Prostate Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

An Introduction to Prostate Cancer

Prostate cancer (PCa) is the second leading cause of cancer death in American men, behind lung cancer and it predominately affects elderly men with higher incidence. About 1 man in 41 will die of prostate cancer. The development of PCa involves several mutations in prostate epithelial cells, usually linked to developmental changes, such as enhanced resistance to apoptotic death, constitutive proliferation, and, in some cases, to differentiation into an androgen deprivation-resistant phenotype, leading to the appearance of castration-resistant PCa (CRPCa), which leads to a poor prognosis in patients. A prostatectomy usually leads to an excellent prognosis with low risk of death from PCa after surgery. However, deregulated production and secretion of growth factors by stromal cells within the PCa microenvironment, as well as mutations in androgen signaling pathway components and further physiological modifications, including angiogenesis, local migration, invasion, intravasation, circulation, and extravasation of the tumor, potentially lead to systemic recurrence of the cancer, including the appearance of focal tumor in advanced stage. Prostate cancer can often be found early by testing for prostate-specific antigen (PSA) levels in a man's blood. Another way to find prostate cancer early is the digital rectal exam (DRE). However, neither the PSA test nor the DRE is 100% accurate. Therefore, optimizing early diagnosis and developing targeted therapy of prostate cancer are the key to improving the benefit of screening and the survival rate of patients. At present, the main molecular signaling pathways of prostate cancer including androgen receptor (AR) mediated signaling pathway, NF-κB signaling pathway, RTK signaling pathway, JAK/STAT signaling pathway and Wnt signaling pathway.

1 Main Signaling Pathways in Prostate Cancer Therapy

Fig.1 Prostate cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in red) and those in preclinical or early phase development (colored in green) for the treatment of advanced stage prostate cancer.

With the rapid development of molecular biology and genomics, it is known that molecular pathogenesis of prostate cancer is extremely complicated. The role of AR signaling in PCa development and progression has been well established and it influences the transition of PCa cells from androgen dependent to castration resistant (CR) stage. Activation of NF-κB pathway and subsequent downstream targets contributes to the progression and metastasis of PCa and blockade of NF-κB pathway of is associated with suppression of angiogenesis, invasion, and metastasis. EGFR/Akt or EGFR/MARP signaling pathways are critical for maintaining cell survival in PCa and disrupting the EGFR activation can inhibit tumor growth and neovascularization. Deregulation in the expression of downstream effectors of PI3K/ and RAS/mitogen-activated protein kinase (MAPK) pathways are reported to contribute significantly to PCa growth and development. Wnt5, a critical ligand that activates the β-catenin-independent pathway in Wnt signaling promotes the rapid proliferation and metastasis of PCa. Overexpression of insulin receptor IGF1R promoted tumor growth and enhanced angiogenesis in human PCa cells. Above molecular mechanisms provide multiple potential targets for the treatment of prostate cancer.

1.1 Androgen receptor (AR) mediated signaling pathway

The AR signaling is vital for normal functioning of the prostate, and initiation and maintenance of spermatogenesis. AR is a member of the steroid hormone receptor family of ligand-activated nuclear transcription factors. Under physiological conditions, both testosterone and DHT can bind to and activate AR signaling. Binding of ligand to the AR induces conformational changes in the LBD facilitating intramolecular and intermolecular interaction between NTD and CTD. This subsequently results in AR homo-dimerization, phosphorylation and nuclear translocation. Deregulated AR signaling is common during PCa development and CRPC progression due to overexpression of AR arising due to amplification/mutations, co-activator and co-repressor modifications, aberrant activation/post-translational modification, altered steroidogenesis, and generation of AR splice variants. Aberrant activation of AR also occurs via alterations in the steroidogenesis pathways which permits PCa cells to bypass testosterone and utilize adrenal androgens to generate functionally potent DHT via the 5α-dione pathway. Besides localization in the nucleus, some AR variants also exhibit exclusive cytoplasmic function that is sufficient enough for transcriptional effects. In addition, ARVs can freely enter the nucleus without association with the Hsp90 chaperone complex. Aside from the above described mechanisms that induce changes in the activity of AR, various transcription factors such as the protooncogene c-Myc, c-Jun, Sp1, FOXO3a, lymphoid enhancer binding factor 1 (LEF1), NF-κB and twist-1 also have a crucial role in promoting AR expression via gene regulation.

