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Mechanisms behind growth of castration-resistant prostate cancer bone metastases
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: The first-line treatment for patients with advanced prostate cancer (PC) is androgen deprivation therapy. This therapy is initially effective, but after some time tumors relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors show androgen receptor (AR) activity despite castrate levels of circulating testosterone. AR activity could be caused by several mechanisms including; intratumoral androgen synthesis, AR amplification, AR mutations and expression of AR splice variants. The mechanisms controlling CRPC growth in the clinically most relevant metastatic site, the bone, are not fully identified. The purpose of this thesis was therefore to explore AR expression and possible mechanisms behind CRPC growth in PC bone metastases in order to find mechanisms that could be targeted for treatment and/or predict response to certain therapies.

Materials and Methods: We have examined hormone-naïve and CRPC bone metastases samples obtained from patients at metastasis surgery, non-malignant and malignant prostate samples obtained from patients at radical prostatectomy, and PC cell lines cultured in vitro. Analysis has been performed using RT-PCR, whole-genome expression arrays, immunohistochemistry, western blotting, FISH, copy number assays and gene ontology analysis. Functional studies have been made by protein overexpression and knock-down in PC cells in vitro and effects studied by evaluation of cell viability, migration, and invasion.

Results: We found that high nuclear AR immunostaining (presumed to reflect high AR activity) in bone metastases from CRPC patients was associated with a particularly poor prognosis, while no difference in AR staining was observed between hormone-naïve and CRPC metastases. Further, expression of AR splice variants (AR-V7, AR-V567es) was associated with a high nuclear AR immunostaining score and shown to be increased in CRPC compared to hormone-naïve bone metastases. High levels (levels in the upper quartile) of AR splice variants in CRPC bone metastases was related to disturbed cell cycle regulation and short patients survival. No differences in steroidogenic enzyme levels were detected between CRPC and hormone-naïve bone metastases. Higher levels of enzymes involved in late steps of androgen synthesis (adrenal gland steroid conversion) were observed in bone metastases than in non-malignant and/or malignant prostate tissue, while the enzyme levels in earlier steps (de novo steroidogenesis) were lower in bone metastases. A subgroup of metastases expressed very high levels of AKR1C3, indicating that this group may have an induced capacity of converting adrenal-gland derived steroids into more potent androgens. This was not associated to CRPC but merely with the advanced stage of metastasis. High protein levels of AR splice variants were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR variant levels. Furthermore, about half of the CRPC bone metastases showed androgen receptor gene amplification which was associated with co-amplification of YIPF6, and a gene expression pattern that pointed at decreased osteoclast activity, and consequently decreased bone resorption.

Conclusions: The majority of CRPC bone metastases show high nuclear AR immunostaining that seems to be associated with a particularly unfavorable outcome after metastasis surgery. Subgroups of CRPC bone metastases could be identified according to presence of AR amplification and expression levels of AKR1C3 or AR splice variants, which might have clinical relevance for treatment of PC patients.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2013. , 43 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1620
Keyword [en]
Prostate cancer, castration-resistance, bone metastases, androgen receptor, intratumoral steroidogenesis, androgen receptor splice variants, androgen receptor amplification
National Category
Cancer and Oncology Other Basic Medicine
Research subject
Pathology
Identifiers
URN: urn:nbn:se:umu:diva-83956ISBN: 978-91-7459-775-2 (print)OAI: oai:DiVA.org:umu-83956DiVA: diva2:678136
Public defence
2014-01-10, Sal B, 9tr, By 1D Tandläkarhögskolan, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Note

Författaren är även publicerad med efternamnet Hörnberg.

Available from: 2013-12-18 Created: 2013-12-11 Last updated: 2014-01-15Bibliographically approved
List of papers
1. Nuclear androgen receptor staining in bone metastases is related to a poor outcome in prostate cancer patients
Open this publication in new window or tab >>Nuclear androgen receptor staining in bone metastases is related to a poor outcome in prostate cancer patients
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2010 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 17, no 4, 885-895 p.Article in journal (Refereed) Published
Abstract [en]

Androgen receptors (ARs) are probably of importance during all phases of prostate cancer (PC) growth, but their role in bone metastases is largely unexplored. Bone metastases were therefore collected from hormone-naive (n=11), short-term castrated (n=7) and castration-resistant PC (CRPC, n=44) patients by biopsy (n=4) or at surgery to alleviate symptoms from metastases complications (metastasis surgery, n=58), and immunostained for nuclear ARs, Ki67, active caspase-3, prostate-specific antigen (PSA) and chromogranin A, and results were related to serum PSA, treatments and outcome. Nuclear AR immunostaining was decreased and apoptosis was increased, but cell proliferation remained largely unaffected in metastases within a few days after surgical castration. In CRPC patients, nuclear AR staining of metastases was increased when compared to short-term castrated patients. The nuclear AR staining score was related to tumour cell proliferation, but it was not associated with other downstream effects of AR activation such as apoptosis and PSA staining, and it was only marginally related to the presence of neuroendocrine tumour cells. Serum PSA at metastasis surgery, although related to outcome, was not associated with AR staining, markers of metastasis growth or PSA staining in metastases. High nuclear AR immunostaining was associated with a particularly poor prognosis after metastasis surgery in CRPC patients, suggesting that such men may benefit from the potent AR blockers now tested in clinical trials.

