New cell model helping to build an arsenal against ovarian cancer
By Lindsay Key, Fralin Life Science Institute
It's no wonder that sphingolipids were named after the mythical, riddle-me-this creature known as the Sphinx. Since their discovery in cellular membranes in the 1870s, they've continued to challenge scientists. However, members of the Ovarian Cancer Research Group at Virginia Tech are testing the mysterious compounds for their potential to treat ovarian cancer.
Found in every cell, sphingolipids are involved in cell recognition, signal transmission, and the regulation of cell growth, differentiation, and cell death. Variations in the way the long-chain molecules assemble can generate thousands of distinct sphingolipids that perform unique functions in different cell types.
"You really have to start anew with every cell, and it appears that the sphingolipid effect is context-dependent. So, in a liver cell they induce different effects than in a colon cell or an ovarian cancer cell," says Eva Schmelz, a researcher with the Fralin Life Science Institute at Virginia Tech. "Depending on the function of the cell, there is a distinctive pattern of sphingolipids. There are literally thousands of sphingolipids in your body. I think we have only begun to understand the complexity of this whole field."
Schmelz became interested in sphingolipids as a postdoctoral associate at Emory University in Atlanta, Ga., in the early 1990s. After completing a Ph.D. in nutrition and human biology at Justus-Liebig University in Giessen, Germany, she began working in the laboratory of Al Merrill, then professor of biochemistry at Emory University, investigating the connection between dietary sphingolipids and colon cancer. (Merrill is now professor and chair of molecular cell biology at Georgia Tech.)
Because sphingolipids are found in all cells, they are also found in food, and are most abundant in milk, cheese, meats, and some vegetable products.
For the Emory University study, sphingolipids from milk were added to the diet of mice that carry a mutation that also causes colon cancer in humans. Compared to the mice that did not receive any sphingolipids, tumor development and growth was slowed by 50 to 80 percent in the mice that received the sphingolipid diet (Cancer Research, 1996 and 2001. Journal of Nutrition, 2000). While effective against cancer cells, dietary sphingolipids caused no side effects in the mice, most likely because the approach slows cancer cells without immediately killing them, says Schmelz.
The implications of this discovery — that cancer could be stymied into a chronic disease that exhibits fatal effects only after the typical life span — were so exciting that the research team extended the studies to include cancers outside the intestinal tract. They documented that the dietary sphingolipids slowed the progression of breast cancer in mice (Food and Function, 2010) and did not cause side effects commonly associated with advanced drug therapy regimens.
Schmelz was hooked. After completing her post-doctorate and a two-year research assistant professorship at Emory University, she accepted a position as assistant professor at the Karmanos Cancer Institute in the pathology department at Wayne State University School of Medicine in Detroit. There, she continued her work on colon and breast cancer prevention.
Confronting ovarian cancer
At Wayne State University, Schmelz teamed up with Chris Roberts, an assistant professor in immunology and microbiology, who was working to develop a mouse cell model for ovarian cancer that would broaden and energize ovarian cancer research. With the new mouse cell model, researchers can see the early, early/intermediate, intermediate, and late stages of ovarian cancer (Neoplasia, 2005).
Ovarian cancer is the fifth leading cause of death among all cancers in women in western countries and is the leading cause of death from female reproductive tract malignancies. It is usually detected late due to the lack of early symptoms; by the time 62 percent of patients are diagnosed, the cancer has spread to other parts of the body (metastasized).
Using the model, the researchers generated cell lines from mouse ovaries for each stage of the disease. "Just by looking at the cells through a microscope you can tell the difference between early and late stages," Schmelz says. "The late cells grow much faster than early cells, and are smaller and more mobile."
Schmelz and Roberts began to use the model to explore whether a sphingolipid-rich diet could prevent the progression of ovarian cancer. "Since ovarian cancer is very aggressive and not likely to respond to preventive compounds in the diet, I thought that we could use the model that shows us ovarian cancer at earlier stages to see if we can actually define the potential and also the limitations of this approach," she says.
At this point, in 2007, Roberts and Schmelz moved to Virginia Tech — Roberts as an associate professor of biomedical sciences and pathobiology in the Virginia-Maryland Regional College of Veterinary Medicine and Schmelz as an associate professor in human nutrition, foods, and exercise in the College of Agriculture and Life Sciences.
