CD9 Negatively Regulates CD26 Expression and Inhibits CD26-Mediated Enhancement of Invasive Potential of Malignant Mesothelioma Cells

<div><p>CD26/dipeptidyl peptidase IV is a cell surface glycoprotein which consists of multiple functional domains beside its ectopeptidase site. A growing body of evidence indicates that elevated expression of CD26 correlates with disease aggressiveness and invasive potential of selected malignancies. To further explore the molecular mechanisms involved in this clinical behavior, our current work focused on the interaction between CD26 and CD9, which were recently identified as novel markers for cancer stem cells in malignant mesothelioma. We found that CD26 and CD9 co-modulated and co-precipitated with each other in the malignant mesothelioma cell lines ACC-MESO1 and MSTO-211H. SiRNA study revealed that depletion of CD26 led to increased CD9 expression, while depletion of CD9 resulted in increased CD26 expression. Consistent with these findings was the fact that gene transfer of CD26 into CD26-negative MSTO-211H cells reduced CD9 expression. Cell invasion assay showed that overexpression of CD26 or gene depletion of CD9 led to enhanced invasiveness, while CD26 gene depletion resulted in reduced invasive potential. Furthermore, our work suggested that this enhanced invasiveness may be partly mediated by α5β1 integrin, since co-precipitation studies demonstrated an association between CD26 and α5β1 integrin. Finally, gene depletion of CD9 resulted in elevated protein levels and tyrosine phosphorylation of FAK and Cas-L, which are downstream of β1 integrin, while depletion of CD26 led to a reduction in the levels of these molecules. Collectively, our findings suggest that CD26 potentiates tumor cell invasion through its interaction with α5β1 integrin, and CD9 negatively regulates tumor cell invasion by reducing the level of CD26-α5β1 integrin complex through an inverse correlation between CD9 and CD26 expression. Our results also suggest that CD26 and CD9 serve as potential biomarkers as well as promising molecular targets for novel therapeutic approaches in malignant mesothelioma and other malignancies.</p></div

CD26 potentiates tumor cell invasion.

<p>(A). Flow cytometric analysis of CD26 and CD9 expressions in MESO1. (B). Sorting of CD26⁻CD9⁺ cells and CD26⁺CD9⁺ cells, and immunoprecipitation was performed with humanized anti-CD26 mAb and anti-CD9 mAb (5H9), then probed with anti-CD26 polyclonal antibody and anti-CD9 mAb (5H9). (C and E).Tumor cell invasion was measured with the Boyden chamber-based cell invasion assay for 24 h. Number of invaded cells was represented as means ± SE (n = 5).*p<0.01, **p<0.001. (D). Flow cytometric analysis of CD26 and CD9 expressions in MESO1 and sorting of CD26⁺CD9⁺ cells and CD26⁺CD9⁻ cells. These results were also confirmed by 3 separate experiments.</p

CD26 associates with CD9 in an inverse manner.

<p>(A). Heat map representing color-coded expression levels of differentially expressed genes. CD26/Depletion: control siRNA- and CD26 siRNA-transfectedMESO1. CD26/Over expression: MSTO-Wild and MSTO-CD26 (+) cells. Upregulated (red) or downregulated (green). (B and C). MESO1 transfectants of control siRNA, CD26 siRNA, and CD9 siRNA, or MSTO-Wild and MSTO-CD26 (+) cells were stained with anti-CD26-FITC or anti-CD9-FITC and subjected to flow cytometry. (D). MESO1 transfectants with control siRNA, CD26 siRNA, and CD9 siRNA, or MSTO-Wild and MSTO-CD26 (+) cells were lysed and probed with anti-CD26 polyclonal antibody, anti-CD9 mAb (5H9) and anti-β-actin polyclonal antibody. (E). RT-PCR was carried out for analysis of CD26 and CD9 gene expressions on MESO1 transfectants with controlsiRNA, CD26siRNA, and CD9siRNA, or on MSTO-Wild and MSTO-CD26 (+) cells. GAPDH amplification was used as internal control. These results were also confirmed by 5 separate experiments.</p

CD26 potentiates invasiveness through α5β1 integrin.

