Tasnim Ara

Interleukin-6 in Bone Metastasis and Cancer Progression

The bone and bone marrow are among the most frequent sites of cancer metastasis. It is estimated that 350,000 patients die with bone metastases annually in the United States. The ability of tumor cells to colonize the bone marrow and invade the bone is the result of close interactions between tumor cells and the bone marrow microenvironment. In this article, we review the contribution of interleukin-6 (IL-6) produced in the bone marrow microenvironment to bone metastasis. This cytokine has a strong pro-tumorigenic activity due to its multiple effects on bone metabolism, tumor cell proliferation and survival, angiogenesis, and inflammation. These effects are mediated by several signaling pathways, in particular the Janus kinase/signal transducer and transcription activator (JAK/STAT-3), Ras/mitogen activated protein kinase (MAPK), and phosphoinositol-3 kinase (PI3K)-protein kinase B/Akt (PkB/Akt), which are activated by IL-6 and amplified in the presence of soluble IL-6 receptor (sIL-6R). Supporting the role of IL-6 in human cancer is the observation of elevated serum levels of IL-6 and sIL-6R in patients with bone metastasis and their association with a poor clinical outcome. Over the last decade several large (monoclonal antibodies) and small (inhibitors of IL-6 mediated signaling) molecules that inhibit IL-6 activity in preclinical models have been developed. Several of these inhibitors are now undergoing phases I and II clinical trials, which will determine their inclusion in the list of effective targeted agents in the fight against cancer.

Vα24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages

Tumor infiltration with Valpha24-invariant NKT cells (NKTs) associates with favorable outcome in neuroblastoma and other cancers. Although NKTs can be directly cytotoxic against CD1d+ cells, the majority of human tumors are CD1d-. Therefore, the role of NKTs in cancer remains largely unknown. Here, we demonstrate that CD68+ tumor-associated monocytes/macrophages (TAMs) represented the majority of CD1d-expressing cells in primary human neuroblastomas. TAMs stimulated neuroblastoma growth in human cell lines and their xenografts in NOD/SCID mice via IL-6 production. Indeed, TAMs produced IL-6 in primary tumors and in the BM of patients with metastatic neuroblastoma. Gene expression analysis using TaqMan low-density arrays of 129 primary human neuroblastomas without MYCN amplification revealed that high-level expression of TAM-specific genes (CD14, CD16, IL6, IL6R, and TGFB1) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing of monocytes was CD1d restricted because it was inhibited by a CD1d-specific mAb. Cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and suppressed tumor growth compared with mice transferred with monocytes alone. Thus, killing of TAMs reveals what we believe to be a novel mechanism of NKT antitumor activity that relates to the disease outcome.

Interleukin-6 in the Bone Marrow Microenvironment Promotes the Growth and Survival of Neuroblastoma Cells

Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells. An analysis of the expression of IL-6 and its receptor, IL-6R, in 11 neuroblastoma cell lines indicated the expression of IL-6 in 4 cell lines and of IL-6R in 9 cell lines. Treatment of IL-6R-positive cells with recombinant human IL-6 resulted in signal transducer and activator of transcription-3 and extracellular signal-regulated kinase-1/2 activation. Culturing IL-6R-positive neuroblastoma cells in the presence of BMSC or recombinant human IL-6 increased proliferation and protected tumor cells from etoposide-induced apoptosis, whereas it had no effect on IL-6R-negative tumor cells. In vivo, neuroblastoma tumors grew faster in the presence of a paracrine source of IL-6. IL-6 induced the expression of cyclooxygenase-2 in neuroblastoma cells with concomitant release of prostaglandin-E2, which increased the expression of IL-6 by BMSC. Supporting a role for stromal-derived IL-6 in patients with neuroblastoma bone metastasis, we observed elevated levels of IL-6 in the serum and bone marrow of 16 patients with neuroblastoma bone metastasis and in BMSC derived from these patients. Altogether, the data indicate that stromal-derived IL-6 contributes to the formation of a bone marrow microenvironment favorable to the progression of metastatic neuroblastoma.

