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Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans -regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality. Co-dependency mapping assays have revealed genetic dependencies in cancer and could shed light on chromatin crosstalk mechanisms. Here, the authors establish a pipeline to integrate co-dependency mapping screens with molecular information in pan-cancer cell lines in order to reveal chromatin complexes and potential drug targets.

FRY is an evolutionarily-conserved gene involved in cellular differentiation, division, morphology, polarity, and adhesion. Decreased expression of the FRY protein in breast cancer cell lines reduced the expression of gene networks associated with epithelial differentiation, morphology, polarity and adhesion. Ectopic expression of the wild-type rat FRY gene in human breast cancer cells restored a gene expression profile associated with differentiation, suppression of epithelial-mesenchymal transition. Breast cancer cells expressing wild-type FRY at physiological levels reacquired a non-transformed morphology in vitro and a non-tumorigenic in vivo phenotype in the nude mouse xenograft model. We further determined that expression of FRY mRNA and protein correlated with breast cancer phenotypes such as Elston tumor grade, estrogen receptor, progesterone receptor, and Her2 receptor status. Despite these finding, significant gaps in knowledge remain in our understanding of how FRY expression is decreased during tumor progression. Based on preliminary data, we hypothesized modulation of FRY expression occurs through epigenetic mechanisms which include DNA methylation, histone modification and chromatin remodeling, and the activity/interaction of non-coding RNAs. To understand how DNA methylation regulates FRY transcription in breast tumors, we examined publicly available RNA-seq and DNA methylation data from The Cancer Genome Atlas (TCGA). Analysis of FRY promoter methylation revealed no significant changes in DNA methylation in normal vs. breast tumors. However, when we subdivided the tumors by hormone receptor status, we observed an increase in FRY promoter methylation in ER- and PR- breast tumors. Treatment of various breast cancer cell lines with the DNMT inhibitor, 5-Aza-2-deoxycytidine, resulted in upregulation of FRY mRNA and protein expression. These results indicate that loss of FRY expression may in part be due to increased methylation. To elucidate the role of chromatin in the role of FRY expression we searched publicly available chromatin immunoprecipitation sequencing data from the Encyclopedia of DNA elements (ENCODE) to identify key chromatin markers and transcription factors that bind to the FRY promoter. Using this data, we demonstrated that FRY has a canonical gene promoter characterized by low levels of H3K4me1 and high levels H3K4me2/me3. Furthermore, using data from the ENCODE, we identified 28 transcription factors predicted to binds to the FRY gene promoter, the most significant of which were estrogen receptor 1 (ESR1) and progesterone receptor (PR). To determine if histone deacteylases (HDACs) impacted FRY expression, we dosed various breast cancer cell lines with commonly used the HDAC inhibitors, Trichostatin-A and Panobinostat (LBH589). Through treatment of our cell lines with these HDAC inhibitors, we were able to modulate FRY expression, indicating that its expression is in part regulated by protein acetylation. Next, we explored whether transcriptional repressor, enhancer of Zeste Homolog 2 (EZH2) can bind to the FRY promoter to down regulate its expression. Using Chromosome Immunoprecipitation DNA sequencing (ChIP-seq) data from the ENCODE database, we identified that EZH2 binds to the FRY promoter in both normal human mammary epithelial cells (HMECs) and in the breast cancer derived MCF-7 cell line. Furthermore, when we treated cells with EZH2 inhibitors, GSK343 and DZNep, we induce the expression of FRY mRNA and protein in the MDA-MB231, MDA-MB-468, MCF-7, T47-D and HCC1954 cell lines. Our results suggest that elevated expression of EZH2 contributes to decreased FRY expression in breast cancer cells. One unique feature of the FRY gene promoter is that it also encodes an antisense long coding RNA (lncRNA), FRY-AS1. Many lncRNAs have been shown to be biologically active and have been implicated various cellular processes such as transcriptional interference, the induction of chromatin remodeling, and modulation of protein activity. To date, no studies have focused on the functional characterization of FRY-AS1. In this present study, we analyzed publicly available RNA-Seq data. We found that FRY-AS1 is differentially expressed in a variety of human tissue, and that FRY mRNA expression is strongly correlated with FRY-AS1 expression. Next, using RNA-seq data from TCGA BRCA dataset, we found that FRY-AS1 expression was significantly decreased in basal-like tumors, and in estrogen negative, and progesterone negative tumors. In vitro studies indicated that FRY-AS1 was decreased in 4 of 5 cell lines examined relative to the levels non-tumorigenic mammary epithelial cell line. Cellular fractionation studies revealed that FRY-AS1 is primarily localized to the nucleus. Ectopic over-expression of FRY-AS1 in the MDA-MB-231 induced differentiation to a more differentiated epithelial phenotype in vitro and induced the formation the formation acinar structures in 3D-Matrigel cultures. Significantly, overexpression of FRY-AS1 also increased endogenous FRY expression, indicating that the lncRNA regulates FRY expression in trans. Lastly, we treated the MDA-MB-231, MDA-MB-468, MCF-7, HCC1954, and T47-D cell lines with HDAC, DNMT, or EZH2 inhibitors. In these studies, we were able to induce FRY-AS1 expression in all cancer cell lines examined, which indicates that the expression of this lncRNA is regulated in part through multiple epigenetic mechanisms. Taken together, our results demonstrate that FRY-AS1 is a bona-fide lncRNA with functional significance in regulation of the FRY carcinoma susceptibility gene, and hence may be a target therapeutic intervention.

