Hormone molecular biology and clinical investigation (Horm Mol Biol Clin Investig). The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) triggers apoptosis in breast cancer cells and adipocytes via the induction of the apoptotic Ca2+ signal - a sustained increase in concentration of. Gastrointestinal Hormones/chemistry; Gastrointestinal Hormones/genetics; Gastrointestinal Hormones/metabolism.; Gene Expression Regulation; Humans.
Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole-genome RNA sequencing, gene set enrichment analysis, and immunohistochemistry. Our analyses revealed 5 mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: AR-high tumors (ARPC), AR-low tumors (ARLPC), amphicrine tumors composed of cells coexpressing AR and NE genes (AMPC), double-negative tumors (i.e., AR –/NE –; DNPC), and tumors with small cell or NE gene expression without AR activity (SCNPC).
RE1 silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion.
We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the 5 mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design. The androgen receptor (AR) regulates cellular programs that promote the survival and proliferation of prostate cancer (PC) cells. Consequently, first-line treatment for metastatic PC centers on inhibiting AR activity through androgen deprivation therapy (ADT), resulting in the suppression of AR target genes and clinical remissions that generally last several years ( ).
However, ADT is not curative. PC recurs as castration-resistant prostate cancer (CRPC), typically with reactivated AR signaling. Second-generation AR pathway inhibitors (ARIs), such as enzalutamide (ENZ) and abiraterone (ABI), were designed to further repress AR signaling and are primarily used to treat CRPC.
Although these agents extend survival, durable complete responses are rare and these therapies also eventually fail (, ).Typically, the vast majority of metastatic CRPC (mCRPC) tumors progress with rising prostate-specific antigen (PSA/KLK3) levels despite standard of care treatment. Moreover, most mCRPC tumors are adenocarcinomas, which have robust AR program activity ( ). Though rigorous epidemiological data are lacking, recent studies report that a substantial number of mCRPC tumors progressing on ARIs have lost AR signaling ( ). Paralleling increased use of ARIs has been an increase in the proportion of treatment-resistant CRPC metastases that have AR-null phenotypes, i.e. Tumors with diffuse small cell or neuroendocrine (NE) characteristics (SCNPC) or the recently described double-negative (DNPC) phenotype that lacks both NE and AR activity ( ).
A contemporary study evaluating the histology and molecular characteristics of 202 men with mCRPC found that 17% of the evaluable tumors were classified as SCNPC and this phenotype was associated with shortened survival ( ). Notably, a subset of tumors exhibited discordance between the pathological assessment of SCNPC and gene expression programs. Other tumors exhibited mixed phenotypes of NE features concurrent with AR activity, raising questions of intratumoral heterogeneity due to mixtures of cell types ( ).In addition to histology, the classification of cancers into subtypes with distinct functional features has been supplemented with transcript profiles. For example, transcript signatures subdivide breast cancers into groups that exhibit distinct outcomes ( ). This subclassification, based on gene expression levels, has also been applied to localized PC and shown to associate with treatment outcomes (, ).
In this study, we sought to characterize the phenotypic diversity of treatment-refractory mCRPC using both histological assessments and gene expression profiling. In addition to SCNPC and DNPC, we identified adenocarcinomas that had high AR activity (ARPC), measurable but low AR activity (ARLPC), and amphicrine tumors comprised of cells exhibiting both AR and NE activity (AMPC). Notably, a subset of DNPC tumors exhibited squamous differentiation. We determined that the relationships of 2 major factors regulating cell differentiation states, AR and RE1 silencing transcription factor (REST), associated with these mCRPC subtypes. We generated a 26-gene transcriptional signature that could be clinically useful for classifying mCRPC phenotypes and prioritizing treatment. Treatment-refractory mCRPC exhibits diverse phenotypes. To assess the diversity of phenotypes present following resistance to therapeutics used to treat metastatic PC, we evaluated 98 tumors obtained at rapid autopsy from 55 men between 2003 and 2017.
