Identification of cancer stem cell markers in human malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24⁺ cells proliferated by asymmetric cell division-like manner. In addition, CD9⁺ and CD24⁺ cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases.     

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.     

Activation of matrix metalloproteinase-26 by HOXA10 promotes embryo adhesion in vitro

Successful embryonic implantation requires an effective maternal–embryonic molecular dialogue. However, the detailed mechanisms of epithelial-embryo adhesion remain poorly understood. Here, we report that matrix metalloproteinase-26 (MMP-26) is a novel downstream target gene of homeobox a 10 (HOXA10) in human endometrial cells. HOXA10 binds directly to a conserved TTAT unit (−442 to −439) located within the 5′ regulatory region of the MMP-26 gene and regulates the expression and secretion of MMP-26 in a concentration-dependent manner. Moreover, the adenovirus-mediated overexpression of MMP-26 in Ishikawa cells markedly increased BeWo spheroid adhesion. An antibody blocking assay further demonstrated that the promotion of BeWo spheroid adhesion by HOXA10 and MMP-26 was significantly inhibited by pre-treatment with a specific antibody against MMP-26. These results demonstrate that the HOXA10-mediated expression of MMP-26 promotes embryo adhesion during the process of embryonic implantation.     

Post-Translational Regulation of CD133 by ATase1/ATase2-Mediated Lysine Acetylation

The CD133 cell-surface protein expresses the AC133 epitope that is associated with cancer progenitor cells and cancer resistance to traditional anticancer therapies. We report that the endoplasmic reticulum Golgi intermediate compartment residing acetyltransferases, ATase1 (NAT8B) and ATase2 (NAT8), can physically interact with CD133 to acetylate the protein on three lysine residues predicted to reside on the first extracellular loop of CD133. Site-directed mutagenesis of these residues mimicking a loss of acetylation and downregulation or inhibition of ATase1/ATase2 resulted in near-complete abolishment of CD133 protein expression. We also demonstrate that targeting ATase1/ATase2 results in apoptosis of CD133 expressing acute lymphoblastic leukemia cells. Taken together, we suggest that lysine acetylation on predicted extracellular residues plays a key role in expression and trafficking of CD133 protein to the cell surface and can be targeted to disrupt CD133 regulation and function.     

CD26/DPPIV cell membrane expression and DPPIV activity in plasma of patients with acute leukemia

CD26/DPPIV (dipeptidil peptidase IV) displays an array of diverse functional properties, with a role in the development of several human cancers. This enzyme is found mainly anchored in the membrane of cells although it also has an enzymatically active plasma isoform. The regulation of biological activities of cytokines by DPP IV activity has a potential role in the homeostatic regulation of hematopoiesis. In this study, we analyzed the CD26 antigen cell membrane expression by flow cytometry and the DPPIV activity in plasma of patients of acute leukemia. The results showed that the plasma DPPIV activity is significantly higher in leukemia patients and could be 100% inhibited by Januvia? (Merck Sharp & Dohme) a selective DPPIV inhibitor. Although CD26 expression on immune cells were not leukemia-dependent the analysis of the correlation between CD26 expression and the DPPIV plasma activity were statistically significant (p < 0.01) in acute lymphoid leukemia (B-ALL and T-ALL).     

Neuroimmunomodulative properties of dipeptidyl peptidase IV/CD26 in a TNBS-induced model of colitis in mice

Causal connections between dipeptidyl peptidase IV, also known as CD26 molecule (DPP IV/CD26) and inflammatory bowel disease (IBD) have been shown, but mechanisms of these interactions are unclear. Our hypothesis was that DPP IV/CD26 could affect the neuroimmune response during inflammatory events. Therefore, we aimed to evaluate its possible role and the relevance of the gut-brain axis in a model of IBD in mice. Trinitrobenzenesulfonic acid-induced (TNBS) colitis was induced in CD26-deficient (CD26(-/-) ) and wild-type (C57BL/6) mice. Pathohistological and histomorphometrical measurements were done. Concentrations and protein expressions of DPP IV/CD26 substrates neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined. Concentrations of IL-6 and IL-10 were evaluated. Investigations were conducted at systemic and local levels. Acute inflammation induced increased serum NPY concentrations in both mice strains, more enhanced in CD26(-/-) mice. Increased NPY concentrations were found in colon and brain of C57BL/6 mice, while in CD26(-/-) animals only in colon. VIP and IL-6 serum and tissue concentrations were increased in both mice strains in acute inflammation, more pronouncedly in CD26(-/-) mice. IL-10 concentrations, after a decrease in serum of both mice strains, increased promptly in CD26(-/-) mice. Decreased IL-10 concentration was found in brain of C57BL/6 mice, while it was increased in colon of CD26(-/-) mice in acute inflammation. DPP IV/CD26 deficiency affects the neuroimmune response at systemic and local levels during colitis development and resolution in mice. Inflammatory changes in the colon reflected on investigated parameters in the brain, suggesting an important role of the gut-brain axis in IBD pathogenesis.     

