Probes for INS

Abstract

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition, and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high grade prostatic intraepithelial neoplasia or PIN), and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in 8 cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma, and its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target.

Kinase activation by chromosomal translocations is a common mechanism that drives tumorigenesis in spitzoid neoplasms. To explore the landscape of fusion transcripts in these tumors, we performed whole-transcriptome sequencing using formalin-fixed, paraffin-embedded (FFPE) tissues in malignant or biologically indeterminate spitzoid tumors from 7 patients (age 2-14 years). RNA sequence libraries enriched for coding regions were prepared and the sequencing was analyzed by a novel assembly-based algorithm designed for detecting complex fusions. In addition, tumor samples were screened for hotspot TERT promoter mutations, and telomerase expression was assessed by TERT mRNA in situ hybridization (ISH). Two patients had widespread metastasis and subsequently died of disease, and 5 patients had a benign clinical course on limited follow-up (mean: 30 months). RNA sequencing and TERT mRNA ISH were successful in six tumors and unsuccessful in one disseminating tumor because of low RNA quality. RNA sequencing identified a kinase fusion in five of the six sequenced tumors: TPM3-NTRK1 (2 tumors), complex rearrangements involving TPM3, ALK, and IL6R (1 tumor), BAIAP2L1-BRAF (1 tumor), and EML4-BRAF (1 disseminating tumor). All predicted chimeric transcripts were expressed at high levels and contained the intact kinase domain. In addition, two tumors each contained a second fusion gene, ARID1B-SNX9 or PTPRZ1-NFAM1. The detected chimeric genes were validated by home-brew break-apart or fusion fluorescence in situ hybridization (FISH). The two disseminating tumors each harbored the TERT promoter -124C>T (Chr 5:1,295,228 hg19 coordinate) mutation, whereas the remaining five tumors retained the wild-type gene. The presence of the -124C>T mutation correlated with telomerase expression by TERT mRNA ISH. In summary, we demonstrated complex fusion transcripts and novel partner genes for BRAF by RNA sequencing of FFPE samples. The diversity of gene fusions demonstrated by RNA sequencing defines the molecular heterogeneity of spitzoid neoplasms.

Telomerase is reactivated in most cancers and is possibly an early driver event in melanoma. Our aim was to test a novel in situ hybridization technique, RNAscope, for the detection of human telomerase reverse transcriptase (hTERT) mRNA in archival formalin-fixed, paraffin-embedded (FFPE) tissue and to compare the mRNA expression of melanomas and benign naevi. Furthermore, we wanted to see if hTERT mRNA could be a diagnostic or prognostic marker of melanoma. In situ hybridization for the detection of hTERT mRNA was performed on FFPE tissue of 17 melanomas and 13 benign naevi. We found a significant difference in the expression of hTERT mRNA between melanomas and benign naevi (P<0.001) and the expression of hTERT mRNA correlated with Breslow thickness (ρ=0.56, P=0.0205) and the Ki67 proliferation index (ρ=0.72, P=0.001). This study showed that RNAscope was a reliable in situ hybridization method for the detection of hTERT mRNA in FFPE tissue of melanomas and benign naevi. hTERT mRNA was more abundantly expressed in melanomas compared with benign naevi, but cannot be used solely as a diagnostic marker due to an overlap in expression. The hTERT mRNA expression in melanomas correlated with the prognostic markers Breslow thickness and the Ki67 index indicating a prognostic potential of hTERT mRNA.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited.

Prostate cancer remains dependent on androgen receptor signaling even after castration. Aberrant androgen receptor signaling in castration resistant prostate cancer is mediated by mechanisms such as alterations in the androgen receptor and activation of interacting signaling pathways. Clinical evidence confirms that resistance to the next generation anti-androgen, enzalutamide, may be mediated to a large extent by alternative splicing of the androgen receptor to generate constitutively active splice variants such as AR-V7. The splice variants AR-V7 and Arv567es have been implicated in the resistance to not only enzalutamide, but also to abiraterone and other conventional therapeutics such as taxanes. Numerous studies including ours suggest that splicing factors such as hnRNPA1 promote the generation of AR-V7, thus contributing to enzalutamide resistance in prostate cancer cells. In the present study, we discovered that quercetin, a naturally occurring polyphenolic compound, reduces the expression of hnRNPA1, and consequently, that of AR-V7. The suppression of AR-V7 by quercetin resensitizes enzalutamide-resistant prostate cancer cells to treatment with enzalutamide. Our results indicate that quercetin downregulates hnRNPA1 expression, downregulates the expression of AR-V7, antagonizes androgen receptor signaling, and resensitizes enzalutamide-resistant prostate cancer cells to enzalutamide treatment in vivo in mouse xenografts. These findings demonstrate that suppressing the alternative splicing of the androgen receptor may have important implications in overcoming the resistance to next-generation anti-androgen therapy.

