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Chemokines and their cognate receptors comprise an intricate signaling network that becomes high-jacked by cancer cells for uncontrollable tumor growth and dissemination. ACKR3 (Atypical Chemokine Receptor 3), traditionally called CXCR7, is up-regulated in many cancers, including advanced prostate cancer, and represents promising targets for therapeutic intervention. Unlike typical G protein-coupled receptors such as CXCR4, CXCR7, once bound by its cognate ligand CXCL12, initiates the recruitment of β-arrestin instead of G proteins, and results in rapid internalization and degradation of CXCL12, functioning as a scavenger receptor. However, recent evidence suggests that CXCR7 may be more than a scavenger or auxiliary receptor of CXCR4 and that it may play essential roles in regulating cancer progression, some of which are independent of CXCR4 and its ligands, such as CXCL12. Constitutively active CXCR7 binds to β-arrestin. This protein complex internalizes to form a scaffold for assembling and activating various cytoplasmic kinases necessary for cell survival and tumor growth. Here we review and discuss the up-to-date knowledge on CXCR7 regulation and function and how this new understanding guides the development of CXCR7 inhibitors, focusing on prostate cancer.

Androgen receptor (AR) pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance to therapy is common. Here, we used a CRISPR activation screen in metastatic castration-sensitive prostate cancer cells to identify genes that promote resistance to AR inhibitors. Activation of the TGFβ target gene paired-related homeobox2 (PRRX2) promoted enzalutamide resistance. PRRX2 expression was the highest in double-negative prostate cancer (DNPC), which lack AR signaling and neuroendocrine differentiation, and a PRRX2-related gene signature identified a subset of patients with DNPC with reduced overall survival. PRRX2-expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, treatment with CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing, castration-resistant tumors to enzalutamide. Overall, PRRX2 was identified as a driver of enzalutamide resistance. The PRRX2 signature merits investigation as a biomarker of enzalutamide resistance in prostate cancer that could be reversed with CDK4/6 and BCL2 inhibitors. Significance: PRRX2 mediates enzalutamide resistance via activation of the E2F and BCL2 pathways, which can be targeted with CDK4/6 and BCL2 inhibitors to reverse resistance.

Supplementary Table from A Genome-Wide CRISPR Activation Screen Identifies PRRX2 as a Regulator of Enzalutamide Resistance in Prostate Cancer

siRNA sequence used in this study

Onset of castration-resistance prostate cancer (CRPC) after long-term androgen deprivation therapy remains a major obstacle in the treatment of prostate cancer. The peptidylarginine deiminase PADI2 has been implicated in chronic inflammatory diseases and cancer. Here we show that PADI2 is an androgen-repressed gene and is upregulated in CRPC. PADI2 expression was required for survival and cell-cycle progression of prostate cancer cells, and PADI2 promoted proliferation of prostate cancer cells under androgen-deprived or castration conditions in vitro and in vivo. Cytoplasmic PADI2 protected the androgen receptor (AR) against proteasome-mediated degradation and facilitated AR binding to its target genes after nuclear translocation and citrullination of histone H3 amino acid residue R26. In contrast, mutant PADI2 D180A failed to affect AR stability, nuclear translocation, or transcriptional activity. PADI2 mediated AR control in a manner dependent on its enzymatic activity and nuclear localization, as correlated with increased histone H3 citrullination. Notably, coadministration of the PADI inhibitor Cl-Amidine and the AR signaling inhibitor enzalutamide synergized in inhibiting CRPC cell proliferation in vitro and tumor growth in vivo. Overall, our results establish PADI2 as a key mediator for AR in prostate cancer progression, especially CRPC, and they suggest PADI as novel therapeutic targets in this disease setting. Cancer Res; 77(21); 5755–68. ©2017 AACR.

Effects of truncate PADI2 on AR activity. Various truncated PADI2 were constructed and their cytoplasmic/nuclear locations differ. PADI2 in its different lengthen could activate AR signaling through citrullination.

