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HOXB13 suppresses de novo lipogenesis through HDAC3-mediated epigenetic reprogramming in prostate cancer

Nature Genetics volume 54, pages 670–683 (2022)Cite this article

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Abstract

HOXB13, a homeodomain transcription factor, critically regulates androgen receptor (AR) activities and androgen-dependent prostate cancer (PCa) growth. However, its functions in AR-independent contexts remain elusive. Here we report HOXB13 interaction with histone deacetylase HDAC3, which is disrupted by the HOXB13 G84E mutation that has been associated with early-onset PCa. Independently of AR, HOXB13 recruits HDAC3 to lipogenic enhancers to catalyze histone deacetylation and suppress lipogenic regulators such as fatty acid synthase. Analysis of human tissues reveals that the HOXB13 gene is hypermethylated and downregulated in approximately 30% of metastatic castration-resistant PCa. HOXB13 loss or G84E mutation leads to lipid accumulation in PCa cells, thereby promoting cell motility and xenograft tumor metastasis, which is mitigated by pharmaceutical inhibition of fatty acid synthase. In summary, we present evidence that HOXB13 recruits HDAC3 to suppress de novo lipogenesis and inhibit tumor metastasis and that lipogenic pathway inhibitors may be useful to treat HOXB13-low PCa.

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Fig. 1: HOXB13 WT, but not G84E mutant, inhibits lipogenic programs in PCa.
Fig. 2: HOXB13 interacts with HDAC3 protein through its MEIS domain.
Fig. 3: HOXB13 recruits HDAC3 to catalyze histone deacetylation.
Fig. 4: HDAC3 is required for HOXB13-mediated suppression of de novo lipogenesis.
Fig. 5: HOXB13 is hypermethylated and downregulated in CRPC.
Fig. 6: HOXB13 loss promotes cell motility and PCa metastasis.
Fig. 7: Therapeutic targeting of HOXB13-low tumors with an FASN inhibitor.

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Data availability

All sequencing data (RNA-seq and ChIP–seq) generated for the study have been deposited in the Gene Expression Omnibus (GSE153586) at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE153586. The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE60 partner repository with the dataset identifier PXD030810. Source data are provided with this paper.

Code availability

The code for NGS analyses performed in this paper has been uploaded to https://github.com/JYULAB/HOXB13_project.

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Acknowledgements

We thank Y. Jia and W. Xie for providing some technical support for in vivo experiments. We thank all members of the Yu laboratory for helpful discussions. This work was partially supported by the Northwestern University Pathology Core Facility, the Center for Advanced Microscopy/Nikon Imaging Center and the Robert H. Lurie Comprehensive Cancer Center Support Grant (grant no. NCI P30CA060553). NGS was done at the University of Chicago Genomics Facility and lipid profiling at the Mass Spectrometry Core, University of Illinois at Chicago. IP-MS was done at the Taplin Mass Spectrometry Facility of Harvard Medical School. We thank the patients and their families, C. Higano, E. Yu, E. Mostaghel, H. Cheng, P. Nelson, B. Montgomery, M. Schweizer, A. Hsieh, J. Wright, D. Lin, F. Vakar-Lopez, X. Zhang, M. Roudier, L. True and the rapid autopsy teams for their contributions to the University of Washington Medical Center Prostate Cancer Donor Rapid Autopsy Program and the Development of the LuCaP PDX models. Funding supports for the work include the NIH/NCI training grant no. T32CA09560 (to G.G., L.T.B.); prostate cancer SPORE grant no. P50CA180995 (to W.J.C., J.Y., X.Y.); grants no. R01CA257446 and no. R01CA227918 (to J.Y.); grant no. R50CA211271 (to J.C.Z.); NIH-R35 grant no. CA197532 (to N.S.C.); grant no. R01CA251555 (to M.L.F.); Prostate Cancer Foundation grant no. 2017CHAL2008 (to J.Y.); and the Department of Defense grants no. W81XWH-19-1-0565 and no. W81XWH-21-1-0234 (to M.L.F.) and no. W81XWH-17-1-0405 and no. W81XWH-17-1-0578 (to J.Y.). Generation and maintenance of the LuCaP PDX models were partially funded by NIH awards no. P50CA97186 and no. P01CA163227.