Patient demographics and clinical data are summarized in; supplemental material available online with this article; All patients received ADT. The median duration of treatment was 4.2 years (range, 0.3–15.1 years). Patients also received a variety of other drugs including docetaxel ( n = 44; 80%), abiraterone ( n = 8; 15%), enzalutamide ( n = 4; 7%) or both abiraterone and enzalutamide ( n = 17; 31%). Bone, lymph nodes, and liver were the most frequent sites of metastasis.To provide an initial evaluation of phenotypic diversity, we used histological assessments and immunohistochemical (IHC) analysis of proteins associated with AR-active adenocarcinomas (AR and PSA) and NE differentiation (chromogranin A, CHGA; synaptophysin, SYP). Molecular profiling of mCRPC reveals a heterogeneous disease.
( A) IHC of 5 mCRPC sites from patients using antibodies to AR, PSA, CHGA, and SYP. Scale bars: 20 μM.
( B) RNA-Seq heatmap of mCRPC specimens acquired through rapid autopsy from 2003–2017 ( n = 98). REST-repressed NE genes are listed in the NEURO I panel (top), NE transcription factors are listed in the NEURO II panel (middle), and AR-associated genes are listed in the AR panel (bottom). Results are expressed as log 2 fragments per kilobase of transcript per million mapped reads (FPKM) and colored according to scale. ( C) Venn diagram showing the number of unique and shared upregulated genes between phenotypes relative to ARPC (up 3-fold; P 3-fold and P. Disease progression is a continuum in mCRPC specimens. ( A) IHC of different mCRPC sites from patient 13-084. Site PP7 (bone; ARPC), II2 (bone; ARLPC), PP7 (bone; DNPC), and H1 (liver, SCNPC).
Primary antibodies were directed toward pan-cytokeratin, AR, PSA, CHGA, and SYP. Insets for AR and PSA staining are images of the same section using the ×400 objective lens. Original magnification 40×. ( B) IHC of LuCaP 173.2 tumor sections from passages 2, 4, 7, 8, and 11 using a SYP antibody. Black arrows point to clusters of cells with SYP positivity. Magnification 100×. ( C) RNA-Seq heatmap and NEURO score of LuCaP 173.1 and serial passages from LuCaP 173.2.
Results are expressed as log 2 FPKM or as enrichment scores and are colored according to scale.The amphicrine phenotype and relationship with REST expression. Historically, SCNPC was considered to lack AR activity but recent studies have identified atypical tumors with NE features that express AR and exhibit AR-mediated signaling ( ). In addition, classification of the AMPC phenotype based on bulk RNA-Seq or IHC using single markers may be due to tumors comprised of multiple cell types (AR +/NE – and AR –/NE +) or may be due to tumors comprised of individual cells expressing both AR and NE differentiation markers simultaneously. Thus, to establish the existence of AMPC cells in our clinical specimens (i.e., cells with both AR transcriptional output and neuroendocrine features; AR +/NE +), we stained LuCaP PDX tumors and patient metastases with immunofluorescent (IF) antibodies to PSA and SYP. AMPC LuCaP 77CR tumors contained numerous cells coexpressing PSA and SYP. Though PSA and SYP coexpression normally occurred throughout LuCaP 77CR tumors, we also identified a subset of tumors with focal SYP expression (unpublished observations).
Furthermore, we used IF to characterize 6 AMPC metastases from 4 patients. The patient tissues used for IF analysis were adjacent to the specimens used for RNA-Seq. Importantly, IF demonstrated clear PSA and SYP coexpression in patient tumor cells.
However, patient 13-042 site M3 and patient 17-033 site J1 showed regions that were mixed of distinct populations of ARPC and SCNPC tumor cells. REST splicing occurs in AMPC and SCNPC phenotypes. ( A) Immunofluorescence of an AMPC LuCaP 77CR tumor using PSA (green) and SYP (red) antibodies. Sections were counterstained with DAPI (blue) and top panels represent LuCaP 77CR PDX sections stained with secondary antibody only.
Scale bars: 20 μM. ( B) Immunoblot of LuCaP PDX specimens probing for REST, AR, and SYP. ACTB was used as a loading control. Short, 10-second film exposure; long, 5-minute film exposure. ( C) PCR of LuCaP PDX specimens using primers specific to REST shows the REST4 insertion sequence appearing in AMPC (LuCaP 77CR) and SCNPC (LuCaP 93, 145.2, and 173.1) but not in DNPC (LuCaP 173.2) or ARPC (LuCaP 86.2 and 73). ( D) RNA-Seq heatmap of VCaP cells displaying NE-associated genes (NEURO I and NEURO II) and AR-associated genes.