Primer sequence used in this study

Supplementary Data from A Genome-Wide CRISPR Activation Screen Identifies PRRX2 as a Regulator of Enzalutamide Resistance in Prostate Cancer

Castration-resistant prostate cancer (CRPC) that has developed resistance to the new-generation androgen receptor (AR) antagonist enzalutamide is a lethal disease. Transcriptome analysis of multiple prostate cancer models identified CXCR7, an atypical chemokine receptor, as one of the most upregulated genes in enzalutamide-resistant cells. AR directly repressed CXCR7 by binding to an enhancer 110 kb downstream of the gene and expression was restored upon androgen deprivation. We demonstrate that CXCR7 is a critical regulator of prostate cancer sensitivity to enzalutamide and is required for CRPC growth in vitro and in vivo. Elevated CXCR7 activated MAPK/ERK signaling through ligand-independent, but β-arrestin 2-dependent mechanisms. Examination of patient specimens showed that CXCR7 and pERK levels increased significantly from localized prostate cancer to CRPC and further upon enzalutamide resistance. Preclinical studies revealed remarkable efficacies of MAPK/ERK inhibitors in suppressing enzalutamide-resistant prostate cancer. Overall, these results indicate that CXCR7 may serve as a biomarker of resistant disease in patients with prostate cancer and that disruption of CXCR7 signaling may be an effective strategy to overcome resistance.Significance:These findings identify CXCR7-mediated MAPK activation as a mechanism of resistance to second-generation antiandrogen therapy, highlighting the therapeutic potential of MAPK/ERK inhibitors in CRPC.

Androgen receptor (AR) pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance to therapy is common. Here, we used a CRISPR activation screen in metastatic castration-sensitive prostate cancer cells to identify genes that promote resistance to AR inhibitors. Activation of the TGFβ target gene paired-related homeobox2 (PRRX2) promoted enzalutamide resistance. PRRX2 expression was the highest in double-negative prostate cancer (DNPC), which lack AR signaling and neuroendocrine differentiation, and a PRRX2-related gene signature identified a subset of patients with DNPC with reduced overall survival. PRRX2-expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, treatment with CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing, castration-resistant tumors to enzalutamide. Overall, PRRX2 was identified as a driver of enzalutamide resistance. The PRRX2 signature merits investigation as a biomarker of enzalutamide resistance in prostate cancer that could be reversed with CDK4/6 and BCL2 inhibitors.Significance:PRRX2 mediates enzalutamide resistance via activation of the E2F and BCL2 pathways, which can be targeted with CDK4/6 and BCL2 inhibitors to reverse resistance.

PADI2 inhibition decreases the transcriptional activity of PSA. Treatment with siPADI2 or Cl-amidine suppresses the transcriptional activity of PSA in LNCaP and C4-2B cells.

Supplementary Figure 1. Generation of Enz-resistant prostate cancer cell lines. Supplementary Figure 2. AR directly represses CXCR7 gene expression. Supplementary Figure 3. CXCR7 expression is required for C4-2B Enz-resistant prostate cancer cell survival and invasiveness. Supplementary Figure 4. CXCR7 activates MAPK pathway independently of CXCR4 and CXCL12. Supplementary Figure 5. ARRB2 expression is required for Enz-resistant prostate cancer cell growth and invasion. Supplementary Figure 6. CXCR7 is upregulated in metastatic prostate cancer. Supplementary Figure 7. MEK inhibitor trametinib suppresses Enz-resistant prostate cancer growth in vitro and in vivo. Supplementary Table S1: Oligonucleotides that were utilized in this study.

Legends for Supplementary Figures and Tables.

PADI2 makes no significant effect on the mRNA expression level of AR, but stabilizes AR protein, facilitates its nuclear translocation and activity. PADI2 stabilizes AR at protein, but not mRNA level, and facilitates AR nuclear translocation and activity.

Enrichment of citrullinated H3R2+R8+R17 and H4R3 on AR target gene. No visible citrullination of H3R2+R8+R17 and H4R3 on AR target gene was observed after treatment with silPADI2 or Cl-amidine.

Characterization of PADI2 and PADI4 in PCa cells with the stimulation of androgen and the effect of siPADI4 on cell viability of PCa cells. Androgen suppresses the expression of PADI2, but not PADI4 in PCa cells and silencing PADI4 makes no obvious effect on cell viability of PCa cells.

The Polycomb Group (PcG) protein EZH2 is a critical component of a multiprotein complex that methylates Lys27 of histone 3 (H3K27), which consequently leads to the repression of target gene expression. We have previously reported that EZH2 is overexpressed in metastatic prostate cancer and is a marker of aggressive diseases in clinically localized solid tumors. However, the global set of genes directly regulated by PcG in tumors is largely unknown, and thus how PcG mediates tumor progression remains unclear. Herein we mapped genome-wide H3K27 methylation in aggressive, disseminated human prostate cancer tissues. Integrative analysis revealed that a significant subset of these genes are also targets of PcG in embryonic stem cells, and their repression in tumors is associated with poor prognosis. By stepwise cross-validation, we developed a “Polycomb repression signature” composed of 14 direct targets of PcG in metastatic tumors. Notably, solid tumor subtypes in which this gene signature is repressed show poor clinical outcome in multiple microarray data sets of tumors including breast and prostate cancer. Taken together, our results show a fingerprint of PcG-mediated transcriptional repression in metastatic prostate cancer that is reminiscent of stem cells and associated with cancer progression. Therefore, PcG proteins play a central role in the epigenetic silencing of target genes and functionally link stem cells, metastasis, and cancer survival. [Cancer Res 2007;67(22):10657–63]

Supplementary Figure 2 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion. Cancer Res; 70(21); 8347–56. ©2010 AACR.