Mechanisms of invasion and metastasis in human neuroblastoma

Neuroblastoma is the second most common solid tumor in children that is metastatic in 70% of patients at the time of diagnosis. The ability of neuroblastoma cells to colonize distant organs like the bone marrow and the bone is the result of close interactions between tumor cells and the microenvironment. Significant progress has been recently made in our understanding of the mechanisms that promote the colonization and invasion of the bone by neuroblastoma cells and these mechanisms are reviewed in this article. How this understanding is now allowing us to test new therapeutic agents specifically targeted at interfering with neuroblastoma metastasis is then discussed.

Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma

Valpha24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.

Critical Role of STAT3 in IL-6–Mediated Drug Resistance in Human Neuroblastoma

Drug resistance is a major cause of treatment failure in cancer. Here, we have evaluated the role of STAT3 in environment-mediated drug resistance (EMDR) in human neuroblastoma. We determined that STAT3 was not constitutively active in most neuroblastoma cell lines but was rapidly activated upon treatment with interleukin (IL)-6 alone and in combination with the soluble IL-6 receptor (sIL-6R). Treatment of neuroblastoma cells with IL-6 protected them from drug-induced apoptosis in a STAT3-dependent manner because the protective effect of IL-6 was abrogated in the presence of a STAT3 inhibitor and upon STAT3 knockdown. STAT3 was necessary for the upregulation of several survival factors such as survivin (BIRC5) and Bcl-xL (BCL2L1) when cells were exposed to IL-6. Importantly, IL-6-mediated STAT3 activation was enhanced by sIL-6R produced by human monocytes, pointing to an important function of monocytes in promoting IL-6-mediated EMDR. Our data also point to the presence of reciprocal activation of STAT3 between tumor cells and bone marrow stromal cells including not only monocytes but also regulatory T cells (Treg) and nonmyeloid stromal cells. Thus, the data identify an IL-6/sIL-6R/STAT3 interactive pathway between neuroblastoma cells and their microenvironment that contributes to drug resistance.

A phase I study of zoledronic acid and low‐dose cyclophosphamide in recurrent/refractory neuroblastoma: A new approaches to neuroblastoma therapy (NANT) study

Zoledronic acid, a bisphosphonate, delays progression of bone metastases in adult malignancies. Bone is a common metastatic site of advanced neuroblastoma. We previously reported efficacy of zoledronic acid in a murine model of neuroblastoma bone invasion prompting this Phase I trial of zoledronic acid with cyclophosphamide in children with neuroblastoma and bone metastases. The primary objective was to determine recommended dosing of zoledronic acid for future trials.

Sorafenib inhibits endogenous and IL-6/S1P induced JAK2-STAT3 signaling in human neuroblastoma, associated with growth suppression and apoptosis

Neuroblastoma is the most common extracranial solid tumor in the pediatric population. Sorafenib (Nexavar), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in certain types of cancers. Here, we tested antitumor effects of sorafenib (≤ 10 µM) on four human neuroblastoma cell lines, CHLA255, CHLA171, CHLA90 and SK-N-AS. Sorafenib inhibited cell proliferation and induced apoptosis of neuroblastoma tumor cells in a dose-dependent manner. Sorafenib inhibited phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) proteins at Tyr705 in these cells, associated with inhibition of phosphorylated JAK2, an upstream kinase that mediates STAT3 phosphorylation. Expression of a constitutively-activated STAT3 mutant (pSTAT3-C) partially blocked the antitumor effects of sorafenib on neuroblastoma cells. Sorafenib also inhibited the phosphorylation of STAT3 induced by IL-6 and sphingosine-1-phosphate (S1P), a recently identified regulator for STAT3, in these tumor cells. Moreover, sorafenib downregulated phosphorylation of MAPK (p44/42) in neuroblastoma cells, consistent with inhibition of their upstream regulators MEK1/2. Sorafenib inhibited expression of cyclin E, cyclin D1/D2/D3, key regulators for cell cycle, and the antiapoptotic proteins Mcl-1 and survivin. Finally, sorafenib suppressed the growth of human neuroblastoma cells in a mouse xenograft model. Taken together, these findings suggest the potential use of sorafenib for the treatment of pediatric neuroblastomas.

