STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma

We previously demonstrated that human mesenchymal stem cells (MSCs) promote the growth of osteosarcoma in the bone microenvironment. The aim of the present study was to further determine the effect of IL-6/STAT3 signaling on the progression of osteosarcoma. First, conditioned medium from MSCs was used to stimulate the growth of osteosarcoma cells (Saos-2) in vitro. We found that STAT3 was activated and that the activation could be blocked by an IL-6-neutralizing antibody. The inhibition of STAT3 in Saos-2 cells by siRNA or AG490 decreased cell proliferation, migration and invasion, down-regulated the mRNA expression of Cyclin D, Bcl-xL and Survivin and enhanced the apoptotic response. Furthermore, a nude mouse osteosarcoma model was established by injecting luciferase-labeled Saos-2 cells into the tibia, and the effect of STAT3 on tumor growth was determined by treating the mice with AG490. In vivo bioluminescence images showed that tumor growth was dramatically reduced in the AG490 group. In addition, STAT3 inhibition decreased the lung metastasis rate and prolonged the survival of these mice. After treatment with AG490, the protein levels of IL-6, p-STAT3 and PCNA were decreased, and the level of apoptosis in the tumor was increased. Altogether, these data indicate that MSCs in the bone microenvironment might promote the progression of osteosarcoma and protect tumor cells from drug-induced apoptosis through IL-6/STAT3 signaling.     

c-MYC functions as a molecular switch to alter the response of human mammary epithelial cells to oncostatin M

Cytokines play an important role in creating an inflammatory microenvironment, which is now considered a hallmark of cancer. Although tumor cells can exploit cytokine signaling to promote growth, invasion, and metastasis, the response of normal and premalignant epithelial cells to cytokines present in a developing tumor microenvironment remains unclear. Oncostatin M (OSM), an IL-6 family cytokine responsible for STAT3 activation, has been implicated in cancer development, progression, invasion, and metastasis. Paradoxically, OSM can also suppress the growth of normal cells and certain tumor-derived cell lines. Using isogenic human mammary epithelial cells (HMEC) at different stages of neoplastic transformation, we found that OSM signaling suppressed c-MYC expression and engaged a p16- and p53-independent growth arrest that required STAT3 activity. Inhibition of STAT3 activation by expressing a dominant-negative STAT3 protein or a STAT3-shRNA prevented the OSM-mediated arrest. In addition, expression of c-MYC from a constitutive promoter also abrogated the STAT3-mediated arrest, and strikingly, cooperated with OSM to promote anchorage-independent growth (AIG), a property associated with malignant transformation. Cooperative transformation by c-MYC and OSM required PI3K and AKT signaling, showing the importance of multiple signaling pathways downstream of the OSM receptor in defining the cellular response to cytokines. These findings identify c-MYC as an important molecular switch that alters the cellular response to OSM-mediated signaling from tumor suppressive to tumor promoting.     

Direct gap junction communication between malignant glioma cells and astrocytes

Gap junctions are intercellular channels that connect the interiors of coupled cells. We sought to determine the extent to which malignant glioma cells form gap junction channels with astrocytes from either adult human brain or rat forebrain. The astrocytic gap junction protein, connexin 43 (Cx43), was identified in immunoreactive plaques at areas of cell-to-cell contact between cocultured glioma cells and astrocytes. These gap junction plaques were composed of functional channels, because extensive dye coupling was evident between the glioma cells and astrocytes from both human and rat brain. Calcium signaling was also readily transmitted from glioma cells to astrocytes and vice versa. In live rat brain, injection of glioma cells prelabeled with the gap junction tracer, dicarboxy-dichlorofluorescein, revealed extensive dye transfer to host cells, demonstrating that malignant glioma cells directly couple with normal brain cells. These observations suggest that intercellular communication via gap junctions may play a role in regulating cellular interactions during tumor invasion. In fact, the presence of gap junctions between astrocytes and glioma cells was sufficient to induce a transformation of astrocytic phenotype. Astrocytes cocultured with C6 glioma cells overexpressing Cx43 were significantly smaller and expressed a lower level of glial fibrillary acidic protein than astrocytes cocultured with otherwise identical mock-transfected, gap junction-deficient C6 cells. Thus, direct cellular coupling with glioma cells result in a phenotypic transformation of astrocytes that may contribute to the susceptibility of surrounding tissue to glioma invasion.     

