Publications by authors named "Ladan Fazli"

220 Publications

A PRC2-independent function for EZH2 in regulating rRNA 2'-O methylation and IRES-dependent translation.

Nat Cell Biol 2021 Apr 1;23(4):341-354. Epub 2021 Apr 1.

Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Dysregulated translation is a common feature of cancer. Uncovering its governing factors and underlying mechanism are important for cancer therapy. Here, we report that enhancer of zeste homologue 2 (EZH2), previously known as a transcription repressor and lysine methyltransferase, can directly interact with fibrillarin (FBL) to exert its role in translational regulation. We demonstrate that EZH2 enhances rRNA 2'-O methylation via its direct interaction with FBL. Mechanistically, EZH2 strengthens the FBL-NOP56 interaction and facilitates the assembly of box C/D small nucleolar ribonucleoprotein. Strikingly, EZH2 deficiency impairs the translation process globally and reduces internal ribosome entry site (IRES)-dependent translation initiation in cancer cells. Our findings reveal a previously unrecognized role of EZH2 in cancer-related translational regulation.
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http://dx.doi.org/10.1038/s41556-021-00653-6DOI Listing
April 2021

A noncanonical AR addiction drives enzalutamide resistance in prostate cancer.

Nat Commun 2021 03 9;12(1):1521. Epub 2021 Mar 9.

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN, USA.

Resistance to next-generation anti-androgen enzalutamide (ENZ) constitutes a major challenge for the treatment of castration-resistant prostate cancer (CRPC). By performing genome-wide ChIP-seq profiling in ENZ-resistant CRPC cells we identify a set of androgen receptor (AR) binding sites with increased AR binding intensity (ARBS-gained). While ARBS-gained loci lack the canonical androgen response elements (ARE) and pioneer factor FOXA1 binding motifs, they are highly enriched with CpG islands and the binding sites of unmethylated CpG dinucleotide-binding protein CXXC5 and the partner TET2. RNA-seq analysis reveals that both CXXC5 and its regulated genes including ID1 are upregulated in ENZ-resistant cell lines and these results are further confirmed in patient-derived xenografts (PDXs) and patient specimens. Consistent with the finding that ARBS-gained loci are highly enriched with H3K27ac modification, ENZ-resistant PCa cells, organoids, xenografts and PDXs are hyper-sensitive to NEO2734, a dual inhibitor of BET and CBP/p300 proteins. These results not only reveal a noncanonical AR function in acquisition of ENZ resistance, but also posit a treatment strategy to target this vulnerability in ENZ-resistant CRPC.
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http://dx.doi.org/10.1038/s41467-021-21860-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943793PMC
March 2021

Androgen receptor (AR) antagonism triggers acute succinate-mediated adaptive responses to reactivate AR signaling.

EMBO Mol Med 2021 Mar 11:e13427. Epub 2021 Mar 11.

Vancouver Prostate Centre, Vancouver, BC, Canada.

Treatment-induced adaptive pathways converge to support androgen receptor (AR) reactivation and emergence of castration-resistant prostate cancer (PCa) after AR pathway inhibition (ARPI). We set out to explore poorly defined acute adaptive responses that orchestrate shifts in energy metabolism after ARPI and identified rapid changes in succinate dehydrogenase (SDH), a TCA cycle enzyme with well-known tumor suppressor activity. We show that AR directly regulates transcription of its catalytic subunits (SDHA, SDHB) via androgen response elements (AREs). ARPI acutely suppresses SDH activity, leading to accumulation of the oncometabolite, succinate. Succinate triggers calcium ions release from intracellular stores, which in turn phospho-activates the AR-cochaperone, Hsp27 via p-CaMKK2/p-AMPK/p-p38 axis to enhance AR protein stabilization and activity. Activation of this pathway was seen in tissue microarray analysis on prostatectomy tissues and patient-derived xenografts. This adaptive response is blocked by co-targeting AR with Hsp27 under both in vitro and in vivo studies, sensitizing PCa cells to ARPI treatments.
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http://dx.doi.org/10.15252/emmm.202013427DOI Listing
March 2021

Plasma ctDNA is a tumor tissue surrogate and enables clinical-genomic stratification of metastatic bladder cancer.

Nat Commun 2021 01 8;12(1):184. Epub 2021 Jan 8.

Department of Urologic Sciences, Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada.

Molecular stratification can improve the management of advanced cancers, but requires relevant tumor samples. Metastatic urothelial carcinoma (mUC) is poised to benefit given a recent expansion of treatment options and its high genomic heterogeneity. We profile minimally-invasive plasma circulating tumor DNA (ctDNA) samples from 104 mUC patients, and compare to same-patient tumor tissue obtained during invasive surgery. Patient ctDNA abundance is independently prognostic for overall survival in patients initiating first-line systemic therapy. Importantly, ctDNA analysis reproduces the somatic driver genome as described from tissue-based cohorts. Furthermore, mutation concordance between ctDNA and matched tumor tissue is 83.4%, enabling benchmarking of proposed clinical biomarkers. While 90% of mutations are identified across serial ctDNA samples, concordance for serial tumor tissue is significantly lower. Overall, our exploratory analysis demonstrates that genomic profiling of ctDNA in mUC is reliable and practical, and mitigates against disease undersampling inherent to studying archival primary tumor foci. We urge the incorporation of cell-free DNA profiling into molecularly-guided clinical trials for mUC.
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http://dx.doi.org/10.1038/s41467-020-20493-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794518PMC
January 2021

A polymeric paste-drug formulation for local treatment of upper tract urothelial carcinoma.

