Journal: Cell Genomics

Article Title: CXCR4-LASP1-G9a-SNAIL axis drives NEPC transdifferentiation via induction of EMT and downregulation of REST

doi: 10.1016/j.xgen.2025.100916

Figure Lengend Snippet: Elevated LASP1 expression in NEPC (A) Schematic summary of identification of five potential early epigenetic drivers of NEPC transdifferentiation. The data were analyzed between PDX Adeno and NEPC, and LTL331 post-castration week 8 compared to pre-castration (Pre-Cx). (B) Heatmap displaying consistent increased expression of five potential early epigenetic drivers in LTL331 post-castration week 8 and week 12 compared to Pre-Cx). (C) Consistent increased expression of LASP1 mRNA in LTL331 post-castration week 6, 8, and 10 compared to Pre-Cx. Data show mean ± SEM from three biological replicates. The p value was calculated with unpaired two-tail Student’s t tests. (D) Representative images of IHC staining of LASP1, CgA, NCAM1, SYP, and AR in adenocarcinoma tumors (Adeno) and neuroendocrine tumors (NEPC) of both clinical samples and PDX samples. (E) Western blot analysis of LASP1, ENO2, and REST expression in Adeno and NEPC PDX samples.

Article Snippet: Membranes were blocked in PBS containing 5% BSA, or TBS Odyssey buffer (Licor, 927–60001) for probing with phospho-antibodies or 5% non-fat milk for total and loading control antibodies against Vinculin (Sigma Aldrich, V4139, 1:2000 dilution), LASP1 (Santa Cruz,sc-374059, 1:500 dilution), ENO2 (Cell signaling, 8171, 1:1000 dilution), REST (abcam, ab21635, 1:1000 dilution), total H3 (Abcam, ab1791, 1:10000 dilution), H3K9me1 (abcam, ab176880, 1:1000 dilution), H3K9me2 (abcam, ab1220,1:1000 dilution), H3K9me3 (abcam, ab8898, 1:1000 dilution), pLASP1(a kind gift from Dr.

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Cell Genomics

Article Title: CXCR4-LASP1-G9a-SNAIL axis drives NEPC transdifferentiation via induction of EMT and downregulation of REST

doi: 10.1016/j.xgen.2025.100916

Figure Lengend Snippet: LASP1 drives NE transdifferentiation (A) Real-time qPCR analysis of LASP1 , SYP , ENO2 , and NCAM1 expression in 42D ENZR cells compared to 16D CRPC cells. (B) Western blot analysis of LASP1, SYP, ENO2, and NCAM1 expression in 42D ENZR cells compared to 16D CRPC cells. (C) Real-time qPCR analysis of LASP1 , SYP , ENO2 , and NCAM1 expression in 16D CRPC cells castrated for 3, 7, and 14 days. Cells were cultured in RPMI + 10% fetal bovine serum (FBS), RPMI + 10% charcoal-stripped serum (CSS), or RPMI + 10% CSS+10 μM enzalutamide (ENZ). (D) Western blot analysis of LASP1, SYP, ENO2, and NCAM1 expression in 16D CRPC cells castrated for 3, 7, and 14 days. Cells were cultured in RPMI + 10% FBS, RPMI + 10% CSS, or RPMI + 10% CSS +10 μM ENZ. (E) Western blot analysis of LASP1 levels in LASP1-OE 16D CRPC stable cell lines. (F) Real-time qPCR analysis of SYP , ENO2 , and NCAM1 expression in exogenously expressing vector or LASP1 in 16D CRPC cells. LASP1-OE 16D CRPC or mock-transduced 16D CRPC were cultured in RPMI + 10% FBS, RPMI + 10% CSS, or RPMI + 10% CSS +10 μM ENZ. (G) Western blot analysis of ENO2 and NCAM1 expression in exogenously expressing either mock or LASP1 in 16D CRPC cells. LASP1-OE 16D CRPC or mock-transduced 16D CRPC were cultured in RPMI + 10% FBS, RPMI + 10% CSS, or RPMI + 10% CSS +10 μM ENZ. (H) Representative IHC images of NCAM1, ENO2, AR, and LASP1 and hematoxylin and eosin (H&E) staining for LASP1-OE 16D CRPC or mock-transduced 16D CRPC xenograft tumors. (I) GSEA analysis of LASP1-OE 16D CRPC tumor-derived cells versus mock-transduced 16D CRPC cells. (J) Heatmap displaying increased NE marker expression in LASP1-OE 16D CRPC tumor-derived cells versus mock-transduced 16D CRPC cells. Data show mean ± SEM from three biological replicates. The p values (A and F) were calculated with unpaired two-tail Student’s t tests. Densitometry analysis data are in .

Article Snippet: Membranes were blocked in PBS containing 5% BSA, or TBS Odyssey buffer (Licor, 927–60001) for probing with phospho-antibodies or 5% non-fat milk for total and loading control antibodies against Vinculin (Sigma Aldrich, V4139, 1:2000 dilution), LASP1 (Santa Cruz,sc-374059, 1:500 dilution), ENO2 (Cell signaling, 8171, 1:1000 dilution), REST (abcam, ab21635, 1:1000 dilution), total H3 (Abcam, ab1791, 1:10000 dilution), H3K9me1 (abcam, ab176880, 1:1000 dilution), H3K9me2 (abcam, ab1220,1:1000 dilution), H3K9me3 (abcam, ab8898, 1:1000 dilution), pLASP1(a kind gift from Dr.

