Probes for INS

Abstract

Abstract

Abstract

BACKGROUND:

Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer.

METHOD:

We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively.

RESULTS:

Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection.

DISCUSSION:

These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Androgen deprivation therapy (ADT) has been used to treat salivary duct carcinoma (SDC). The androgen receptor splice variant-7 (AR-V7) has been detected in castration-resistant prostate cancer (CRPC) and implicated in resistance to androgen receptor (AR)-targeted therapies. Given the potential role of AR/AR-V7 in SDC treatment, this study focuses on AR/AR-V7 expression in SDC specimens collected prior to ADT. RNA in situ hybridization (ISH) and immunohistochemistry (IHC) to detect total AR and AR-V7 were performed on formalin-fixed, paraffin-embedded SDC specimens from 23 patients. Full length AR (AR-FL) and AR-V7 transcripts were quantified in a subset of tumors by reverse transcription polymerase chain reaction (RT-PCR). Twenty SDCs were positive for total AR by ISH and IHC. Among AR positive SDCs, 70% (14/20) were positive for AR-V7 mRNA by ISH, while 15% (3/20) were positive for AR-V7 protein by IHC. The three SDCs which expressed the highest levels of AR-V7 were all from female patients; one of them expressed significant amount of AR-V7 and barely detectable AR-FL transcripts by RT-PCR. Immunohistochemistry expression of Forkhead box protein A1, prostate-specific antigen, prostatic acid phosphatase, NKX3.1 was observed in some SDCs regardless of patient gender. Five SDCs demonstrated strong human epidermal growth factor receptor 2 (HER2) expression. We conclude that treatment-naïve SDCs may express AR-V7 at levels comparable to or even exceeding the levels detected in CRPC. Our data support the feasibility to incorporate AR-V7 assessment via ISH and/or IHC in the ongoing clinical trials evaluating the therapeutic benefit of AR targeted therapies in SDC patients.

The presence and extent of cribriform pattern of prostate cancer portend recurrence and cancer death. Therelative expressions within this morphology of the prognostically adverse loss of PTEN, and the downstream inactivation of cell cycle inhibitor p27/Kip1 had been uncertain. In this study, we examined 52 cases of cribriform cancer by immunohistochemistry (IHC) for PTEN, p27, and CD44 variant (v)7/8, and a subset of 17 casesby chromogenic in situ hybridization (ISH) using probe for PTEN or CDKN1B (gene for p27). The fractions of epithelial pixels positive by IHC and ISH were digitally assessed for benign acini, high grade prostatic intraepithelial neoplasia (PIN), and 8 morphological patterns of cancer. Immunostaining results demonstrated that: 1. PTEN loss was significant for fused small acini, cribriform-central cells, small cribriform acini, and Gleason grade 5 cells in comparison with other acini. 2. p27 loss was significant only for cribriform-peripheral cells; and borderline-significant for fused small acini in comparison with benign acini. 3. CD44v7/8 showed expression loss in cribriform-peripheral cells; other comparisons were not significant. ISH showed thatcribriform cancer had significant PTEN loss normalized to benign acini (P < .02), while Gleason 3 cancer or fused small acini did not. With CDKN1B, the degree of signal loss among various cancer morphologies was insignificant. In conclusion, molecular disparities emerged between the fused small acini and cribriform patterns of Gleason 4 cancer. PTEN or p27 loss as prognostic factors demand distinct assessment in the varieties of Gleason 4 cancer, and in the biphenotypic peripheral versus central populations in cribriform structures.

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3,theactive form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted "Swiss cheese-like" cribriform morphology (CM) comprising multiple abnormal "back to back" lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.

Abstract

Background
The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide.

Objective
To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC.

Design, setting, and participants
Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis.

Outcome measurements and statistical analysis
Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined.

Results and limitations
Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5–90) compared to CRPC (HS 135, IQR 80–157.5; p < 0.0001), and in biopsy tissue taken before (HS 80, IQR 30–136.3) compared to after (HS 140, IQR 105–167.5; p = 0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004–1.020; p = 0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins.

Conclusions
We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC.

Patient summary
In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time.

Advanced prostate cancer initially responds to androgen deprivation therapy (ADT), but the disease inevitably recurs as castration-resistant prostate cancer (CRPC). Although CRPC initially responds to abiraterone and enzalutamide, the disease invariably becomes non-responsive to these agents. Novel approaches are required to circumvent resistance pathways and extend survival, but the mechanisms underlying resistance remain poorly defined. Our group previously showed the histone lysine-N-methyltransferase EZH2 to be overexpressed in prostate cancer and quantitatively associated with progression and poor prognosis. In this study, we screened a library of epigenetic inhibitors for their ability to render CRPC cells sensitive to enzalutamide and found that EZH2 inhibitors specifically potentiated enzalutamide-mediated inhibition of proliferation. Moreover, we identified antisense oligonucleotides (ASO) as a novel drug strategy to ablate EZH2 and AR expression, which may have advantageous properties in certain settings. RNA-seq, ChIP-seq, and ATAC-seq demonstrated that EZH2 inhibition altered the AR cistrome to significantly upregulate AR signaling, suggesting an enhanced dependence of CRPC cells on this pathway following inhibition of EZH2. Combination treatment with ASO targeting EZH2 and AR transcripts inhibited prostate cancer cell growth in vitro and in vivo better than single agents. In sum, this study identifies EZH2 as a critical epigenetic regulator of ADT resistance and defines ASO-based co-targeting of EZH2 and AR as a promising strategy for treatment of CRPC.

Immunohistochemical staining for Phosphatase and Tensin Homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC with 56% of samples on a mixed tumour tissue microarray (TMA) showing high expressionby ISH compared to 42% by IHC. On a prostate TMA 49% of cases showed high expression by ISH compared to 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumours clear over expression of PTEN mRNA on malignant epithelium compared to benign epithelium was frequently observed and quantified. The use of Spot Studio software in the mixed tumour TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumour samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumours (upto 3 fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumour can be explored with more confidence.