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

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

Discovery and development of ODM-204: A Novel nonsteroidal compound for the treatment of castration-resistant prostate cancer by blocking the androgen receptor and inhibiting CYP17A1

We report the discovery of a novel nonsteroidal dual-action compound, ODM-204, that holds promise for treating patients with castration-resistant prostate cancer (CRPC), an advanced form of prostate cancer characterised by high androgen receptor (AR) expression and persistent activation of the AR signaling axis by residual tissue androgens. For ODM-204, has a dual mechanism of action. The compound is anticipated to efficiently dampen androgenic stimuli in the body by inhibiting CYP17A1, the prerequisite enzyme for the formation of dihydrotestosterone (DHT) and testosterone (T), and by blocking AR with high affinity and specificity. In our study, ODM-204 inhibited the proliferation of androgen-dependent VCaP and LNCaP cells in vitro and reduced significantly tumour growth in a murine VCaP xenograft model in vivo. Intriguingly, after a single oral dose of 10-30 mg/kg, ODM-204 dose-dependently inhibited adrenal and testicular steroid production in sexually mature male cynomolgus monkeys. Similar results were obtained in human chorionic gonadotropin-treated male rats. In rats, leuprolide acetate-mediated (LHRH agonist) suppression of the circulating testosterone levels and decrease in weights of androgen-sensitive organs was significantly and dose-dependently potentiated by the co-administration of ODM-204. ODM-204 was well tolerated in both rodents and primates. Based on our data, ODM-204 could provide an effective therapeutic option for men with CRPC.</p

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

Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A.

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases

Androgen receptor therapy resistance in prostate cancer: From the disease model to the mechanisms

Androgens act through the androgen receptor (AR) and are critical regulators of prostate differentiation and function, as well as prostate cancer (PCa) growth and survival. The development of treatment resistance in castration-resistant prostate cancer (CRPC) is a major clinical concern in PCa. Although several mechanisms contribute to the treatment resistance, the AR remains a key factor in progression of the disease. In this study, VCaP xenograft bearing mice were surgically operated by performing orchiectomy (ORX) and/or adrenalectomy (ADX) and treated with antiandrogen to model the different hormonal therapies used. Until now, it has been assumed that, unlike human, the murine adrenal cortex does not produce androgens. Our data, however, clearly indicates a contribution of the mouse adrenal gland to the intratumoral steroids in the castration-resistant VCaP tumors and to their androgen dependent growth. This is significant as it demonstrates a similarity between rodents and humans and indicates that the data obtained from the mouse models likely translate to humans better than previously anticipated. Using VCaP tumor xenografts, we have demonstrated that the antiandrogen (enzalutamide) first stabilized tumor size, decreased serum PSA levels, and reduced intratumoral androgens, but after the treatment resistance, tumors continued to grow and intratumoral testosterone and DHT elevated back to the level observed in non-treated castration-resistant tumors. Interestingly, the tumor growth rate after ADX was slower in comparison with tumors with enzalutamide treatment and no up-regulation of DHT was observed. In summary, the data suggest that antiandrogen therapy resistance in the VCaP xenografts is associated with high intratumoral DHT production and active androgen action. Consequently, resistance to hormonal treatments is mediated by continuous activation of the AR signaling pathway caused by e.g., increased expression of the AR, AR splice variants and reactivation of tumor androgen synthesis, which together ultimately leads to DHT-mediated receptor activation.Androgeenireseptoriin liittyvä hoitoresistenssi eturauhassyövässä -Tutkimusmallista mekanismiin Eturauhasen kasvu ja toiminta ovat riippuvaista androgeeneista eli miessukupuolihormoneista, joita tuotetaan kivesten lisäksi pieniä määriä lisämunuaisissa. Androgeenit vaikuttavat kudoksissa androgeenireseptorin (AR) kautta ja ne ovat välttämättömiä elimistön normaaleille toiminnoille, mutta myös eturauhassyövän kasvulle. Edenneen eturauhassyövän ensisijainen hoitomuoto onkin jo vuosikymmenien ajan ollut kemiallinen tai kirurginen kastraatio, mutta ajan mittaan vaste hoidolle usein häviää ja syöpä muuttuu kastraatioresistentiksi eturauhassyöväksi. Resistenssin kehittyminen on merkittävä kliininen ongelma eturauhassyövän hoidossa. Vaikka sen taustalla on useita mekanismeja, AR ja androgeenit ovat edelleen tärkeitä tekijöitä taudin etenemisessä. Tässä tutkimuksessa hiiriin inokuloitiin ihmisen VCaP syöpäsoluja ja muodostuneita kasvaimia hoidettiin kivesten ja lisämunuaisten poistolla sekä antiandrogeenilla hormonaalisten hoitojen mallintamiseksi. Vastoin aikaisempaa yleistä olettamusta tutkimuksemme osoittaa, että hiiren lisämunuaisilla on vaikutusta kasvaimen androgeenipitoisuuksiin ja kasvuun. Havainto on tärkeä, sillä se osoittaa, että hiirimalleilla saadut tulokset ovat paremmin verrattavissa potilaiden tilaan. VCaP ksenografti-tutkimusmallilla olemme osoittaneet antiandrogeenin (entsalutamidin) hidastavan kasvaimen kasvua, vähentävän seerumin PSA pitoisuutta ja kasvaimen androgeeni pitoisuuksia väliaikaisesti, mutta hoitoresistenssin muodostuttua kasvaimen testosteroni sekä DHT pitoisuudet nousevat hoitoa edeltävälle tasolle ja kasvaimet jatkavat kasvuaan. Mielenkiintoista on, että kastraation ja lisämunuaisten poiston yhdistelmällä, vastaavaa androgeenien nousua ei kasvaimissa havaittu ja niiden kasvu oli hitaampaa. Yhteenvetona tulokset viittaavat siihen, että resistenssi hormonaalisille hoidoille välittyy jatkuvan AR signalointireitin kautta, joka AR-reseptorin muutosten sekä kasvaimen androgeenisynteesin aktivoitumisen vuoksi johtavat DHT-välitteiseen reseptoriaktivaatioon