Urol Oncol 2021 03 26;39(3):194.e1-194.e7. Epub 2020 Nov 26.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Background: Intravesical instillation of chemo- or immunotherapy is commonly used in bladder cancer. Upper tract urothelial carcinoma (UTUC) shares similar pathological features, but current formulations are not suitable for direct instillation to the upper urinary tract.

Objective: To evaluate in vivo applicability, characteristics and toxicity of ST-UC, a mucoadhesive polymeric paste formulation of gemcitabine, for upper urinary tract instillation.

Material And Methods: Three pigs received 10 ml of ST-UC (100 mg/ml gemcitabine) retrogradely into 1 renal pelvis for pharmacokinetic studies. Four days later, a second injection into the contralateral renal pelvis was followed by serial euthanasia of the pigs and nephroureterectomy after 1, 3, and 6 hours. Adverse effects were monitored. Urine, serum, and tissue gemcitabine concentrations were measured, along with histologic examination of the upper urinary tract.

Results: Retrograde instillation of ST-UC was well tolerated with mild, completely receding hydronephrosis. Urine gemcitabine concentrations were highest in the first 3-hour collection interval. Hundred percent of gemcitabine was recovered in the urine within 24 hours. Serum peak concentrations (c) of gemcitabine were low at 5.5 µg/ml compared to the 10 to 30 µg/ml levels observed after a single intravenous dose of 1,000 mg/m gemcitabine. The formulation was still traceable after one hour and gemcitabine tissue concentrations are supportive of this extended drug exposure. No major histopathological changes were observed. The main limitation of this study is the lack of antitumor activity data.

Conclusion: This preclinical evaluation of ST-UC demonstrated feasible instillation in the renal pelvis, no significant safety concerns, and sustained release of gemcitabine.
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http://dx.doi.org/10.1016/j.urolonc.2020.07.028DOI Listing
March 2021

Improving prostate cancer classification in H&E tissue micro arrays using Ki67 and P63 histopathology.

Comput Biol Med 2020 Dec 14;127:104053. Epub 2020 Oct 14.

Electrical and Computer Engineering, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Histopathology of Hematoxylin and Eosin (H&E)-stained tissue obtained from biopsy is commonly used in prostate cancer (PCa) diagnosis. Automatic PCa classification of digitized H&E slides has been developed before, but no attempts have been made to classify PCa using additional tissue stains registered to H&E. In this paper, we demonstrate that using H&E, Ki67 and p63-stained (3-stain) tissue improves PCa classification relative to H&E alone. We also show that we can infer PCa-relevant Ki67 and p63 information from the H&E slides alone, and use it to achieve H&E-based PCa classification that is comparable to the 3-stain classification. Reported improvements apply to classifying benign vs. malignant tissue, and low grade (Gleason group 2) vs. high grade (Gleason groups 3,4,5) cancer. Specifically, we conducted four classification tasks using 333 tissue samples extracted from 231 radical prostatectomy patients: regression tree-based classification using either (i) 3-stain features, with a benign vs malignant area under the curve (AUC = 92.9%), or (ii) real H&E features and H&E features learned from Ki67 and p63 stains (AUC = 92.4%), as well as deep learning classification using either (iii) real 3-stain tissue patches (AUC = 94.3%) and (iv) real H&E patches and generated Ki67 and p63 patches (AUC = 93.0%) using a deep convolutional generative adversarial network. Classification performance was assessed with Monte Carlo cross validation and quantified in terms of the Area Under the Curve, Brier score, sensitivity, and specificity. Our results are interpretable and indicate that the standard H&E classification could be improved by mimicking other stain types.
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http://dx.doi.org/10.1016/j.compbiomed.2020.104053DOI Listing
December 2020

Paternally Expressed Gene 10 (PEG10) Promotes Growth, Invasion, and Survival of Bladder Cancer.

Mol Cancer Ther 2020 10 26;19(10):2210-2220. Epub 2020 Aug 26.

The Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

() has been associated with neuroendocrine muscle-invasive bladder cancer (MIBC), a subtype of the disease with the poorest survival. In this work, we further characterized the expression pattern of in The Cancer Genome Atlas database of 412 patients with MIBC, and found that, compared with other subtypes, mRNA level was enhanced in neuroendocrine-like MIBC and highly correlated with other neuroendocrine markers. PEG10 protein level also associated with neuroendocrine markers in a tissue microarray of 82 cases. In bladder cancer cell lines, PEG10 expression was induced in drug-resistant compared with parental cells, and knocking down of PEG10 resensitized cells to chemotherapy. Loss of PEG10 increased protein levels of cell-cycle regulators p21 and p27 and delayed G-S-phase transition, while overexpression of PEG10 enhanced cancer cell proliferation. PEG10 silencing also lowered levels of SLUG and SNAIL, leading to reduced invasion and migration. In an orthotopic bladder cancer model, systemic treatment with PEG10 antisense oligonucleotide delayed progression of T24 xenografts. In summary, elevated expression of in MIBC may contribute to the disease progression by promoting survival, proliferation, and metastasis. Targeting PEG10 is a novel potential therapeutic approach for a subset of bladder cancers.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-1031DOI Listing
October 2020

LIN28B promotes the development of neuroendocrine prostate cancer.

