By Rachel Brand

Jennifer Richer, Andrew Thorburn and Kate Horwitz are three of 42 CU Cancer Center members doing breast cancer research.

Breast cancer

To those outside medicine, it seems like one disease. But scientists believe that breast cancer may contain as many as 40 subtypes, each with its own fingerprint that may reveal exactly what genetic mutations, receptors, signaling pathways or other cellular processes may be at play in their formation.

If different tumor types have different fingerprints, that rules out a magic bullet to treat everyone. But it also points the way to tests and treatments based on individual tumor types. Those treatments will rely on targeted therapies that attack the cancer cells and nothing else—a paradigm shift from traditional therapies that are broadly used and that wipe out all fast-dividing cells. Tomorrow’s cancer treatments will hone in on the genetic mutations or mistakes in cancer cells, fix them or render them irrelevant. But what exactly should doctors target?

Receptors or faulty genes? Signaling pathways? All of the above? Forty two of the University of Colorado Cancer Center’s members are working to answer these questions about breast cancer. They are seeking specific genes and other cell qualities that make tumors different from normal cells and other tumors. Some of these genes make tumors grow faster or make them immune to therapy. Other genes play a part in how tumors evolve as they progress and respond (or don’t) to treatment. And some cancer-specific genes may do nothing at all.

“What we have to sort out is which markers and genes drive breast cancer growth and survival, and what are just innocent bystanders,” said Anthony Elias MD, professor of Medical Oncology at the University of Colorado Denver School of Medicine (CU med school) and CU Cancer Center associate director for clinical research. “We need to figure out what is allowing the tumor to succeed—that’s what can be a therapeutic target.”

Hormones and breast cancer

It’s widely known that 70 percent of breast cancer tumors are so-called “estrogen receptor positive.” Receptors are specialized protein molecules that can be thought of as locks on or inside a cell that accept certain proteins and hormones, among other things. The hormone fits into the receptor like a key into a lock, sending a signal to tell the tumor cell to grow.

Estrogen is a generic term for a group of hormones involved in female sexual maturation. Estrogen is also the most important hormone in hormone replacement therapy (HRT), the treatment to stave off hot flashes, brittle bones and other symptoms of menopause. While estrogen doesn’t cause cells to become cancerous, it does provide the environment in which cancer cells grow. Provide an environment rich in estrogen, and these breast tumors with estrogen receptors grow faster and migrate into surrounding tissue. Cut off the supply of estrogen, and the tumors starve to death.

In general, patients with estrogen receptor positive tumors are good candidates for anti-estrogen therapy, which can shrink tumors in three out of four patients. These therapies range from Tamoxifen, a drug that directly blocks the effect of estrogen in breast cancer cells; to aromatase inhibitors, drugs that stop post-menopausal women from converting other hormones to estrogen, in effect cleansing the cell environment of estrogen. Both drugs work well on their own.

When Elias, a breast cancer physician and researcher, arrived at CU Cancer Center in 2001, he quickly set to work on answering two long-standing questions: What would happen if he combined Tamoxifen and the aromatase inhibitor exemestane? Would the combination be a double whammy of a treatment?

Elias started a two-arm cancer clinical trial in 2002 of post-menopausal women with estrogen receptor positive tumors. Some patients received Tamoxifen alone, and others received the combination. But just eleven patients into the study, he cancelled the drug-combination arm. It appeared the combination did less than the medicines alone: it didn’t shrink tumors as well.

“It was counter to our original hypothesis,” he said. “But we didn’t understand why. At the earliest opportunity, we decided this was worth analyzing and formally studying.”

Dr. Horwitz

Enter Kathryn Horwitz, PhD, distinguished professor of Endocrinology, Diabetes and Metabolism at CU medical school and a CU Cancer Center basic researcher who was the first person to identify the role of progesterone receptors— another female hormone—in breast cancer, and Jennifer Richer, PhD, assistant professor of Pathology at CU medical school, who became expert in breast cancer in Horwitz’s lab.

