Publications

How the breast ducts, the branching tubes that will carry milk, grow during normal breast development during puberty is a major question being studied. To expand current knowledge of how mammary branching takes place in animals and humans, this research group took time-lapse movie images that monitored branching initiation, elongation, and splitting. 

The investigators examined previously identified “crucial” proteins called growth factors and found that mammary ducts only branched when these proteins were close by.  They also discovered that branching only takes place in special sites where there are multiple layers of tissue. Interestingly, similar multiple layers of cells are an early event in breast tumors formation. The new video methods developed for use in this study will be extremely useful in future studies that monitor cellular growth and proliferation and in understanding tumor progress. 

Progesterone acting through two isoforms of the progesterone receptor (PR), PRA and PRB, regulates proliferation and differentiation in the normal mammary gland in mouse, rat and human. Progesterone and PR have also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is on recent advances in understanding the role of the PR isoform specific functions in the normal breast and in breast cancer. Also discussed is information obtained from rodent studies on the hormonal regulation of PR isoform expression in mammary gland, recent progress in unraveling the molecular mechanisms of P action via PR isoforms, comparison of human, mouse, and rat PR isoform expression, and possible relationship of PR isoform expression to normal mammary gland development in human, rat, and mouse. Studies of P action and PR isoform-specific functions in normal mammary gland and during tumor development in animal models can provide an important insight into breast cancer etiology. Most importantly, such studies may provide novel preventive, diagnostic, and therapeutic strategies to fight breast cancer.

Patients with metastatic breast cancer (cancer that spreads from the breast to other sites in the body), have an increased risk of mortality.  Presently, estrogen-receptor status is the most common predictor of breast cancer survival.  A tumor which responds to estrogen (estrogen-receptor positive) may be more easily treated with anti-estrogenic therapies, while a tumor which does not respond to estrogen (estrogen-receptor negative) may be more difficult to treat.  New discoveries concerning a special protein called GATA-3 have provided clues about breast cancer prognosis and offer a possible explanation of carcinogenesis. GATA-3 levels in breast tissue are a more powerful predictor of breast cancer status than the current estrogen-receptor approach.

The research team studying the GATA factors found that low GATA-3 expression led to advanced cancers that are more prone to metastasis, while high GATA-3 expression was correlated with smaller tumor size that did not show metastatic capabilities.  Replacing the GATA-3 in the low GATA-3 tumor stopped their metastasis. A better understanding of the GATA pathway offers hope in finally understanding the molecular mechanisms of breast cancer.

Stat5a is a protein that is important for controlling growth and development of the mammary gland, and has also been implicated in breast cancer. Stat5a functions as a transcription factor that induces expression of genes, but in order to do so it must first be activated. In mammary cells, Stat5a is activated primarily via the hormone prolactin. Although the mechanisms that activate Stat5a are well understood, very little is known about those that regulate its expression in the mammary gland. In this report, sections of mouse mammary glands were stained for Stat5a protein in order to examine its levels throughout development. We found that it is not present before puberty, when estrogen (E) and progesterone (P) levels are low, but appears during puberty and is maintained in adult animals. When both E and P were eliminated from mice by removing the ovaries, Stat5a disappeared, and treatment with E+P was necessary to restore its expression to the level seen in intact, mature animals. Stat5a positive cells also contained receptors for both E and P, suggesting that its expression is directly regulated by these receptors in response to hormone treatment. Thus, these results identify a novel mechanism of in which E and P induce Stat5a expression, allowing for the protein to be subsequently activated by prolactin. The active protein is then able to regulate downstream genes in order to promote mammary gland development.

Transduction is the process of transferring genetic material from one organism to another with the help of a virus. This technique allows exclusive study of a single gene and enables researchers to determine its unique role. The mouse mammary gland is ideally suited for studying the function of individual genes, and a group of researchers have come up with a new way to study them. 

The team infected the cell of breast ducts, called mammary epithelial cells, with a virus containing the individual gene of study. Their method turned out to be much more practical and effective than the existing approach, while also being more cost effective.  Information gained from studies using this model supports the current theory that during formation of the extensively branched network of tubes during development of the breast, many mammary branches can arise from a single breast stem cell or several different stem cells can contribute to the formation of a single tube. This understanding will help us understand the origins of the breast tubes and how abnormal cells contribute to the pattern during the early stages of tumor formation.

