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This file contains additional data including additional mRNA analyses of TCGA and SPORE datasets, validation of antibodies and siRNAs, ATO dose finding studies, analyses of mutations in PI3K pathway in human cancers. The file also contains 2 tables of Taqman probe information and human and mouse specific qPCR primers.

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo. Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K–mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448–59. ©2017 AACR.

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs’ recapitulation of human tumors., Patient-derived xenograft models (PDX) have been extensively used to study the molecular and clinical features of cancers. Here the authors present a cohort of 536 PDX models from 25 cancers, as well as their genomic and evolutionary profiles and their suitability for clinical trials.

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

SummaryIntermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes including signal transduction and gene expression patterns arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here we quantified over 100 metabolic features, mostly from 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

Although non-small cell lung cancer (NSCLC) patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs. Fil: Dalvi, Maithili P.. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Wang, Lei. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Zhong, Rui. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Kollipara, Rahul K.. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Park, Hyunsil. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Bayo Fina, Juan Miguel. University of Texas. Southwestern Medical Center; Estados Unidos. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentina Fil: Yenerall, Paul. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Zhou, Yunyun. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Timmons, Brenda C.. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Rodriguez Canales, Jaime. University of Texas; Estados Unidos Fil: Behrens, Carmen. Md Anderson Cancer Center; Estados Unidos Fil: Mino, Barbara. University of Texas; Estados Unidos Fil: Villalobos, Pamela. University of Texas; Estados Unidos Fil: Parra, Edwin R.. University of Texas; Estados Unidos Fil: Suraokar, Milind. University of Texas; Estados Unidos Fil: Pataer, Apar. University of Texas; Estados Unidos Fil: Swisher, Stephen G.. University of Texas; Estados Unidos Fil: Kalhor, Neda. University of Texas; Estados Unidos Fil: Bhanu, Natarajan V.. University of Pennsylvania; Estados Unidos Fil: Garcia, Benjamin A.. University of Pennsylvania; Estados Unidos Fil: Heymach, John V.. University of Texas; Estados Unidos Fil: Coombes, Kevin. University of Texas; Estados Unidos Fil: Xie, Yang. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Girard, Luc. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Gazdar, Adi F.. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Kittler, Ralf. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Wistuba, Ignacio I.. University of Texas; Estados Unidos Fil: Minna, John D.. University of Texas. Southwestern Medical Center; Estados Unidos Fil: Martinez, Elisabeth D.. University of Texas. Southwestern Medical Center; Estados Unidos

Summary RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.

Globally, an estimated 13 million preterm babies are born each year. These babies are at increased risk of infant mortality and life-long health complications. Interventions to prevent preterm birth (PTB) require an understanding of processes driving parturition. Prostaglandins (PGs) have diverse functions in parturition, including regulation of uterine contractility and tissue remodeling. Our studies on cervical remodeling in mice suggest that although local synthesis of PGs are not increased in term ripening, transcripts encoding PG-endoperoxide synthase 2 (Ptgs2) are induced in lipopolysaccharide (LPS)-mediated premature ripening. This study provides evidence for two distinct pathways of cervical ripening: one dependent on PGs derived from paracrine or endocrine sources and the other independent of PG actions. Cervical PG levels are increased in LPS-treated mice, a model of infection-mediated PTB, consistent with increases in PG synthesizing enzymes and reduction in PG-metabolizing enzymes. Administration of SC-236, a PTGS2 inhibitor, along with LPS attenuated cervical softening, consistent with the essential role of PGs in LPS-induced ripening. In contrast, during term and preterm ripening mediated by the antiprogestin, mifepristone, cervical PG levels, and expression of PG synthetic and catabolic enzymes did not change in a manner that supports a role for PGs. These findings in mice, supported by correlative studies in women, suggest PGs do not regulate all aspects of the parturition process. Additionally, it suggests a need to refocus current strategies toward developing therapies for the prevention of PTB that target early, pathway-specific processes rather than focusing on common late end point mediators of PTB.

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo. Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K–mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448–59. ©2017 AACR.