Patients enrolled in Elias’s study had core needle biopsies of their tumors before treatment with Tamoxifen or Tamoxifen and exemestane. Horwitz and Richer compared the tumors’ pathology before and after treatment. They were able to break the tumors’ genetic codes to understand how the anti-estrogen therapies worked on a molecular level.

They were surprised to find that the drugs effectively canceled each other out. When exemestane kept estrogen away, Tamoxifen stopped doing its job of blocking estrogen in breast cancer cells and began acting like estrogen, fitting into the estrogen receptors on the cells and turning on the genes.

“So the implication is that Tamoxifen is not a pure anti-estrogen,” said Horwitz. “We unmasked its estrogen effects.” This information may push clinicians away from Tamoxifen to newer pure anti-estrogen treatments.

The tumor samples also contained markers that predicted patients’response to anti-estrogen therapy. While 75 percent of women with
estrogen receptor positive breast tumors respond to the therapy, that means 25 percent do not. Why don’t their tumors shrink? While
the larger answer remains a mystery, Richer and Horwitz’s analysesrevealed 50 genes that “predict” a good therapeutic response to
anti-estrogen therapies.

“It’s a big breakthrough,” said Richer, cautioning that the finding needs to be validated with a bigger group of patients.

If the results hold, the researchers could develop a test for patients’ tumors prior to therapy. If a tumor tests negative for a panel of some of those 50 genes, the patient wouldn’t waste her time with anti-estrogen therapy and could move on to more aggressive treatment.

Prostate cancer treatment for breast cancer

Interestingly, Richer also found that the receptor for the male hormone androgen remains turned on high in tumors of patients who don’t
respond to anti-estrogen therapy. Other researchers saw the same thing in estrogen receptor negative tumors, but hadn’t deemed it to be important in driving estrogen receptor positive tumors.

Richer and Elias, however, think the finding may be significant. Tumors are tricky; they evolve in response to their environment. The researchers wonder if, during treatment, some tumors switch from being activated by estrogen to being activated by androgen in order to stay viable. If so, patients who don’t respond to anti-estrogen therapy might benefit from androgen-deprivation therapy, a treatment for prostate cancer. Richer’s lab is testing this hypothesis in mice, and if promising, Elias might modify his clinical trial to test a combination of a prostate cancer drug and exemestane.

“There’s a chance that we have discovered a resistance pathway that may be very important in the treatment of breast cancer,” Elias said. While much of this study is preliminary, it lays the groundwork for therapeutic advances.

“It’s really important to figure out how these hormone receptors work,” Richer said. “Because really, the majority of breast cancers have all the receptors —estrogen, progesterone and androgen.” And while these hormone receptors may act differently in different women’s tumors, “it’s all geared towards more individualized therapy,” Richer added. “What is your tumor, what are its characteristics, and what type of therapy would work best for you?”

Another answer: stem cells

Horwitz is looking in another direction to answer the question of why some estrogen receptor positive breast cancers don’t respond to
anti-estrogen treatment: cancer stem cells. Breast cancer stem cells are a group of rare, slow-growing cells that resist current therapies. Horwitz’s research team is testing the hypothesis that breast cancers grow from a tiny group of cancerous stem cells, or cancer cells with stem cell-like properties.

“These stem cells are entirely different,” she said. “So you could take a tumor that is 99.9 percent estrogen receptor positive and administer anti-estrogen therapy, and the vast majority of the cancer would be killed, but the rare stem cells would survive.”

Further she is researching how women’s hormones may stimulate these cells, causing cancers to enlarge or recur.“This is a highly controversial area,” Horwitz said. “The idea is that you have a very large cancer, but only a few cells in that cancer are really feeding its development. It’s such a new area that even defining what a breast cancer stem cell is, is up for discussion.”

In a recent study, Horwitz and her collaborator, Carol Sartorius, PhD, assistant professor of Medicine at CU medical school, found that progesterone (a hormone given in conjunction with estrogen in HRT) helped to increase the number of breast cancer stem cells.

“There is no question that estrogens increase the growth of breast cancers that contain estrogen or progesterone receptors,” Horwitz
explained. “If we treat the cancers with progesterone, nothing much happens to growth. But now we’re seeing that, instead, progesterone increases the number of stem-like cells.”

