Mutant PIK3CA controls DUSP1-dependent ERK 1/2 activity to confer response to AKT target therapy.

BACKGROUND: Alterations in the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway are frequent in urothelial bladder cancer (BLCA) and thus provide a potential target for novel therapeutic strategies. We investigated the efficacy of the AKT inhibitor MK-2206 in BLCA and the molecular determinants that predict therapy response. METHODS: Biochemical and functional effects of the AKT inhibitor MK-2206 were analysed on a panel of 11 BLCA cell lines possessing different genetic alterations. Cell viability (CellTiter-Blue, cell counts), apoptosis (caspase 3/7 activity) and cell cycle progression (EdU incorporation) were analysed to determine effects on cell growth and proliferation. cDNA or siRNA transfections were used to manipulate the expression of specific proteins such as wild-type or mutant PIK3CA, DUSP1 or CREB. For in vivo analysis, the chicken chorioallantoic membrane model was utilised and tumours were characterised by weight and biochemically for the expression of Ki-67 and AKT phosphorylation. RESULTS: Treatment with MK-2206 suppressed AKT and S6K1 but not 4E-BP1 phosphorylation in all cell lines. Functionally, only cell lines bearing mutations in the hotspot helical domain of PIK3CA were sensitive to the drug, independent of other genetic alterations in the PI3K or MAPK signalling pathway. Following MK-2206 treatment, the presence of mutant PIK3CA resulted in an increase in DUSP1 expression that induced a decrease in ERK 1/2 phosphorylation. Manipulating the expression of mutant or wild-type PIK3CA or DUSP1 confirmed that this mechanism is responsible for the induction of apoptosis and the inhibition of tumour proliferation in vitro and in vivo, to sensitise cells to AKT target therapy.Conclusion or interpretation:PIK3CA mutations confer sensitivity to AKT target therapy in BLCA by regulating DUSP1 expression and subsequent ERK1/2 dephosphorylation and can potentially serve as a stratifying biomarker for treatment.

[The impact of the androgen receptor splice variant AR-V7 on the prognosis and treatment of advanced prostate cancer]. / Bedeutung der Androgenrezeptor-Spleißvariante AR-V7 für Prognose und Therapie des fortgeschrittenen Prostatakarzinoms.

A recently discovered mechanism enabling prostate cancer cells to escape the effects of endocrine therapies consists in the synthesis of C-terminally truncated, constitutively active androgen receptor (AR) splice variants (AR-V). Devoid of a functional C-terminal hormone/ligand binding domain, various AR-Vs are insensitive to therapies targeting the androgen/AR signalling axis. Preliminary studies suggest that AR-V7, the most common AR-V, is a promising predictive tumour marker and a relevant selection marker for the treatment of advanced prostate cancer. This review critically outlines recent advances in AR-V7 diagnostics and presents an overview of current AR-V7 targeted therapies.

[(11)C]choline PET/CT in prostate cancer patients with biochemical recurrence after radical prostatectomy.

OBJECTIVE: To evaluate [(11)C]choline positron emission tomography/computed tomography ([(11)C]choline PET/CT) for the detection of a biochemical recurrence of prostate cancer after radical prostatectomy. METHODS: Retrospective analysis of [(11)C]choline PET/CT performed in 41 consecutive prostate cancer patients with a rising PSA. The mean time to biochemical relapse was 24 months. PSA levels were determined at time of examination, and patients received either a targeted biopsy or surgery. Histopathology reports served as reference for the evaluation of the [(11)C]choline PET/CT findings. RESULTS: Mean PSA in [(11)C]choline PET/CT positive patients was 3.1 ng/ml (median 2.2 ng/ml, range 0.5-11.6 ng/ml) and 0.86 ng/ml in [(11)C]choline PET/CT negative patients (median 0.83 ng/ml, range 0.41-1.40 ng/ml). Six of 12 patients with PSA < 1.5 ng/ml [(11)C]choline PET/CT revealed a pathological uptake. Histopathology was positive in 6/12 patients in this group. At PSA levels ranging from 1.5 to 2.5 ng/ml all [(11)C]choline PET/CT were positive (n = 16), a positive histology was found in 12/16 patients (75%) and at PSA 2.5-5 ng/ml [(11)C]choline PET/CT was positive in 8/8 patients, confirmed by histology in 7/8 patients. Finally, at PSA higher than 5 ng/ml [(11)C]choline PET/CT identified 5/5 patients positive all confirmed by histology. The sensitivity of [(11)C]choline PET/CT for the detection of recurrence at PSA < 2.5 ng/ml was 89% with a positive predictive value of 72%. CONCLUSION: [(11)C]choline PET/CT is useful for re-staging of prostate cancer in patients with rising PSA even at levels below 1.5 ng/ml. Our study confirms results from other published studies on [(11)C]choline PET/CT in prostate cancer relapse.

Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression.

Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.

[Role of androgen receptors in hormone-refractory prostate cancer: molecular basics and experimental therapy approaches]. / Die Rolle des Androgenrezeptors im hormonrefraktären Prostatakarzinom : Molekulare Grundlagen und experimentelle Therapieansätze.

The development of hormone-refractory prostate cancer cells is one of the major causes for the progression and high mortality rates in advanced prostate cancer (PCA). While the loss of the androgen receptor (AR) is the predominant mechanism for development of a hormone-insensitive disease in vitro, the first in vivo studies showed that the AR is still expressed or is even overexpressed in hormone-refractory PCA. In view of the increasing cases of PCA in the industrialized Western countries, a series of cell and molecular biological studies has led to the identification of various new factors and mechanisms that play a role during the development of hormone-refractory tumors. These findings should lead to the development of new therapeutic strategies.

[Inhibitors of the androgen receptor N­terminal domain : Therapies targeting the Achilles' heel of various androgen receptor molecules in advanced prostate cancer]. / Inhibitoren des Androgenrezeptor-N-Terminus' : Zielgerichtete Therapien gegen die Achillesferse verschiedener Androgenrezeptormoleküle im fortgeschrittenen Prostatakarzinom.

Although prostate cancer responds well to primary endocrine therapies, tumor progression with castration resistant tumor cells almost invariably occurs within a few years. Unfortunately, some CRPC patients do not respond to second-line therapies with abiraterone or enzalutamide. Moreover, patients who initially responded well to second-line hormone therapy develop resistance to abiraterone and/or enzalutamide within a short period of time. Besides an increase of intracellular androgen receptor (AR) levels, the predominant resistance mechanisms include AR aberrations (point mutations, AR splice variants) occurring predominantly at the androgen or ligand binding domain of the AR. The following review delineates recent progress in the development of AR inhibitors that do not depend on androgen binding and represent a putative third generation of AR inhibitors.

Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor.

Aberrant activation of the androgen receptor (AR) may be one of the mechanisms which contribute to progression of prostatic carcinoma to an androgen-independent stage. We investigated effects of growth factors on stimulation of the AR-mediated gene transcription in human prostatic tumor cell lines. DU-145 cells, which do not contain endogenous AR, were cotransfected with an androgen-inducible chloramphenicol acetyltransferase (CAT) reporter gene and an AR expression vector. The reporter gene (CAT) was driven either by artificial promoters consisting of one or two androgen-responsive elements in front of a TATA box or by the promoter of the prostate-specific antigen (PSA) gene, a naturally occurring androgen-inducible promoter. Insulin-like growth factor-I (IGF-I), at a concentration of 50 ng/ml, stimulated AR-mediated reporter gene transcription to the same extent as the synthetic androgen methyltrienolone. This growth factor was effective irrespective of the nature of the androgen-inducible promoter. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF), at concentrations of 50 ng/ml, activated CAT reporter gene transcription only in experiments in which the artificial promoter with two androgen-responsive elements was used. Insulin-like growth factor-II and basic fibroblast growth factor displayed no effect on AR-mediated gene transcription. None of the growth factors stimulated reporter gene activity in control experiments when added to cells cotransfected with the CAT gene and an empty expression vector. AR activation by IGF-I, KGF, and EGF was completely inhibited by the pure AR antagonist casodex, showing that these effects are AR mediated. Activation of endogenous AR by growth factors was studied in the LNCaP cell line by determination of PSA secretion. IGF-I, at a concentration of 50 ng/ml, increased the PSA level in the supernatant of this cell line 5-fold. Again, the IGF-I effect on PSA secretion was blocked by casodex. Our results provide evidence that IGF-I, KGF, and EGF directly activate the AR in the absence of androgens, which means that the androgen-signaling chain may be activated by growth factors in an androgen-depleted environment. These findings may have implications for endocrine therapy for metastatic prostatic carcinoma.

Gamma-interferon reduces expression of the protooncogene c-erbB-2 in human ovarian carcinoma cells.

The overexpression of the protooncogene c-erbB-2 (HER-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that, in three of four ovarian carcinoma cell lines, there is a gamma-interferon-mediated reduction in c-erbB-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relation between the absolute amount of c-erbB-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of gamma-interferon. Other chemotherapeutic agents did not affect c-erbB-2 expression, although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in three gamma-interferon-sensitive human breast cancer cell lines. Expression of the oncogene c-erbB-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which gamma-interferon inhibits cell proliferation.

