Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.


Bioinformatics analysis of proteomics data
To evaluate HSP27 client protein's involvement in reported biological processes, pathways enrichment analysis on the identified proteins using KEGG Pathway database was performed.
We used the R Bioconductor package "Pathview" to retrieve, integrate and visualize pathway enrichments.

Antibodies used for Western blot analysis and Immunoprecipitation (IP)
For western blot, we used 1:5000 rabbit anti-HSP27 polyclonal antibody (Enzo Life Science, (n=53), G4 (n=69), and G5 (n=49). The second "NHT" TMA includes 94 patients exposed to neoadjuvant hormone therapy (NHT) before surgery includes Naïve (n=76), NHT treatment (n=42) and transurethral resection (TURP) of CRPC (n=70). Immunohistochemistry (IHC) staining was conducted by Ventana Autostainer Model Discover XT (Ventana Medical System, Tuscan, Arizona). Enzyme-labeled biotin-streptavidin system and solvent-resistant 3,3′-Diaminobenzidine (DAB) Map kit was used with 1/50 mouse anti-Menin monoclonal antibody (Santa Cruz Biotechnology, CA, USA). Digital images were obtained using the Leica SCN400 scanning system (Concord Leica Microsystems, Ontario, Canada), and scoring was performed visually by an anatomopathologist (Ladan Fazli). Nuclear immunoreactivity of Menin was evaluated using a 3-point scale scoring system by assigning zero for negative, one for weak to moderate, and two for strong staining intensity in the majority of tumor cells.

Menin mRNA expression correlates with prostate cancer progression
We perfoExpression analysis of MEN1 mRNA expression in a pooled series of 2,081 clinical tissue samples publicly available, including 272 "normal" samples (normal tissue and benign   prostate hyperplasia: BPH), 1,643 PC primary tumor samples, and 90 PC metastatic  was performed with enrichR. A value of p<0.05 was considered statistically significant.

Computational analyses
Reads with a Phred quality score less than 30 were filtered out. For Menin ChIP-seq, reads were mapped to Homo sapiens genome assembly (GRCh37/hg19) using default parameters of Bowtie2 (v2.3.4.1) (1). Duplicate tags were removed and mapped tags were processed for further analysis. High confidence binding sites were determined using MACS2 peak caller (2): broad mode (broad-cutoff=0.01 -p 0.01). Inputs were used as controls. To get rid of noisy signal issues, peaks located less than 2500 bp from each other were stitched together and peaks smaller than 500 bp wide were removed. To link ChIP-seq data with gene expression, only peaks located at genes TSS (TSS defined from -2kb to +5kb) were filtered in using BEDTools intersect (v2.17.0) with a minimum overlap of 1-base (3). For ChIP-seq quantitative analysis, mapped tags were counted within TSS-intersected peak coordinates (defined previously by MACS2) using featureCounts (4) and then normalized as "reads per million mapped reads" using their respective library size.
For RNA-seq, PC3 and LNCaP expression tables were obtained from the publicly available Gene Expression Omnibus (GEO) repository (accession number: GSE59009) and merged. The supervised analysis was carried out using Welch's t-test and FDR adjustment using p. adjust function.
For ChIP-seq and RNA-seq crossing, only Menin target genes (i.e. genes bound by Menin on their TSS) were analyzed. The biological significance of Differentially Expressed and Menin-Bound Genes (DEBGs) was explored by GO term enrichment analysis including biological process (BP) and molecular function (MF), based on Bioconductor packages clusterProfiler (6) (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html). KEGG pathway enrichment analysis of DEBGs was performed with clusterProfiler as well. A value of P<0.05 was considered statistically significant.

Mass spectrometry analysis for Menin interactome
The immunoprecipitated samples were stacked on NuPAGE™ 4-12% Bis-tris acrylamide gels according to the manufacturer's instructions (Invitrogen, Life Technologies). Protein containing bands were stained with Thermo Scientific Imperial Blue, cut from the gel, and following reduction and iodoacetamide alkylation, digested with high sequencing grade trypsin