Joyce C. Solheim

Down-Regulation of Vascular Endothelial Cell Growth Factor-C Expression Using Small Interfering RNA Vectors in Mammary Tumors Inhibits Tumor Lymphangiogenesis and Spontaneous Metastasis and Enhances Survival

Tumor production of vascular endothelial cell growth factor (VEGF)-C is associated with tumor lymphangiogenesis and lymph node metastasis. In this study, we examined the effects of small interfering RNA (siRNA)-mediated inhibition of VEGF-C on murine mammary tumor growth, metastasis, and survival. The mRNA and protein expression of VEGF-C in murine mammary tumor cells stably transfected with a VEGF-C siRNA vector were significantly lower compared with VEGF-C-control vector-transfected cells. Cl66-siVEGFC tumors had lower levels of lymphangiogenesis and lymph node and spontaneous lung metastasis than Cl66-control tumors. However, we did not observe significant differences in primary tumor growth and experimental lung metastasis between mice injected with Cl66-siVEGFC and Cl66-control cells. In addition, mice bearing Cl66-siVEGFC tumors lived significantly longer than mice bearing Cl66-control tumors. Furthermore, our data suggest that inhibition of VEGF-C modulates immune cell infiltration and their function, which might be critical in tumor immunity. In summary, our data show that inhibition of VEGF-C expression using siRNA-mediated gene silencing vectors reduces lymphangiogenesis and lymph node and spontaneous lung metastasis, and enhances survival.

Class I MHC molecules: Assembly and antigen presentation

Summary: Several years ago, the only factor known to be necessary for the assembly and surface expression of class I MHC was pjm; even for β2m, it was unclear at what point in class I maturation its role was played. Recent experiments that employed attachment of an endoplasmic reticulum (ER) retention signal to β2m have shown that the point of time at which β2m is required is while the class I heavy chain is in the ER. Later association between β2m and class I is not vital in order for properly folded class I to be expressed at β2m cell surface. After crystallization of the first class I MHC molecule, it was reahed that not only is antigen presented by class I, but that antigen is presented in the form of a peptide that stabilizes the class I structure and allows its transit to the cell surface. Class I allelic differences influence interactions with both peptide and β2m, with likely consequences for the ability of the class I heavy chains to present antigen through alternative pathways. Furthermore, it is now also clear that formation of appropriate disulfide bonds in the class I heavy chain is needed before class I can bind peptide antigen securely, a process that may he assisted by an ER chaperone. Many different proteins that are resident in the ER, such as calnexin, transporter associated with antigen processing (TAP), caheticulin, and tapasin, have been found to be integral to class I assembly. TAP, tapasin, and calreticulin hind preferentially to the open form of class I, which can be distinguished with the use of a monoclonal antibody specific for this form. Calreticulin and calnexin contrast in their interactions with class I, despite other similarities between these two chaperones. Overall, class I MHC assembly is now understood to involve the interplay of multiple intra‐ and intermolecular events in a defined chronological order which ensure continual reporting of cellular contents to cytotoxic T lymphocytes.

Virus Evasion of MHC Class I Molecule Presentation

The MHC class I H chain and the L chain that assembles with it, β2-microglobulin (β2m),[3][1] are cotranslationally inserted into the lumen of the endoplasmic reticulum (ER) ([1][2]). In the ER, the MHC/β2m heterodimer binds peptide that is generated by proteasomal protein degradation in the

