**2. Endocrine and molecular biology and controls in prostate cancer**

#### **2.1. Hormones and prostate cancer**

progressively leads from to an acute high-grade PIN (HGPIN) to superficial cancers, and then to invasive disease [11, 12] (See Figure 1). The most common form of PCa (around 95%) originates in the glandular/epithelial tissue, and is coined prostatic adenocarcinoma; thus, this term has emerged synonymously with PCa [13]. The residual forms of PCa are termed nonadenocarcinoma, and present as less commonly occurring cancers, but are often more aggressive. Such can be categorized as epithelial (such as squamous cell carcinoma) and nonepithelial (such as osteo- and angio-sarcoma), and also include others which rarely develop in the prostate and are derived from primary tumors of the bladder and urethra (such as transitional cell carcinoma) [13]. The diagram below gives a general overview of prostate location, the division of zones and the general progression of PCa as the cell and tissue phenotype changes from normal to A. prostatic intraepithelial neoplasia (PIN), to B. increasing and severe high-grade PIN (HGPIN), then to C. superficial cancers, and finally to D. invasive

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

**A**

While the specific causes of PCa remain unknown, the only established risk factors for PCa are age, race and family history [16]. However, it is well accepted that the stimulation of androgens over a prolonged period contributes to the development of PCa [17]; thus, the androgen receptor (AR), along with its various cofactors play an important role in PCa. Nonetheless, progression of PCa to androgen independence (or the hormone refractory state) is one of the primary reasons for PCa-related deaths today [18]. PCa is the sixth most commonly diagnosed cancer worldwide [19]. For Canadian men, PCa is the most prevalent (based on 15 years) and frequently diagnosed non-dermatological cancer [20]. Like most other solid malignancies, PCa

**B C** 

**D** 

disease [11, 12, 15].

**Figure 1.** GA Tobin, 2011. An Overview of the Pathogenesis of PCa.

2

Charles Huggins introduced the phenomenon of androgen removal as a plausible means for prostate cancer treatment in men after his observation that castration in canines caused shrinkage of prostate tumors [37]. Similar to normal prostate cells, prostate cancer cells are androgen dependent at the outset with a normally functioning androgen receptor (the receptor only responds to androgen) [37]. These initial studies on the dependence of the prostate gland on androgens formed the principle behind androgen deprivation therapy. There are several avenues that can be utilized to remove androgens, and the decision to do so is largely de‐ pendent on the stage or severity of cancer. Early cancers commonly target 5alpha(α)-reductase inhibition in an effort to decrease levels of the active hormone dihydrotestosterone (DHT). In fact, in the Prostate Cancer Prevention Trial (PCPT), finasteride (a 5α-reductase inhibitor) has been reported to lower the incidence of prostate cancer in men by 25% [38]. The initial concern about finasteride causing more severe cancers was refuted. However, despite this, the FDA has published advice concerning the use of finasteride and an increased risk of high-grade PCa [39]. Interestingly, the PCPT study is the one which Brasky *et al.* used to extrapolate data for their recent findings regarding n-3 PUFA and prostate cancer [40]. AR antagonists can also be used to block both testicular and adrenally produced androgens from binding to the androgen receptor and initiating gene transcription [41]. Additionally, testicular-derived androgen production can be blocked by luteinizing hormone (LH) inhibition [42]. For more aggressive cancers, these previously mentioned methods can be used in combination blocking both androgen production and their actions [41].