Supplementary Figure 3 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

Supplementary Table 5 from Autoantibody Profiles Reveal Ubiquilin 1 as a Humoral Immune Response Target in Lung Adenocarcinoma

Supplementary Figure 4 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

Supplementary Data, Methods, Figures 1-5, Tables 1-5 from A Polycomb Repression Signature in Metastatic Prostate Cancer Predicts Cancer Outcome

Supplementary Figure 1 from Cdc42-Interacting Protein 4 Promotes Breast Cancer Cell Invasion and Formation of Invadopodia through Activation of N-WASp

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Supplementary Table 1, Figures 1-3 from A Fluorescence In situ Hybridization Screen for E26 Transformation–Specific Aberrations: Identification of DDX5-ETV4 Fusion Protein in Prostate Cancer

PDF file, 272K, Figure S1. ERG induces Wnt signaling in prostate cells, which is mediated by LEF1. Figure S2. ERG and beta-catenin synergistically induce Wnt signaling. Figure S3. LEF1 is a critical mediator in ERG-induced Wnt signaling. Table S1. Antibodies that were used in this study. Table S2. Primers that were used in this study.

FoxA1 (FOXA1) is a pioneering transcription factor of the androgen receptor (AR) that is indispensible for the lineage-specific gene expression of the prostate. To date, there have been conflicting reports on the role of FoxA1 in prostate cancer progression and prognosis. With recent discoveries of recurrent FoxA1 mutations in human prostate tumors, comprehensive understanding of FoxA1 function has become very important. Here, through genomic analysis, we reveal that FoxA1 regulates two distinct oncogenic processes via disparate mechanisms. FoxA1 induces cell growth requiring the AR pathway. On the other hand, FoxA1 inhibits cell motility and epithelial-to-mesenchymal transition (EMT) through AR-independent mechanism directly opposing the action of AR signaling. Using orthotopic mouse models, we further show that FoxA1 inhibits prostate tumor metastasis in vivo. Concordant with these contradictory effects on tumor progression, FoxA1 expression is slightly upregulated in localized prostate cancer wherein cell proliferation is the main feature, but is remarkably downregulated when the disease progresses to metastatic stage for which cell motility and EMT are essential. Importantly, recently identified FoxA1 mutants have drastically attenuated ability in suppressing cell motility. Taken together, our findings illustrate an AR-independent function of FoxA1 as a metastasis inhibitor and provide a mechanism by which recurrent FoxA1 mutations contribute to prostate cancer progression. Cancer Res; 73(12); 3725–36. ©2013 AACR.

There is considerable evidence that the presence of cancer can elicit a humoral immune response to specific proteins in the host, and these resulting autoantibodies may have potential as noninvasive biomarkers. To characterize the autoantibody repertoire present in the sera of patients with lung adenocarcinoma, we developed a high-density peptide microarray derived from biopanning a lung cancer phage display library. Using a 2,304-element microarray, we interrogated a total of 250 sera from Michigan lung cancer patients and noncancer controls to develop an “autoantibody profile” of lung adenocarcinoma. A set of 22 discriminating peptides derived from a training set of 125 serum samples from lung adenocarcinoma patients and control subjects was found to predict cancer status with 85% sensitivity and 86% specificity in an independent test set of 125 sera. Sequencing of the immunoreactive phage-peptide clones identified candidate humoral immune response targets in lung adenocarcinoma, including ubiquilin 1, a protein that regulates the degradation of several ubiquitin-dependent proteasome substrates. An independent validation set of 122 serum samples from Pittsburgh was examined using two overlapping clones of ubiquilin 1 that showed 0.79 and 0.74 of the area under the receiver operating characteristics curve, respectively. Significantly increased levels of both ubiquilin 1 mRNA and protein, as well as reduced levels of the phosphorylated form of this protein, were detected in lung tumors. Immunofluorescence using anti–ubiquilin 1 antibodies confirmed intracellular expression within tumors cells. These studies indicate that autoantibody profiles, as well as individual candidates, may be useful for the noninvasive detection of lung adenocarcinoma. [Cancer Res 2007;67(7):3461–7]

