Emma Jernberg

Characterization of prostate cancer bone metastases according to expression levels of steroidogenic enzymes and androgen receptor splice variants.

Background 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. Methodology/Principal Findings Untreated, hormone-naïve (HN, n = 9) and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. 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. 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 than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. 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. Conclusions/Significance 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 2nd line androgen-deprivation therapy.

Subgroups of castration-resistant prostate cancer bone metastases defined through an inverse relationship between androgen receptor activity and immune response

BACKGROUND Novel therapies for men with castration-resistant prostate cancer (CRPC) are needed, particularly for cancers not driven by androgen receptor (AR) activation. OBJECTIVES To identify molecular subgroups of PC bone metastases of relevance for therapy. DESIGN, SETTING, AND PARTICIPANTS Fresh-frozen bone metastasis samples from men with CRPC (n=40), treatment-naïve PC (n=8), or other malignancies (n=12) were characterized using whole-genome expression profiling, multivariate principal component analysis (PCA), and functional enrichment analysis. Expression profiles were verified by reverse transcription-polymerase chain reaction (RT-PCR) in an extended set of bone metastases (n=77) and compared to levels in malignant and adjacent benign prostate tissue from patients with localized disease (n=12). Selected proteins were evaluated using immunohistochemistry. A cohort of PC patients (n=284) diagnosed at transurethral resection with long follow-up was used for prognostic evaluation. RESULTS AND LIMITATIONS The majority of CRPC bone metastases (80%) was defined as AR-driven based on PCA analysis and high expression of the AR, AR co-regulators (FOXA1, HOXB13), and AR-regulated genes (KLK2, KLK3, NKX3.1, STEAP2, TMPRSS2); 20% were non-AR-driven. Functional enrichment analysis indicated high metabolic activity and low immune responses in AR-driven metastases. Accordingly, infiltration of CD3+ and CD68+ cells was lower in AR-driven than in non-AR-driven metastases, and tumor cell HLA class I ABC immunoreactivity was inversely correlated with nuclear AR immunoreactivity. RT-PCR analysis showed low MHC class I expression (HLA-A, TAP1, and PSMB9 mRNA) in PC bone metastases compared to benign and malignant prostate tissue and bone metastases of other origins. In primary PC, low HLA class I ABC immunoreactivity was associated with high Gleason score, bone metastasis, and short cancer-specific survival. Limitations include the limited number of patients studied and the single metastasis sample studied per patient. CONCLUSIONS Most CRPC bone metastases show high AR and metabolic activities and low immune responses. A subgroup instead shows low AR and metabolic activities, but high immune responses. Targeted therapy for these groups should be explored. PATIENT SUMMARY We studied heterogeneities at a molecular level in bone metastasis samples obtained from men with castration-resistant prostate cancer. We found differences of possible importance for therapy selection in individual patients.

High Lysyl Oxidase (LOX) in the Non-Malignant Prostate Epithelium Predicts a Poor Outcome in Prostate Cancer Patient Managed by Watchful Waiting

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.

Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck

BackgroundWilms’ tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu.MethodsSilencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN.ResultsWe found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples.ConclusionsOur findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth.

Extratumoral Heme Oxygenase-1 (HO-1) Expressing Macrophages Likely Promote Primary and Metastatic Prostate Tumor Growth

Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1). To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RT-PCR showed that the main site of HO-1 synthesis was HO-1+ macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1+ macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1+ macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1+ macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1+ macrophages may have an important role in prostate cancer.

Clinical relevance of androgen receptor alterations in prostate cancer

Prostate cancer (PC) remains a leading cause of cancer-related deaths among men worldwide, despite continuously improved treatment strategies. Patients with metastatic disease are treated by androgen deprivation therapy (ADT) that with time results in the development of castration-resistant prostate cancer (CRPC) usually established as metastases within bone tissue. The androgen receptor (AR) transcription factor is the main driver of CRPC development and of acquired resistance to drugs given for treatment of CRPC, while a minority of patients have CRPC that is non-AR driven. Molecular mechanisms behind epithelial AR reactivation in CRPC include AR gene amplification and overexpression, AR mutations, expression of constitutively active AR variants, intra-tumoural and adrenal androgen synthesis and promiscuous AR activation by other factors. This review will summarize AR alterations of clinical relevance for patients with CRPC, with focus on constitutively active AR variants, their possible association with AR amplification and structural rearrangements as well as their ability to predict patient resistance to AR targeting drugs. The review will also discuss AR signalling in the tumour microenvironment and its possible relevance for metastatic growth and therapy.

High levels of the AR‐V7 Splice Variant and Co‐Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases

The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration‐resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid‐converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long‐term goal of identifying novel molecular targets for treatment.

