Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.

Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.

Control of DNA replication in vitro using a reversible replication barrier.

A major obstacle to studying DNA replication is that it involves asynchronous and highly delocalized events. A reversible replication barrier overcomes this limitation and allows replication fork movement to be synchronized and localized, facilitating the study of replication fork function and replication coupled repair. Here we provide details on establishing a reversible replication barrier in vitro and using it to monitor different aspects of DNA replication. DNA template containing an array of lac operator (lacO) sequences is first bound to purified lac repressor (LacR). This substrate is then replicated in vitro using a biochemical replication system, which results in replication forks stalled on either side of the LacR array regardless of when or where they arise. Once replication forks are synchronized at the barrier, isopropyl-ß-D-thiogalactopyranoside can be added to disrupt LacR binding so that replication forks synchronously resume synthesis. We describe how this approach can be employed to control replication fork elongation, termination, stalling and uncoupling, as well as assays that can be used to monitor these processes. We also explain how this approach can be adapted to control whether replication forks encounter a DNA lesion on the leading or lagging strand template and whether a converging fork is present. The required reagents can be prepared in 1-2 weeks and experiments using this approach are typically performed over 1-3 d. The main requirements for utilizing the LacR replication barrier are basic biochemical expertise and access to an in vitro system to study DNA replication. Investigators should also be trained in working with radioactive materials.

Replication fork uncoupling causes nascent strand degradation and fork reversal.

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts and validated this approach with experiments in human cells. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. Notably, the replicative helicase remains bound during NSD and fork reversal. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and elucidate key events that precede fork reversal.

RTEL1 and MCM10 overcome topological stress during vertebrate replication termination.

Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.

Approaches to Monitor Termination of DNA Replication Using Xenopus Egg Extracts.

DNA replication is crucial for cell viability and genome integrity. Despite its crucial role in genome duplication, the final stage of DNA replication, which is termed termination, is relatively unexplored. Our knowledge of termination is limited by cellular approaches to study DNA replication, which cannot readily detect termination. In contrast, the Xenopus laevis egg extract system allows for all of DNA replication to be readily detected. Here we describe the use of this system and assays to monitor replication termination.

Replication fork collapse in vitro using Xenopus egg extracts.

When a replication fork encounters a nick in the parental DNA, the replisome dissociates and the replication fork structure is lost. This outcome is referred to as replication fork "collapse." Collapsed forks can be highly cytotoxic and mutagenic if not appropriately repaired by the cell. However, the events that occur during and after replication fork collapse are unclear. Here, we describe an in vitro system to induce site specific, strand specific replication fork collapse using Xenopus egg extracts, which contain the full set of DNA replication and repair enzymes. We also describe simple assays to monitor the stability of DNA nicks and the different structures formed during replication fork collapse. This methodology permits detailed mechanistic analysis of collapsed forks in vitro.

Enhancing progestin therapy via HDAC inhibitors in endometrial cancer.

Uterine endometrial cancer (EC) incidence and deaths are on the rise. Hormone therapy, a traditional treatment regimen for this disease, uses progesterone and its synthetic analogue, progestin, to induce cell differentiation, apoptosis, and inhibition of invasion. This therapy is highly effective for progesterone receptor (PR) positive tumors in the short term. However, responsiveness decreases over time due to loss of PR expression; acquired resistance leads to treatment failure and poor prognosis. Primary resistance occurs in advanced, PR-negative tumors. Regardless, progestin therapy can be effective if the PR downregulation mechanism is reversed and if functional PR expression is restored. Using histone deacetylase inhibitors (HDACi), we inhibited cell proliferation in three EC cell lines and restored functional PR expression at the mRNA and protein levels. Two HDACi were tested using an endometrial xenograft tumor model: entinostat, an oral drug, and romidepsin, an IV drug. In vitro and in vivo studies support that entinostat decreased EC tumor growth, induced differentiation, and increased expression of the PR-targeted gene, PAEP. These findings supported the approval of a new NIH NCTN clinical trial, NRG-GY011, which concluded that dual treatment of MPA and entinostat, decreased expression of the proliferation marker, Ki67, but did not increase PR expression relative to single treatment with MPA in this short-term study. Therefore, a more potent HDACi, romidepsin, was investigated. Romidepsin treatment inhibited tumor growth and enhanced progestin treatment efficacy. More importantly, PR, PAEP, and KIAA1324 expressions were upregulated. Using a chromatin immunoprecipitation assay, we verified that HDACi can reverse PR downregulation mechanisms in mice models. Other potential drug efficacy markers, such as CD52, DLK1, GALNT9, and GNG2, were identified by transcriptome analysis and verified by q-PCR. Many of the upregulated drug efficacy markers predict favorable patient outcomes, while downregulated genes predict worse survival. Here, our current data suggests that romidepsin is a more potent HDACi that has the potential to achieve more robust upregulation of PR expression and may be a more promising candidate for future clinical trials.