Despite the fact that most prostate cancers respond well to androgen deprivation, prostate cancer cells develop survival mechanisms involving the androgen receptor that can render these treatments ineffective; this represents the progression from androgen sensitive to androgen insensitive disease. Central to this role, the AR develops abnormal signaling functions that are not seen in normal AR signaling in androgen sensitive prostate cancer progression. There are several mechanisms through which the androgen receptor can partic‐ ipate in androgen insensitive prostate cancer. Amplification of the AR creates a hypersensi‐ tivity to even low amounts of circulating androgens [43]. The AR can also become promiscuous; mutations of the ligand binding domain can allow for aberrant AR activation by factors other than androgens (i.e: estrogen or other steroid hormones) and increased AR activity can also occur when such mutations are found in coregulators [44]. The AR can become an outlaw receptor - receptive to transactivation, phosphorylation and activation by other signaling pathways and peptide growth factors (i.e: insulin growth factor -1 (IGF-1) in the absence of androgen [45]. Other modified functions of the AR include activation of other proproliferative and anti-apoptotic factors that can bypass the AR pathway, eliminating any dependence on androgens for these physiological effects. For example: bcl-2, an anti-apoptotic protein, is not expressed in normal prostate epithelium, however in the absence of AR function, bcl-2 is produced [46]. Thus, effective treatment is dependent in the ability of the AR to develop these modified survival mechanisms.

The topic of testosterone production is worthy outside the realm of AR function in androgen insensitive prostate cancer. It is well known that the testes are the main site of testosterone production. Therapies that reduce testosterone production by targeting the testes do not affect adrenal androgen synthesis which is increased in androgen insensitive prostate cancer, thus compensating for the reduced testicular androgen production. In such cases, a broad spectrum cytochrome P450 inhibitor may be recommended, although their use may introduce side effects so extensive that their utility is limited [47].

#### **2.2. Molecular biology of prostate cancer**

Hanahan and Weinberg, in 2011, summarize the unique biological capabilities of cancer [48]. The hallmarks of cancer outlined within include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angio‐ genesis, and activating invasion and metastasis. Interestingly, in this updated review, genomic stability and inflammation are noted as underlying these characteristics as they expedite acquisition and foster multiple functions, respectively. Additionally, two emerging hallmarks - reprogramming of energy metabolism and evading immune destruction are discussed, as well as the dimensions of tumor complexity including the ability of these acquired traits to create the tumor microenvironment [48]. We agree that the recognition of all of these concepts, alone or in combination, provide guidance in the development of new cancer treatments. The paragraphs to follow are meant to provide a basic overview of some of the aforementioned concepts and their relevance in prostate cancer. While we have not specifically discussed tumor microenvironment or two of the above-mentioned emerging hallmarks and their relevance to prostate cancer in this chapter, such topics of importance subsequently emerge in broader areas of discussion.

#### *2.2.1. Sustaining proliferative signaling*

receptor and initiating gene transcription [41]. Additionally, testicular-derived androgen production can be blocked by luteinizing hormone (LH) inhibition [42]. For more aggressive cancers, these previously mentioned methods can be used in combination blocking both

Despite the fact that most prostate cancers respond well to androgen deprivation, prostate cancer cells develop survival mechanisms involving the androgen receptor that can render these treatments ineffective; this represents the progression from androgen sensitive to androgen insensitive disease. Central to this role, the AR develops abnormal signaling functions that are not seen in normal AR signaling in androgen sensitive prostate cancer progression. There are several mechanisms through which the androgen receptor can partic‐ ipate in androgen insensitive prostate cancer. Amplification of the AR creates a hypersensi‐ tivity to even low amounts of circulating androgens [43]. The AR can also become promiscuous; mutations of the ligand binding domain can allow for aberrant AR activation by factors other than androgens (i.e: estrogen or other steroid hormones) and increased AR activity can also occur when such mutations are found in coregulators [44]. The AR can become an outlaw receptor - receptive to transactivation, phosphorylation and activation by other signaling pathways and peptide growth factors (i.e: insulin growth factor -1 (IGF-1) in the absence of androgen [45]. Other modified functions of the AR include activation of other proproliferative and anti-apoptotic factors that can bypass the AR pathway, eliminating any dependence on androgens for these physiological effects. For example: bcl-2, an anti-apoptotic protein, is not expressed in normal prostate epithelium, however in the absence of AR function, bcl-2 is produced [46]. Thus, effective treatment is dependent in the ability of the AR to develop

