Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

(A,B) End-point PCR (A) and Western blot analysis (B) of TRPM8 in LNCaP (WT), RWPE-1 (WT) and PC-3 (WT and MM) cell lines.GAPDH was used as loading control.(C) Immunoblotting analysis of TRPM8 protein with the Alomone (#ACC-049) antibody in wild type and genetically modified (M8, CAS, MM) LNCaP and RWPE-1 cell lines.(D) RT-qPCR analysis of TRPM8, KLK3 and TMPRSS2 genes in both wild type (WT) and genetically engineered (MM, two independent clones) LNCaP cell lines expressing reduced levels of TRPM8 RNA compared to clones described in Fig. 1C.(E) Immunoblot analysis of NF-kB p65 in cytosolic and nuclear fractions of CAS, WT, and MM (described in D) engineered LNCaP cell lines.β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as cytoplasmic and nuclear markers, respectively.(F,G) RT-qPCR analyses of canonical NF-kB and IRF3 targets genes in both LNCaP and RWPE-1 CAS and MM cells.Data Information: In (D,E,G,H) data are presented as mean ± SD of n = 4 (D), n = 2 (E), and n = 3 (G,H) independent biological replicates.*P ≤ 0.05; **P ≤ 0.01; ns, not statistically significant (Two-tailed Student's t-test).Source data are available online for this figure.

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Alessandro Alaimo et al EV4 The EMBO Journal Alessandro Alaimo et al

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Figure EV2 .
Figure EV2.Pro-inflammatory activity of the mutant non-coding RNA of TRPM8.

Figure EV3 .
Figure EV3.TRPM8 RNA secretion modulates TLR3/TAK1 signal cascade.(A)RT-qPCR analysis of TRPM8 transcript released by WT and MM LNCaP cells into extracellular vesicles (EVs).Specificity and sequence of amplicons obtained with sets of primers spanning different exons (Ex) of the coding sequence of TRPM8 RNA are shown.Y3 transcript was used to normalize the data.(B) Nucleus/Cytosol fractionation and Western blot analysis of NF-kB p65 and PSA in LNCaP CAS cells conditioned with the supernatants of LNCaP WT cells.β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as markers of the cytosolic and nuclear fractions, respectively.(C) Nucleus/Cytosol fractionation and Western blot analysis of NF-kB p65 and PSA in RWPE-1 CAS cells conditioned with the supernatants of RWPE-1 (CAS, M8, and MM).β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as markers of the cytosolic and nuclear fractions, respectively.(D,E) Nucleus/Cytosol fractionation and Western blot analysis of NF-kB p65 and PSA in RWPE-1 MM and M8 (D) and LNCaP MM and M8 (E) cell lines in the presence or absence of Takinib (TAK1 inhibitor; 10 μM, 24 h).β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as markers of the cytosolic and nuclear fractions, respectively.Data Information: In (A) data are presented as mean ± SD of n = 3 biological replicates.Source data are available online for this figure.

Figure EV4 .
Figure EV4.Prediction of TRPM8 RNA structures highlights putative TLR3 binding motifs.(A)TLRs expression in LNCaP and RWPE-1 prostate cell lines based on publicly available datasets.(B) In silico prediction of the secondary structure of human TRPM8 transcript.Highly stable dsRNA stems with bulge/internal loops are indicated in red.(C) Mountain plot of (B).(D) Nucleus/Cytosol fractionation and Western blot analysis of NF-kB p65 and PSA in RWPE-1 MM and M8 cell lines in the presence or absence of TLR3/dsRNA Complex Inhibitor (TLR3 inhibitor; 20 μM, 24 h).β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as markers of the cytosolic and nuclear fractions, respectively.(E) Nucleus/Cytosol fractionation and Western blot analysis of NF-kB p65, c-Rel, phospho-p50, STAT1, IRF3 and PSA in LNCaP CAS cells conditioned with the supernatants of LNCaP MM cells.β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as markers of the cytosolic and nuclear fractions, respectively.(F) Nucleus/Cytosol fractionation and Western blot analysis of NF-kB p65 in LNCaP CAS cells transfected with Poly(I:C) (2 μg/mL, 24 h) and treated or not with the the combination of the RIG1 inhibitor RIG012 (2 μM, 6 h) and the PKR inhibitor C16 (2 μM, 6 h).Untransfected and untreated LNCaP CAS cells served as control for the activation of NF-kB by Poly(I:C).β-Tubulin (β-TUB) and Fibrillarin (FBL) were used as markers of the cytosolic and nuclear fractions, respectively.(G) PCR analysis of TLR3-bound TRPM8 transcript (native RIP) in LNCaP WT cells treated or not with TLR3/dsRNA complex inhibitor.Sets of primers spanning different exons (Ex) of the coding sequence of TRPM8 RNA are shown.Data Information: In (A) the heatmap was plotted with the pheatmap package, scaling gene expression by row.All the analyses were done with R 4.0.3.Source data are available online for this figure.

Figure
Figure EV5.TRPM8 RNA secretion alters fibroblasts functions.(A,B) Immunolocalization of PSA in FFPE sections of LNCaP WT (n = 3 xenografts/5 sections) and MM (n = 2 xenografts/4 sections) tumors (A), coupled with the percentage of PSA+ cells (total number of cells counted: WT = 142.293;MM = 106.013)(B).(C) Quantification of total PSA (free and complexed) circulating in the plasma of xenografted mice (WT, n = 4; MM, n = 4).(D) Quantification of Collagen Type-I (percentage of blue area to total section area; n = 6 sections per genotype), relative to Fig. 4D.(E) Quantification of Vimentin and α-Sma in FFPE sections of LNCaP WT and LNCaP MM xenograft (a minimum of 5 different areas per genotype were analyzed), relative to Fig. 4E.(F) Western blot analysis of Vimentin and α-SMA proteins in NAF, CAF, LNCaP and PC-3 cell lines.GAPDH was used as loading control.(G) Inverse correlation in PCa of the expression of P3H3 (left panel) and COLGALT1 (right panel) with respect to TRPM8 (TCGA, cBioportal).(H) RT-qPCR analysis of the indicated genes of the metabolism of type I collagen in NAF cells.(I) RT-qPCR analysis of PH3, PH4, COLGALT1, and COLGALT2 genes expression in CAFs conditioned with the supernatant of LNCaP MM cells and treated or not with the TLR3 inhibitor (20 μM, 24 h).(J) Western blot analysis of COL1A1 levels in CAFs conditioned with the supernatant of LNCaP MM cells and treated or not with the TLR3 inhibitor (XX μM, 24 h).GAPDH was used as loading control.(K,L) RT-qPCR analysis of canonical NF-kB targets genes in NAF (K) and CAF (L) cells conditioned with the supernatants of LNCaP (CAS, WT, MM) cell lines.(M) Magnification images of MHCII, CD45R (B220), IBA1 and NKp46 immunolocalization in FFPE sections of LNCaP wild type (WT) and LNCaP MM xenografts.Scale bars, 20 μm.Data Information: In (B-E) box-plots elements indicate the median (center line), upper and lower quartiles (box limits).Whiskers extend from the minimum to the maximum.*P ≤ 0.05; **P ≤ 0.01.(Student's ttest).In (H,I,K,L) data are presented as mean ± SD of n = 3 independent biological replicates.*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.(Two-tailed Student's t-test).Source data are available online for this figure.