J Clin Invest 2020 10;130(10):5338-5348

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Therapy-induced neuroendocrine prostate cancer (t-NEPC) is a highly aggressive subtype of prostate cancer with poor patient survival. Emerging evidence indicates that t-NEPC can develop when prostate adenocarcinoma cells acquire cancer stem-like cell signaling in the presence of androgen receptor inhibition, followed by redifferentiation toward neuroendocrine lineage and subsequent t-NEPC progression. Whether the stem-like signaling is controlled by the core pluripotency stem cell genes (e.g., LIN28 and SOX2) remains unknown. Here, we report that the transcription of the LIN28B isoform and SOX2 were co-upregulated in t-NEPC patient tumors, patient-derived xenografts, transgenic mice, and cell models. Immunohistochemistry validated that LIN28B and SOX2 protein expression were elevated in t-NEPC patient biopsies. Using prostate adenocarcinoma and t-NEPC cell models, we demonstrated that LIN28B induced a stem-like gene network, neuroendocrine biomarkers, and neuroendocrine cell morphology. LIN28B depletion by CRISPR inhibited t-NEPC tumorigenesis and xenograft growth. These LIN28B functions were mediated mainly through the suppression of let-7 miRNA expression, resulting in de-repression of the transcription factor HMGA2 and HMGA2-mediated SOX2 expression. This study revealed a mechanism by which t-NEPC can develop through the LIN28B/let-7/SOX2 axis that regulates a cancer cell stem-like gene network, highlighting LIN28B as a potential therapeutic target in t-NEPC.
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http://dx.doi.org/10.1172/JCI135373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524485PMC
October 2020

p300-Mediated Acetylation of Histone Demethylase JMJD1A Prevents Its Degradation by Ubiquitin Ligase STUB1 and Enhances Its Activity in Prostate Cancer.

Cancer Res 2020 08 10;80(15):3074-3087. Epub 2020 Jun 10.

Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland.

The androgen receptor (AR) pathway plays a central role in the development of castration-resistant prostate cancer (CRPC). The histone demethylase JMJD1A has been shown to regulate activities of AR and c-Myc transcription factors and promote prostate cancer progression. Here, we report that JMJD1A protein stability is controlled by the ubiquitin ligase STUB1. High levels of JMJD1A were strongly correlated with low STUB1 levels in human CRPC specimens. STUB1 inhibited AR activity, AR-V7 levels, and prostate cancer cell growth partly through degradation of JMJD1A. Furthermore, the acetyltransferase p300 acetylated JMJD1A at lysine (K) 421, a modification that recruits the BET family member BRD4 to block JMJD1A degradation and promote JMJD1A recruitment to AR targets. Increased levels of both total and K421-acetylated JMJD1A were observed in prostate cancer cells as they developed resistance to the AR antagonist enzalutamide. Treatment of prostate cancer cells with either p300 or BET inhibitors destabilized JMJD1A, and enzalutamide-resistant prostate cancer cells were more sensitive than parental cells to these inhibitors. Together, our findings identify a critical role for acetylation of JMJD1A in regulating JMJD1A stability and AR activity in CRPC. These newly identified mechanisms controlling JMJD1A protein stability provide potential druggable targets to encourage the development of additional therapies for advanced prostate cancer. SIGNIFICANCE: Identification of mechanisms regulating JMJD1A protein stability reveals new strategies to destabilize JMJD1A and concomitantly inhibit AR activities as potential prostate cancer therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415556PMC
August 2020

Histone demethylase JMJD1A promotes expression of DNA repair factors and radio-resistance of prostate cancer cells.

Cell Death Dis 2020 04 1;11(4):214. Epub 2020 Apr 1.

Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, USA.

The DNA damage response (DDR) pathway is a promising target for anticancer therapies. The androgen receptor and myeloblastosis transcription factors have been reported to regulate expression of an overlapping set of DDR genes in prostate cancer cells. Here, we found that histone demethylase JMJD1A regulates expression of a different set of DDR genes largely through c-Myc. Inhibition of JMJD1A delayed the resolution of γ-H2AX foci, reduced the formation of foci containing ubiquitin, 53BP1, BRCA1 or Rad51, and inhibited the reporter activity of double-strand break (DSB) repair. Mechanistically, JMJD1A regulated expression of DDR genes by increasing not only the level but also the chromatin recruitment of c-Myc through H3K9 demethylation. Further, we found that ubiquitin ligase HUWE1 induced the K27-/K29-linked noncanonical ubiquitination of JMJD1A at lysine-918. Ablation of the JMJD1A noncanonical ubiquitination lowered DDR gene expression, impaired DSB repair, and sensitized response of prostate cells to irradiation, topoisomerase inhibitors or PARP inhibitors. Thus, development of agents that target JMJD1A or its noncanonical ubiquitination may sensitize the response of prostate cancer to radiotherapy and possibly also genotoxic therapy.
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http://dx.doi.org/10.1038/s41419-020-2405-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113292PMC
April 2020

Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma.

