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African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function. Significance: Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.

In this study, we used whole-exome sequencing of a cohort of 45 advanced-stage, treatment-naïve Nigerian (NG) primary prostate cancer tumors and 11 unmatched nontumor tissues to compare genomic mutations with African American (AA) and European American (EA) The Cancer Genome Atlas (TCGA) prostate cancer. NG samples were collected from six sites in central and southwest Nigeria. After whole-exome sequencing, samples were processed using GATK best practices. BRCA1 (100%), BARD1 (45%), BRCA2 (27%), and PMS2(18%) had germline alterations in at least two NG nontumor samples. Across 111 germline variants, the AA cohort reflected a pattern [BRCA1 (68%), BARD1 (34%), BRCA2 (28%), and PMS2 (16%)] similar to NG samples. Of the most frequently mutated genes, BRCA1 showed a statistically (P ≤ 0.05) higher germline mutation frequency in men of African ancestry (MAA) and increasing variant frequency with increased African ancestry. Disaggregating gene-level mutation frequencies by variants revealed both ancestry-linked and NG-specific germline variant patterns. Driven by rs799917 (T>C), BRCA1 showed an increasing mutation frequency as African ancestry increased. BRCA2_rs11571831 was present only in MAA, and BRCA2_rs766173 was elevated in NG men. A total of 133 somatic variants were present in 26 prostate cancer–associated genes within the NG tumor cohort. BRCA2 (27%), APC (20%), ATM (20%), BRCA1 (13%), DNAJC6 (13%), EGFR (13%), MAD1L1 (13%), MLH1 (11%), and PMS2 (11%) showed mutation frequencies >10%. Compared with TCGA cohorts, NG tumors showed statistically significant elevated frequencies of BRCA2, APC, and BRCA1. The NG cohort variant pattern shared similarities (cosign similarities ≥0.734) with Catalogue of Somatic Mutations in Cancer signatures 5 and 6, and mutated genes showed significant (q < 0.001) gene ontology (GO) and functional enrichment in mismatch repair and non-homologous repair deficiency pathways. Here, we showed that mutations in DNA damage response genes were higher in NG prostate cancer samples and that a portion of those mutations correlate with African ancestry. Moreover, we identified variants of unknown significance that may contribute to population-specific routes of tumorigenesis and treatment. These results present the most comprehensive characterization of the NG prostate cancer exome to date and highlight the need to increase diversity of study populations. Significance: MAA have higher rates of prostate cancer incidence and mortality, however, are severely underrepresented in genomic studies. This is the first study utilizing whole-exome sequencing in NG men to identify West African ancestry-linked variant patterns that impact DNA damage repair pathways.

The sodium/iodide symporter (NIS) frequently shows diminished plasma membrane (PM) targeting in differentiated thyroid cancer (DTC), resulting in suboptimal radioiodide (RAI) treatment and poor prognosis. The mechanisms which govern the endocytosis of NIS away from the PM are ill-defined. Here, we challenged the hypothesis that new mechanistic understanding of NIS endocytosis would facilitate prediction of patient outcomes and enable specific drug modulation of RAI uptake in vivo. Through mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET, we identify an acidic dipeptide within the NIS C-terminus which mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. We propose that NIS internalisation is orchestrated by the interaction of a C-terminal diacidic motif with AP2σ2, together with the proto-oncogene PBF acting via AP2μ2. Given that NIS internalisation was specifically druggable in vivo, our data provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer.SummaryWe delineate the role of endocytic genes in regulating NIS activity at the plasma membrane and highlight the potential for systemic targeting of endocytosis to enhance radioiodine effectiveness in radioiodine-refractory cancer cells.

SF_6 RNA-Seq PCA

ST_12 ChIP-Seq to RNA-Seq

SF_8 RNA-Seq GSEA

SF_13 GSEA tumors

Supplementary Table _2 - RIME II

ST_3 ATAC-Seq

SF_9 overlap miRNA

African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function.Significance:Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.

