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. 2024 Oct 29;221(11):e20231832. doi: 10.1084/jem.20231832

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© 2024 Hsu et al.

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Figure 5. — Impa1 deficiency abrogates CRPC progression in TRAMP mouse model and IMPA1/inositol is upregulated in prostate cancer patients and CRPC patients and correlates with poor survival outcome. (A) Immunoblotting of prostate tissue from male C57BL/6J WT (n = 3) and TRAMP (n = 3) mice of 8 mo with indicated antibodies. LE, long exposure; SE, short exposure. Immunoblotting data were verified in at least two independent experiments. (B) Immunoblotting of prostate tissue from Impa1^FL/FL and Impa1^FL/FL/PB-Cre4 with indicated antibodies. Impa1^FL/FL, Impa1^FL/FL/PB-Cre4, TRAMP/PB-Cre4, and TRAMP/Impa1^FL/FL/PB-Cre4 mice were generated from four generations of backcrossing to maintain in C57BL/6J background. All mice were intercrossed to generate pups, followed by genotyping to group the age-matched mice for each experimental group. Immunoblotting data were verified in at least two independent experiments. (C) Representative images of AP, VP, and DLP lobes of the prostate from Impa1^FL/FL, Impa1^FL/FL/PB-Cre4, TRAMP/PB-Cre4, and TRAMP/Impa1^FL/FL/PB-Cre4 mice at the age of 7 mo. (D) H&E staining of AP and DLP lobes of prostate from Impa1^FL/FL, Impa1^FL/FL/PB-Cre4, TRAMP/PB-Cre4, and TRAMP/Impa1^FL/FL/PB-Cre4 mice at the age of 7 mo with 4× and 10× magnification (three mice for each group). Scale bar for 4× magnification, 500 μm; scale bar for 10× magnification, 100 μm. (E) ALDH1A1 cell population from the prostate tissue of WT, TRAMP/PB-Cre4 or TRAMP/Impa1^FL/FL/PB-Cre4 mice was determined by flow cytometry analysis using isotype and ALDH1A1 antibody. (F) The percentage of ALDH1A1 (ALDH^high) cells from prostate tissue was quantified from E shown as the mean ± SEM of three independent experiments for each group (three mice for each group). TRAMP, TRAMP/PB-Cre4 mice, and TRAMP/Impa1 KO, TRAMP/Impa1^FL/FL/PB-Cre4 mice. ***, P < 0.001 by two-tailed unpaired t test. (G) Kaplan–Meier survival plots of Impa1^FL/FL, Impa1^FL/FL/PB-Cre4, TRAMP/PB-Cre4, and TRAMP/Impa1^FL/FL/PB-Cre4 mice (15 mice for each group). P < 0.0001 by log–rank test. (H) The levels of inositol in the sera of normal male people (n = 20) and prostate cancer patients (n = 29) were determined by K-INOSL assay kit. ***, P < 0.001 by two-tailed unpaired t test. (I) IMPA1 protein expression from adjacent normal and prostate cancer patient with low or high Gleason score in our in-house prostate cancer samples was determined by IHC staining. Scale bar, 20 μm. (J) Box plot represents IMPA1 expression in adjacent normal (80 cases) and prostate cancer patients with high Gleason score and advanced stage (80 cases). ***, P < 0.001 by Mann–Whitney U test. (K) Disease-specific survival outcome with IMPA1 low or high expression was shown by Kaplan–Meier plots in prostate cancer patients with high Gleason score and advanced stage (80 cases). **, P = 0.0091 by long–rank test. Source data are available for this figure: SourceData F5.