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Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma.

Yamamoto J, Ohnuma K, Hatano R, Okamoto T, Komiya E, Yamazaki H, Iwata S, Dang NH, Aoe K, Kishimoto T, Yamada T, Morimoto C - Br. J. Cancer (2014)

Bottom Line: We also indicates that SSTR4-mediated cytostatic effects are regulated by SHP-2 PTP, and that this inhibitory effect by SST agonist is enhanced via lipid raft clustering of associated molecules following crosslinking of anti-CD26 antibody.Finally, using an in vivo xenograft model, we demonstrate that the anti-tumour effect of anti-CD26 mAb is enhanced when combined with SSTR4 agonist treatment, and that SSTR4 is highly coexpressed with CD26 on epithelioid or biphasic types of MPM tissues obtained from patients' surgical specimens.Combination therapy with humanised anti-CD26 mAb and SSTR4 agonist may therefore potentiate anti-tumour effect on MPM.

View Article: PubMed Central - PubMed

Affiliation: Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

ABSTRACT

Background: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm arising from mesothelial lining of pleura. CD26 molecules preferentially expressed on epithelioid type of MPM. This study investigates the molecular mechanisms of CD26 regulating MPM cells in vitro and in vivo.

Methods: Biochemical and cell biological approaches were used for identifying a novel molecular target of MPM. Its contribution to tumour expansion has been also assessed using animal models. The clinical samples of MPM were also assessed for its expression.

Results: We identify that cytostatic effects in MPM are mediated by somatostatin (SST) receptor 4 (SSTR4), being inhibited by the interaction of CD26 molecules. We also indicates that SSTR4-mediated cytostatic effects are regulated by SHP-2 PTP, and that this inhibitory effect by SST agonist is enhanced via lipid raft clustering of associated molecules following crosslinking of anti-CD26 antibody. Finally, using an in vivo xenograft model, we demonstrate that the anti-tumour effect of anti-CD26 mAb is enhanced when combined with SSTR4 agonist treatment, and that SSTR4 is highly coexpressed with CD26 on epithelioid or biphasic types of MPM tissues obtained from patients' surgical specimens.

Conclusions: Combination therapy with humanised anti-CD26 mAb and SSTR4 agonist may therefore potentiate anti-tumour effect on MPM.

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Related in: MedlinePlus

The cytoplasmic region of CD26 is required for cell migration, invasion and colony formation. (A) Cells were seeded on top of a Boyden chamber. The number of cells that migrated through the uncoated filter in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells migrating through the filter were shown in the lower panels (crystal violet staining). Scale bars indicate 200 μm. (B) Cells were seeded on top of Matrigel-coated chamber inserts. The number of cells that invaded through the Matrigel in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells invading through the filter were shown in the lower panels (crystal violet staining). Scale bar indicates 200 μm. (C) Cells were plated in a cell suspension agar matrix between layers of base agar matrix. After 1 week, the agar matrix was solubilised and the cells were stained with MTT solution. Absorbance at 570 nm was measured (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells grown in soft agar just before solubilisation to indicate cell size and morphology were shown in the lower panels (phase-contrast imaging). Original magnification, × 8. Scale bars indicate 50 μm. (D) SCID mice were injected i.p. with 1 × 105 luciferase-expressing MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi cells. Tumour growth was measured by in vivo bioluminescence photometry, with imaging data of each cohort being indicated as total flux of photons per second (mean±s.e.m.; n=20). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. Representative optical bioluminescence imaging of each cohort mice was shown with intensity of luminescence as heat maps in the lower panels. The full colour version of this figure is available at British Journal of Cancer online.
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fig1: The cytoplasmic region of CD26 is required for cell migration, invasion and colony formation. (A) Cells were seeded on top of a Boyden chamber. The number of cells that migrated through the uncoated filter in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells migrating through the filter were shown in the lower panels (crystal violet staining). Scale bars indicate 200 μm. (B) Cells were seeded on top of Matrigel-coated chamber inserts. The number of cells that invaded through the Matrigel in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells invading through the filter were shown in the lower panels (crystal violet staining). Scale bar indicates 200 μm. (C) Cells were plated in a cell suspension agar matrix between layers of base agar matrix. After 1 week, the agar matrix was solubilised and the cells were stained with MTT solution. Absorbance at 570 nm was measured (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells grown in soft agar just before solubilisation to indicate cell size and morphology were shown in the lower panels (phase-contrast imaging). Original magnification, × 8. Scale bars indicate 50 μm. (D) SCID mice were injected i.p. with 1 × 105 luciferase-expressing MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi cells. Tumour growth was measured by in vivo bioluminescence photometry, with imaging data of each cohort being indicated as total flux of photons per second (mean±s.e.m.; n=20). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. Representative optical bioluminescence imaging of each cohort mice was shown with intensity of luminescence as heat maps in the lower panels. The full colour version of this figure is available at British Journal of Cancer online.

