Fibroblast Growth Factor-8 (FGF-8) is the most complex member of the fibroblast growth factor (FGF) family, which is comprised of nine genes in mammals encoding 23 fibroblast growth factor proteins. These proteins have a very similar amino acid sequence and share approximately 35-50% of their amino acid sequence. As part of a signalling pathway, these encoded proteins are secreted to the extracellular matrix, thereby interacting with FGF tyrosine kinase receptors to trigger downstream functions. FGF-8 was originally isolated from a mouse mammary gland carcinoma cell line and characterized as an androgen-induced growth factor. FGF-8 normally induces cell migration, proliferation, and differentiation. ^[1] This gene is a crucial component for normal development of brain, limbs, eyes, ears, and gonadotropin-releasing hormone (GnRH) during the process of embryogenesis. ^[2] More specifically, its functions include regulation of mesodermal, neuroectodermal, and epithelial cells in embryonic development.^[3] FGF-8 has also been detected at high levels in human lactating mammary glands, malignant tumor growth in prostate cancer, and angiogenesis.

Some of the functions of FGF-8 at the embryonic stage include body axis development, brain development, outgrowth and patterning of the facial primordia, pharyngeal development, limb development.

The continued expression of FGF-8 RNA in early formation of the primitive streak (E6.5) and its subsequent expression in the tail bud along with two other members of the FGF family (FGF-3 and FGF-4) suggest that FGF-8 activity is necessary for normal development of the body axis^[4] . Furthermore, the early expression of the FGF-8 prior to gastrulation in the region of epiblast containing cells eventually forms the mesoderm and endoderm^[5] . More specifically, FGF-8 regulates the expression of other genes that specify the fates of the mesodermal cells ^[5]. In this regulatory mechanism, the expressed FGF-8 protein targets homeobox-containing genes including Evx1 (in Drosophila)^[6] , Xhox3 (in Xenopus)^[7] , GSc (in Drosophila gooseberry)^[8] and bicoid that are involved in determining the posterior/ventral and anterior/dorsal cell fates.

The presence of FGFR1 and FGFR2 receptors in the early stages of the neural tube formation indicates that fibroblast growth factors may play a role in early stages of normal brain development^[9] . In these stages, the FGF-8 protein is mostly expressed in the forebrain (telencephalon commissural plate), the isthmus (at the mesencephalon-metencephalon boundary), the ventral midline of the hypothalamus, and the optic stalk ^[5]. This widespread expression of FGF-8 along with the FGF receptor proteins also suggests the regulatory effect of FGF-8 protein on patterning those regions of the brain. Further research shows cooperative function of FGF-8 with another signaling molecule called WNT1 in the development of the midbrain/hindbrain regions and the dorsal and ventral diencephalon ^[5]. The expression of each of the two genes is regulated by the other gene, as their domains of expression appear to be mutually exclusive. ^[5]

FGF-8 expression in patches of surface ectoderm overlying the facial primordia (at stage E9.5) covers the mandibular and maxillary areas, and the frontonasal mass ^[5]. Its interactions with FGFR1 receptor in the mesenchyme cells results in production of an epithelium-derived signal that involves in regulating the outgrowth and patterning of the facial primordia ^[10] . For example, according to Lumsden’s research, tooth formation is initiated by the inductive signals provided in ectodermal patches containing high levels of FGF-8 RNA localizations^[11] .

In early stages of mice embryo development (E8.0), the FGF-8 RNA is expressed in symmetrical bands along the bilateral walls of the foregut endoderm, on top of surface ectoderm, and within lateral mesoderm. ^[5] Specifically, FGF-8 RNA localization happens at extreme lateral endodermal regions of each pharyngeal pouch and the overlying surface ectoderm of the pharyngeal groove ^[5]. The ectodermal surface covering the mandibular and maxillary prominences derived from the first pharyngeal arch as well as the frontonasal mass also possesses high levels of FGF-8 RNA. ^[5]

In situ hybridization during stage 1 (E9.0-E9.5) of limb bud formation in chick embryos shows FGF-8 RNA localization in ectodermal layers ventral to the limb bud ^[5] In fact the fate of the cells involved in initial outgrowth of the limb might be determined by early expression of the FGF-8 protein locally.^[5] As the thickening of the ectodermal layer begins at the distal tip of the limb bud (stage 2-3), FGF-8 expression is restricted to limb regions where the Apical Ectodermal Ridge (AER) signals are also present ^[12] .