1.2 NF-κB signaling pathway

NF-κB is a protein complex that regulates expression of key genes required for innate and adaptive immunity, cell proliferation and survival, and lymphoid organ development. In humans, the NF-κB family comprises of five proteins namely p65 (RelA), RelB, p105/p50 (NF-κB1), p100/p52 (NF-κB2) and c-Rel. These proteins associate with each other to form homo- or heterodimeric complexes that are transcriptionally active. In the canonical pathway of activation, degradation of the IκB inhibitory protein occurs through phosphorylation at specific serine residues by IκB kinase complex (composed of the catalytic subunits IKKα and IKKβ, and the regulatory scaffolding protein NEMO). As a consequence, free NF-κB dimer enters the nucleus, binds to κB enhancer sites in the DNA and activates transcription of a wide array of genes participating in the immune and inflammatory response, cell growth, adhesion, metastasis, and apoptosis evasion. In prostate tumor cells, NF-κB is found frequently stimulated due to augmented levels of receptors such as TNF that radically increase IκB degradation. In androgen-independent prostate tumors, NF-κB expression is increased at both mRNA and protein level due to increased interleukin 6 (IL-6) expression that occurs as a result of constitutive NF-κB activation promoted by signal transduction via NF-κB inducing kinase (NIK) and IKK. NF-κB also targets a transcription regulatory element of the prostate specific antigen PSA, a vital marker for development and progression of PCa. NF-κB signaling in PCa cells also correlates with cancer progression, chemoresistance, and PSA recurrence. Reports also indicate that NF-κB activation contributes to soft-tissue or bone metastasis in prostate cancer. Furthermore, p65 of NF-κB could increase endogenous AR expression and its associated downstream target genes enhancing growth and survival in human PCa cells.

1.3 RTK signaling pathway

RTK signaling pathway has two major signaling pathway branches, PI3K/AKT and Ras/MAPK pathways. PI3K/AKT pathway, a chief intracellular signal transduction mechanism that links diverse classes of membrane receptors essentially plays a central role in cellular quiescence, cell growth, proliferation, differentiation, motility, survival and angiogenesis. Following stimulation by tyrosine kinase growth factor receptors, PI3K induces conscription and stimulation of the serine/threonine-specific protein kinase AKT and subsequently binds to AKT resulting in membrane translocation and its activation via phosphorylation. Activated AKT in turn phosphorylates and galvanizes several other proteins including mTOR ultimately inducing and regulating a wide array of cellular processes. PI3K/AKT pathway is often augmented due to the loss of tumor suppressor PTEN. In PCa cells, aberrant PI3K/AKT pathway disturbs the action of ERKs thereby favoring AR-independent growth. Congruently, AR target genes might impede PI3K/AKT pathway to favor AR-dependent growth, invasion and metastasis in PCa cells. Mitogen-activated protein kinases (MAPKs) comprise three distinct groups specifically ERKs, JNKs, and p38 isoforms. MAPK signaling links extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, migration, apoptosis and transformation. Overexpression of EGF, FGF, IGF, and KGFs in PCa frequently results in activation of endogenous Ras and MAPK pathways. In addition, p38 signaling chiefly activated at later stages of PCa increases the expression of aquaporins which are pore-forming proteins thereby enabling PCa cells survive through hypoxia.

1.4 JAK/STAT signaling pathway

JAK/STAT pathway is an imperative and pleiotropic membrane-to-nucleus cascade that transduces multitude of signals for normal development, cellular homeostasis, cell proliferation, differentiation, migration and apoptosis following stimulation by a wide variety of stimuli including reactive oxygen species, cytokines, and growth factors. Briefly, activation of JAK/STAT pathway occurs when ligand binding induces multimerization of receptor subunits resulting in signal propagation via phosphorylation of receptor-associated JAK tyrosine kinases (JAK1, JAK2, JAK3 and Tyk2). Activated JAKs subsequently induce phosphorylation of other additional targets comprising both the receptors and STAT proteins. Phosphorylation in turn induces dimerization of STATs through conserved SH2 domain subsequently allowing their entry into the nucleus through importin α-5 and the Ran nuclear import pathway. In the nucleus, STATs bind to specific sequences in the DNA to stimulate or suppress transcription of target genes. The DNA repair gene BRAC1 can induce cell proliferation and inhibit apoptotic cell death in PCa cells through interaction with JAK1/2 and STAT3 phosphorylation. In addition, activation of STAT3 in PCa cells also stimulates various other genes that are associated with cell cycle progression, anti-apoptosis, angiogenesis and tumor invasion. STAT5a/b dimerization and ensuing nuclear translocation where the dimers binding to specific response elements of target genes promotes prostate cancer growth, tumor progression and distant metastases. Components of JAK-STAT pathway specifically pJAK-1 and pSTAT-3 function as predictors of biochemical relapse and poor prognosis of PCa.