Abstract 508: Signal transduction and activator of transcription (STAT) 3 is necessary for environment-mediated drug resistance

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL There is recent evidence that STAT3 plays a pivotal and central role in controlling inflammatory pathways that promote malignant transformation and progression, including the ability of tumor cells to escape drug-induced apoptosis. Using neuroblastoma as a model, our laboratory had previously demonstrated that bone marrow mesenchymal stem cells through their production of interleukin-6 are a source of environment-mediated drug resistance. Here we have examined the role of STAT3, a downstream target of IL-6, in this effect. We first demonstrate that in contrast to many other cancers, STAT3 is not constitutively active in most neuroblastoma cells. However, it is rapidly phosphorylated after 15 minutes of exposure to IL-6 and its soluble receptor (sIL6-R). Activation of STAT3 is associated with its nuclear translocation and DNA binding. Treatment of CHLA-255 and SK-N-SH neuroblastoma cells with IL-6 increases their resistance to Etoposide and Melphalan in a dose-dependent manner. IL-6 inhibits the release of cytochrome C from the mitochondria and upregulates several pro-survival factors like Bcl-XL, XIAP and survivin without affecting the expression of Bax and Bad. The protective effect of IL-6 on drug-induced apoptosis is significantly inhibited in the presence of the STAT3 inhibitor Stattic (2.5 μM) or upon downregulation of STAT3 by siRNA, indicating that STAT3 is necessary for drug resistance. We also show that sIL-6R, produced by monocytes, enhances STAT3 activation and promotes drug resistance. In vivo, IL-6 and sIL-6R increase STAT3 activation in CHLA-255 neuroblastoma cells and their proliferation. Finally we observed elevated expression of phosphoSTAT3 in bone marrow samples of patients with metastatic neuroblastoma cells. Altogether, the data demonstrate that in neuroblastoma STAT3 activation by IL-6 in the tumor microenvironment plays a key role in environment-mediated drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2011-508

Abstract 4340: Sorafenib induces growth arrest and apoptosis in neuroblastoma cells via inhibition of JAK2/STAT3 and MEK1/2/MAPK (p44/42) signaling pathways

Neuroblastoma is the most common extracranial solid tumor in the pediatric population. It accounts for 6-10% of all childhood cancers. Currently, there is a critical need to find new drugs that are less toxic and target cell signaling pathways implicated as key mediators in the formation of neuroblastoma. Sorafenib (Nexavar), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in certain types of cancers. Here, we tested antitumor effects of sorafenib (≤ 10 µM) on three human neuroblastoma cell lines, CHLA255, CHLA171 and SK-N-AS. Sorafenib inhibited cell proliferation and induced apoptosis in these neuroblastoma tumor cells in a time- and dose-dependent manner. Sorafenib inhibited phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) proteins at Tyr705 in these cells as early as 15 min after treatment, associated with inhibition of phosphorylated JAK2, an upstream kinase that mediates STAT3 phosphorylation. Expression of a constitutively-activated STAT3 mutant (pSTAT3-C) partially blocked the antitumor effects of sorafenib on neuroblastoma cells. Sorafenib also inhibited the phosphorylation of STAT3 induced by IL-6 in these tumor cells. Moreover, sorafenib downregulated phosphorylation of MAPK (p44/42) in these neuroblastoma cells after 5 min treatment, consistent with inhibition of their upstream regulators MEK1/2. Finally, sorafenib inhibited expression of cyclin E, cyclin D1, cyclin D2 and cyclin D3, key regulators for cell cycle, and the antiapoptotic protein Mcl-1, which was accompanied by induction of growth arrest and apoptosis in neuroblastoma cells. Taken together, these findings suggest the potential use of sorafenib for the treatment of pediatric neuroblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4340. doi:10.1158/1538-7445.AM2011-4340