Results are expressed as log 2 FPKM and colored according to scale. ( E) Immunoblot of C4-2B, VCaP, and LuCaP 93 whole-cell extracts using antibodies against AR, REST, SYP, and ACTB. ACTB was used as a loading control. ( F) PCR of C4-2B, VCaP, and NCIH660 cells using primers specific to REST. The upper band represents the REST4 splice variant.Previously, we interrogated SCNPC patient tumors and PDX lines and determined that SCNPC is associated with loss of REST repressor activity ( ). However, the role of REST in other mCRPC phenotypes has not been evaluated.
Using representative PDX lines from ARPC (LuCaP 35CR and 96CR), AMPC (LuCaP 77CR), ARLPC (LuCaP 176), DNPC (LuCaP 173.2), and SCNPC (LuCaP 173.1 and 93), we found that full-length REST protein was decreased in both AMPC and SCNPC tumors. Interestingly, we observed REST species with our REST C-terminus antibody at both approximately 120 KDa and approximately 200 KDa in PDX lysates from ARPC, ARLPC, and DNPC. Full-length REST protein is predicted to be 116 KDa but can be O-glycosylated and readily detected at approximately 200 KDa (, ). Additionally, the REST transcript has multiple splice variants that produce truncated proteins ( ).
Nevertheless, both REST proteins with intact C-terminus repressor domains were diminished in AMPC and SCNPC PDX models. Moreover, AMPC LuCaP 77CR showed robust expression of both AR and SYP protein, whereas ARLPC LuCaP 176 had low AR protein expression.Next, we examined alternative splicing of the REST transcript. The RNA splicing factor SRRM4 splices the REST transcript to REST4, resulting in the loss of the C-terminus repressor domain and diminished REST transcriptional repression (, ). To examine SRRM4-mediated splicing of REST in mCRPC phenotypes, we conducted PCR using REST primers spanning the SRRM4 splice site and determined that REST4 splicing events occurred exclusively in AMPC and SCNPC LuCaP PDX models. However, instead of a previously reported 62 bp insertion into the REST transcript ( ), band sequencing identified a 50 bp insertion, suggesting a mechanism of REST4 splicing in AMPC and SCNPC phenotypes similar to small cell lung cancer ( ). Taken together, these data indicate that the AMPC phenotype arises from the loss of REST transcriptional repression.To further support the existence of the AMPC phenotype, we interrogated the VCaP PC cell line, which exhibits an amphicrine-like transcript profile.
RNA-Seq confirmed that VCaP cells express AR-associated genes and the REST-repressed NEURO I genes but do not express the NEURO II transcription factors that drive the SCNPC phenotype. Immunoblot analysis determined that VCaP cells express considerable AR and SYP protein and have diminished full-length REST protein expression compared with C4-2B cells. Immunofluorescence validated that both PSA and SYP are coexpressed in the same VCaP cell and PCR analysis of REST determined that REST transcripts are alternatively spliced to REST4, similar to neuroendocrine NCIH660 cells. Notably, the growth of VCaP cells is inhibited by ADT or exposure to the AR antagonist enzalutamide ( ). In addition, 22Rv1 cells also exhibit features of AMPC as they have attenuated REST expression and appreciable SYP and AR protein expression ( ). Taken together, these results confirm the existence of AMPC cells in patient specimens and in CRPC models in vitro and in vivo, and suggest that the AMPC phenotype is still driven by AR activity and is responsive, at least transiently, to AR pathway repression.REST knockdown in CRPC cells promotes an amphicrine phenotype.
To examine the impact of the loss of REST activity in AR-expressing and AR-null cell lines, we conducted knockdown studies using siRNAs directed to REST (siREST) or a negative control (siNCT) in C4-2B, PC-3, and PacMet AR-null cells. PacMetUT1 cells were modified using CRISPR-Cas9–mediated editing to knockout AR expression and were characterized previously (, ). REST knockdown led to increases in the NE-associated protein SYP. Interestingly, REST depletion in AR-expressing C4-2B cells did not alter AR protein expression or the magnitude of AR transcriptional output. However, this does not rule out the possibility that complete loss of REST could alter the AR transcriptional output.