Mesenchymal stem cells enhance lung cancer initiation through activation of IL-6/JAK2/STAT3 pathway

Background

The role of mesenchymal stem cells (MSCs) and IL-6 in lung cancer has not been well-addressed. We aimed to determine if MSCs can enhance the ability of tumor initiation of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway.

Materials and methods

Lung cancer cell lines A549 and CL1-5 were directly or indirectly cocultured with MSCs. Spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. Cells without and with MSCs were injected into NOD/SCID mice. The percentage of tumor formation was determined. The influence of the IL-6/JAK2/STAT3 signaling pathway in cancer cell sphere formation and tumor growth were investigated.

Results

A very small number of lung cancer cells, when mixed with otherwise non-tumorigenic MSCs, obtained de novo tumorigenicity when injected subcutaneously and allowed to form a tumor xenograft. Secretion of IL-6 from MSCs increased activation of the JAK2/STAT3 pathway in cancer cells, and enhanced sphere formation and tumor initiation. A reduced capacity of tumor formation of A549 and CL1-5 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 and CL1-5 admixed with MSCs.

Conclusions

Culture of A549 or CL1-5 lung cancer cells with MSCs increased sphere formation, drug resistance, and overexpression of pluripotency markers through activation of the IL-6/JAK2/STAT3 pathway. MSCs enhanced the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Blockade of the IL-6/JAK2/STAT3 pathway attenuated the capability of A549 and CL1-5 cells to form tumors.     

p38 MAPK inhibits breast cancer metastasis through regulation of stromal expansion

p38 MAPK signaling controls cell growth, proliferation and the cell cycle under stress conditions. However, the function of p38 activation in tumor metastasis is still not well understood. We report that p38 activation in breast cancer cells inhibits tumor metastasis but does not substantially modulate primary tumor growth. Stable p38 knockdown in breast cancer cells suppressed NF‐κB p65 activation, inhibiting miR‐365 expression and resulting in increased IL‐6 secretion. The inhibitory effect of p38 signaling on metastasis was mediated by suppression of mesenchymal stem cell (MSC) migration to the primary tumor and sites of metastasis, where MSCs can differentiate into cancer‐associated fibroblasts to promote tumor metastasis. The migration of MSCs to these sites relies on CXCR4‐SDF1 signaling in the tumor microenvironment. Analysis of human primary and metastatic breast cancer tumors showed that p38 activation was inversely associated with IL‐6 and vimentin expression. This study suggests that combination analysis of p38 MAPK and IL‐6 signaling in patients with breast cancer may improve prognosis and treatment of metastatic breast cancer.     

Interleukin 6 augments lung cancer chemotherapeutic resistance via ataxia‐telangiectasia mutated/NF‐kappaB pathway activation

Although it is known that ataxia‐telangiectasia mutated (ATM) and interleukin 6 (IL‐6) contribute to multiple drug resistance (MDR) in tumor chemotherapy, the exact role of ATM activation in MDR resulting from increased IL‐6 expression is still unclear. In the present study, we demonstrate that the activation of the ATM‐NF‐kappaB pathway, resulting from increased IL‐6 expression, plays a central role in augmented chemoresistance in lung cancer cell lines. This result was supported by the increased expressions of Bcl‐2, Mcl‐1, Bcl‐xl, and the upregulation of MDR‐associated protein ABCG2. The higher level of IL‐6 reveals not only higher ATM/NF‐kappaB activity but also increased expressions of ABCG2, Bcl‐2, Mcl‐1 and Bcl‐xl. Most importantly, lung cancer cells themselves upregulated IL‐6 secretion by activating the p38/NF‐kappaB pathway through treatment with cisplatin and camptothecin. Taken together, these findings demonstrate that chemotherapeutic agents increase IL‐6 expression, hence activating the ATM/NF‐kappaB pathway, augmenting anti‐apoptotic protein expression and contributing to MDR. This indicates that both IL‐6 and ATM are potential targets for the treatment of chemotherapeutic resistance in lung cancer.     