Methods and Materials, Tables 1-4, Figures 1-6

The enhancer pioneer transcription factor FoxA1 is a global mediator of steroid receptor (SR) action in hormone-dependent cancers. In castration-resistant prostate cancer (CRPC), FoxA1 acts as an androgen receptor cofactor to drive G2 to M-phase cell-cycle transit. Here, we describe a mechanistically distinct SR-independent role for FoxA1 in driving G1 to S-phase cell-cycle transit in CRPC. By comparing FoxA1 binding sites in prostate cancer cell genomes, we defined a codependent set of FoxA1-MYBL2 and FoxA1-CREB1 binding sites within the regulatory regions of the Cyclin E2 and E2F1 genes that are critical for CRPC growth. Binding at these sites upregulate the Cyclin E2 and Cyclin A2 genes in CRPC but not in earlier stage androgen-dependent prostate cancer, establishing a stage-specific role for this pathway in CRPC growth. Mechanistic investigations indicated that FoxA1, MYBL2, or CREB1 induction of histone H3 acetylation facilitated nucleosome disruption as the basis for codependent transcriptional activation and G1 to S-phase cell-cycle transit. Our findings establish FoxA1 as a pivotal driver of the cell-cycle in CRPC which promotes G1 to S-phase transit as well as G2 to M-phase transit through two distinct mechanisms. Cancer Res; 71(21); 6738–48. ©2011 AACR.

Supplementary Material - PDF file 1368K, Figure S1. FoxA1 promotes cell cycle progress ion but inhibits cell motility. Figure S2. FoxA1 promotes cell pro liferation through the AR. Figure S3. FoxA1 inhibits PC-3M cell inva sion independently of the AR. Figure S4. WB analysis of Vimentin in FoxA1 knockdown LNCaP cells. Figure S5. FoxA1 directly suppresses SLUG expression. Figure S6. FoxA1 expression is up-regulated in localized PCa, but is significantly down- regulated in metastatic PCa. Figure S7. FoxA1 mutants fail to bind the SLUG enhancer. Table S1. GO analysis of genes repressed by shFoxA1 (i .e. FoxA1-induced genes) in LNCaP cells. Table S2. GO analysis of genes induced by shFoxA1 (i.e. FoxA1-repressed) in LNCaP cells. Table S3. GO analysis of genes repressed by siFoxA1 (i .e. FoxA1-induced genes) in LNCaP cells. Table S4. GO analysis of genes induced by siFoxA1 (i .e. FoxA1-repressed) in LNCaP cells. Table S5. GO analysis of genes repressed by shFoxA1 (i .e. FoxA1-induced genes) in 22RV1 cells. Table S6. GO analysis of genes induced by shFoxA1 (i.e. FoxA1-repressed) 22RV1 cells. Table S7. Sequences of oligonucleotid es used in this study

The Polycomb group (PcG) protein Enhancer of Zeste Homolog 2 (EZH2) is one of the three core subunits of the Polycomb Repressive Complex 2 (PRC2). It harbors histone methyltransferase activity (MTase) that specifically catalyze histone 3 lysine 27 (H3K27) methylation on target gene promoters. As such, PRC2 are epigenetic silencers that play important roles in cellular identity and embryonic stem cell maintenance. In the past two decades, mounting evidence supports EZH2 mutations and/or over-expression in a wide array of hematological cancers and solid tumors, including prostate cancer. Further, EZH2 is among the most up-regulated genes in neuroendocrine prostate cancers, which become abundant due to the clinical use of high-affinity androgen receptor pathway inhibitors. While numerous studies have reported epigenetic functions of EZH2 that inhibit tumor suppressor genes and promote tumorigenesis, discordance between EZH2 and H3K27 methylation has been reported. Further, enzymatic EZH2 inhibitors have shown limited efficacy in prostate cancer, warranting a more comprehensive understanding of EZH2 functions. Here we first review how canonical functions of EZH2 as a histone MTase are regulated and describe the various mechanisms of PRC2 recruitment to the chromatin. We further outline non-histone substrates of EZH2 and discuss post-translational modifications to EZH2 itself that may affect substrate preference. Lastly, we summarize non-canonical functions of EZH2, beyond its MTase activity and/or PRC2, as a transcriptional cofactor and discuss prospects of its therapeutic targeting in prostate cancer.