Other studies have shown that women who take HRT containing estrogens and progestins–synthetic progesterone–are at higher risk for breast cancer than women taking HRT with estrogens alone. (The progestin is necessary to protect against uterine cancers). Studies also show that some women have small, dormant and undiagnosed breast cancers—tumors too small to be detected by mammography in four out of five cases. Horwitz believes the stem cells in these tiny tumors may become activated by the progestins in combination HRT.

The same may be true for breast cancer survivors. Horwitz believes they should avoid HRT. The progestins in the therapy may reawaken tiny  amounts of dormant tumor, and the estrogens would then cause the cancer to grow again. “We need to give progestins in ways that don’t target the breast,” she said. “These are our current ideas. Now we’re thinking about ways to test them and, importantly, to find drugs that might kill breast cancer stem cells,” she said. “But also we need to have much better diagnostic procedures to find tiny, early stage breast cancers. Such women should probably avoid progestins.”

Six1 and Breast Cancer

Another approach to targeted breast cancer therapy lies in the partnership of reproductive scientist Heide Ford, PhD, and pharmacologist Andrew Thorburn, PhD. They are investigating genetic abnormalities associated with cancer cells and searching for ways to make existing therapies more effective. These basic researchers are interested in the Six1 gene, a “master regulator” of human development. In normal development, the gene is responsible for cell growth and survival, cell migration and invasion and determining cells’ fate. Turned on (or expressed) in embryos, the gene plays a key role in the development of our kidneys, muscles and other organs.

“All cells in the body have the same genes,” said Ford, associate professor of Obstetrics and Gynecology at the University of Colorado
School of Medicine. “They get switched on or off by proteins in different sequences depending on what the cell is supposed to do. But no one knows why a gene like Six1 gets switched on again or how to turn it off. This is one of the big questions we’re trying to answer.”

Ford discovered that Six1 is turned off in most normal adult cells, but it is reactivated in about 50 percent of breast tumors and 90 percent of metastatic breast lesions. Tumors that express the Six1 gene correlate with worse prognosis, shortened survival and increased metastasis and relapse—in short, more aggressive cancer.

Ford has also been able to prove, using mouse models, that Six1 plays a role in creating cancer, and that it seems to be involved in
promoting tumor-initiating cells—stem-like cells that make up 1 percent of breast tumors, but may be responsible for creating the
other 99 percent of cells in a tumor. She has also shown in mouse models that Six1 caused tumors to metastasize to organs that are relevant to human breast cancer.

“Metastases are what kill most people,” she said. “So if you could find a way to remove the primary tumor and inhibit metastases, you might be able to prolong life and stop the tumor from coming back.”

That means finding a drug that changes the volume on Six1. You can’t design a drug until you solve the gene’s structure, an intricate task that uses a process called X-ray crystallography. Ford is working with Rui Zhao, PhD, assistant professor of Biochemistry and Molecular Genetics at UCD SOM and an expert in structural biology, to solve its structure. Once the structure is solved—and Ford and Zhao are close to doing so—Ford, Zhao and Thorburn can begin working to develop a drug that tones down Six1’s effects.

Thorburn said that Six1 also makes cells selectively resistant to a key signaling pathway the body’s immune system uses to cause cells to die. Called TRAIL, this pathway is significant in many types of cancer. At least six drugs are in development that will use it to enhance the body’s natural immune system so it can more effectively fight cancer. You can’t kill cancer cells that express Six1 with TRAIL-promoting drugs, Thorburn explained, and the TRAIL-promoting drugs could prove to be very useful in aggressive cancer, especially if it comes back after initial treatment. Figuring out how Six1 blocks TRAIL is important to identifying which people the TRAIL-promoting drugs will help or not.

“It’s critical that we put together all the disparate and unconnected bits of how this particular part of biology works, to really understand it,” said Thorburn, professor and vice-chair of Pharmacology at CU medical school and associate director for basic research at CU Cancer Center. “We are basic research scientists. We don’t treat patients. But ultimately, we want to help people who have cancer, so we need to be able to translate what we know into new therapies and tests.”

Rachel Brand is a Denver-based writer who covers health care, policy and biotechnology.