Antiandrogens Reduce Intratumoral Androgen Concentrations and Induce Androgen Receptor Expression in Castration-Resistant Prostate Cancer xenografts

The development of castration-resistant prostate cancer (CRPC) is associated with the activation of intratumoral androgen biosynthesis and an increase in androgen receptor (AR) expression. We recently demonstrated that, similarly to the clinical CRPC, orthotopically grown castration-resistant VCaP (CR-VCaP) xenografts express high levels of AR and retain intratumoral androgen concentrations similar to tumors grown in intact mice. Herein, we show that antiandrogen treatment (enzalutamide or ARN-509) significantly reduced (10-fold, P <0.01) intratumoral testosterone and dihydrotestosterone concentrations in the CR-VCaP tumors, indicating that the reduction in intratumoral androgens is a novel mechanism by which antiandrogens mediate their effects in CRPC. Antiandrogen treatment also altered the expression of multiple enzymes potentially involved in steroid metabolism. Identical to clinical CRPC, the expression levels of the full-length AR (twofold, P <0.05) and the AR splice variants 1 (threefold, P <0.05) and 7 (threefold, P <0.01) were further increased in the antiandrogen-treated tumors. Nonsignificant effects were observed in the expression of certain classic androgen-regulated genes, such as TMPRSS2 and KLK3, despite the low levels of testosterone and dihydrotestosterone. However, other genes recently identified to be highly sensitive to androgen-regulated AR action, such as NOV and ST6GalNAc1, were markedly altered, which indicated reduced androgen action. Taken together, the data indicate that, besides blocking AR, antiandrogens modify androgen signaling in CR-VCaP xenografts at multiple levels.Peer reviewe

PP2A-based triple-strike therapy overcomes mitochondrial apoptosis resistance in brain cancer cells

Mitochondrial glycolysis and hyperactivity of the phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway are hallmarks of malignant brain tumors. However, kinase inhibitors targeting AKT (AKTi) or the glycolysis master regulator pyruvate dehydrogenase kinase (PDKi) have failed to provide clinical benefits for brain tumor patients. Here, we demonstrate that heterogeneous glioblastoma (GB) and medulloblastoma (MB) cell lines display only cytostatic responses to combined AKT and PDK targeting. Biochemically, the combined AKT and PDK inhibition resulted in the shutdown of both target pathways and priming to mitochondrial apoptosis but failed to induce apoptosis. In contrast, all tested brain tumor cell models were sensitive to a triplet therapy, in which AKT and PDK inhibition was combined with the pharmacological reactivation of protein phosphatase 2A (PP2A) by NZ-8-061 (also known as DT-061), DBK-1154, and DBK-1160. We also provide proof-of-principle evidence for in vivo efficacy in the intracranial GB and MB models by the brain-penetrant triplet therapy (AKTi + PDKi + PP2A reactivator). Mechanistically, PP2A reactivation converted the cytostatic AKTi + PDKi response to cytotoxic apoptosis, through PP2A-elicited shutdown of compensatory mitochondrial oxidative phosphorylation and by increased proton leakage. These results encourage the development of triple-strike strategies targeting mitochondrial metabolism to overcome therapy tolerance in brain tumors.Peer reviewe

Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.ISSN:2041-172