Mammary tumor heterogeneity in the expansion of myeloid-derived suppressor cells

The tumor microenvironment is heterogeneous for the expansion and infiltration by myeloid derived suppressor cells (MDSCs) which has been hypothesized to be dependent on tumor burden. We report a relationships between tumor size, MDSCs and T-cells; using four murine mammary tumors to assess tumor growth, infiltration and gene expression. Our analysis of cellular infiltration into tumors and gene expression used collagenase dissociated tumors and density gradient isolation of non-parenchymal cells (NPCs). The frequency of splenic and peripheral blood (PB) MDSCs was tumor dependent resulting in a significantly increased number of MDSCs. The MDSC frequency inversely correlated with the frequency of CD3+ lymphocytes in the spleen, independent of the tumor studied and directly correlated with tumor burden. Tumor growth up-regulated cyclooxygenase-2 (COX-2), vascular endothelial growth factor-A (VEGF-A), granulocyte (G-) and granulocyte-monocyte-colony stimulating factor (GM-CSF), arginase-1 (ARG-1), and nitric oxide synthase-2 (NOS-2) transcription in the tumor and spleens (not VEGF-A). The frequency of splenic MDSCs directly correlated with splenic COX-2, NOS-2, and ARG-1 message levels, while COX-2 and NOS-2 transcript levels inversely correlated with splenic CD3+ cell frequency. COX-2 mRNA levels also directly correlated with the ARG-1 and NOS-2 transcript levels from tumor-infiltrating leukocytic cells, supporting prostaglandin E2 as a regulator of ARG-1 and NOS-2 transcription. In summary, MDSC numbers in the spleen and tumor microenvironment are tumor dependent, directly correlating with tumor size and inversely correlating with T-cell number. MDSCs are also directly associated with VEGF-A and G-CSF transcript levels suggesting multiple mechanisms for MDSC regulation and COX-2, NOS-2 and ARG-1 supporting multiple mechanisms of T-cell suppression.

HLA-B polymorphism affects interactions with multiple endoplasmic reticulum proteins.

To explore the nature of amino acid substitutions that influence association with TAP, we compared a site‐directed mutant of HLA‐B*0702 (Y116D) to unmutated HLA‐B7 in regard to TAP interaction. We found that the mutant had stronger association with TAP, and, in addition, with tapasin and calreticulin. These data confirm the importance of position 116 for TAP association, and indicate that (1) an aspartic acid at the 116 position can facilitate the interaction, and (2) association with tapasin and calreticulin is affected along with TAP. Furthermore, we tested three natural subtypes of HLA‐B15, and found that a B15 subtype with a tyrosine at position 116 (B*1510) was strongly associated not only with TAP, but also with tapasin and calreticulin. In contrast, two B15 subtypes with a serine at position 116 (B*1518 and B*1501) exhibited very little or no association with any of these proteins. Thus, very closely related HLA‐B subtypes can differ in regard to interaction with the entire assembly complex. Interestingly, when their surface expression was tested by flow cytometry, the HLA‐B15 subtypes with little to no detectable intracellular assembly complex association had a slightly, yet consistently, higher level of the open heavy chain form than did the B15 subtype with intracellular assembly complex association. These data suggest that the relatively low strength or short length of interaction between endoplasmic reticulum proteins and natural HLA class I molecules can decrease their surface stability.

HLA class I polymorphism has a dual impact on ligand binding and chaperone interaction.

This article will describe coordinated analyses of how amino acid substitutions in the HLA class I antigen binding groove modify chaperone interaction and peptide ligand presentation. By parallel testing of ligand presentation and chaperone interaction with a series of natural HLA-B subtypes, this study has discovered that position 116 of the HLA-B15 class I heavy chain is pivotal in both peptide selection and control of interaction between the assembly complex and the class I heavy chain. Correlated with these qualitative differences in peptide selection and chaperone association are quantitative differences in the expression levels of the HLA molecules at the cell surface. These parallel studies, therefore, demonstrate that particular HLA class I polymorphisms can simultaneously influence ligand presentation and interaction with intracellular chaperones.

