Two Genes Control Breast Cancer Stem Cell Proliferation and Tumor Properties

When mothers breastfeed their babies, they depend upon a unique interaction of genes and hormones to produce milk and deliver it to their hungry little tyke. Unfortunately, this same cocktail of genes and hormones can also lead to breast cancer, especially if the mother has her first pregnancy after age 30.

A medical research group at the Medical College of Georgia at Georgia Regents University has established that a gene called DNMT1 plays a central role in the maintenance of the breast (mammary gland) stem cells that enable normal rapid growth of the breasts during pregnancy. This same gene, however, can also maintain those cancer stem cells that enable breast cancer. According to their work, the DNMT1 gene is highly expressed in the most common types of breast cancer.

Also, another gene called ISL1, which encodes a protein that puts the brakes on the growth of breast stem cells, is nearly silent in the breasts during pregnancy and in breast cancer stem cells.

Dr. Muthusamy Thangaraju, a biochemist at the Medical College of Georgia, who is the corresponding author of this study, which was published in the journal Nature Communications, said, “DNMT1 directly regulates ISL1. If the DNMT1 expression is high, this ISL1 gene is low.” Thangaraju and his team first observed the connection between DNMT1 and ISL1 when they knocked out the DNMT1 gene mice and noted an increase in the expression of ISL1. These results inspired them to examine the relationship of these two genes in human breast cancer cells.

Thangaraju and his co-workers discovered that ISL1 is silent in most human breast cancers. Furthermore, they demonstrated that restoring higher levels of ISL1 to human breast cancer cells dramatically reduced cancer stem cell populations, the growth of these cells, and their ability to spread throughout the tissue; all of which are hallmarks of cancer.

Conversely, Thangaraju and his team knocked out the DNMT1 gene in a breast-cancer mouse model, the breast will not develop as well. However, according to Thangaraju, this same deletion will also prevent the formation of about 80 percent of breast tumors. In fact, DNMT1 also down-graded super-aggressive, triple-negative breast cancers, which are negative for the estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-).

The findings from this work also point toward new therapeutic targets for breast cancer and new strategies to diagnose early breast cancer. For example, a blood test for ISL1 might provide a marker for the presence of early breast cancer. Additionally, the anti-seizure medication valproic acid is presently being used in combination with chemotherapy to treat breast cancer, and this drug increases the expression of ISL1. This might explain why valproic acid works for these patients, according to Thangaraju. Workers in Thangaraju’s laboratory are already screening other small molecules that might work as well or better than valproic acid.

Mammary stem cells maintain the breasts during puberty as well as pregnancy, which are both periods of dynamic breast cell growth. During pregnancy, breasts may generate 300 times more cells as they prepare for milk production. Unfortunately, these increased levels of cell growth might also include the production of tumor cells, and the mutations that lead to breast cancer increase in frequency with age. If the developing fetus dies before she comes to term, immature breast cells that were destined to become mature mammary gland cells can more easily become cancer, according to Rajneesh Pathania, a GRU graduate student who is the first author of this study.

DNMT1 is essential for maintaining a variety of stem cell types, such as hematopoietic stem cells, which produce all types of blood cells. However, the role of DNMT1 in the regulation of breast-specific stem cells that make mammary gland tissue and may enable breast cancer has not been studied to this point.

For reasons that unclear, there is an increased risk of breast cancer if the first pregnancy occurs after age 30 or if mothers lose their baby during pregnancy or have an abortion. Women who never have children also are at increased risk, but multiple term pregnancies further decrease the risk, according to data compiled and analyzed by the American Cancer Society.

Theories to explain these phenomena include the coupling of the hormone-induced maturation of breast cells that occurs during pregnancy with an increase in the potential to produce breast cancer stem cells. Most breast cancers thrive on estrogen and progesterone, which are both highly expressed during pregnancy and help fuel stem cell growth. During pregnancy, stem cells also divide extensively and as their population increases, DNMT1 levels also increase.