Clin Genitourin Cancer 2020 08 8;18(4):322-331.e2. Epub 2020 Jan 8.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, British Columbia, Canada; Department of Medical Oncology, BC Cancer, British Columbia, Canada.

Background: There is a lack of molecularly-informed biomarkers for patients with metastatic renal cell carcinoma (RCC). Plasma cell-free DNA (cfDNA) sequencing is a minimally-invasive alternative to tissue for profiling the genome in other cancers but relevance in metastatic RCC remains unclear.

Materials And Methods: Whole blood was collected from 55 patients with metastatic RCC. Plasma cfDNA and leukocyte DNA were subjected to targeted sequencing across 981 cancer genes. Matched tumor tissue from 14 patients was analyzed.

Results: Thirty-three percent of patients had evidence for RCC-derived circulating tumor DNA (ctDNA), significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential, were detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. CtDNA-positive patients had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of clonal hematopoiesis of indeterminate potential had an intermediate prognosis compared with ctDNA-positive and -negative patients.

Conclusions: CfDNA sequencing enables straightforward characterization of the somatic RCC genome in a minority of patients with metastatic RCC. Owing to low ctDNA abundance, and the presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.
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http://dx.doi.org/10.1016/j.clgc.2019.12.018DOI Listing
August 2020

Cancer Cells Employ Nuclear Caspase-8 to Overcome the p53-Dependent G2/M Checkpoint through Cleavage of USP28.

Mol Cell 2020 03 22;77(5):970-984.e7. Epub 2020 Jan 22.

Experimental Dermatology, Department of Dermatology, TU-Dresden, Dresden 01307, Germany; Center for Regenerative Therapies Dresden, TU-Dresden, Dresden 01307, Germany. Electronic address:

Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
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http://dx.doi.org/10.1016/j.molcel.2019.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060810PMC
March 2020

Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

Clin Cancer Res 2020 04 9;26(7):1678-1689. Epub 2020 Jan 9.

Vancouver Prostate Centre, Vancouver, British Columbia, Canada.

Purpose: Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression.

Experimental Design: Unique prostate cancer cell models that capture AR stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored.

Results: Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells.

Conclusions: Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0098DOI Listing
April 2020

Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models.

J Clin Invest 2020 02;130(2):699-714

Department of Urologic Sciences, Vancouver Prostate Centre, and.

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.
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http://dx.doi.org/10.1172/JCI130819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994194PMC
February 2020

Characterization of a Prostate- and Prostate Cancer-Specific Circular RNA Encoded by the Androgen Receptor Gene.

Mol Ther Nucleic Acids 2019 Dec 24;18:916-926. Epub 2019 Oct 24.

Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. Electronic address:

The linear mRNAs transcribed under alternative RNA splicing and overexpression/amplification of the androgen receptor (AR) gene are poor prognostic biomarkers of castrate-resistant prostate cancer (PCa). Whether the AR gene also transcribes non-coding circular RNAs that are associated with PCa development and tumor progression remains unclear. Here, we identified and characterized an AR circular RNA, called circAR3, that is widely expressed in PCa cell models and prostate tumors. circAR3 can be secreted into culture media of PCa cell lines and is detectable in the serum from mice bearing PCa xenografts. In PCa patient tissues, circAR3 is highly expressed in benign prostate and hormone naive PCa but downregulated when tumors were treated with neoadjuvant hormone therapy and further reduced when tumors progressed to the castrate-resistant stage. However, circAR3 levels in plasma are extremely low in patients with benign prostate, are upregulated in PCa patients with high Gleason scores and lymph node metastasis, and become undetectable in men after radical prostatectomy. circAR3 does not affect AR signaling, PCa cell proliferation, and invasion rates. Our results demonstrated that the origin of the detectable plasma circAR3 is from the prostate/PCa. Plasma circAR3 may be developed to be a PCa biomarker to monitor PCa development and tumor progression.
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http://dx.doi.org/10.1016/j.omtn.2019.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883311PMC
December 2019

Identification of Hypermutation and Defective Mismatch Repair in ctDNA from Metastatic Prostate Cancer.

Clin Cancer Res 2020 03 19;26(5):1114-1125. Epub 2019 Nov 19.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, British Columbia, Canada.

Purpose: DNA mismatch repair defects (MMRd) and tumor hypermutation are rare and under-characterized in metastatic prostate cancer (mPC). Furthermore, because hypermutated MMRd prostate cancers can respond to immune checkpoint inhibitors, there is an urgent need for practical detection tools.

Experimental Design: We analyzed plasma cell-free DNA-targeted sequencing data from 433 patients with mPC with circulating tumor DNA (ctDNA) purity ≥2%. Samples with somatic hypermutation were subjected to 185 × whole-exome sequencing and capture of mismatch repair gene introns. Archival tissue was analyzed with targeted sequencing and IHC.

Results: Sixteen patients (3.7%) had somatic hypermutation with MMRd etiology, evidenced by deleterious alterations in , or , microsatellite instability, and characteristic trinucleotide signatures. ctDNA was concordant with mismatch repair protein IHC and DNA sequencing of tumor tissue. Tumor suppressors such as , and were inactivated by mutation rather than copy-number loss. Hotspot mutations in oncogenes such as , and were common, and the androgen receptor ()-ligand binding domain was mutated in 9 of 16 patients. We observed high intrapatient clonal diversity, evidenced by subclonal driver mutations and shifts in mutation allele frequency over time. Patients with hypermutation and MMRd etiology in ctDNA had a poor response to AR inhibition and inferior survival compared with a control cohort.

