Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.

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.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

The effect of oral metformin on insulin sensitivity in insulin-resistant ponies

Metformin may be an effective therapeutic option for insulin-resistant (I-R) horses/ponies because, in humans, it reportedly enhances insulin sensitivity (SI) of peripheral tissues without stimulating insulin secretion. To determine the effect of metformin on insulin and glucose dynamics in I-R ponies, six ponies were studied in a cross-over design by Minimal Model analysis of a frequently-sampled intravenous glucose tolerance test (FSIGT). Metformin was administered at 15. mg/kg bodyweight (BW), orally, twice-daily, for 21. days to the metformin-treated group. The control group received a placebo. A FSIGT was conducted before and after treatment. The Minimal Model of glucose and insulin dynamics rendered indices describing SI, glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and the disposition index (DI). The body condition score (BCS), BW and cresty neck score (CNS) were also assessed. There was no significant change in SI, Sg, AIRg, DI, BW, BCS or CNS in response to metformin, or over time in the control group. There were no measurable benefits of metformin on SI, consistent with recent work showing that the bioavailability of metformin in horses is poor, and chronic dosing may not achieve therapeutic blood concentrations. Alternatively, metformin may only be effective in obese ponies losing weight or with hyperglycaemia.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n = 4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n = 4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and 48 h (n = 4), and in normal control horses (n = 4). The only change in gene expression observed was an upregulation of MMP-9 (p < 0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p < 0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Insulin increases De Novo Steroidogenesis in prostate cancer cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

The interactions between insulin and androgens in progression to castrate resistant prostate cancer

An association between the metabolic syndrome and reduced testosterone levels has been identified, and a specific inverse relationship between insulin and testosterone levels suggests that an important metabolic crosstalk exists between these two hormonal axes; however, the mechanisms by which insulin and androgens may be reciprocally regulated are not well described. Androgen-dependant gene pathways regulate the growth and maintenance of both normal and malignant prostate tissue, and androgen-deprivation therapy (ADT) in patients exploits this dependence when used to treat recurrent and metastatic prostate cancer resulting in tumour regression. A major systemic side effect of ADT includes induction of key features of the metabolic syndrome and the consistent feature of hyperinsulinaemia. Recent studies have specifically identified a correlation between elevated insulin and high-grade PCa and more rapid progression to castrate resistant disease. This paper examines the relationship between insulin and androgens in the context of prostate cancer progression. Prostate cancer patients present a promising cohort for the exploration of insulin stabilising agents as adjunct treatments for hormone deprivation or enhancers of chemosensitivity for treatment of advanced prostate cancer.

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

Ghrelin is a multifunctional hormone, with roles in stimulating appetite and regulating energy balance, insulin secretion and glucose homeostasis. The ghrelin gene locus (GHRL) is highly complex and gives rise to a range of novel transcripts derived from alternative first exons and internally spliced exons. The wild-type transcript encodes a 117 amino acid preprohormone that is processed to yield the 28 amino acid peptide ghrelin. Here, we identified insulin-responsive transcription corresponding to cryptic exons in intron 2 of the human ghrelin gene. A transcript, termed in2c-ghrelin (intron 2-cryptic), was cloned from the testis and the LNCaP prostate cancer cell line. This transcript may encode an 83 AA preproghrelin isoform that codes for the ghrelin, but not obestatin. It is expressed in a limited number of normal tissues and in tumours of the prostate, testis, breast and ovary. Finally, we confirmed that in2c-ghrelin transcript expression, as well as the recently described in1-ghrelin transcript, is significantly upregulated by insulin in cultured prostate cancer cells. Metabolic syndrome and hyperinsulinaemia has been associated with prostate cancer risk and progression. This may be particularly significant after androgen deprivation therapy for prostate cancer, which induces hyperinsulinaemia, and this could contribute to castrate resistant prostate cancer growth. We have previously demonstrated that ghrelin stimulates prostate cancer cell line proliferation in vitro. This study is the first description of insulin regulation of a ghrelin transcript in cancer, and should provide further impetus for studies into the expression, regulation and function of ghrelin gene products.

Signaling of receptor tyrosine kinases in the nucleus

Since the discovery of the first receptor tyrosine kinase (RTK) proteins in the late 1970s and early 1980s, many scientists have explored the functions of these important cell signaling molecules. The finding that these proteins are often deregulated or mutated in diseases such as cancers and diabetes, together with their potential as clinical therapeutic targets, has further highlighted the necessity for understanding the signaling functions of these important proteins. The mechanisms of RTK regulation and function have been recently reviewed by Lemmon & Schlessinger (2010) but in this review we instead focus on the results of several recent studies that show receptor tyrosine kinases can function from subcellular localisations, including in particular the nucleus, in addition to their classical plasma membrane location. Nuclear localisation of receptor tyrosine kinases has been demonstrated to be important for normal cell function but is also believed to contribute to the pathogenesis of several human diseases.