In five different types of human breast cancer, researchers found high levels of DNMT1 and ISL1 turned off. Even in a laboratory dish, when they reestablished normal expression levels of ISL1, human breast cancer cells and stem cell activity were much reduced, Thangaraju said.

How Our Own Immune Systems Aid the Spread of Breast Cancer

Our immune systems help us fight off diseases and invasions of our bodies by foreign organisms. How surprising might it be to learn that our immune systems actually help tumors spread through our bodies?

Dr. Karin de Visser and her team at the Netherlands Cancer Institute have discovered that breast tumors cells induce certain immune cells to enable the spread of cancer cells. They published their findings online on March 30 in the journal Nature.

About one in eight women will develop breast cancer in Western countries. Of those women who die of this disease, 90 percent of them die because the cancer has spread to other parts of their body and formed metastases. Given these grim facts, cancer researchers are spending a good deal of time, treasure and energy to understand how metastasis occurs. A few years ago, several cancer biologists reported that breast cancer patients who showed high numbers of immune cells called neutrophils in their blood show an increased risk of developing metastatic breast cancers. Immune cells like neutrophils are supposed to protect our body. Why then are high neutrophil levels linked to worse outcome in women with breast cancer?

Neutrophils in a blood smear amidst red blood cells.


Dr. Karin de Visser, group leader at the Netherlands Cancer Institute, and her team discovered that certain types of breast tumors use a signaling molecule called Interleukin-17 to initiate a domino effect of reactions within the immune system. The tumor cells stimulate the body to produce lots of neutrophils, which typically occurs during an inflammatory reaction. However, these tumor-induced neutrophils behave differently from normal neutrophils. These tumor-induced neutrophils block the actions of other immune cells, known as T cells. T cells are the cells that can (sometimes) recognize and kill cancer cells.

De Visser and her team went on to define the role of the signaling protein called interleukin-17 (or IL-17) in this process. “We saw in our experiments that IL-17 is crucial for the increased production of neutrophils”, says De Visser. “And not only that, it turns out that this is also the molecule that changes the behavior of the neutrophils, causing them to become T cell inhibitory.”

The first author of the Nature paper, postdoctoral researcher Seth Coffelt, showed the importance of the IL-17-neutrophil pathway when he inhibited the IL-17 pathway in a mouse model that mimics human breast cancer metastasis. When these neutrophils were inhibited, the animals developed much less metastases than animals from the control group, in which the IL-17-neutrophil route was not inhibited. “What’s notable is that blocking the IL-17-neutrophil route prevented the development of metastases, but did not affect the primary tumor,” De Visser comments. “So this could be a promising strategy to prevent the tumor from spreading.”

Inhibiting neutrophils would not be a prudent clinical strategy, since drugs that inhibit neutrophils would make patients susceptible to all kinds of infections. However, Inhibition of IL-17 might be a safer strategy. Fortunately, drugs that inhibit IL-17 already exist.  Presently, anti-IL-17 drugs are being tested in clinical trials as a treatment for inflammatory diseases, like psoriasis and rheumatism. Last month, the first anti-IL-17 based therapy for psoriasis patients was approved by the U.S. Federal Drug Administration (FDA). “It would be very interesting to investigate whether these already existing drugs are beneficial for breast cancer patients. It may be possible to turn these traitors of the immune system back towards the good side and prevent their ability to promote breast cancer metastasis,” De Visser says.

Breast Cancer Clinical Trial Targets Cancer Stem Cells

Even though my previous posts about cancer stem cells have generated very little interest, understanding cancer as a stem cell-based disease has profound implications for how we treat cancer. If the vast majority of the cells in a tumor are slow-growing and not dangerous but only a small minority of the cells are rapidly growing and providing the growth the most of the tumor, then treatments that shave off large numbers of cells might shrink the tumor, but not solve the problem, because the cancer stem cells that are supplying the tumor are still there. However, if the treatment attacks the cancer stem cells specifically, then the tumor’s cell supply is cut off and the tumor will wither and die.