Cervical epithelia have numerous functions that include proliferation, differentiation, maintenance of fluid balance, protection from environmental hazards, and paracellular transport of solutes via tight junctions (TJs). Epithelial functions must be tightly regulated during pregnancy and parturition as the cervix undergoes extensive growth and remodeling. This study evaluated TJ proteins, as well as markers of epithelial cell differentiation in normal and cervical ripening defective mice to gain insights into how the permeability barrier is regulated during pregnancy and parturition. Although numerous TJ proteins are expressed in the nonpregnant cervix, claudins 1 and 2 are temporally regulated in pregnancy. Claudin 1 mRNA expression is increased, whereas claudin 2 expression declines. The cellular localization of claudin 1 shifts at the end of pregnancy (gestation d 18.75) to the plasma membrane in a lattice pattern, consistent with TJs in the apical cells. The timing of claudin 1-enriched TJs coincides with initiation of terminal differentiation of cervical squamous epithelia as evidenced by the increased expression of genes by differentiated epithelia late on gestation d 18. The cervical ripening defective steroid 5α-reductase type 1 deficient mouse, which has an elevated local progesterone concentration, also has aberrant claudin 1 and 2 expressions, fails to form claudin 1-enriched TJs, and lacks normal expression of genes involved in epithelial terminal differentiation. These data suggest that changes in permeability barrier properties during cervical ripening are, in part, negatively regulated by progesterone, and that dynamic changes in barrier properties of the cervix occur during pregnancy and parturition.

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1−/−), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1−/− mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, ...

The molecular mechanisms controlling the initiation of parturition remain largely undefined. We report a new animal model in which parturition does not occur. A line of mice expressing a human apolipoprotein B (APOB) gene fail to deliver their young if the transgene is present in homozygous (Tg/Tg), but not in heterozygous (Tg/Wt), form. Cloning and mapping of the transgene insertion locus indicate that 10 copies of the 80-kilobase APOB genomic fragment inserted into mouse chromosome 6 result in a small, 390-base pair deletion of mouse genomic DNA. Nine other lines expressing the transgene have normal labor, suggesting that transgene insertion in this mutant line disrupted a mouse gene crucial for successful parturition. The pathophysiology of parturition failure in these animals was defined using physiological, endocrinological, and morphological techniques. Results indicate that luteolysis occurs in Tg/Tg mice but is delayed by 1 day. Delivery did not occur in mutant mice at term after spontaneous luteolysis or even after removal of progesterone action by ovariectomy or antiprogestin treatment. Biomechanical and functional studies of the uterus and cervix revealed that the primary cause of failed parturition in Tg/Tg mice was not inadequate uterine contractions of labor but, rather, a rigid, inelastic cervix at term that was abnormally rich in neutrophils and tissue monocytes. Characterization of the transgene insertional mutant, Tg/Tg, indicates that progesterone withdrawal is insufficient to complete parturition in the presence of inadequate cervical ripening at term.

A high-virulence clone (HVC) was proposed as causing much of the morbidity and mortality when a collection of group B Streptococcus (GBS) isolates was examined by multi-locus enzyme electrophoresis. HVC isolates could be further distinguished by their inability to grow at 40 degrees C, and a temperature-sensitive aldolase was identified as responsible for this characteristic. In the present study, the HVC was sought in a collection of 57 GBS isolates by hybridization with a probe containing a putative aldolase gene on genomic DNA restriction enzyme digests. Isolates were initially classified as HVC or non-HVC by their inability to grow at 40 degrees C. Three serotype III invasive isolates had the HVC control restriction/hybridization pattern. They were also unable to grow at 40 degrees C. The remaining 11 invasive and all carrier isolates showed a pattern identical to that of the non-HVC control. These results provide additional support for the existence of a highly virulent clonal group among serotype III isolates and suggest that hybridization with a probe containing the aldolase gene on DNA restriction enzyme digests can be an alternative method for identifying highly virulent isolates.

