Exploration of Pregnancy-Related Changes in Breast Tissue

Originally from the São Paulo state of Brazil, dos Santos was introduced to genetic and molecular biology during her undergraduate education in Brazil and recognized that as her future career path.

“The idea that we have all of that genetic molecular information inside ourselves and yet we don’t have it figured it out completely blows my mind,” she noted. “Yet all of this information is constantly at work inside us, all of the time. I felt a need to better understand it.”

Following the second year of her PhD studies related to gene regulation, dos Santos was invited to do a fellowship in a lab at The Children’s Hospital of Philadelphia. While there she published several papers and met her future husband, Chris Vakoc, MD, PhD, then an MD/PhD student, working in an adjacent lab. (Vakoc was profiled in Oncology Times in June 2017).

“We were the only people working weekends and late hours, so we ate a lot of dinners together,” she said with a laugh, “and that’s how we fell in love.” After dos Santos returned to Brazil to complete her PhD, the couple moved together to Cold Spring Harbor Laboratory. They are now married and the proud parents of two sons, 8 and 6.

When she arrived at Cold Spring Harbor, she was hunting for a project. Initially in the lab of Gregory Hannon, PhD, “I started learning about breast development and realized how very little we knew about the things that happen in breast cells,” recalled dos Santos. “That was very puzzling to me because it is such a relevant issue to nursing, to supporting offspring, and to breast cancer which ranks among the top three cancers killing women. I then started working on how to isolate cells from the breast in order to devise a technology, an approach, wherein we could really ask what kind of genes are in each one of those cells.”

Dos Santos spent a lot of time—and two pregnancies—working on the project. “By the time I had my second child, we were in a position to ask more biologically relevant questions. And that is when I started working with pregnancy-related changes specifically. I could relate to it having just been pregnant myself. I knew, for example, that milk production was easier in the second pregnancy than the first. I had experienced it. I knew things happened biologically and I set about finding the molecular basis for that biology.”

Exploring Breast Cells

Today, dos Santos has her own lab that maintains two prime objectives. “The first is to understand normal breast development, milk production, all of the signals that happen during pregnancy that really shapes up a different type of organ from the one a woman had when she was not pregnant,” she explained.

“We look at the nucleus of the cells, what happens when genes switch on and then switch off, and how that transfers into a different kind of functioning of the cells before, during, and after pregnancy. When we look at the tissue morphology, we see that the tissue totally goes back to how it looked before pregnancy occurred.

“However, we now understand that the cells are not the same; they are substantially different. Even how they react during a second pregnancy is different. We call it a ‘memory of pregnancy.’ Certain genes that are turned on by a first pregnancy dim down at the end of the pregnancy, but they never turn off. So when a second pregnancy occurs, they turn back on a hundred-fold faster than they did in the first pregnancy.”

The second major quest in her lab is for greater understanding about breast cancer risk, and particularly how pregnancy changes that risk. “We approach this by understanding the pregnancy-related changes in the genes that turn on and off. We correlate that with the fact women who are pregnant before the age of 25 years have a decreased risk in the development of breast cancer, while women who are pregnant after age 40 gain a greater risk of breast cancer.”

The dos Santos lab, however, specifically looks at the decrease in risk, not the increase in risk. “By examining the normal progression of cells, from normal to cancer, and how cells that had previously experienced pregnancy blocked that progression, we hope to identify better biomarkers that are more able to predict one’s predisposition to breast cancer,” the researcher explained. “We know there are a lot of genetic mutations that have been associated with breast cancer development, [and for] many cases of breast cancer cases there is still no known genetic basis for why a woman develops it. We know, for example, that women who carry the BRCA gene mutations are at a higher risk to develop those cancers, but a certain subset of those women will not develop breast cancer. Something is blocking it. What is it?”

The notion that better markers for women of any age, any pregnancy stage, and at any genetic risk could better predict the likelihood of cancer development is a powerful driving force in the dos Santos lab. To realize such a goal could have immense impact on patient outcomes.

“Perhaps clinicians would be able to say, ‘You have a 50 percent risk increase, so we should increase the frequency of your mammograms,’” said dos Santos. “If researchers can come up with better markers and gene signatures that can better predict disease development, they will guide clinicians in caring for patients.”

How Work Is Conducted

The dos Santos lab uses mouse models for much of the work, and for many reasons. “We can mimic in mice the same phenomena that happen in humans, not only allowing us to understand which changes after pregnancy can block cancer progression, but to manipulate those genes as well. If we find that a particular gene is associated with cells that do not turn into cancer, we can manipulate the gene and ask the question, ‘Would loss of that gene then eradicate or reduce the preventive aspect that pregnancy brings?’ It also gives us hints to developing biomarkers for screening as well as possibly developing a vaccination step that could be given to women to block cancer development,” dos Santos told Oncology Times.