Growing demands in high data capacity and low energy consumption have driven the development of high-performance optical interconnects for many commercial applications, such as links for long-haul, intra-/inter-datacenter, and 5G communication. Typically, the photonic devices used in these environments are optimized for operation at or above room temperature, however there is an existing and growing need for optimized photonic devices to operate in cryogenic and/or high-radiation environments. Applications of these optical interconnects range from control and readout from superconducting integrated circuits for quantum computing, to readout of tracking detectors in high-energy physics (HEP) particle accelerators, to readout of next-generation infrared (IR) focal plane array (FPA) detectors. Key to the success of these optical interconnects is the high-performance and ruggedization of the electro-optic modulator (EOM), typically implemented either as a remoted external device or as a directly modulated light source. This PhD dissertation addresses the challenges of optical interconnects for harsh environments in the following manner: (1) a novel and wavelength division multiplexed (WDM) scalable optical interconnect architecture has been developed—whereby the remoted EOM has been implemented as a microring resonator—and reduces both system complexity and energy consumption, (2) a high-speed optical link operating at cryogenic temperatures has been demonstrated—utilizing a silicon photonic based EOM—in addition to laser wavelength locking of the microring resonator, and (3) a semiconductor physics based model of the EOM has been developed to address the temperature dependent effects of the silicon p-n junction embedded in the microring resonator—based on the free-carrier plasma dispersion effect in doped silicon—to aid in the device design and optical interconnect link optimization for various harsh environment applications. Lastly, an outlook and suggested areas for future research—in the area of optimized silicon photonic devices for harsh environments—is given.

Inactivating, monoallelic mutations in histone acetyltransferases (HATs) / are common in germinal center (GC) B-cell lymphomas and are implicated in derangements of the GC reaction, evasion of immune surveillance, and disease initiation. This study evaluates a first-in-class HAT activator, YF2, as a way to allosterically induce the functional HAT allele. YF2 binds to the bromo/RING domains of CREBBP/p300, increasing enzyme auto-acetylation and activation, and is selectively cytotoxic in HAT-mutated lymphoma cell lines. YF2 induces CREBBP/p300-mediated acetylation of putative targets including H3K27, p53, and BCL6. Treatment with YF2 transcriptionally activates numerous immunological pathways and increases markers of antigen presentation. Furthermore, YF2 modulates the GC reaction and increases B-cell maturation. YF2 is well tolerated and improves survival in cell line- and patient-derived xenograft lymphoma mouse models. In summary, pharmacological activation of the functional HAT allele using YF2 effectively counteracts monoallelic / mutations in GC B-cell lymphoma.