Despite advances in targeted agents and immunotherapy, non-small cell lung cancer (NSCLC) remains the number one cause of cancer-related death. To identify new therapeutic targets in NSCLC, we performed an siRNA screen directed against genes involved in chromatin remodeling. This screen showed that RUVBL1 and RUVBL2 (herein collectively referred to as RUVBL1/2) were universally but differentially required for the viability of 24 NSCLC cell lines, which was an on-target effect. Various independent gene expression datasets/platforms show that NSCLC patient tumors have increased levels of RUVBL1 and RUVBL2 mRNAs, in comparison to normal lung, and that patients with high levels of RUVBL1 or RUVBL2 have a poorer prognosis, suggesting that RUVBL1/2 may play an important role in NSCLC tumors. To confirm this at the protein level, we validated an antibody against RUVBL1 for immunohistochemistry, stained clinically annotated NSCLC tissue microarrays for RUVBL1, and found that patients with higher levels of RUVBL1 protein also have a poorer prognosis. To better understand the role of RUVBL1/2 in NSCLC at a molecular level, we measured the distribution of cells in the cell cycle following RUVBL1/2 KD, determined RUVBL1/2 interacting proteins by immunoprecipitation followed by tandem mass-spec (IP-MS/MS), and measured gene expression changes following RUVBL1/2 KD by RNA-seq. Depletion of RUVBL1/2 arrested cells in S-phase and promoted pan-γH2AX positivity, IP-MS/MS showed an over-representation of proteins involved in DNA repair and replication, and gene set enrichment analysis of the RNA-seq data displayed a downregulation of transcripts involved in DNA replication and repair, strongly implicating RUVBL1/2 in these processes. To further probe the effects of RUVBL1/2 loss, we performed low-level knock down (KD) of RUVBL1/2, such that viability is largely unaffected, and then measured the viability of cells in response to various drugs (n=35). These drugs target a wide variety of biological processes; however, the only drugs with increased efficacy in the presence of RUVBL1/2 KD were those that damage DNA, target ATR or its downstream partner CHEK1, or target some mitotic proteins, further implicating RUVBL1/2 in DNA replication and/or repair. Due to their roles in replication, we reasoned that RUVBL1/2 KD may enhance the effects of ionizing radiation, a treatment frequently given to NSCLC patients. Low level KD of RUVBL1/2 decreased the clonogenic potential of multiple NSCLC cell lines, which can be phenocopied by depleting independent subunits of RUVBL1/2-containing chromatin remodeling complexes. Finally, we show that RUVBL1/2 depend upon their ATPase activity to support NSCLC viability, suggesting that small molecule inhibitors of this protein may be efficacious in the treatment of NSCLC, especially when combined with radiation therapy. Citation Format: Paul Yenerall, Rahul Kollipara, Amit Das, Pamela Villalobos, Long Shan Li, Brenda Timmons, Luc Girard, Jaime Rodriguez-Canales, Ignacio Wistuba, John Minna, Ralf Kittler. The chromatin remodelers RUVBL1 and RUVBL2 are prognostic factors and therapeutic targets in non-small cell lung cancer due to their roles in DNA replication, repair, and radiosensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2017-1111

Cancer-derived exosomes are intracellular signaling organelles that can act via inter-cellular communication to help cancer cells invade neighboring tissues and prime metastatic sites for cancer cell spread. Lung cancer is a highly metastatic disease, with metastases often occurring when tumors are in a clinically early stage. Little is known of how lung cancer exosomes influence the migratory/ invasive phenotype of lung cancer cells. We studied migration and invasion mechanisms by transferring cancer-derived exosomes to recipient cells including cancer cells, human bronchial epithelial cells (HBECs) cells, and HBECs that we had modified with common lung cancer oncogenic changes. Exosomes isolated from lung cancer line H1299 cells (p53null, NRAS mutant) and non-malignant, immortalized HBEC3-KT cells were characterized for exosome content and type by nanoparticle tracking analysis, electron microscopy, western blotting with antibodies directed at HSP70 and CD63, and generation of H1299 and HBEC3-KT derivatives stably transfected with CD63-GFP, releasing green fluorescence exosomes for tracking purposes. Exosomes were isolated using a variety of published methods and the amount quantified: 106 H1299 and HBEC-3KT cells produced 9 x 109 and 7 x 109 exosomes of similar size (125.4nm and 129.8nm). Effect of exosomes from cancer and HBECs were tested on: 1. cell motility and invasiveness of HBECs and their oncogenic derivatives determined by transwell chamber migration assay and scratch assay; 2. vascular leakiness properties in the lung by evaluating mouse lung endothelial permeability after exosomes were injected (an early event in metastatic spread for the migrated cells); and 3. xenograft tumor formation. We found CD63-GFP exosomes from H1299 and HBEC3-KT are actively incorporated by all of the cell types. However, only H1299 exosomes (but not HBEC3-KT exosomes) induced migratory/invasive phenotypic and morphologic changes and they did this in a concentration-dependent manner in lung cancer cells and HBECs with oncogenic changes (HBEC sh-p53+KRASv12 and sh-p53+KRASv12+c-MYC manipulated HBECs) but not in the parental HBEC3-KT cells. Also, H1299 exosomes but not HBEC3-KT exosomes enhanced the lung endothelial permeability as evaluated by the extravasated dextran leaking. NSCLC H1299 cells when injected with its own exosomes produced better xenograft takes compared to H1299 cells alone. Lung cancer-derived exosomes compared to exosomes derived from immortalized but non-malignant human bronchial epithelial cells induced an increased migratory/invasive phenotype with lung vascular leakiness and higher xenograft tumor take rates. However, they induced these changes only in the context of key oncogenic changes. These results suggest understanding and targeting the mechanism of such lung cancer derived exosome behavior could lead to novel therapeutic strategies. Citation Format: Yoshihisa Shimada, Paul Yenerall, Kimberly Avila, Hyunsil Park, Brenda Timmons, Kenneth Huffman, Boning Gao, Dhruba Deb, Maithili Dalvi, John D. Minna. Uptake of lung cancer exosomes induces migratory and invasive phenotypic changes in lung epithelial cells in an oncogene context dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2017-907

Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35–55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNγ transcripts be detected 10 days post immunization for EAE. IFNγ is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.

Objective We sought to determine if endothelial microparticles (EMPs), markers of endothelial damage, are associated with soluble fms-like tyrosine kinase 1 (sFlt1), soluble endoglin, and placental growth factor (PlGF) in women with preeclampsia. Study Design A prospective cohort study was conducted on 20 preeclamptic women and 20 controls. EMPs by flow cytometry, sFlt1, soluble endoglin, and PlGF were measured at time of enrollment, 48-hours postpartum, and 1-week postpartum. Results Preeclamptic CD31 + /42 − , CD62E + , and CD105 + EMP levels were significantly elevated in preeclamptics vs controls at time of enrollment. The sFlt1:PlGF ratio was correlated with CD31 + /42 − and CD105 + EMPs (r = 0.69 and r=0.51, respectively) in preeclampsia. Levels of CD31 + /42 − EMPs remained elevated 1-week postpartum ( P = .026). Conclusion EMPs are elevated in preeclampsia. The correlation of EMPs and the sFlt1:PlGF ratio suggests that antiangiogenesis is related to apoptosis of the endothelia. Endothelial damage persists 1 week after delivery.

In the current study, the mechanisms of premature cervical ripening in murine models of preterm birth resulting from infection or early progesterone withdrawal were compared with the process of term cervical ripening. Tissue morphology, weight, gene expression, and collagen content along with immune cell populations were evaluated. Premature ripening induced by the progesterone receptor antagonist mifepristone results from an acceleration of processes in place during term ripening as well as partial activation of proinflammatory and immunosuppressive processes observed during postpartum repair. In contrast to term or mifepristone-induced preterm ripening, premature ripening induced in an infection model occurs by a distinct mechanism which is dominated by an influx of neutrophils into the cervix, a robust proinflammatory response and increased expression of prostaglandin-cyclooxygenase-endoperoxide synthase 2, important in prostaglandin biosynthesis. Key findings from this study confirm that cervical ripening can be initiated by more than one mechanism and is not necessarily an acceleration of the physiologic process at term. These results will influence current strategies for identifying specific etiologies of preterm birth and developing subsequent therapies.

Appropriate and timely cervical remodeling is key for successful birth. Premature cervical opening can result in preterm birth which occurs in 12.5% of pregnancies. Research focused on the mechanisms of term and preterm cervical remodeling is essential to prevent prematurity. This review highlights recent findings that better define molecular processes driving progressive disorganization of the cervical extracellular matrix. This includes studies that redefine the role of immune cells and identify diverse functions of the cervical epithelia and hyaluronan in remodeling. New investigations proposing that infection-induced premature cervical remodeling is distinct from the normal process are presented. Recent advances in our understanding of term and preterm cervical remodeling provide new directions for investigation and compel investigators to reevaluate currently accepted models.

A greater understanding of the processes that regulate cervical remodeling during pregnancy, parturition, and the postpartum period is required to understand causes of preterm and posterm birth in which abnormal cervical function is the primary culprit. In the current study, gene expression patterns unique to cervical ripening as compared with cervical dilation and/or postpartum repair are identified in a mouse model. Genes differentially regulated from gestation day 15 to late day 18 reveal processes important for cervical ripening. Genes differentially regulated from late day 18 to 2 hours after birth reveal processes that could be important during cervical dilation or the postpartum recovery period. Based on expression patterns, cervical ripening requires a downregulation of collagen assembly genes; increased synthesis of glycosaminoglycans that disrupt the matrix, such as hyaluronan; increased metabolism of progesterone; and changes in epithelial barrier properties. The latter phases of dilation and postpartum recovery are associated with increased assembly of mature collagen, synthesis of matrix proteins that promote a dense connective tissue, activation of inflammatory responses, prostaglandin synthesis, and further changes in epithelial barrier properties and differentiation. Processes/gene expression required for cervical ripening are distinct from those important in latter phases of cervical remodeling and highlight the importance of timing of tissue collection for understanding the molecular mechanisms of cervical ripening.

