CXCR4 Mediated Chemotaxis Is Regulated by 5T4 Oncofetal Glycoprotein in Mouse Embryonic Cells

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Targeting Wnt/EZH2/microRNA-708 signaling pathway inhibits neuroendocrine differentiation in prostate cancer

Prostate cancer (PC) castration resistance has been linked to the differentiation of PC luminal cells into hormone-refractory neuroendocrine (NE) cells. However, the molecular mechanisms controlling the emergence of lethal NE prostate cancer (NEPC) remain unclear. The present study aimed to investigate the mechanisms underlying the transition from prostate adenocarcinoma to NEPC. The microRNA miR-708 was involved in NE differentiation and was downregulated in NEPC cells and tumor specimens. miR-708 targeted Sestrin-3 to inhibit Forkhead Box O1 (FOXO1) phosphorylation, resulting in apoptosis of prostate adenocarcinoma cells and AKT-inactivated NEPC cells, the latter of which was consistent with the progression of tumor xenografts in mice under miR-708 treatment. In silico analysis of PC and NEPC tumor specimens suggested that the polycomb repressive complex subunit Enhancer of zeste homolog 2 (EZH2) was particularly overexpressed in NEPC. Notably, EZH2 bound to the miR-708 promoter and induced its silencing in NEPC. Inhibition of EZH2 prevented NE differentiation of PC cells. EZH2 expression was regulated by both Cyclin Dependent Kinase 1 (CDK1) and Wnt signaling. Silencing transcription factor 4 (TCF4), as a key protein in Wnt signaling, prevented NEPC formation. These results provide a molecular basis for the roles of miR-708 and EZH2 in NE differentiation in PC and highlight a new paradigm in NEPC formation and survival.Other Information Published in: Cell Death Discovery License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41420-019-0218-y</p

Characterization of m5T4 specific mAbs.

<p>(A), Summarizes the IgG subclasses of five m5T4 specific monoclonal antibodies (mAb, made in 5T4KO mice) recognizing distinct epitopes in the proximal and distal 5T4 extracellular LRR containing domains. (B), Shows titration of mAb activity in m5T4 specific ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009982#pone.0009982-Marchese1" target="_blank">[27]</a>. (C), Shows titration of mAb cell surface labeling of B16m5T4 tumor cells by flow cytometry. (D), Western blot analysis of mAb probing against recombinant m5T4-pIg showing recognition of m5T4 by all mAbs except B1C3.</p

Influence of 5T4 on the chemokine receptors CXCR6 and CXCR3 in ES cells.

<p>(A), The expression and cellular localization of 5T4 and CXCR6 molecules on undifferenetiated and differentiating WT-ES cells was determined. Both molecules can be detected at the cell surface in differentiating cells with some areas of co-localization (5T4  =  green; CXCR6  =  red; composite, co-localisation  =  yellow; co-localized areas shown in separate channel). (B), Undifferentiated (white columns) and differentiating (black columns) ES cells were placed in a gradient of chemokine CXCL16 or not. (C), Immunofluorescence detection of 5T4 and CXCR3 in differentiating WT and 5T4KO-ES cells (5T4  =  green, CXCR4  =  red). Cell surface expression of 5T4 is present only on differentiating WT-ES cells whilst cell surface expression of CXCR3 is evident in both undifferentiated and differentiating WT and KO ES cells. (D), Undifferentiated WT (white columns) and 5T4KO (black columns) ES cells exhibit no CXCL10 dependent chemotaxis but differentiating WT and 5T4KO-ES cells, acquire significant chemotaxis towards CXCL10.</p

Inhibition of chemotaxis by monoclonal antibodies recognizing 5T4.