In the case of breast cancer, the tumors return after treatment and spread to other parts of the body because radiation and current chemotherapy treatments do not kill the cancer stem cells.

This premise constitutes the foundation of a clinical trial operating from the University of Michigan Comprehensive Cancer Center and two other sites. This clinical trial will examine a drug that specifically attacks breast cancer stem cells. The drug, reparixin, will be used in combination with standard chemotherapy.

Dr. Anne Schott, an associate professor of internal medicine at the University of Michigan and principal investigator of this clinical trial, said: “This is one of only a few trials testing stem cell directed therapies in combination with chemotherapy in breast cancer. Combining chemotherapy in breast cancer has the potential to lengthen remission for women with advanced breast cancer.”

Cancer stem cells are the small number of cells in a tumor that fuel its growth and are responsible for metastasis of the tumor. This phase 1b study will test reparixin, which is given orally, with a drug called paclitaxel in women who have HER2-negative metastatic breast cancer. This study is primarily designed to test how well patients tolerate this particular drug combination. However, researchers will also examine how well reparixin appears to affect various cancer stem cells indicators and signs of inflammation. The study will also examine how well this drug combination controls the cancer and affects patient survival.

This clinical trial emerged from laboratory work at the University of Michigan that showed that breast cancer stem cells expressed a receptor on their cell surfaces called CXCR1. CXCR1 triggers the growth of cancer stem cells in response to inflammation and tissue damage. Adding reparixin to cultured cancer stem cells killed them and reparixin works by blocking CXCR1.

Mice treated with reparixin or the combination of reparixin and paclitaxel had significantly fewer (dramatically actually) cancer stem cells that those treated with paclitaxel alone. Also, riparixin-treated mice developed significantly fewer metastases that mice treated with chemotherapy alone (see Ginestier C,, et al., J Clin Invest. 2010, 120(2):485-97).

Isolating Mammary Gland Stem Cells

Female mammary glands are home to a remarkable population of stem cells that grow in culture as small balls of cells called “mammospheres.” Clayton and others were able to identify these stem cells in 2004 (Clayton, Titley, and Vivanco, Exp Cell Res 297 (2004): 444-60), and Max Wicha’s laboratory at the University of Michigan showed that a signaling molecule called Sonic Hedgehog and a Polycomb nuclear factor called Bmi-1 are necessary for the self-renewal of normal and cancerous mammary gland stem cells (Lui, et al., Cancer Res June 15, 2006 66; 606). The biggest problem with mammary gland stem cells is isolating them from the rest of the mammary tissue.

Mammary gland stem cells or MaSCs are very important for mammary gland development and during the induction of breast cancer. Getting cultures of MsSCs is really tough because the MaSCs share cell surface markers with normal cells and they are also quite few in number.

Gregory Hannon and his co-workers at Cold Spring Harbor Laboratory used a mouse model to identify a novel cell surface protein specific to MaSCs. By exploiting this unusual marker, Hannon and his team were able to isolate MaSCs from mouse mammary glands of rather high purity.

Camila Do Santos, the paper’s first author, said that “We are describing a marker called Cd1d.” Cd1d is also found on the surfaces of cells of the immune system, but is specific to MaSCs in mammary tissue. Additionally, MaSCs divide slower than the surrounding cells. Do Santos and her colleagues used this feature to visually isolate MaSCs from cultured mammary cells.

They used a mouse strain that expresses a green glowing protein in its cells and then made primary mammary cultures from these green glowing mice. After shutting of the expression of the green glowing protein with doxycycline, the cultured cells divided, and diluted the quantity of green glow protein in the cells. This caused them to glow less intensely. However, the slow-growing MaSCs divided much more slowly and glowed much more intensely. Selecting out the most intensely glowing cells allowed Dos Santos and her colleagues to enrich the culture for MaSCs.

“The beauty of this is that by stopping GFP expression, you can directly measure the number of cell divisions that have happened since the GFP was turned off,” said Dos Santos. She continued: “The cells that divide the least will carry GFP the longest and are the ones we characterized.”

