Figure 6.
SIRT1 promotes NEPC in human prostate cancer cell models. (A) Visual depiction of a subset of the SIRT1 regulon showing NEPC MRs it is predicted to up-regulate (red) or down-regulate (blue) in prostate cancer. (B) Expression of SIRT1 protein in human prostate cancer. IHC for SIRT1 was performed on seven independent patient tumors (see Materials and methods). Scale bars represent 100 µm. (C and D). Loss-of-function of SIRT1 in LNCaP cells. (C) Schematic diagram showing that LNCaP cells can be induced toward neuroendocrine differentiation by treatment with db-cAMP and IBMX (abbreviated +cAMP). shRNA silencing SIRT1 is predicted to inhibit this transition. (D) Heatmap representation of relative expression levels of the SIRT1 regulon (left) and known markers of NEPC or androgen signaling (right). Data show the results of real-time PCR analyses from day 0 (before NEPC induction) and day 7 (7 days after induction) for the shControl and two independent shRNA for SIRT1. Experiments were done with a minimum of five independent RNA samples for each condition. (E–H) Gain-of-function of SIRT1 in 22RV1 cells. (E) Schematic representation showing treatment of 22RV1 cells with +cAMP for induction toward neuroendocrine differentiation, as in panel C. SIRT1 expression was induced in 22RV1 cells using with CRISPRa and analyzed in vitro (F and G) and in vivo (H–J). (F) Western blot showing protein expression levels of SIRT1 in the 22RV1 cells. Actin, control for protein loading; positions of molecular weight markers are shown. The experiments were repeated a minimum of three times with three biological replicates in each. (G) Heatmap representation of relative gene expression levels of known markers of NEPC. (H–J) Results of 22RV1 cells grown orthotopically in host nude mice (n = 5/group). (H) Representative images of the 22RV1 prostate tumors. Shown are H&E staining, immunofluorescence (IF) staining for SIRT1 and synaptophysin (SYP). Scale bars represent 20 µm for H&E, and 10 µm for IF images. (I) Prostate tumors collected at the time of sacrifice. (J) Summary of tumor weights. See also Fig. S5. Source data are available for this figure: SourceData F6. Refer to the image caption for details. Panel A: A diagram showing the predicted regulon of SIRT1, with genes it is predicted to up-regulate in red and down-regulate in blue. Panel B: Immunohistochemistry images comparing adenocarcinoma and neuroendocrine prostate cancer. Panel C: A schematic diagram illustrating the experimental setup for loss-of-function of SIRT1 in LNCaP cells induced toward neuroendocrine differentiation by treatment with db-cAMP and IBMX. Panel D: Heatmaps showing relative expression levels of the SIRT1 regulon and known markers of NEPC or androgen signaling, analyzed by real-time PCR at day 0 and day 7 post-induction. Panel E: A schematic representation of the experimental setup for gain-of-function of SIRT1 in 22RV1 cells treated with db-cAMP and IBMX. Panel F: A Western blot image showing protein expression levels of SIRT1 in 22RV1 cells, with actin as a control for protein loading. Panel G: A heatmap representation of relative gene expression levels of known markers of NEPC. Panel H: Representative images of 22RV1 prostate tumors grown orthotopically in host nude mice, showing hematoxylin and eosin (H and E) staining, and immunofluorescence staining for SIRT1 and Synaptophysin (SYP). Panel I: Images of prostate tumors collected at the time of sacrifice. Panel J: A bar graph summarizing tumor weights.

SIRT1 promotes NEPC in human prostate cancer cell models. (A) Visual depiction of a subset of the SIRT1 regulon showing NEPC MRs it is predicted to up-regulate (red) or down-regulate (blue) in prostate cancer. (B) Expression of SIRT1 protein in human prostate cancer. IHC for SIRT1 was performed on seven independent patient tumors (see Materials and methods). Scale bars represent 100 µm. (C and D). Loss-of-function of SIRT1 in LNCaP cells. (C) Schematic diagram showing that LNCaP cells can be induced toward neuroendocrine differentiation by treatment with db-cAMP and IBMX (abbreviated +cAMP). shRNA silencing SIRT1 is predicted to inhibit this transition. (D) Heatmap representation of relative expression levels of the SIRT1 regulon (left) and known markers of NEPC or androgen signaling (right). Data show the results of real-time PCR analyses from day 0 (before NEPC induction) and day 7 (7 days after induction) for the shControl and two independent shRNA for SIRT1. Experiments were done with a minimum of five independent RNA samples for each condition. (E–H) Gain-of-function of SIRT1 in 22RV1 cells. (E) Schematic representation showing treatment of 22RV1 cells with +cAMP for induction toward neuroendocrine differentiation, as in panel C. SIRT1 expression was induced in 22RV1 cells using with CRISPRa and analyzed in vitro (F and G) and in vivo (H–J). (F) Western blot showing protein expression levels of SIRT1 in the 22RV1 cells. Actin, control for protein loading; positions of molecular weight markers are shown. The experiments were repeated a minimum of three times with three biological replicates in each. (G) Heatmap representation of relative gene expression levels of known markers of NEPC. (H–J) Results of 22RV1 cells grown orthotopically in host nude mice (n = 5/group). (H) Representative images of the 22RV1 prostate tumors. Shown are H&E staining, immunofluorescence (IF) staining for SIRT1 and synaptophysin (SYP). Scale bars represent 20 µm for H&E, and 10 µm for IF images. (I) Prostate tumors collected at the time of sacrifice. (J) Summary of tumor weights. See also Fig. S5. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal