Mesenchymal stem cell-derived interleukin-6 and vascular endothelial growth factor promote breast cancer cell migration

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF‐7, T47D, and SK‐Br‐3 cells), we analyzed the role of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF) in the cross‐talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs‐CM), in absence or in presence of the anti‐VEGF antibody bevacizumab or an anti‐IL‐6 antibody, alone or in combination. We found that anti‐VEGF and anti‐IL‐6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL‐6 proteins on breast cancer cell growth and migration. IL‐6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL‐6 significantly increased the ability to migrate of MCF‐7, T47D and SK‐Br‐3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL‐6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC‐secreted IL‐6 and VEGF may act as paracrine factors to sustain breast cancer cell migration. J. Cell. Biochem. 113: 3363–3370, 2012. © 2012 Wiley Periodicals, Inc.     

VEGF111b,a new member of VEGFxxxb isoforms and induced by mitomycin C,inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA_3.1 empty vector, pcDNA_3.1-VEGF111b or pcDNA_3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.     

Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF(165/121) antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.     

Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.     

Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.     

The effects of adipose-derived stem cells on CD133-expressing bladder cancer cells

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine. They are also considered as a preferred cell source for urinary tract reconstruction. However, as MSCs exhibit affinity to tumor microenvironment, possible activation of tumor-initiating cells remains a major concern in the application of stem cell-based therapies for patients with a bladder cancer history. To analyze the influence of adipose-derived stem cells (ASCs) on bladder cancer cells with stem cell-like properties, we isolated CD133-positive bladder cancer cells and cultured them in conditioned medium from ASCs (ASC-CM). Our results showed that parental 5637 and HB-CLS-1 cells showed induced clonogenic potential when cultured in ASC-CM. Soluble mediators secreted by ASCs increased proliferation and viability of unsorted cells as well as CD133+ and CD133− subpopulations. Furthermore, incubation with ASC-CM modulated activation of intracellular signaling pathways. Soluble mediators secreted by ASCs increased phosphorylation of AKT1/2/3 (1.4-fold, P < 0.05), ERK1/2 (1.6-fold, P < 0.02), and p70 S6K (1.4-fold) in CD133+ cells isolated from 5637 cell line. In turn, decreased phosphorylation of those three proteins involved in PI3K/Akt and MAPK signaling was observed in CD133+ cells isolated from HB-CLS-1 cell line. Our results revealed that bladder cancer stem-like cells are responsive to signals from ASCs. Paracrine factors secreted by locally-delivered ASCs may, therefore, contribute to the modulation of signaling pathways involved in cancer progression, metastasis, and drug resistance.     

Vascular endothelial growth factor principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is essential for bone formation. However, the effect of VEGF on osteoblastic cells during osteoblastogenesis is still controversial. The aim of this study was to clarify the relationship between osteoblastic cells derived from human mesenchymal stem cells (MSCs) and VEGF in the early stage of osteoblastic differentiation. Continuous dexamethasone treatment with a low concentration stimulated osteoblastogenesis of MSCs and the expression of VEGF121 mRNA. The VEGF secretion from osteoblastic cells also increased along with osteoblastogenesis. Neuropilin-1, which mainly binds VEGF165, was detected at all stages during early osteoblastogenesis, but VEGF receptor-1 and -2 were not detected on RT-PCR analyses. In this study, VEGF had no direct effect on the proliferation of osteoblastic cells. However, the secreted VEGF in the conditioned medium of osteoblastic cells exhibited high angiogenic power as to endothelial cell proliferation. Our findings indicated that VEGF121 principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis. The present study also demonstrated the differential expression of VEGF121 during osteoblastogenesis. The increase of VEGF in the early stage might be a useful marker of induction of bone formation due to human MSCs.     

Interleukin 6 mediated recruitment of mesenchymal stem cells to the hypoxic tumor milieu

Mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic precursor cells predominantly found in the bone marrow. They have been recently reported to home towards the hypoxic tumor microenvironment in vivo. Interleukin-6 is a multifunctional cytokine normally involved in the regulation of the immune and inflammatory response. In addition to its normal function, IL-6 signaling has been implicated in tumorigenesis. Solid tumors develop hypoxia as a result of inadequate O₂ supply. Interestingly, tumor types with increased levels of hypoxia are known to have increased resistance to chemotherapy as well as increased metastatic potential. Here, we present evidence that under hypoxic conditions (1.5% O₂) breast cancer cells secrete high levels of IL-6, which serve to activate and attract MSCs. We now report that secreted IL-6 acts in a paracrine fashion on MSCs stimulating the activation of both Stat3 and MAPK signaling pathways to enhance migratory potential and cell survival. Inhibition of IL-6 signaling utilizing neutralizing antibodies leads to attenuation of MSC migration. Specifically, increased migration is dependent on IL-6 signaling through the IL-6 receptor. Collectively, our data demonstrate that hypoxic tumor cells specifically recruit MSCs, which through activation of signaling and survival pathways facilitate tumor progression.     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

Protein kinase C zeta regulates interleukin-8-mediated stromal-derived factor-1 expression and migration of human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are bone marrow-derived cells with multipotent differentiation capability that are mobilized into the circulation in response to injury and localize to areas of tissue damage including solid tumors. They have the capacity to adopt a phenotype similar to carcinoma-associated fibroblasts (CAFs) and, like CAFs, promote tumor growth. The molecular communication between tumor cells and MSCs has not been well defined. However, MSCs have increased expression of the chemokine stromal-derived factor 1 (SDF-1) when exposed to conditioned medium from tumor cells. Additionally, SDF-1 has been shown to be important in the promotion of tumor growth by CAFs. These data suggest that the SDF-1 signaling axis is a key feature of the tumor microenvironment. In this report, we demonstrate that interleukin 8 (IL-8) induces an increase in SDF-1 expression by MSCs. The increase in SDF-1 expression in response to IL-8 is mediated by the activation of the protein kinase C (PKC) zeta isoform. In a functional assay, activation of PKC is required for in vitro MSC migration in response to tumor conditioned medium. These results indicate that IL-8-mediated SDF-1 production by MSCs requires PKC zeta activation. This signaling pathway provides insight into possible molecular targets for cancer therapy aimed at disrupting the interaction between components of the tumor microenvironment.     

Characteristics of the PI3K/AKT and MAPK/ERK pathways involved in the maintenance of self-renewal in lung cancer stem-like cells

Lung cancer is the leading cause of cancer-related mortality worldwide due to its early asymptomatic and late metastasis. While cancer stem cells (CSCs) may play a vital role in oncogenesis and development of lung cancer, mechanisms underlying CSCs self‐renewal remain less clear. In the present study, we constructed a clinically relevant CSCs enrichment recognition model and evaluated the potential functions of phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT) and mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathways in lung cancer via bioinformatic analysis, providing the basis for in depth mechanistic inquisition. Experimentally, we confirmed that PI3K/AKT pathway predominantly promotes proliferation through anti-apoptosis in lung adenocarcinoma cells, while MAPK/ERK pathway has an overwhelming superiority in regulating the proliferation in lung CSCs. Further, utilizing stemness score model, LLC-Symmetric Division (LLC-SD) cells and mouse orthotopic lung transplantation model, we elucidated an intricate cross-talk between the oncogenic pathway and the stem cell reprograming pathway that impact stem cell characteristics as well as cancer biology features of lung CSCs both in vitro and in vivo. In summary, our findings uncovered a new insight that PI3K/AKT and MAPK/ERK pathways as oncogenic signaling pathway and/or stem cell signaling pathway act distinctively and synergistically to regulate lung CSCs self-renewal.     

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell‐derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non‐MP secreted factors (Sup) were isolated from serum‐free medium conditioned by human microvascular ECs (HMEC‐1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP‐containing MPs were isolated from cells transduced with CMV‐GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP‐MPs, but not free GFP. Thus, only MP‐associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP‐1, MMP‐3, CCL‐2/MCP‐1 and IL‐6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF‐κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms.     

Monocytes conditioned media stimulate fibronectin expression and spreading of inflammatory breast cancer cells in three-dimensional culture: A mechanism mediated by IL-8 signaling pathway

Background

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer characterized by invasion of carcinoma cells into dermal lymphatic vessels where they form tumor emboli over expressing adhesion molecule E-cadherin. Although invasion and metastasis are dynamic processes controlled by complex interaction between tumor cells and microenvironment the mechanisms by which soluble mediators may regulate motility and invasion of IBC cells are poorly understood. The present study investigated the effect of media conditioned by human monocytes U937 secreted cytokines, chemokines and growth factors on the expression of adhesion molecules E-cadherin and fibronectin of human IBC cell line SUM149. Furthermore, cytokines signaling pathway involved were also identified.

