The+Molecular+Basis+of+Pregnancy's+Protective+Role+Against+Breast+Cancer

An Introduction To Breast Cancer
Megan Carlson and Christina Wood

Breast Cancer takes the lives of thousands of women and hundreds of men each year. As the second leading toc cause of death in women one in eight, or about 12.3% of all women will be diagnosed with some type of breast cancer over the course of her lifetime. The National Cancer Institute estimates that 235,030 people will be diagnosed with breast cancer in 2014 — 40,430 will die of it this year (SEER). Surprisingly the incidence of breast cancer in the United States has remained somewhat constant over the last 20 years, and we have made remarkable progress in providing a higher quality of care and quality of life for those diagnosed with breast cancer; the 5-year survival rate from 2004-2010 was 89% (SEER). Breast cancer is a group of normal mammary epithelial cells that have acquired genetic mutations that support rapid and uncontrolled cellular growth. These cells are termed hyperplastic and as the tumor grows, divides, and begins to overtake normal tissue, it can metastasize and form new 2º tumors in additional regions of the body. Eventually these tumors place a fatal physiological strain on the patient. (Read Christina's Blog Post for more here!)

So what causes breast cancer? Because only 1% of the cases occur in men, we know that hormones play a major role in the pathogenesis of breast tissue specific cancers. When most people think about hormones one of the first topics that comes to mind is pregnancy. This is where we began our research. For decades we have known that women who bear children (parous), particularly young in life seem to be less vulnerable to breast cancer than those who do not bear children (nulliparous). In 1968 by Joseph Fraumeni Jr. studied cancer mortality rates of catholic nuns in the United States between 1950 and 1954. He concluded that nulliparity (never bearing children) and the associated lack of hormonal changes that come with pregnancy had a significant impact on the development of cancers, especially cancers of reproductive organs such as the breast(Whitaker, Fraumeni). From this we aim to delve a bit deeper into the molecular and cellular mechanisms that give parous women an edge over nulliparous women in terms of cancer development.

Glossary
To clarify any terms that may be new or foreign to those unfamiliar with Breast Cancer.

=Anatomy and Dynamics of Breast Tissue=

**The Female Breast and Cell Types**
Much like any other organ, the breasts of female fetuses begin to develop during gestation in which the primary mammary bud and rudimentary mammary gland forms (Javen). After the child is born, the cellular architecture of the breast remains unchanged until she reaches puberty and hormones such as estrogen begin to stimulate development. At this point, the breast undergoes an increase in adipose and connective tissues (structural stromal cells) as well as ductal elongation and branching of the glandular parenchymal cells. However, this ductal development does not mark the end of breast development (Javen). We recognize three distinct structures that make up the functional unit of the female breast; in order from least to most complex we have terminal end buds (TEBs), terminal ducts (TD), and alveolar buds (AB) (see Figure 1). As women undergo puberty the ductules begin to elongate and branch, and upon further hormonal stimulation will reach their final state of development in which clusters of ABs will carry out lactation. However, the female breast, whose primary function is to produce milk for breastfeeding, is not fully developed at birth or even after puberty. In fact the breast is not fully developed until after the completion of a woman’s first pregnancy. The hormonal changes that occur in pregnancy stimulate cellular changes which terminally differentiate the cells discussed below. This added developmental milestone has been implicated as one of the major factors that allows pregnancy to decrease a woman’s risk of developing breast cancer in her lifetime. Figure 1 helps to visualize the development of structures in the breast.

In addition to the three recognized breast structures, we also recognize four unique breast tissue types, all of which can be found at the various stages of development. As the breast develops the ratios of these cells change as these cells begin to differentiate. Lobule type 1 cells (lob 1) are the most undifferentiated type of breast tissue, and are present at a high density in underdeveloped breasts. These cells actively differentiate into lobule type 2 (lob 2) cells, which are more complex and begin to branch as the ductules form. From here the cells further differentiate into lobule type 3 cells (lob 3) and lobule type 4 cells (lob 4), which are even more complex in terms of branching and contain numerous clusters of ABs. Interestingly, puberty induces differentiation of lob 1 to lob 2 and lob 3 tissues, but differentiation into lob 4 tissues requires pregnancy. Only pregnancy can stimulate the breast to begin lactation. In women who never have children, their breasts have a high proportion of lob 1 tissue in comparison to those that do have children. These lob 1 cells have a high rate of proliferation, so they are constantly dividing, whi ch is why they make a prime target for cancer. They have a higher rate of replication of DNA and therefore an increased risk of mutation. (Read Megan's Blog Post for more!)