SF_3 motifs in ATAC-Seq

ST_4 ChIP-Seq

ST_5 ChromHMM

ST_7 ATAC-Seq to RIME

SF_5 ChIP-Seq motif

SF_2 Ancestry cell lines

SF_4 VDR cistrome

ST_6 ChIP-Seq Motif

ST_8 ATAC/ChIP-Seq to splicing

SF_7 RNA-Seq volcanos

In this study, we used whole-exome sequencing of a cohort of 45 advanced-stage, treatment-naïve Nigerian (NG) primary prostate cancer tumors and 11 unmatched nontumor tissues to compare genomic mutations with African American (AA) and European American (EA) The Cancer Genome Atlas (TCGA) prostate cancer. NG samples were collected from six sites in central and southwest Nigeria. After whole-exome sequencing, samples were processed using GATK best practices. BRCA1 (100%), BARD1 (45%), BRCA2 (27%), and PMS2(18%) had germline alterations in at least two NG nontumor samples. Across 111 germline variants, the AA cohort reflected a pattern [BRCA1 (68%), BARD1 (34%), BRCA2 (28%), and PMS2 (16%)] similar to NG samples. Of the most frequently mutated genes, BRCA1 showed a statistically (P ≤ 0.05) higher germline mutation frequency in men of African ancestry (MAA) and increasing variant frequency with increased African ancestry. Disaggregating gene-level mutation frequencies by variants revealed both ancestry-linked and NG-specific germline variant patterns. Driven by rs799917 (T>C), BRCA1 showed an increasing mutation frequency as African ancestry increased. BRCA2_rs11571831 was present only in MAA, and BRCA2_rs766173 was elevated in NG men. A total of 133 somatic variants were present in 26 prostate cancer–associated genes within the NG tumor cohort. BRCA2 (27%), APC (20%), ATM (20%), BRCA1 (13%), DNAJC6 (13%), EGFR (13%), MAD1L1 (13%), MLH1 (11%), and PMS2 (11%) showed mutation frequencies >10%. Compared with TCGA cohorts, NG tumors showed statistically significant elevated frequencies of BRCA2, APC, and BRCA1. The NG cohort variant pattern shared similarities (cosign similarities ≥0.734) with Catalogue of Somatic Mutations in Cancer signatures 5 and 6, and mutated genes showed significant (q < 0.001) gene ontology (GO) and functional enrichment in mismatch repair and non-homologous repair deficiency pathways. Here, we showed that mutations in DNA damage response genes were higher in NG prostate cancer samples and that a portion of those mutations correlate with African ancestry. Moreover, we identified variants of unknown significance that may contribute to population-specific routes of tumorigenesis and treatment. These results present the most comprehensive characterization of the NG prostate cancer exome to date and highlight the need to increase diversity of study populations.Significance:MAA have higher rates of prostate cancer incidence and mortality, however, are severely underrepresented in genomic studies. This is the first study utilizing whole-exome sequencing in NG men to identify West African ancestry-linked variant patterns that impact DNA damage repair pathways.

Supplementary Table 1. Known Germline Variants. Supplementary Table 2. Germline Variants. Supplementary Table 3. Known Somatic Variants. Supplementary Table 4. Somatic Vars in NG Normal. Supplementary Table 5. NG WES QC Metrics. Supplementary Table 6. NG Cohort Description. Supplementary Table 7. TCGA Cohort Description. Supplementary Table 8. AA Germ Var Frequency. Supplementary Table 9. Somatic Variants. Supplementary Table 10. NG Somatic ClueGo Results.

Supplementary Figure 1. WES Analysis Workflow. Supplementary Figure 2. Variant Strand Bias. Supplementary Figure 3. Somatic Variant Filtering. Supplementary Figure 4. Manual Somatic Variant Inspection. Supplementary Figure 5. Genetic Admixture Analysis. Supplementary Figure 6. Comparison of somatic variant count based on normal sample usage. Supplementary Figure 7. TCGA EA Somatic Variant Lollipop Plots. Supplementary Figure 8. NG PCa Novel Somatic Variants. Supplementary Figure 9. Nigerian Germline Variant – Normal/Tumor Comparison Lollipop Plots. Supplementary Figure 10. TCGA PCa Somatic Variant COSMIC Signature Analysis.

Video 3. N+/E- cells 3D reconstruction animation shows localization of ENT1 (red) and DAPI (blue)

Epithelial–mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.

Video 2. Animation displays 3D reconstruction of PANC-1 cells grown in monolayer immunostained for ENT1 (red) and DAPI (blue)

Video 4. E+/N- cells animation displays 3D reconstruction of ENT1 (red) and DAPI (blue)

Video 5. PANC-1 cells stably overexpressing EpCAM are shown in this animation of a 3D reconstruction of compiled z-stack images. ENT1 (red), EpCAM (green), DAPI (blue).

Video 1. PANC-1 cells stably overexpressing E-cadherin are shown in this animation of a 3D reconstruction of compiled z-stack images. ENT1 (red), E-cadherin (green), DAPI (blue).

A) Representative images of hematoxylin and eosin (H&E) staining of MCFDCIS, MCFDCIS-Î"4 and mixed (4:1 respectively) tumors that were injected in the mammary fat pad of SCID/Beige mice after two or five weeks. MCFDCIS-Î"4 tumors were not collected at two weeks due to small tumor size. Scale bar=50μm. B) Growth of subcutaneous tumors of DCIS or DCIS-Î"4-hy in athymic nude mice. n=5. **p

A list of differentially expressed genes in tumors and cells.

The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes—ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)—controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP—a principal component of endoplasmic reticulum (ER)–associated degradation—governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs.Significance:These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer.

Significant co-localization and trafficking of ARF4-dsRED and NIS-GFP proteins in co-incident vesicles at the plasma membrane in HeLa cells

Fletcher A et al Supplementary Information

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an “enabler” phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population.Significance:A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

Supplementary Methods

Fletcher A et al Data Source File