Intratumoral androgen biosynthesis in prostate cancer: Evidence from preclinical models and clinical specimens

Androgens regulate the growth and development of normal prostate and prostate cancer. Blocking the production and effects of androgens, either by castration or medication, has been the most efficient strategy for treating metastatic prostate cancer for decades. Although most patients respond to the therapy, in many of them the disease progresses to castration-resistant prostate cancer (CRPC) that cannot be cured with current therapies. Intratumoral androgen biosynthesis has been identified as one of the mechanisms leading to castration resistance. Recent studies have confirmed that prostate tumors can synthesize androgens by themselves to maintain tumor growth in androgen-deprived conditions. Thus, suppressing intratumoral androgen biosynthesis has become an attractive option for drug development. To understand the mechanisms of intratumoral androgen biosynthesis and develop new CRPC therapies, better preclinical models for CRPC are needed. In this study, we developed an orthotopic VCaP xenograft model suitable for studying the progression of CRPC and the mechanisms of intratumoral androgen biosynthesis in vivo. The VCaP model exhibited the clinical features of CRPC, including the activation of intratumoral androgen biosynthesis and the overexpression of androgen receptor (AR) and its splice variants. Furthermore, novel antiandrogens enzalutamide and ARN-509 reduced intratumoral androgen levels and altered steroidogenic enzyme expression in the VCaP model. In addition to preclinical studies, androgen levels were analyzed in prostate and serum specimens obtained from prostate cancer patients. Intraprostatic androgen levels in cancerous and benign samples were highly variable between the patients. Distinct intratumoral androgen levels and altered AR target gene expression were associated with TMPRSS2-ERG fusion gene expression

Applying mass spectrometric methods to study androgen biosynthesis and metabolism in prostate cancer

Recent development of gas chromatography and liquid chromatography-tandem mass spectrometry (GC-MS/MS, LC-MS/MS) has provided novel tools to define sex steroid concentrations. These new methods overcome several of the problems associated with immunoassays for sex steroids. With the novel MS-based applications we are now able to measure small concentrations of the steroid hormones reliably and with high accuracy in both body fluids and tissue homogenates. The sensitivity of the tandem mass spectrometry assays allows us also for the first time to reliably measure picomolar or even femtomolar concentrations of estrogens and androgens. Furthermore, due to a high sensitivity and specificity of MS technology, we are also able to measure low concentrations of steroid hormones of interest in the presence of pharmacological concentration of other steroids and structurally closely related compounds. Both of these features are essential for multiple preclinical models for prostate cancer. The MS assays are also valuable for the simultaneous measurement of multiple steroids and their metabolites in small sample volumes in serum and tissue biopsies of prostate cancer patients before and after drug interventions. As a result, novel information about steroid hormone synthesis and metabolic pathways in prostate cancer has been obtained. In our recent studies, we have extensively applied a GC-MS/MS method to study androgen biosynthesis and metabolism in VCaP prostate cancer xenografts in mice. In the present review, we shortly summarize some of the benefits of the GC-MS/MS and novel LC-MS/MS assays, and provide examples of their use in defining novel mechanisms of androgen action in prostate cancer.Peer reviewe

A Comprehensive Panel of Three-Dimensional Models for Studies of Prostate Cancer Growth, Invasion and Drug Responses

Peer reviewe

High intratumoral dihydrotestosterone is associated with antiandrogen resistance in VCaP prostate cancer xenografts in castrated mice

Antiandrogen treatment resistance is a major clinical concern in castration-resistant prostate cancer (CRPC) treatment. Using xenografts of VCaP cells we showed that growth of antiandrogen resistant CRPC tumors were characterized by a higher intratumor dihydrotestosterone (DHT) concentration than that of treatment responsive tumors. Furthermore, the slow tumor growth after adrenalectomy was associated with a low intratumor DHT concentration. Reactivation of androgen signaling in enzalutamide-resistant tumors was further shown by the expression of several androgen-dependent genes. The data indicate that intratumor DHT concentration and expression of several androgen-dependent genes in CRPC lesions is an indication of enzalutamide treatment resistance and an indication of the need for further androgen blockade. The presence of an androgen synthesis, independent of CYP17A1 activity, has been shown to exist in prostate cancer cells, and thus, novel androgen synthesis inhibitors are needed for the treatment of enzalutamide-resistant CRPC tumors that do not respond to abiraterone.Peer reviewe