Conclusions: Hypermutated MMRd mPC is associated with oncogene activation and subclonal diversity, which may contribute to a clinically aggressive disposition in selected patients. In patients with detectable ctDNA, cell-free DNA sequencing is a practical tool to prioritize this subtype for immunotherapy..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1623DOI Listing
March 2020

The molecular function of kallikrein-related peptidase 14 demonstrates a key modulatory role in advanced prostate cancer.

Mol Oncol 2020 01 28;14(1):105-128. Epub 2019 Nov 28.

Australian Prostate Cancer Research Centre-Queensland (APCRC-Q), Institute of Health & Biomedical Innovation, Queensland University of Technology, Woolloongabba, Australia.

Kallikrein-related peptidase 14 (KLK14) is one of the several secreted KLK serine proteases involved in prostate cancer (PCa) pathogenesis. While relatively understudied, recent reports have identified KLK14 as overexpressed during PCa development. However, the modulation of KLK14 expression during PCa progression and the molecular and biological functions of this protease in the prostate tumor microenvironment remain unknown. To determine the modulation of KLK14 expression during PCa progression, we analyzed the expression levels of KLK14 in patient samples using publicly available databases and immunohistochemistry. In order to delineate the molecular mechanisms involving KLK14 in PCa progression, we integrated proteomic, transcriptomic, and in vitro assays with the goal to identify substrates, related-signaling pathways, and functional roles of this protease. We showed that KLK14 expression is elevated in advanced PCa, and particularly in metastasis. Additionally, KLK14 levels were found to be decreased in PCa tissues from patients responsive to neoadjuvant therapy compared to untreated patients. Furthermore, we also identified that KLK14 expression reoccurred in patients who developed castrate-resistant PCa. The combination of proteomic and transcriptomic analysis as well as functional assays revealed several new KLK14 substrates (agrin, desmoglein 2, vitronectin, laminins) and KLK14-regulated genes (Interleukin 32, midkine, SRY-Box 9), particularly an involvement of the mitogen-activated protein kinase 1 and interleukin 1 receptor pathways, and an involvement of KLK14 in the regulation of cellular migration, supporting its involvement in aggressive features of PCa progression. In conclusion, our work showed that KLK14 expression is associated with the development of aggressive PCa suggesting that targeting this protease could offer a novel route to limit the progression of prostate tumors. Additional work is necessary to determine the benefits and implications of targeting/cotargeting KLK14 in PCa as well as to determine the potential use of KLK14 expression as a predictor of PCa aggressiveness or response to treatment.
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http://dx.doi.org/10.1002/1878-0261.12587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944120PMC
January 2020

Deep Learning-Based Gleason Grading of Prostate Cancer From Histopathology Images-Role of Multiscale Decision Aggregation and Data Augmentation.

IEEE J Biomed Health Inform 2020 05 30;24(5):1413-1426. Epub 2019 Sep 30.

Visual inspection of histopathology images of stained biopsy tissue by expert pathologists is the standard method for grading of prostate cancer (PCa). However, this process is time-consuming and subject to high inter-observer variability. Machine learning-based methods have the potential to improve efficient throughput of large volumes of slides while decreasing variability, but they are not easy to develop because they require substantial amounts of labeled training data. In this paper, we propose a deep learning-based classification technique and data augmentation methods for accurate grading of PCa in histopathology images in the presence of limited data. Our method combines the predictions of three separate convolutional neural networks (CNNs) that work with different patch sizes. This enables our method to take advantage of the greater amount of contextual information in larger patches as well as greater quantity of smaller patches in the labeled training data. The predictions produced by the three CNNs are combined using a logistic regression model, which is trained separately after the CNN training. To effectively train our models, we propose new data augmentation methods and empirically study their effects on the classification accuracy. The proposed method achieves an accuracy of [Formula: see text] in classifying cancerous patches versus benign patches and an accuracy of [Formula: see text] in classifying low-grade (i.e., Gleason grade 3) from high-grade (i.e., Gleason grades 4 and 5) patches. The agreement level of our automatic grading method with expert pathologists is within the range of agreement between pathologists. Our experiments indicate that data augmentation is necessary for achieving expert-level performance with deep learning-based methods. A combination of image-space augmentation and feature-space augmentation leads to the best results. Our study shows that well-designed and properly trained deep learning models can achieve PCa Gleason grading accuracy that is comparable to an expert pathologist.
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http://dx.doi.org/10.1109/JBHI.2019.2944643DOI Listing
May 2020

Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling.

Cell Commun Signal 2019 09 18;17(1):120. Epub 2019 Sep 18.

Department of Urology, Upstate Cancer Center, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, New York, 13210, USA.

Background: Prostate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear.

Methods: To investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines.

Results: Low ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor.

Conclusions: ABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.
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http://dx.doi.org/10.1186/s12964-019-0410-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6749699PMC
September 2019

BMI1 is directly regulated by androgen receptor to promote castration-resistance in prostate cancer.

Oncogene 2020 01 28;39(1):17-29. Epub 2019 Aug 28.

Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, 77030, USA.

B lymphoma Mo-MLV insertion region 1 (BMI1) has been reported to be an oncoprotein. BMI1 represses tumor suppressors to promote cell proliferation, epithelial-mesenchymal transition (EMT), and cancer progression. Although it is known that the expression of BMI1 is increased in many cancer types, the mechanism of BMI1 upregulation is not yet clear. We performed integrative analysis for 3 sets of prostate cancer (PCa) genomic data, and found that BMI1 and androgen receptor (AR) were positively correlated, suggesting that AR might regulate BMI1. Next, we showed that dihydrotestosterone (DHT) upregulated both mRNA and protein levels of BMI1 and that BMI1 was increased in castration-resistant prostate cancer (CRPC) from both human patients and a mouse xenograph model. We further identified an AR binding site in the promoter/enhancer region of BMI1, and confirmed BMI1 as the direct target of AR using gene-editing technology. We also demonstrated that high expression of BMI1 is critical for the development of castration-resistance. Our data also suggest that BMI1-specific inhibitors could be an effective treatment of CRPC.
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http://dx.doi.org/10.1038/s41388-019-0966-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386438PMC
January 2020

Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.

Oncogene 2019 11 13;38(45):7060-7072. Epub 2019 Aug 13.

Department of Structural and Cellular Biology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, LA, USA.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.
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http://dx.doi.org/10.1038/s41388-019-0947-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842090PMC
November 2019

Regulation of the unfolded protein response through ATF4 and FAM129A in prostate cancer.

Oncogene 2019 08 16;38(35):6301-6318. Epub 2019 Jul 16.

Department of Biosciences, University of Oslo, Oslo, Norway.

Cancer cells exploit many of the cellular adaptive responses to support their survival needs. One such critical pathway in eukaryotic cells is the unfolded protein response (UPR) that is important in normal physiology as well as disease states, including cancer. Since UPR can serve as a lever between survival and death, regulated control of its activity is critical for tumor formation and growth although the underlying mechanisms are poorly understood. Here we show that one of the main transcriptional effectors of UPR, activating transcription factor 4 (ATF4), is essential for prostate cancer (PCa) growth and survival. Using systemic unbiased gene expression and proteomic analyses, we identified a novel direct ATF4 target gene, family with sequence similarity 129 member A (FAM129A), which is critical in mediating ATF4 effects on prostate tumorigenesis. Interestingly, FAM129A regulated both PERK and eIF2α in a feedback loop that differentially channeled the UPR output. ATF4 and FAM129A protein expression is increased in patient PCa samples compared with benign prostate. Importantly, in vivo therapeutic silencing of ATF4-FAM129A axis profoundly inhibited tumor growth in a preclinical PCa model. These data support that one of the canonical UPR branches, through ATF4 and its target gene FAM129A, is required for PCa growth and thus may serve as a novel therapeutic target.
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http://dx.doi.org/10.1038/s41388-019-0879-2DOI Listing
August 2019

Selective targeting of PARP-2 inhibits androgen receptor signaling and prostate cancer growth through disruption of FOXA1 function.

Proc Natl Acad Sci U S A 2019 07 2;116(29):14573-14582. Epub 2019 Jul 2.

Division of Urology, Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;

Androgen receptor (AR) is a ligand-activated transcription factor and a key driver of prostate cancer (PCa) growth and progression. Understanding the factors influencing AR-mediated gene expression provides new opportunities for therapeutic intervention. Poly(ADP-ribose) Polymerase (PARP) is a family of enzymes, which posttranslationally modify a range of proteins and regulate many different cellular processes. PARP-1 and PARP-2 are two well-characterized PARP members, whose catalytic activity is induced by DNA-strand breaks and responsible for multiple DNA damage repair pathways. PARP inhibitors are promising therapeutic agents that show synthetic lethality against many types of cancer (including PCa) with homologous recombination (HR) DNA-repair deficiency. Here, we show that, beyond DNA damage repair function, PARP-2, but not PARP-1, is a critical component in AR transcriptional machinery through interacting with the pioneer factor FOXA1 and facilitating AR recruitment to genome-wide prostate-specific enhancer regions. Analyses of PARP-2 expression at both mRNA and protein levels show significantly higher expression of PARP-2 in primary PCa tumors than in benign prostate tissues, and even more so in castration-resistant prostate cancer (CRPC) tumors. Selective targeting of PARP-2 by genetic or pharmacological means blocks interaction between PARP-2 and FOXA1, which in turn attenuates AR-mediated gene expression and inhibits AR-positive PCa growth. Next-generation antiandrogens act through inhibiting androgen synthesis (abiraterone) or blocking ligand binding (enzalutamide). Selective targeting of PARP-2, however, may provide an alternative therapeutic approach for AR inhibition by disruption of FOXA1 function, which may be beneficial to patients, irrespective of their DNA-repair deficiency status.
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http://dx.doi.org/10.1073/pnas.1908547116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642419PMC
July 2019

Validation of the prognostic value of NF-κB p65 in prostate cancer: A retrospective study using a large multi-institutional cohort of the Canadian Prostate Cancer Biomarker Network.

PLoS Med 2019 07 2;16(7):e1002847. Epub 2019 Jul 2.

Centre de recherche du Centre hospitalier de l'Université de Montréal, Centre hospitalier de l'Université de Montréal, Montreal, Quebec, Canada.