Effects of WNT/beta-catenin pathway activation on signaling through T-cell factor and androgen receptor in prostate cancer cell lines.

Dysregulation of the WNT/beta-catenin pathway is thought to contribute to prostate cancer progression. Mutations of beta-catenin occurring in 5-7% of advanced prostate cancers may act by stimulating TCF-dependent and/or androgen receptor (AR)-dependent transcription. Using a reporter gene approach we found overexpressed mutated beta-catenin to enhance AR-regulated probasin-promoter activity in the AR-positive prostate cancer cell line 22Rv1, particularly at low androgen levels. In 22Rv1 cells mutated beta-catenin was able to stimulate TCF-dependent transcription but was unable to do so in LNCaP cells where it activates the AR. Since beta-catenin mutations are rare in vivo, we studied further possible routes of WNT-pathway modulation. Higher concentrations of LiCl, a GSK3beta-inhibitor, were required to activate TCF-dependent rather than AR-dependent reporter constructs. In 22Rv1 overexpression of E-cadherin repressed androgen-dependent transcription, but did not inhibit transcription of TCF-dependent reporter genes as in bladder cancer cell lines. Interestingly, Wnt-3a stimulated proliferation selectively in the AR-positive prostate cancer cell lines 22Rv1 and LNCaP, even though TCF-dependent reporter gene transcription was not induced in LNCaP cells. In summary, the data from our study support the idea that activation of WNT/beta-catenin signaling in AR-positive prostate cancer cells may predominantly act through AR-dependent mechanisms rather than classical TCF-dependent mechanisms.

Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone.

Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for the synthetic androgen mibolerone was 3 nM for both receptors. There was also no significant difference in binding of other steroids and nonsteroidal antiandrogens as revealed by competition binding assays. However, transfection experiments to determine the trans-activation potential of the mutant receptor produced differences in the action of this receptor compared to the wild type receptor. Dihydrotestosterone and the synthetic androgens methyltrienolone (R1881) and mibolerone were equally proficient in conferring trans-activation activity to both the mutant and wild type receptors. Adrenal androgens such as dehydroepiandrosterone and androstenedione, as well as progesterone mediated a higher trans-activation through the mutant than through the wild type receptor. These data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone. This pattern of ligand-dependent trans-activation may have significance in the process controlling the progression of prostatic carcinoma.

Antioxidant enzymes in malignant prostate cell lines and in primary cultured prostatic cells.

The antioxidant enzymes catalase, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were determined in the androgen-response LNCaP and androgen-nonresponsive PC-3 and DU 145 cells as well as in prostatic epithelial cell cultures of benign and malignant human prostatic tissue. There were no differences between the enzyme activities of the human primary cell cultures from cancerous tissue and their normal counterparts. The enzyme activities of the three permanent cell lines were either higher (SOD, catalase, GR) or lower (GST, GPx) than in the primary cell cultures. In LNCaP cells catalase and GR were significantly higher, GST, in contrast, was significantly lower than in PC-3 and DU 145 cells. GST in PC-3 and DU 145 cells, and SOD in all the three cell lines showed no significant differences. Catalase, GPx and GR values were significantly different in the three permanent cell lines. The different enzymatic equipment of the prostate cancer cell lines provides the basis for experimental testing of new concepts of cancer treatment with the help of systematic modulations of the antioxidant defence systems in prostate cancer.

The androgen receptor in hormone-refractory prostate cancer: relevance of different mechanisms of androgen receptor signaling (Review).

The last decade has brought increased awareness to prostate cancer as a significant health problem. Prostate cancer is very heterogeneous in its etiology and progression, but androgen signaling appears to be a common key element in its development and progression. Blocking of androgen signaling results in a decrease in tumor volume as well as a decline in serum PSA in the majority of patients with prostate cancer. Today, endocrine therapy involves androgen depletion by orchiectomy or by treatment with LHRH-analoga as well as blockade of the androgen receptor (AR) with anti-androgens. However, during these treatments almost all tumors relapse to a hormone-insensitive state. The mechanisms that lead from initially androgen-sensitive to androgen-unresponsive tumor cell growth have been partly elucidated by new insights into the molecular mechanisms of androgen receptor signaling over the past several years. In addition to androgen receptor mutations that broaden the ligand-specificity of the AR, androgen-independent transactivation of the AR by peptide growth factors such as epidermal growth factor and insulin-like growth factor-I has been discovered. Furthermore, analysis of proteins that interact with the AR led to the isolation of coactivator proteins that mediate transcriptional activation by the AR. The following review will discuss the elements involved in androgen receptor signaling and summarize the present knowledge of their biological and clinical relevance in advanced prostate cancer.