The topic of testosterone production is worthy outside the realm of AR function in androgen insensitive prostate cancer. It is well known that the testes are the main site of testosterone production. Therapies that reduce testosterone production by targeting the testes do not affect adrenal androgen synthesis which is increased in androgen insensitive prostate cancer, thus compensating for the reduced testicular androgen production. In such cases, a broad spectrum cytochrome P450 inhibitor may be recommended, although their use may introduce side

Hanahan and Weinberg, in 2011, summarize the unique biological capabilities of cancer [48]. The hallmarks of cancer outlined within include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angio‐ genesis, and activating invasion and metastasis. Interestingly, in this updated review, genomic stability and inflammation are noted as underlying these characteristics as they expedite acquisition and foster multiple functions, respectively. Additionally, two emerging hallmarks - reprogramming of energy metabolism and evading immune destruction are discussed, as well as the dimensions of tumor complexity including the ability of these acquired traits to create the tumor microenvironment [48]. We agree that the recognition of all of these concepts, alone or in combination, provide guidance in the development of new cancer treatments. The

androgen production and their actions [41].

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

these modified survival mechanisms.

effects so extensive that their utility is limited [47].

**2.2. Molecular biology of prostate cancer**

Although the host may provide a source of growth factors, such are not essential for the survival of cancer cells. Cancer cells can evade the requirement for exogenous growth factors in several ways, including the production of their own growth factors. Cancer cell mutations can result in constitutively active growth factors and growth factor receptor pathways. Thus the initiation of the signaling cascade occurs without the required binding of the growth factor ligand to the receptor; cell growth occurs in the absence of ligand. In this regard, mutations in both the ras family and epidermal growth factor (EGF) receptors are seen in prostate cancer [49]. Another example of such includes autocrine and/or paracrine production of IGF-1 by prostate cancer cells [50], as stromal prostate cancer cells and the adjacent epithelium produces IGF-1 and expresses IGF-1 receptor, respectively.

#### *2.2.2. Evading growth suppressors*

Pathway hypersensitivity can result when cancer cells over-express growth factor receptors in the presence of normal physiological levels of a growth factor. PCa cells frequently increase their expression of Human Epidermal Growth Factor Receptor (HER)2 and the androgen receptor [51]. In this way, small amounts of androgen that can direct large increases in the expression of pro-carcinogenic genes or AR regulated gene expression can even occur in the absence of androgens.

#### *2.2.3. Resisting cell death*

The body eliminates damaged or dysregulated cells through intrinsic mechanisms including tumor suppressor pathways responsible for the mediation of DNA repair, cell cycle arrest, apoptosis and senescence [52]. However, some of these damaged cells harbor mutations and/ or have the ability to produce factors that allow them to survive. Proliferation of these cells, if not kept in check, results in tumors. The expression of Bcl-2, an anti-apoptotic protein indicates that a cell may not respond to cell death cues. Production of Bcl-2 is high in prostate cancer cells [46], rendering them resistant to apoptosis.

#### *2.2.4. Enabling replicative immortality*

The balance between growth and anti-growth signaling results in normal cell growth. Differ‐ entiation of cells occurs only when cells halt the growth program. Thus, rapidly proliferating (undifferentiated) cells form a mass of cells commonly referred to as a tumor. Such unimpeded growth is usually less differentiated than normal cells. Under normal conditions, transforming growth factor beta (TGFβ) is an anti-growth factor stimulating epithelial cells to a terminally differentiated state. However, this property is dysregulated in prostate cancer development [53]. As TGF-β signals through retinoblastoma (Rb) tumor suppressor gene [54], it is assumed to cause an increase in cell cycle inhibitors, such as p21. Thus, the loss of Rb in prostate cancer renders the pro-differentiation, anti-proliferation effects of TGF-β null [55].

The ability of cancer cells to replicate many more times over their natural lifespan distinguishes them from normal cells. Inherent defects in prostate cancer cells, such as losses of cell cycle inhibitors, including p21 or p27 enable this phenomenon. As with other cancer cell types, dysregulated telomere maintenance provides another explanation. The small DNA fragments at the ends of chromosomes are known as telomeres [56]. Telomere shortening to a desired length, following cell reproduction, signals a cell to stop replicating. However, cancer cells express elevated levels of telomerase, thus allowing cancer cells to replicate beyond their normal programmed number of replications by maintaining telomere length [38].