Background We sought to determine the sites of recurrence and identify predicting factors for recurrence and survival in patients who underwent gastrectomy for adenocarcinoma at an institution where preoperative therapy is commonly used for advanced gastric cancer. Methods We collected clinicopathologic data and sites of recurrence from a prospectively maintained database of patients who underwent potentially curative resection of gastric or gastroesophageal adenocarcinoma at our institution in 1995–2014, and we assessed associations between these characteristics and recurrence patterns and survival. Results We identified 488 patients who underwent R0 resection of localized gastric cancer. The median age was 63 years (interquartile range 53–71 years), and 60% were male. The most common T and N categories, per endoscopic ultrasonography, were T3 (58%) and N0 (61%). Preoperative treatment was used in 61% of patients. A total of 125 (26%) patients experienced recurrence during follow-up. Recurrences were locoregional in 19 patients (15%), peritoneal in 61 (49%), and nonperitoneal distant in 67 (54%). The peritoneum also was the most common organ of recurrence (49%), followed by the liver (21%). The median time from primary resection to recurrence was 2.7 years for locoregional, 1.3 years for peritoneal, and 0.6 years for nonperitoneal distant recurrence ( p  = 0.01). Median overall survival was markedly shorter after peritoneal and nonperitoneal distant recurrences than after locoregional recurrences. Conclusions The peritoneum was a common site of recurrence after curative resection of gastric cancer and was associated with poor survival. Prophylactic treatment targeting the peritoneal cavity might improve survival of advanced gastric cancer.

Background This study aimed to identify the yield of staging laparoscopy with peritoneal lavage cytology for gastric cancer patients and to track it over time. Methods The medical records of patients with gastric or gastroesophageal adenocarcinoma who underwent pretreatment staging laparoscopy at the authors’ institution from 1995 to 2012 were reviewed. The yield of laparoscopy was defined as the proportion of patients who had positive findings on laparoscopy, including those with macroscopic carcinomatosis, positive cytology, or other clinically important findings. To compare the yield of laparoscopy over time, the patients were divided into three 6-year ranges based on the date of diagnosis. Associations between clinicopathologic factors and peritoneal disease were examined using uni- and multivariate analyses. Results The study included 711 patients. Among these patients, 43.5 % had gastroesophageal junction tumors, 72.9 % had poorly differentiated adenocarcinoma, and 53 % had signet ring cell morphology. Endoscopic ultrasound had most commonly identified T3 (83.9 %) and N-positive (66.4 %) tumors. At laparoscopy, 148 (20.8 %) patients had been found to have macroscopic peritoneal carcinomatosis. Among 514 macroscopically negative patients who underwent peritoneal lavage cytologic analysis, 68 (13.2 %) had positive cytology results for malignancy. The total laparoscopy yield was 36 %, which did not change over time ( p = 0.58). Multivariate analysis demonstrated that positive cytology or carcinomatosis was associated with poorly differentiated histology, linitis plastica, and equivocal computed tomography findings. Conclusions Laparoscopy remains a useful staging procedure to evaluate for peritoneal spread when treatment or surgery is considered, even with the current availability of high-quality imaging.

Intratumoral heterogeneity (ITH) is a fundamental property of cancer; however, the origins of ITH remain poorly understood. We performed single-cell transcriptome profiling of peritoneal carcinomatosis (PC) from 15 patients with gastric adenocarcinoma (GAC), constructed a map of 45,048 PC cells, profiled the transcriptome states of tumor cell populations, incisively explored ITH of malignant PC cells and identified significant correlates with patient survival. The links between tumor cell lineage/state compositions and ITH were illustrated at transcriptomic, genotypic, molecular and phenotypic levels. We uncovered the diversity in tumor cell lineage/state compositions in PC specimens and defined it as a key contributor to ITH. Single-cell analysis of ITH classified PC specimens into two subtypes that were prognostically independent of clinical variables, and a 12-gene prognostic signature was derived and validated in multiple large-scale GAC cohorts. The prognostic signature appears fundamental to GAC carcinogenesis and progression and could be practical for patient stratification. Single-cell analysis of gastric cancer samples tracks the cell of origin of metastatic lesions and identifies an independent prognostic signature of the clinical outcome.

