Supplementary Figure 2 from Breast Cancer Cells Stimulate Neutrophils to Produce Oncostatin M: Potential Implications for Tumor Progression

Supplementary Figure 1 from Breast Cancer Cells Stimulate Neutrophils to Produce Oncostatin M: Potential Implications for Tumor Progression

Breast cancer cell-response to inflammatory cytokines such as interleukin-6 (IL-6) and oncostatin M (OSM) may affect the course of clinical disease in a cancer subtype-dependent manner. Furthermore, vascular endothelial growth factor A (VEGF) secretion induced by IL-6 and OSM may also be subtype-dependent. Utilizing datasets from Oncomine, we show that poor survival of invasive ductal carcinoma (IDC) breast cancer patients is correlated with both high VEGF expression and high cytokine or cytokine receptor expression in tumors. Importantly, epidermal growth factor receptor-negative (HER2-), but not HER2-positive (HER2+), patient survival is significantly lower with high tumor co-expression of VEGF and OSM, OSMRβ, IL-6, or IL-6Rα compared to low co-expression. Furthermore, assessment of HER2- breast cancer cells in vitro identified unique signaling differences regulating cytokine-induced VEGF secretion. The levels of VEGF secretion were analyzed by ELISA with siRNAs for hypoxia inducible factor 1 α (HIF1α) and signal transducer and activator of transcription 3 (STAT3). Specifically, we found that estrogen receptor-negative (ER-) MDA-MB-231 cells respond only to OSM through STAT3 signaling, while ER+ T47D cells respond to both OSM and IL-6, though to IL-6 to a lesser extent. Additionally, in the ER+ T47D cells, OSM signals through both STAT3 and HIF1α. These results highlight that the survival of breast cancer patients with high co-expression of VEGF and IL-6 family cytokines is dependent on breast cancer subtype. Thus, the heterogeneity of human breast cancer in relation to IL-6 family cytokines and VEGF may have important implications in clinical treatment options, disease progression, and ultimately patient prognosis.

Chronic pancreatitis is an inflammatory disease that causes progressive destruction of pancreatic acinar cells and, ultimately, loss of pancreatic function. We investigated the role of IκB kinase α (IKKα) in pancreatic homeostasis. Pancreas-specific ablation of IKKα (Ikkα(Δpan)) caused spontaneous and progressive acinar cell vacuolization and death, interstitial fibrosis, inflammation, and circulatory release of pancreatic enzymes, clinical signs resembling those of human chronic pancreatitis. Loss of pancreatic IKKα causes defective autophagic protein degradation, leading to accumulation of p62-mediated protein aggregates and enhanced oxidative and ER stress in acinar cells, but none of these effects is related to NF-κB. Pancreas-specific p62 ablation prevented ER and oxidative stresses and attenuated pancreatitis in Ikkα(Δpan) mice, suggesting that cellular stress induced by p62 aggregates promotes development of pancreatitis. Importantly, downregulation of IKKα and accumulation of p62 aggregates were also observed in chronic human pancreatitis. Our studies demonstrate that IKKα, which may control autophagic protein degradation through its interaction with ATG16L2, plays a critical role in maintaining pancreatic acinar cell homeostasis, whose dysregulation promotes pancreatitis through p62 aggregate accumulation.

Saturated fatty acids (FA) exert adverse health effects and are more likely to cause insulin resistance and type 2 diabetes than unsaturated FA, some of which exert protective and beneficial effects. Saturated FA, but not unsaturated FA, activate Jun N-terminal kinase (JNK), which has been linked to obesity and insulin resistance in mice and humans. However, it is unknown how saturated and unsaturated FA are discriminated. We now demonstrate that saturated FA activate JNK and inhibit insulin signaling through c-Src activation. FA alter the membrane distribution of c-Src, causing it to partition into intracellular membrane subdomains, where it likely becomes activated. Conversely, unsaturated FA with known beneficial effects on glucose metabolism prevent c-Src membrane partitioning and activation, which are dependent on its myristoylation, and block JNK activation. Consumption of a diabetogenic high-fat diet causes the partitioning and activation of c-Src within detergent insoluble membrane subdomains of murine adipocytes.

Epidemiological studies indicate that overweight and obesity are associated with increased cancer risk. To study how obesity augments cancer risk and development, we focused on hepatocellular carcinoma (HCC), the common form of liver cancer whose occurrence and progression are the most strongly affected by obesity among all cancers. We now demonstrate that either dietary or genetic obesity is a potent bona fide liver tumor promoter in mice. Obesity-promoted HCC development was dependent on enhanced production of the tumor-promoting cytokines IL-6 and TNF, which cause hepatic inflammation and activation of the oncogenic transcription factor STAT3. The chronic inflammatory response caused by obesity and enhanced production of IL-6 and TNF may also increase the risk of other cancers.