The variant rs77559646 associated with aggressive prostate cancer disrupts ANO7 mRNA splicing and protein expression

Prostate cancer is among the most common cancers in men, with a large fraction of the individual risk attributable to heritable factors. A majority of the diagnosed cases does not lead to a lethal disease, and hence biological markers that can distinguish between indolent and fatal forms of the disease are of great importance for guiding treatment decisions. Although over 300 genetic variants are known to be associated with prostate cancer risk, few have been associated with the risk of an aggressive disease. One such variant is rs77559646 located in ANO7. This variant has a dual function. It constitutes a missense mutation in the short isoform of ANO7 and a splice region mutation in full-length ANO7. In this study, we have analyzed the impact of the variant allele of rs77559646 on ANO7 mRNA splicing using a minigene splicing assay and by performing splicing analysis with the tools IRFinder (intron retention finder), rMATS (replicate multivariate analysis of transcript splicing) and LeafCutter on RNA sequencing data from prostate tissue of six rs77559646 variant allele carriers and 43 non-carriers. The results revealed a severe disruption of ANO7 mRNA splicing in rs77559646 variant allele carriers. Immunohistochemical analysis of prostate samples from patients homozygous for the rs77559646 variant allele demonstrated a loss of apically localized ANO7 protein. Our study is the first to provide a mechanistic explanation for the impact of a prostate cancer risk SNP on ANO7 protein production. Furthermore, the rs77559646 variant is the first known germline loss-of-function mutation described for ANO7. We suggest that loss of ANO7 contributes to prostate cancer progression.</p

Identification of an H-Ras nanocluster disrupting peptide

AbstractThe Ras-MAPK pathway is critical to regulate cell proliferation and differentiation. Its dysregulation is implicated in the onset and progression of numerous types of cancers. To be active, Ras proteins are membrane anchored and organized into nanoclusters, which realize high-fidelity signal transmission across the plasma membrane. Nanoclusters therefore represent potential drug targets. However, targetable protein components of signalling nanoclusters are poorly established.We previously proposed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering by stabilizing stacked dimers of H-Ras and Raf via a direct interaction of dimeric Gal1 with the Ras binding domain (RBD) in particular of B-Raf. Here, we provide further supportive evidence for this model. We establish that the B-Raf preference emerges from divergent regions of the Raf RBDs that were proposed to interact with Gal1. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B-Raf-RBD. Its 23-mer core fragment is thus sufficient to interfere with Gal1-enhanced H-Ras nanocluster, reduce MAPK-output and cell viability inHRAS-mutant cancer cell lines.Our data therefore suggest that the interface between Gal1 and the RBD of B-Raf can be targeted to disrupt Gal1-enhanced H-Ras nanoclustering. Collectively, our results support that Raf-proteins are integral components of active Ras nanoclusters

Optimized design and analysis of preclinical intervention studies in vivo

Recent reports have called into question the reproducibility, validity and translatability of the preclinical animal studies due to limitations in their experimental design and statistical analysis. To this end, we implemented a matching-based modelling approach for optimal intervention group allocation, randomization and power calculations, which takes full account of the complex animal characteristics at baseline prior to interventions. In prostate cancer xenograft studies, the method effectively normalized the confounding baseline variability, and resulted in animal allocations which were supported by RNA-seq profiling of the individual tumours. The matching information increased the statistical power to detect true treatment effects at smaller sample sizes in two castration-resistant prostate cancer models, thereby leading to saving of both animal lives and research costs. The novel modelling approach and its open-source and web-based software implementations enable the researchers to conduct adequately-powered and fully-blinded preclinical intervention studies, with the aim to accelerate the discovery of new therapeutic interventions.Peer reviewe

Adrenals Contribute to Growth of Castration-Resistant VCaP Prostate Cancer Xenografts