The aim of this study was to compare nasogastric (NG) feeding with nasojejunal (NJ) feeding when treating pediatric patients with acute pancreatitis (AP). We performed a single-center, prospective, randomized, active-controlled trial involving 77 pediatric patients with AP from April 2014 to December 2017. The patients were randomized into two groups: the NG tube feeding group (34 patients) and the NJ tube feeding group (33 patients). The primary outcome measures included the enteral nutrition intolerance, the length of tube feeding time, the recurrent pain of pancreatitis and complications. A total of 62 patients with AP (31 patients for each group) came into the final analysis. No differences were found in baseline characteristics, pediatric AP score and computed tomography severity score between the two groups. Three (9.7%) patients in the NG group and one (3.2%) patient in the NJ group developed intolerance (relative risk = 3.00, 95% confidence interval 0.33–27.29, P = 0.612). The tube feeding time and length of hospital stay of the NG group were significantly shorter than those of the NJ group (P = 0.016 and 0.027, respectively). No patient died in the trial. No significant differences were found in recurrent pain, complications, nutrition delivery efficacy, and side effects between the two groups. NG tube feeding appears to be effective and safe for acute pediatric pancreatitis compared with NJ tube feeding. In addition, high qualified, large sample sized, randomized controlled trials in pediatric population are needed.

Gut dysbiosis and associated bile acid (BA) metabolism play an important role in the pathogenesis of Crohn's disease (CD). We investigated the impacts of the exclusive enteral nutrition treatment (EEN) on the gut microbiome (GM) and BAs metabolism for patients with CD.Targeted metabolomics analysis and metagenomics analysis were performed in feces to investigate the BA and GM changes of patients before and after 2-months EEN therapy. The Pediatric Crohn's Disease Activity Index (PCDAI) and fecal calprotectin were used to evaluate the severity and mucosal inflammation of CD.A total of 27 newly diagnosed pediatric patients with CD and 27 healthy controls were recruited in this study. Both GM structure and the secondary BA metabolism were significantly impaired in patients, which could return towards normal levels after EEN treatment. The most abundant taxa Firmicutes and 11 BAs were found closely associated with the PCDAI score and fecal calprotectin. Meanwhile, the close interactions between Firmicute bacteria and BAs might contribute to the remission of CD after EEN treatment. The qPCR data further confirmed that the relative expressions of Firmicutes phylum, and genus Flavonifractor and Clostridium V were improved after EEN treatment.Firmicutes bacteria and the balance of primary and secondary BA compositions in the gut were closely associated with the health status of CD disease indicated by the PCDAI score and fecal calprotectin. Understanding the recovery process of gut microbiome and BA metabolism will help us to explore the potential mechanisms of EEN therapy.

Members of the NSD protein family (NSD1, NSD2, and NSD3) are histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. H3K36 modifications play an important role in regulating the function and structure of chromatin, affecting transcription, replication, and repair. Abnormal H3K36 methylation is often detected during tumor development and progression. Inactivating NSD1 mutations are frequent in head and neck squamous cell carcinoma (HNSCC). They commonly occur in HPV-negative oropharyngeal (OP) and laryngeal (LC) carcinomas, and define a prognostic subtype in LC, associated with significantly improved overall and progression-free survival. Notably, the SCC4 cell line, carrying a damaging mutation in the NSD1 gene demonstrated reduced dimethylation level of H3K36 compared to NSD1 wild-type HNSCC counterparts. To explore the biological impact of the NSD1/NSD2 loss of function in HNSCC, we established cell lines with doxycycline-inducible shRNA knockdown of NSD1 and NSD2 in the set of HNSCC cell lines originating from different sites (JHU011, JHU022, Cal27, and FaDu cell lines). The depletion of NSD1 and NSD2 led to reduction of K36me2, significant decrease in cell growth as measured by cell titer blue (CTB) and clonogenic assays. NSD1/NSD2 depletion in these HNSCC cells also caused a significant increase in apoptosis. Gene Set Enrichment Analysis (GSEA) of RNA-seq for NSD1 wt versus knockdown cells indicates that NSD1 knockdown reduced expression of E2F target genes. Among the E2F transcription factor family, E2F2 gene expression was significantly decreased in all NSD1 knockdown cell lines. NSD1 knockdown also activated gene pathways related to autophagy and response to starvation. NSD1 knockdown reduced the levels of autophagy initiation gene ULK1 at both mRNA and protein levels. We also probed for protein signaling in HNSCC cells following NSD1 depletion using a reverse protein phase array (RPPA) approach, and validated Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C), as NSD1-regulated. PIP4K2B was regulated at the mRNA level by NSD1 as well. The CHIP-qPCR assay demonstrated the loss of H3K36me2 at the promoter of the PIP4K2B gene in NSD1 knockdown cells, suggesting direct regulation by NSD1. Moreover, PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation, which suggests that the NSD1 may regulate proliferative activity through PIP4K2B. Taken together, while this data supports the suggestion that NSD histone methyltransferases have multiple downstream targets, the underlying mechanism remain to be investigated in more detail. Further, NSD proteins are attractive targets for drug development for improving treatment strategies for HNSCC. Citation Format: Iuliia Topchu, Igor Bychkov, Petr Makhov, Evgeny Izumchenko, John Karanicolas, Jindan Yu, Jochen Lorch, Yanis Boumber. NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4756.

