**5. Molecular and endocrine controls**

was the responsibility of genetic changes of malignant cells in the primary tumor [69]. In 2008, Husemann *et al*, used transgenic mice to show systemic dissemination (specifically to lungs and bone marrow) of mammary tissue derived premalignant cells prior to the emergence of mammary tumors [70]. Additionally, this research reported that systemic dissemination of tumor cells can occur in pre-invasive stages of tumor progression as observed in female patients with ductal carcinoma *in situ*. A complementary accumulation of evidence supports the evolution of an early dissemination model, where malignant cells outside the primary lesion can also migrate to distal sites (such as lung and bone marrow) and cause tumors via various genetic programs [54, 71, 72]. Such a model is inclusive to "self-seeding", the term coined when cancer cells not only seeds regional (lymph nodes) and distant sites but also fuel

**Figure 1. (Tobin, GA, 2011) Physiological Aspects of Disease Progression in Breast Cancer:** The development of breast cancer has been proposed as a multi-step process. The most deadly aspect of breast cancer is metastasis and involves a cascade of reactions. Although the molecular mechanisms underlying this process are not fully understood,

Thus, breast cancer is not a single disease, but rather an assortment of diseases with diverse characteristics and clinical outcomes which will likely always require a variety and/or combination of treatments or alternatively, a broad spectrum application. Combine this with the fact that, despite major advances in our understanding of the biology of cancer, further research is required to improve our understanding of tumor establishment, progression and dissemination - the principal cause of mortality. Then, our goal in breast cancer research

the growth of the original tumor itself [73].

350 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

any disruption along the cascade could arrest disease progression.

#### **5.1. BRCA and other gene expression signatures associated with increased risk of breast cancer**

Less time, low cost and new sequencing technologies are all accomplishments that yield impact on molecular biomedical research. Recently, for example, such advances (combined with some prospective epidemiology studies) have allowed researchers to compare the DNA from healthy breast tissue, initial tumor cells and then cells obtained nine years later when the breast cancer had metastasized [74]. The 32 DNA mutations reported in the metastasized cells are a prime example of how technological advances can serve to provide us with ample data regarding gene expression that may offer insights into the progression of breast cancer disease. However, consider that gene expression profiling / signatures of primary breast tumors can only be used as a predictor of susceptibility or disease progression in breast cancer, particularly metastasis. Currently, it is not possible to accurately predict the risk of metastasis or prevent it. As a result, more than ½ of the patients treated with adjuvant chemotherapy are needlessly exposed to harmful side effects [5]. This presents another compelling reason to further study drug targets that are specific to, and have potential for, treatment in metastatic breast cancer.

Nonetheless, based on current knowledge, and aside from the many identified factors that could impact the risk of developing breast cancer (including personal and environmental), genetic mutations in critical cancer genes (both tumor suppressor and oncogenes) have been identified for their increased or associated risk with breast cancer [75]. Of the many that have been reported throughout the history of cancer genetics, Table 2 below captures those that stand out principally for their research and/or clinical significance in relation to breast cancer and summarizes major function, encoded proteins and known disease associations or risk factors. Such genetic aberrations are acquired over a person's lifetime; or less commonly, are inherited. While it is estimated that only 5% to 10% of breast cancers are hereditary, some gene variations are associated with both hereditary and somatic mutations [75]. The tumor sup‐ pressor genes *BRCA1* and *BRCA2* are the major genes related to hereditary breast cancer [76], and mutations in these and other *BRCA* genes in women are associated with a 60-80% risk of developing breast cancer throughout their life span [77]. Other genes with inherited altera‐ tions, including *CDH1*, *PTEN*, *TP53, CHEK 2*, and *ATM* have been noted to increase or are associated with the risk of developing breast cancer [78]. Most notably, the latter three have presented the strongest evidence related to the risk of developing breast cancer [79]. Somatic mutations mostly reference *ERBB2/HER2(neu)* in breast cancer, however *TP53* genes and others have been associated with some cases of breast cancer in this manner [80]. It is note‐ worthy that not all people who inherit mutations in these genes will develop cancer.