<p>(A), The chemotactic migration exhibited by differentiating WT-ES cells towards CXCL12 was abolished in the presence of the m5T4 specific mAb B1C3 (10 µg) but not in presence of mAb P1C9 or P1H10 (10 µg) or an irrelevant control antibody (10 µg). MAbs B3F1 and B5C9 (10 µg) reduced the chemotactic response. (−  =  no CXCL12, +  =  10ng CXCL12). (B), MAb dose response of inhibition of chemotaxis towards CXCL12 in differentiating WT-ES cells. (C), The chemotactic migration exhibited by primary WT MEF was abolished in the presence of the m5T4 specific mAb B1C3 (10 µg) but not in presence of mAb P1C9 (10 µg) or an irrelevant control antibody (10 µg). MAbs B3F1 and B5C9 (10 µg) reduced the chemotactic response.</p

Effects of cytoskeleton, microtubule and Golgi disruption on the co-localisation pattern of 5T4 and CXCR4.

<p>Primary murine embryonic fibroblasts were assessed for their pattern of 5T4 and CXCR4 expression by immunofluorescence following 24 hours disruption of either the cytoskeleton (cytochalasin D), Golgi (brefeldin A) or microtubules (nocodazole) and 1 hour after drug washout. Cell surface expression of 5T4 (green) and CXCR4 (red) with regions of co-localization of the two antigens (seen as yellow) (also shown by co-localization analysis) are depicted.</p

Role of 5T4 expression in the CXC12/CXCR4 axis in MEF.

<p>(A), MEF derived from wild-type, (WT, black columns), 5T4 heterozygote, (HET, grey columns) and 5T4 null, (5T4KO, white columns) embryos show 5T4 gene dose related CXCLl2 chemotaxis. (B), Chemotaxis of 5T4KO MEF following mock infection, (black columns), or infection with RAd-eGFP, (grey columns), or RAd-m5T4, (white columns); CXCL12 chemotaxis is only restored by RAd-m5T4. (+ or – 30 ng CXCL12). (C), Pattern of expression of CXCR4, (green) and 5T4, (red) in WT and 5T4KO MEF. In WT cells, CXCR4 and 5T4 are seen at the cell surface and clearly co-localize (CXCR4  =  green; 5T4  =  red; composite: co-localization  =  yellow; co-localized areas shown in separate channel) while in 5T4KO cells CXCR4 is located intracellularly around the nucleus; compare to DAPI labeling (blue). (D), 5T4KO MEF infected with RAd-m5T4 exhibit cell surface expression of both 5T4 and CXCR4 also displayed by co-localization (5T4 = red; CXCR4  = green; composite co localization =  yellow; co-localized areas shown in separate channel). RAd-GFP had no effect on CXCR4 expression (not shown).</p

5T4 is not required for CXCR7 surface expression.

<p>Immunofluorescence detection of 5T4 and CXCR7 in undifferentiated and differentiating WT and 5T4KO-ES cells (5T4  =  green, CXCR7  =  red). Cell surface expression of CXCR7 is high in undifferentiated ES cells (either WT or 5T4KO). In 3 day differentiating WT-ES cells, CXCR7 is relatively downregulated from the cell surface whereas in differentiating 5T4KO-ES surface CXCR7 is retained.</p

The transmembrane domain of 5T4 is necessary for CXCR4 cell surface expression.

<p>(A), 5T4KO MEF were transduced with retroviral vectors encoding both eGFP and full length or truncated 5T4 or chimeric 5T4/CD44 constructs. Successful infection was assessed by GFP expression and the location of CXCR4, assessed in these cells. Cell surface expression of CXCR4 is only seen with constructs containing 5T4 TM (viii, xvi, xxiv); the extracellular and cytoplasmic domains of 5T4 are not required. (B), Consistent with this CXCL12 chemotaxis of the retrovirally transduced GFP+ 5T4 null MEF with 5T4 extracellular domain, (dark grey), 5T4 extracellular domain CD44 transmembrane and cytosolic domains, (grid) and mock infected, (white) showing no affect whereas full length 5T4, (light grey), 5T4 extracellular and transmembrane domains, (spots) and CD44 extracellular domain 5T4 transmembrane and cytosolic domains, (stripes) show comparable levels to wild-type (black columns).</p