Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic

Objectives  

IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV).     

Increased lymphocyte infiltration in patients with head and neck cancer treated with the IRX-2 immunotherapy regimen

Twenty-seven subjects with squamous cell cancer of the head and neck received the neoadjuvant IRX-2 immunotherapy regimen prior to surgery in a Phase 2 trial. Pretreatment tumor biopsies were compared with the primary tumor surgical specimens for lymphocyte infiltration, necrosis and fibrosis, using hematoxylin and eosin stain and immunohistochemistry in 25 subjects. Sections were examined by three pathologists. Relative to pretreatment biopsies, increases in lymphocyte infiltration (LI) were seen using H and E or immunohistochemistry. CD3+ CD4+ T cells and CD20+ B cells were primarily found in the peritumoral stroma and CD3+ CD8+ T cells and CD68+ macrophages were mainly intratumoral. LI in the surgical specimens were associated with reductions in the primary tumor size. Improved survival at 5?years was correlated with high overall LI in the tumor specimens. Neoadjuvant IRX-2 immunotherapy regimen may restore immune responsiveness presumably by mobilizing tumor infiltrating effector lymphocytes and macrophages into the tumor.     

TG2, a novel extracellular protein with multiple functions

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca(2+)-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for β integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCα. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.     

New insights in antigen processing and epitope selection: development of novel immunotherapeutic strategies for cancer, autoimmunity and infectious diseases

Current evidence suggests that MHC class II-restricted CD4+ T-cells play a crucial role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Antigens must be processed within endosomal and lysosomal compartments of antigen presenting cells (APC) before binding to MHC class II molecules for display to T-cells. Only a limited number of processed peptides termed immunodominant are selected for display by MHC class II molecules and prove capable of inducing strong T-cell responses. Thus processing reactions within APC are of central importance for the development of effective vaccines as they modulate the number of peptide: class II complexes by enhancing or disrupting epitope formation and display. Studies suggest that there are substantial gaps in our knowledge of how antigen processing and presentation by APC regulates epitope selection and immunodominance in disease situations. Here we describe new insights in antigen processing and epitope selection with relevance to immunotherapeutic strategies for cancer, autoimmunity and infectious diseases.     

hOLFML1, a novel secreted glycoprotein, enhances the proliferation of human cancer cell lines in vitro

We describe a novel secreted protein, named hOLFML1 (human olfactomedin-like protein 1), with an olfectamine domain in its C-terminus, mainly expressed in the small intestine, liver, lung and heart. Immunohistochemical staining on human small intestine indicated that the protein localizes preferentially in the intestinal villi. Interestingly, ectopic hOLFML1 promoted proliferation of HeLa cells and increased the percentage of cells in S phase. In contrast, knock down of hOLFML1 protein expression by siRNA inhibited cell proliferation and delayed the entry of cells into S phase. Our data also revealed that hOLFML1 is N-glycosylated and its secretion is triggered by serum. Taken together, these findings suggest that hOLFML1 may play a significant role in the regulation of cell proliferation in vitro.     

Regulated expression patterns of IRX-2, an Iroquois-class homeobox gene, in the human breast

In the mouse mammary gland, homeobox gene expression patterns suggest roles in development and neoplasia. In the human breast, we now identify a family of Iroquois-class (IRX) homeobox genes. One gene, IRX-2, is expressed in discrete epithelial cell lineages being found in ductal and lobular epithelium, but not in myoepithelium. Expression is absent from associated mesenchymal adipose stroma. During gland development, expression is concentrated in terminal end buds and terminal lobules and is reduced in a subset of epithelial cells during lactation. In contrast to observations for many homeobox genes in the mouse mammary gland in which homeobox gene expression is lost on neoplastic progression, IRX-2 expression is maintained in human mammary neoplasias. Data suggest IRX-2 functions in epithelial cell differentiation and demonstrate regulated expression during ductal and lobular proliferation as well as lactation.     

LcFIN2, a novel chloroplast protein gene from sheepgrass,enhances tolerance to low temperature in Arabidopsis and rice

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA‐sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing‐induced protein 2, encoded a chloroplast‐targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non‐photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal‐related and photosynthesis‐related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low‐temperature tolerance in crops.     

FBXO2, a novel marker for metastasis in human gastric cancer

FBXO2 belongs to the F-box family of proteins, is a cytoplasmic protein and ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins. Recently published studies indicate that other members of the F-box family, such as SKP2 and FBXW7, are involved in the development of gastric cancer. The role of FBXO2 in the process of tumorigenesis, including gastric cancer, is still unknown. In this study, we show that the level of FBXO2 is highly correlated with lymph node metastasis, and that overall survival (OS) of patients with high FBXO2 expression is significantly shorter than patients with low FBXO2 expression. FBXO2 promoted the proliferation and migration of human gastric cancer cells, whereas knockdown of FBXO2 by siRNA led to a decrease in those activities. Down-regulating FBXO2 reduced epithelial-mesenchymal transition (EMT) in gastric cancer cells, with increased expression of E-cadherin and decreased expression of N-cadherin and vimentin. In summary, our findings suggest that FBXO2-regulated EMT led to carcinogenicity in gastric cancer and may be a novel target in the diagnosis and treatment of gastric cancer.     