In the normal mouse mammary gland progesterone (P) can cause cells to divide or change form through two progesterone receptor (PR) isoforms, PRA and PRB. PRA is the predominant isoform expressed in the adult virgin mouse, and PRB is predominantly expressed during pregnancy. In order to study the control of the PR isoforms by hormones and to examine the different functions of PRA and PRB, we removed the ovaries, which are the major source of hormone production, from adult mice and the mice were treated for 3, 5, or 10 days with estrogen (E), P, or estrogen + progesterone (E+P). We used a technique called immunohistochemistry to stain thin sections of mammary tissue with antibodies specific for PRA or PRB. This technique allowed us to investigate the regulation of PRA and PRB by hormones and to determine the role of PRA and PRB in cell turnover and overall changes in organization of the mammary gland. E treatment caused limited cell turnover that was only present after 5 days and was localized to the ends of ducts. P-induced cell turnover led to the formation of small buds, called sidebranches, off of the ducts and formation of grape-like clusters of cells called alveoli. However, the effect of E+P on sidebranching and formation of alveoli was greater. E increased PRA level, while P decreased PRA level. PRB expression was only detected following treatment with P or E+P. During sidebranching, PRA was the predominant PR isoform expressed. Cell turnover of both PRA expressing and PRA negative cells was responsible for P-induced sidebranching. In contrast, PRB was the predominant PR isoform expressed during formation of alveoli. Cell turnover of both PRB expressing and PRB negative cells was responsible for P-induced alveolar expansion. These results demonstrate that control of PRA and PRB levels by hormones in vivo occur differently and suggest that P induces cell turnover through PRA and PRB by direct and indirect mechanisms.

Progesterone (P), acting through progesterone receptor (PR) isoforms A and B, plays an important role in normal mammary gland development and is implicated in the etiology of breast cancer. Because of notable similarities between human and rat mammary gland development and hormonal responsiveness of mammary cancers in both the human and rat we undertook the analysis of progesterone action in the rat mammary gland. Using antibodies that detect only PRA or only PRB by immunohistochemistry, we investigated PRA and PRB expression at various mammary gland developmental stages (puberty, sexual maturity, pregnancy, lactation and after postlactational involution), and their functional roles in the regulation of proliferation. The percentage of PRA positive (PRA+) cells decreased from puberty to adulthood and further decreased after pregnancy, the percentage of PRB expressing cells was relatively constant at all developmental stages. Interestingly, during all developmental stages there was a significant proportion of cells that expressed only PRB. We found that the majority of PRA+ cells co-expressed PRB.  In the pubertal and adult virgin mammary gland, PRA+PRB+ cells also expressed nuclear cyclin D1, protein that is essential for proliferation, but these cells did not contain the proliferation marker BrdU. Additionally, we found that PRA+PRB+ cells in the adult gland lacked expression of phospho-Rb, another protein required for proliferation, but expressed high levels of CDK inhibitors, p21 and  p27 that capable of halting the cell proliferation. From these observations we conclude that it is likely that PRA+ PRB+ cells are cell cycle arrested and do not proliferate. These results imply that if P acts to promote proliferation in the virgin gland through its action in PRA+PRB+ cells it most likely does so through a paracrine mechanism(s). At various developmental stages, especially during pregnancy, a high percentage of cells that expressed only PRB were positive for BrdU. From this observation, we conclude these cells proliferate and that P acting through PRB may directly stimulate proliferation. Our study demonstrates that the rat mammary gland can be a superb animal model to study P action because, similarly to the adult human breast, it expresses PRB and both PRA and PRB are highly co-expressed in the same cell.

For many years, the areas surrounding cancerous cell growth have largely been ignored, while the cancer cells have received all the research attention.  Recent work, however, demonstrates that the type of tissue surrounding tumors makes a big difference in regards to how fast or slow a cancer grows. 