NSD1, NSD2, and NSD3 constitute the nuclear receptor–binding SET Domain (NSD) family of histone 3 lysine 36 (H3K36) methyltransferases. These structurally similar enzymes mono- and di-methylate H3K36, which contribute to the maintenance of chromatin integrity and regulate the expression of genes that control cell division, apoptosis, DNA repair, and epithelial-mesenchymal transition (EMT). Aberrant expression or mutation of members of the NSD family is associated with developmental defects and the occurrence of some types of cancer. In this review, we discuss the effect of alterations in NSDs on cancer patient’s prognosis and response to treatment. We summarize the current understanding of the biological functions of NSD proteins, focusing on their activities and the role in the formation and progression in solid tumors biology, as well as how it depends on tumor etiologies. This review also discusses ongoing efforts to develop NSD inhibitors as a promising new class of cancer therapeutic agents.

Data concerning of the effectiveness of infliximab in very early onset Crohn’s disease patients are rare. To assess the effectiveness and safety issues of infliximab treatment for this rare cohort. The pediatric Crohn’s disease activity index, Crohn’s disease endoscopic index score, height, and weight were retrospectively recorded at baseline, week 14, and week 54. The rates of clinical remission and mucosal healing and growth of patients were compared between patients younger and older than 6 years of age. Loss of response or non-response to infliximab and adverse events were assessed during the entire treatment period. Sixty-five patients were enrolled in the study. Sixty-four percent and 40.0% of very early onset Crohn’s disease patients achieved clinical remission and mucosal healing after induction therapy. Adjusted for the covariances, very early disease onset had no association with primary non-response (p = 0.360) or mucosal healing (p = 0.361). Early disease onset was associated with discontinuation of infliximab due to adverse events (hazard ratio [HR] 7.15, 95% CI 1.73–29.51, p = 0.006). Patients < 6 years had lower body mass index for age z score improvement during the induction phase (p = 0.04). Very early onset Crohn’s disease patients had similar non-response rates and mucosal healing rates as those who were 6 years or older during induction therapy. Greater discontinuation of infliximab due to adverse events was observed in very early onset Crohn’s disease patients.

HOXB13, a homeodomain transcription factor, critically regulates androgen receptor (AR) function and promotes androgen-dependent prostate cancer (PCa) growth. However, the functions of HOXB13 in an AR-independent context remain elusive. Here we report an essential role of HOXB13 in directly suppressing lipogenic transcriptional programs in both AR-positive and -negative PCa cells. The MEIS domain (aa70-150) of HOXB13 interacts with the histone deacetylase HDAC3, which is disrupted by HOXB13 G84E mutation that has been associated with early-onset PCa. Thus, HOXB13 wildtype (WT), but not G84E mutant, recruits HDAC3 to lipogenic enhancers to catalyze histone de-acetylation and suppress lipogenic programs. HOXB13 knockdown unleashes the expression of key lipogenic regulators such as fatty acid synthase (FASN), requiring HDAC3. Analysis of human tissues revealed that HOXB13 is lost in about 30% of metastatic castration-resistant PCa, at least in part, through DNA hypermethylation. Functionally, loss of HOXB13 leads to massive lipid accumulation in PCa cells, thereby promoting cell motility in vitro and fueling xenograft tumor metastasis in vivo, which is mitigated by pharmaceutical inhibitors of FASN. In summary, our study discovers an essential AR-independent function of HOXB13 in repressing de novo lipogenesis and inhibiting tumor metastasis and defines a subclass of PCa that may benefit from lipogenic pathway inhibitors.