Mentions: We previously showed that CD26-mediated tumour cell proliferation of T-lymphoma and MPM cell lines was exerted via β1 integrin, in relation to the process of cell adhesion (Sato et al, 2005; Okamoto et al, 2014). Meanwhile, with the proximal signalling events associated with the cytoplasmic region of CD26 being shown in normal human T lymphocytes (Ohnuma et al, 2007), it is conceivable that similar CD26-mediated proximal events may have a role in MPM cell biology. To define the crucial role of the CD26 cytoplasmic region in regulating migratory, invasive or proliferative activity of MPM cells, we used a mutant construct of CD26 in which its cytoplasmic region was replaced with that of human CD10 (CD26-CD10 chimaeric receptor), which was shown to abrogate CD26-mediated costimulation in T cells (Ohnuma et al, 2007). CD10, as is the case with CD26, is a type II transmembrane glycoprotein with a relatively short cytoplasmic tail containing signal sequence that has an expected membrane topology similar to CD26 (Ogata et al, 1989; Maguer-Satta et al, 2011). We then transfected CD26-negative parental MSTO cells with full-length human CD26 or CD26-CD10 chimaeric receptor to establish MSTO-CD26WT or MSTO-CD26/10Chi, respectively (Supplementary Figure S1A). As shown in Figure 1A, a significant increase in migration was observed in MSTO-CD26WT as compared with MSTO-Mock (P<0.0001) or MSTO-CD26/10Chi (P<0.0001). Similarly, an increase in invasion was also observed in MSTO-CD26WT as compared with MSTO-Mock or MSTO-CD26/10Chi (Figure 1B). To study the process of tumour formation in MPM cells, we conducted colony formation assay as a model of anchorage-independent cell growth. As shown in Figure 1C, a significant increase in colony formation was observed in MSTO-CD26WT as compared with MSTO-Mock (P<0.0001) or MSTO-CD26/10Chi (P<0.0001). To extend the above in vitro results to in vivo experimentation, we conducted the cell growth assay using tumour-transplant mice. A significant increase in in vivo tumour growth was observed with MSTO-CD26WT as compared with MSTO-Mock (P<0.0001) or MSTO-CD26/10Chi (P<0.0001) (Figure 1D). These results suggest that the cytoplasmic region of CD26 is important for CD26 function in such biological processes of MPM as cell migration, invasion and anchorage-independent cell growth as well as in vivo tumour growth using xenograft mouse model.


Regulation of somatostatin receptor 4-mediated cytostatic effects by CD26 in malignant pleural mesothelioma.

Yamamoto J, Ohnuma K, Hatano R, Okamoto T, Komiya E, Yamazaki H, Iwata S, Dang NH, Aoe K, Kishimoto T, Yamada T, Morimoto C - Br. J. Cancer (2014)

The cytoplasmic region of CD26 is required for cell migration, invasion and colony formation. (A) Cells were seeded on top of a Boyden chamber. The number of cells that migrated through the uncoated filter in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells migrating through the filter were shown in the lower panels (crystal violet staining). Scale bars indicate 200 μm. (B) Cells were seeded on top of Matrigel-coated chamber inserts. The number of cells that invaded through the Matrigel in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells invading through the filter were shown in the lower panels (crystal violet staining). Scale bar indicates 200 μm. (C) Cells were plated in a cell suspension agar matrix between layers of base agar matrix. After 1 week, the agar matrix was solubilised and the cells were stained with MTT solution. Absorbance at 570 nm was measured (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells grown in soft agar just before solubilisation to indicate cell size and morphology were shown in the lower panels (phase-contrast imaging). Original magnification, × 8. Scale bars indicate 50 μm. (D) SCID mice were injected i.p. with 1 × 105 luciferase-expressing MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi cells. Tumour growth was measured by in vivo bioluminescence photometry, with imaging data of each cohort being indicated as total flux of photons per second (mean±s.e.m.; n=20). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. Representative optical bioluminescence imaging of each cohort mice was shown with intensity of luminescence as heat maps in the lower panels. The full colour version of this figure is available at British Journal of Cancer online.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4007235&req=5

