Fig 2: Chondroitin sulfate enables cell motility and metastases in castration-resistant prostate cancer.a CSA measurements in LNCaP cells after 72 h of Fluorosamine (FL) treatment using rVAR2 binding on flow cytometry; linear regression followed by Ancova test; p < 0.0001, F = 18.59, DFn = 3, DFd = 12. b LC-MS quantification of C4S in LNCaP cells in AD condition after 72 h of FL treatment (50 μM). Data are shown as mean ± SEM; n = 4 biological replicates; p = 0.0041 by two-tailed t test. c FL EC50 values in AR + and AR- PC cell lines. Data are shown as EC50 plus upper and lower limits; n = 8 biological replicates. d Growth (IncuCyte) of LNCaP cells in FBS or AD conditions ± FL treatments. Data are shown as mean ± SEM, n = 4 biologically independent cell cultures; after 100 h p < 0.0001 by two-way ANOVA followed by Tukey’s test. e Cell death measurement of LNCaP cells treated as in d using propidium iodide (red staining; Incucyte) (Left panel). Data are shown as mean ± SEM, n = 4 independent samples; after 100 h p < 0.05 by two-way ANOVA followed by Tukey’s test. Representative image of Incucyte at endpoint (Right panel) f Representative images of wound healing closure in PC3 exposed to FL treatments, yellow show original scratch wound limits. Blue shows closed wound margins. g Scratch width measurements of f in μm. Data are shown as mean ± SD, n = 9 biologically independent replicates; after 6 h p < 0.001 by two-way ANOVA followed by Tukey’s test. h Invasion capacity (IncuCyte; Chemotaxis assay) of PC3 in presence and absence of FL treatment. Results are expressed as phase object count normalized to initial top value and shown as mean ± SD, n = 3 biologically independent replicates; after 26 h p < 0.05, after 46 h p < 0.0001 by two-way ANOVA followed by Tukey’s test. i Protocol schematic of PC3 metastatic seeding capacity in mice. j Representative H&E and HOXB13 staining of PC3 bone and lung metastases. Scale bar represents 100 μm k IVIS imaging of mice for metastases detection at humane or experimental endpoint. D is the day of termination. l Number of mice with jaw/bone or lung metastases in PBS or FL treated group. m Kaplan–Meier estimates of metastasis-free survival of mice injected with PC3 cells pretreated with FL 25 μM or Mock; Log-rank test. ns: not significant; **p < 0.01; ****p < 0.0001. Source data are provided as a Source Data file.

Fig 3: HOXB13 interacts with HDAC3 protein through its MEIS domain.a. Co-IP of HDAC1 and HDAC3 shows interaction with HOXB13. Whole-cell lysates from LNCaP cells stably expressing Flag-tagged HDAC1 or HDAC3 were subjected to co-IP using anti-Flag antibody. The eluted co-IP complex was analyzed by WB, along with input controls. * indicates non-specific bands detected by anti-Flag antibody.b. Cell lysates from LNCaP stably expressing HA-HOXB13 WT or G84E mutant were subjected to co-IP using an anti-HA antibody and then WB, along with input controls.c. HOXB13 interaction with HDAC3 is not dependent on AR. Whole-cell lysates from LNCaP with control (siCtrl) or AR knockdown (siAR) were subjected to co-IP using an anti-HOXB13 antibody and then WB, along with input controls.d. AR-negative PC-3 cells were subjected to co-IP using anti-HOXB13 or IgG control antibodies and then WB, along with input controls.e-f. Co-IP of HA-HDAC3 FL or deletion constructs (e) transfected into 293T cells with coexpression of HOXB13 (f).g-h. Co-IP of Flag-HOXB13 FL or deletion constructs (g) transfected into 293T cells with HDAC1 and HDAC3 co-expression (h).i. A model depicting the interactions between HOXB13 and HDAC3/NCoR complex. CTD: C-terminal domain of HDAC3; MEIS: the MEIS domain of HOXB13; and DAD: the DAD domain of SMRT/NCoR.