A Systems Approach to Prostate Cancer Classification : Letter

A molecular classification system with clinical impact has been lacking for prostate cancer until You and colleagues published “Integrated Classification of Prostate Cancer Reveals a Novel Luminal Subtype with Poor Prognosis” ([1][1]). Gene expression profiles were used to stratify localized

Bone Cell Activity in Clinical Prostate Cancer Bone Metastasis and Its Inverse Relation to Tumor Cell Androgen Receptor Activity

Advanced prostate cancer frequently metastasizes to bone and induces a mixed osteoblastic/osteolytic bone response. Standard treatment for metastatic prostate cancer is androgen-deprivation therapy (ADT) that also affects bone biology. Treatment options for patients relapsing after ADT are limited, particularly in cases where castration-resistance does not depend on androgen receptor (AR) activity. Patients with non-AR driven metastases may, however, benefit from therapies targeting the tumor microenvironment. Therefore, the current study specifically investigated bone cell activity in clinical bone metastases in relation to tumor cell AR activity, in order to gain novel insight into biological heterogeneities of possible importance for patient stratification into bone-targeting therapies. Metastasis tissue obtained from treatment-naïve (n = 11) and castration-resistant (n = 28) patients was characterized using whole-genome expression analysis followed by multivariate modeling, functional enrichment analysis, and histological evaluation. Bone cell activity was analyzed by measuring expression levels of predefined marker genes representing osteoclasts (ACP5, CTSK, MMP9), osteoblasts (ALPL, BGLAP, RUNX2) and osteocytes (SOST). Principal component analysis indicated a positive correlation between osteoblast and osteoclast activity and a high variability in bone cell activity between different metastases. Immunohistochemistry verified a positive correlation between runt-related transcription factor 2 (RUNX2) positive osteoblasts and tartrate-resistant acid phosphatase (TRAP, encoded by ACP5) positive osteoclasts lining the metastatic bone surface. No difference in bone cell activity was seen between treatment-naïve and castration-resistant patients. Importantly, bone cell activity was inversely correlated to tumor cell AR activity (measured as AR, FOXA1, HOXB13, KLK2, KLK3, NKX3-1, STEAP2, and TMPRSS2 expression) and to patient serum prostate-specific antigen (PSA) levels. Functional enrichment analysis indicated high bone morphogenetic protein (BMP) signaling in metastases with high bone cell activity and low tumor cell AR activity. This was confirmed by BMP4 immunoreactivity in tumor cells of metastases with ongoing bone formation, as determined by histological evaluation of van Gieson-stained sections. In conclusion, the inverse relation observed between bone cell activity and tumor cell AR activity in prostate cancer bone metastasis may be of importance for patient response to AR and/or bone targeting therapies, but needs to be evaluated in clinical settings in relation to serum markers for bone remodeling, radiography and patient response to therapy. The importance of BMP signaling in the development of sclerotic metastasis lesions deserves further exploration.

Abstract A11: Categorization of castration-resistant prostate cancer bone metastases according to expression of steroid-converting enzymes and constitutively active androgen receptor variants

Abstract Patients with advanced prostate cancer (PC) are given androgen ablation therapy. After a period of initial remission tumors eventually relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors express the androgen receptor (AR) and AR regulated genes. Possible mechanisms behind AR activity in CRPC include steroid synthesis by tumor cells and expression of constitutively active AR variants. We have previously found high levels of cholesterol in prostate cancer bone metastases that theoretically could be the substrate for CYP11 and intra-tumoral synthesis of steroids (Thysell et al, 2010). Steroid-converting enzymes have, as far as we know, not been thoroughly examined in PC bone metastases, and the aim of this study was therefore to analyze the expression of the main enzymes involved in the conversion of cholesterol into testosterone (T) and dihydrotestosterone (DHT) in hormone-naive (HN) and CRPC bone metastases in comparison to primary tumors. We have previously also shown that the constitutively active AR variant AR-V7 is expressed at high levels in CRPC bone metastases in patients with particularly poor prognosis (Hornberg et al, 2011). Another aim of this study was therefore to evaluate if CRPC bone metastases could be differentiated according to expression of steroid-converting enzymes and AR-V7 into sub-groups with potentially different mechanisms behind castration-resistance. Expression levels of the enzymes included in the classical and backdoor steroidogenesis pathways were analyzed by applying an Illumina whole-genome array to total RNA extracted from frozen biopsies of prostate tissue obtained at radical prostatectomy and metastasis surgery, respectively. Levels were compared between HN bone metastases (n=10), CRPC bone metastases (n=30), and non-malignant (n=12) and malignant (n=13) areas of the primary tumors. Results were verified using RT-PCR and immunohistochemistry in an extended series of samples. Significant increased gene expression levels of HSD17B10 were found in CRPC compared to HN bone metastases, which possibly indicated enhanced conversion of androstanediol to DHT in CRPC metastases. The CRPC metastases did however not show higher levels of CYP11, CYP17, AKR1C3, HSD3B2, or SRD5A1/2 than the HN metastases, indicating no general induction of the classical steroidogenesis pathway in CRPC bone metastases. The expression levels of AKR1C3 and AKR1C2 were significantly higher, and of SRD5A2 significantly lower, in bone metastases than in primary tumors, with highly variable levels found for AKR1C3. About 20% of the CRPC metastases expressed extremely high levels of AKR1C3 which suggested the likelihood of androstenedione conversion into T in those tumors. Another 20% of the CRPC metastases expressed extreme values of the constitutively active AR-V7. In conclusion, AKR1C3 and AR-V7 expression in bone metastases were able to categorize CRPC patients according to potentially different mechanisms behind castration-resistance, and may furthermore also influence patient response to different types of anti-androgen therapies. Citation Format: Emma Jernberg, Anders Widmark, Sead Crnalic, Anders Bergh, Pernilla Wikstrom. Categorization of castration-resistant prostate cancer bone metastases according to expression of steroid-converting enzymes and constitutively active androgen receptor variants [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A11.