Paula J. Hurley

ESR1 mutations in circulating plasma tumor DNA from metastatic breast cancer patients

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA

OBJECTIVESnCirculating plasma DNA is being increasingly used for biomedical and clinical research as a substrate for genetic testing. However, cell lysis can occur hours after venipuncture when using standard tubes for blood collection, leading to an increase in contaminating cellular DNA that may hinder analysis of circulating plasma DNA. Cell stabilization agents can prevent cellular lysis for several days, reducing the need for immediate plasma preparation after venipuncture, thereby facilitating the ease of blood collection and sample preparation for clinical research. However, the majority of cell stabilizing reagents have not been formally tested for their ability to preserve circulating plasma tumor DNA.nnnDESIGN & METHODSnIn this study, we compared the properties of two cell stabilizing reagents, the cell-free DNA BCT tube and the PAXgene tube, by collecting blood samples from metastatic breast cancer patients and measuring genome equivalents of plasma DNA by droplet digital PCR. We compared wild type PIK3CA genome equivalents and also assayed for two PIK3CA hotspot mutations, E545K and H1047R.nnnRESULTSnOur results demonstrate that blood stored for 7 days in BCT tubes did not show evidence of cell lysis, whereas PAXgene tubes showed an order of magnitude increase in genome equivalents, indicative of considerable cellular lysis.nnnCONCLUSIONSnWe conclude that BCT tubes can prevent lysis and cellular release of genomic DNA of blood samples from cancer patients when stored at room temperature, and could therefore be of benefit for blood specimen collections in clinical trials.

Wnt signaling though beta-catenin is required for prostate lineage specification

Androgens initiate a complex network of signals within the UGS that trigger prostate lineage commitment and bud formation. Given its contributions to organogenesis in other systems, we investigated a role for canonical Wnt signaling in prostate development. We developed a new method to achieve complete deletion of beta-catenin, the transcriptional coactivator required for canonical Wnt signaling, in early prostate development. Beta-catenin deletion abrogated canonical Wnt signaling and yielded prostate rudiments that exhibited dramatically decreased budding and failed to adopt prostatic identity. This requirement for canonical Wnt signaling was limited to a brief critical period during the initial molecular phase of prostate identity specification. Deletion of beta-catenin in the adult prostate did not significantly affect organ homeostasis. Collectively, these data establish that beta-catenin and Wnt signaling play key roles in prostate lineage specification and bud outgrowth.

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

Significance Despite the widespread use and success of tamoxifen for treating ER-positive breast cancers, overcoming resistance to this drug remains an unmet need in clinical breast oncology. The results presented in this study demonstrate that overexpression of a novel gene, MACROD2, can mediate tamoxifen resistance and estrogen independent growth in human breast cancers, and that amplification of MACROD2 in primary breast tumors is associated with worse overall survival. Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

A human prostatic bacterial isolate alters the prostatic microenvironment and accelerates prostate cancer progression

Inflammation is associated with several diseases of the prostate including benign enlargement and cancer, but a causal relationship has not been established. Our objective was to characterize the prostate inflammatory microenvironment after infection with a human prostate‐derived bacterial strain and to determine the effect of inflammation on prostate cancer progression. To this end, we mimicked typical human prostate infection with retrograde urethral instillation of CP1, a human prostatic isolate of Escherichia coli. CP1 bacteria were tropic for the accessory sex glands and induced acute inflammation in the prostate and seminal vesicles, with chronic inflammation lasting at least 1 year. Compared to controls, infection induced both acute and chronic inflammation with epithelial hyperplasia, stromal hyperplasia, and inflammatory cell infiltrates. In areas of inflammation, epithelial proliferation and hyperplasia often persist, despite decreased expression of androgen receptor (AR). Inflammatory cells in the prostates of CP1‐infected mice were characterized at 8 weeks post‐infection by flow cytometry, which showed an increase in macrophages and lymphocytes, particularly Th17 cells. Inflammation was additionally assessed in the context of carcinogenesis. Multiplex cytokine profiles of inflamed prostates showed that distinct inflammatory cytokines were expressed during prostate inflammation and cancer, with a subset of cytokines synergistically increased during concurrent inflammation and cancer. Furthermore, CP1 infection in the Hi‐Myc mouse model of prostate cancer accelerated the development of invasive prostate adenocarcinoma, with 70% more mice developing cancer by 4.5u2009months of age. This study provides direct evidence that prostate inflammation accelerates prostate cancer progression and gives insight into the microenvironment changes induced by inflammation that may accelerate tumour initiation or progression. Copyright

TWIST1-WDR5-Hottip regulates Hoxa9 chromatin to facilitate prostate cancer metastasis.

TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR.

NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability

Significance The mechanism of how loss of the tumor suppressor p53 can lead to genomic instability is not fully understood. This study demonstrates that under physiologic low levels of proliferation, homozygous loss of tumor protein 53 (TP53) via genome editing, but not common p53 missense mutations, results in an inability to increase expression of N-Myc down-regulated gene 1 (NDRG1). In turn, failure to upregulate NDRG1 protein under low proliferative states leads to supernumerary centrosome formation, a known mechanism of aneuploidy. These results provide a mechanistic link between loss of TP53, proliferation, NDRG1, and genomic instability and help explain how cells with a low proliferative index and p53 loss can acquire additional genetic alterations that lead to cancer. The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.

PIK3CA mutations and TP53 alterations cooperate to increase cancerous phenotypes and tumor heterogeneity

Background/purposeThe combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies.Experimental design/MethodsWe used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies.ResultsCompared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic.ConclusionsThis cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

Combining immune check-point blockade and cryoablation in an immunocompetent hormone sensitive murine model of prostate cancer

BackgroundProstate cancer remains the second leading cause of cancer related death in men. Immune check point blocking antibodies have revolutionized treatment of multiple solid tumors, but results in prostate cancer remain marginal. Previous reports have suggested that local therapies, in particular cryoablation might increase tumor immunogenicity. In this work, we examine potential synergism between tumor cryoabalation and check point blocking antibodies.MethodsFVB/NJ mice were injected subcutaneously into each flank with either 1u2009×u2009106 or 0.2u2009×u2009106 isogenic hormone sensitive Myc-Cap cells to establish synchronous grafts. Mice were treated with four intraperitoneal injections of anti-PD-1 (10u2009mg/kg), anti-CTLA-4 (1u2009mg/kg), or isotype control antibody with or without adjuvant cryoablation of the larger tumor graft and with or without neo-adjuvant androgen deprivation with degarelix (ADT). Mouse survival and growth rates of tumor grafts were measured. The immune dependency of observed oncological effects was evaluated by T cell depletion experiments.ResultsTreatment with anti-CTLA-4 antibody and cryoablation delayed the growth of the distant tumor by 14.8 days (pu2009=u20090.0006) and decreased the mortality rate by factor of 4 (pu2009=u20090.0003) when compared to cryoablation alone. This synergy was found to be dependent on CD3+u2009and CD8+u2009cells. Combining PD-1 blockade with cryoablation did not show a benefit over use of either treatment alone. Addition of ADT to anti-PD1 therapy and cryoablation doubled the time to accelerated growth in the untreated tumors (pu2009=u20090.0021) and extended survival when compared to cryoablation combined with ADT in 25% of the mice. Effects of combining anti-PD1 with ADT and cryoablation on mouse survival were obviated by T cell depletion.ConclusionTrimodal therapy consisting of androgen deprivation, cryoablation and PD-1 blockade, as well as the combination of cryoablation and low dose anti-CTLA-4 blockade showed that local therapies with cryoablation could be considered to augment the effects of checkpoint blockade in prostate cancer.

Distinct Pathways Involved in S-Phase Checkpoint Control

The S-phase checkpoint is activated when DNA damage occurs during DNA synthesis or when DNA replication intermediates accumulate. Depending on the type and magnitude of damage, cells activate one of the three distinct S-phase checkpoint pathways: (1) an intra-S-phase checkpoint induced by double strand break, (2) a replication checkpoint by the stalled replication fork, and (3) a S–M checkpoint to block premature mitosis. These checkpoint pathways coordinate a network of signaling molecules and are thought to ensure the fidelity of the replicating genome.