Cytokine-induced osteopoietic differentiation of transplanted marrow cells.

Transplantation of whole bone marrow (BMT) leads to engraftment of both osteoprogenitor cells and hematopoietic cells; however, the robust osteopoietic chimerism seen early after BMT decreases with time. Using our established murine model, we demonstrate that a post-BMT regimen of either granulocyte-colony stimulating factor, growth hormone, parathyroid hormone, or stem cell factor each stimulates greater donor osteoblast chimerism at 4 months posttransplantation than saline-treated controls and approximates the robust osteopoietic chimerism seen early after BMT; however, only growth hormone led to significantly more donor-derived osteocytes than controls. Importantly, there were no adverse hematologic consequences of the different treatments. Our data demonstrate that these cytokines can stimulate the differentiation of transplanted donor marrow cells into the osteopoietic lineage after BMT. Post-BMT cytokine therapy may generate durable osteopoietic engraftment, which should lead to sustained clinical benefit and render BMT more applicable to bone disorders.

Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells.

Human multipotent mesenchymal stromal cells (MSCs) suppress proliferation and alloreactivity of T cells. Several signaling molecules and enzymes contribute to this effect. We focused on carbohydrate-protein interactions and investigated whether lectins are involved in immune modulation by MSC. Gene expression profiling of MSCs revealed that one of the most important lectins in this setting, galectin-1, was highly expressed. Galectin-1 protein was detected intracellularly and on the cell surface of MSCs. In addition, galectin-1 was released into the cell culture supernatant by MSCs. To analyze the functional role of galectin-1, a stable knockdown of galectin-1 in MSCs with use of a retroviral transfection system was established. Galectin-1 knockdown in MSCs resulted in a significant loss of their immunomodulatory properties, compared with MSCs infected with nontargeting control sequences. The galectin-1 knockdown partially restored the proliferation of CD4(+) and CD8(+) T cells. By contrast, the effect of MSCs on nonalloreactive natural killer (NK) cells was unaffected by down-regulation of galectin-1 expression. Furthermore, MSC-derived galectin-1 significantly modulated the release of cytokines involved in graft-versus-host disease (GVHD) and autoimmunity (eg, tumor necrosis factor-α [TNFα], IFNγ, interleukin-2 [IL-2], and IL-10. These results identify galectin-1 as the first lectin mediating the immunomodulatory effect of MSCs on allogeneic T cells.

Expression of Slug is regulated by c-Myb and is required for invasion and bone marrow homing of cancer cells of different origin.

In metastatic cancer cells, the process of invasion is regulated by several transcription factors that induce changes required for migration and resistance to apoptosis. Slug (SNAI2, Snail2) is involved in epithelial mesenchymal transition in physiological and in pathological contexts. We show here that in embryonic kidney, colon carcinoma, chronic myeloid leukemia-blast crisis, and in neuroblastoma cells, expression of Slug is transcriptionally regulated by c-Myb via Myb binding sites in the 5'-flanking region and in the first intron of the slug gene. In embryonic kidney and neuroblastoma cells, c-Myb induced vimentin, fibronectin, and N-cadherin expression and membrane ruffling via actin polymerization consistent with the acquisition of a mesenchymal-like phenotype. Furthermore, down-regulation of endogenous c-Myb levels in colon carcinoma cells led to increased expression of E-cadherin and reduced levels of vimentin. Some of these changes are predominantly Slug-dependent as Slug silencing via RNA interference (RNAi) reverts the cells to a quasi-parental condition. Changes in gene expression and morphology induced by c-Myb-activated Slug correlated with increased ability to migrate (embryonic kidney) and to invade through a Matrigel membrane (embryonic kidney, colon carcinoma, neuroblastoma). c-Myb-dependent Slug expression was also essential for the homing of chronic myeloid leukemia K562 cells to the bone marrow. In summary, we show here that the proto-oncogene c-Myb controls Slug transcription in tumor cells of different origin. Such a regulatory pathway contributes to the acquisition of invasive properties that are important for the metastatic process.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation.

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility.

BACKGROUND: Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. RESULTS: We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. CONCLUSION: We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

Impact of a single nucleotide polymorphism in the MDM2 gene on neuroblastoma development and aggressiveness: results of a pilot study on 239 patients.

