Fig 2: Arf-WRC interaction is direct and can be greatly enhanced by Rac1.(A) Coomassie blue–stained SDS-PAGE gels showing GST-Arf1 pull down of WRC FL (left) and ∆WRC230 (right) in the presence or absence of untagged Rac1QP. In the schematic of WRCs, dotted lines indicate unstructured sequences. Both the A and D sites for Rac1 binding are indicated. (B) Coomassie blue–stained SDS-PAGE gels showing pull down of ∆WRC230 by GST-Arf1 in indicated nucleotide states or in the presence of the Arf1-binding protein EspG. (C) EPD assay to measure the binding affinity of the Arf1-WRC interaction in the presence of indicated Rac1 variants. On the left is the quantification of the data pooled from two to three independent experiments for each condition and globally fitted to obtain the binding isotherms. The derived Kd and fitting errors are shown in the table. On the right are representative Coomassie blue–stained SDS-PAGE gels of the supernatant samples used for quantification. (D) Coomassie blue–stained SDS-PAGE gels showing GST-Arf1 pull down of WRC subcomplexes in the presence or absence of Rac1QP. The dimer is the Sra1/Nap1 subcomplex. The trimer is the WAVE1(1-230)/Abi2(1-158)/HSPC300 subcomplex. Asterisks indicate weak binding signals. (E) Arf1 binding to the WRC is sensitive to pH and salt concentration. Shown is Coomassie blue–stained SDS PAGE from GST-Arf1 pull down of ∆WRC230 in indicated buffer conditions, in the presence or absence of Rac1QP. Red asterisk indicates increased background binding to GST beads at pH 6, to avoid which we use pH 7 and 50 mM NaCl throughout this study. (F) Coomassie blue–stained SDS-PAGE gel showing pull down of ∆WRC230 by different GST-tagged Arf-family members with or without Rac1QP.

Fig 3: RWPE-1 ABI1 KO spheroids exhibit invasive migratory phenotype in 3D Matrigel cultures. Time-lapse images acquired overnight of representative spheroids showing key features of the spheroids. Cells were plated in 50% Matrigel and allowed to grow for 6 days before live overnight imaging (every 5 min for 14 h). (a) RWPE-1 parental, (b) Control clone 16, (c) ABI1 KO clone 12, (d) ABI1 KO clone 35, (e) ABI1 rescue Isoform 2, (f) ABI1 rescue Isoform 3 cells. (g-h) Analysis of the morphology and behavior of the spheroids was performed by quantifying the percentage of organoids exhibiting the following characteristics: spinning, spherical shape, grape-like morphology, projections and cell budding/migration from the spheroid. The control spheroids show a spherical shape, while the KO spheroids do not (black outline). KO spheroids feature projections (black filled arrows) and cells trying to escape from the organoid (white arrows). The data represent n = 3 experiments (18–25 spheroids per cell line per experiment) and were normalized to the parental RWPE-1 cell data. The graph was generated with Prism software, and the results are presented as the mean ± SEM, unpaired Student’s t-test with p-values * (p < 0.05), ** (p < 0.01), *** (p < .001), and **** (p < 0.0001)

