Meaning of "steroidogenic" in the English dictionaryAlthough the human sebaceous gland can synthesize cholesterol from acetate steroidogenic enzymes definition can further metabolize steroids such as dehydroepiandrosterone into potent androgens, the de novo production of steroids from cholesterol has not been demonstrated in human skin. The goal of this study was to delineate the steroidogenic pathway upstream from dehydroepiandrosterone by steroidogenic enzymes definition the presence of tren ace veins of the P side chain cleavage system Pscc. This system catalyzes the initial step in steroid hormone synthesis following translocation of cholesterol to the inner mitochondrial membrane. In concert with its cofactors, adrenodoxin and adrenodoxin reductase, and the transcription factor steroidogenic factor 1, Pscc converts cholesterol to pregnenolone. An SV40 immortalized human sebaceous gland steroisogenic line SEB-1 was established steroidogenic enzymes definition order to facilitate investigation of the Pscc system. The sebaceous phenotype of SEB-1 sebocytes was confirmed using immunohistochemistry, Oil Red O staining, and gene array expression analysis. Presence of Pscc, adrenodoxin reductase, cytochrome P hydroxylase Pc17and steroidogenic factor 1 was documented steroixogenic human facial skin, human sebocytes, and SEB-1 sebocytes.
Steroidogenic - definition of steroidogenic by The Free Dictionary
Intra-tumoral steroidogenesis and constitutive androgen receptor AR activity have been associated with castration-resistant prostate cancer CRPC. This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants AR-Vs and to clinical and pathological variables.
Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay.
No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases.
Induced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis.
Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2 nd line androgen-deprivation therapy. May 14, ; Accepted: September 2, ; Published: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have declared that no competing interests exist. The first-line treatment for patients with advanced prostate cancer PC is androgen deprivation therapy ADT. This therapy is effective in most patients, but after a period of initial remission tumors generally relapse, predominantly within the bone, and are then termed castration-resistant PC CRPC.
In spite of low circulating levels of androgens in castrated men the majority of CRPC tumors show androgen receptor AR activity and expression of AR regulated genes .
Possible mechanisms behind AR activity in CRPC include AR gene amplification and mutations, intra-tumoral synthesis of androgens, and expression of constitutively active AR splice variants  , . Notably some CRPC tumors, and individual tumor cells in most patients, show very low or absence of nuclear AR immunostaining, and factors down-stream the AR are not necessarily up-regulated in those cases  ,  ,  , .
Some CRPC patients however respond to 2 nd line ADT and this might be due to intra-tumoral steroidogenesis and production of testosterone, dihydrotestosterone DHT , or other androgens at levels high enough for AR activation, as reported by others  ,  ,  , . It is, however, not known when these steroidogenic enzymes are up-regulated. Are they increased already in previously untreated HN metastases, or as the AR-Vs  increased as result of castration treatment?
One aim of this study was therefore to analyze expression of steroid-converting enzymes potentially involved in synthesis of testosterone and DHT in a set of HN and CRPC bone metastases obtained from patients at metastasis surgery.
Furthermore, enzyme expression levels were analyzed in relation to expression of AR-Vs, in order to identify potential different mechanisms behind CRPC in individual bone metastases.
Due to the acute situation when bone metastasis surgery is performed in order to relief spine symptoms and paresis, logistics do not always allow written consent and the local ethic review board therefore specifically approved also verbal consent. Verbal consent is documented by the physician in the patient journal. Clinical characteristics and therapies are summarized in Table 1 , and this patient series has been previously described in Crnalic et al  ,  , .
Median age for the patients was 60 years range 48—68 years and median PSA were 6. Four of the tumors were in stage T2 and nine in stage T3. For cases were DNA and protein were analyzed in parallel to RNA, nucleic acids and protein were isolated from the same tissue sections using the AllPrep method.
Primer sequences are shown in Table S1. Samples were fixed in buffered formalin, decalcified in formic acid, and embedded in paraffin . A total score ranging from 0—12 was obtained by multiplying the staining intensity and fraction scores.