PALI1 is a newly identified accessory protein of the Polycomb repressive complex 2 (PRC2) that catalyzes H3K27 methylation. However, the roles of PALI1 in cancer are yet to be defined. Here, we report that PALI1 is upregulated in advanced prostate cancer (PCa) and competes with JARID2 for binding to the PRC2 core subunit SUZ12. PALI1 further interacts with the H3K9 methyltransferase G9A, bridging the formation of a unique G9A-PALI1-PRC2 super-complex that occupies a subset of G9A-target genes to mediate dual H3K9/K27 methylation and gene repression. Many of these genes are developmental regulators required for cell differentiation, and their loss in PCa predicts poor prognosis. Accordingly, PALI1 and G9A drive PCa cell proliferation and invasion in vitro and xenograft tumor growth in vivo. Collectively, our study shows that PALI1 harnesses two central epigenetic mechanisms to suppress cellular differentiation and promote tumorigenesis, which can be targeted by dual EZH2 and G9A inhibition.

Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration-resistant prostate cancers become androgen receptor (AR) signaling independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome, and interactome using in vivo, in vitro, and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR cofactors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc–induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for enhancer of zeste homolog 2 (EZH2) inhibition to reverse the N-Myc–induced suppression of epithelial lineage genes. Altogether, our data provide insights into how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.

Castration-resistant prostate cancer (CRPC) that has developed resistance to the new-generation androgen receptor (AR) antagonist enzalutamide is a lethal disease. Transcriptome analysis of multiple prostate cancer models identified CXCR7, an atypical chemokine receptor, as one of the most upregulated genes in enzalutamide-resistant cells. AR directly repressed CXCR7 by binding to an enhancer 110 kb downstream of the gene and expression was restored upon androgen deprivation. We demonstrate that CXCR7 is a critical regulator of prostate cancer sensitivity to enzalutamide and is required for CRPC growth in vitro and in vivo. Elevated CXCR7 activated MAPK/ERK signaling through ligand-independent, but β-arrestin 2-dependent mechanisms. Examination of patient specimens showed that CXCR7 and pERK levels increased significantly from localized prostate cancer to CRPC and further upon enzalutamide resistance. Preclinical studies revealed remarkable efficacies of MAPK/ERK inhibitors in suppressing enzalutamide-resistant prostate cancer. Overall, these results indicate that CXCR7 may serve as a biomarker of resistant disease in patients with prostate cancer and that disruption of CXCR7 signaling may be an effective strategy to overcome resistance. Significance: These findings identify CXCR7-mediated MAPK activation as a mechanism of resistance to second-generation antiandrogen therapy, highlighting the therapeutic potential of MAPK/ERK inhibitors in CRPC.

Prostate cancer (PCa) is the most commonly diagnosed cancer among males in the United States. The mainstay treatment for metastatic PCa patients is androgen-deprivation therapies, including high-affinity androgen receptor antagonists, such as enzalutamide. Unfortunately, most patients develop resistance and relapse with castration-resistant PCa (CRPC). We have previously reported up-regulation of CXCR7, an atypical chemokine receptor, in CRPC. CXCR7 interacts with beta-arrestin (ARRB2), a cytoplasmic scaffold protein, and translocates to the cytoplasm to induce signal transduction and PCa progression. However, much remains to be known regarding CXCR7-driven downstream signaling and its therapeutic targeting in advanced PCa. To unveil CXCR7-downstream signaling, we utilized RNA-seq to profile gene expression. Gene ontology analyses revealed remarkable enrichment of CXCR7-regulated genes in G2/M cell cycle progression. Comprehensive phospho-proteomics analysis of CXCR7-knockdown PCa cells showed markedly reduced phosphorylation of AURKA substrates. AURKA, a serine/threonine-protein kinase, is a key regulator of mitosis and G2/M progression and is frequently upregulated in CRPC and the late-stage neuroendocrine PCa (NEPC). Activated AURKA experiences autophosphorylation and phosphorylates downstream substrates, e.g., TACC3, that mediate proper cell division. Immunoblotting confirmed that CXCR7 depletion reduces phosphorylation of AURKA and TACC3. Co-immunoprecipitation revealed protein-protein interaction between CXCR7 and AURKA, which is mediated by ARRB2. Accordingly, ARRB2-knockdown similarly inhibits AURKA phosphorylation and arrests cell proliferation. Immunofluorescence coupled with confocal imaging demonstrated that CXCR7 is packed into clathrin-coated vesicles and locates in the plasma membrane, cytoplasm, and perinuclear Golgi complex, which surrounds the centrosomal AURKA and ARRB2. We found that CXCR7 translocates to the Golgi complex along microtubule-dependent trafficking. Finally, we found that pharmacological inhibitors of AURKA abolish CXCR7-driven PCa cell proliferation in vitro and tumor growth in vivo. Conclusions: Our results describe for the first time that the CXCR7-ARRB2 complex interacts with AURKA and promotes its activation. Our data suggest that AURKA-targeting is a promising therapeutic strategy for CRPC and NEPC with CXCR7 up-regulation. Citation Format: Galina Gritsina, Zhuoyuan Lin, Ka Wing Fong, Xiaodong Lu, Changsheng Zhao, Jindan Yu. CXCR7 drives prostate cancer growth through AURKA activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB032.