The role of adrenal androgens as drivers for castration-resistant prostate cancer (CRPC) growth in humans is generally accepted; however, the value of preclinical mouse models of CRPC is debatable, because mouse adrenals do not produce steroids activating the androgen receptor. In this study, we confirmed the expression of enzymes essential for de novo synthesis of androgens in mouse adrenals, with high intratissue concentration of progesterone (P-4) and moderate levels of androgens, such as androstenedione, testosterone, and dihydrotestosterone, in the adrenal glands of both intact and orchectomized (ORX) mice. ORX alone had no effect on serum P-4 concentration, whereas orchectomized and adrenalectomized (ORX + ADX) resulted in a significant decrease in serum P-4 and in a further reduction in the Low levels of serum androgens (androstenedione, testosterone, and dihydrotestosterone), measured by mass spectrometry. In line with this, the serum prostate-specific antigen and growth of VCaP xenografts in mice after ORX + ADX were markedly reduced compared with ORX alone, and the growth difference was not abolished by a glucocorticoid treatment. Moreover, ORX + ADX altered the androgen-dependent gene expression in the tumors, similar to that recently shown for the enzalutamide treatment. These data indicate that in contrast to the current view, and similar to humans, mouse adrenals synthesize significant amounts of steroids that contribute to the androgen receptor dependent growth of CRPC.Peer reviewe

Marketing communications plan for Mobilat

The purpose of this thesis is to find possible media for communicating benefits of Mobilat to young consumers and to analyse consumer behaviour differences between younger and older consumers regarding pain gel usage. The main beneficiary of this thesis is STADA Nordic, which is a part of STADA Arzneimittel, an international pharmaceutical company. STADA Nordic tasked this thesis to find best alternatives for advertising their pain gel product Mobilat to young consumers. According to STADA Nordic most of its customers using Mobilat are older consumers and in order to keep the product profitable, the age variation within this customer base must be diversified. The knowledge base of this thesis consists of three separate parts: communication theory, communications in marketing and marketing communication planning. Communication theory defines the term communication and explores most significant models in the field. Communication in marketing lists the common media of communication used for marketing purposes and advertising. Marketing communication planning -section details SOSTAC® -process model and other theory related to planning effective communications. For collecting data two methods are used. Survey is conducted to receive data of consumer behaviour of Finnish consumers’ pain medication usage. The sample consists of personnel and students of Laurea University of Applied Sciences. The data is analysed and conclusions of the results of the survey are made. The second method of data collection is studying possible marketing channels and briefly analysing their suitability for STADA’s purposes. This thesis’s survey provides information of consumer preference, when self-medicating joint and muscle pains. Analysis of marketing channels provides details such as key demographics of those channels. Both, the results of the survey and the analysis of channels, are brought together and concluded in the last chapter

Exchange Students’ Health Security in Africa

Exchange programs have been popular among university students already for over 30 years. Through Erasmus+ program, over 90 000 exchange students were sent from Finland to all around the world. The Erasmus+ program supports cooperation between different uni-versities for students, teachers, and other staff mobilities. Nursing students have a lot of pos-itive experiences about their exchange periods for example from cultural, language and so-cial skills perspective. Benefits of the exchange periods are considered important as in per-sonal growth, context-sensitivity, and citizenship. Employers also give value to a nursing student who has been abroad. This thesis was implemented as a functional thesis as a request to Health Africa - network. The aim of the thesis was to improve knowledge of health security during the exchange study period for social- and health field students. The purpose of the thesis was to create a digital databank for the training of social- and health field students. The research question that the thesis addressed was: What aspects should social- and health field students con-sider before an exchange period? Inductive content analysis was the chosen research method. A total of 12 students participated in the Webropol survey with open questions. The data analysis resulted in the formulation of three upper category classes in total, which were physical health security, psychological health security, and social health security. These categories were used as topics when Moodle platform was created for future ex-change students. It is important to have needed information on the same platform and easily accessible. Furthermore, education is an important factor, especially from the point of view of health promotion and safety. This thesis provides help for future exchange students by offering updated information from the perspective of health security