#### *2.2.5. Inducing angiogenesis, activating invasion and metastasis*

In order to expand in size, cancer cells require the support of a vasculature system. Judah Folkman's pioneering work in cancer revealed this unconditional requirement for sustained angiogenesis in cancer [57], accompanied by increased requirements for oxygen and nutrient supply, as well as waste removal; the latter is due to the ability of cancer cells to rapidly proliferate. In prostate cancer, and other cancers, vascular endothelial growth factor (VEGF) production is elevated [58]. This paracrine factor stimulates nearby endothelial cells and epithelial cells to form irregular vasculature associated with tumorigenesis. This confirms the notion that cancer cells produce factors that recruit endothelial progenitor cells and epithelial cells are capable of forming primitive vascular channels.

To further survive along the cancer cascade, and even in the presence of increased angiogen‐ esis, cells must metastasize as the tissue of origin is capacity limiting with respect to tumor burden [57]. Metastasis of cancer cells involves degradation of the basement membrane at the site of origin and penetration to the bloodstream and/or lymph. Although few cells survive this journey through the lymph and blood stream, those that penetrate into the tissue of destination complete the metastatic cascade by invading and colonizing to secondary sites.

Cadherins, a class of membrane receptors, can undergo isoform switching (known as "cad‐ herin switching") during normal development which allows cell types to segregate from one another. Within this family, particular cadherin members promote cell motility and invasion control via growth factor receptor signaling and pathway signaling components [59]. In tumor cells, this activity ceases resulting in aggressive tumor cells with an ability to escape the origin of the tumor and metastasize.

Therefore, the functional significance of these specific cadherins and cadherin switching provides insight into the molecular mechanism underlying tumor progression and offers an opportunity for the development of novel molecular targets for anti-cancer therapy. Integrity of the basement membrane and maintenance of epithelial integrity involves E-cadherin (epithelial calcium-dependent adhesion), a Type 1 transmembrane protein. The extracellular component of E-cadherin is responsible for homophilic interactions, while the cytoplasmic component of E-cadherin binds to beta (β-) and gamma (γ-) catenin [60]. While the loss of Ecadherin signifies the ability of a cancer cell to leave the prostate [61], it would be remiss to think that this loss alone is responsible for tumor cell invasion considering the dimensions of tumor microenvironment [48]. Aside from changes in cell-to-cell and cell-to-matrix adhesion, tumor invasion involves additional cellular events including cell migration and proteolytic degradation of the extracellular matrix (ECM), and other events which alone or in combination can affect cell signaling pathways. The initial suggestion that E-cadherin downregulation may result in the activation of specific signaling pathways triggering tumor cell invasion has been explored. Because of its dual role as a cytoplasmic cellular adhesion complex component and its fundamental role in Wnt-mediated signal transduction, β-catenin has emerged as a prime contender for activating such signaling pathways. Additionally, levels of matrix metallopro‐ teases (MMPs) also increase in prostate cancer and degrade stromal and basement membrane components, enabling cancer cells to escape the tissue of origin [62]. Overall, such factors that allow the progression of the metastatic cascade, rather than the original tumor, are responsible for cancer death.