Transcript profiling by RNA-Seq and subsequent GSEA showed that REST ablation in C4-2B, PC-3, and PacMet AR-null cells led to significant upregulation of known REST-repressed genes (up 3-fold; P. REST knockdown in AR-expressing and AR-null CRPC cell lines. ( A) Immunoblot of REST, AR, SYP, and ACTB using C4-2B, PC-3, and PacMet AR-null cells transfected with either REST siRNA (siREST) or negative control siRNA (siNCT). ( B) AR activity scores assessed in C4-2B cells transfected with siNCT ( n = 2) or siREST ( n = 2) by RNA-Seq.
( C) RNA-Seq heatmap of the 24 common upregulated genes (up 3-fold; P 3-fold; P. DNPC can convert to a squamous phenotype. ( A) H&E staining of mCRPC tissues from LuCaP 173.2 and patient 13-084.
Black arrows point to squamous pearl structures. ( B) Expression of squamous cell lung cancer associated genes from RNA-Seq of LuCaP 173.2 DNPC cells and squamous pearl (SP) cells isolated by laser capture microdissection. Results are expressed as log 2 FPKM and colored according to scale. ( C) IHC of specimens from LuCaP 173.2 and patient 13-084 using KRT6 antibody or IgG as a negative control.
( D) H&E staining (left panels) and KRT6 IHC (right panels) of DNPC tumor sections from patients 11-028 and 13-099. Scale bars: 20 μM.Examination of 4 other patients with DNPC metastases identified squamous pearls with positive KRT6 staining in 2 patients: 11-028 and 13-099. Interestingly, patient 11-028 had an adenocarcinoma phenotype in the initial prostate biopsy and was subsequently treated with diethylstilbesterol (DES) for 13 months prior to cystoprostatectomy. At the time of cystoprostatectomy, histology and IHC revealed adenocarcinoma with focal basaloid and squamous differentiation in several sections of the prostate as well as a left axillary lymph node that was consistent with squamous carcinoma. The other 2 patients with KRT6-positive metastases, patients 13-084 and 13-099, had primary prostate cancers with no evidence of squamous differentiation, and subsequent hormone therapy led to the appearance of squamous mCRPC. Although DES and hormone therapies have been linked to the development of squamous cancer in the prostate with subsequent squamous metastases ( – ), this report provides evidence for hormone therapy–mediated conversion of ARPC to squamous DNPC at metastatic sites.We compared the top significantly (FDR. Expression of squamous markers is associated with DNPC and ARLPC.
RNA-Seq heatmap of patient specimens ( n = 98) highlighting AR-regulated genes and genes associated with squamous pearl cells (SQUAM). Results are expressed as log 2 FPKM and colored according to scale.Transcript signatures define the molecular phenotypes of mCRPC. The variability in expression of any single marker, both at the biological level and technical level makes tumor classification by immunohistochemistry challenging. Transcript panels for tumor classification have been explored as predictive and prognostic biomarkers for treatment decision-making (, ).
We leveraged the data generated from the patient metastases and LuCaP PDX models to develop a 26-gene transcriptomic signature for defining treatment-refractory mCRPC phenotypes. Using the aforementioned AR, NEURO I, and NEURO II gene panels, as well as a squamous panel (SQUAM) that includes KRT5, KRT6A, KRT6B, and FGFBP1, we conducted multidimensional scaling (MDS) and cluster analysis of the patient metastases and LuCaP PDX models ( and ). The MDS demonstrated clear distinction between the 5 mCRPC phenotypes (ARPC, ARLPC, AMPC, DNPC, and SCNPC) in both patient specimens and LuCaP PDX models.
Cluster analysis using AR, NE, and squamous gene expression profiles segregates mCRPC specimens and LuCaP PDX models into the different phenotypes. ( A) RNA sequencing of mCRPC specimens acquired between 2003–2017 ( n = 98; modified from ). NE genes listed in the NEURO I and NEURO II panels, AR and AR-regulated genes are listed in the AR panel, and squamous associated genes are shown in SQUAM panel. Results are expressed as log 2 FPKM and colored according to scale. Multidimensional scaling and cluster analysis of ( B) mCRPC specimens ( n = 98) and ( C) LuCaP PDX models using the 26-gene set depicted in A.