Cervical remodeling during pregnancy and parturition is a single progressive process that can be loosely divided into four overlapping phases termed softening, ripening, dilation/labor, andpost partumrepair. Elucidating the molecular mechanisms that facilitate all phases of cervical remodeling is critical for an understanding of parturition and for identifying processes that are misregulated in preterm labor, a significant cause of perinatal morbidity. In the present study, biomechanical measurements indicate that softening was initiated between gestation days 10 and 12 of mouse pregnancy, and in contrast to cervical ripening on day 18, the softened cervix maintains tissue strength. Although preceded by increased collagen solubility, cervical softening is not characterized by significant increases in cell proliferation, tissue hydration or changes in the distribution of inflammatory cells. Gene expression studies reveal a potentially important role of cervical epithelia during softening and ripening in maintenance of an immunomucosal barrier that protects the stromal compartment during matrix remodeling. Expression of two genes involved in repair and protection of the epithelial permeability barrier in the gut (trefoil factor 1) and skin (serine protease inhibitor Kazal type 5) were increased during softening and/or ripening. Another gene whose function remains to be elucidated, purkinje cell protein 4, declines in expression as remodeling progressed. Collectively, these results indicate that cervical softening during pregnancy is a unique phase of the tissue remodeling process characterized by increased collagen solubility, maintenance of tissue strength, and upregulation of genes involved in mucosal protection.

Purpose: To develop a robust real-time PCR-based assay with improved accuracy for quantifying the relative fractions of human and mouse DNA in heterogeneous samples such as patient-derived cancer xenografts or cultures. Background: Patient derived mouse xenografts (PDXs), or irradiated mouse fibroblast conditionally reprogrammed cancer cultures (CRCs) provide valuable resources for studying cancers, but as mouse tissue in heterogeneous samples can significantly confound downstream functional and molecular assays (such as next-generation sequencing) it is important to determine the fraction of mouse DNA in mixed samples. Methods: Using an approach different from a previously reported PCR assay (Alcoser SY et al BMC Biotech 2011, 11:124), we established a Taqman real-time PCR assay employing mouse- and human-specific primers that amplified the same chromosomal region of PTGER2 on human chromosome 14q22.1 and mouse chromosome 14 22.68 cM. We also developed a method to enrich human tumor cells from PDXs using enzymatic digestion with Liberase (Roche), followed by differential centrifugation and adherence. Results: Utilizing serial dilutions of heterogeneous DNA mixtures of mouse spleen DNA and normal human white blood cell DNA at varied DNA ratios we demonstrated the assay to be linear (Pearson r = 0.9984, human DNA; r = 0.9998, mouse DNA; p6.4 pg DNA/reaction). The assay was evaluated in 66 samples of human tumors (lung, ovary, neuroblastoma, colon, pancreas, stomach and melanoma) including 25 PDXs before and after tumor cell enrichment, 12 non-enriched PDXs, 2 CRC-derived xenografts and 2 CRCs. Of the PDXs 28 were subcutaneous tumors and 9 were malignant ascites. A wide range of human:mouse DNA ratios were found from zero to 99% among all samples, with two PDXs and 2 CRC-derived xenografts lacking detectable human DNA. The highest human cell fractions were in the malignant ascites samples. Of the enriched samples, 18 of 25 (72%) showed tumor cell enrichment of 2 to 71-fold as compared with the value of matching non-enriched PDX. Conclusions: We developed a robust real-time PCR assay for measuring the relative fraction of species-specific DNA in human:mouse heterogenous samples. The human:mouse DNA ratios in PDX samples varied widely and the human tumor cell populations in PDXs can be effectively enriched in vitro. Assessing human tumor cell content with this assay will facilitate molecular studies with PDXs and mouse-human cocultivated cells. Citation Format: Yu-An Zhang, Victor Stastny, Mahboubeh Papari-Zareei, Heather Davidson, Boning Gao, Brenda Timmons, Jingsheng Yan, C Patrick Reynolds, John D. Minna, Adi F. Gazdar. Assessing and enriching human tumor cell content in patient-derived cancer xenografts and co-cultures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 225. doi:10.1158/1538-7445.AM2015-225

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1-/-), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1-/- mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.