1.5 Wnt signaling pathway

Wnt/β-catenin pathway is a highly conserved developmental signaling pathway comprising of secreted glycoproteins that play a vital role in tissue homeostasis, cell proliferation, differentiation, migration, and epithelial- mesenchymal communications, polarity and asymmetric cell division. Based on the ability to stabilize the multifunction protein β-catenin, Wnt signaling is β-catenin predominantly exists in the cytosol in complex with APC, Axin, CK1, and GSK-3β. However, free β-catenin accumulates in the perinuclear region and ultimately interact with lymphoid enhancer factor/T cell factor (LEF/TCF) in the DNA to stimulate transcription of various target genes including that of c-Myc, p300, Foxo, Bcl9-2, c-Jun, CtBP, and cyclin D1. Increased expression of β-catenin occurs quite commonly in PCa and is associated with growth, proliferation and metastasis of prostate cancer cells.

2 Prostate cancer diagnosis

Since the introduction of serum prostate-specific antigen (PSA) screening of asymptomatic populations, prostate cancer incidence rates have increased dramatically, as has the number of men undergoing radical prostatectomy and radiation therapy for this disease. However, false positives for PSA continue to be a significant problem resulting in unnecessary biopsies, and the value of broad-based PSA testing regarding predicting surgical cures has recently come into question. Currently, there are no markers that differentiate clinically relevant from clinically benign disease. Better indicators of prostate cancer presence and progression are needed to avoid unnecessary treatment, predict disease course, and develop more effective therapy. A variety of putative prostate cancer markers have been described in human serum, urine, seminal fluid, and histological specimens. These markers exhibit varying capacities to detect prostate cancer and to predict disease course.

2.1 Molecular Markers for Prostate cancer

Current advancements in proteomics, tissue microarray, DNA microarray, immunohistochemical staining, and other biotechnologies have paved the way and have significantly increased the pace at which novel biomarkers are being discovered and developed. The genetic landscape of prostate cancer was intensely explored in the last few years with NGS, whole genome expression analyses and analyses of epigenetic alterations. There is a relatively low rate of mutations in PCa compared to other tumors and prevalence of non-random copy number variations (CNV) in most PCa tumors involving well-known prostate oncogenes or tumor suppressors. Consequently, several published studies have supported the usefulness of the prostate cancer antigen 3 (PCA3 or DD3) gene as a biomarker in the diagnosis of prostate cancer stage and grading. Hypermethylation of the PDLIM4 gene has been shown to be a sensitive molecular tool in detecting prostate tumorigenesis and can be used as a biomarker to predict the biochemical, local, and systemic recurrence of prostate cancer. Patients with expression of ERG in high-grade prostatic intraepithelial neoplasia are more likely to develop prostate cancer. Homozygous loss of PTEN is causative in progression to aggressive metastatic phenotype and castration resistance and there is a strong oncogenic interaction between high levels of ERG and PTEN loss. Meanwhile, MAGI2/3 mutations and PIK3CA mutations are enriched in tumors positive. NKX3.1 is frequently mutated or lost in localized PCa, MED12 is mutated in 5% of prostate cancer, classical tumor suppressors TP53 inactivates, as well as CDKN1B (p27/KIP) and RB1 occur in primary PCa and castrate resistant disease (CRPC). miRNAs are expressed in a tissue- and function-specific manner and are protected from nuclease degradation in the bloodstream. This makes them new candidate biomarkers for detecting cancers. MiR-141 has an elevated level in the blood of patients having metastatic prostate cancer. In addition, there are several differentially expressed microRNAs (miR-200c, miR-21, miR-210, miR-205, miR-20a, miR-143∗, miR-143, and miR-16) that can be used as biomarkers.