1637 Oncostatin M (OSM), a multifunctional cytokine belonging to the IL-6 family, is produced by many cell types including human T-lymphocytes, neutrophils, macrophages and monocytes. OSM has been shown to inhibit the proliferation of breast and other tumor cell lines in vitro, and attention has been given to OSM as a potential cancer therapeutic. Recent studies, however, have shown that OSM stimulates cell detachment and invasion in vitro, and induces vascular endothelial growth factor (VEGF) in endothelial and astroglioma cells, which suggests that OSM may play a role in angiogenesis and tumor progression. Here we demonstrate that OSM induces VEGF by approximately 4-fold in several human breast cancer cell lines, including MDA-MB-231 and T47D, and that this induction is dose- and time-dependent. Additionally, we demonstrate a novel property of OSM in the up-regulation of HIF1α, a potent pro-angiogenic transcription factor, and provide new evidence that OSM-induced VEGF expression is dependent on both the STAT3 and HIF1 signaling pathways. We show that conditioned media from OSM-treated breast cancer cells enhances endothelial cell formation by ∼150%, as measured by an increase in branch points in an in vitro angiogenesis assay. In addition, OSM-induced VEGF causes dramatic in vivo neovascularization, as measured by a 12-fold increase in hemoglobin levels in a Matrigel plug assay. This is the first direct evidence that OSM-induced VEGF in human breast cancer cells occurs through activation of STAT3 and HIF1α, and has a potent pro-angiogenic effect on endothelial cells in vitro and in vivo. These results suggest the importance of designing therapeutics to inhibit OSM during breast cancer progression and subsequent metastasis.

BACKGROUND. Hepatocyte growth factor/scatter factor (HGF/SF) facilitates intercellular communication between the epithelial carcinoma and its surrounding stromal tissue during metastatic invasion through interaction with its proto-oncogenic receptor, Met, found on carcinoma cells. This study utilizes the C3(1)/Tag transgenic mouse prostate cancer cell line model in an attempt to characterize the interaction between HGF/SF and Met on the metastatic potential of prostate cancer. METHODS. Exogenous HGF was supplied to the prostate adenocarcinoma cell line (Pr-14) and metastatic cell line (Pr-14c) to evaluate mitogenicity by proliferation assays, morphological characteristics on an extracellular matrix substrate, and motogenic properties using the scatter assay, invasion chambers, and zymogram studies to analyze secretory enzymes produced by the cell lines. RESULTS. RNA and protein analyses show that the cell lines express similar amounts of Met. Pr-14 cells have an increased growth rate following HGF/SF treatment, whereas the metastatic Pr-14c cells show little change. Morphological studies of Pr-14c cells on extracellular matrix demonstrate negligible changes when compared to the tubular formation of Pr-14 cells after HGF/SF stimulation. Motility studies of the metastatic cells following HGF/SF treatment reveal a potentially faulty signaling pathway downstream of Met activation in the metastatic prostate cells. CONCLUSIONS. Our studies suggest that proliferation, invasion, and cell morphological characteristics may be induced independently from the HGF/SF-Met pathway in C3(1)/Tag metastatic prostate cancer cells. (c) 2005 Wiley-Liss, Inc.

Abstract Tumor-associated and tumor-infiltrating neutrophils (TAN) and macrophages (TAM) can account for as much as 50% of the total tumor mass in invasive breast carcinomas. It is thought that tumors secrete factors that elicit a wound-repair response from TAMs and TANs and that this response inadvertently stimulates tumor progression. Oncostatin M is a pleiotropic cytokine belonging to the interleukin-6 family that is expressed by several cell types including activated human T lymphocytes, macrophages, and neutrophils. Whereas oncostatin M can inhibit the proliferation of breast cancer cells in vitro, recent studies suggest that oncostatin M may promote tumor progression by enhancing angiogenesis and metastasis. In addition, neutrophils can be stimulated to synthesize and rapidly release large quantities of oncostatin M. In this article, we show that human neutrophils secrete oncostatin M when cocultured with MDA-MB-231 and T47D human breast cancer cells. Neutrophils isolated from whole blood or breast cancer cells alone express little oncostatin M by immunocytochemistry and ELISA, but neutrophils express and release high levels of oncostatin M when they are cocultured with breast cancer cells. In addition, we show that granulocyte-macrophage colony-stimulating factor produced by breast cancer cells and cell-cell contact are both necessary for the release of oncostatin M from neutrophils. Importantly, neutrophil-derived oncostatin M induces vascular endothelial growth factor from breast cancer cells in coculture and increases breast cancer cell detachment and invasive capacity, suggesting that neutrophils and oncostatin M may promote tumor progression in vivo.