Techniques: Expressing, Western Blot, Cell Culture, Stable Transfection, Plasmid Preparation, Staining, Derivative Assay, Marker

Journal: Cell Genomics

Article Title: CXCR4-LASP1-G9a-SNAIL axis drives NEPC transdifferentiation via induction of EMT and downregulation of REST

doi: 10.1016/j.xgen.2025.100916

Figure Lengend Snippet: LASP1 interacting with SNAIL regulates REST expression (A) SNAIL forms a complex with LASP1 as measured by coIP in NCI-H660 cells. (B) Schematic diagram of protein-structure domains of LASP1 and SNAIL constructs. (C) LASP1 forms a complex with SNAIL through LIM domain and SNAG domain, respectively, measured by coIP in 293T cells transfected with mutant constructs. (D) Western blot analysis of phospho-LASP1 in LNCaP cells. LNCaP cells were serum starved overnight, followed by 10 μM forskolin stimulation at different time points. (E) Representative immunofluorescence images of LASP1 in LNCaP cells stimulated with 10 μM forskolin at different time points. (F) Representative immunofluorescence images of LASP1 and SNAIL in LNCaP cells stimulated with 10 μM forskolin at different time points. (G) Representative images of proximity-ligation assay measuring LASP1 and SNAIL interaction in LNCaP cells after forskolin stimulation. (H) Representative images of proximity-ligation assay measuring phospho-LASP1 and SNAIL interaction in LNCaP cells after forskolin stimulation. (I) LASP1 forms a complex with SNAIL, co-occupying E-box motif in the REST promoter region measured by ChIP-PCR. (J) REST promoter luciferase reporter analysis in LNCaP cells with increased doses of ectopic expression of LASP1 or SNAIL. (K) REST promoter luciferase reporter analysis in 293T cells with ectopic expression of LASP1 and SNAIL mutant constructs. (L) Real-time qPCR and western blot analysis of LASP1, REST, and ENO2 expression in NCI-H660 cells transfected with siRNAs against LASP1 or negative-control siRNA. (M) Real-time qPCR and western blot analysis of LASP1, REST, and ENO2 expression in LNCaP transfected with LASP1 or control vector. Data show mean ± SEM from three biological replicates. The p values (G–M) were calculated with unpaired two-tail Student’s t tests. Densitometry analysis data are in .

Article Snippet: Membranes were blocked in PBS containing 5% BSA, or TBS Odyssey buffer (Licor, 927–60001) for probing with phospho-antibodies or 5% non-fat milk for total and loading control antibodies against Vinculin (Sigma Aldrich, V4139, 1:2000 dilution), LASP1 (Santa Cruz,sc-374059, 1:500 dilution), ENO2 (Cell signaling, 8171, 1:1000 dilution), REST (abcam, ab21635, 1:1000 dilution), total H3 (Abcam, ab1791, 1:10000 dilution), H3K9me1 (abcam, ab176880, 1:1000 dilution), H3K9me2 (abcam, ab1220,1:1000 dilution), H3K9me3 (abcam, ab8898, 1:1000 dilution), pLASP1(a kind gift from Dr.

Techniques: Expressing, Construct, Transfection, Mutagenesis, Western Blot, Immunofluorescence, Proximity Ligation Assay, Luciferase, Negative Control, Control, Plasmid Preparation

Journal: Cell Genomics

Article Title: CXCR4-LASP1-G9a-SNAIL axis drives NEPC transdifferentiation via induction of EMT and downregulation of REST

doi: 10.1016/j.xgen.2025.100916

Figure Lengend Snippet: Inhibition of CXCR4 induced phenotypic reversion in NEPC cells (A) Elevated CXCR4 mRNA expression in neuroendocrine tumors (NEPC) from PDX samples and two clinical cohorts. (B) Representative IHC staining of CXCR4 in NEPC tumors of PDX and clinical samples. (C) CXCR4 forms a complex with LASP1 measured by coIP in 16D CRPC cells under serum-starvation conditions. (D) Representative immunofluorescence of LASP1 in 16D CRPC cells stimulated with CXCL12. 16D CRPC cells were serum starved overnight, followed by stimulation of 50 nM CXCL12. (E) Real-time qPCR analysis of NCAM1 and ENO2 in 16D CRPC cells treated with CXCL12. Data show mean ± SEM from three biological replicates. The p values were calculated with unpaired two-tail Student’s t tests. (F) Representative immunofluorescence of LASP1 in 16D CRPC cells stimulated with CXCL12 ± AMD3100. 16D CRPC cells were serum starved overnight, followed by stimulation of 50 nM CXCL12 with or without 5 μg/mL AMD3100. (G) RNA expression levels of NE markers and REST in LTL331R-2D cells treated with AMD3100 versus control. (H) Heatmap displaying luminal genes and Nelson response to androgen up genes in LTL331R-2D cells treated AMD3100 versus control.

Article Snippet: Membranes were blocked in PBS containing 5% BSA, or TBS Odyssey buffer (Licor, 927–60001) for probing with phospho-antibodies or 5% non-fat milk for total and loading control antibodies against Vinculin (Sigma Aldrich, V4139, 1:2000 dilution), LASP1 (Santa Cruz,sc-374059, 1:500 dilution), ENO2 (Cell signaling, 8171, 1:1000 dilution), REST (abcam, ab21635, 1:1000 dilution), total H3 (Abcam, ab1791, 1:10000 dilution), H3K9me1 (abcam, ab176880, 1:1000 dilution), H3K9me2 (abcam, ab1220,1:1000 dilution), H3K9me3 (abcam, ab8898, 1:1000 dilution), pLASP1(a kind gift from Dr.

Techniques: Inhibition, Expressing, Immunohistochemistry, Immunofluorescence, RNA Expression, Control