A note on trader Sharpe Ratios

Traders in the financial world are assessed by the amount of money they make and, increasingly, by the amount of money they make per unit of risk taken, a measure known as the Sharpe Ratio. Little is known about the average Sharpe Ratio among traders, but the Efficient Market Hypothesis suggests that traders, like asset managers, should not outperform the broad market. Here we report the findings of a study conducted in the City of London which shows that a population of experienced traders attain Sharpe Ratios significantly higher than the broad market. To explain this anomaly we examine a surrogate marker of prenatal androgen exposure, the second-to-fourth finger length ratio (2D:4D), which has previously been identified as predicting a trader's long term profitability. We find that it predicts the amount of risk taken by traders but not their Sharpe Ratios. We do, however, find that the traders' Sharpe Ratios increase markedly with the number of years they have traded, a result suggesting that learning plays a role in increasing the returns of traders. Our findings present anomalous data for the Efficient Markets Hypothesis.

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 expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

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.

Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

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.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. © 2012 Sieh et al.

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.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) slows cancer development but increases metastases in TRAMP prostate cancer prone mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1fms and syngeneic C57BL/6 mice. Whilst TRAMPfmsmic-1 survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.

Paradoxical roles of tumour necrosis factor-alpha in prostate cancer biology

Tumour necrosis factor (TNF) is a pleiotropic cytokine with dual roles in cancer biology including prostate cancer (PCa). On the one hand, there is evidence that it stimulates tumour angiogenesis, is involved in the initiation of PCa from an androgen-dependent to a castrate resistant state, plays a role in epithelial to mesenchymal plasiticity, and may contribute to the aberrant regulation of eicosanoid pathways. On the other hand, TNF has also been reported to inhibit neovascularisation, induce apoptosis of PCa cells, and stimulate anti-tumour immunity. Much of the confusion surrounding its seemingly paradoxical roles in cancer biology stems from the dependence of its effects on the biological model within which TNF is investigated. This review will address some of these issues, and also discuss on the therapeutic implications.

IGF2 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.