High expression levels of IKKα and IKKβ are necessary for the malignant properties of liver cancer

IKK‐NF‐κB signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy. Therefore, in this study, we analyzed the expression of mRNAs encoding components and targets of NF‐κB signaling including IKKα, IKKβ, RANK, RANKL, OPG, CyclinD3, mammary serine protease inhibitor (Maspin), CyclinD1, c‐FLIP, Bcl‐xl, Stat3, Cip1 and Cip2 by real‐time PCR in 40 patients with liver cancer. After statistical analysis, 7 indices including IKKα, IKKβ, RANK, Maspin, c‐FLIP, Cip2 and cyclinD1 were found to show significant differences between tumor tissue and its corresponding adjacent tissue. When IKKα and IKKβ were downregulated in the hepatocellular carcinoma (HCC) cell lines of MHCC‐97L and MHCC‐97H in vitro, the numbers of BrdU positive cells were decreased in both IKKα and IKKβ knockdown cells. Levels of apoptosis were also investigated in IKKα and IKKβ knockdown cells. The growth of HCC was inhibited in the subcutaneous implantation model, and lung metastatogenesis was also significantly inhibited in the kidney capsule transplantation model. Downregulation of IKKα and IKKβ in HCC cultured in vitro revealed that increased Maspin, OPG and RANKL expression was associated with metastasis of HCC. These findings were associated with downregulation of Bcl‐XL and c‐FLIP, which may be the reason for increased apoptosis. The therapeutic effect of IKKα and IKKβ downregulation depends on extent of NF‐κB inhibition and the malignant nature of the HCC. We anticipate that IKK‐targeted gene therapy can be used in the treatment of HCC, a cancer that is notoriously resistant to radiation and chemotherapy.     

IL‐6 enriched lung cancer stem‐like cell population by inhibition of cell cycle regulators via DNMT1 upregulation

Tumors are influenced by a microenvironment rich in inflammatory cytokines, growth factors and chemokines, which may promote tumor growth. Interleukin‐6 (IL‐6) is a multifunctional cytokine and known as a regulator of immune and inflammation responses. IL‐6 has also been reported to be associated with tumor progression and chemoresistance in different types of cancers. In our study, we demonstrated that IL‐6 enriches the properties of lung cancer stem‐like cells in A549 lung cancer cells cultured in spheroid medium. IL‐6 also promotes sphere formation and stem‐like properties of A549 cells by enhancing cell proliferation. Methylation‐specific polymerase chain reaction (PCR) was performed and revealed that IL‐6 increased methylation of p53 and p21 in A549 cancer cells. Western blot analysis and quantitative real‐time PCR demonstrated that IL‐6 increased the expression of DNA methyltransferase 1 (DNMT1) in A549 cells cultured in spheroid medium, but not the expression of DNMT3a or DNMT3b. Knockdown of DNMT1 eliminated IL‐6‐mediated hypermethylation of cell cycle regulators and enrichment of lung cancer stem‐like properties. In conclusion, our study, for the first time, shows that the IL‐6/JAK2/STAT3 pathway upregulates DNMT1 and enhances cancer initiation and lung cancer stem cell (CSC) proliferation by downregulation of p53 and p21 resulting from DNA hypermethylation. Upon blockage of the IL‐6/JAK2/STAT3 pathway and inhibition of DNMT1, the proliferation of lung CSCs was reduced and their formation of spheres and ability to initiate tumor growth were decreased. These data suggest that targeting of the IL‐6/JAK2/STAT3 signaling pathway and DNMT1 may become important strategies for treating lung cancer.     