Using this strategy, Dos Santos and others selected stem cells from the mammary glands in order to examine their gene expression signature. They also confirmed that by exploiting Cd1d expression in the MaSCS, in combination with other techniques, they could enhance the purity of the cultures several fold.

Hannon added, “With this advancement, we are now able to profile normal and cancer stem cells at a very high degree of purity, and perhaps point out which genes should be investigated as the next breast cancer drug targets.”

Will we be able to use these cell for therapeutic purposes some day?  Possibly, but at this time, more must be known about them and MaSCs must be better characterized.

A Protein Responsible for Cancer Stem Formation Provides a Drug Target

Eighty-five percent of all tumors are carcinomas, which are tumors that form in layers of cells that line surfaces.  Such cell layers are known as an epithelium. When carcinomas form, they undergo an “epithelial-mesenchymal” transformation” or EMT.  EMT means that cells go from being closely aligned and tightly bound to each other in a an organized layer to cells that have little to do with each other and grow in unorganized clumps.  Is there a molecule that unites the carcinomas and if so is this molecule a potential drug target for cancer treatments?

Mammary Carcinoma
Mammary Carcinoma

Researchers at the University of Texas MD Anderson Cancer Center have identified a protein that seems to play a pivotal role in EMT.  This protein, FOXC2, may lay at the nexus of why some carcinomas resist chemotherapy and grow uncontrollably and spread.  FOXC2 could, conceivably represent a novel drug target for chemotherapy.

Sendurai Mani, assistant professor of Translational Molecular Pathology and co-director of the Metastasis Research Center at MD Anderson, said, “We found that FOXC2 lies at the crossroads of the cellular properties of cancer stem cells and cells that have undergone EMT, a process of cellular change associated with generating cancer stem cells.”

Cancer stem cells are fewer in number than other tumor cells, yet research has tied them to cancer progression and resistance to treatment.  Abnormal activation of EMT can actually create cancer stem cells, according to Mani.

Mani continued, “There are multiple molecular pathways that activate EMT.  We found many of these pathways also activate FOXC2 expression to launch this transition, making FOXC2 a potentially efficient check point to block EMT from occurring. ”  Mani’s research group used experiments with cultured cells and mice to discover these concepts, but the next step will require assessing the levels of FOXC2 expression in human tumors samples.

In the meantime, these new data from Mani’s research team may have profound implication for the treatment of particular types of carcinomas that have proven to be remarkably stubborn.  Breast cancers, for example, are typically carcinomas of the mammary gland ductal system.  A specific group of breasts cancers are very notoriously resistant to treatment, and FOXC2 seems to be at the center of such breast cancers.

The anti-cancer drug sunitinib, which is marketed under the trade name Sutent, has been approved by the US Food and Drug Administration (US FDA) for three different types of cancers.  In this study, sunitinib proved effective against these particularly stubborn types of breast cancer; the so-called “triple-negative, claudin-low” breast cancers.


Mani explained why such breast cancers are so resistant to treatment:  “FOXC2 is a transcription factor, a protein that binds to DNA in the promoter region of genes to activate them.  For a variety of reasons, transcription factors are hard to target with drugs.”

However, sunitinib seems to target these triple-negative breast cancers.  When mice with triple-negative breast cancer were treated with sunitinib, the treated mice had smaller primary tumors, longer survival, and fewer incidences of metastasis.  The cancer cells also showed a marked decreased in their ability to form “mammospheres,” or balls of cancer stem cells (this is an earmark of cancer stem cells).  Thus sunitinib seem to attack cancer stem cells.

As it turns out, FOXC2 activates the expression of the platelet-derived growth factor receptor-beta (PDGFRc-beta).  Activation of PDGFRc-beta drives cell proliferation in FOXC2-positive cells, and sunitinib inhibits PDGFRc-beta and inhibits cells that have active FOXC2 and undergoing EMT.