Histone modifications play important role in regulating the function and structure of chromatin. Abnormal histone methylation is often detected during tumor development and progression. NSD1, NSD2, and NSD3 are key histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. Inactivating NSD1 mutations are frequent in head neck squamous cell carcinoma (HNSCC) commonly occur in HPV-negative oropharyngeal (OP) carcinoma and laryngeal carcinomas (LC), and define a novel prognostic subtype in LC, where they associate with dramatically improved overall and progression-free survival. Here, we explored the biological impact of the loss of function of NSD1 in head neck squamous carcinoma (HNSCC). First, we discovered that HNSCC cells with a damaging mutation in NSD1 have reduced K36me2 methylation levels relative to NSD1 wild-type HNSCC cells. Second, we also found slower cell proliferation in NSD1 mutant cell line (SCC4) in comparison with other NSD1 WT cell lines. To further investigate the biologic impact of NSDs, we knocked down NSD1 and NSD2 with shRNA in different histologic subtypes of HNSCC cell lines (JHU011, JHU022, Cal27, and FaDu cell lines). We discovered that depletion of NSD1 and NSD2 results in a reduction of K36me2 and a significant decrease in cell proliferation and clonogenic formation in HNSCC, but not in lung cancer cells. Next, we performed a flow cytometry-based assay and found that NSD1/NSD2 depletion in HNSCC cells causes a significant increase in apoptosis level. We also probed for gene expression and signaling in HNSCC cells following NSD1 depletion using RNA sequencing and reverse protein phase array (RPPA) approaches. From a list of RPPA candidate targets of NSD1, we confirmed the decrease in the protein and mRNA level of Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C). PIP4K2B may regulate the ratio of lipid messengers PI5P and PI(4,5)P2 (substrate and reaction product, respectively). The level of phosphorylation of mitogen-activated kinase p70S6 was also decreased under NSD1 knockdown. PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation. Taken together, this data supports the idea that NSD1 is required for HNSCC cell proliferation via PIP4K2B. Downstream signaling, gene expression effects, and possible cell cycle regulation by NSD enzymes remain to be investigated in more detail. Further, NSDs might be attractive targets for drug development, and targeting NSD1/NSD2 enzymes may be a new strategy for improving outcomes in HNSCC patients. Citation Format: Iuliia Topchu, Rajendra Pangeni, Igor Bychkov, Petr Makhov, John Karanicolas, Jindan Yu, Erica Golemis, Jochen Lorch, Yanis Boumber. NSD histone methyltransferases drive cell proliferation in HPV-negative head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3718.

Androgen receptor pathway inhibitors are the mainstay treatment for advanced prostate cancer, but resistance is common. Here, we used a CRISPR activation screen to identify genes that promote enzalutamide resistance in the metastatic castration-sensitive prostate cancer cell line LNCaP. We found that activation of the TGF-β target gene, PRRX2, promoted enzalutamide resistance. PRRX2 expression is highest in double negative prostate cancer (DNPC) patients and a PRRX2-related gene signature identified a subset of DNPC patients with lower overall survival. PRRX2 expressing cells showed alterations in the CDK4/6/Rb/E2F and BCL2 pathways. Accordingly, CDK4/6 and BCL2 inhibitors sensitized PRRX2-expressing enzalutamide-resistant tumors to enzalutamide. High PRRX2 expression/signature in prostate cancer merit investigation as markers of enzalutamide resistance that could be reversed with CDK4/6 and BCL2 inhibitors. Citation Format: Yara R. Rodriguez, Kenji Unno, Mihai Truica, Zachary R. Chalmers, Young A Yoo, Rajita Vatapalli, Vinay Sagar, Jindan Yu, Barbara Lysy, Huiying Han, Sarki A. Abdulkadir. Genome-wide CRISPR activation screen identifies PRRX2 as an enzalutamide resistance gene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB082.

Prostate cancer (PC) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation antiandrogen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and a basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable upregulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of the TGF-β pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of the TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 downregulation induces TGF-β signaling, EMT, and cell motility, which is effectively blocked by the TGF-β receptor I inhibitor galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-β signaling, indicated by SMAD2 phosphorylation, in CRPC as compared with primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PC cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-β signaling, and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.