to combat hypoxia, by activating multiple genes involved in angiogenesis and glucose metabolism. Hypoxia inducible factor-1 (HIF-1) is a constitutively expressed basic helix–loop–helix transcription factor, formed by the assembly of HIF-1α and HIF-1β (Arnt), that is stablized in response to hypoxia, and rapidly degraded under normoxic conditions. It activates the transcription of genes important for maintaining oxygen homeostasis. Here, we demonstrate that engineered down-regulation of HIF-1α by intratumoral gene transfer of an antisense HIF-1α plasmid leads to the down-regulation of VEGF, and decreased tumor microvessel density. Antisense HIF-1α monotherapy resulted in the complete and permanent rejection of small (0.1 cm in diameter) EL-4 tumors, which is unusual for an anti-angiogenic agent where transient suppression of tumor growth is the norm. It induced NK cell-dependent rejection of tumors, but failed to stimulate systemic T cell-mediated anti-tumor immunity, and synergized with B7–1-mediated immunotherapy to cause the NK cell and CD8 T cell-dependent rejection of larger EL-4 tumors (0.4 cm in diameter) that were refractory to monotherapies. Mice cured of their tumors by combination therapy resisted a rechallenge with parental tumor cells, indicating systemic antitumor immunity had been achieved. In summary, whilst intensive investigations are in progress to target the many HIF-1 effectors, the results herein indicate that blocking hypoxia-inducible pathways and enhancing NK-mediated antitumor immunity by targeting HIF-1 itself may be advantageous, especially when combined with cancer immunotherapy.

Mouse B7-H3 induces antitumor immunity

Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8+ T and NK cells, with no apparent contribution from CD4+ T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.

β7 integrins contribute to demyelinating disease of the central nervous system

A role for alpha4 integrins in different forms of the multiple sclerosis-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated, but the individual contributions of alpha4beta1, alpha4beta7, and the related alphaEbeta7 integrin have not been determined. The P7 integrins alpha4beta7 and alphaEbeta7 play a central role in chronic inflammation, mediating the trafficking, entry, and/or adhesion of lymphocytes in the inflamed pancreas and gut, and their ligands MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial cells and/or on microvessels in the inflamed central nervous system. Here, we show that an antibody directed against the beta7 subunit greatly attenuates a non-remitting form of EAE, induced by adoptive transfer of myelin oligodendrocyte peptide (MOG35-55)-stimulated T cells. Combinational treatment with both anti-beta7 and alpha4 integrin subunit antibodies led to more rapid and complete remission than that obtained with anti-alpha4 antibody alone, potentially implicating a role for alphaEbeta7 in disease progression. Remission correlated with the down-regulation of the vascular addressins VCAM-1. MAdCAM-1, and ICAM-1 on cerebral blood vessels. Attenuated forms of disease were induced by adoptive transfer of either wild-type encephalitogenic T cells to beta7-deficient gene knockout mice, or of beta7-/-encephalitogenic T cells to wild-type recipients. The former finding indicates that beta7 + ve recruited cells contribute to disease progression. Thus alpha4beta1, alpha4beta7, and alphaEbeta7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis.

The β7 integrin gene (Itgb-7) promoter is responsive to TGF-β1: defining control regions

Abstract The β7 integrins LPAM-1 (α4β7) and M290 (αEβ7) mediate the homing of lymphocytes to gut-associated lymphoid tissue, and the proposed retention of intraepithelial lymphocytes (IEL), respectively. Here we show that the gut mucosal cytokine TGF- β1 increases the expression of β7 and αE subunit mRNA transcripts and the cell-surface expression of M290 on T cells, and that it decreases the level of α4 integrin transcripts. Induced β7 integrin gene expression was inhibited by the protein tyrosine kinase inhibitor genistein, implicating a role for tyrosine phosphorylation. An analysis of the β7 integrin gene promoter revealed three DNAse I hypersensitivity sites, two of which mapped to the 5′ and 3′ ends of a promoter fragment (nucleotides +690 to +63) that directed both the basal and the TGF-β1-induced expression of a heterologous reporter gene. Deletion analysis identified two TGF-β1 response regions encompassing nucleotides –509 to –398 (TGFBRR1), and –122 to +32 (TGFBRR2). TGFBRR1 interacted with at least five protein complexes, whose binding could be induced with TGF-β1 stimulation and could be antagonized by TGFBRR2 which harbored both similar and distinctive cis-elements. TGFBRR2 interacted specifically with at least two major nuclear protein complexes, whose binding was phosphorylation dependent. These data provide new insights into the mechanism by which TGF-β may switch LPAM-1+ve migrating T cells to express M290, facilitating their retention in the gut.

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha.