**Pregnancy and Breast Development[[file:25690_ftp.pdf]]**
So where does that leave us? We know that as the breast develops the structures and ti ssue types become more complex, and women who are nuliparous have a higher concentration of the less differentiated tissue type. But why should this matter in terms of cancer? Lob 1 tissue contains TEBs, TDs, and ABs, but TEB’s are overrepresented. The cells of the TEBs are responsible for differentiating into the remaining 3 breast tissue types and the remaining 2 breast structures (TDs and ABs). The lob 1 breast cells have the highest rate of proliferation out of the four cell types, and because of this they are at the highest risk of developing into ductal carcinomas. Therefore the breasts of women who never have children and are not capable of terminally differentiating into the lob 4 tissues will have more of the highly proliferative lob 1 cells. The first review paper we researched on the topic of pregnancy and breast cancer suggested that two cell types are responsible for developing or not developing cancer [ref]. This suggests that breast tissue can be divided into two categories — those that are susceptible to carcinogenesis and those that are immune to carcinogenesis. But which of the above cells fit this criteria? Which are the most susceptible? We have evidence to support the fact that lob 1 tissues, and more specifically the TEBs, are the most susceptible to carcinogenesis. This is a result of their active proliferation and differentiation into the more developed breast tissue. Tissue that can reach the later stages of development such as the alveolar buds stage are generally more resistant to carcinogens because these cells do not proliferate as often — essentially their cellular kinetics and cell cycle timing sets them apart. TEBs decrease in frequency with age due to the fact that the breasts will continue to develop, however without a full term pregnancy TEBs exist at a detectable level; parous women on the other hand do not have TEBs (Russo, 1982).

=**The Cellular and Molecular Basis of Protection**=

Cell Cycle Kinetics
Loss of cell cycle control is one if the hallmarks of cancer. In the case of parity and breast cancer, there is no “loss of control” associated with having or not having children, but instead it is thought that having children changes the dynamics of the cell cycle. In Table 19, Russo et al. (1983) analyzed the length of each component of the cell cycle (G2, S, M, G1, and C or complete cycle) in three various structures in the female breast of rats. They were either young virgin rats (YV), old virgin rats (OV), or parous rats (P). Most of the structures had similar cell cycle component lengths (see interesting notes for complete table breakdown) excluding the G1 phase which appears to be longer in all breast structures of parous rats in comparison to both virgin groups. Russo and fellow researchers determined that rats who were parous had TDs and ABs whose G1 phase is about 2 times longer than those who are or remain virgins throughout their lives. But why is this important? Cells require the proper environment and pro-growth signals in order to grow, divide, and in some cases differentiate. Along with the fact that these more differentiated structures are not required to divide as often as cells like TEBs, the cell cycle is tightly controlled with numerous checkpoints to ensure that the cells are ready to divide. Among these checkpoints, and the most important to our discussion, are those that monitor DNA damage. The adjacent image depicts the cell cycle as it pertains to DNA damage checkpoints, and how the cell will eventually progress past Start and divide. As mentioned earlier, the phase in which we are most interested is in the lengthened G1 phase. Here any damage inflicted upon the DNA (due to carcinogens, UV rays, etc.) must be repaired in order for the cell to divide. If in fact the G1 phase is longer we hypothesize that the cell will have more time to repair that damage before the cell must commit to division.