Aranorosin and a novel derivative inhibit the anti-apoptotic functions regulated by Bcl-2

Bcl-2 is an intracellular membrane protein that prevents cells from undergoing apoptosis in response to various cell-death signals. It negatively regulates mitochondrial outer membrane permeabilization, which is responsible for the release of apoptogenic factors and the subsequent activation of caspases. A microbial metabolite, aranorosin, was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Based on its structure, a more potent derivative, K050, was synthesized. Apoptosis could be induced in a cell line that overexpressed Bcl-2 when cells were treated with an anti-Fas antibody in addition to K050, at sub-micromolar concentrations. Furthermore, K050 inhibited anti-apoptotic functions regulated by Bcl-2, resulting in a Fas-triggered mitochondrial transmembrane potential loss, the activation of caspase-9, and a morphological change to apoptosis. Inhibition of cell-based function of Bcl-2 and its anti-apoptotic effects could serve as useful pharmacological effects. Thus, a novel aranorosin derivative, K050, could be a potent therapeutic agent against Bcl-2-overexpressing human malignancies.     

Group V sPLA2: classical and novel functions

Group V sPLA(2) is unique among the family of secretory sPLA(2) enzymes in being able to bind to cell membranes through both interfacial-binding and through binding to proteoglycan. The function of group V sPLA(2) as an enzyme and its cross-talk with cPLA(2)alpha in initiating eicosanoid generation is well documented. Evidence, though, is emerging on the ability of this molecule to act as a regulator of several intracellular and extracellular pathways independently of its ability to provide arachidonic acid for eicosanoid generation, acting within the cell or as a secreted enzyme. In this article we will provide an overview of the properties of the enzyme and how they relate to our current understanding of its function.     

ROS enhances CXCR4-mediated functions through inactivation of PTEN in prostate cancer cells

Inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is heavily implicated in the tumorigenesis of prostate cancer. Conversely, the upregulation of the chemokine (CXC) receptor 4 (CXCR4) is associated with prostate cancer progression and metastasis. Studies have shown that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells. Loss of PTEN function is typically due to genetic and epigenetic modulations, as well as active site oxidation by reactive oxygen species (ROS); likewise ROS upregulates CXCR4 expression. Herein, we show that ROS accumulation permitted CXCR4-mediated functions through PTEN catalytic inactivation. ROS increased p-AKT and CXCR4 expression, which were abrogated by a ROS scavenger in prostate cancer cells. ROS mediated PTEN inactivation but did not affect expression, yet enhanced cell migration and invasion in a CXCR4-dependent manner. Collectively, our studies add to the body of knowledge on the regulatory role of PTEN in CXCR4-mediated cancer progression, and hopefully, will contribute to the development of therapies that target the tumor microenvironment, which have great potential for the better management of a metastatic disease.     

Glypican-3, a novel prognostic marker of hepatocellular cancer, is related with postoperative metastasis and recurrence in hepatocellular cancer patients

Metastasis/recurrence has been the most fundamental characteristic of hepatocellular cancer (HCC) and the ultimate cause of most HCC-related deaths. However, there are still a limited number of reliable tumor markers that can be used to predict the possibility of metastasis/recurrence in an HCC patient after operation. Recently, much evidence has shown that glypican-3 (GPC3) can be a useful tool to identify the early development of HCC, but little research has been done to test its usefulness as a prognostic marker related to post-operative metastasis/recurrence in HCC patients. In this study, the expression of GPC3 and its relationship with clinicopathological factors were determined by immunohistochemical analysis in 61 primary HCC patients. The potential prognostic value of GPC3 was investigated by comparing the survival time between HCC patients with high and low GPC3 expression. The results demonstrated that GPC3 expression was closely related with metastasis/recurrence in an HCC patient who can receive the operation. The risk of metastasis/recurrence after surgery in an HCC patient with high GPC3 expression was increased to 3.214 as compared to that of an HCC patient with low GPC3 expression. Survival analysis showed that HCC patients with high GPC3 expression had a significantly shorter overall survival time than HCC patients with low GPC3 expression (P = 0.003). Further, multivariate analysis showed that GPC3 expression was a significant, independent prognostic parameter (P = 0.030) for HCC patients. Overall, the study indicates that GPC3 might be a valuable marker closely related with prognosis and post-operative metastasis/recurrence in HCC patients.     

Decrease of PDSS2 expression,a novel tumor suppressor,in non-small cell lung cancer

Background: Prenyl diphosphate synthase subunit 2 (PDSS2) gene has recently been reported as a potential tumor suppressor. The association of PDSS2 and non-small cell lung cancer (NSCLC) has not been known. Methods: To investigate its association with NSCLC, we examined the expression level of PDSS2 in 28 paired clinical samples of non-small cell lung cancer tissues and surrounding normal tissues. Results: PDSS2 was constitutionally expressed in normal lung tissues regardless of sex, race and smoking history. An overall decreased PDSS2 expression was found in the tumor tissues compared to surrounding normal tissues. Decrease in PDSS2 expression was more severe in poorly and poor-to-moderately differentiated lung cancers, while the decrease was not significant in moderately to well-differentiated tumors. Moreover, the expression of PDSS2 decreased more in higher pathological stage, and in patients with lymph node metastasis. The decrease in PDSS2 expression in tumor tissues was not related to sex or histological type of NSCLC, but was related to smoking history. No correlation has been found between PDSS2 and the clinical factors of EGRF, Ki-67 and p53. Conclusion: Taken together, decreased expression of PDSS2 in NSCLC was evident. This is an initial report for the expression of PDSS2 in relation to different factors in lung cancer. Loss of PDSS2 could serve as a potential biomarker in NSCLC development. The role of PDSS2 as a tumor suppressor, and the mechanism of its potential anti-tumor action in NSCLC warrant further investigation.