Characterization of class I MHC folding intermediates and their disparate interactions with peptide and β2-microglobulin

Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin (beta 2m), and bind peptide ligand to complete the trimeric complex. Although each of these initial events is thought to be critical for class I folding, their sequential order and effect on class I structure are unknown. Using mAb specific for distinct conformations of H-2Ld and Lq, we have defined folding intermediates of class I molecules. We show here that non-peptide-associated forms of Ld or Lq, detected by mAb 64-3-7 and designated L alt, lack numerous conformational epitopes surrounding their ligand binding sites. These results support the notion that L alt molecules have an open conformation. Interestingly, a significant proportion of L alt molecules were detected in association with beta 2m and these L alt/beta 2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain/beta 2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the Ld/beta 2m/peptide trimeric complex in vivo. The characteristics of L alt molecules were investigated further by ascertaining the disulfide bond status of these molecules and their association with beta 2m and peptide. Treatment of cells with dithiothreitol (DTT), a membrane-permeable reducing agent, demonstrated that L alt molecules constitute a heterogeneous population including reduced, partially reduced and native class I molecules. Furthermore, partially reduced Ld alt molecules, in a cell line expressing a mutant Ld molecule lacking the alpha 2 domain disulfide bond, accumulated intracellularly, were not beta 2m-associated and displayed marginal peptide-induced folding in vitro. In accordance with this latter finding, peptide was found to preferentially convert fully disulfide-bonded forms of Ld alt to conformed Ld. Thus, we propose that intrachain disulfide bond formation precedes the association of class I heavy chain with beta 2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.

Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice.

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147–579) and 329 (161–523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4+ T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-γ, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.

A Charged Amino Acid Residue in the Transmembrane/Cytoplasmic Region of Tapasin Influences MHC Class I Assembly and Maturation

Tapasin influences the quantity and quality of MHC/peptide complexes at the cell surface; however, little is understood about the structural features that underlie its effects. Because tapasin, MHC class I, and TAP are transmembrane proteins, the tapasin transmembrane/cytoplasmic region has the potential to affect interactions at the endoplasmic reticulum membrane. In this study, we have assessed the influence of a conserved lysine at position 408, which lies in the tapasin transmembrane/cytoplasmic domain. We found that substitutions at position K408 in tapasin affected the expression of MHC class I molecules at the cell surface, and down-regulated tapasin stabilization of TAP. In addition to affecting TAP interaction with tapasin, the substitution of alanine, but not tryptophan, for the lysine at tapasin position 408 increased the amount of tapasin found in association with the open, peptide-free form of the HLA-B8 H chain. Tapasin K408A was also associated with more folded, β2-microglobulin-assembled HLA-B8 molecules than wild-type tapasin. Consistent with our observation of a large pool of tapasin K408A-associated HLA-B8 molecules, the rate at which HLA-B8 migrated from the endoplasmic reticulum was slower in tapasin K408A-expressing cells than in wild-type tapasin-expressing cells. Thus, the alanine substitution at position 408 in tapasin may interfere with the stable acquisition by MHC class I molecules of peptides that are sufficiently optimal to allow MHC class I release from tapasin.

Active YAP promotes pancreatic cancer cell motility, invasion and tumorigenesis in a mitotic phosphorylation-dependent manner through LPAR3

The transcriptional co-activator Yes-associated protein, YAP, is a main effector in the Hippo tumor suppressor pathway. We recently defined a mechanism for positive regulation of YAP through CDK1-mediated mitotic phosphorylation. Here, we show that active YAP promotes pancreatic cancer cell migration, invasion and anchorage-independent growth in a mitotic phosphorylation-dependent manner. Mitotic phosphorylation is essential for YAP-driven tumorigenesis in animals. YAP reduction significantly impairs cell migration and invasion. Immunohistochemistry shows significant upregulation and nuclear localization of YAP in metastases when compared with primary tumors and normal tissue in human. Mitotic phosphorylation of YAP controls a unique transcriptional program in pancreatic cells. Expression profiles reveal LPAR3 (lysophosphatidic acid receptor 3) as a mediator for mitotic phosphorylation-driven pancreatic cell motility and invasion. Together, this work identifies YAP as a novel regulator of pancreatic cancer cell motility, invasion and metastasis, and as a potential therapeutic target for invasive pancreatic cancer.