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.

Treatment with androgen-targeted therapies can induce upregulation of epithelial plasticity pathways. Epithelial plasticity is known to be important for metastatic dissemination and therapeutic resistance. The goal of this study is to elucidate the functional consequence of induced epithelial plasticity on AR regulation during disease progression to identify factors important for treatment-resistant and metastatic prostate cancer. We pinpoint the epithelial plasticity transcription factor, Snail, at the nexus of enzalutamide resistance and prostate cancer metastasis both in preclinical models of prostate cancer and in patients. In patients, Snail expression is associated with Gleason 9-10 high-risk disease and is strongly overexpressed in metastases as compared to localized prostate cancer. Snail expression is also elevated in enzalutamide-resistant prostate cancer cells compared to enzalutamide-sensitive cells, and downregulation of Snail re-sensitizes enzalutamide-resistant cells to enzalutamide. While activation of Snail increases migration and invasion, it is also capable of promoting enzalutamide resistance in enzalutamide-sensitive cells. This Snail-mediated enzalutamide resistance is a consequence of increased full-length AR and AR-V7 expression and nuclear localization. Downregulation of either full-length AR or AR-V7 re-sensitizes cells to enzalutamide in the presence of Snail, thus connecting Snail-induced enzalutamide resistance directly to AR biology. Finally, we demonstrate that Snail is capable of mediating-resistance through AR even in the absence of AR-V7. These findings imply that increased Snail expressionduring progression to metastatic disease may prime cells for resistance to AR-targeted therapies by promoting AR activity in prostate cancer.

Abstract

BACKGROUND:

Androgen receptor splice variant 7 (AR-V7) has been implicated in resistance to abiraterone and enzalutamide treatment in men with metastatic castration-resistant prostate cancer (mCRPC). Tissue- or cell-based in situ detection of AR-V7, however, has been limited by lack of specificity.

OBJECTIVE:

To address current limitations in precision measurement of AR-V7 by developing a novel junction-specific AR-V7 RNA in situ hybridization (RISH) assay compatible with automated quantification.

DESIGN, SETTING, AND PARTICIPANTS:

We designed a RISH method to visualize single splice junctions in cells and tissue. Using the validated assay for junction-specific detection of the full-length AR (AR-FL) and AR-V7, we generated quantitative data, blinded to clinical data, for 63 prostate tumor biopsies.

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:

We evaluated clinical correlates of AR-FL/AR-V7 measurements, including association with prostate-specific antigen progression-free survival (PSA-PFS) and clinical and radiographic progression-free survival (PFS), in a subset of patients starting treatment with abiraterone or enzalutamide following biopsy.

RESULTS AND LIMITATIONS:

Quantitative AR-FL/AR-V7 data were generated from 56 of the 63 (88.9%) biopsy specimens examined, of which 44 were mCRPC biopsies. Positive AR-V7 signals were detected in 34.1% (15/44) mCRPC specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 11.9% (range 2.7-30.3%). Positive detection of AR-V7 was correlated with indicators of high disease burden at baseline. Among the 25 CRPC biopsies collected before treatment with abiraterone or enzalutamide, positive AR-V7 detection, but not higher AR-FL, was significantly associated with shorter PSA-PFS (hazard ratio 2.789, 95% confidence interval 1.12-6.95; p=0.0081).

CONCLUSIONS:

We report for the first time a RISH method for highly specific and quantifiable detection of splice junctions, allowing further characterization of AR-V7 and its clinical significance.

PATIENT SUMMARY:

Higher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer.

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.