Inflammatory Cytokines Promote Melanoma Proliferation and Migration

Mawusi Kamassa, M.S., Maxwell Schwam, Omama Ahmad, Yassmin Hegazy, Zachariah Burns, Elizabeth Brandon, Ph.D.Department of Biological Sciences, Mississippi College, Clinton, MS

References

Summary

Background

Experimental and clinical data indicate that obesity increases the risk for colorectal, esophageal, pancreatic, endometrial, renal cell carcinoma, and post-menopausal breast cancer. We found that melanoma grows much faster in obese mice than in normal body weight mice. Several metabolic, endocrine, and immune system factors are positively correlated with a poor prognosis and shorter disease-free survival times in obesity associated cancers. The paracrine signaling between tumor cells and stromal cells is a hallmark of cancer that has only recently become appreciated as a catalyst for mutagenesis, immune evasion, and metastasis. Although some mechanistic knowledge has been obtained from a “one growth factor, one effect” approach, this must be paired with a systems analysis to gain an understanding of the interactions between stromal cells and melanoma cells. Our data reveals that the inflammatory cytokines, resistin and IL-6, promote melanoma proliferation (6.1x105 cells/day and 1.0x106 cells/day v. 5.5x105 cells/day for controls, p=0.05). There is still a wide gap in our knowledge of how obesity promotes melanoma metastasis. Individual inflammatory cytokines had no effect on melanoma migration, but co-culturing cells with macrophages significantly increases migration. Preliminary data also suggests that co-culture with adipocytes increases melanoma migration. The influence of inflammation on tumors is well known, but the mechanisms by which fat cells affect melanomas is unknown. These results suggest that there could be many stromal cells that affect melanoma growth and metastasis in obese individuals.

• Physiologic concentrations of resistin and IL-6 stimulate melanoma cell proliferation.

• Macrophages significantly increase melanoma cell migration while individual cytokines have no

effect.• Previous results from our lab revealed a role for the

adipokine leptin in melanoma tumor growth in obese mice. These results further our

understanding of the mechanisms by which obesity increases melanoma growth by indentifying a link between inflammation melanoma cell proliferation and migration.

• Cytokines and Growth FactorsWhen treating with an individual cytokine or growth factor, the concentrations were as follows: resistin and IL-6 both at 100 ng/mL and 10 ng/mL and IL-4 was used at 10 ng/mL. Cytokines were recombinant mouse proteins purchased from R & D Systems (Minneapolis, MN).• Proliferation24 well plates were plated with 20,000 cells/well in 1 mL of RPMI w/ 10% serum. After an overnight incubation, the medium was aspirated and 1 mL of RPMI w/ 0.5% serum was added. After 24 hours to synchronize, medium was aspirated and 1 mL of treatment medium was added. Medium containing 10% serum was added as positive control. 24 hours later, cells for the first time point were aspirated and then trypsinized with 200ul trypsin for 10 minutes. Cells were resuspended and counted using a Cellometer Auto T4 (Nexcelcom, Lawrence, MA). Cells reserved for the 48 hour time point were fresh medium. These cells were collected and counted the same as the 24 hour cells.

• Mueller MM, Fusenig NE. Friends or foes- bipolar effects of the tumour stroma in cancer. Nature 2004. Volume 4; 839-849.

• Chiang AC, Massagué J. Molecular basis of metastasis. N Engl J Med 2008; 359: 2814-23.• Lee, Yi-Chen, et al. Resistin expression in breast cancer tissue as a marker of prognosis and

hormone therapy stratification. Gynecologic Oncology 125.3 (2012): 742-750.• Gabay, Cem, et al. Leptin directly induces the secretion of interleukin 1 receptor antagonist in human

monocytes. Journal of Clinical Endocrinology & Metabolism 86.2 (2001): 783-791.• Mantzoros Giuseppe Matarese, Stergios Moschos and Christos S. Leptin in Immunology. J Immunol

2005; 174:3137-3142.• Lord, G. M., G. Matarese, J. K. Howard, R. J. Baker, S. R. Bloom, and R. I. Lechler. 1998. Leptin

modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394:897

• Martin-Romero, C., J. Santos-Alvarez, R. Goberna, and V. Sanchez-Margalet. 2000. Human leptin enhances activation and proliferation of human circulating T lymphocytes. Cell. Immunol. 199:15.

• Brandon E., Gu J., Cantwell L., Zhi H., Wallace G., Hall J.E.(2009) Obesity promotes melanoma tumor: growth: role of leptin. Cancer Biology & Therapy 8:19, 1871-1879.

Obesity is a serious health risk in America, approximately 32% of adults are obese, 65% are overweight and 18.8% of 6 to 11 year old children are obese. There is a strong, positive correlation between BMI and the occurrence of several types of cancer, such as colon, gastric, breast, ovarian, uterine, endometrial, renal cell carcinoma, prostate, and melanoma. The increasing incidences of these cancers is well correlated to the increasing trend in obesity in the last 30 years (Calle, 2003). Few studies have demonstrated a direct cause and effect relationship between obesity and cancer and the epidemiology of cancer suggests that the response to obesity is not the same for all tumors. Consequently, the mechanisms by which obesity may influence carcinogenesis are poorly understood. What is clear from these studies is that when obese patients get cancer, they have a poorer prognosis and shorter survival times. Metastatic disease significantly increases mortality and there is accumulating experimental and clinical evidence that obesity increases metastasis.