Expression of the Stem Cell Factor Nestin in Malignant Pleural Mesothelioma Is Associated with Poor Prognosis

Background

The epithelioid and sarcomatoid histologic variants of malignant pleural mesothelioma (MPM) can be considered as E- and M-parts of the epithelial-mesenchymal transition (EMT) axis; the biphasic being an intermediate. EMT is associated with an increase of stem cell (SC) traits. We correlated the neural crest SC marker nestin and the EMT marker periostin with histology, type of neo-adjuvant chemotherapy (CT) and overall survival (OS) of MPM patients.

Patients and Methods

Tumor tissues of a historic cohort 1 (320 patients) and an intended induction chemotherapy followed by extrapleural pneumonectomy (EPP) cohort 2 (145 patients) were immunohistochemically H-scored (intensity of immunoreactivity multiplied by frequency of stained cells). Paired chemo-naïve biopsies and -treated surgical specimens were available for 105/145 patients. CT included platinum/gemcitabine (Pla/Gem) or platinum/pemetrexed (Pla/Pem).

Results

Expression of any cytosolic nestin progressively increased from epithelioid to biphasic to sarcomatoid MPM in cohort 1, whereas the diagnostic markers calretinin and podoplanin decreased. In cohort 2, Pla/Pem CT increased the expression level of nestin in comparison to Pla/Gem, whereas the opposite was found for periostin. In Pla/Pem treated patients, nestin was higher in biphasic MPM compared to epithelioid. In addition to non-epithelioid histology, any expression of nestin in chemo-naïve biopsies (median overall survival: 22 vs. 17 months) and chemo-treated surgical specimens (18 vs. 12 months) as well as high periostin in biopsies (23 vs. 15 months) were associated with poor prognosis. In the multivariate survival analysis, any nestin expression in chemo-naïve biopsies proved to be an independent prognosticator against histology. In both pre- and post-CT situations, the combination of nestin or periostin expression with non-epithelioid histology was particularly/ dismal (all p-values <0.05).

Conclusions

The SC marker nestin and the EMT marker periostin allow for further prognostic stratification among histologic variants of MPM. Their expression level is influenced by neo-adjuvant chemotherapy.     

Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26

Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2epsilon, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2epsilon to CD26 on the cell surface induces a Ca(2+) signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2epsilon binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2epsilon. As a result, the plasminogen 2epsilon-induced Ca(2+) signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel by 1-LN cells is blocked. Angiostatin 2epsilon is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2epsilon may also directly block tumor metastasis.     

Structural details of monoclonal antibody m971 recognition of the membrane-proximal domain of CD22

Cluster of differentiation-22 (CD22) belongs to the sialic acid–binding immunoglobulin (Ig)-like lectin family of receptors that is expressed on the surface of B cells. It has been classified as an inhibitory coreceptor for the B-cell receptor because of its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function make it an attractive target for B-cell depletion in cases of B-cell malignancies. Genetically modified T cells with chimeric antigen receptors (CARs) derived from the m971 antibody have shown promise when used as an immunotherapeutic agent against B-cell acute lymphoblastic leukemia. A key aspect of the efficacy of this CAR-T was its ability to target a membrane-proximal epitope on the CD22 extracellular domain; however, the molecular details of m971 recognition of CD22 have thus far remained elusive. Here, we report the crystal structure of the m971 fragment antigen-binding in complex with the two most membrane-proximal Ig-like domains of CD22 (CD22_d6–d7). The m971 epitope on CD22 resides at the most proximal Ig domain (d7) to the membrane, and the antibody paratope contains electrostatic surfaces compatible with interactions with phospholipid head groups. Together, our data identify molecular details underlying the successful transformation of an antibody epitope on CD22 into an effective CAR immunotherapeutic target.     

Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD

Background

Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface.

Method

Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers.

Results

In smokers with normal lung function, the expression of CD25⁺CD4⁺ was increased, whereas the proportions of FoxP3⁺ and CD127⁺ were unchanged compared to never-smokers. Among CD4⁺ cells expressing high levels of CD25, the proportion of FoxP3⁺ cells was decreased and the percentage of CD127⁺ was increased in smokers with normal lung function. CD4⁺CD25⁺ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers.

Conclusion

The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25⁺ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development.     

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.