Author information

Authors and Affiliations

  1. Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Xiaodong Lu, Ka-wing Fong, Galina Gritsina, Fang Wang, Lourdes T. Brea, Jonathan C. Zhao & Jindan Yu

  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Sylvan C. Baca, Jacob E. Berchuck, Sandor Spisak & Matthew L. Freedman

  3. Department of Pathology, Northwestern University, Chicago, IL, USA

    Jenny Ross & Ximing Yang

  4. Department of Urology, University of Washington, Seattle, WA, USA

    Colm Morrissey & Eva Corey

  5. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA

    Navdeep S. Chandel, William J. Catalona, Ximing Yang & Jindan Yu

  6. Division of Pulmonary and Critical Care, Department of Medicine, Northwestern University, Chicago, IL, USA

    Navdeep S. Chandel

  7. Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL, USA

    Navdeep S. Chandel & Jindan Yu

  8. Department of Urology, Northwestern University, Chicago, IL, USA

    William J. Catalona

  9. Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA

    Matthew L. Freedman

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  1. Xiaodong Lu
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Contributions

J.Y. and X.L. conceived the project and designed the experiments. J.C.Z., S.C.B., M.L.F. and J.Y. conducted bioinformatic and statistical analyses. F.W. and G.G. assisted with in vivo mouse experiments. K.F. performed IP-MS experiments and tissue microarray acquisition. J.R., X.L. and X.Y. performed HOXB13 and FASN IHC scoring. L.T.B. validated key experiments. J.E.B. carried out MeDIP-seq experiments. S.S. generated G84E isogenic 22Rv1. E.C. provided LuCaP PDX tissues. C.M. generated CRPC TMAs. X.L., J.C.Z. and J.Y. wrote the original manuscript. N.S.C., W.J.C. and M.L.F. consulted on the project and edited the manuscript.

Corresponding authors

Correspondence to Jonathan C. Zhao or Jindan Yu.

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Extended data

Extended Data Fig. 1 HOXB13 WT, but not G84E mutant, inhibits lipogenic programs in PCa.

a. Heatmap showing HOXB13-induced gene expression in PCa cells with HOXB13 KD and/or rescue. HOXB13-induced (n = 276) genes were derived by comparing shHOXB13 with pGIPZ using FDR < 0.05 and fold change > =2.5. b. GO analysis of HOXB13-induced genes identified in a. GO analyses was performed by DAVID, top enriched molecular concepts are shown. The X-axis indicates enrichment significance. One-sided Fisher’s Exact test was performed and -log10 (p-value) are shown. c. QRT-PCR validation of cell cycle gene regulation by HOXB13 in LNCaP cells. d. RT-qPCR validation of PSA and other key lipogenic gene regulation by HOXB13 in PC-3, C4-2B, and 22Rv1 cells. e. IGV view of the percentage of HOXB13 WT (C) and G84E alleles (T) (left) and genome browser view of mRNA expression of FASN (right, chr17:80,036,214-80,056,106, hg19, hg19) in WT and five isogenic G84E clones of 22Rv1. The isogenic G84E cells of HOXB13 were generated by CRISPR editing. f. WB validating the expression of FASN, PSA, AR, and HOXB13 in control and HOXB13-KD LNCaP cells. LNCaP cells were infected with control shRNA (pGIPZ) or shRNA targeting HOXB13 (shHOXB13) for four days, followed by hormone starvation (Ethl) or regular medium (10% FBS) for three days. g. Pie chart showing the genomic distribution of HOXB13 binding sites in LNCaP cells. h-i. ChIP-qPCR analyses of HOXB13 at lipogenic gene enhancers in LNCaP (h) cells with HOXB13 KD and/or rescue and PC-3 (i) cells with HOXB13 KD. Data in c,d and h,i were shown as technical replicates from one of three (n = 3) independent experiments. Data shown are mean ±s.e.m, P values by unpaired two-sided t-test.