FGF-8 is mainly expressed in the steroid hormone target tissues in the reproductive and genitourinary tract organs of adults. ^[13] In kidneys, breasts, prostates and testes, FGF-8 is expressed at very low levels.^{[14] [15] [16]} Recently, it is discovered that FGF-8 is expressed in peripheral blood leukocytes and human bone marrow cells, suggesting that FGF-8 is also involved in normal hematopoiesis.^[17] It is also suggested that FGF-8 plays a role in the embryonic development of bone marrow stem cells. ^[18]

In humans, the FGF-8 gene is located on the long side of chromosome 10 at position 24 (10q24) from base pair 103,529,886 to base pair 103,540,125. There are five different transcript variants of this gene which form different protein isoforms with distinct functions: isoform A, isoform E, isoform F, isoform B, and isoform G. ^[2]

The FGF-8 protein consists of 233 amino acids with a weight of about 26 kD. ^[13]

Fibroblast Growth Factors (FGF) initiate their function by binding to Fibroblast Growth Factor Receptors (FGFR) which are located on the cell membrane.^[19] There are four FGFR genes in total that encode for four specific FGFRs (FGFR1-FGFR4.^[19] These FGFR are immunoglobulin tyrosine kinase cell surface receptors which include two or three immunoglobulin domain (D1, D2 and D3), a transmembrane domain and a cytoplasmic domain containing two kinase regions. ^[20] Presence of serine-rich acidic region, called the “acidic box” located between the first and second immunoglobulin domains, is one of the distinguishing characteristics of FGFRs (D1 and D2). ^[19] D2 and D3 domains have a crucial role in FGF binding and determine the specificity of the receptor; however, D1 and acid box domain are believed to have an autoinhibitory role in receptor binding. ^[21] Exon skipping and alternative splicing leads to creation of distinct isotopes of each receptor. ^[19]

Extracellular surface of the target cells of growth factors contain a highly sulphated region called Heparine Sulphate Glycosaminoglycan(HSGAG) which binds to the Heparine Binding site(HBS) located on the FGF and further increases the affinity of FGF to binds to its receptor.(FGFR)^[19] Binding of the FGF and HSGAGs to FGFR leads to dimerization of FGFRs and subsequent transphosphorylation of intracellular tyrosine residues. ^{[22] [23]} During embryonic development, FGF signal transduction proceeds via three distinct pathways as follows:

This is the most common pathway governing the function of FGF during early embryonic development. In this pathway, phosphorylation of the tyrosine residue of a protein on the surface of membrane (FRS2) initiates the Ras-MAPK pathway. ^[24] Phosphorylated FRS2 activates Grb2 which subsequently activates another protein, called Ras by GTP exchange mechanism. ^[25] Activated Ras further activates other effector proteins such as Raf which eventually activate MAPK signaling cascade. MAPK signalling pathway leads to further phosphorylation and activation of other transcription factors. ^[26]

The PLCᵞ/Ca2+pathway is initiated by binding of PLCᵞ to phosphorylated tyrosine. Activated PLCᵞ hydrolyzes phosphatidylinositol-4,5-diphosphate and leads to the formation of inositol-1,4,5-triphosphate and diacylglycerol as secondary messenger. ^[24] Diacylglycerol activates protein kinase C and inositol-1,4,5-triphosphate leads to the release of intracellular Ca2+ ^[24]

This pathway is mostly studied in mesoderm development in Xenopus. ^[24] Very little information is known about this pathway.

In men, prostate cancer is the second cause of cancer-causing death ^[27] . Previous research has shown that the signaling pathways of fibroblast growth factors play a significant role in inducing proliferation of prostate cancer cells, most specifically in mediation of stromal-epithelial interactions. Manipulation of the FGF-8 protein levels at the genetic level has proved to directly affect cancer progression ^[28] . Of the four FGF-8 isoforms, over-expression of the FGF-8b is thought to be the most important in carcinogenesis in prostate tissues ^[29] . Previous studies have shown that in stages T1, T2, T3, and T4 of prostate cancer, the FGF-8b expression has been approximately 40%, 67%, 94%, 100%, respectively. FGF-8 mRNA is overexpressed in 60-70% of newly diagnosed prostate cancers ^[30]. Some of the receptors that have been expressed in malignant prostate cancer cells are found to be FGFR2, FGFR3 and FGFR4 ^[31] .