<p>(A).MSTO-Wild, MSTO-CD26 (+) cells were subjected to flow cytometry for CD26, α5, and β1. (B). MESO1 and MSTO-CD26 (+) cells were subjected to immunoprecipitation with control IgG, humanized anti-CD26 mAb, anti-CD9 mAb (5H9), anti-α5 mAb (2H6), or anti-β1 mAb (4B4). The immunoblot was probed with anti-CD26 polyclonal antibody, anti-CD9 mAb (5H9), anti-α5 mAb (2H6), or anti-β1 mAb (4B4). (C). Boyden chamber-based cell invasion assay of MESO1 and MSTO-CD26 (+) cells treated with anti-α5, and β1 antibodies for 24 h. (n = 5). *p<0.01, **p<0.005. (D).The cell migration assay of MESO1 and MSTO-CD26 (+) cells treated with anti-α5, and β1 antibodies.(n = 5). *p<0.05, **p<0.01.</p

CD9 negatively regulates CD26-mediated invasion.

<p>(A-E).Cells were analyzed by the cell invasion assay for 24 h. Number of invaded cells were represented as means ± SE (n = 5). (A). MSTO-Wild,MSTO-CD26 (+), and MESO1 cells. Invaded cells stained are shown in the top panel. **p<0.005. (B). MSTO-CD26 (+) or MESO1 cells transfected with controlsiRNA or CD26siRNA. **p<0.005, ***p<0.001. (C). MSTO-Wild,MSTO-CD26 (+), and MESO1 cells transfected with control-siRNA or CD9siRNA. Invaded cells stained are shown in the right panel. **p<0.005, ***p<0.001. (D). MSTO-Wild, MSTO-CD26 (+), and MESO1 cells treated with control IgG or anti-CD9 mAb (10 µg/ml).**p<0.005, ***p<0.001. (E). NCI-H226 was transfected with pMX vector control or pMX-CD9. After staining with CD26-FITC and CD9-FITC, cells were subjected to flow cytometry.Boyden chamber-based cell invasion assay was performed with NCI-H226 transfected with pMX vector control or pMX-CD9 for 24 h. Number of invaded cells/well was represented as means ± SE (n = 5).*p<0.05.</p

Downregulation of CD9 enhances CD26-mediated invasive potential.

<p>(A). MESO1 and MSTO-CD26 (+) cells transfected with control siRNA and CD26-siRNA were subjected to immunoblotting using anti-α5 (2H6), anti-β1 (4B4) mAbs, anti-CD26 polyclonal antibody, anti-β-actin polyclonal antibody. (B). The same cells transfected with control-siRNA and CD9-siRNA. Anti-CD9 mAb (5H9) was used for immunoblotting. (C). MESO1 and MSTO-CD26 (+) cells were subjected to immunoprecipitation to anti-β1 mAb (4B4), anti-FAK mAb (10G2), and anti-Cas-L Ab (TA248). Immunoblotting was performed with anti-FAK (10G2), and anti-Cas-L Ab (TA248). (D). MESO1 and MSTO-CD26 (+) cells were transfected with control siRNA, CD26 siRNA or CD9 siRNA, then subjected to immunoprecipitation with anti-FAK mAb (10G2), or anti-Cas-L Ab (TA248). Immunoblotting was performed with anti-FAK mAb (10G2) or anti-Cas-L Ab (TA248). (E). MESO1 and MSTO-CD26 (+) transfectants with control siRNA or CD9 siRNA were immunoprecipitated with anti-FAK mAb (10G2) or anti-Cas-L Ab (TA248). Immunoblotting was performed with anti-phosphotyrosine mAb (4G10). Similar results were observed by 3 separate experiments.</p

CD26 associates with CD9.

<p>(A). Flow cytometric analysis of CD26 and CD9 expression on MESO1, MSTO-Wild or MSTO-CD26 (+) cells. (B). MESO1 or MSTO-CD26 (+) cells were incubated up to 72 h at 37 °C with either control IgG (10 µg/ml) or humanized anti-CD26 mAb (10 µg/ml). These cells were stained with anti-CD26-FITC (5K76) or with anti-CD9-FITC, and subjected to flow cytometry. Intensity of modulation was indicated by mean fluorescence intensity (MFI). (C). MESO1 or MSTO-CD26 (+) cells were subjected to immunoprecipitation with control IgG, humanized anti-CD26 mAb, and anti-CD9 mAb (5H9). Immunoblot was conducted with anti-CD26 polyclonal antibody, and anti-CD9 mAb (5H9). These results were also confirmed by 5 separate experiments.</p

Multidimensional Screening Platform for Simultaneously Targeting Oncogenic KRAS and Hypoxia-Inducible Factors Pathways in Colorectal Cancer

