Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted.

In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

A smoking-associated 7-gene signature for lung cancer diagnosis and prognosis.

Smoking is responsible for 90% of lung cancer cases. There is currently no clinically available gene test for early detection of lung cancer in smokers, or an effective patient selection strategy for adjuvant chemotherapy in lung cancer treatment. In this study, concurrent coexpression with multiple signaling pathways was modeled among a set of genes associated with smoking and lung cancer survival. This approach identified and validated a 7-gene signature for lung cancer diagnosis and prognosis in smokers using patient transcriptional profiles (n=847). The smoking-associated gene coexpression networks in lung adenocarcinoma tumors (n=442) were highly significant in terms of biological relevance (network precision = 0.91, FDR<0.01) when evaluated with numerous databases containing multi-level molecular associations. The gene coexpression network in smoking lung adenocarcinoma patients was confirmed in qRT-PCR assays of the identified biomarkers and involved signaling pathway genes in human lung adenocarcinoma cells (H23) treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Furthermore, the western blotting results of p53, phospho­p53, Rb and EGFR in NNK-treated H23 and transformed normal human lung epithelial cells (BEAS-2B) support their functional involvement in smoking-induced lung cancer carcinogenesis and progression.

Bone marrow osteoblast damage by chemotherapeutic agents.

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-ß1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-ß1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts.

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.

Cellular elements of the subarachnoid space promote ALL survival during chemotherapy.

CNS infiltration by leukemic cells remains a problematic disease manifestation of acute lymphoblastic leukemia (ALL). Prophylactic regimens for CNS leukemia including intrathecal chemotherapeutics have decreased CNS involvement in ALL, but are not without toxicities. Using co-culture models, we show that astrocytes, choroid plexus epithelial cells, and meningeal cells protect ALL cells from chemotherapy-induced cell death using drugs included in prophylactic regimens-cytarabine, dexamethasone, and methotrexate. Understanding how ALL cells survive in the CNS remains invaluable for designing strategies to prevent CNS leukemia and minimizing the need for treatment in this sensitive anatomical site where treatment-induced toxicity is of significant concern.

Extracorporeal membrane oxygenation induced cardiac dysfunction in newborn lambs.

Extracorporeal membrane oxygenation (ECMO) is routinely used to support cardiopulmonary failure in infants and children. Suboptimal outcomes for primary cardiac support suggest a need for investigation of the impact of ECMO on the heart. Twenty-four newborn lambs received a brief period of ECMO support to investigate the hypothesis that ECMO produces cardiac dysfunction in newborn lamb. Dorset newborn lambs, 4-7 days of age, were exposed to ECMO for 5 min at a 100 ml/kg flow rate and quickly weaned off. Measurements included echocardiographic mean left ventricular (LV) velocity of circumferential fiber shortening corrected for heart rate (mVCFc), LV shortening fraction, and peak systolic wall stress plus hemodynamic measurement of LV maximum rate of pressure change with time (LV dp/dt max), maximum rate of pressure change divided by developed pressure (LV dp/dtP), right atrial pressure, pulmonary capillary wedge pressure, mean pulmonary artery pressure, LV peak and end-diastolic pressure, and aortic pressure. These measures were also obtained after an exposure to 5 min of ECMO and immediate disconnect for 5 min, followed by ECMO administration for 1 h again, followed by discontinuation of ECMO. LV mVCFc is decreased after exposure to 5 min of ECMO support despite a decrease in LV peak systolic wall stress that provides afterload reduction. LV mVCFc is inversely related to peak systolic wall stress at a significance level of p < 0.0001. The time period after initiation of ECMO is a significant factor in the model (p = 0.0097). Time [baseline] was different from the other time points with p = 0.0010. Average mVCFc at baseline is 1.27 ± 0.35 and decreases to 1.01 ± 0.42 after 5 min of ECMO that is then withdrawn. Peak systolic wall stress decreases from 36.0 ± 13.1 at baseline to 29.8 ± 12.1 after 5 min of ECMO. LV dp/dt max decreases from 1,769 ± 453 mmHg/s at baseline to 1,311 ± 513 mmHg/s after exposure to 5 min of ECMO (p = 0.0005). Baseline LV dp/dt max is different from each point after start of ECMO. Diastolic LVdp/dt min increased from -1,340 ± 477 mmHg/s to -908 ± 393 mmHg/s at 5 min. Echocardiographic mVCFc, when considered in isolation or as a function of LV peak systolic wall stress, shows diminished LV function after ECMO. Hemodynamic measurement of LV dp/dt max and LV dp/dt min confirms the observation. Separation of the humoral from mechanical effect of ECMO with the short exposure to the extracorporeal circuit shows that an immediate decrement of LV function occurs at initiation of ECMO, a finding that has not been stressed with previous studies of extracorporeal support. This implies a potentially outcome-limiting deleterious effect for the patient who requires ECMO support for the heart rather than the lungs. We should continue to strive to understand and ameliorate this deleterious effect of the extracorporeal circulation circuit.