Loss of progesterone receptor through epigenetic regulation is associated with poor prognosis in solid tumors.

BACKGROUND: Hormonal therapy using progestins, acting through the progesterone receptor (PR), is a well-established method to treat uterine endometrial hyperplasia and carcinoma. Recent population studies indicate that progestin exposure significantly reduces the incidence of ovarian, pancreatic and lung cancers in addition to endometrial cancer in women. This unexpected differentiating function of progestin in organs outside of the reproductive system led us to hypothesize that progestins/PR are protective against cancer development and progression in many tumor types. METHODS: The Cancer Genome Atlas, Oncomine and Prognostic Databases were searched to determine the relative expression of PR in tumors from multiple sites, and clinical outcomes linked to PR expression were determined. In addition, mRNA and protein expression were evaluated using real-time PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was performed to understand the PR downregulation mechanisms in tumor cells and patient samples. Methylation-specific PCR was conducted to survey the PR methylation status. The Student's t-test were performed to determine significance. Flow cytometry was used to quantify apoptotic cells. RESULTS: Low PR expression levels were consistently linked to less favorable clinical outcomes in endometrial, pancreatic, ovarian and non-small cell lung cancers. Clinical specimens and cell lines from these cancers demonstrate low levels of PR, and we now report that the mechanism for loss of PR is mediated through epigenetic repression. However, PR silencing can be overcome with epigenetic modulators. Histone deacetylase inhibitor (LBH589) and hypomethylating agent (5-aza-decitabine) restored functional PR expression at both the mRNA and protein levels and promoted marked cell death through induction of apoptosis in the presence of progesterone. CONCLUSIONS: Our studies support the possibility that progestin therapy in combination with epigenetic modulators, a concept we term "molecularly enhanced progestin therapy", is an approach worthy of study for malignancies originating from tissues outside of the reproductive tract.

Inverse Relationship between Progesterone Receptor and Myc in Endometrial Cancer.

Endometrial cancer, the most common gynecologic malignancy, is a hormonally-regulated disease. Response to progestin therapy positively correlates with hormone receptor expression, in particular progesterone receptor (PR). However, many advanced tumors lose PR expression. We recently reported that the efficacy of progestin therapy can be significantly enhanced by combining progestin with epigenetic modulators, which we term "molecularly enhanced progestin therapy." What remained unclear was the mechanism of action and if estrogen receptor α (ERα), the principle inducer of PR, is necessary to restore functional expression of PR via molecularly enhanced progestin therapy. Therefore, we modeled advanced endometrial tumors that have lost both ERα and PR expression by generating ERα-null endometrial cancer cell lines. CRISPR-Cas9 technology was used to delete ERα at the genomic level. Our data demonstrate that treatment with a histone deacetylase inhibitor (HDACi) was sufficient to restore functional PR expression, even in cells devoid of ERα. Our studies also revealed that HDACi treatment results in marked downregulation of the oncogene Myc. We established that PR is a negative transcriptional regulator of Myc in endometrial cancer in the presence or absence of ERα, which is in contrast to studies in breast cancer cells. First, estrogen stimulation augmented PR expression and decreased Myc in endometrial cancer cell lines. Second, progesterone increased PR activity yet blunted Myc mRNA and protein expression. Finally, overexpression of PR by adenoviral transduction in ERα-null endometrial cancer cells significantly decreased expression of Myc and Myc-regulated genes. Analysis of the Cancer Genome Atlas (TCGA) database of endometrial tumors identified an inverse correlation between PR and Myc mRNA levels, with a corresponding inverse correlation between PR and Myc downstream transcriptional targets SRD5A1, CDK2 and CCNB1. Together, these data reveal a previously unanticipated inverse relationship between the tumor suppressor PR and the oncogene Myc in endometrial cancer.