In addition to a lengthened G1 phase which increases the opportunity to repair DNA damage, the ABs seen mostly in parous rats also have larger non-proliferative compartments and a reduced capacity for DNA-DMBA binding [What does this mean? It's the first time you introduce DMBA.]. This means that a much lower percentage of the cells in the breast are actively proliferating, mostly due to the fact that they have reached maximal complexity and therefore do not need to further differentiate into new tissue types, so they turn off that function. Because the cells of the ABs are not actively proliferating like the TEBs, the DMBA carcinogen has a much more limited target to bind to in the great tissue of parous rats [ I suspect that DMBA has a fine time binding to DNA regardless of differentiation. The difference is that because the AB cells are post-mitotic, DNA-damage induced replication errors are less likely to induce mutations. It has nothing to do with the affinity of the carcinogen to DNA.] The fact that carcinogens such as DMBA do not bind as well to the DNA of cells that are not actively proliferating provides further support to the argument that parous rats are less susceptible to breast cancer. However, in order for these cell kinetics to have a significant protective effect against tumor formation, a pregnancy must be carried out fully and lactation must occur to ensure full differentiation of the TEB to ABs and lobules has occurred. If this terminal differentiation event has not happened or is stopped early (miscarriage), the structures that could have become fully differentiated will regress slightly and remain more proliferative until a full term pregnancy coupled with lactation occurs (Russo, 1983).

The terminal end buds were only measured in the young virgin rats. But why? As mentioned in the anatomy and dynamics section, as rats (and people) age the breast continues to develop and those TEB structures will also differentiate and branch. While TEBs were found in old virgin rats, it is likely that there were not enough of the cells in order to preform a complete cell cycle experiment. Instead they measured the TDr or terminal duct regression structures. If the female never becomes pregnant or delivers a child some of the differentiated structures will regress. This is where TEBs may be located, but they most likely do not make up a majority of these structures. This is supported by the fact that parous rats, whose breast never have to go through the regression process, only have terminally differentiated structures.

While the data is included for all phases of the cell cycle, the only one that we are interested in the the G1 phase. This is the phase responsible for repairing the type of DNA damage induced by our carcinogen so while there is more interesting data in the other cell cycle phase lengths, they are not crucial to our discussion. As you can see the length of each cell cycle for each structure is essentially the same across the board excluding the G2 phase.

My best guess as to why the G2 phase of parous rats is longer in OV rats would be that it compensates for the incredibly short G1 phase. If our theories are correct and more damage can be repaired in the G1 phase of parous rats, perhaps the OV rats have adapted by increasing their G2 phase. If mutations are allowed to pass though G1 they will likely result in DNA damage or mutations during the process of replication in S phase. In order to compensate for the potential increase in mutations/DNA damage perhaps the longer G2 phase (which is primarily utilized as a checkpoint and timer for un-repaired DNA damage to be fixed) acts in a similar manner as the lengthened G1 phase in order to increase the amount of time to catch mistakes before the cell proceeds with mitosis. Call it your last line of defense.

More Effective DNA Repair
In the same paper as described above, the level of DNA repair was measured in rats who were young virgins, old virgins, and parous. In parous rats, the induction of unscheduled DNA synthesis (UDS) was 4-fold higher that that of virgin rats. UDS is the cell utilizing nucleotide excision repair to fix any mutations that the carcinogen may have caused in the genetic material of the cell and replacing the incorrect nucleotide with the right one, while outside of S phase of the cell cycle. Figure 5 shows the results of this experiment using varying concentrations of DMBA in order to induce UDS.This was measured by the level of 3 H-thymidine incorporation into the cell nucleus. In parous rats, a higher rate of 3 H-thymidine incorporation was detected, meaning more nucleotides were being utilized to synthesize the DNA that needs to be fixed. It is clear that on average a higher percentage of cells from parous rats were induced to undergo UDS, and this was consistent across each of the increasing concentrations of DMBA used. This means that the breast tissue cells of a parous rat can repair mutations and or DNA damage 4 times more effectively and rapidly than virgin rats can. This ties back to the previous discussion of cell cycle kinetics and the variant length of the G1 phase in these rats. When the cell has more time to repair the damage, we see an increase in the activity of the DNA repair mechanisms.