fig1: The cytoplasmic region of CD26 is required for cell migration, invasion and colony formation. (A) Cells were seeded on top of a Boyden chamber. The number of cells that migrated through the uncoated filter in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells migrating through the filter were shown in the lower panels (crystal violet staining). Scale bars indicate 200 μm. (B) Cells were seeded on top of Matrigel-coated chamber inserts. The number of cells that invaded through the Matrigel in the lower chamber was counted. The mean number of cells per field was determined from five fields per filter (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells invading through the filter were shown in the lower panels (crystal violet staining). Scale bar indicates 200 μm. (C) Cells were plated in a cell suspension agar matrix between layers of base agar matrix. After 1 week, the agar matrix was solubilised and the cells were stained with MTT solution. Absorbance at 570 nm was measured (mean±s.e.m.; n=5 experiments with triplicates). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. NS denotes ‘not significant'. Representative microphotographs of cells grown in soft agar just before solubilisation to indicate cell size and morphology were shown in the lower panels (phase-contrast imaging). Original magnification, × 8. Scale bars indicate 50 μm. (D) SCID mice were injected i.p. with 1 × 105 luciferase-expressing MSTO-Mock, MSTO-CD26WT or MSTO-CD26/10Chi cells. Tumour growth was measured by in vivo bioluminescence photometry, with imaging data of each cohort being indicated as total flux of photons per second (mean±s.e.m.; n=20). A significant increase in MSTO-CD26WT is indicated as P<0.0001 (vs MSTO-Mock or MSTO-CD26/10Chi), as calculated by ANOVA with Tukey–Kramer post hoc test. Representative optical bioluminescence imaging of each cohort mice was shown with intensity of luminescence as heat maps in the lower panels. The full colour version of this figure is available at British Journal of Cancer online.
Mentions: We previously showed that CD26-mediated tumour cell proliferation of T-lymphoma and MPM cell lines was exerted via β1 integrin, in relation to the process of cell adhesion (Sato et al, 2005; Okamoto et al, 2014). Meanwhile, with the proximal signalling events associated with the cytoplasmic region of CD26 being shown in normal human T lymphocytes (Ohnuma et al, 2007), it is conceivable that similar CD26-mediated proximal events may have a role in MPM cell biology. To define the crucial role of the CD26 cytoplasmic region in regulating migratory, invasive or proliferative activity of MPM cells, we used a mutant construct of CD26 in which its cytoplasmic region was replaced with that of human CD10 (CD26-CD10 chimaeric receptor), which was shown to abrogate CD26-mediated costimulation in T cells (Ohnuma et al, 2007). CD10, as is the case with CD26, is a type II transmembrane glycoprotein with a relatively short cytoplasmic tail containing signal sequence that has an expected membrane topology similar to CD26 (Ogata et al, 1989; Maguer-Satta et al, 2011). We then transfected CD26-negative parental MSTO cells with full-length human CD26 or CD26-CD10 chimaeric receptor to establish MSTO-CD26WT or MSTO-CD26/10Chi, respectively (Supplementary Figure S1A). As shown in Figure 1A, a significant increase in migration was observed in MSTO-CD26WT as compared with MSTO-Mock (P<0.0001) or MSTO-CD26/10Chi (P<0.0001). Similarly, an increase in invasion was also observed in MSTO-CD26WT as compared with MSTO-Mock or MSTO-CD26/10Chi (Figure 1B). To study the process of tumour formation in MPM cells, we conducted colony formation assay as a model of anchorage-independent cell growth. As shown in Figure 1C, a significant increase in colony formation was observed in MSTO-CD26WT as compared with MSTO-Mock (P<0.0001) or MSTO-CD26/10Chi (P<0.0001). To extend the above in vitro results to in vivo experimentation, we conducted the cell growth assay using tumour-transplant mice. A significant increase in in vivo tumour growth was observed with MSTO-CD26WT as compared with MSTO-Mock (P<0.0001) or MSTO-CD26/10Chi (P<0.0001) (Figure 1D). These results suggest that the cytoplasmic region of CD26 is important for CD26 function in such biological processes of MPM as cell migration, invasion and anchorage-independent cell growth as well as in vivo tumour growth using xenograft mouse model.

Bottom Line: We also indicates that SSTR4-mediated cytostatic effects are regulated by SHP-2 PTP, and that this inhibitory effect by SST agonist is enhanced via lipid raft clustering of associated molecules following crosslinking of anti-CD26 antibody.Finally, using an in vivo xenograft model, we demonstrate that the anti-tumour effect of anti-CD26 mAb is enhanced when combined with SSTR4 agonist treatment, and that SSTR4 is highly coexpressed with CD26 on epithelioid or biphasic types of MPM tissues obtained from patients' surgical specimens.Combination therapy with humanised anti-CD26 mAb and SSTR4 agonist may therefore potentiate anti-tumour effect on MPM.

View Article: PubMed Central - PubMed

Affiliation: Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

ABSTRACT

Background: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm arising from mesothelial lining of pleura. CD26 molecules preferentially expressed on epithelioid type of MPM. This study investigates the molecular mechanisms of CD26 regulating MPM cells in vitro and in vivo.

Methods: Biochemical and cell biological approaches were used for identifying a novel molecular target of MPM. Its contribution to tumour expansion has been also assessed using animal models. The clinical samples of MPM were also assessed for its expression.

Results: We identify that cytostatic effects in MPM are mediated by somatostatin (SST) receptor 4 (SSTR4), being inhibited by the interaction of CD26 molecules. We also indicates that SSTR4-mediated cytostatic effects are regulated by SHP-2 PTP, and that this inhibitory effect by SST agonist is enhanced via lipid raft clustering of associated molecules following crosslinking of anti-CD26 antibody. Finally, using an in vivo xenograft model, we demonstrate that the anti-tumour effect of anti-CD26 mAb is enhanced when combined with SSTR4 agonist treatment, and that SSTR4 is highly coexpressed with CD26 on epithelioid or biphasic types of MPM tissues obtained from patients' surgical specimens.

Conclusions: Combination therapy with humanised anti-CD26 mAb and SSTR4 agonist may therefore potentiate anti-tumour effect on MPM.

Show MeSH
Related in: MedlinePlus