PURPOSE: MDM2 is a key negative regulator of p53 activity, and a single nucleotide polymorphism (SNP309, T>G change; rs 2279744) in its promoter increases the affinity for the transcription factor SP1, enhancing MDM2 expression. We carried out a pilot study to investigate the effect of this polymorphism on development and behavior of neuroblastoma, an extracranial pediatric tumor with unfrequent genetic inactivation of p53. EXPERIMENTAL DESIGN: We genotyped the MDM2-SNP309 alleles of tumor DNA from 239 neuroblastoma patients and peripheral blood DNA from 237 controls. In 40 of 239 neuroblastomas, the MDM2-SNP309 alleles were also genotyped in peripheral blood DNA. Data were analyzed by two-sided Fisher's exact test, log-rank test, and Kaplan-Meier statistics. Where appropriate, data are reported with 95% confidence intervals (CI). RESULTS: The frequency of both the T/G and G/G genotypes or the G/G or T/G genotype only was higher in neuroblastoma DNA samples than in controls: 60.3% (95% CI, 54.1-66.5) versus 47.3% (95% CI, 40.9-53.6), 30.4% (95% CI, 22.4-37.8) versus 15.0% (95% CI, 9.2-20.7), and 52.0% (95% CI, 45.0-59.9) versus 41.9% (95% CI, 35.3-48.5), respectively; Two-Sided Fisher's Exact Test P values were 0.006, 0.003, and 0.048, respectively; Odds ratios were 1.69 (95% CI, 1.18-2.43), 2.45 (95% CI, 1.37-4.39) and 1.51 (95% CI, 1.02-2.22), respectively. A significant association (P = 0.016) between heterozygous (T/G)/homozygous (G/G) genotypes at SNP309 and advanced clinical stages was also shown. Homozygous/heterozygous SNP309 variant carriers had a shorter 5-year overall survival than patients with the wild-type allele (P = 0.046; log-rank test). A shorter overall survival in patients with heterozygous/homozygous SNP309 was also observed in the subgroups with age at diagnosis >1 year and adrenal primary tumor (P = 0.024 and P = 0.014, respectively). CONCLUSIONS: Data from this pilot study suggest that the MDM2 G/G and T/G-SNP309 alleles are markers of increased predisposition to tumor development and disease aggressiveness in neuroblastoma. However, additional studies with larger patient cohorts are required for a definitive assessment of the clinical relevance of these data.

Understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the Mesenkillers.

A tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surrounding cells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development, local invasion and metastases has become increasingly clear allowing the identification of TS as one of the possible ways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts (TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development, several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts, via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymal progenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identify multimodal approaches to target TS by biomolecules, monoclonal antibodies, and more recently via cell based strategies. These latter strategies appear extremely promising, although still associated with debated and unclear findings. This review discusses crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma, pancreatic and breast carcinoma, where stroma plays distinct paradigmatic roles. The recognition of these distinct stromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TS by novel compounds and/or MSC having specific killing activities.

Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

A degradation-resistant c-Myb mutant cooperates with Bcl-2 in enhancing proliferative potential and survival of hematopoietic cells.

The c-myb gene is preferentially expressed in primitive hematopoietic cell and plays a central role in the control of cell proliferation, differentiation and survival by regulating the transcription of several genes implicated in these processes including the antiapoptotic Bcl-2. We show here that, compared to wild-type c-Myb, overexpression of a degradation resistant c-Myb mutant [Delta(358-452) c-Myb] enhances the clonogenic potential of hematopoietic progenitors as indicated by increased cytokine-dependent primary and secondary colony formation of Lin(-) Sca-1(+) Kit(+) mouse marrow cells. Moreover, proliferation assays of IL-3 dependent myeloid precursor 32Dcl3 cells co-expressing Bcl-2 and c-Myb indicate that these cells continue to proliferate in the absence of IL-3 and this effect is more apparent in cells expressing the degradation resistant Delta(358-452) c-Myb. Interestingly, overexpression of Delta(358-452) c-Myb is by itself sufficient to protect 32Dcl3 cells from apoptosis induced by IL-3 deprivation; moreover, these cells are also increased in number which most likely reflects the enhanced proliferative potential conferred by Delta(358-452) c-Myb to apoptosis-resistant cells.

Enhanced proliferative potential of hematopoietic cells expressing degradation-resistant c-Myb mutants.

The c-myb gene encodes a transcription factor required for proliferation, differentiation, and survival of hematopoietic cells. Expression of c-Myb is often increased in hematological malignancies, but the underlying mechanisms are poorly understood. We show here that c-Myb has a longer half-life (at least 2-fold) in BCR/ABL-expressing than in normal hematopoietic cells. Such enhanced stability was dependent on a phosphatidylinositol 3-kinase (PI-3K)/Akt/GSKIIIbeta pathway(s) as indicated by the suppression of c-Myb expression upon treatment with PI-3K inhibitors or co-expression with dominant negative Akt or constitutively active GSKIIIbeta. Moreover, inhibition of GSKIIIbeta by LiCl enhanced c-Myb expression in parental 32Dcl3 cells. Compared with wild type c-Myb, three mutants (delta(358-452), delta(389-418), and L389A/L396A c-Myb) of the leucine zipper domain had increased stability. However, only expression of delta(358-452) was not affected by inhibition of the PI-3K/Akt pathway and was not enhanced by a proteasome inhibitor, suggesting that leucine zipper-dependent and -independent mechanisms are involved in the regulation of c-Myb stability. Indeed, delta(389-418) carrying four lysine-to-alanine substitutions (delta(389-418) K387A/K428A/K442A/K445A) was as stable as delta(358-452) c-Myb. Compared with full-length c-Myb, constitutive expression of delta(358-452) and delta(389-418) c-Myb in Lin-Sca-1+ mouse marrow cells increased cytokine-dependent primary and secondary colony formation. In K562 cells, expression of delta(358-452), delta(389-418), and L389A/L396A c-Myb led to enhanced proliferation after STI571 treatment. Thus, enhanced stability of c-Myb by activation of PI-3K-dependent pathway(s) might contribute to the higher proliferative potential of BCR/ABL-expressing and, perhaps, other leukemic cells.