The LuCaP analysis was conducted on 18 distinct PDX lines ( n = 2 for each line). ARPC (AR +/NE –; green), ARLPC (AR low/NE –; purple), DNPC (AR –/NE –; blue), AMPC/mixed (AR +/NE +; red), SCNPC (AR –/NE +; yellow).We further evaluated our 26-gene signature using PolyA RNA-Seq landscapes from 270 CRPC metastases in the Stand Up To Cancer (SU2C) data set ( ). Although the SU2C data set contains RNA-Seq from mCRPC tumors earlier in disease progression and from tumors that are responding to treatment, the transcriptional signature segregated the tumors into the 5 mCRPC phenotypes. Interestingly, we detected expression of the squamous-associated genes in 2 DNPC tumors and 2 ARLPC tumors but also observed marked squamous marker expression in 4 ARPC tumors and 1 SCNPC tumor. Thus, removing the squamous genes from the analysis showed a more effective clustering of the tumors into their respective phenotypes.
We do not know if the ARPC and SCNPC specimens with squamous aspects represent tumors containing 2 different phenotypes or single phenotypes. The clinical phenotyping of mCRPC has been limited to morphologic and immunohistochemical analyses.
Although adenocarcinoma (AR and PSA) and NE (CHGA and SYP) biomarkers have provided some clarity for pathologic classification, the complexity of tumor heterogeneity, and the emergence of new treatment-resistant phenotypes have catalyzed a need for deeper understanding of the mCRPC disease continuum. Moreover, anaplastic tumors or aggressive variants are a clinically defined group of small cell metastatic/CRPC phenotypes with varying degrees of both AR expression (generally AR-null) and NE differentiation (, ). The classifications prompted a call for further elucidation of underlying mCRPC biology and more accurate nomenclature that limits confusion between research and medical fields ( ). At the transcriptome level, expression signatures for classifying SCNPC have been demonstrated ( ), but an encompassing signature that appreciates the spectrum of mCRPC phenotypes has not been identified. In this report, we interrogated end-stage mCRPC patient specimens and treatment-resistant LuCaP PDX models and demonstrated that transcriptome analysis in conjunction with IHC is a powerful method for phenotyping mCRPC in the current era.
Our approach led to the characterization of 5 distinct mCRPC phenotypes (AR-high/ARPC, AR-low/ARLPC, amphicrine/AMPC, double-negative/DNPC, and small cell or NE tumors lacking AR expression/SCNPC) and ultimately resulted in a clinically relevant 26-gene transcriptional signature to classify mCRPC biospecimens. Schematic of the mCRPC disease continuum. The proposed mechanisms, molecular drivers, and cellular differentiation states following AR pathway inhibition therapy. ADT, androgen deprivation therapy; ABI, abiraterone; ENZ, enzalutamide; PC, hormone-sensitive prostate cancer; ARPC, AR-high prostate cancer; ARLPC, AR-low prostate cancer; SCNPC, small cell or NE prostate cancer; DNPC, double-negative prostate cancer; AMPC, amphicrine prostate cancer; SQUAPC, squamous prostate cancer.The analyses of DNPC tumors and the LuCaP 173.2 PDX model unexpectedly revealed the appearance of squamous cell pearls within the mass of DNPC tumor cells. RNA-Seq and staining for KRT6 confirmed the molecular nature of the squamous pearls.
We hypothesize that only the most differentiated squamous carcinoma cells stain positive for KRT6 and display a cornified GSEA profile and that there exists a proliferating DNPC/squamous intermediary. Support for this hypothesis comes from RNA-Seq and IHC that shows ARLPC metastases and LuCaP 176 significantly enriched for squamous transcriptional profiles but negative for squamous pearl structures and KRT6 staining. The mechanisms of ARLPC/DNPC to squamous transition remain unclear, but future research examining the parallels between prostate and lung cancer lineage plasticity is warranted. For example, prostate and lung epithelial cells can be reprogrammed to small cell NE cancers through induction of the same transcription factor pathways ( ). Moreover, lung adenocarcinomas can transition to squamous cell carcinomas through LKB1-loss ( ).