Source data

Extended Data Fig. 2 HOXB13 interacts with HDAC3 protein through its MEIS domain.

a. Heatmap showing the number of peptides of AR-cofactors and HDAC3/NCoR complex enriched by HOXB13 WT or G84E mutant mass spectrometry. The complete lists are included in Supplemental Table 6. b-c. Co-IP of V5-NcoR1 full-length (FL) or deletion mutants (b) expressed in 293 T cells along with HOXB13, with or without HDAC3 co-expression (c). Co-IP using whole-cell lysates showed no interactions between HOXB13 and NCoR1 in cells without HDAC3 co-expression. d-e. Co-IP of HA-HDAC3 FL or deletion mutants (d) expressed in 293 T cells along with HOXB13 (e). Whole-cell lysates were subjected to co-IP using an anti-HA antibody. f. Fractionation assay showing cellular localization of HOXB13 WT and mutants in LNCaP cells. WT, G84E and ΔMEIS HOXB13 were detected on the chromatin, whereas ΔHOX and WFQ-3A have impaired ability to bind chromatin. Asterisk indicates endogenous HOXB13. g-h. Schematic illustration of a series of HOXB13 deletion mutants (g) and their interaction with HDAC3 (h). Whole-cell lysates from 293 T cells co-transfected with HA-HDAC3 along with Flag-tagged HOXB13 FL or its deletion mutants were subjected to co-IP using an anti-Flag antibody. Asterisk indicates the size of corresponding mutants. i. The interaction between HOXB13 and MEIS1 is not interrupted by HDAC3. Whole-cell lysates from 293 T cells co-transfected with Flag-HOXB13 along with HA-tagged MEIS1 and gradually increased amount of HA-tagged HDAC3 were subjected to co-IP using an anti-Flag antibody.

Source data

Extended Data Fig. 3 HOXB13 recruits HDAC3 to catalyze histone deacetylation.

a-b. MA plot showing differential HOXB13 (a) and HDAC3 ChIP-seq enrichment (b) in control (pGIPZ) and HOXB13 KD (shHOXB13) LNCaP cells. Color encodes the intensity of HOXB13 ChIP-seq in control cells. Dotted lines represent 2-fold differences. c. Venn diagram showing the overlap between HDAC3 and HOXB13 cistromes in PC-3 and LNCaP cells (top). Bottom: H3K27ac ChIP-seq was performed in PC-3 cells with control or HOXB13 KD, and their average intensity plots centered (±5 kb) on co-occupied sites are shown. d-e. ChIP-qPCR analyses of HDAC3 (d) and H3K27ac (e) at lipogenic gene enhancers in PC3 cells with control or HOXB13 KD. Data were normalized to 2% of input DNA. Shown are the mean ±s.e.m of technical replicates from one of three (n = 3) independent experiments. P values were calculated by unpaired two-sided t-test. f-h. ChIP-qPCR analysis of HOXB13 (f), HDAC3 (g), and H3K27ac (h) at PSA and FASN enhancer in 7 CRPC PDX tumors. Data were normalized to 5% of input DNA. Shown are the mean ±s.e.m of technical replicates from one of three (n = 3) independent experiments. Unpaired two-sided t-test was performed between indicated groups.

Extended Data Fig. 4 HDAC3 is required for HOXB13-mediated suppression of de novo lipogenesis.

a. Venn Diagram showing the overlap between HOXB13- or HDAC1/3-induced or –repressed genes in LNCaP cells. b. GO analysis of HDAC1/3-repressed (left) and -induced (right) genes in LNCaP cells. Top enriched molecular concepts are shown. p values were calculated by one-sided Fisher’s exact test. c. QRT-PCR analysis of SREBF2, FASN, and SCD expression in PC-3 cells with KD of HOXB13, HDAC3, or both. Data were normalized to GAPDH. Shown are mean ±s.e.m of technical replicates from one of three (n = 3) independent experiments. P values were calculated by unpaired two-sided t-test. d. WB analysis of HOXB13 and HDAC1/3 co-regulated genes in LNCaP cells with HOXB13 KD and rescue by HOXB13 WT or mutants. HOXB13 depletion expectedly led to up-regulation of FASN, SREBF1, and PSA, which were again repressed by re-introduction of WT HOXB13. HOXB13 ΔHOX/WFQ-3A mutants, although capable of interacting with HDAC3, are unable to bind DNA17 and consequently failed to repress lipogenic genes. Importantly, ΔMEIS and G84E mutants, which have impaired ability to interact with HDAC3, also failed to fully repress these genes. e-f. ORO staining and quantification of lipid accumulation in 22Rv1 (e) and PC-3 (f) with shHOXB13 and/or rescue. Scale bar: 30 µm. Quantification data are the mean ±s.d of technical replicates from one of two (n = 2) independent experiments. P values were calculated by unpaired two-sided t-test. h. LipidSpot staining and quantification of lipid droplets in HOXB13 WT and five clones of isogenic G84E 22Rv1. Scale bar:20 µm. LipidSpot staining intensity was quantified and calculated as the fold change of LipidSpot staining in G84E mutant cells compared with WT controls (right). Each data point represents an average of 6 (n = 6) fields per independent experiment by image J. Data are the mean ±s.d. Unpaired two-sided t-test was performed between indicated groups as show in figure.