Nuclear EGFRvIII‐STAT5b complex contributes to glioblastoma cell survival by direct activation of the Bcl‐XL promoter

Aberrant EGFR signaling strongly promotes glioma malignancy and treatment resistance. The most prevalent mutation, ΔEGFR/EGFRvIII, is an in‐frame deletion of the extracellular domain, which occurs in more than 25% of glioblastomas and enhances growth and survival of tumor cells. Paradoxically, the signaling of the potent oncogene ΔEGFR is of low intensity, raising the question of whether it exhibits preferential signaling to key downstream targets. We have observed levels of phosphorylation of STAT5 at position Y699 in cells expressing ΔEGFR that are similar or higher than in cells that overexpress EGFR and are acutely stimulated with EGF, prompting us to investigate the role of STAT5 activation in glioblastoma. Here, we show that in human glioblastoma samples, pSTAT5 levels correlated positively with EGFR expression and were associated with reduced survival. Interestingly, the activation of STAT5b downstream of ΔEGFR was dependent on SFKs, while the signal from acutely EGF‐stimulated EGFR to STAT5b involved other kinases. Phosphorylated STAT5b and ΔEGFR associated in the nucleus, bound DNA and were found on promoters known to be regulated by STAT5 including that of the Aurora A gene. ΔEGFR cooperated with STAT5b to regulate the Bcl‐XL promoter and knockdown of STAT5b suppressed anchorage independent growth, reduced the levels of Bcl‐XL and sensitized glioblastoma cells to cisplatin. Together these results delineate a novel association of nuclear ΔEGFR with STAT5b, which promotes oncogenesis and treatment resistance in glioblastoma by direct regulation of anti‐apoptotic gene, Bcl‐XL.     

Loss of cooperativity of secreted CD40L and increased dose–response to IL4 on CLL cell viability correlates with enhanced activation of NF‐kB and STAT6

Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non‐malignant B‐lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non‐malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment‐dependent anti‐apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF‐1, BAFF, APRIL, anti‐IgM, interleukin‐4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose–response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non‐malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high‐throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF‐kB pathway in the malignant cells. Thus, we propose that the anti‐apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF‐kB signaling due to an increased affinity of sCD40L to its receptor.     

The invasive potential of human melanoma cell lines correlates with their ability to alter fibroblast gene expression in vitro and the stromal microenvironment in vivo

The tumor microenvironment is thought to play an important role in invasion and metastasis. Previously, we have shown that signaling from melanoma cells can alter the gene expression profiles of fibroblasts in vitro and in vivo. To investigate whether the capacity to signal fibroblasts and alter host gene expression profiles is correlated to the invasive potential of specific human melanoma cell lines, we assayed changes in gene expression of fibroblasts when cocultured with the human melanoma cell lines BLM, MV3, A2058, SK‐mel28 and WM164. Results indicated that the gene expression of key chemokines and cytokines, such as IL‐1B, IL‐8, IL‐6 and CCL2/MCP1, was significantly upregulated in fibroblasts cocultured with the invasive melanoma lines BLM and MV3 compared to fibroblasts cocultured with noninvasive WM164 cells. The results were verified by quantitative RT‐PCR as well as by protein assay and supported by immunohistochemistry of human invasive melanoma. Furthermore, a role for fibroblast‐secreted IL‐1B in the invasion of melanoma was demonstrated in vitro, where siRNA silencing of IL‐1B in melanoma‐stimulated fibroblasts resulted in a diminution of melanoma invasion. Although CCL2/MCP1, a chemoattractant for macrophages, was shown to be upregulated in fibroblasts cocultured with metastatic melanoma cell lines, immunohistochemical analysis of human melanoma also indicated CCL2/MCP1 production associated with the melanoma. In summary, these experiments indicate that the invasiveness of melanoma can partly be correlated to its ability to stimulate host stromal fibroblasts to give rise to the secretion of chemokines that generate a microenvironment that is conductive for melanoma invasion and metastasis. © 2009 UICC