VE-cadherin Regulates Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Sensitivity to Apoptosis.

The mechanisms by which the bone marrow microenvironment regulates tumor cell survival are diverse. This study describes the novel observation that in addition to Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cell lines, primary patient cells also express Hypoxia Inducible Factor-2α (HIF-2α) and Vascular Endothelial Cadherin (VE-cadherin), which are regulated by Abl kinase. Tumor expression of the classical endothelial protein, VE-cadherin, has been associated with aggressive phenotype and poor prognosis in other models, but has not been investigated in hematopoietic malignancies. Targeted knockdown of VE-cadherin rendered Ph+ ALL cells more susceptible to chemotherapy, even in the presence of bone marrow stromal cell (BMSC) derived survival cues. Pre-treatment of Ph+ ALL cells with ADH100191, a VE-cadherin antagonist, resulted in increased apoptosis during in vitro chemotherapy exposure. Consistent with a role for VE-cadherin in modulation of leukemia cell viability, lentiviral-mediated expression of VE-cadherin in Ph- ALL cells resulted in increased resistance to treatment-induced apoptosis. These observations suggest a novel role for VE-cadherin in modulation of chemoresistance in Ph+ ALL.

Ph+/VE-cadherin+ identifies a stem cell like population of acute lymphoblastic leukemia sustained by bone marrow niche cells.

Although leukemic stem cells (LSCs) show a symbiotic relationship with bone marrow microenvironmental niches, the mechanism by which the marrow microenvironment contributes to self-renewal and proliferation of LSCs remains elusive. In the present study, we identified a unique subpopulation of Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) cells coexpressing markers of endothelial cells (including VE-cadherin, PECAM-1, and Flk-1) and committed B-lineage progenitors. After long-term coculture with bone marrow stromal cells, tumor cells formed hematopoietic colonies and cords, expressed early stem- cell markers, and showed endothelial sprouting. Gene expression profiles of LSCs were altered in the presence of stromal cell contact. Stromal cell contact promoted leukemic cell VE-cadherin expression, stabilized beta-catenin, and up-regulated Bcr-abl fusion gene expression. Our study indicates that these specific tumor cells are uniquely positioned to respond to microenvironment-derived self-renewing and proliferative cues. Ph(+)/VE-cadherin(+) tumor subpopulation circumvents the requirement of exogenous Wnt signaling for self-renewal through stromal cell support of leukemic cell VE-cadherin expression and up-regulated Bcr-abl tyrosine kinase activity. These data suggest that strategies targeting signals in the marrow microenvironment that amplify the Bcr-abl/VE-cadherin/beta-catenin axis may have utility in sensitizing drug-resistant leukemic stem cells.

Chemotherapy disrupts activity of translational regulatory proteins in bone marrow stromal cells.