Results

U937 secreted cytokines, chemokines and growth factors were characterized by cytokine antibody array. The major U937 secreted cytokines/chemokines were interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1/CCL2). When SUM149 cells were seeded in three dimensional (3D) models with media conditioned by U937 secreted cytokines, chemokines and growth factors; results showed: 1) changes in the morphology of IBC cells from epithelial to migratory spindle shape branched like structures; 2) Over-expression of adhesion molecule fibronectin and not E-cadherin. Further analysis revealed that over-expression of fibronectin may be mediated by IL-8 via PI3K/Akt signaling pathway.

Conclusion

The present results suggested that cytokines secreted by human monocytes may promote chemotactic migration and spreading of IBC cell lines. Results also indicated that IL-8 the major secreted cytokine by U937 cells may play essential role in fibronectin expression by SUM149 cells via interaction with IL-8 specific receptors and stimulation of PI3K/Akt signaling pathway.     

Proteomic studies of rat tibialis anterior muscle during postnatal growth and development

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.     

miR-126 enhances the sensitivity of non-small cell lung cancer cells to anticancer agents by targeting vascular endothelial growth factor A

Increasing evidence suggests that hsa-miR-126 (miR-126) is down-regulated in non-small cell lung cancer (NSCLC) cell lines and the restoration of miR-126 impairs tumor cell proliferation, migration, invasion, and survival by targeting specific molecules. Here, we reported for the first time that miR-126 was involved in regulating the response of NSCLC cells to cancer chemotherapy. After transfected A549 cells with miR-126 mimic or inhibitor, we found that an elevated level of miR-126 was significantly associated with a decreased half maximal inhibitory concentration of adriamycin (ADM) and vincristine, an increased accumulation of ADM, down-regulation of vascular endothelial growth factor A (VEGFA) and multidrug resistance-associated protein 1 (MRP1), and inactivation of the Akt signaling pathway. Furthermore, enhanced expression of miR-126 suppressed the growth of A549 xenograft and inhibited the expression of VEGFA and MRP1. miR-126 could efficiently down-regulate VEGFA expression through the interaction with the VEGFA 3'-untranslated region, whereas restoration of VEGFA could partially attenuate the suppression of MRP1 by miR-126. However, LY294002, an inhibitor of the PI3K/Akt signaling pathway, diminished this effect, suggesting that enhanced expression of miR-126 increased the sensitivity of NSCLC cells to anticancer agents through negative regulation of a VEGF/PI3K/Akt/MRP1 signaling pathway.     

p38 MAPK mediates gamma-irradiation-induced endothelial cell apoptosis, and vascular endothelial growth factor protects endothelial cells through the phosphoinositide 3-kinase-Akt-Bcl-2 pathway

Therapeutic radiation is widely used in cancer treatments. The success of radiation therapy depends not only on the radiosensitivity of tumor cells but also on the radiosensitivity of endothelial cells lining the tumor vasculature. Vascular endothelial growth factor (VEGF) plays a critical role in protecting endothelial cells against a number of antitumor agents including ionizing radiation. Strategies designed to overcome the survival advantage afforded to endothelial cells by VEGF might aid in enhancing the efficacy of radiation therapy. In this report we examined the signaling cascade(s) involved in VEGF-mediated protection of endothelial cells against gamma-irradiation. gamma-Irradiation-induced apoptosis of human dermal microvascular endothelial cells (HDMECs) was predominantly mediated through the p38 MAPK pathway as an inhibitor of p38 MAPK (PD169316), and dominant negative mutants of p38 MAPK could significantly enhance HDMEC survival against gamma-irradiation. Inhibition of the PI3K and MAPK pathways markedly up-regulated gamma-irradiation-mediated p38 MAPK activation resulting in enhanced HDMEC apoptosis. In contrast, VEGF-treated HDMECs were protected from gamma-irradiation-induced apoptosis predominantly through the PI3K/Akt pathway. Bcl-2 expression was markedly elevated in VEGF-treated HDMECs, and it was significantly inhibited by the PI3K inhibitor LY294002. HDMECs exposed to irradiation showed a significant decrease in Bcl-2 expression. In contrast, VEGF-stimulated HDMECs, when irradiated, maintained higher levels of Bcl-2 expression. Taken together our results suggest that gamma-irradiation induces endothelial cell apoptosis predominantly via the activation of p38 MAPK, and VEGF protects endothelial cells against gamma-irradiation predominantly via the PI3K-Akt-Bcl-2 signaling pathway.