Our RNA-Seq data show no evidence LKB1/STK11-loss across patient samples but other mechanisms of adenocarcinoma-squamous differentiation are likely. We speculate that there are common molecular pathways driving lung and prostate cancer lineage switching. Taken together, our data demonstrate that treatment-induced ARPC-DNPC-squamous conversion is one potential pathway to bypass AR-suppression strategies.There are currently no standard treatments for SCNPC, DNPC, and ARLPC phenotypes. However, RNA-Seq and GSEA between mCRPC phenotypes revealed biologically relevant pathways that could be further interrogated for therapeutic benefit. Significantly upregulated genes common to the AR-null and ARLPC phenotypes were enriched for cell adhesion processes, and delving into the pathway revealed CEACAM5 as a top hit. CEACAM5 has been identified as a surface marker of potential utility in directing chimeric antigen T cells in SCNPC ( ), and our data reveal that this therapy could also be clinically effective against subsets of ARLPC and DNPC tumors. Moreover, upregulated genes common to ARLPC and AR-null phenotypes are enriched in immune system, inflammatory, and defense responses.
Since the phenotype comparisons were relative to ARPC, these data support the notion that the immune cell content of ARLPC and AR-null tumors is strikingly different from that of ARPC tumors. Notably, DNPC-specific immune-related genes included IL8 and CXCR1, which have been reported to promote CRPC metastasis and angiogenesis ( ), and genes such as TGFB and RUNX2, which support tumor growth in bone ( ). Whether associated with the tumor cells, immune cells, or stromal cells, the presence of enriched immunomodulatory signaling pathways suggests that ARLPC, DNPC, and SCNPC tumors could be more receptive to immunotherapies.Our group and others have previously demonstrated that loss of REST repressor activity promotes the SCNPC phenotype (, ). Here, we confirmed the existence of AMPC cells in vivo and in vitro and demonstrated that AMPC cells express a limited set of neuronal genes that are REST-repressed (NEURO I panel) and maintain AR signaling.
Furthermore, transcriptome analysis of mCRPC and LuCaP PDX tumors and siRNA-mediated knockdown of REST in AR-expressing and AR-null cell lines provided compelling evidence that loss of REST repressor activity is critical for conversion to the AMPC/mixed phenotype but does not necessarily drive the SCNPC phenotype. We realize that siRNA treatments transiently relieve REST-mediated transcriptional repression, and that the impact of sustained REST-ablation remains to be determined. However, we hypothesize that epigenetic factors are preventing the expression of core SCNPC transcription factors (NEURO II panel of genes) that would permit SCNPC transdifferentiation in AR-null and AR-expressing cells with REST knockdown.
Indeed, the epigenomes of NE and adenocarcinoma tumors are significantly different and EZH2 inhibitors have been shown to reactivate AR expression in some SCNPC models (, ). On the other hand, SRRM4-mediated splicing of REST and other neuronal regulators can drive ARPC-SCNPC transition ( ).
Our results demonstrated that SRRM4-mediated splicing of REST occurs in both AMPC and SCNPC PDX models and in AMPC VCaP cells, suggesting that SRRM4 expression alone is not sufficient to drive SCNPC conversion in all cases. Concordantly, overexpression of SRRM4 converted DU145 cells to a classical SCNPC phenotype but failed to do so in PC-3, 22Rv1, and LNCaP cells ( ), implying that multiple hits to the cellular blueprint are required for complete lineage switching. Furthermore, REST directly represses SRRM4 expression and loss of REST activity has been proposed as a feed-forward mechanism for SCNPC conversion (, ). Whether loss of REST activity mediates increased SRRM4 expression or increased SRRM4 activity mediates loss of REST requires further examination.
Nevertheless, our data clearly show that the homeostatic regulation of the SRRM4-REST axis is required for epithelial differentiation and function.In summary, our comprehensive analysis of end-stage mCRPC highlights the use of AR and REST transcriptional programs to categorize mCRPC phenotypes in the abiraterone/enzalutamide era. In addition, the data generated in this report could be exploited through biopsy or blood-based biomarkers in future therapeutic studies to define inclusion criteria. This approach could stratify patients according to mCRPC phenotypes and account for the mCRPC disease continuum to implement targeted therapies. Tissue acquisition. Biospecimens were obtained within 8 hours of death from patients who died of metastatic CRPC. Visceral metastases were identified at the gross level, bone biopsies were obtained according to a previously described template ( ) from 16–20 different sites, and metastases were identified at a histological level. LuCaP PDX lines were established from specimens acquired at either radical prostatectomy or at autopsy, implanted, and maintained by serial passage in immune compromised male mice ( ).Cell lines.