Colorectal cancer (CRC) is a genetic disease, due to progressive accumulation of mutations in oncogenes and tumor suppressor genes. Large scale genomic sequencing projects revealed >100 mutations in any individual CRC. Many of these mutations are likely passenger mutations, and fewer are driver mutations. Of these, activating mutations in RAS proteins are essential for cancer initiation, progression, and/or resistance to therapy. There has been significant interest in developing drugs targeting mutated cancer gene products or downstream signaling pathways. Due to the number of mutations involved and inherent redundancy in intracellular signaling, drugs targeting one mutation or pathway have been either ineffective or led to rapid resistance. We have devised a strategy whereby multiple cancer pathways may be simultaneously targeted for drug discovery. For proof-of-concept, we targeted the oncogenic KRAS and HIF pathways, since oncogenic KRAS has been shown to be required for cancer initiation and progression, and HIF-1α and HIF-2α are induced by the majority of mutated oncogenes and tumor suppressor genes in CRC. We have generated isogenic cell lines defective in either oncogenic KRAS or both HIF-1α and HIF-2α and subjected them to multiplex genomic, siRNA, and high-throughput small molecule screening. We have identified potential drug targets and compounds for preclinical and clinical development. Screening of our marine natural product library led to the rediscovery of the microtubule agent dolastatin 10 and the class I histone deacetylase (HDAC) inhibitor largazole to inhibit oncogenic KRAS and HIF pathways. Largazole was further validated as an antiangiogenic agent in a HIF-dependent manner in human cells and <i>in vivo</i> in zebrafish using a genetic model with activated HIF. Our general strategy, coupling functional genomics with drug susceptibility or chemical-genetic interaction screens, enables the identification of potential drug targets and candidates with requisite selectivity. Molecules prioritized in this manner can easily be validated in suitable zebrafish models due to the genetic tractability of the system. Our multidimensional platform with cellular and organismal components can be extended to larger scale multiplex screens that include other mutations and pathways

Phase 1 study of inotuzumab ozogamicin combined with R-GDP for the treatment of patients with relapsed/refractory CD22+ B-cell non-Hodgkin lymphoma

<p><b>Objective</b>: To evaluate safety, tolerability, and preliminary activity of inotuzumab ozogamicin (InO) plus rituximab, gemcitabine, dexamethasone, and cisplatin (R-GDP) in patients with relapsed/refractory CD22+ B-cell non-Hodgkin lymphoma (NHL).</p> <p><b>Methods</b>: Patients received InO plus R-GDP (21-day cycle; six-cycle maximum) using up-and-down dose-escalation schema for gemcitabine and cisplatin to define the highest dosage regimen(s) with acceptable toxicity (Part 1; <i>n</i> = 27). Part 2 (<i>n</i> = 10) confirmed safety and tolerability; Part 3 (<i>n</i> = 18) evaluated preliminary efficacy.</p> <p><b>Results:</b> Among 55 patients enrolled, 42% were refractory at baseline (median 2 [range, 1–6] prior therapies); 38% had diffuse large B-cell lymphoma (DLBCL). The highest dosage regimen with acceptable toxicity was InO 0.8 mg/m², rituximab 375 mg/m², cisplatin 50 mg/m², gemcitabine 500 mg/m² (day 1 only) and dexamethasone 40 mg (days 1–4); this was confirmed in Part 2, in which three patients had dose-limiting toxicities (grade 4 thrombocytopenia [<i>n</i> = 2], febrile neutropenia [<i>n</i> = 2]). Most frequent treatment-related adverse events were thrombocytopenia (any grade, 85%; grade ≥3, 75%) and neutropenia (69%; 62%). Overall (objective) response rate (ORR) was 53% (11 complete, 18 partial responses); ORR was 71%, 33%, and 62% in patients with follicular lymphoma (<i>n</i> = 14), DLBCL (<i>n</i> = 21), and mantle cell lymphoma (<i>n</i> = 13), respectively.</p> <p><b>Conclusions:</b> InO 0.8 mg/m² plus R-GDP was associated with manageable toxicity, although gemcitabine and cisplatin doses were lower than in the standard R-GDP regimen due to hematologic toxicity. Evidence of antitumor activity was observed; however, these exploratory data should be interpreted with caution due to the small sample size and short follow-up duration (Clinicaltrials.gov number: NCT01055496).</p