Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling.

BACKGROUND: The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. METHODS: The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM-transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. RESULTS: ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. CONCLUSIONS: The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior.

Hepatocellular Carcinoma Cells Are Protected From Immunolysis by Mesenchymal Stromal Cells Through Indoleamine 2,3 Dioxygenase.

B7 family proteins serve as checkpoint molecules that protect tumors from T cell mediated lysis. Tryptophan degrading enzymes indoleamine 2,3 dioxygenase (IDO) and tryptophan 2,3 dioxygenase (TDO) also induce T cell immune tolerance. However, little is known about the relative contribution of B7 molecules, tryptophan degrading enzymes, as well as the impact of tumor and stromal cell interactions to the development of immunosuppressive tumor microenvironment. To investigate such interactions, we used a tripartite model of human hepatocellular carcinoma cell line (HepG2) and mesenchymal stromal cells (MSCs) co-cultured with peripheral blood mononuclear cells (PBMCs). Co-culture of HepG2 cells and activated PBMCs demonstrate that HepG2 cells undergo PBMC mediated cytolysis, despite constitutive expression of B7-H3 and upregulation of PD-L1 by IFNγ. Knockdown of B7-H3, PD-L1 or IDO does not modulate PBMC mediated lysis of HepG2 cells. However, TNFα preactivation enhances lysis of HepG2 cells, and blocking of TNFα production from PBMCs protects HepG2 cells. On the other hand, MSCs protect HepG2 cells from PBMC mediated lysis, even in the presence of TNFα. Further investigation showed that MSC mediated protection is associated with the unique secretome profile of upregulated and downregulated cytokines and chemokines. IFNγ activated MSCs are superior to TNFα activated or control MSCs in protecting HepG2 cells. Blockade of IFNγ driven IDO activity completely abolishes the ability of MSCs to protect HepG2 cells from cytolysis by PBMCs. These results suggest that inhibition of IFNγ activation of IDO induction in stromal cells, combined with usage of TNFα, could be a novel immunotherapeutic strategy to induce regression of hepatocellular carcinoma.

Ruxolitinib Inhibits IFNγ Licensing of Human Bone Marrow Derived Mesenchymal Stromal Cells.

Ruxolitinib is a JAK2/JAK1 inhibitor that blocks the inflammatory JAK-STAT signaling pathway. Ruxolitinib has been demonstrated to be effective in the treatment of steroid-resistant acute graft-versus-host disease (GVHD). Ruxolitinib's effect on inflammatory cells of hematopoietic origin is known. However, its effect on nonhematopoietic cell types with immune-modulating and antigen-presenting cell competency plausibly involved in pathogenesis of GVHD has not been explored. Mesenchymal stromal cells (MSCs) are CD45- nonhematopoietic cells of the bone marrow with immune modulatory functions in vivo. MSCs' immunobiology largely depends on their responsiveness to IFNγ. We aimed to define the effect of ruxolitinib on the immunobiology of MSCs that are modulated by IFNγ. Human bone marrow derived MSCs, peripheral blood mononuclear cells (PBMCs), and primary bone marrow aspirates were analyzed for their sensitivity to ruxolitinib-mediated blocking of IFNγ-induced STAT-1 phosphorylation and downstream effector molecules, utilizing Western blot, flow cytometry, secretome analysis, and phosflow techniques. IFNγ-induced cytostatic effects on MSCs are reversed by ruxolitinib. Ruxolitinib inhibits IFNγ and secretome of activated peripheral PBMC-induced STAT-1 phosphorylation on human bone marrow derived MSCs. In addition, ruxolitinib inhibits IFNγ-induced pro-GVHD pathways on MSCs, which includes HLAABC(MHCI), HLADR(MHCII), CX3CL1, and CCL2. IFNγ-induced immunosuppressive molecules IDO and PDL-1 were also inhibited by ruxolitinib on MSCs. Comparative analysis with PBMCs has demonstrated that MSCs are as equal as to HLADR+ PBMC populations in responding to ruxolitinib-mediated inhibition of IFNγ-induced STAT-1 phosphorylation. Ex vivo analysis of human marrow aspirates has demonstrated that ruxolitinib blocks IFNγ-induced STAT-1 phosphorylation in CD45+/-HLADR+/- populations at different levels, which is depending on their sensitivity to IFNγ responsiveness. These results inform the hypothesis that ruxolitinib's immune-modulatory effects in vivo may pharmacologically involve marrow and tissue-resident MSCs. Ruxolitinib affects the immunobiology of MSCs equivalent to professional HLADR+ antigen presenting cells, which collectively mitigate GVHD.

Percutaneous correction (humeroplasty) of humeral head defects (Hill-Sachs) associated with anterior shoulder instability: a cadaveric study.

PURPOSE: The goal of our study was to determine whether a bone tamp could be used to correct a Hill-Sachs lesion by a unique technique that we developed termed "humeroplasty." TYPE OF STUDY: In vitro cadaveric study. METHODS: We created reproducible Hill-Sachs lesions in 14 cadaveric humerii, which we then reduced with a curved bone tamp using our proposed technique. Paired t tests, with the significance set at P < .05, were then used to compare the prereduction and postreduction depths, widths, lengths, and volumes of the lesions. RESULTS: The average prereduction defect measurements were 8.9 mm deep, 16.3 mm wide, 24.1 mm long, and 1,755 mm3 in volume. The average postreduction defect measurements were 1.6 mm deep, 6.2 mm wide, 10.4 mm long, and 50.3 mm3 in volume. A statistically significant improvement (P < .001) was seen in all parameters. CONCLUSIONS: Our technique was able to consistently and significantly restore all measured parameters of the Hill-Sachs lesions using a curved bone tamp. This reduction to a "small" defect may reduce the higher rate of recurrence normally found with the surgical treatment of shoulder instability when "large" Hill-Sachs lesions are present. CLINICAL RELEVANCE: Humeroplasty potentially might reduce the higher rate of recurrence observed when the surgical treatment of shoulder instability involves the presence of large Hill-Sachs lesions.

ODAM Expression Inhibits Human Breast Cancer Tumorigenesis.

We have posited that Odontogenic Ameloblast Associated Protein (ODAM) serves as a novel prognostic biomarker in breast cancer and now have investigated its potential role in regulating tumor growth and metastasis. Human breast cancer MDA-MB-231 cells were transfected with a recombinant ODAM plasmid construct (or, as a control, the plasmid vector alone). ODAM expression increased adhesion and apoptosis of the transfected MDA-MB-231 cells and suppressed their growth rate, migratory activity, and capability to invade extracellular matrix-coated membranes. Implantation of such cells into mouse mammary fat pads resulted in significantly smaller tumors than occurred in animals that received control cells; furthermore, ODAM-expressing cells, when injected intravenously into mice, failed to metastasize, whereas the control-transfected counterparts produced extensive lung lesions. Our finding that induction of ODAM expression in human breast cancer cells markedly inhibited their neoplastic properties provides further evidence for the regulatory role of this molecule in tumorigenesis and, consequently, is of potential clinical import.

Association of breast cancer and immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) and breast cancer are common disorders. Only six cases in which patients have had both diseases have been reported. We describe a 40-year-old woman who had ITP while responding to therapy for metastatic breast cancer. Given the few reported cases, the diverse presentations of thrombocytopenia during the course of each patient's breast cancer and the variable therapeutic responses of ITP, the association of breast cancer and ITP is probably coincidental.