Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase.

DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by DNA primase. Primase contains a redox-active 4Fe-4S cluster in the C-terminal domain of the p58 subunit (p58C). The redox state of this 4Fe-4S cluster can be modulated via the transport of charge through the protein and the DNA substrate (redox switching); changes in the redox state of the cluster alter the ability of p58C to associate with its substrate. The efficiency of redox switching in p58C can be altered by mutating tyrosine residues that bridge the 4Fe-4S cluster and the nucleic acid binding site. Here, we report the effects of mutating bridging tyrosines to phenylalanines in yeast p58C. High-resolution crystal structures show that these mutations, even with six tyrosines simultaneously mutated, do not perturb the three-dimensional structure of the protein. In contrast, measurements of the electrochemical properties on DNA-modified electrodes of p58C containing multiple tyrosine to phenylalanine mutations reveal deficiencies in their ability to engage in DNA charge transport. Significantly, this loss of electrochemical activity correlates with decreased primase activity. While single-site mutants showed modest decreases in activity compared to that of the wild-type primase, the protein containing six mutations exhibited a 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for charge transport in yeast p58C exist, but inhibiting these pathways together diminishes the ability of yeast primase to generate primers. These results support a model in which redox switching is essential for primase activity.

Mechanism of action of nucleotide excision repair machinery.

Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.

XPA tumor variant leads to defects in NER that sensitize cells to cisplatin.

Nucleotide excision repair (NER) reduces efficacy of treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of the NER genes Excision Repair Cross Complementation Group 1 and 2 (ERCC1 and ERCC2) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair. In this study, we report in-depth analyses of a subset of the predicted variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to improve variant effect prediction. Broadly, these findings suggest XPA tumor variants should be considered when predicting chemotherapy response.

Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis.

Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and ß-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. ß-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals ß-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.

XPA tumor variants lead to defects in NER that sensitize cells to cisplatin.

Nucleotide excision repair (NER) neutralizes treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of either of the NER genes Excision Repair Cross Complementation Group 1 and 2 ( ERCC1 and ERCC2 ) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of such mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER scaffold protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair activity on a UV-damaged substrate. In this study, we report in-depth analyses of a subset of the predicted NER-deficient XPA variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation resulting from tumor missense mutation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to further improve variant effect prediction efforts. More broadly, these findings suggest XPA tumor variants should be considered when predicting patient response to Pt-based chemotherapy. Significance: A destabilized, readily degraded tumor variant identified in the NER scaffold protein XPA sensitizes cells to cisplatin, suggesting that XPA variants can be used to predict response to chemotherapy.

An Active Learning Framework Improves Tumor Variant Interpretation.

SIGNIFICANCE: A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.

Uropathogenic Escherichia coli subverts mitochondrial metabolism to enable intracellular bacterial pathogenesis in urinary tract infection.

Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.

Lineage plasticity-mediated therapy resistance in prostate cancer.

Therapy resistance is a significant challenge for prostate cancer treatment in clinic. Although targeted therapies such as androgen deprivation and androgen receptor (AR) inhibition are effective initially, tumor cells eventually evade these strategies through multiple mechanisms. Lineage reprogramming in response to hormone therapy represents a key mechanism that is increasingly observed. The studies in this area have revealed specific combinations of alterations present in adenocarcinomas that provide cells with the ability to transdifferentiate and perpetuate AR-independent tumor growth after androgen-based therapies. Interestingly, several master regulators have been identified that drive plasticity, some of which also play key roles during development and differentiation of the cell lineages in the normal prostate. Thus, further study of each AR-independent tumor type and understanding underlying mechanisms are warranted to develop combinational therapies that combat lineage plasticity in prostate cancer.

RUNX2 overexpression and PTEN haploinsufficiency cooperate to promote CXCR7 expression and cellular trafficking, AKT hyperactivation and prostate tumorigenesis.

Rationale: The overall success rate of prostate cancer (PCa) diagnosis and therapy has been improved over the years. However, genomic and phenotypic heterogeneity remains a major challenge for effective detection and treatment of PCa. Efforts to better classify PCa into functional subtypes and elucidate the molecular mechanisms underlying prostate tumorigenesis and therapy resistance are warranted for further improvement of PCa outcomes. Methods: We generated Cre+;Runx2-cTg;Ptenp/+ (Runx2-Pten double mutant) mice by crossbreeding Cre+;Runx2-cTg males with Pten conditional (Ptenp/p) females. By using Hematoxylin and Eosin (H&E) staining, SMA and Masson's Trichrome staining, we investigated the effect of PTEN haploinsufficiency in combination with Runx2 overexpression on prostate tumorigenesis. Moreover, we employed immunohistochemistry (IHC) to stain Ki67 for cell proliferation, cleaved caspase 3 for apoptosis and AKT phosphorylation for signaling pathway in prostate tissues. Chromatin immunoprecipitation coupled quantitative PCR (ChIP-qPCR), reverse transcription coupled quantitative PCR (RT-qPCR), western blot (WB) analyses and immunofluorescence (IF) were conducted to determine the underlying mechanism by which RUNX2 regulates CXCR7 and AKT phosphorylation in PCa cells. Results: We demonstrated that mice with prostate-specific Pten heterozygous deletion and Runx2 overexpression developed high-grade prostatic intraepithelial neoplasia (HGPIN) and cancerous lesions at age younger than one year, with concomitant high level expression of Akt phosphorylation and the chemokine receptor Cxcr7 in malignant glands. RUNX2 overexpression induced CXCR7 transcription and membrane location and AKT phosphorylation in PTEN-deficient human PCa cell lines. Increased expression of RUNX2 also promoted growth of PCa cells and this effect was largely mediated by CXCR7. CXCR7 expression also positively correlated with AKT phosphorylation in PCa patient specimens. Conclusions: Our results reveal a previously unidentified cooperative role of RUNX2 overexpression and PTEN haploinsufficiency in prostate tumorigenesis, suggesting that the defined RUNX2-CXCR7-AKT axis can be a viable target for effective treatment of PCa.

TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer.

Purpose: Deletions or mutations in PTEN and TP53 tumor suppressor genes have been linked to lineage plasticity in therapy-resistant prostate cancer. Fusion-driven overexpression of the oncogenic transcription factor ERG is observed in approximately 50% of all prostate cancers, many of which also harbor PTEN and TP53 alterations. However, the role of ERG in lineage plasticity of PTEN/TP53-altered tumors is unclear. Understanding the collective effect of multiple mutations within one tumor is essential to combat plasticity-driven therapy resistance.Experimental Design: We generated a Pten-negative/Trp53-mutated/ERG-overexpressing mouse model of prostate cancer and integrated RNA-sequencing with ERG chromatin immunoprecipitation-sequencing (ChIP-seq) to identify pathways regulated by ERG in the context of Pten/Trp53 alteration. We investigated ERG-dependent sensitivity to the antiandrogen enzalutamide and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib in human prostate cancer cell lines, xenografts, and allografted mouse tumors. Trends were evaluated in TCGA, SU2C, and Beltran 2016 published patient cohorts and a human tissue microarray.Results: Transgenic ERG expression in mice blocked Pten/Trp53 alteration-induced decrease of AR expression and downstream luminal epithelial genes. ERG directly suppressed expression of cell cycle-related genes, which induced RB hypophosphorylation and repressed E2F1-mediated expression of mesenchymal lineage regulators, thereby restricting adenocarcinoma plasticity and maintaining antiandrogen sensitivity. In ERG-negative tumors, CDK4/6 inhibition delayed tumor growth.Conclusions: Our studies identify a previously undefined function of ERG to restrict lineage plasticity and maintain antiandrogen sensitivity in PTEN/TP53-altered prostate cancer. Our findings suggest ERG fusion as a biomarker to guide treatment of PTEN/TP53-altered, RB1-intact prostate cancer. Clin Cancer Res; 24(18); 4551-65. ©2018 AACR.

Loss of FOXO1 Cooperates with TMPRSS2-ERG Overexpression to Promote Prostate Tumorigenesis and Cell Invasion.

E26 transformation-specific transcription factor ERG is aberrantly overexpressed in approximately 50% of all human prostate cancer due to TMPRSS2-ERG gene rearrangements. However, mice with prostate-specific transgenic expression of prostate cancer-associated ERG alone fail to develop prostate cancer, highlighting that ERG requires other lesions to drive prostate tumorigenesis. Forkhead box (FOXO) transcription factor FOXO1 is a tumor suppressor that is frequently inactivated in human prostate cancer. Here, we demonstrate that FOXO1, but not other FOXO proteins (FOXO3 and FOXO4), binds and inhibits the transcriptional activity of prostate cancer-associated ERG independently of FOXO1 transcriptional activity. Knockdown of endogenous FOXO1 increased invasion of TMPRSS2-ERG fusion-positive VCaP cells, an effect completely abolished by ERG knockdown. Patient specimen analysis demonstrated that FOXO1 and ERG protein expression inversely correlated in a subset of human prostate cancer. Although human ERG transgene expression or homozygous deletion of Foxo1 alone in the mouse prostate failed to promote tumorigenesis, concomitant ERG transgene expression and Foxo1 deletion resulted in upregulation of ERG target genes, increased cell proliferation, and formation of high-grade prostatic intraepithelial neoplasia. Overall, we provide biochemical and genetic evidence that aberrantly activated ERG cooperates with FOXO1 deficiency to promote prostate tumorigenesis and cell invasion. Our findings enhance understanding of prostate cancer etiology and suggest that the FOXO1-ERG signaling axis can be a potential target for treatment of prostate cancer. Cancer Res; 77(23); 6524-37. ©2017 AACR.

BET bromodomain-mediated interaction between ERG and BRD4 promotes prostate cancer cell invasion.

Prostate cancer (PCa) that becomes resistant to hormone castration and next-generation androgen receptor (AR)-targeted therapies, called castration-resistant prostate cancer (CRPC), poses a significant clinical challenge. A better understanding of PCa progression and key molecular mechanisms could bring novel therapies to light. One potential therapeutic target is ERG, a transcription factor aberrantly up-regulated in PCa due to chromosomal rearrangements between androgen-regulated gene TMPRSS2 and ERG. Here we show that the most common PCa-associated truncated ERG T1-E4 (ERGΔ39), encoded by fusion between TMPRSS2 exon 1 and ERG exon 4, binds to bromodomain-1 (BD1) of bromodomain containing protein 4 (BRD4), a member of the bromodomain and extraterminal domain (BET) family. This interaction is partially abrogated by BET inhibitors JQ1 and iBET762. Meta-analysis of published ERG (T1-E4) and BRD4 chromatin immunoprecipitation-sequencing (ChIP-seq) data demonstrates overlap in a substantial portion of their binding sites. Gene expression profile analysis shows some ERG-BRD4 co-target genes are upregulated in CRPC compared to hormone-naïve counterparts. We provide further evidence that ERG-mediated invasion of PCa cells was significantly enhanced by an acetylation-mimicking mutation in ERG that augments the ERG-BRD4 interaction. Our findings reveal that PCa-associated ERG can interact and co-occupy with BRD4 in the genome, and suggest this druggable interaction is critical for ERG-mediated cell invasion and PCa progression.

Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, ß-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only ß-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.