\*\* ERBB2 (erythroblastosis oncogene B), HER2/neu (Human Epidermal growth factor Receptor2, neu: derived from a rodent (neu)ral tumor), VEGF (vascular endothelial growth factor).

**Table 2.** Familiar Genetic Mutations, Encoding Proteins and their Major Functions in Relation to the Associated and Increased Risk of Developing Breast Cancer.

Those listed in Table 2, as mentioned, represent familiar genes associated with breast cancer. However, others genes that are applicable in breast cancer progression will be reviewed in terms of their associated cell signaling pathways. Considering the profound implications that the CSC theory has for cancer chemoprevention and therapy, combined with our interest in plant based molecules, we will also examine gene function in the context of opportunities for natural product compounds in CSC self-renewal.

#### **5.2. Endocrine controls**

**Gene Major Function: Associated Proteins Risk Factor Ref.**

Strong evidence indicating a 60 – 80% risk of developing breast cancer for people with mutations.

Reduced levels of the BRCA2 protein may cause Fanconi anemia. Patients with such are prone to several types of cancers, including reproductive system

Confers susceptibility and linked to an modest increase (up to 2 times)

Causes Li-Fraumeni syndrome: results in higher-than-average-risk of breast cancer and several other

Causes Li-Fraumeni syndrome. Can double breast cancer risk.

Causes Cowden syndrome which presents a higher risk of both benign and cancerous tumors in the breast, digestive tract, thyroid,

Increased risk of breast cancer, particularly invasive lobular breast

Amplification occurs in 15-30% of

human breast cancers.

uterus, and ovaries.

cancer.

\*\* ERBB2 (erythroblastosis oncogene B), HER2/neu (Human Epidermal growth factor Receptor2, neu: derived from a

**Table 2.** Familiar Genetic Mutations, Encoding Proteins and their Major Functions in Relation to the Associated and

associated tumors.

of breast cancer.

cancers.

[81 - 86]

[87] [88]

[89-93]

[94-96]

[97-99]

[100, 101]

[102-104]

[34] [80]

Encodes breast cancer type 1 susceptibility protein; responsible for DNA repair,

352 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

transcriptional regulation and cell cycle check

Encodes BRCA2 susceptibility protein involved in the repair of chromosomal damage,

especially in the error-free repair of DNA double strand breaks. As with BRCA1, indicates a high

Encodes protein with a phosphatidylinositol 3 kinase (PI3K)–like domain which plays a central role in the complex processes that repair DNA double-strand breaks. Also involved in regulation of cell cycle progression and the maintenance of genomic stability.

Encodes p53 protein and regulates cell cycle,

Encodes a serine/threonine-protein kinase which plays a critical role in DNA damage signaling pathways. Phosphorylates and regulates the functions of p53 and BRCA1.

Encodes phosphatase and tensin homolog protein, namely phosphatidylinositol-3,4,5 trisphosphate 3-phosphatase, which is involved

Encodes E-cadherin protein. Down-regulation decreases the strength of cellular adhesion, increases cellular motility; allowing cell invasion.

Encodes a transmembrane receptor with constitutive tyrosine-kinase activity.

rodent (neu)ral tumor), VEGF (vascular endothelial growth factor).

Increased Risk of Developing Breast Cancer.

in the regulation of the cell cycle.

preventing tumor growth.

point control.

degree of risk.

BRCA1 (BReast CAncer gene one)

BRCA2 (BReast CAncer gene two)

ATM (Ataxia telangiectasia mutation)

p53 (TP53) (Tumor Protein p53)

(Checkpoint kinase

PTEN (Phosphatase and tensin homolog)

CDH1 (Cadherin 1 Gene) or Ecadherin (epithelial cadherin)

\*\* ERBB2, (HER2/neu), or VEGF

CHEK2

2)