Fig 4: Therapeutic targeting of HOXB13-low tumors with a FASN inhibitor.a. IHC staining of FASN in human primary PCa and CRPC. Representative images of FASN staining in primary tumors (top panels) and metastatic CRPC (bottom panels) are shown in low- (40×) and high-magnification (200×).b. Quantification of FASN staining intensities in human PCa. y-axis shows the percentage of tumors with none, weak, moderate, and intense IHC staining.c. Pearson correlation between FASN and HOXB13 IHC scores in 72 sites from 25 CRPC patients. A two-sided P value for the T-distribution was shown.d. IVIS imaging of intravenous PC-3M xenograft tumors at week 0 (left) and 5 (right) after inoculation. Heatmap shows IVIS signal intensity color scale.e. Kaplan-Meier analyses of metastasis-free survival of pGIPZ and HOXB13 KD mice (n = 4 mice for pGIPZ vehicle group, n = 5 mice for the rest groups) treated with vehicle or TVB-2640. Metastasis was defined as a whole-mouse IVIS signal higher than 1 × 105 photons/s after three weeks of inoculation. P values were determined by a log-rank test.f. ex vivo IVIS analysis and quantification of PC-3M tumor metastasis to the lung. The number of mice in each group is the same as in e. y-axis shows the normalized luciferase intensity. Indicated P values were shown by an unpaired two-sided t-test.g. Luciferase and Pan-keratin IHC were used to identify metastasized PC-3M cells in the lungs of the mice. Representative images of H&E, Luciferase, and pan-keratin IHC staining in the indicated groups (3 mice in each group) are shown. Scale bar: 30 µm. Insets are shown at higher magnification. Red arrows indicate metastases in mouse lungs.h. Tumors in the lung were identified by GFP (Green) by IF (bottom row), and an adjacent section was used for Oil Red O staining (top). The IF images (bottom) have a Scale bar of 30 µm, and the Oil Red O staining images (top) have a Scale bar of 20 µm.

Fig 5: HOXB13 is hypermethylated and down-regulated in CRPCa. HOXB13 gene methylation levels in a variety of cancer types and corresponding benign (normal) samples using TCGA methylation data. Blue arrow indicates PCa (PRAD).*marks tumor types of significant differences compared with matched benign samples by Wilcoxon rank sum test. The sample sizes were included in Supplemental Table 7.b. Average methylation levels of Illumina Infinium probes targeting different regions of the HOXB13 gene (chr17:46,802,127–46,806,111. hg19) based on TCGA methylation data. Probes in green fonts target CpG islands. *probes with significantly differential methylation between normal and prostate tumor samples (Wilcoxon rank sum test with adjusted p-value < 0.05).c-d. Genome browser view of RNA-seq data (c) of HOXB13 (chr17:48,724,763–48,728,750, hg38) and average methylation levels (d) of 3 CpG islands within the HOXB13 gene loci (chr17:44,157,125–44,161,110. hg18) in LNCaP, 22Rv1, PC-3, DU145, and RWPE-1 cells.e. Expression of HOXB13 in Log2(FKPM) in LuCaP PDXs based on RNA-seq data. Blue indicates high- and red for low-HOXB13 samples.f. IGV track showing methylation at HOXB13 gene (chr17:46,802,127–46,806,111. hg19) in LuCaP PDX tumors with high or low HOXB13 as indicated in e. HOXB13 gene methylation in LuCaP PDX tumors was analyzed using MeDIP-seq data.g-h. RT-qPCR and WB analyses of PC-3M cells subjected to EZH2 or GFP-DNMT3A overexpression. PCR data shown are mean ±s.e.m. of technical replicates from one of three (n=3) independent experiments and analyzed by unpaired two-sided t-test.i. WB analyses of LNCaP cells treated with dCas9-SunTag-DNMT3A system with gRNAs targeting the 4 CpGs within the HOXB13 gene loci. The gRNAs were cloned into the pLKO5.sgRNA.EFS.tRFP657 vector and an sgRNA (NC) that had no cognate target in the human genome was used as a negative control. WB was performed 7 days after co-infection of indicated constructs.