Ki67 were scored by evaluating — tumour epithelial cells per patient, as described earlier . The assays were run according to the manufacturer's description, with RNaseP as reference gene, and analyzed using the Copy Caller software Applied Biosystems. Filters were then stripped, according to standard procedures, and analyzed for actin protein levels using the rabbit anti-actin antibody from SIGMA Saint Louis.
The intensity of the 80 kDa band in each patient sample was analyzed and expressed in relation to the corresponding full length AR band intensity. The 22Rv1 cell extract was run as a positive control sample on each gel. Correlations between variables were analyzed using Spearman rank test. Groups were compared using the Mann-Whitney U test for continuous variables and the Chi-square test for categorical variables.
Kaplan-Meier survival analysis was performed with death of PC as event and follow-up time as time between metastasis surgery and the latest follow-up examination December A P-value less or equal to 0. Steroids secreted by the adrenal gland are highlighted in gray. None of the analyzed enzymes in the steroidogenesis pathway Figure 1 and 2 showed significantly different mRNA expression levels between HN and CRPC bone metastases for comparison between metastases and prostate tissue see below.
The variability between different CRPC metastases was however large and individuals with particularly high expression of specific steroidogenic enzymes were observed in the CRPC group Figure 2. Open circles indicate outliers and extremes. X indicates extreme levels outside the scale. Transcript levels of many enzymes involved in later steps of androgen synthesis were increased in metastases compared to non-malignant prostate and primary tumor tissue Figure 2.
Taken together those results indicate that PC bone metastases in patients may have induced capacity to convert androgens with low AR affinity into more potent androgens, while de novo synthesis of androgens from cholesterol is less likely. Although none of the examined steroidogenic enzymes showed significantly increased expression levels in CRPC compared to HN bone metastases, individual CRPC metastases were noted to express very high transcript levels Figure 2.
As this enzyme has the capacity to convert circulating DHEA and androstenedione synthetized in the adrenals into 5-androstenediol and testosterone, it was considered to be of particular interest. To determine whether the AKR1C3 mRNA levels in the metastases were reflected in the corresponding protein levels, we assessed the AKR1C3 protein expression by immunohistochemistry and by using a scoring system that took both staining intensity and distribution into account Figure 3.
Cytoplasmic AKR1C3 immunostaining was demonstrated in most tumor epithelial cells while nuclear staining was heterogeneously observed and not scored. All metastases showed strong positive staining in the endothelial cells, whereas variable staining was observed in fibroblasts and hematopoietic cells in the bone marrow.
A total score ranging from 0—12 was obtained by multiplying the staining intensity score 0—3 in the tumor epithelial cells with the positive fraction score 1—4. In contrast to this, there was no correlation found between the AKR1C3 immunostaining and cancer-specific survival of CRPC patients after metastasis surgery data not shown.
There was also no association observed between the AKR1C3 immunostaining score and the PSA staining score data not shown or the tumor cell proliferation index Ki67 staining score, data not shown. The AR protein levels were studied by immunoblot analysis of 29 CRPC bone metastases selected based on AR immunohistochemistry results to include AR low to AR high score metastases, and where adequate tissue remained to allow immunoblot analysis.
Analysis was performed using an antibody targeting the N-terminal domain of the AR. The 22Rv1 cell extract was included as a positive control. Interestingly, only one out of 13 7. We further hypothesized that tumor cell expression of AKR1C3 and constitutively active AR variants could be of relevance for individual tumor response to 2 nd line ADT and clinical progression  , and the 28 bone metastases evaluated for both AKR1C3 and AR-V expression were therefore divided according to their AR-V and AKR1C3 protein expression levels, as shown in Figure 5 , in order to demonstrate sub-groups of possible clinical relevance.
Notably, none of the metastases with homogeneous AKR1C3 staining, i. This observation deserves to be further examined, as it might indicate that high AR-V levels are induced preferable in tumors cells without endogenous steroidogenesis.
Unfortunately, we were not able to study this hypothesized heterogeneity among tumor cells as antibodies for immunohistochemical evaluation of AR-Vs were not available. In this study we compared expression levels of steroidogenic enzymes in CRPC bone metastases with levels in metastases obtained from un-treated patients and, surprisingly, found no induction of the examined enzymes in CRPC compared to HN metastases.