Radiation exposure is known to disrupt normal breast tissue.  Changes in this tissue may ultimately increase the rate of tumor progression.  To perform their studies, members of the Bay-area research team used ionizing radiation to change the tissue environment in normal breast cells and caused tumors to grow faster.  This experiment first proves that disrupting normal tissue promotes cancer progression to a tumor, and second that ionizing radiation is capable of disrupting that tissue.  In the future it is possible that cancer cells may be controlled by monitoring the tissue in their immediate environment.

Transforming Growth Factor β (TGFβ) is an abundant signaling molecule which plays an important role in cellular regulation. TGFβ acts a both a tumor suppressor and a tumor promoter, making it a very complicated molecule to study. 

The Bay-area research team discusses how TGFβ coordinates gene expression.  They review the literature that during the beginning stages of cancer growth, TGFβ acts as a suppressor by stabilizing the chromosomes, while during the later stages of cancer growth TGFβ increases the rate of cancer cell invasion and migration.  If TGFβ holds any therapeutic potential, more research is necessary to understand how the timing of these contradictory effects are controlled.

During development particular cells are given specific jobs; this is known as differentiation.  The undifferentiated cells of a given population tend to multiply very quickly and have a higher propensity toward becoming cancerous.  GATA-3 is known to play a role in differentiation by transforming undifferentiated cells in breast ducts into mature cells.  Relatively little is known about the differentiation process undergone by mammary epithelial cells (the principal cells implicated in breast cancer), but recent research has determined that GATA-3 is a significantly player. 

Multiple studies have demonstrated that breast tumors with high GATA-3 expression have a good prognosis since the cells tend to be better differentiated.  Breast cancers with low GATA-3 expression tend to be diffuse, metastatic, and have a worse prognosis than others.  This research demonstrates that GATA-3 expression levels may serve as a reliable prognostics tool in the fight against breast cancer.

In young animals and girls, the mammary gland develops during puberty in a process known as branching morphogenesis. The tip of a growing mammary bud repeatedly divides to generate an extensively branched network of ducts.  This group of researchers decided to try and identify the genes that control the branching process.  Gene analysis studies were used to reveal that 1074 genes increased at the tip of the growing bud, 222 genes increased in the surrounding tissue environment, and 385 genes increased in both.

The identification strategy designed for use in this study was shown to accurately predict known markers of cancer prevalence, and was then used to confirm the expression of supplementary genes.  The new method also had the ability to pinpoint where exactly in the mammary gland the genes were being expressed.  Therapeutic action to stop cancer growth may be possible if misregulated genes can be identified.

The process known as branching morphogenesis is a fundamental biological process involved in creating an extensively branched network of tubes in breast, lung, kidney and salivary gland in mammals, but also in organs in lower animals such as fruit flies.  This review by a Bay Area research team compared the differences and similarities between branching in the mouse and fly.  The team highlighted the importance of the tissue surrounding the regions of uncontrolled cancerous cell growth, and used a time-lapse microscopy technique to watch the ducts branch in real time.  The group identified a molecule known as FGF (fibroblast growth factor) as a key modulator of developmental activity.  Using original state-of-the-art microscopy techniques, the team discovered that nearly 18% of all branching events were trifurcations, meaning three ducts were made from one original duct, instead of just two. 

Several other studies have determined that the same mechanisms which govern fly tracheal branching also control mammary branching.  Due to the similarities between the two systems, animal models remain essential to the continued study of human breast cancer.

Mammary branching refers to the creation of an extensive network of milk-carrying ducts. The pattern and cycle of ductal branching has been shown to be regulated by hormonal control by altering the immediate cellular environment. Endocrine hormones circulate throughout the entire body and are known to initiating branching. Locally, cyclic ovarian hormones like estrogen and progesterone are responsible for remodeling the original branching patterns. Cellular communication between the two tissue types functions to define growth boundaries and allow for reorganization of ductal branches.

This article summarizes the hormones and regulators involved in branching morphogenesis, emphasizing the fact that more research needs to be performed in order to understand how these molecules interact to control such a complicated process.

Mammary branching refers to the formation of an extensive network of milk carrying ducts in the breast of female mammals. During adulthood these branches are maintained while new branches continue to form. This paper compares the similarities between methods of branching in human, mouse, and fly models. Mouse and fly models are useful when doing research because mice have mammary glands like humans do, and flies have similar branching organs and a very short lifecycle. It is necessary to understand normal breast development so doctors and researchers can identify when things become abnormal.