National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-36843 (URN)10.1677/ERC-10-0059 (DOI)000284490000009 ()20688881 (PubMedID)
Available from: 2010-10-12 Created: 2010-10-12 Last updated: 2017-12-12Bibliographically approved
2. Expression of androgen receptor splice variants in prostate cancer bone metastases is associated with castration-resistance and short survival
Open this publication in new window or tab >>Expression of androgen receptor splice variants in prostate cancer bone metastases is associated with castration-resistance and short survival
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 4, e19059- p.Article in journal (Refereed) Published
Abstract [en]

Background: Constitutively active androgen receptor variants (AR-V) lacking the ligand binding domain (LBD) may promote  the development of castration-resistant prostate cancer (CRPC). The expression of AR-Vs in the clinically most important metastatic site, the bone, has, however, not been well documented. Our aim was therefore to compare levels of AR-Vs in hormone-naive (HN) and CRPC bone metastases in comparison to primary PC and non-malignant prostate tissue, as well as in relation to AR protein expression, whole-genome transcription profiles and patient survival.

Methodology/Principal Findings: Hormone-naı¨ve (n = 10) and CRPC bone metastases samples (n = 30) were obtained from  40 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomized men. Levels of full length AR (ARfl) and AR-Vs termed AR-V1, AR-V7, and AR-V567es mRNA were measured with RT-PCR and whole-genome transcription profiles with an Illumina Beadchip array. Protein levels were examined by Western blotting and immunohistochemistry. Transcripts for ARfl, AR-V1, and AR-V7 were detected in most primary tumors and metastases, and levels were significantly increased in CRPC bone metastases. The AR-V567es transcript was detected in 23% of the CRPC bone metastases only. A sub-group of CRPC bone metastases expressed LBD-truncated AR proteins at levels comparable to the ARfl. Detectable AR-V567es and/or AR-V7 mRNA in the upper quartile, seen in 1/3 of all CRPC bone metastases, was associated with a high nuclear AR immunostaining score, disturbed cell cycle regulation and short survival.

Conclusions/Significance: Expression of AR-Vs is increased in CRPC compared to HN bone metastases and associated with a particularly poor prognosis. Further studies are needed to test if patients expressing such AR-Vs in their bone metastases benefit more from drugs acting on or down-stream of these AR-Vs than from therapies inhibiting androgen synthesis.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-43913 (URN)10.1371/journal.pone.0019059 (DOI)000290020700019 ()21552559 (PubMedID)
Available from: 2011-05-16 Created: 2011-05-16 Last updated: 2017-12-11Bibliographically approved
3. Characterization of prostate cancer bone metastases according to expression levels of steroidogenic enzymes and androgen receptor splice variants
Open this publication in new window or tab >>Characterization of prostate cancer bone metastases according to expression levels of steroidogenic enzymes and androgen receptor splice variants
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, e77407- p.Article in journal (Refereed) Published
Abstract [en]

Background: Intra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated withcastration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels ofsteroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation toexpression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables.

Methodology/Principal Findings: Untreated, hormone-naıve (HN, n = 9) and CRPC bone metastases samples (n = 45) wereobtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry andimmunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases.Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases thanin non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNAlevels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which wasnot due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only oneexception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with highAKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individualbone metastases.

Conclusions/Significance: Induced capacity of converting adrenal-gland derived steroids into more potent androgens wasindicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage ofmetastasis. 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 2nd line androgen-deprivation therapy.

Place, publisher, year, edition, pages
Public Library of Science, 2013
Keyword
prostate cancer, bone metastases, androgen receptor splice variants, steroidogenic enzymes
National Category
Basic Medicine Cancer and Oncology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-83840 (URN)10.1371/journal.pone.0077407 (DOI)000327162900006 ()24244276 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2013-12-10 Created: 2013-12-10 Last updated: 2017-12-06Bibliographically approved
4. Molecular features of prostate cancer bone metastases harboring androgen receptor gene amplification
Open this publication in new window or tab >>Molecular features of prostate cancer bone metastases harboring androgen receptor gene amplification
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The relation between AR amplification and other mechanisms behind castration-resistance in prostate cancer, such as increased expression of AR splice variants and steroid-converting enzymes in CRPC metastases, has been poorly examined. Specific aims of this study were therefore to examine AR amplification in hormone-naïve and castration-resistant prostate cancer (CRPC) bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying molecular targets for treatment of CRPC bone metastases. AR amplification was assessed by fluorescence in situ hybridization and verified in 16 (53 %) of the CRPC bone metastases (n=30), and in none of the untreated bone metastases (n=10). AR amplification was associated with increased expression of AR and its constitutively active AR-V7 splice variant as well as with co-amplification of genes in the AR proximity at Xq12, such as of YIPF6. Furthermore, gene expression pattern pointed at decreased osteoclast activity, and consequently decreased bone resorption and increased bone mineral density in AR amplified metastases. In conclusion, our results indicated a sclerotic phenotype in CRPC bone metastases with AR amplification that may be of both biological and clinical relevance. This is a novel hypothesis that requires to be thoroughly examined.

National Category
Basic Medicine Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-83841 (URN)
Available from: 2013-12-10 Created: 2013-12-10 Last updated: 2016-08-22Bibliographically approved

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