BackgroundThe American Joint Committee on Cancer (AJCC) recently released its 8th edition staging system, which created a separate staging system for gastric cancer patients who have undergone preoperative therapy (ypStage). The objective of this retrospective study was to apply the new ypStage to patients who have undergone preoperative therapy and potentially curative gastrectomy.MethodsWe collected data from a prospectively maintained institutional database of gastric cancer patients who underwent potentially curative gastrectomy after preoperative therapy (1995–2015). Kaplan–Meier survival estimations and log-rank tests were performed to compare survival. Univariable and multivariable analyses were performed to determine risk factors for overall survival.ResultsA total of 354 patients met our criteria. Most patients completed planned preoperative therapy (94%; 332/354) and received chemoradiation therapy (75%; 265/354). Although clinical stage (cStage) provided a poor discrimination of survival, postneoadjuvant pathological stage (ypStage) identified significant variation in survival (p < 0.001). Multivariable analysis showed the following factors were associated with survival after adjustment for ypStage: Asian race (HR 0.52; p = 0.028), linitis plastica (HR 1.66; p = 0.037), and R1 resection (HR 1.91; p = 0.016). Survival was not longer in ypT0N0 patients than in ypStage I patients (HR 1.29; p = 0.377).ConclusionsThe AJCC 8th edition staging system for gastric cancer demonstrated reasonable survival prediction by ypStage, but not cStage, in patients who had undergone preoperative therapy. ypT0N0 patients, although not defined in the 8th edition, may be considered for inclusion in the ypStage I group.

Purpose The aim of this phase II study was to perform neoadjuvant hyperthermic intraperitoneal chemoperfusion (HIPEC) via a minimally invasive approach without cytoreduction for patients with gastric cancer and positive peritoneal cytology or low-volume peritoneal carcinomatosis. Methods Patients with gastric or gastroesophageal adenocarcinoma and positive peritoneal cytology or radiologically occult peritoneal carcinomatosis after systemic chemotherapy received laparoscopic HIPEC with mitomycin C 30 mg and cisplatin 200 mg. Patients whose peritoneal disease resolved were offered gastrectomy. The primary endpoint was overall survival (OS), with secondary endpoints of HIPEC complications and gastrectomy rate. Results We enrolled 19 patients (6 with positive peritoneal cytology only and 13 with peritoneal carcinomatosis) and treated them with 38 laparoscopic HIPEC procedures. Patients had received a median of 8 cycles (range 3–12) of systemic chemotherapy prior to enrollment. Fourteen patients were also treated with chemoradiotherapy before or between cycles of HIPEC. The complication rate for HIPEC was 11% (4 of 38 procedures), the 30-day mortality rate was 0%, and the median length of hospital stay after HIPEC was 3 days (range 2–6). Five patients went on to receive gastrectomy. The median follow-up was 18.9 months, the median OS from the date of diagnosis of metastatic disease was 30.2 months, and the median OS from the first laparoscopic HIPEC was 20.3 months. Conclusions Laparoscopic HIPEC was well tolerated, and an encouraging number of patients demonstrated an absence of peritoneal disease after HIPEC and were able to undergo gastrectomy. Comparative studies will be required to clarify survival benefits.

Background Linitis plastica due to gastric adenocarcinoma is a condition with a long history, but still lacks a standardized definition and is commonly confused with Borrmann type IV, Lauren diffuse, and signet-cell type gastric cancer. The absence of a clear definition is a problem when investigating its biological characteristics and role as a possible independent factor for prognosis. Nevertheless, the biological behavior for linitis plastica, which is unique, may be valuable in risk stratification and have implications for treatment. A definition of linitis plastica based on molecular or genomic criteria could represent a useful starting point for investigating new targeted therapies. Main body This literature review of linitis plastica will focus on the current classifications for gastric cancer, illustrating how the concept of linitis plastica relates to them in most cases and identifying a clear and reproducible definition. Moreover, the review will highlight the diagnostic challenges associated with linitis plastica, its prognostic implications, and the therapeutic options available. Future perspectives for its management are also addressed. Conclusion Linitis plastica is a carcinoma with a scirrhous stroma, involving the submucosal and muscular layers of the stomach even in the absence of mucosal alteration. In most cases, the primary cancer cells are signet-ring cells or scattered cells in the context of a poorly differentiated carcinoma. Diagnosis is challenging. Staging should be thorough, including diagnostic laparoscopy in all cases due to the high incidence of peritoneal involvement. The prognostic significance of linitis plastica is still controversial. Curative-intent surgery, when feasible, should be performed, with a multimodality treatment approach. Cancer-stroma interactions are important features of this disease, and represent attaining potential target for future therapies. Future pathologic assessments of gastric cancer should report the stromal reaction in order to allow better characterization of the tumor.