Studies in all three models- humans, mice, and flies- show that hormones control proper tissue organization. Localized growth in certain areas is additionally regulated by small molecules which can either increase or decrease branching. The way in which a few of these small molecules work is well understood, while many other important molecules have yet to be identified. Some molecules specifically control making the ducts longer, while others are in charge of making sure the branches successfully split from the primary duct.

The goal over the next five years is to understand how the growing tissue cells communicate with the regulatory molecules and how disruption of this interaction can lead to varying diseased states like cancer.

In Petri dish studies, a molecule known as Mfge8 has been shown to be an official garbage collector, responsible for clearing old and dead cells. In the developing mammary gland, Mfge8 molecules are critical because they are required to clear the spaces inside the new milk ducts. Without the orderly removal of the cellular waste by Mfge8, the tissue can become inflamed and prevent proper development.

The research team demonstrated that Mfge8 has the same job in a live animal as it does in a Petri dish. The team showed that mammary gland tissue reorganization was significantly delayed in mice without any Mfge8 due to an excess of uncleared cellular garbage.

Progesterone (P) is capable of inducing cell turnover in the mammary gland. P acts through binding to the progesterone receptor (PR), which exists as two isoforms, PRA and PRB, that have different functions. In this study we used sections of mammary gland tissue stained with antibodies specific for PRA or PRB to show that PRA and PRB expression in mammary epithelial cells is spatially and temporally separated during normal mammary gland development in the mouse. In the virgin mammary gland when ductal development occurs, PRA is the predominant PR isoform expressed. PRB was the predominant PR isoform expressed during the formation of alveoli in response to pregnancy, while PRA expression decreased during pregnancy. Both PRA and PRB were expressed together in only a small percentage of cells; usually, mouse mammary epithelial cells expressed either PRA only or PRB only. During pregnancy, PRB expressing cells proliferated, whereas PRA expressing cells did not. These results indicate that different actions of P are mediated in PRA positive versus PRB positive cells in vivo. The separation of PR isoform expression into different cells and different stages of development in the mouse mammary gland provides a unique opportunity to further examine the specific functions of PRA versus PRB in vivo.

A new model of cancer as “a phenomenon of tissues” rather than just abnormal single cells is proposed to much more accurately describe how cancer actually develops. To test the new model, this group of Bay-area researchers documented cancerous growth from mice both exposed and unexposed to radiation. The irradiated tissue became very disorganized and exhibited excess cellular growth, while unirradiated tissue was two to four times less likely to show abnormal growth.

The authors discuss the literature that shows that the dose of radiation, the intensity of the radiation, and the type of tissue being exposed all play a part in determining the lasting effect of exposure. Fortunately, preliminary reports reason that restoring the health of the tissue surrounding the cancer may prevent detrimental effects.

It has been repeatedly shown that the areas surrounding breast cancer growth are just as, if not more, important than the site of the cancer growth.  This study concentrated on the ability of an abnormal cellular neighborhood to induce and support cancerous cell growth.  Experiments in which supportive tissue was treated with a cancer causing agent showed that tumors actually developed in the adjacent cells not treated with the carcinogen! 

The research team is studying how blocking an important receptor pocket on surrounding tissue can cause a dramatic increase in the number of cancerous cells.  The team argues that monitoring the state of tissue surrounding cancerous cells can serve as an effective means of monitoring and predicting cancerous activity.

Ionizing Radiation (IR) acts as both a carcinogen and a therapeutic agent. Low dose exposure can increase an individual’s risk of developing cancer, while high dose exposure is capable of slowing or stopping cancer growth. Radiation is known to damage the mammary tissue in two ways: directly and indirectly. IR induced DNA damage can lead to cell death, which is thought to be the basis of its therapeutic effects. DNA damage can also lead to mutations, which contribute to cancer. 