Results from experiments in which mouse models of obesity were used to test the hypothesis that obesity is associated with increased melanoma growth identified a positive correlation between obesity and melanoma growth. Tumor growth was partly dependent on the adipokine leptin. The ratio of tumor weight to body weight was significantly lower in obese leptin-deficient mice (ob/ob) compared to the ratio in obese leptin replete animals (Brandon, 2009). Tumors from the obese mice had significant necrosis, which is a potent stimulator of macrophage infiltration and inflammation. Obesity causes inflammation in adipose tissue and many tumors exhibit large numbers of immune cells in the tumor stroma. Inflammation promotes the growth and metastasis of many kinds of cancers, but its role in obesity-associated melanoma is unknown.

Many factors contribute to metastasis and they are interrelated. Angiogenesis, the extracellular matrix, interactions between tumor cells and stromal cells, and inflammation are all associated with metastasis (Mueller, 2004). Obesity is also associated with inflammation of adipose tissue and this may explain the poor prognosis of many obese cancer patients. Leptin production increases with BMI and stimulates expression of several inflammatory cytokines: the endogenous antagonist of the IL-1 receptor, IL-1Ra (Gabay, 2001), TNF-α, IL-6, and IL-12 (reviewed in Mantzoros, 2005). Leptin also promotes the proinflammatory T helper cell type 1 (Th1) differentiation (Lord, 1998; Martin-Romero, 2000). Resistin levels also rise in obesity and it is also associated with inflammation. Macrophages secrete resistin, which acts via positive feedback to increase the secretion of more resistin, TNF-α and IL-6. Some data indicate that resistin is correlated with breast, prostate, and colorectal cancers and high resistin levels in breast tumors is correlated with poor prognosis and survival (Lee, 2012). Therefore, a major goal of our research is to discern the mechanisms by which inflammation affects melanoma migration and invasion in vitro in order to identify points of intervention that may aid in slowing metastasis in vivo.

• Cell Culture: B16F10 mouse melanoma cancer cells were grown in an incubator at 37°C with 5% CO2 in RPMI with 10% fetal bovine serum (FBS), 4.5 g/L glucose, 2 mM sodium pyruvate, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 1% penicillin/streptomycin—hereafter referred to as normal RPMI. RAW 264.7 cells were cultured using the same conditions. These cells differentiated when exposed to melanoma cells.

• Migration: 80,000 cells were plated on inserts (8 µm pores, Millicell) and put in 700 ul of RPMI w/ 10% serum in 24-well plates. For the co-culture experiments, 150,000 cells were plated into the bottoms of 24-well plates and allowed to attach overnight. After 24 hours, inserts were removed from wells and medium was carefully pipetted out. Inserts were then place in 700 µL of treatment medium or in wells containing cells. Cells were incubated for 24 hours. Inserts were removed from plates, fixed in methanol, and stained with hematoxylin and eosin. Cells were removed from the proximal side of the insert with wetted cotton swabs and then migrated cells were counted on an inverted Nikon Eclipse Ti microscope with the 10x objective. Images were taken on the 20x objective with a Nikon DS-Fi2 camera.

Day 1: 80,000 cells were plated in inserts and 150,000 macrophages were plated in 24-well plates. Day 2: Inserts containing melanoma cells were transferred to wells containing macrophages and the cells were incubated overnight in regular RPMI. Day 3: Inserts were removed and stained. A. The graph represents the means of 2 independent experiments. A Student’s T-test was used to compare control and co-cultured cells. Error bars = SEM. B. and C. are representative images taken of the inserts.

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Cytokine induced proliferation of melanoma cells. Melanoma cells were seeded in 24-well plates (20K/well). Synchronized cells were treated with mouse resistin or IL-6 in regular RPMI. Cells were trypsinized and counted at 24 and 48 hrs with a T4 automated cell counter. Graphs show the means of 4 independent experiments for each treatment. Student’s T-test used to compare growth rates of treated cells to RPMI 10% serum at 48 hrs. Error bars = SEM.

We would like to thank Dr. Stephanie Carmicle for the RAW 264.7 mouse macrophages. This work was supported by the Mississippi INBRE, funded by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103476.

Acknowledgments

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Figure 3

60,000 cells/insert were plated and incubated overnight in regular RPMI. Cytokines were prepared in regular RPMI and added to the lower chambers. The medium in the upper chamber was replaced with fresh regular RPMI and the inserts were incubated for 24 hours. The graph represents the means of 3 independent experiments. ANOVA was used to compare treatments with control cells. Error bars = SEM.

Figure 4

B. Melanoma Alone C. Melanoma with Macrophages

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Day 1: 80,000 macrophages were plated in inserts and 150,000 melanoma cells were plated in 24-well plates. Day 2: Inserts containing macrophages were transferred to wells containing melanoma cells and the cells were incubated overnight in regular RPMI. Day 3: Inserts were removed and stained. A. The graph shows data from one preliminary experiment. B. and C. are representative images taken of the inserts.

Abstract: P1919

Abstract Methods

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