Over the years, and more notably since the discovery of suitable breast cancer cell lines and animal models, the symbiotic relationship between lab research and clinical investigations have advanced our knowledge of endocrine action. Breast cancer is influenced and highly regulated by several sex and growth hormones (including estrogens, androgens, progesterone, prolactin and insulin-like growth factors) and each of the sub-types and gene expression patterns of breast cancer are characterized by both unique and specific endocrine controls [105]. Particularly, estrogens and progesterone have received the greatest attention likely because of their involvement in normal and neoplastic mammary tissue and the scale of their associated risk estimation for breast cancer. Research into the role of androgens has likely been a consideration in breast cancer research as androgens are necessary precursors to all endog‐ enous estrogens. The role of prolactin in the pathogenesis of breast cancer remains unclear given the scarcity of studies to date. Similarly, although well studied, the role of Insulin-like growth factor (IGF) in breast cancer is inconsistent.

There are challenges in defining the role of progesterone in breast cancer and the role of progesterone receptor (PR) action in breast cancer remains divisive. In breast cancer, proges‐ terone has biphasic effects (both proliferative and inhibitory) on breast cancer cell lines grown *in vitro* [106, 107]. Depending upon cellular context and/or the presence of secondary agents, there may be a role for progesterone as a priming agent with growth promoting activity [106].

While progesterone is found as a single hormone, the major endogenous estrogens in fe‐ males include estrone (E1), estradiol (E2), and estriol (E3) and are primarily produced dur‐ ing menopause, in non-pregnant and pregnant females, respectively [108]. A variety of synthetic (xenoestrogens) and natural substances have been identified that also possess es‐ trogenic activity including those derived from plant products (phytoestrogens) and fungi (mycoestrogens) [108]. The actions of estrogens are mediated by their respective receptors binding to specific DNA sequences to activate the transcription of estrogen receptor (ER) regulated genes, including direct target genes [109]. Approximately 80% of breast cancers demonstrate expression of PR and/or ER [110]. Once established, these breast cancers which are classified as either hormone-sensitive or hormone-receptor-positive cancers are reliant on hormones to grow. Thus, treatment includes the suppression of hormone production in the body for these specific breast cancers. Therefore, this section will provide an overview of estrogens, their past indications in the treatment of breast cancer and their potential role in relation to naturally occurring dietary compounds.

#### *5.2.1. The role of estrogens and breast cancer*

The link involving estrogen and breast cancer can be traced back to as early as 1896 when a surgeon in England reported an improvement in condition in three young female breast cancer patients after the removal of their ovaries [111]. Since estrogen had not yet been discovered, the surgeon had unknowingly removed the source of estrogen that promotes the survival and division of cancer cells [111]. Since that time, a lot of evidence has shown that interactions between estrogens and their receptors influence the pathogenesis of breast cancer. Estrogen promotes proliferative effects on cultured human breast cancer cells [112]. Estradiol affects breast cancer risk by controlling the mitotic rate of breast epithelial cells and high levels of estradiol in post-menopausal women are also known to increase the risk of breast cancer [113]. Estradiol has also been shown to increase breast cancer risk via its metabolite, catechol estrogen 4-hydroxyestradiol, causing direct DNA damage through the formation of free radicals [114]. Estradiol has also been shown to modulate breast cancer cell apoptosis [115]. ER is a major determinant of the cellular response of estrogen and has been indicated in breast cancer promotion [116, 117]. The binding of estrogen to the ER modulates the transcription of a series of genes, including those coding for proliferation.

The close relationship between the etiology of breast cancer and exposure to estrogen warrants examination of key variables that may affect estrogen homeostasis, in particular those exhibiting anti-estrogen activity. Because of their impact on the primary and metastatic aspects of disease, anti-hormonal drugs are the mainstay breast cancer treatment. The goal in treating hormone receptor +ve breast cancers is to utilize drugs which suppress production of estrogen in the body. Estrogens in naturally occurring dietary compounds such as soy are used as an alternative to hormone therapy because of their anti-proliferative effects. This practice, in breast cancer treatment, is widely known as hormonal therapy, or anti-estrogen therapy, but is not representative of the term hormone replacement therapy [118].