Various studies reveal that ionizing radiation also leads to rapid changes in extracellular signals produced of exposed tissue, indicating that IR action may be two-fold: through DNA damage and altered signaling. The authors postulate that these two effects interact both in the response to high dose radiotherapy and the increase cancer risk following low dose radiation exposures. With an enhanced awareness of the dual properties of radiation, it may be possible to further optimize radiotherapy and limit its detrimental effect on cancer risk.

It is recognized that mice lacking the gene regulator C/EBPβ (which makes a molecule that regulates the life cycle of cells) are ten times less responsive to pregnancy hormones than are mice with the receptor.  Studies show that the growing mammary ducts of mice without the C/EBPβ regulator are unable to receive hormonal messages.  The research team studying these mice found that those lacking the regulator were also deficient in regulator E, a molecule responsible for controlling cellular growth. 

This discovery provides an understanding of how breast cancer disrupts the normal cell cycle and causes excessive, tumorigenic, cell growth.  A better understanding of normal cell cycle regulation will be able to offer additional insight into the molecular methods used to prevent tumor growth.  With such knowledge, physicians and researchers may be capable of recognizing when cancerous growth is likely to occur, and prevent it from occurring as a result.

For tumors to grow and spread, a cancer cell must be able to generate new blood vessels in order to support its increasing size. Ionizing radiation is used therapeutically to treat breast cancer. However, ionizing radiation can cause growth of new blood vessels. Even though ionizing radiation has been shown to cause the growth of new blood vessels, how it does this is poorly understood. 

Members of the Bay Area research team decided to conduct a series of experiments to figure out how radiation caused the growth of new blood vessels. They discovered that a specific type of cell, known as a mast cell (which usually is involved in allergic responses), was responsible for promoting the generation of new blood vessels. When the researchers added molecules that inactivated the mast cells, no new blood vessels grew. This finding is important because it reveals that mast cells may be required for significant tumor growth.

Cancer is a result of uncontrolled cell growth and a loss of cell-cycle control.  It has long been known that telomeres, or specialized DNA segments, play a vital role in the prevention of unwanted chromosomal breakdown.  When telomeres become damaged or do not work properly, any organism is at a much higher risk of developing cancer. 

The research team studied three binding proteins known to be involved in the regulation of telomeres.  They discovered that the cancerous cells were direct descendents of cells who had lost appropriate telomere function.  The team hypothesizes that the main cause of telomere dysfunction is damage from oxidative free radicals, unstable molecules which can cause damage to the body.

The genes that control growth of the breast cells during pubertal development are of great interest because the same genes may be appropriated during breast tumor growth.  The major growth factor that targets the mammary epidermal growth factor receptor (EGFR), a key receptor in tumor progression, is a membrane protein called amphiregulin (AREG). To work to stimulate cell division the factor needs to be clipped to release it so it can reach EGFR on cells further away. Previous studies have reported impaired mammary development in mice lacking AREG and EGFR. A third molecule, ADAM17, is an enzyme that clips AREG and acts as a messenger between the two molecules.  The research team studying mice lacking these factors looked at how the three above mentioned molecules regulate mammary growth and why they are important in breast development. 

The team figured out that one type of tissue, the epithelial cells of the ducts, produces AREG and another type of tissue (the neighboring fibroblasts) with the proper receptors (EGFRs) responds to AREG. Before the second type of tissue can receive the AREG message, the third molecule (ADAM17) is required to remove it from the site where it was produced. ADAM17 acts like a pair of scissors that cuts and releases AREG from the layer of tissue where it is produced so it can move across to the layer where it can act. The team is continuing to investigate what initiates the production of AREG.

Appropriate mammary gland development and production of milk fat is required for proper milk production. Lipids are the primary components of fat, and the team hypothesizes that lower lipid levels lead to ambiguous classification of mammary cells, consequently increasing the risk of developing breast cancer. It has been previously demonstrated that mice lacking an enzyme known as DGAT1, which is implicated in fat production, are unable to produce milk. The team studying DGAT1 deficient mice explain that mice without this enzyme have abnormal lipid levels in their supporting cells, which increases their risk of breast cancer.

DGAT1 expression is required in proper mammary gland development during pregnancy, ultimately in milk production. Breast tissue of mice without the DGAT1 enzyme develops improperly and may lead to abnormal cancerous growth.