OBJECTIVE: Bone marrow stromal cell function is a critical influence on hematopoietic reconstitution following progenitor or stem cell transplantation. Stromal cells support hematopoietic cell migration, survival, and proliferation. We have previously reported that stromal cell matrix metalloproteinase-2 (MMP-2) is necessary for optimal support of pro-B-cell chemotaxis through its regulation of stromal cell-derived factor-1 (CXCL12) release. Following exposure to the topoisomerase II inhibitor, etoposide (VP-16), stromal cell MMP-2 protein expression is reduced. The current study investigated the mechanism by which VP-16 may alter translation of MMP-2 in bone marrow stromal cells. MATERIALS AND METHODS: Bone marrow stromal cells were exposed to chemotherapeutic agents etoposide, melphalan, and 4-hydroperoxycyclophosphamide (4HC) and evaluated for MMP-2 expression by enzyme-linked immunosorbent assay and support of pro-B-cell chemotaxis by chemotaxis assay. Western blot analyses were completed to evaluate phosphorylation of stromal cell translational regulatory proteins 4E binding protein-1 (4EBP-1), P70(S6K), and S6 or MMP-2 in the presence of chemotherapy, or the chemical inhibitors rapamycin or LY294002. RESULTS: Rapid dephosphorylation of 4EBP-1, P70(S6K), and S6 following VP-16 exposure was observed, consistent with blunted translational efficiency. We also observed that inhibition of stromal cell mammalian target of rapamycin with rapamycin, or phosphatidylinositol 3 kinase with LY294002, resulted in inhibition of stromal cell MMP-2 protein. In addition we found that the chemotherapeutic agents melphalan and 4HC disrupt bone marrow stromal cell MMP-2 protein expression and support of chemotaxis. CONCLUSIONS: These data suggest that one mechanism by which chemotherapy may alter stromal cells of the bone marrow microenvironment is through disrupted translation of proteins.

MMP-2 is required for bone marrow stromal cell support of pro-B-cell chemotaxis.

OBJECTIVE: We have previously demonstrated that bone marrow stromal cells (BMSCs) exposed to etoposide (VP-16) have reduced support of CXCR4(+) cell chemotaxis and diminished stromal cell derived factor-1 (CXCL12) in the supernatants. Based on the identification of CXCL12 as a matrix metalloproteinase-2 (MMP-2) substrate, we investigated potential dysregulation of MMP-2 expression or activity in chemotherapy-treated BMSCs. METHODS: BMSCs exposed to VP-16 were evaluated for MMP-2 expression by gelatin zymography, ELISA, and western blot. Chemotaxis assays were completed to evaluate pro-B cell chemotaxis toward either MMP-2(-/-) BMSCs or BMSCs exposed to MMP-2 inhibitors. RESULTS: BMSC exposure to VP-16 resulted in an immediate, transient, increase in MMP-2, followed by reduced MMP-2 protein expression. MMP-2 reduction correlated with diminished CXCL12 protein and reduced support of pro-B cell chemotaxis. BMSCs derived from MMP-2 knockout mice had less chemotactic support of CXCR4(+) cells than wild-type controls. Inhibition of BMSC MMP-2 activity by OA-Hy also reduced chemotactic support and CXCL12 protein detected in BMSC supernatants. VP-16-induced reduction of BMSC support of hematopoietic cell migration was restored by supplementing cultures with recombinant MMP-2 protein. CONCLUSIONS: These data suggest that MMP-2 is sensitive to chemotherapy-induced stress, and may regulate BMSC support of pro-B cell chemotaxis. Increased MMP-2 expression during the acute phase of chemotherapy exposure potentially inactivates CXCL12. Subsequently, chronic exposure to chemotherapy, with the associated downregulation of MMP-2, interrupts CXCL12 release from the extracellular matrix, also blunting BMSC support of pro-B cell migration.

Activation of transforming growth factor-beta1/p38/Smad3 signaling in stromal cells requires reactive oxygen species-mediated MMP-2 activity during bone marrow damage.