All cells were maintained at 37°C in humidified Steri-Cult CO 2 incubators (Thermo Fisher Scientific). C4-2B (gift from L. Chung, Cedars-Sinai Medical Center, Los Angeles, CA), PC-3 (ATCC), and PacMet AR-null cells ( ) were maintained in RPMI-1640 Media (Gibco, Life Technologies) with 10% fetal bovine serum (Atlanta Biologicals). VCaP cells (ATCC) were maintained in DMEM (ATCC) with 10% fetal bovine serum (Atlanta Biologicals).Transient transfections.
Cells were suspended in Nucleofector Solution V (Lonza) and 5 μL of 50-μM ON-TARGETplus pooled REST siRNA or control siRNA (Dharmacon). Cell suspensions were electroporated using the Nucleofector II device (Lonza) and program T-027 and then replated in complete media. Forty-eight hours after transfection, cells were harvested for RNA or protein for subsequent analysis.Immunohistochemistry. The antibodies used in this study are listed in. Five-micron-thick sections of the TMAs were deparaffinized and rehydrated in sequential xylene and graded ethanol. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 30 minutes.
Endogenous peroxidase and avidin/biotin were blocked respectively (Vector Laboratories). Sections were then blocked with 5% normal goat-horse-chicken serum, incubated with primary antibody , incubated with biotinylated secondary antibody (Vector Laboratories), followed by ABC reagent (Vector Laboratories), and stable DAB (Thermo Fisher Scientific). All sections were lightly counterstained with hematoxylin and mounted with Cytoseal XYL (Richard Allan Scientific). Mouse or rabbit IgG was used as negative control.
IHC scores were represented as values between 0–200 and were determined as previously described ( ).Immunofluorescence. Formalin-fixed, paraffin-embedded (FFPE) patient and LuCaP PDX tumors were prepared according to the above methods up to and including antigen retrieval. For cells in culture, VCaP cells were seeded on chamber slides (Nunc Lab-Tek; Thermo Fisher Scientific) in complete media 24 hours prior to fixing. Cells were fixed and permeabilized with ice-cold methanol for 10 minutes and then washed in PBS. Cells and sections were blocked for 1 hour with 5% normal goat-horse-chicken serum, incubated for 1 hour (cells) or overnight (sections) with primary antibodies in blocking buffer, washed, and then incubated with fluorescent secondary antibodies for 45 minutes in the dark. All incubations occurred at room temperature and antibodies used are listed in. Slides were mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and then visualized and imaged at ×20 on an Olympus BX41 Fluorescence Microscope.Immunoblot analysis.
Protein extracts from LuCaP PDXs and cell lines were obtained using the Nuclear Extract Kit (Active Motif) according to the manufacturer’s protocols. Quantification of total protein was determined using the ProStain Protein Quantification Kit (Active Motif) according to the manufacturer’s protocols. Ten to twenty micrograms of total protein lysate was electrophoresed on 4%–15% Bis-Tris gels (Bio-Rad Laboratories) with 1× Tris/Glycine/SDS Buffer (Bio-Rad Laboratories). The proteins were transferred to PVDF that was blocked with 5% Blotting-Grade Blocker (Bio-Rad Laboratories) in TBS/0.1% Tween-20 and subsequently probed with primary and secondary antibodies. Proteins were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories).RNA isolation. Total RNA was isolated from 98 tissue samples of CRPC metastases, which had been frozen in OCT (Tissue-Tek) with RNA STAT-60 (Tel-Test).
Using an H&E-stained slide for each sample for orientation, 1-mm core punches of tumor were obtained. Alternatively, multiple sections enriched for tumor were cut using a Leica CM3050S cryostat. Total RNA was isolated from flash-frozen LuCaP PDX tissues or cell lines with RNA STAT-60 (Tel-Test) followed by purification with RNeasy Mini Kit (Qiagen) using the manufacturer’s recommended in-solution DNase digestion (Qiagen). The purity and yield of the RNA were determined on a NanoDrop 2000 (Thermo Fisher Scientific). RNA integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies).PCR and sequencing. First-strand cDNA synthesis was performed with 1 μg RNA using an Advantage RT-for-PCR Kit (Clontech Laboratories).
PCR was performed using either Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen; real-time PCR) or HotStarTaq Plus Master Mix (Qiagen; PCR for sequencing) on a Rotor-Gene Q (Qiagen). PCR primers (Integrated DNA Technologies) specific for REST were adapted from Raj et al.