The cause of cancer cell growth and proliferation, among other contributing factors, is the overexpression of the FGF-8b protein in prostate cancer cells ^[32] . Ideally, the expression of FGF-8b should be down-regulated extensively in prostate cancer cells in order to prevent rapid proliferation of cancerous cells. ^[28] This down-regulation can be at the level of controlling expression of other factors which influence the expression of FGF-8b.

The three main cell lines used in prostate cancer research are LNCaP, DU-145, and PC-3 ^[33] . For the purposes of this experiment, only LNCaP cell lines will be used. The primary experimental objective would be to test whether androgen expression induces FGF-8 expression in cancer cells. Furthermore, two experiments can be done to confirm that FGF-8b expression is increased in the presence of androgen ^[28]. From previous experiments it can be hypothesized that androgen expression upregulates the expression of FGF-8b in prostate tumour cells ^[28]. Prostate Specific Androgen (PSA) is used to measure the amounts of androgens present in cancer cells; therefore, its expression will also be considered ^[34] .

In order to test for the coexpression of FGF-8b, androgen, and PSA in vitro, LNCaP cell lines are used since their proliferation is increased by the presence of androgens. Sample cells are treated with a media containing androgen. Untreated LNCaP cells were used as control for this test.

Another way to confirm this coexpression is the use of immunohistochemistry techniques in vivo to compare levels of expression of FGF-8b and androgen receptor in both castrated mice and mice treated with testosterone using LNCap cell lines. More specifically, immunohistochemistry is used to detect the prostate specific antigen (PSA) through the use of specific fluorescently labeled antibodies.

Once the coexpression of FGF-8b and androgen has been confirmed, a loss of function experiment can be done to decrease androgen levels in order to down-regulate FGF-8b. LNCaP cell lines are cultured in androgen-positive media, and are co-transfected with androgen receptors and the FGF-8.luc reporter gene. Each sample of cells is exposed to different doses of bicalutamide (a non-steroidal anti-androgen drug that binds to androgen receptors and prevents their activation). For example, a 10-fold and 100-fold molar excess of bicalutamide is used compare to that of the androgen amount. The controls are LNCaP cancer cells with no manipulations; cell have both androgen and FGF-8b. Levels of androgen can be monitored by measuring the PSA.

^[28]

Coexpression of androgen, PSA, and FGF-8b should be evident in LNCaP cells treated with androgen; these cells should have a higher level of FGF-8b expression compared to the control untreated LNCaP cells. Therefore, it can be concluded that androgen expression induces the expression of FGF-8b.

Immunohistochemistry should show that FGF-8b and androgen receptors both show reduced levels of expression in castrated mice compared to testosterone-treated mice. Castration should prevent cell proliferation due to decreasing androgen receptor, PSA, and FGF-8b. Testosterone expression should increase PSA, androgen receptor, and FGF-8b expression.

The expression level of FGF-8b protein in cell lines containing both androgen receptor FGF.luc should be significantly reduced with higher dosage of bicalutamide. However, adding lower dosages of bicalutamide should not reduce the expression of FGF-8b.

^[28]

Down-regulating the activation of androgen receptors through the application of the anti-androgen bicalutamide decreases the androgen levels, which in turn decreases the expression of FGF-8b. ^[28] Therefore, if bicalutamide is expressed in normal prostate cancer cells of malignant tumours, cell proliferation and growth is suppressed due to the decreased levels of FGF-8b. ^[28] A similar approach can be imposed on preventing the growth of cancerous cells in other areas such as cancerous breast tissues. The above experimental approach can be considered a hormone therapy, which may have the potential to be a therapeutic treatment. More specifically, this hormone therapy can be used when surgery and radiation is no longer effective in situations when the malignant tumour has spread uncontrollably ^[35] . This method may also be used after surgery and radiation has been done and cancer has returned to the prostatic tissues ^[35]. Generally speaking, hormone therapy may have the potential to make cancer treatments more effective at any stage of the disease, though it is often used in late-stage cancers ^[35].

Though this method of approaching cancer cells may seem plausible, the is a wide range of side-effects for using hormone therapy. Some examples of these side effects include ^[35]

• Depression and decreased mental activity
• Loss of muscle mass
• Weight gain
• Fatigue
• Increased cholesterol
• Erectile dysfunction
• Reduced sexual desire
• Hot flashes
• Osteoporosis
• Anemia

In some cases, these side effects can be of great danger to the organ systems and the individual's general health ^[35].