The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1α therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1α and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1α and VHL plasmids. Combination therapy resulted in increased losses of HIF-1α, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1α, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

Cloning of the mucosal addressin MAdCAM-1 from human brain: Identification of novel alternatively spliced transcripts

The mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1). expressed selectively on high endothelial venules (HEV) and lamina propria venules, directs lymphocyte traffic by binding the lymphocyte Peyers patch adhesion molecule‐1 (LPAM‐1, α4β7). Full‐length DNA encoding human MAdCAM‐1 was obtained by combining sequences from an expressed sequence tag (EST) identified in an early stage human brain cDNA library, a polymerase chain reaction‐derived clone, and a MAdCAM‐1 genomic clone. The deduced amino acid sequence revealed an 18 amino acid signal peptide, two N‐terminal immunoglobulin (Ig)‐like domains conserved (59–65%) in sequence with those of the mouse homologue, an 86 amino acid mucin‐like region rich in serine‐threonine residues, a 20 amino acid transmembrane domain and a 43 amino acid charged cytoplasmic domain. No counterpart to the third IgA‐like domain of mouse MAdCAM‐1 was present; however, the serine‐threonine‐rich mucin domain was extended as two distinguishable major and minor mucin regions unrelated to the mouse mucin domain. The major domain is formed from six tandem repeats of an eight amino acid sequence having the MUC‐2‐related consensus DTTSPEP/SP. Human MAdCAM‐1 mRNA transcripts were restricted to small intestine, colon, spleen, pancreas and brain. Alternatively spliced MAdCAM‐1 variants were identified that lack parts of the second Ig domain and all or part of the major mucin domain, indicating that the function of this vascular addressin is regulated by extensive modifications to its multi‐domain structure.

Mucosal vascular addressin cell adhesion molecule-1 is expressed outside the endothelial lineage on fibroblasts and melanoma cells.

Mucosal vascular addressin cell adhesion molecule‐1 (MAdCAM‐1) is predominantly expressed on high endothelial venules in inflamed tissues where it assists with leucocyte extravasation. Here we report that MAdCAM‐1 has the potential to be more widely expressed outside the endothelial cell lineage than previously appreciated. Thus, MAdCAM‐1 RNA transcripts and cell‐surface protein were expressed by NIH 3T3 fibroblasts following activation with tumour necrosis factor‐α (TNF‐α), and by freshly isolated and cultured primary mouse splenic and tail fibroblasts in the absence of TNF‐α stimulation. They were constitutively expressed by B16F10 melanoma cells, and expression was enhanced by cell activation with TNF‐α. Mucosal vascular addressin cell adhesion molecule‐1 was expressed on the apical surface of isolated cells, but became predominantly localized to cell junctions in confluent cell monolayers, suggesting it may play a role in the homotypic aggregation of cells. Tumour necrosis factor‐α enhanced the expression of a firefly luciferase reporter directed by the MAdCAM‐1 promoter in NIH 3T3 and B16F10 cells. A DNA fragment extending from nt −1727 to −673 was sufficient to confer cell‐type selective expression. Mucosal vascular addressin cell adhesion molecule‐1 expressed by NIH 3T3 cells was biologically active, as it supported the adhesion of TK‐1 T cells in an α4β7‐dependent fashion. The expression of MAdCAM‐1 by fibroblasts, and melanomas suggests MAdCAM‐1 may play a role in regulating host responses in the periphery, leucocyte transmigration across nonendothelial boundaries, or the homotypic interactions of some malignant melanomas.

The small GTP-binding proteins Rho and Rac induce T cell adhesion to the mucosal addressin MAdCAM-1 in a hierarchical fashion.

Here we report that an activator (AIF4–) of heterotrimeric GTP‐binding proteins (G‐proteins) and inhibitors (lovastatin and C3 exoenzyme) of small GTP‐binding proteins regulate the induction of α4β7‐mediated adhesion of TK‐1 T lymphoma cells (α4+β7+β1–) to the mucosal addressin cell adhesion molecule MAdCAM‐1. Activation of cell adhesion by AIF4– was abrogated by lovastatin, thereby establishing a link between heterotrimeric G‐proteins and small GTP‐binding proteins in the regulation of α4β7‐mediated cell adhesion. Increased numbers of cells bound MAdCAM‐1‐coated microspheres following activation with AIF4–, discounting an obligatory role for cell spreading in α4β7‐mediated cell adhesion. MAdCAM‐1‐Fc dimers triggered ligand‐induced clustering of α4β7 in response to AIF4– and Mn2+‐induced activation of integrins. Hence α4β7 cluster formation may be responsible, at least in part, for inducing cell adhesion in response to both extracellular and intracellular signals that impact on integrin function. Electroporation of constitutively active V14RhoA and V12Rac1 recombinant proteins into TK‐1 cells revealed that both RhoA and Rac1 induce α4β7 adhesion to MAdCAM‐1. Activation is hierarchical since Rac1 is unable to directly activate α4β7, but induces cell adhesion via RhoA, whereas the transient induction of cell adhesion mediated by RhoA is dependent on the activities of protein tyrosine kinases and protein kinase(s) C.