The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

The role of insulin in advanced prostate cancer

Advanced prostate cancer is a common and generally incurable disease. Androgen deprivation therapy is used to treat advanced prostate cancer with good benefits to quality of life and regression of disease. Prostate cancer invariably progresses however despite ongoing treatment, to a castrate resistant state. Androgen deprivation is associated with a form of metabolic syndrome, which includes insulin resistance and hyperinsulinaemia. The mitogenic and anti-apoptotic properties of insulin acting through the insulin and hybrid insulin/IGF-1 receptors seem to have positive effects on prostate tumour growth. This pilot study was designed to assess any correlation between elevated insulin levels and progression to castrate resistant prostate cancer. Methods: 36 men receiving ADT for advanced prostate cancer were recruited, at various stages of their treatment, along with 47 controls, men with localised prostate cancer pre-treatment. Serum measurements of C-peptide (used as a surrogate marker for insulin production) were performed and compared between groups. Correlation between serum C-peptide level and time to progression to castrate resistant disease was assessed. Results: There was a significant elevation of C-peptide levels in the ADT group (mean = 1639pmol/L)) compared to the control group (mean = 1169pmol/L), with a p-value of 0.025. In 17 men with good initial response to androgen deprivation, a small negative trend towards earlier progression to castrate resistance with increasing C-peptide level was seen in the ADT group (r = -0.050), however this did not reach statistical significance (p>0.1). Conclusions: This pilot study confirms an increase in serum C-peptide levels in men receiving ADT for advance prostate cancer. A non-significant, but negative trend towards earlier progression to castrate resistance with increasing C-peptide suggests the need for a formal prospective study assessing this hypothesis.

Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine : involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

Neuronal nicotinic acetylcholine receptors : neuroplastic changes underlying alcohol and nicotine addictions

Addictive drugs can activate systems involved in normal reward-related learning, creating long-lasting memories of the drug's reinforcing effects and the environmental cues surrounding the experience. These memories significantly contribute to the maintenance of compulsive drug use as well as cue-induced relapse which can occur even after long periods of abstinence. Synaptic plasticity is thought to be a prominent molecular mechanism underlying drug-induced learning and memories. Ethanol and nicotine are both widely abused drugs that share a common molecular target in the brain, the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are ligand-gated ion channels that are vastly distributed throughout the brain and play a key role in synaptic neurotransmission. In this review, we will delineate the role of nAChRs in the development of ethanol and nicotine addiction. We will characterize both ethanol and nicotine's effects on nAChR-mediated synaptic transmission and plasticity in several key brain areas that are important for addiction. Finally, we will discuss some of the behavioral outcomes of drug-induced synaptic plasticity in animal models. An understanding of the molecular and cellular changes that occur following administration of ethanol and nicotine will lead to better therapeutic strategies.

Targeting aurora kinases : a novel approach to curb the growth and chemoresistance of androgen refractory prostate cancer

Aurora Kinase (AK) based therapy targeting AK-A & B is effective against some cancers. We have explored its potential against previously unreported incurable, metastatic androgen depletion independent Prostate Cancer (ADIPC). We used androgen sensitive (AS) and ADI lines derived from Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. The relevance of this model was unequivocally established through focussed array, quantitative PCR and western blotting studies; significantly greater alteration of genes (fold change and number) representing major cancer pathways was shown in ADI cells compared to AS lines. A marked enhancement of in vivo growth of the ADI subline showing the greatest degree of gene modulations [TRAMP C1 (TC1)-T5: TC1-T5] reflected this. In contrast to the parental AS TC1 line, TC1-T5 cells grew with 100% incidence in the prostate, as lung pseudometastases and migrated to the bone and other soft tissues. The potential involvement of AKs in this transition was indicated by the significant upregulation of AK-A/B and their downstream regulators, survivin and phosphorylated-histone H3 in TC1-T5 cells compared to TC1 cells. This led to enhanced sensitivity of TC1-T5 cells to the pan-AK inhibitor, VX680 and to significant reduction in in vivo tumour growth rates when AK-A and/or B were downregulated in TC1-T5 cells. This cell growth inhibition was markedly enhanced when both AKs were downregulated and also led to substantially greater sensitivity of these cells to docetaxel, the only chemotherapeutic with activity against ADI PC. Finally, use of VX680 with docetaxel led to impressive synergies suggesting promise for treating clinical ADI metastatic PC.

Kallikrein 14 is down-regulated by androgen receptor signalling and harbours genetic variation that is associated with prostate tumour aggressiveness

Kallikrein 14 (KLK14) has been proposed as a useful prognostic marker in prostate cancer, with expression reported to be associated with tumour characteristics such as higher stage and Gleason score. KLK14 tumour expression has also shown the potential to predict prostate cancer patients at risk of disease recurrence after radical prostatectomy. The KLKs are a remarkably hormone-responsive family of genes, although detailed studies of androgen regulation of KLK14 in prostate cancer have not been undertaken to date. Using in vitro studies, we have demonstrated that unlike many other prostatic KLK genes that are strictly androgen responsive, KLK14 is more broadly expressed and inversely androgen regulated in prostate cancer cells. Given these results and evidence that KLK14 may play a role in prostate cancer prognosis, we also investigated whether common genetic variants in the KLK14 locus are associated with risk and/or aggressiveness of prostate cancer in approximately 1200 prostate cancer cases and 1300 male controls. Of 41 single nucleotide polymorphisms assessed, three were associated with higher Gleason score (≥7): rs17728459 and rs4802765, both located upstream of KLK14, and rs35287116, which encodes a p.Gln33Arg substitution in the KLK14 signal peptide region. Our findings provide further support for KLK14 as a marker of prognosis in prostate cancer.

Identification of GABA receptors in chick cornea

Purpose: The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods: Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results: Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions: These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question.

Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.