Triple-negative breast cancer cells lack receptors that are used by the most common anti-cancer drugs.  These deficiencies are responsible for the resistance of these cancers to treatment.  Such cancer cells also tend to under go EMT because they lack the protein claudin, which binds epithelial cells together.  Without claudin, these cancer cells become extremely aggressive.

Since cells undergoing EMT are heavily expressing FOXC2, Mani and his colleagues used a small RNA molecule that makes a short hairpin and inhibits FOXC2 synthesis.  Unfortunately, blocking FOXC2 had no effect on cell growth, but it did alter the physical appearance of the cells and reduced their expression of genes associated with EMT and increased the expression of E-cadherin, a protein necessary for epithelial cell organization.  Breast cancer cells also became less invasive when FOXC2 was inhibited, and they down-regulated CD44 and CD24, which are markers of cancer stem cells..  Additionally, triple-negative breast cancer cells that had FOXC2 inhibited had a reduced ability to make mammospheres.  Thus, FOXC2 expression is elevated in cancer stem cells, and inhibition of FOXC2 decreased the ability of the cancer stem cells to behave as cancer stem cells.


Mani’s group also approached these experiments from another approach by overexpressing FOXC2 in malignant mammary epithelial cells.  This forced FOXC2 expression drove cells to undergo EMT and become much more aggressive and metastatic (the cancer spread to the liver, hind leg, lungs, and brain).  Breast cancer cells without forced FOXC2 overexpression showed no tendency to metastasize.

Finally, Mani’s group examined metastatic mammary tumors that were highly aggressive when implanted into nude mice (mice that cannot reject transplants).  Two of the tumors were claudin-negative and both of these tumors showed elevated FOXC2 expression.  When FOXC2 expression was blocked by Mani’s hairpin RNA, the claudin-negative tumors became less aggressive and grew more as mesenchymal cells.  The cells that underwent EMT also showed high levels of PDGF-RC-beta expression.

Mani said of these data: “We thought PDGF-B might be a drugable target in these FOXC2-expressing cells.”  Mani’s group also showed that suppressing FOXC2 reduced the expression of PDGFRC-Beta.  Thus, this small molecule might be an effective therapeutic strategy for treating these hard-to-treat breast cancers.

MD Anderson has filed a patent application connected to this study.

See Hollier B.G., Tinnirello A.A., Werden S.J., Evans K.W., Taube J.H., Sarkar T.R., Sphyris N., Shariati M., Kumar S.V., Battula V.L., Herschkowitz J.I., Guerra R., Chang J.T., Miura N., Rosen J.M., and Mani S.A.,. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Research. e-Pub 2/2013.

What Does Breast Cancer Have to Do With Skin Stem Cells?

BRCA1 is a gene that plays a huge role in breast cancer. Particular mutations in BRCA1 predispose women increased risks of breast cancer cervical, uterine, pancreatic, and colon cancer and men to increased risks of pancreatic cancer, testicular cancer, and early-onset prostate cancer.

BRCA1 encodes a protein that helps repair damage to chromosomes. When this protein product does not function properly, cells cannot properly repair acquired chromosomal damage, and they die or become transformed into cancer cells.

What does this have to do with stem cells? A study led by Cédric Blanpain from the Université libre de Bruxelles showed that BRCA1 is critical for the maintenance of hair follicle stem cells.

Peggy Sotiropoulou and her colleagues in Blanpain’s laboratory showed that when BRCA1 is deleted, hair follicle cells how very high levels of DNA damage and cell death. This accumulated DNA damaged drives the follicle stem cells to divide furiously until they burn themselves out. This is in contrast to the other stem cell populations in the skin, particularly those in the sebaceous glands and epidermis, which are maintained and seem unaffected by deletion of BRCA1.

Sotiropoulou said of these results: “We were very surprised to see that distinct types of cells residing within the same tissue may exhibit such profoundly different responses to the deletion of the same crucial gene for DNA repair.”