The removal of bulky adducts (DNA covalently bound to a chemical) from the DNA was also used as a measurement of DNA repair employed in protecting rats against tumor formation. The removal of these DMBA-DNA adducts is much faster and more efficient in the breast tissue of parous rats, showing that 38% of the adducts are removed after 48 hours. This is compared to the relatively slow adduct removal rate of virgin rats, which only removed 12% of the adducts after 48 hours. The increased ability of adduct excision results in the lower tumor incidence in parous rats and could possibly be due to the increased differentiation of the ABs in parous rats. When all of these repair mechanisms and the longer G1 portion of the cell cycle are taken together, it is easy to see how the cells of parous rats, that contain more ABs than TEBs, are able to employ many tactics to reduce the rate and or risk of mutation acquisition thereby decreasing the likelihood of tumor formation. It is also important to note that these are //in vitro// studies preformed on rat breast tumor cells in culture. While this data is exciting as it provides support for our hypotheses about cell cycle kinetics, the next step may be to attempt an //in vivo// study as well.

=Hormones and Epigenetic Factors=

Many researchers have postulated that there are molecular and epigenetic factors stemming from pregnancy hormones that confer resistance to breast cancer. In a study done by Blakely and colleagues, pregnancy in rats was shown to induce alterations in gene expression that had protective effects against tumor growth (Blakely et al., 2014). Four different rat strains were tested by treating parous and nulliparous rats from each of them with estradiol and progesterone at levels equivalent to pregnancy. After doing microchip profiling on tissue from each strain, there are 70 genes that are consistently altered by pregnancy hormones. The genes that were found to be unregulated or down regulated fit five distinct categories: those involved in differentiation, immune response,TGF- b, cell growth, and structure of the extracellular matrix. Overall, the parous rats and rats treated with estradiol and progesterone showed an increase in tumor free survival rate compared to nulliparous rats (Blakely). The figure below shows the levels of up regulation and down regulation of a few key genes impacted by hormone levels.

Genes involved in immune system function and differentiation were shown to be up regulated by pregnancy hormones estrogen and progesterone. Examples of genes that fall in this category are TGF-b3, which is up-regulated by 1.3-fold and IgHalpha, which is up-regulated 25-fold (Blakely). TGF-b3 is transforming growth factor and is involve d in cell differentiation to allow for lob 4 tissue during lactation. It also has the capacity to induce apoptosis in cells with damage or in response to signal molecules. This gene plays a significant role in maintaining a functional cell and in many breast cancer types resistance to TGF-b3 is seen. IgHalpha is a gene that codes for the heavy chains that compromise antibodies. It's role is to recognize foreign cells and coordinate an immune response to potential threats. Other immune related genes showed an increase in macrophages, T-cells, and antibacterial activity in the breast tissue to protect from foreign invasion and destroy any unfamiliar cells.

There are also genes shown to be down regulated by pregnancy hormones, for example igf-1 and ghr, which play a role in cell growth. Igf-1 has the task of cell proliferation and prevention of apoptosis, so when it is down regulated, a higher level of apoptosis can occur, thereby preventing the take over of cancer cells (Blakely). The decrease in Igf-1 is accompanied by a decrease in ghr, growth hormone receptor, as well.

The Extracellular matrix (ECM) is also regulated by pregnancy hormones. Most of the ECM genes coding for structural components like collagen are down regulated, as well as genes coding for cell to cell interactions. A few of these genes are sparc and Lgals1, and Serpinh1.

One of the protein products that was shown to be upregulated in the cells of parous rats was RbAp46. This is a retinoblastoma associated protein that has been shown to have many functions, some of which include apoptosis, histone deacetylation, control of cell growth, and interactions with many other signaling pathways and proteins. One specifically being GADD45, which when interacting with RbAp46 plays the role of stopping cell growth due to DNA damage (Ginger 2009).