Dose-escalated chemotherapy has proven utility in a variety of treatment settings, including preparative regimens before bone marrow or hematopoietic stem cell transplantation. However, the potential damage imposed by aggressive regimens on the marrow microenvironment warrants further investigation. In the present study, we tested the hypothesis that dose-escalated chemotherapy, with etoposide as a model chemotherapeutic agent, activates the transforming growth factor beta-1 (TGF-beta1) signaling pathway in bone marrow stromal cells. After high-dose etoposide exposure in vitro, Smad3 protein was phosphorylated in a time-and dose-dependent manner in marrow-derived stromal cells, coincident with the release of active and latent TGF-beta1 from the extracellular matrix. Phosphorylation was modulated by p38 kinase, with translocation of Smad3 from the cytoplasm to the nucleus subsequent to its phosphorylation. Etoposide induced activation of TGF-beta1 followed the generation of reactive oxygen species and required matrix metalloproteinase-2 (MMP-2) protein availability. Chemotherapy effects were diminished in MMP-2(-/-) knockout stromal cells and TGF-beta1 knockdown small interfering RNA-transfected stromal cells, in which phosphorylation of Smad3 was negligible after etoposide exposure. Stable transfection of a human MMP-2 cDNA into bone marrow stromal cells resulted in elevated phosphorylation of Smad3 during chemotherapy. These data suggest TGF-beta1/p38/Smad3 signaling cascades are activated in bone marrow stromal cells after dose-escalated chemotherapy and may contribute to chemotherapy-induced alterations of the marrow microenvironment.

Stromal cell protection of B-lineage acute lymphoblastic leukemic cells during chemotherapy requires active Akt.

Several studies document ALL cell response to survival signals from bone marrow stromal cells. The current study suggests a requirement for active Akt in ALL cells for optimal stromal cell protection during chemotherapy. ALL cells expressing dominant negative Akt were not efficiently rescued from Ara-C or etoposide-induced apoptosis by stromal cell co-culture. In addition, inhibition of ALL cell PI-3 kinase activity diminished stromal cells support of tumor cells during treatment. ALL cell lines co-cultured with bone marrow stromal cells during chemotherapy maintained higher levels of phosphorylated Akt protein and reduced PP2A activity when compared to ALL cells treated in medium alone. Chemotherapy-induced PARP and Bcl-2 cleavage was reduced in ALL cells cultured with a stromal cell layer compared to tumor cells exposed to drug in medium alone. However, interaction with stromal cells was not able to efficiently block treatment-induced PARP or Bcl-2 cleavage in leukemic cells with blunted Akt activity. These data suggest a pivotal role for Akt in mediating stromal cell regulation of ALL cell apoptosis.

Stromal cells expressing elevated VCAM-1 enhance survival of B lineage tumor cells.

In the current study we generated murine bone marrow stromal cells that constitutively express human VCAM-1. These stromal cell lines allow investigation of the contribution of VCAM-1 initiated signaling to tumor cell survival. Co-culture of ALL cell lines with stromal cells engineered to over express VCAM-1 enhanced survival of leukemic cells in a PI-3 kinase-dependent manner, compared to co-culture with parental stromal cells expressing only endogenous VCAM-1. These observations suggest that modulation of stromal cell VCAM-1 by specific chemotherapeutic drugs may have utility in decreasing residual disease. In addition, these novel lines provide an in vitro model in which other tumor types that interact with stromal cells in the bone marrow microenvironment may be evaluated to determine the contribution of VCAM-1 initiated signaling to modulation of treatment response.

Chemotherapy induces bcl-2 cleavage in lymphoid leukemic cell lines.

Bcl-2 is the major anti-apoptotic protein evaluated in studies aimed at understanding programmed cell death. Recent work suggests that the biological activity of Bcl-2 is modulated by proteolytic cleavage, with a 23 kDa cleaved Bcl-2 product having pro-apoptotic activity. In the current study we evaluated the effect of chemotherapy on Bcl-2 cleavage in B lineage leukemic cell lines. JM-1, SUP-B 15 and RS4 leukemic cell lines cleaved Bcl-2 to its 23 kDa form when exposed to the chemotherapeutic agents 1-beta-D-arabinofuranosyl-cytosine (Ara-C) or etoposide (VP-16). Chemotherapy induced Bcl-2 cleavage was blunted by inhibition of caspase activity. Co-culture of leukemic cells with bone marrow stromal cells during chemotherapy exposure resulted in reduced levels of 23 kDa Bcl-2 protein. These observations suggest that the bone marrow microenvironment may contribute to maintenance of residual leukemic disease during treatment by reducing generation of pro-apoptotic 23 kDa Bcl-2.