Previously, oncostatin M (OSM) has been shown to inhibit the proliferation of breast cancer cells in vitro. Circumstantial evidence, however, suggests that OSM could be involved in the development of a metastatic phenotype in vivo. We examined the effects of OSM on the proliferation and metastatic potential of the murine mammary carcinoma cell lines M6 (adenocarcinoma) and M6c (metastatic adenocarcinoma). OSM inhibits the proliferation of both cell lines by 43%, but also causes a loss of cell-cell and cell-substratum adhesion that culminates in cell detachment from monolayer culture. OSM treatment results in a 258% and 550% increase in the detachment of M6 and M6c, respectively, in 32 hours. This effect was abrogated by the selective Cox-2 inhibitor NS-398, and by the anti-inflammatory glucocorticoid dexamethasone. Exogenous prostaglandin E2 (PGE2) partially reverses NS-398's inhibition of OSM-induced cell detachment, indicating Cox-2 involvement. In addition, OSM induces the expression of Cox-2 mRNA, and of the 74 kDa form of Cox-2 protein. M6 and M6c cells detached by OSM are viable and will re-adhere and proliferate in the absence of OSM. OSM-detached cells (M6DET and M6cDET) were collected and maintained in culture and their invasiveness was assessed in vitro. Importantly, M6DET and M6cDET are both significantly more invasive that their respective parental cells. These data suggest that OSM could contribute to the development of a metastatic phenotype in vivo, which would render OSM unsuitable as a cancer therapy and suggest that OSM itself is a potential therapeutic target.

Oncostatin M (OSM), an IL-6 family cytokine, has previously been shown to increase migration of several breast cancer cell lines in vitro. Our studies report additional effects of OSM treatment on the human breast carcinoma cell line T-47D. OSM treatment alters T-47D cell morphology from a normal epithelial phenotype to a mesenchymal-like phenotype that is associated with cell detachment from substratum. These effects are also seen with H3922 human breast cancer cells. OSM treatment of T-47D cells for 5–8 days leads to a three-fold increase in cell detachment. OSM-induced detachment of T-47D cells is blocked by the protein kinase inhibitors UO126 and bisindolylmaleimide, indicating a role for MAP kinases and protein kinase C in OSM signaling events that regulate cell detachment. T-47D cells induced to detach by OSM have a reduced capacity to re-adhere to laminin in comparison to other extracellular matrix components. Detached multi-cell aggregates of T-47D cells are viable, whereas detached single cells appear apoptotic. In addition, OSM treatment induces the secretion of the lysosomal proteases cathepsins D and L from T-47D cells, which have been implicated in invasion and metastasis. Importantly, OSM-treated T-47D cells show a 250% increase in invasive capacity as measured by the Matrigel invasion chamber assay. Collectively, these data demonstrate that OSM induces a motile/invasive phenotype in T-47D cells in vitro, and suggest that OSM may enhance metastasis in vivo. Our results suggest that OSM itself may be a valid therapeutic target.

We have developed four new mammary adenocarcinoma cell lines from the C3(1)/SV40 Large T-antigen (Tag) transgenic mouse model: M28N2 and M27H4 (weakly tumorigenic), M6 (carcinoma), and M6C (metastatic). The C3(1) promoter directs Tag expression to the mammary epithelium and 100% of female C3(1)/Tag transgenic mice develop mammary adenocarcinoma in a predictable and progressive manner. The cell lines we developed from this model are demonstrated to be of epithelial origin and display growth rates, both in vitro and following subcutaneous inoculation into nude mice, that are consistent with their representative stage of tumor progression. The more tumorigenic cell lines, M6 and M6C, both express the sodium/iodide symporter, a mammary carcinoma cell marker with potential therapeutic and diagnostic applications. All of the cell lines express estrogen receptor (ER) α and ER β mRNA, and Western blot analysis demonstrates that the ER α protein is down-regulated in the M6 and M6C cell lines. M28N2 cells also express progesterone receptor (PgR), which is very unusual in a mouse mammary carcinoma cell line. In addition, all of the cell lines display growth inhibition when plated in media supplemented with charcoal-stripped fetal calf serum (CS FBS). When CS FBS is supplemented with β estradiol or the progestin MPA, no significant difference in growth rates is observed relative to growth in CS FBS. The development and characterization of a progressive series of new mammary carcinoma cell lines will aid in the study of mammary carcinoma progression both in vitro and in vivo.