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Extended Data Fig. 5 HOXB13 KD induces de novo lipogenesis in PCa cells cultured in either lipid-free or regular medium.

a-e. LipidSpot staining and quantification of lipid droplets in control (sgNC) or HOXB13 KD (sgHOXB13) LNCaP (a-c) and PC-3M (d-f) cultured in lipid-free or regular medium (10% FBS). WB (a,d) validating FASN up-regulation in HOXB13-KD cells cultured in lipid-free medium, confirming de novo lipogenesis. Data (b,e) shown are representative images of LipidSpot staining with a scale bar of 20 µm. LipidSpot staining intensity was quantified and calculated as fold change of LipidSpot staining in each condition normalized to sgNC non-target control under lipid-free medium (c,f). Each data point represents an average of 6 (n = 6) fields per independent experiment by image J. Data are the mean ±s.d. Unpaired two-sided t-test were performed between indicated groups as show in figure. g. Quantification of lipid accumulation of PDXs in Fig.4i. Six (n = 6) representative areas per PDX were quantified using Image J. Data are shown as mean±s.d. Unpaired one-sided t-test was performed between indicated groups as show in figure.

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Extended Data Fig. 6 HOXB13 is hypermethylated and down-regulated in CRPC.

a. 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.

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Extended Data Fig. 7 HOXB13 loss induces PCa cell motility in vitro and tumor metastasis in vivo.

a. Colony formation assays were performed in LNCaP, C4-2B and PC-3 cells with HOXB13 de-regulation or indicated treatment. The data showed that androgen-dependent LNCaP cell growth was abolished by HOXB13 KD, which could be restored by re-expression of either WT or G84E HOXB13, suggesting an AR-dependent effect. By contrast, C4-2B is only partially sensitive to HOXB13 KD, whereas the growth of C4-2B cells pre-treated by enzalutamide (ENZ) and the AR-negative PC-3 cells is unaffected by HOXB13 KD. b. Cell invasion assays of control or HOXB13-KD PC-3M cells cultured in lipid-free or regular medium. Representative images are shown (left panels), and the number of invaded cells are quantified (right panel). Scale bar: 50 µm. Quantified data shown are mean ±s.d. of three representative fields from one of three (n = 3) independent experiments. P values were calculated by unpaired two-sided t-test. c. Cell migration assays of PC-3 cells with control or shHOXB13. Images shown were taken at 0 and 48 hours after a scratch was created on the cell monolayer. d. Tumor volume was measured by IVIS after two weeks of intra-prostate inoculation of PC-3M cells. Y-axis shows the normalized luciferase intensity. Statistical significance was evaluated by one-way ANOVA test (P = 0.836). e. HOXB13 de-regulation did not affect PC-3M xenograft tumor growth. Tumor volume was measured every week by IVIS live mice imaging. Y-axis shows the normalized luciferase intensity. Data shown in each time point are mean ± s.d. Statistical significance was evaluated by one-way ANOVA test (P = 0.365).

Extended Data Fig. 8 Effect of various lipogenic inhibitors on PCa cells.

a. FASN mRNA levels in publically available PCa gene expression profiling datasets. Data shown (Y-axis) are log2-transformed microarray expression values for GSE6919 and GSE35988 and FPKM values for TCGA-dbGaP dataset, which combines prostate samples from dbGaP datasets with accession#: phs000178(TCGA), phs000443, phs000915, and phs000909. P values between primary and metastatic PCa were calculated using unpaired two-sided t-test. Boxplots represent the median and bottom and upper quartiles; Whisker edges indicate minimum and maximum values. b. Cell invasion of control or HOXB13-KD cells treated with various lipid inhibitors. PC-3M cells were treated with 5 µM TVB-2640, 5 µM TOFA, 2 µM Fatostatin or 2 µM Simvastatin for 3 days. C4-2B cells were treated with 1 µM of indicated inhibitors for 3 days. Representative images are shown for C4-2B with a Scale bar of 50 µm and PC-3M with a Scale bar of 30 µm. c. Quantification of cell invasion shown in b. Data shown are the mean ±s.d of three representative fields from one of two (n = 2) independent experiments. P values were calculated by unpaired two-sided t-test comparing inhibitor groups with DMSO in control or shHOXB13 cells. d-e. Representative images of ORO staining (d) and quantification (e) of lipid accumulation in HOXB13-KD PC-3M cells treated with DMSO or 5 μM TVB-2640 for 3 days. Scale bar: 30 µm. Data shown are the mean ± s.d of triplicate wells from one of two (n = 2) independent experiments. Unpaired two-sided t test was performed between indicated groups as shown in figures.