2.2 Protein Markers for Prostate cancer

Serum prostate-specific antigen PSA, discovered in 1971, is considered the most important biomarker for detecting, staging, and monitoring cancer of the prostate in its early stage. PSA testing was initially used for monitoring prostate cancer patients and became extensively used for screening and diagnosing the disease. The main advantage of PSA testing is its superior sensitivity while its main disadvantage is that it is not very specific. In an effort to find ways of improving specificity, several variations on the basic PSA test have been proposed. Of these the prostate health index PHI, which is based on a molecular isoform of free PSA, is the most developed and has been shown to have greater specificity than use of total PSA or % free PSA. Adding the Kallikrein protein hK2 to PSA based markers has also been shown to improve the specificity of PSA based assays.

Human prostatic acid phosphatase (PAP) (or serum acid phosphatase (AP) was reportedly the first serum biomarker for prostate cancer. Although AP, with an elevated level in more than 70% of patients, was linked early with prostate cancer that had spread, both AP and prostate-specific AP (PAP, its subtype) are not sensitive enough for screening. Ki-67, a cell-proliferation associated marker, has been described as one of the most promising biomarkers of prostate cancer. Ki-67 has been suggested as a prolific predictive biomarker for men who have low-grade, low-stage prostate cancer for their PSA relapse after radical prostatectomy. Immunodetectable serum autoantibodies generated in response to the AMACR tumor-associated antigen may also be useful in preliminary diagnosis, especially if combined with PSA screening. Glutathione S-transferase π is an example of a biomarker that has been extensively studied in prostate cancer, primarily as a tissue marker. Past studies suggest GRN-A may be very useful as a prognostic factor in patients with advanced prostate cancer. PSMA has been well characterized as a diagnostic and prognostic marker, but it has limited prostate specificity. In addition, other proteins, prostate stem cell antigen (PSCA), early prostate cancer (EPCA), B7-H3, caveolin-1 (Cav-1), etc., could be the powerful candidate biomarkers for diagnosing and monitoring the status of prostate cancer.

Table 1 Candidate molecular and protein markers in prostate cancer

3 Targeted Therapy for Prostate Cancer

The molecular pathogenesis of prostate cancer is very complex, its occurrence, development and metastasis are closely related to the abnormality of various gene mutations and cell signaling pathways which provide a number of potential key targets for the treatment of prostate cancer. Major components of cell signaling pathways, such as the receptor tyrosine kinases (RTKs), Ras/Raf/mitogen-activated protein kinase cascade, AR pathway proteins and other major signaling cascades. In Table 2-14, selected clinical trials of novel therapeutic targets for the treatment of prostate cancer are presented.

3.1 Prostate cancer therapy for AR mediated signaling pathway

In depth understanding of AR signaling and the mechanisms of castration resistance in PCa has resulted in the development of novel agents that can more efficiently retract AR signaling. Abiraterone acetate (Zytiga) is a selective, oral agent that can irreversibly inhibit the biosynthesis of testosterone. ketoconazole, a broad-spectrum antifungal agent, has been extensively used as testosterone inhibitor and second-line hormonal therapy for PCa. MDV3100 (Enzalutamide) is an inhibitor of the C-terminus ligand-binding domain with multiple effects on AR and it also prevents AR nuclear translocation, AR binding to DNA, and co-activator recruitment. Galeterone (VN/124-1, TOK-001) developed by Tokai Pharmaceuticals, which acts by disrupting the androgen receptor signaling pathway, is a 17- heteroazole steroidal analogue for men with metastatic, castration-resistant PCa or CRPC, whose prostate tumor cells express the AR-V7 splice variant.

Table 2 Clinical trials of testosterone inhibitor Abiraterone acetate

According to statistics, a total of 102 Abiraterone acetate projects targeting prostate cancer testosterone are currently in clinical stage, of which 55 are recruiting, 46 are not recruiting and 1 is Enrolling by invitation.

Table 3 Clinical trials of testosterone inhibitor Ketoconazole

Table 4 Clinical trials of AR inhibitor Enzalutamide

According to statistics, a total of 148 Abiraterone acetate projects targeting prostate cancer testosterone are currently in clinical stage, of which 77 are recruiting, 69 are not recruiting and 2 is Enrolling by invitation.