Mice in the dos Santos Lab run the gamut from those that have never been pregnant and those that have been pregnant, to those that are pregnant now. “We look at how their DNA bends around—opens or closes—much like a yoyo,” dos Santos described. “When it is rolled up tight, genes are prevented from being switched on because proteins are so compacted inside. When the DNA rolls open—we call that open chromatin—the genes are all exposed and factors in our cells can turn on those genes.

“So we look at this yoyo effect—whether genes are exposed and on, or compact/closed and off, and how they may or may not switch back and forth to prevent cancer. And we study the ability of DNA-binding proteins to be present and activate gene expression.”

Prior to her current work as described with the lab mice, dos Santos found it necessary to develop research-appropriate mouse models—a task that took her a year and 8 months to complete.

“The need to develop mouse models was very clear in my mind from the beginning,” she noted. “I am not the first person to investigate the effects of pregnancy in breast cancer, but I felt the available models were not suitable for us to ask the right questions. We developed a mouse model that overexpresses an oncogene, very relevant to breast cancer and amplified in over 60 percent of breast cancer cells, that uses those open regions on the DNA to recreate cancer progress. We are able to use genetic tools to pop in genes or take them out. The first question we asked was would virgin female mice develop cancer, and they did.”

Next, “we confirmed that mice that were pregnant once or twice did not develop breast tumors. When we look at the DNA of those mice, we see that cells are turning genes on and off in different ways from the cells from a never-pregnant mouse. We are beginning to realize that the genes that are off and are preventing cancer from developing are controlling cell growth. Those genes can only be turned back on if pregnancy is in place.”

Could this revelation lead to a vaccine? It is indeed what some of the members of dos Santos’ lab are working toward. “We have a few candidate drugs that either inhibit those genes or enhance the function of those genes. We are putting mice into a cohort of treatment now.

“First, they will receive treatment, then we will stop the treatment and see if the mice develop cancer or not,” explained dos Santos. “We have candidates that seem to have an effect, resulting in smaller tumors or delayed tumor growth. We do not have any that result in no tumors at all, and we are trying to understand why that is. However, even having something to delay cancer onset or slow its growth would boost the opportunity to deal with it at a stage when it can be removed, treated, or possibly prevent metastasis. Having such a strategy available would increase life expectancy and improve life quality.”

Of Mice & Men

In the next year, dos Santos and fellow researchers hope to move their work to using human models of cancer with tissue harvested through a patient tissue donation program in cooperation with Northwell Health.

“We have acquired tissue from women undergoing normal cosmetic surgery; we know if they were pregnant early or late in life,” detailed dos Santos. “We also have that information from patients with cancer from whom we get tumor tissue and tissue from the other breast that has no tumor. We have tissue from women who are predisposed to cancer but do not have cancer, and from healthy women who do not have any sign of cancer risks, either familial or genetic. And now we can apply everything we are learning from mouse models, including doing genetic manipulations on human tissue in a dish, to understand if what we are learning in the mouse can be translated into humans.”

When asked to look in the crystal ball of her career, dos Santos sums up her most immediate goal as working out the prognosis aspect of her research by finding better biomarkers that can be used in clinic to understand who is at greater risk of developing breast cancer.

“This is a priority that is totally up our alley. We now have the staff to help us come up with something that is statistically significant and powerful enough to define signatures or cancer predisposition in tissue that is not cancer. There are already kits available to check cancer cells to see if they will metastasize. But we want to be able to test normal cells and predict the likelihood that they could develop into cancer,” detailed dos Santos. “Further down the line, we would like to have a compelling reason to go into human tests, clinical trials, to see if we have the ability to develop a vaccine to decrease risk or delay development of breast cancer in women, and men, too.”

Breast cancer research is not merely a career for dos Santos, but rather a life commitment. “I am passionate about what I am doing. I do not think I would be able to do anything else that requires as much from me. Being a cancer researcher is about resilience. It is about persistence. It is about feeling empowered. It is also about making difficult decisions to drop one line of questioning and pursue another. It must all be for the greater good,” she reflected.

“Cancer is a huge problem in modern medicine. We often focus only on the treatment,” said dos Santos, “but we need to look at prevention as well. It is not only about prevention from having cancer, but prevention in the sense that we can detect it at a stage where available treatment can be more efficient, where the cancer can be suppressed, or the ability of cells to metastasize can be decreased. We need to imagine the type of prevention that can turn a deadly disease into a chronic condition.”

Valerie Neff Newitt is a contributing writer.

Spotlight on Young Investigators