The molecular mechanisms controlling the initiation of parturition remain largely undefined. We report a new animal model in which parturition does not occur. A line of mice expressing a human apolipoprotein B (APOB) gene fail to deliver their young if the transgene is present in homozygous (Tg/Tg), but not in heterozygous (Tg/Wt), form. Cloning and mapping of the transgene insertion locus indicate that 10 copies of the 80-kilobase APOB genomic fragment inserted into mouse chromosome 6 result in a small, 390-base pair deletion of mouse genomic DNA. Nine other lines expressing the transgene have normal labor, suggesting that transgene insertion in this mutant line disrupted a mouse gene crucial for successful parturition. The pathophysiology of parturition failure in these animals was defined using physiological, endocrinological, and morphological techniques. Results indicate that luteolysis occurs in Tg/Tg mice but is delayed by 1 day. Delivery did not occur in mutant mice at term after spontaneous luteolysis or even after removal of progesterone action by ovariectomy or antiprogestin treatment. Biomechanical and functional studies of the uterus and cervix revealed that the primary cause of failed parturition in Tg/Tg mice was not inadequate uterine contractions of labor but, rather, a rigid, inelastic cervix at term that was abnormally rich in neutrophils and tissue monocytes. Characterization of the transgene insertional mutant, Tg/Tg, indicates that progesterone withdrawal is insufficient to complete parturition in the presence of inadequate cervical ripening at term.

Purpose: Non-small cell lung cancer (NSCLC) patients show benefit from neoadjuvant chemotherapy given before surgical resection or adjuvant chemotherapy administered after tumor resection. However, several of these patients still relapse and die with metastatic NSCLC. These relapses represent resistance of the tumors to chemotherapy. NSCLC resistance to standard taxane and platin doublet chemotherapy is a major problem. Identifying and overcoming resistance mechanisms are thus urgently needed and the focus of this research. Methods: (A) NSCLC cell lines were exposed long term to cycles of paclitaxel + carboplatin in clinically relevant dosage ratios, to select and subsequently characterize resistant cells. (B) Subcutaneous xenografts of parental and resistant cell lines were established in NOD/SCID mice to include expression changes that are retained in vivo. (C) Molecularly and clinically annotated dataset of NSCLC patient tumors obtained after neoadjuvant taxane + platin chemotherapy (N = 66; some had long term disease free survival and some had tumor relapses) were subjected to gene expression and survival analyses. Results and Significance: NSCLC cell lines NCI-H1299 and NCI-H1355 treated for 16-18 cycles with paclitaxel + carboplatin developed >50 fold resistance. Cells also developed cross-resistance to doxorubicin and vinorelbine. In addition, these resistant cell lines showed decreased response to docetaxel + cisplatin doublet in vivo. Consistent with the literature, cells showed increased expression of multi-drug transporter ABCB1/MDR1. This corresponded to decreased intracellular docetaxel accumulation in resistant cells. Cell line variants showed reversal in resistance when exposed to MDR1 inhibitors or with ABCB1 knockdown. Reversal in both cases was however partial, suggesting additional resistance mechanisms. This was also indicated by repeating long term drug selection in presence of MDR inhibitor, wherein cells still developed resistance. Microarray profiles of resistant cell lines suggested mRNA expression changes in stem cell pathways, epithelial-to-mesenchymal transition (EMT) genes and certain epigenetic modulators. Cross-comparison of gene hits from cell lines and xenografts yielded 14 commonly up-regulated and 21 down-regulated genes that formed a resistance signature from preclinical models. This 35-gene resistance signature could successfully classify neoadjuvant treated patient tumors into two main clusters with significant differences in tumor recurrence and cancer-free survival. Studies are underway to determine whether this signature is purely prognostic or also has a functional role in causing resistance. Such information will provide both clinically useful biomarkers to type tumor response in patients and also potentially new therapeutic targets to overcome standard taxane-platin drug resistance in NSCLC. Citation Format: Maithili Prafulla Dalvi, Carmen Behrens, Milind Suraokar, Rui Zhong, Brenda Timmons, Luc Girard, Yang Xie, Ignacio Wistuba, John D. Minna. Developing a molecular understanding of non-small cell lung cancer resistance to standard taxane-platin chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3772. doi:10.1158/1538-7445.AM2014-3772