They continued to develop and use the mouse ovarian cancer cell lines to understand basic fundamental mechanisms driving cancer progression. Soon, they were able to identify molecular markers leading to the growth of mouse ovarian tumors, including alterations in cytoskeletal, adhesion, and intercellular communication proteins, all of which are typically seen in human ovarian cancer. Amy Creekmore, a post-doctoral associate working with Schmelz and Roberts, identified stage-specific changes in gene expression leading to fully transformed cancer cells (PLoS One 2011). That finding means that scientists can now explore preventive measures to stop or slow the spread of the disease based on those markers.
"We are currently determining which genes are responsible for driving the progression of these cells to identify the genetic fingerprint responsible for early disease, and establish these genes as a tool for early diagnosis," says Roberts.
Schmelz and Roberts are expanding their sphingolipid work to investigate whether sphingolipid metabolites can either prevent or reverse these gene expression changes, leading to suppression of tumor formation and progression.
The cancer research group
To fully understand ovarian cancer at all stages, Schmelz and Roberts initiated collaborations with scientists Matt Hulver and Madlyn Frisard in human nutrition and foods, biomedical engineers Rafael Davalos and Masoud Agah, all at Virginia Tech, and with physician Dennis R. Scribner Jr., who is section head of gynecologic oncology at Carilion Clinic in Roanoke, Va.
The group is supported by the National Institutes of Health, the College of Veterinary Medicine Internal Research Competition, and the Fralin Life Science Institute. Cancer biology is one of the institute's primary focus areas and Schmelz, Roberts, Hulver, Frisard, Davalos, and Agah are all Fralin-affiliated faculty members.
Roberts' studies include the responses of the immune system during peritoneal metastasis — when cancer spreads to the membrane that lines the inside of the abdomen and covers the uterus and other organs. It is the most aggressive and least responsive stage of the disease, says Roberts. He, Schmelz, Agah (an associate professor of electrical and computer engineering), and Alperen Ketene, who graduated in spring 2011 with a master's degree in mechanical engineering, have determined that the cellular architecture, or cytoskeleton, affects the biomechanical properties of cells (Nanomedicine, 2011). Changes in these properties can be related to cancer cells' ability to move and use energy, and potentially to their ability to invade other cells.
Cell cytoskeleton refers to the cell's shape and mechanical properties, Agah explains. "Any change in the cytoskeletal structure can affect the interaction of cells with their surrounding microenvironments. Biological events in normal cells, such as embryonic development, tissue growth and repair, and immune responses, as well as cancer cell motility and invasiveness, are dependent upon cytoskeletal reorganization," he says.
"When cells undergo changes in their viscoelastic properties, they are increasingly able to deform, squeeze, and migrate through size-limiting pores of tissue or vasculature onto other parts of the body," Agah says.
Their studies showed a mouse's ovarian cells are stiffer and more viscous when they are benign or more normal-like. Increases in cell deformation "directly correlates with the progression from a non-tumor benign cell to a malignant one that can produce tumors and metastases in mice," says Agah.
The team is investigating how the biophysical properties of ovarian cancer cells can be exploited for isolation and characterization, with the hope that the biophysical signature of cancer cells may reveal new ways to prevent them from spreading. In aid of rapid diagnosis, Davalos and his students are designing a device to trap ovarian cancer cells if they are in a sample of peritoneal fluid.
As a first step in translation of cell and animal research to humans, Scribner is helping to evaluate clinical ovarian cancer specimens from humans for some of the hallmarks identified in the mouse model. He and the Virginia Tech researchers are also looking at the interaction of the cancer cells with the immune system to discover early defects in immune response that allow ovarian cancer progression. It is hoped that these ongoing studies will provide new targets for therapeutic intervention of ovarian cancer.
But such interventions are years in the future. In the meantime, preliminary results also suggest that a sphingolipid-rich diet can significantly delay the onset of aggressive disease in mice, Schmelz says. Once aggressive disease is established, "dietary intervention is less effective," Roberts says. "But we hope that dietary intervention may also prove useful in combination with current therapies, perhaps minimizing side effects, while maximizing tumor cell killing."
"The natural extension of our studies begs the question: Is it possible that the sphingolipid-based preventive therapy would also be effective in humans? Could an effective ovarian cancer prevention regimen be achieved in five years, 10 years?" asks Schmelz. "I don't know. But if we can't develop a vaccine or an effective treatment strategy, then the next best option would be to turn ovarian cancer into a manageable chronic disease. Sphingolipid therapy may do just that."
"It's amazing the new insights that emerge through the collective interactions of an interdisciplinary group," says Roberts. "Attacking cancer from multiple perspectives will likely be the only way to manage the disease successfully."