Androgen receptor gene mutations in prostate cancer. Implications for disease progression and therapy.

Recent studies indicate that androgen receptors are present in all histological types of prostatic tumours, in relapsed prostatic carcinomas and in tumour metastases, even those obtained from patients in whom endocrine therapy was unsuccessful. Several research groups have asked whether structurally altered androgen receptors might be present in human prostatic tumours. The first androgen receptor mutation in prostate cancer was detected in the tumour cell line LNCaP. The frequency of androgen receptor mutations in primary tumours of the prostate is relatively low. In contrast, a high frequency of mutations has been reported in bone metastases from patients who did not respond to endocrine therapy. This fact may reflect genetic instability in these late tumour stages. Mutant androgen receptors detected in human prostate cancer cells are 'promiscuous receptors'; that is, they are activated not only by synthetic and testicular androgens, but also by adrenal androgens, products of dihydrotestosterone metabolism, estrogenic and progestagenic steroids, and even by nonsteroidal antiandrogens. Interestingly, the nonsteroidal antiandrogens hydroxyflutamide and nilutamide, but not bicalutamide, have been reported to have agonistic effects at mutant androgen receptors. It is speculated that the existence of androgen receptor mutations may explain, at least in part, the 'antiandrogen withdrawal syndrome': a temporary improvement in a subpopulation of prostate cancer patients following cessation of an antiandrogen from a therapeutic protocol. Further studies on androgen receptor alterations in prostate cancer should focus on metastatic specimens obtained from the late stages of this disease.

[Interferon-gamma suppresses expression of the HER-2 oncogene in ovarian cancer cells]. / Interferon-Gamma supprimiert die Expression des Onkogens HER-2 in Ovarialkarzinomzellen.

The overexpression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that in 7 out of 8 ovarian carcinoma cell lines there is an interferon-gamma-mediated reduction in HER-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relationship between the absolute amount of HER-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of interferon-gamma. Other chemotherapeutic agents did not affect HER-2 expression although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in 3 interferon-gamma sensitive human breast cancer cell lines. Expression of the oncogene HER-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which interferon-gamma inhibits cell proliferation.

Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle.

The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone.

Molecular biology of prostate cancer.

In spite of progress in diagnosis and treatment, prostate cancer has become one of the most frequent lethal cancers in males in many Western industrialized countries. Research on the molecular biology of prostate cancer is expected to reveal those aspects of Western lifestyle contributing to its high incidence with the aims of improving prevention, distinguishing slow-growing from aggressive clinically relevant cancers, and providing targets for treatment, particularly of locally advanced and of metastatic disease. Traditionally, prostate cancer research focused on androgens. More recently, tumour suppressors and proto-oncogenes important in other human cancers have been intensely investigated. Current approaches include the search for genes mutated in familial cases, identification of recurrent chromosomal alterations and their associated potential tumour suppressor genes, determination of gene expression profiles characterizing tumour stages and subclasses, and elucidation of the importance of epigenetic alterations. Results from such studies have begun to be translated into the clinic. Further successful transfer of results from molecular biology to the clinic will, however, require integration of the amassed molecular data into a biological framework model of prostate carcinoma.

Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas.

We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer.

Fibroblast growth factors and their receptors in urological cancers: basic research and clinical implications.

Because therapeutical options for advanced urological cancers are limited, the understanding of key elements responsible for invasion and metastasis is very important. It has been hypothesized that progression to malignant growth is associated with a dysregulation of growth factors and/or their receptors. In the last few years, signaling pathways of the fibroblast growth factor (FGF) family have been subject to intense investigation. Fibroblast growth factors constitute one of the largest families of growth and differentiation factors for cells of mesodermal and neuroectodermal origin. The family comprises two prototypic members, acidic FGF (aFGF) and the basic FGF (bFGF), as well as 21 additionally related polypeptide growth factors that have been identified to date. FGFs are involved in many biological processes during embryonic development, wound healing, hematopoesis, and angiogenesis. In prostate, bladder, and renal cancers, FGFs regulate the induction of metalloproteinases (MMP) that degrade extracellular matrix proteins, thus facilitating tumor metastasis. Probably due to their potent angiogenic properties, aFGF and bFGF have received the most attention. However, there is increasing evidence that other FGFs also play crucial roles in tumors of the prostate, bladder, kidney, and testis. This review will discuss the different elements involved in FGF signaling and summarize the present knowledge of their biological and clinical relevance in urological cancers.