3.2 Prostate cancer therapy for NF-κB signaling pathway

Preclinical studies in prostate cancer xenografts have firmly established that blockade of receptor activator of NF-κB (RANK) signal instigated by binding of RANK ligands (RANK-L) impairs the establishment and progression of bone metastasis. Denosumab, a fully human monoclonal antibody, binds to RANK-L and directly inhibits osteoclasts to abrogate bone reabsorption. BMS-345541 selectively inhibits the catalytic subunit of IKK and can suppress tumor growth. The small molecule inhibitor, bindarit, an indazolic derivative, which may down-regulate NF-κB through reduced phosphorylation of IκBα and p65. The NF-κB specific inhibitor DHMEQ, prevents the nuclear translocation of the transcription factor, and has been shown to have anti-cancer effects in numerous different cancer subtypes. Amlexanox effectively represses PCa cell migration and tumor metastasis in vitro and in vivo by inhibition of the NF-κB signal pathway through specifically targeting IKKɛ and TBK1.

Table 5 Clinical trials of RANK-L mAb Ketoconazole

3.3 Prostate cancer therapy for RTK signaling pathway

Several inhibitors of RTK signaling are also being actively investigated either as single agents or in combination for PCa treatment and therapy. Many drugs that inhibit VEGF signaling have been tested in prostate cancer. The most well-known is bevacizumab, a humanized monoclonal antibody to VEGF. There are other monoclonal antibodies and small molecule inhibitors that target VEGF signaling, including aflibercept, sunitinib, sorafenib and cediranib (AZD2171). The c-Met receptor tyrosine kinase has received considerable attention as a potential therapeutic target for many solid tumors, including prostate cancer. Rilotumumab, cabozantinib (XL184), tivantinib (ARQ197) are promising c-Met inhibitors or monoclonal antibodies in clinical development for the treatment of prostate cancer. Studies of the mTOR inhibitors rapamycin, everolimus, and temsirolimus as single agents and in combination with the androgen receptor antagonist bicalutamide are in progress. Monoclonal antibodies specific to the IGF-1R and small molecules that aim to inhibit its tyrosine kinase activity have been developed, such as cixutumumab (IMC-A12) and linsitinib (fOSI-906). In prostate cancer, Src appears to be involved in the transition to the castration-resistant phenotype. Dasatinib and saracatinib, the Src inhibitors, suppress growth of prostate cancer in cell lines.

Table 6 Clinical trials of VEGF mAb Bevacizumab

Table 7 Clinical trials of VEGFR inhibitor Sunitinib

Table 8 Clinical trials of VEGFR inhibitor Cediranib

Table 9 Clinical trials of c-Met inhibitor Cabozantinib

Table 10 Clinical trials of mTOR inhibitor Rapamycin

Table 11 Clinical trials of mTOR inhibitor Everolimus

Table 12 Clinical trials of mTOR inhibitor Temsirolimus

Table 13 Clinical trials of Src inhibitor Dasatinib

3.4 Prostate cancer therapy for JAK/STAT signaling pathway

The evidence that the JAK/STAT pathway is activated in a large proportion of solid tumors and that its activation contributes to the malignant properties of cancer cells makes the JAK/STAT pathway a promising target for the development of new therapies. The JAK1/2 inhibitor ruxolitinib improves symptoms and prolongs survival. Blockade of activated STAT3, by either siltuximab (CNTO 328) or LLL12, a specific pSTAT3 inhibitor, suppresses the clonogenicity of the stem-like cells in patients with high-grade disease.

Table 14 Clinical trials of JAK1/2 inhibitor Ruxolitinib

3.5 Prostate cancer therapy for Wnt signaling pathway

Activation of the Wnt pathway also contributes to the tumorigenicity of cancer stem cells (CSCs). Therefore, inhibiting this pathway has been a recent focus for cancer research with multiple targetable candidates in development. OMP-54F28 acts as a decoy receptor by sequestering WNT ligands and selectively reduces cancer stem cells. IWR-1, an inhibitor of tankyrase, can stabilize Axin leading to enhancement of β-catenin destruction. 3289-8625 has a binding affinity to DVL and it may be useful for blocking the Wnt signaling pathway at the Fz-Dvl interaction point.