Extended Data Fig. 9 Therapeutic targeting of HOXB13-low tumors with FASN inhibitors in an orthotopic xenograft model.

a. Graph showing tumor volumes after intraprostatic inoculation of PC-3M cells. Tumor formation was confirmed by IVIS one week after inoculation. Then the mice were randomized to receive vehicle (30% PEG400) or TVB-2640 (100 mg/Kg) every day for 30 days. Tumor volumes were measured twice per week by IVIS after two weeks of inoculation. Y-axis shows the normalized luciferase intensity. Data in each time point are mean ±s.d. Statistical significance was evaluated by one-way ANOVA (P = 0.0007) and comparisons between indicated groups by post hoc Tukey test. PC-3M xenograft tumor growth was not affected by HOXB13 KD (p = 0.3212). Importantly, primary tumor growth in both control (pGIPZ, p = 0.0171) and HOXB13-KD (shHOXB13, p = 0.0082) mice was significantly inhibited by TVB-2640. b-c. Representative ex vivo IVIS images (b) and quantifications (c) of PC-3M tumor metastasis to the liver and lung (n = 6 mice per group). Heatmap shows IVIS signal intensity color scale. Indicated p-values were shown by unpaired two-sided t-test. HOXB13 KD significantly promoted PCa metastases, which was abolished by TVB-2640. d. Validation of PC-3M tumor metastasis to the liver by H&E and IHC. Luciferase and Pan-keratin IHC were used to identify metastasized PC-3M cells in mouse liver. Representative images of H&E, Luciferase and Pan-keratin staining in indicated group (n = 3 mice in each group) are shown. Scale bar: 30 µm. “T” indicates tumor and “n” for normal liver. e. Kaplan-Meier analyses of overall survival of pGIPZ and HOXB13 KD mice treated with vehicle or TVB-2640 (n = 6 mice per group). P values were determined by the log-rank test. TVB-2640 treatment prolonged the overall survival of mice in both control (p = 0.017) and HOXB13-KD (p = 0.005) groups.

Extended Data Fig. 10 Therapeutic targeting of HOXB13-low tumors with FASN inhibitors in an intravenous xenograft model.

a. Body weight analysis in mice inoculated by control or HOXB13-KD PC-3M cells and treated with vehicle or TVB-2640. Data in each time point are mean ±s.d. Y-axis shows the percentage of body weight change. b-c. Representative ex vivo IVIS images (b) of PC-3M tumor metastasis to lung, hind leg, liver, and rib and quantification (c) of metastasis to liver, hind leg, and rib (n = 4 mice for pGIPZ vehicle group; n = 5 mice for the rest groups). At the endpoint, lung, hind leg, liver, and rib were collected and analyzed using ex vivo IVIS. Heatmap in b shows IVIS signal intensity color scale. Y-axis in c shows the normalized luciferase intensity. Indicated p values were by unpaired two-sided t test.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Tables

Supplementary Table 1: Antibodies that were used in this study. Supplementary Table 2: Oligonucleotides that were used in this study. Supplementary Table 3: Genes not rescued by G84E and rescued by G84E in Fig. 1a. Supplementary Table 4: Genes involved in fatty acid uptake related to Supplementary Fig. 4. Supplementary Table 5: Key enzymes involved in lipogenesis related to Supplementary Fig. 7. Supplementary Table 6: Interacting proteins in HOXB13 WT and G84E mass spec related to Extended Data Fig. 2a. Supplementary Table 7: The numbers of normal and tumor in different cancer types related to Extended Data Fig. 6a.

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Lu, X., Fong, Kw., Gritsina, G. et al. HOXB13 suppresses de novo lipogenesis through HDAC3-mediated epigenetic reprogramming in prostate cancer. Nat Genet 54, 670–683 (2022). https://doi.org/10.1038/s41588-022-01045-8

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