Fig 4: ABI1 expression is downregulated during prostate tumor progression. Prostate cancer tissue microarray (n = 505 patients) was stained for ABI1 to evaluate its expression during prostate tumor progression. (a) Representative ABI1 expression staining in Gleason 3, 4, and 5 patterns, as indicated; Benign indicates normal prostate epithelium. (b) Comparison of ABI1 expression among Gleason patterns, 3, 4 and 5 indicating significant downregulation of ABI1 in Gleason 5 vs. 3, (p = 0.0012); or 4 (p = 0.0025) (one-way ANOVA, multiple comparison). (c) Analysis of ABI1 expression in the tissue microarray indicating an association between low ABI1 expression and clinically significant events, such as biochemical recurrence, a metastatic event, and death; p = 0.038; 99% confidence interval: 0.03308–0.4292; Linear-by-Linear association exact test (Sytel Studio-9). (d) Representative Western blots showing reduced levels of ABI1 in different patient-derived organoid cell lines (left) and immortalized prostate cancer cell lines (right) compared with non-tumor 26Nb and RWPE-1 cells. Numbers next to bands in the RWPE-1 lane and on the right side of the panel indicate ABI1 isoform designation: top band, isoform 2; middle band, isoform 3; and bottom band, isoform 5 [34, 40]; #, indicates the mutant ABI1 lacking exon 6 in LNCaP [37]. GAPDH was used as the loading control. (e) Identification of mutations in the cDNA of ABI1 from organoid cultures of human metastatic prostate cancer cell lines. (A. and B.) Schematic of full-length ABI1 cDNA (isoform-1) with coding regions of consecutive exons (Ex1-Ex12) indicated (not drawn to scale). The ATG start codon (corresponding to nucleotide 1), TAA stop codon (corresponding to nucleotide 1527), and the T15, E3, and T33 primer sites (filled-in black arrows) are labeled. The T15, E3, and T33 primers [40] were used to selectively amplify ABI1 cDNA via PCR from the MSK-PCa1 and MSK-PCa2 organoid cell lines. PCR products were subcloned into a pCR-Blunt II TOPO vector and sequenced. A. Schematic of mutation identified in cDNA clone-19 sequence from MSK-PCa1 cells. Sequencing of cDNA clone-19 revealed a 248-bp deletion spanning nucleotides 717 to 964 (from the 3' end of Exon6, at the junction between Exon 6 and Exon 7, to the middle of Exon 9) that is predicted to result in a frame shift and subsequent stop codon at nucleotide 1170. B. Schematic of mutation identified in cDNA clone-9 from MSK-PCa2 cells. Sequencing of cDNA clone-9 revealed a 44-bp deletion spanning nucleotides 955 to 998 in the middle of Exon 9 that is predicted to result in a frame shift and subsequent stop codon at nucleotide 1047

Fig 5: Differential gene expression pattern is correlated with EMT pathways and WNT signaling. The DEGs determined by RNA sequencing demonstrated (a) upregulation of EMT pathways and Wnt signaling. Individual genes, fold changes and p-values are listed. Right panel shows representative western blots of several EMT markers and different integrin proteins validate the RNA sequencing findings. (b) Western blots showing upregulation of STAT3 phosphorylation (Y705 and S727) and FYN activation. (c) ABI1 interacts with STAT3 and FYN in RWPE-1 cells. Western blotting results indicating that FYN (top panel) and STAT3 (middle panel) co-immunoprecipitated with Abi1. Input, RWPE-1 cell lysate; Flow-Through, unbound fraction of the lysate; IgG, control immunoprecipitation lacking anti-Abi1 antibody; IP, immunoprecipitation including the anti-Abi1 antibody. Asterisk in top panel indicates the corresponding bands in fractions. (d-f) ABI1 loss promotes nuclear localization of activated STAT3 as determined by pY705 antibody. (d) Western blotting analysis of p-STAT3 Y705 expression in cytoplasmic (left panel) or, nuclear fraction (right panel) of RWPE ABI1 KO, control and ABI1-rescued clones. (e) Immunofluorescence analysis of p-STAT3 Y705 levels in RWPE ABI1 KO clones. (f) Quantification of nuclear p-STAT3 Y705 levels, n = 3, p < 0.001. (g) An example of inverse correlation of ABI1 and pSTAT3 Y705 expression levels in a prostate tumor. Serial tumor sections were immunostained with antibodies to either ABI1 (left panel), or pSTAT3 Y705 (right panel). Arrows depict two example areas of correlation of low ABI1 and high pSTAT. (h) Schematic depicting the proposed mechanism of how Abi1 loss promotes the invasive potential of prostate epithelial cells. Center, ABI1 (ABI1) is critical for cell junction maintenance through WAVE complex-mediated actin polymerization; disruption of ABI1 leads to loss of WAVE complex (WAVE complex) stability, resulting in lower levels of E-cadherin (CDH1) at cell-cell contacts and downregulation of cell-cell adhesion. Right, ABI1 pY421 binds with high affinity to FYN-SH2 domain. Disruption of ABI1 leads to constitutive activation of the SRC family kinase FYN (FYN) to stimulate STAT3 activation and promote its nuclear localization. Nuclear STAT3 promotes transcription of the epithelial-to-mesenchymal transition (EMT) gene program, including extracellular matrix metalloproteinases that promote migration through matrix degradation in the absence of proper cell-cell adhesion. Left, In the absence of ABI1, activated FYN acts on FAK kinase (FAK) to promote integrin signaling, which also supports cell migratory activity. Activated FAK may also directly bind and activate pSTAT3, thus providing another possibility for EMT pathway activation