Background: The identification of patients with high-risk prostate cancer (PC) is a major challenge for clinicians, and the improvement of current prognostic parameters is an unmet clinical need. We and others have identified an association between the nuclear localization of NF-κB p65 and biochemical recurrence (BCR) in PC in small and/or single-centre cohorts of patients.

Methods And Findings: In this study, we accessed 2 different multi-centre tissue microarrays (TMAs) representing cohorts of patients (Test-TMA and Validation-TMA series) of the Canadian Prostate Cancer Biomarker Network (CPCBN) to validate the association between p65 nuclear frequency and PC outcomes. Immunohistochemical staining of p65 was performed on the Test-TMA and Validation-TMA series, which include PC tissues from patients treated by first-line radical prostatectomy (n = 250 and n = 1,262, respectively). Two independent observers evaluated the p65 nuclear frequency in digital images of cancer tissue and benign adjacent gland tissue. Kaplan-Meier curves coupled with a log-rank test and univariate and multivariate Cox regression models were used for statistical analyses of continuous values and dichotomized data (cutoff of 3%). Multivariate analysis of the Validation-TMA cohort showed that p65 nuclear frequency in cancer cells was an independent predictor of BCR using continuous (hazard ratio [HR] 1.02 [95% CI 1.00-1.03], p = 0.004) and dichotomized data (HR 1.33 [95% CI 1.09-1.62], p = 0.005). Using a cutoff of 3%, we found that this biomarker was also associated with the development of bone metastases (HR 1.82 [95% CI 1.05-3.16], p = 0.033) and PC-specific mortality (HR 2.63 [95% CI 1.30-5.31], p = 0.004), independent of clinical parameters. BCR-free survival, bone-metastasis-free survival, and PC-specific survival were shorter for patients with higher p65 nuclear frequency (p < 0.005). As the small cores on TMAs are a limitation of the study, a backward validation of whole PC tissue section will be necessary for the implementation of p65 nuclear frequency as a PC biomarker in the clinical workflow.

Conclusions: We report the first study using the pan-Canadian multi-centre cohorts of CPCBN and validate the association between increased frequency of nuclear p65 frequency and a risk of disease progression.
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http://dx.doi.org/10.1371/journal.pmed.1002847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605640PMC
July 2019

Molecular Characterization of Neuroendocrine-like Bladder Cancer.

Clin Cancer Res 2019 07 5;25(13):3908-3920. Epub 2019 Apr 5.

Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5% to 15% of muscle-invasive bladder cancer (MIBC) have transcriptomic patterns consistent with NE bladder cancer in the absence of NE histology. The clinical implications of this NE-like subtype have not been explored in depth.

Experimental Design: Transcriptome-wide expression profiles were generated for MIBC collected from 7 institutions and clinical-use of Decipher Bladder. Using unsupervised clustering, we generated a clustering solution on a prospective training cohort (PTC; = 175), developed single-sample classifiers to predict NE tumors, and evaluated the resultant models on a testing radical cystectomy (RC) cohort ( = 225). A random forest model was finalized and applied to 5 validation cohorts ( = 1302). Uni- and multivariable survival analyses were used to characterize clinical outcomes.

Results: In the training cohort (PTC), hierarchical clustering using an 84-gene panel showed a cluster of 8 patients (4.6%) with highly heterogeneous expression of NE markers in the absence of basal or luminal marker expression. NE-like tumors were identified in 1% to 6.6% of cases in validation cohorts. Patients with NE-like tumors had significantly worse 1-year progression-free survival (65% NE-like vs. 82% overall; = 0.046) and, after adjusting for clinical and pathologic factors, had a 6.4-fold increased risk of all-cause mortality ( = 0.001). IHC confirmed the neuronal character of these tumors.

Conclusions: A single-patient classifier was developed that identifies patients with histologic urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC, which may require different treatment.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3558DOI Listing
July 2019

RNA Splicing of the BHC80 Gene Contributes to Neuroendocrine Prostate Cancer Progression.

Eur Urol 2019 08 23;76(2):157-166. Epub 2019 Mar 23.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Background: Prostate adenocarcinoma (AdPC) progression to treatment-induced neuroendocrine prostate cancer (t-NEPC) is associated with poor patient survival. While AdPC and t-NEPC share similar genomes, they possess distinct transcriptomes, suggesting that RNA splicing and epigenetic mechanisms may regulate t-NEPC development.

Objective: To characterize the role of alternative RNA splicing of the histone demethylase BHC80 during t-NEPC progression.

Design, Setting, And Participants: The expression of BHC80 splice variants (BHC80-1 and BHC80-2) were compared between AdPC and t-NEPC patient tumors. Regulatory mechanisms of RNA splicing of the BHC80 gene were studied, and the signal pathways mediated by BHC80 splice variants were investigated in t-NEPC cell and xenograft models.

Results: Global transcriptome analyses identified that the BHC80-2 variant is highly expressed in t-NEPC. Compared with the known histone demethylation activities of the BHC80 gene, we discovered a novel nonepigenetic action of BHC80-2, whereby BHC80-2 is localized in the cytoplasm to trigger the MyD88-p38-TTP pathway, which results in increased RNA stability of multiple tumor-promoting cytokines. While BHC80-2 does not induce neuroendocrine differentiation of cancer cells, it stimulates cell proliferation and tumor progression independent of androgen receptor signaling. Blockade of BHC80-2-regulated MyD88 signaling suppresses growth of several t-NEPC cell spheroid and xenograft models.

Conclusions: Gain of function of BHC80-2 through alternative RNA splicing activates immune responses of cancer cells to promote t-NEPC development.

Patient Summary: The main obstacle to develop effective therapies for patients with t-NEPC is the lack of understanding on how t-NEPC is developed. Our study not only identifies a previously unknown BHC80-2-MyD88 signaling pathway that plays an important role during t-NEPC development, but also provides a proof of principle that targeting this signal pathway may offer an avenue to treat t-NEPC.
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http://dx.doi.org/10.1016/j.eururo.2019.03.011DOI Listing
August 2019

Comparison of Artificial Intelligence Techniques to Evaluate Performance of a Classifier for Automatic Grading of Prostate Cancer From Digitized Histopathologic Images.

JAMA Netw Open 2019 03 1;2(3):e190442. Epub 2019 Mar 1.

Department of Electrical and Computer Engineering, University of British Columbia, Vancouver, British Columbia, Canada.

Importance: Proper evaluation of the performance of artificial intelligence techniques in the analysis of digitized medical images is paramount for the adoption of such techniques by the medical community and regulatory agencies.

Objectives: To compare several cross-validation (CV) approaches to evaluate the performance of a classifier for automatic grading of prostate cancer in digitized histopathologic images and compare the performance of the classifier when trained using data from 1 expert and multiple experts.

Design, Setting, And Participants: This quality improvement study used tissue microarray data (333 cores) from 231 patients who underwent radical prostatectomy at the Vancouver General Hospital between June 27, 1997, and June 7, 2011. Digitized images of tissue cores were annotated by 6 pathologists for 4 classes (benign and Gleason grades 3, 4, and 5) between December 12, 2016, and October 5, 2017. Patches of 192 µm2 were extracted from these images. There was no overlap between patches. A deep learning classifier based on convolutional neural networks was trained to predict a class label from among the 4 classes (benign and Gleason grades 3, 4, and 5) for each image patch. The classification performance was evaluated in leave-patches-out CV, leave-cores-out CV, and leave-patients-out 20-fold CV. The analysis was performed between November 15, 2018, and January 1, 2019.

Main Outcomes And Measures: The classifier performance was evaluated by its accuracy, sensitivity, and specificity in detection of cancer (benign vs cancer) and in low-grade vs high-grade differentiation (Gleason grade 3 vs grades 4-5). The statistical significance analysis was performed using the McNemar test. The agreement level between pathologists and the classifier was quantified using a quadratic-weighted κ statistic.

Results: On 333 tissue microarray cores from 231 participants with prostate cancer (mean [SD] age, 63.2 [6.3] years), 20-fold leave-patches-out CV resulted in mean (SD) accuracy of 97.8% (1.2%), sensitivity of 98.5% (1.0%), and specificity of 97.5% (1.2%) for classifying benign patches vs cancerous patches. By contrast, 20-fold leave-patients-out CV resulted in mean (SD) accuracy of 85.8% (4.3%), sensitivity of 86.3% (4.1%), and specificity of 85.5% (7.2%). Similarly, 20-fold leave-cores-out CV resulted in mean (SD) accuracy of 86.7% (3.7%), sensitivity of 87.2% (4.0%), and specificity of 87.7% (5.5%). Results of McNemar tests showed that the leave-patches-out CV accuracy, sensitivity, and specificity were significantly higher than those for both leave-patients-out CV and leave-cores-out CV. Similar results were observed for classifying low-grade cancer vs high-grade cancer. When trained on a single expert, the overall agreement in grading between pathologists and the classifier ranged from 0.38 to 0.58; when trained using the majority vote among all experts, it was 0.60.

Conclusions And Relevance: Results of this study suggest that in prostate cancer classification from histopathologic images, patch-wise CV and single-expert training and evaluation may lead to a biased estimation of classifier's performance. To allow reproducibility and facilitate comparison between automatic classification methods, studies in the field should evaluate their performance using patient-based CV and multiexpert data. Some of these conclusions may be generalizable to other histopathologic applications and to other applications of machine learning in medicine.
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http://dx.doi.org/10.1001/jamanetworkopen.2019.0442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484626PMC
March 2019

BAP1 haploinsufficiency predicts a distinct immunogenic class of malignant peritoneal mesothelioma.

Genome Med 2019 02 18;11(1). Epub 2019 Feb 18.

Vancouver Prostate Centre, 2660 Oak St, Vancouver, BC, V6H 3Z6, Canada.

Background: Malignant peritoneal mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis.

Methods: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multi-omics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular, we examined BAP1 mutation and copy number status and its relationship to immune checkpoint inhibitor activation.

Results: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies.

Conclusions: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phases I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.
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http://dx.doi.org/10.1186/s13073-019-0620-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378747PMC
February 2019

Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion.

J Natl Cancer Inst 2019 09;111(9):970-982

Background: Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.

Methods: By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided.

Results: EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.

Conclusion: This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.
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http://dx.doi.org/10.1093/jnci/djy209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748813PMC
September 2019