After looking at all the data about gene function and how they change during pregnancy, a clear correlation emerges. The [|table] shown in Blakely's article shows just a few of the genes that are up regulated in parous rats and aid in proper cell functioning and cell cycle control. It also shows the genes that are down regulated in pregnant rats, which are mainly growth factors and ECM regulators. With decrease function of these genes comes decreased cell proliferation and control is maintained over the breast tissue, which helps to prevent the breast from succumbing to cancer. The Kaplan-Meier plot below shows the 5 year survival of rats who have been pregnant versus those who have not. It is clear that pregnancy confers some resistance to cancer and extends the lifespan of the rats. == ==  **Table 2.** Mammary tumor incidence for placebo and estradiol and progesterone–treated rats is   plotted for each strain. Cohort sizes for estradiol and progesterone–treated animals were: Lewis  (n = 16), Wistar-Furth (n = 12).Each strain exhibited significantly decreased tumor incidence in  estradiol and progesterone–treated compared with placebo-treated cohorts.

Conclusion
After reviewing the literature on breast cancer in relation to pregnancy, there appears to be a strong correlation suggesting that, in fact pregnancy does have some preventative effect that decreases tumor incidence. However, there is not just one mechanism that can be pinpointed as the reason behind this protective effect. Pregnancy confers a multimodal resistance strategy, which utilizes structural alterations of breast tissue, cell cycle kinetics, advanced repair mechanisms, and hormonal regulation to make it much more difficult for a tumor to form in the breast. The breast structure changes, becoming more and more differentiated and complex, leading to a higher proportion of cells which have a lower mitotic index and rate DNA synthesis. The cells in the breast after pregnancy also have a longer G1 phase, which means more time for mutation correction and this allows the repair mechanisms to function. One of these is unscheduled DNA synthesis to excise incorrect nucleotides and insert new ones. Another repair mechanism is the removal of bulky carcinogen adducts from the DNA, also preventing tumor formation by preserving the original genetic code. Lastly, the hormones that are elevated during pregnancy, more specifically estrogen and progesterone, work to up regulate and down regulate certain genes that can help control cell growth, Extracellular matrix formation, and immune response. This also keeps the cell from growing out of control and helps the body rid of any damaged cells that could lead to a tumor. All of these protective mechanisms taken together are the reason why pregnancy is shown to reduce breast cancer in women by 50% compared to nulliparous women (National Cancer Institute).

Sources:
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"Breast Cancer Prevention (PDQ®)." National Cancer Institute. National Cancer Institute, 22 May 2014. Web. 27 May 2014.

Fraumeni JF Jr, Lloyd JW, Smith EM, et al. “Cancer mortality among nuns: role of marital status in etiology of neoplastic disease in women”. J Natl Cancer Inst 1969;42:455–468.

Ginger, M. R.. "Persistent Changes in Gene Expression Induced by Estrogen and Progesterone in the Rat Mammary Gland." Molecular Endocrinology: 1993-2009. Web. 6 May 2014.

Javed, Asma, and Aida Lteif. "Development of the Human Breast." Seminars in Plastic Surgery 27.01 (2013): 005-12. PubMed. Web. 17 May 2014.

Wang, Zhao-Yi. "Overexpression of RbAp46 facilitates stress-induced apoptosis and suppresses tumorigenicity of neoplastigenic breast epithelial cells." International Journal of Cancer: 762-768. Web. 6 May 2014.

Russo, Jose, Lee K. Tay, and Irma H. Russo. "Differentiation of the Mammary Gland and Susceptibility to Carcinogenesis." Breast Cancer Research and Treatment 2.1 (1982): 5-73. Web.

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Russo, Jose. "The protective role of pregnancy in breast cancer." Breast Cancer Research(2005): 131-142. Web. 5 May 2014.

"Surveillance, Epidemiology, and End Results ProgramTurning Cancer Data Into Discovery."SEER Cancer of the Breast. National Institute of Health, 15 Apr. 2014. Web. 2 May 2014.

Whitaker, Lucy. “The plight of nuns: hazards of nulliparity”J Fam Plann Reprod Health Care (2012) 38 (2): 116