#### *2.2.6. Inflammation*

growth is usually less differentiated than normal cells. Under normal conditions, transforming growth factor beta (TGFβ) is an anti-growth factor stimulating epithelial cells to a terminally differentiated state. However, this property is dysregulated in prostate cancer development [53]. As TGF-β signals through retinoblastoma (Rb) tumor suppressor gene [54], it is assumed to cause an increase in cell cycle inhibitors, such as p21. Thus, the loss of Rb in prostate cancer

The ability of cancer cells to replicate many more times over their natural lifespan distinguishes them from normal cells. Inherent defects in prostate cancer cells, such as losses of cell cycle inhibitors, including p21 or p27 enable this phenomenon. As with other cancer cell types, dysregulated telomere maintenance provides another explanation. The small DNA fragments at the ends of chromosomes are known as telomeres [56]. Telomere shortening to a desired length, following cell reproduction, signals a cell to stop replicating. However, cancer cells express elevated levels of telomerase, thus allowing cancer cells to replicate beyond their

In order to expand in size, cancer cells require the support of a vasculature system. Judah Folkman's pioneering work in cancer revealed this unconditional requirement for sustained angiogenesis in cancer [57], accompanied by increased requirements for oxygen and nutrient supply, as well as waste removal; the latter is due to the ability of cancer cells to rapidly proliferate. In prostate cancer, and other cancers, vascular endothelial growth factor (VEGF) production is elevated [58]. This paracrine factor stimulates nearby endothelial cells and epithelial cells to form irregular vasculature associated with tumorigenesis. This confirms the notion that cancer cells produce factors that recruit endothelial progenitor cells and epithelial

To further survive along the cancer cascade, and even in the presence of increased angiogen‐ esis, cells must metastasize as the tissue of origin is capacity limiting with respect to tumor burden [57]. Metastasis of cancer cells involves degradation of the basement membrane at the site of origin and penetration to the bloodstream and/or lymph. Although few cells survive this journey through the lymph and blood stream, those that penetrate into the tissue of destination complete the metastatic cascade by invading and colonizing to secondary sites.

Cadherins, a class of membrane receptors, can undergo isoform switching (known as "cad‐ herin switching") during normal development which allows cell types to segregate from one another. Within this family, particular cadherin members promote cell motility and invasion control via growth factor receptor signaling and pathway signaling components [59]. In tumor cells, this activity ceases resulting in aggressive tumor cells with an ability to escape the origin

Therefore, the functional significance of these specific cadherins and cadherin switching provides insight into the molecular mechanism underlying tumor progression and offers an opportunity for the development of novel molecular targets for anti-cancer therapy. Integrity

renders the pro-differentiation, anti-proliferation effects of TGF-β null [55].

normal programmed number of replications by maintaining telomere length [38].

*2.2.5. Inducing angiogenesis, activating invasion and metastasis*

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

cells are capable of forming primitive vascular channels.

of the tumor and metastasize.

Although inflammation has been long recognized in the development of cancer [63, 64], *chronic* inflammation has emerged as one of the enabling characteristics of human cancer [48]. Chronic inflammation can be caused by numerous factors including infectious and non-infectious agents and / or other environmental aspects including hormonal changes and dietary inter‐ ventions [65, 66]. Fostering multiple functions related to cancer, inflammation cuts across broad areas of study including genetics, epidemiology, molecular pathology, histopathology, immunology and animal modelling to name a few. In other words, it would be difficult to capture an association between cancer and inflammation by way of any one over-arching theory. A diverse amalgamation of evidence exist which points to the role of the inflammatory response in physiological maintenance including tissue homeostasis and the healing process involved succeeding injury or damage. Rakoff-Nahoum (2006) has provided an excellent overview of this dual role of inflammation in tumor development [52]. The inter-relationship of cancer and inflammation with respect to inflammatory cells (and their mediators) and signaling pathways involved that have been published in the past suggests that the inflam‐ matory system can affect tumor development and inhibit the development of cancer [67-72]. The topic of inflammation in prostate carcinogenesis has been recently updated in a thorough review authored by Sfanoes and DeMarzo (2012) encompassing recent advances in prostate risk and development with respect to prostatic inflammation stimuli, immunobiology, inflammatory pathways and cytokines, proliferative inflammatory atrophy as a risk factor lesion to prostate cancer development, and the role of nutritional or other anti-inflammatory compounds [66]. While this exceptional review articulates the vast effects of inflammation in prostate cancer, we will aim to highlight evidence to date as related to some promising antiinflammatory natural products while focusing on PUFA.