The REST 5′ primer-GAGAACGCCCATATAAATGTGAAC and 3′ primer-CGGGTTACTTCATGTTGATTAGAG were used. The PCR reaction parameters were as follows: 50°C for 2 minutes and 95°C for 2 minutes (one cycle), followed by 30 cycles (standard gels) or 40 cycles (band sequencing) at 95°C for 10 seconds, annealing/extension at T(m) for 30 seconds, and 72°C for 30 seconds; the final extension was 72°C for 7 minutes. REST and REST4 PCR products were visualized after electrophoresis on a 1.2% agarose gel. For sequencing, PCR product bands were dissected out and purified using the QIAquick Gel Extraction Kit (Qiagen). The purified product was then sequenced by Eurofins Genomics using their Standard Read sequencing chemistry.RNA sequencing. RNA-Seq libraries were constructed from 1 μg total RNA using the Illumina TruSeq Stranded mRNA LT Sample Prep Kit according to the manufacturer’s protocol.
Barcoded libraries were pooled and sequenced on the Illumina HiSeq 2500 generating 50 bp paired end reads. Sequencing reads were mapped to the hg38 human using TopHat v2.1.0 ( ). For PDX samples, sequences were also aligned to the mm10 mouse genome and those derived from potential contamination with mouse tissue were removed from the analysis as previously described ( ). Gene level abundance was quantified from the filtered human alignments in R using the GenomicAlignments Bioconductor package ( ). Differential expression was assessed using transcript abundances as inputs to the edgeR Bioconductor package in R ( ).
For edgeR analysis, genes filtered for a minimum expression level of at least 1 count per million reads (CPM) in at least 2 samples were used to calculate expression differences using an exact test with a negative binomial distribution, applying a significance level of 0.05 with Benjamin-Hochberg false discovery rate (FDR) adjustment. The RNA-Seq data from this report can be accessed through GEO at GSE126078.Pathway analysis.
Gene expression results were ranked by their edgeR statistics and used to conduct Gene Set Enrichment Analysis (GSEA) to determine patterns of pathway activity in different treatment groups. We used the curated pathways from within the MSigDBv6.1. Gene ontology (GO) enrichment of Venn diagram groups was computed using GOrilla with default parameters ( ). The target and background list option was used and gene sets with enrichment of P.
MPL, LGB, PSN, and CM conceived and designed the project. IMC, RC, and AK performed sequencing and bioinformatics analyses. MPL, LGB, LK, BL, YCY, and ACH conducted molecular and cell biology experiments. LDT and RMG provided pathology evaluation.
HMN, EC, and CM provided biospecimens and patient-derived xenograft models. CSH, EYY, HHC, EAM, BM, MTS, DWL, and PSN consented patients for the rapid autopsy program and/or provided clinical expertise. MPL, PSN, and CM wrote the manuscript, and all authors reviewed and edited the manuscript.
TY - JOURT1 - Primary umbilical endometriosis successfully treated with dienogestAU - Kah Teik, ChewAU - Norsaadah, SalimAU - Aziz, SurayaAU - Erica Yee Hing, WongAU - Zainuddin, Ani AmeliaAU - Mohamed Ismail, Nor AzlinAU - Abdul Ghani, Nur AzurahPY - 2017/2/1Y1 - 2017/2/1N2 - Primary umbilical endometriosis (PUE) is a rare gynaecological disorder. A majority of the reported cases were treated with surgical excision as medical treatment had been found to be less successful. We reported a case of PUE successfully treated with dienogest without any profound side effects from the drug.AB - Primary umbilical endometriosis (PUE) is a rare gynaecological disorder. A majority of the reported cases were treated with surgical excision as medical treatment had been found to be less successful.
We reported a case of PUE successfully treated with dienogest without any profound side effects from the drug.KW - dienogestKW - endometriosisKW - primary umbilical endometriosis (PUE)UR -10.1515/hmbci-2016-0017DO - 10.1515/hmbci-2016-0017M3 - ArticleVL - 29SP - 67EP - 69JO - Hormone Molecular Biology and Clinical InvestigationJF - Hormone Molecular Biology and Clinical InvestigationSN - 1868-1883IS - 2ER.