Furthermore, we were able to show distinct differences in expression levels of AKR1C3 and levels of AR splice variants in individual cases of CRPC bone metastases, which we hypothesize could be of relevance for patient response to 2 nd line therapies. Tissue androgens might be derived by de novo synthesis from cholesterol  ,  or by conversion of adrenal precursors into more potent steroids .
Our results showing generally high levels of AKR1C3 and SRD5A1 in metastases are in line with previous reports  ,  ,  and may indicate synthesis of testosterone and DHT in one and two steps, respectively, from adrenal-derived androstenedione.
This route for DHT synthesis, which bypasses testosterone, was recently shown by Chang and co-workers to dominate in CRPC cell lines as well as in patient metastases specimens stimulated with androstenedione . Intra-tumoral synthesis of androsterone and androstanediol could stimulate tumor growth not only as intermediate steroids in DHT synthesis, but also as potent AR activators in cases with mutated ARs .
From this we would like to conclude that adrenal-derived androgens probably contribute to growth of CRPC bone metastases by their intra-metastatic conversion into more potent androgens, while de novo synthesis of androgens from cholesterol within metastases is less likely, except maybe in individual cases with correlated expression of CYP11A1, CYP17A1, and HSD3B2.
We found high expression levels of AKR1C3 in a sub-group of PC bone metastases, which confirm results previously reported in castration-resistant primary prostate tumors  ,  and CRPC tissue of different metastatic origin  ,  , . Beneficial effects of high AKR1C3 levels in HN metastases or in metastases with low nuclear AR immunostaining, as demonstrated for some cases in this study, are less clear, but may depend on other reactions driven by this enzyme.
In conclusion, we want to emphasize that increased tumor expression of steroidogenic enzymes in individual patients is not clearly associated with CRPC but merely associated with advanced tumor stage, as it can be seen not only in CRPC tissue of different origin  ,  ,  ,  ,  but also in previously un-treated, HN, bone metastases this study.
Furthermore, this study together with previous studies indicate that CRPC tumors could be classified according to distinct expression patterns of steroidogenic enzymes, and thus probably different mechanisms behind castration-resistance this study and  ,  , . A limitation to this work is the restricted number of bone metastases, and furthermore the single metastasis sample examined from each patients, which obviously make conclusions regarding this heterogeneous disease and overall patient status uncertain.
We are also aware that expression levels not necessarily reflect biological activities and our hypothesis that tumor expression pattern of steroidogenic AKR1C3 and constitutively active AR variants would predict response to 2 nd line therapies of CRPC needs to be tested upfront in clinical studies.
Conceived and designed the experiments: Revision of article draft: Contributed with clinical expertise and follow-up: Click through the PLOS taxonomy to find articles in your field. Abstract Background Intra-tumoral steroidogenesis and constitutive androgen receptor AR activity have been associated with castration-resistant prostate cancer CRPC.
November 7, Copyright: Introduction The first-line treatment for patients with advanced prostate cancer PC is androgen deprivation therapy ADT. Clinical and histological characteristics of prostate cancer patients with bone metastases and treated with metastasis surgery.
Immunohistochemistry Samples were fixed in buffered formalin, decalcified in formic acid, and embedded in paraffin . Immunoblotting 22Rv1 cells were maintained according to manufacturer's instructions ATCC and protein was extracted as above.
Statistical analysis Correlations between variables were analyzed using Spearman rank test. Simplified scheme of the key steps of testosterone and DHT steroidogenesis. Immunohistochemical staining of AKR1C3 in bone metastases. Expression of steroidogenic enzymes in relation to levels of androgen receptor splice variants The AR protein levels were studied by immunoblot analysis of 29 CRPC bone metastases selected based on AR immunohistochemistry results to include AR low to AR high score metastases, and where adequate tissue remained to allow immunoblot analysis.
Author Contributions Conceived and designed the experiments: Am J Pathol View Article Google Scholar 2. J Clin Oncol View Article Google Scholar 3.