This work provides some of the first clues about how DNA repair mechanisms in different types of adult stem cells are employed at different stages of stem cells activation. Blanpain and his group is determining if other stem cells in the body are also affected by the loss of BRCA1. These results might elucidate why mutations in BRCA1 causes cancer in the breast and ovaries, but not in other tissues.

Myriad Genetics Hordes Breast Cancer Data

Kathleen Sloan the president of the National Organization of Women has a troubling article at the Center for Bioethics and Culture website. It tells the story of a biotechnology company called Myriad Genetics and it BRCA1 & 2 test.

What the heck is BRCA1 & 2?  BRCA stands for “breast cancer” and mutations in BRCA 1 or 2 predispose females to breast and ovarian cancer. Mutations in BRCA genes also increase the risk of colon, prostate and pancreatic cancer.  Approximately 7% of breast cancer and 11 – 15% of ovarian cancer cases are caused by mutations in the BRCA genes.  If someone carries a mutation in either BRCA 1 or 2, they have a syndrome called Hereditary Breast and Ovarian Cancer (HBOC) syndrome.

The BRCA genes encode proteins that help repair DNA when it is damaged. Even though BRCA 1 & 2 work with several other proteins to accomplish this repair, mutations in the BRCA genes that compromise the quality of the proteins they encode can diminish the ability of cells to repair their DNA. Loss of efficient DNA repair systems leads to greater numbers of mutations in cells, some of which cause either loss of tumor suppress genes that normally put the brakes of cell proliferation, or activation of proto-oncogenes, which encode proteins that promote cell proliferation. Loss of tumor suppressor genes and activation of proto-oncogenes produces a cancer cell, and mutations in BRCA 1 or 2 and accelerate the onset of cancer cell formation (this is a highly simplified explanation and I apologize to the aficionados out there, but I am trying put the cookies on a nice low shelf).

Myriad Genetics came along and developed a genetic test for cancer-causing mutations in BRCA 1 & 2. This is good news, but Myriad Genetics is presently with holding their data from patients. This is not good news. Myriad Genetics wants to generate a database of mutations found in BRCA 1 and 2 genes from women all over the world. Some of these mutations do not affect the function of the encoded protein and do not predispose the patient to breast cancer, but some do. Which ones are harmful and which ones are not?

At this point things get sticky. Myriad has complied its sequence data on BRCA in order to construct a “variants of unknown significance” or VUS. Such a compilation would be invaluable, since it would help physicians correctly interpret the results of a breast cancer test. According to its present data archive, Myriad Genetics claims that only 3% of its tests fall into the VUS unknown category. However, other testing services report a 20% VUS rate. Who’s right? hard to say, given that Myriad Genetics will not release its data. Apparently they feel that their data has commercial value.

The problem is that lots of outfits that provided data to Myriad Genetics free of charge in order for them to develop their test. These other outfits have all their data available on public databases. What about Myriad Genetics – nope.

According to Ms. Sloan, “Myriad Genetics, producer of the world’s biggest-selling gene test for breast and ovarian cancers, has become synonymous with corporate greed. In an egregious breach of bioethics, the company refuses to share groundbreaking knowledge that could benefit cancer patients.”

Myriad worked hard to develop this test – I do not think anyone is contesting that. Myriad Genetics has every right to make money off their test, but when they start hoarding potentially life-saving data, I think Ms. Sloan is right that they have crossed the line.

Myriad Genetics is also being sued because of their attempts to patent the BRCA genes. An impressive consortium of researchers, genetic counselors, women patients, cancer survivors, breast cancer and women’s health groups, and scientific associations representing 150,000 geneticists, pathologists and laboratory professionals are all plaintiffs in this lawsuit against the U.S. Patent Office, Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the genes.

The lawsuit avers that patents on human genes violate the First Amendment because genes are “products of nature.” Therefore, such things cannot be patented. Such an argument has a strong intuitive appeal, and is almost certainly correct.

Read Ms. Sloan’s article here and see what you think.