Landscape of gene fusions in epithelial cancers: seq and ye shall find

Abstract Enabled by high-throughput sequencing approaches, epithelial cancers across a range of tissue types are seen to harbor gene fusions as integral to their landscape of somatic aberrations. Although many gene fusions are found at high frequency in several rare solid cancers, apart from fusions involving the ETS family of transcription factors which have been seen in approximately 50 % of prostate cancers, several other common solid cancers have been shown to harbor recurrent gene fusions at low frequencies. On the other hand, many gene fusions involving oncogenes, such as those encoding ALK, RAF or FGFR kinase families, have been detected across multiple different epithelial carcinomas. Tumor-specific gene fusions can serve as diagnostic biomarkers or help define molecular subtypes of tumors; for example, gene fusions involving oncogenes such as ERG, ETV1, TFE3, NUT, POU5F1, NFIB, PLAG1, and PAX8 are diagnostically useful. Tumors with fusions involving therapeutically targetable genes such as ALK, RET, BRAF, RAF1, FGFR1–4, and NOTCH1–3 have immediate implications for precision medicine across tissue types. Thus, ongoing cancer genomic and transcriptomic analyses for clinical sequencing need to delineate the landscape of gene fusions. Prioritization of potential oncogenic “drivers” from “passenger” fusions, and functional characterization of potentially actionable gene fusions across diverse tissue types, will help translate these findings into clinical applications. Here, we review recent advances in gene fusion discovery and the prospects for medicine.http://deepblue.lib.umich.edu/bitstream/2027.42/116210/1/13073_2015_Article_252.pd

N-terminal PDZ-like domain of chromatin organizer SATB1 contributes towards its function as transcription regulator

The special AT-rich DNA-binding protein 1 (SATB1) is a matrix attachment region (MAR)-binding protein that acts as a global repressor via recruitment of CtBP1:HDAC1-containing co-repressors to its binding targets. The N-terminal PSD95/Dlg-A/ZO-1 (PDZ)-like domain of SATB1 mediates interactions with several chromatin proteins. In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal PDZ-like domain (amino acid residues 1-204) lacks DNA binding activity, it would fail to recruit the interacting partners of SATB1 to its genomic binding sites and hence would not repress the SATB1-regulated genes. Indeed, in vivo MAR-linked luciferase reporter assay revealed that overexpression of the PDZ-like domain resulted in de-repression, indicating that the PDZ-like domain exerts a dominant negative effect on genes regulated by SATB1. Next, we developed a stable dominant negative model in human embryonic kidney (HEK) 293T cells that conditionally expressed the N-terminal 1-204 region harbouring the PDZ-like domain of SATB1. To monitor the effect of sequestration of the interaction partners on the global gene regulation by SATB1, transcripts from the induced and uninduced clones were subjected to gene expression profiling. Clustering of expression data revealed that 600 out of 19000 genes analysed were significantly upregulated upon overexpression of the PDZ-like domain. Induced genes were found to be involved in important signalling cascades and cellular functions. These studies clearly demonstrated the role of PDZ domain of SATB1 in global gene regulation presumably through its interaction with other cellular proteins

Expression profiling of cervical cancers in I ndian women at different stages to identify gene signatures during progression of the disease

Cervical cancer is the second most common cancer among women worldwide, with developing countries accounting for >80% of the disease burden. Although in the West, active screening has been instrumental in reducing the incidence of cervical cancer, disease management is hampered due to lack of biomarkers for disease progression and defined therapeutic targets. Here we carried out gene expression profiling of 29 cervical cancer tissues from I ndian women, spanning International Federation of Gynaecology and Obstetrics ( FIGO ) stages of the disease from early lesion (IA and IIA) to progressive stages (IIB and IIIA–B), and identified distinct gene expression signatures. Overall, metabolic pathways, pathways in cancer and signaling pathways were found to be significantly upregulated, while focal adhesion, cytokine–cytokine receptor interaction and WNT signaling were downregulated. Additionally, we identified candidate biomarkers of disease progression such as SPP 1, proliferating cell nuclear antigen ( PCNA ), STK 17A, and DUSP 1 among others that were validated by quantitative real‐time polymerase chain reaction ( qRT ‐ PCR ) in the samples used for microarray studies as well in an independent set of 34 additional samples. Integrative analysis of our results with other cervical cancer profiling studies could facilitate the development of multiplex diagnostic markers of cervical cancer progression. Cervical cancer is the leading cause of cancer deaths among women in I ndia, yet it remains poorly characterized at molecular level. This study provides one of the largest molecular profiling efforts from this region involving cervical cancer tissues from well‐defined clinical stages to identify molecular signatures of disease progression, as well as identify novel biomarkers distinguishing early and advanced disease. We expect this study to serve as a template for larger studies, including those based on high‐throughput sequencing, to help develop robust biomarkers of disease progression and potentially identify actionable therapeutic targets.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101800/1/cam4152.pd

Molecular Phylogeny of OVOL Genes Illustrates a Conserved C2H2 Zinc Finger Domain Coupled by Hypervariable Unstructured Regions

OVO-like proteins (OVOL) are members of the zinc finger protein family and serve as transcription factors to regulate gene expression in various differentiation processes. Recent studies have shown that OVOL genes are involved in epithelial development and differentiation in a wide variety of organisms; yet there is a lack of comprehensive studies that describe OVOL proteins from an evolutionary perspective. Using comparative genomic analysis, we traced three different OVOL genes (OVOL1-3) in vertebrates. One gene, OVOL3, was duplicated during a whole-genome-duplication event in fish, but only the copy (OVOL3b) was retained. From early-branching metazoa to humans, we found that a core domain, comprising a tetrad of C2H2 zinc fingers, is conserved. By domain comparison of the OVOL proteins, we found that they evolved in different metazoan lineages by attaching intrinsically-disordered (ID) segments of N/C-terminal extensions of 100 to 1000 amino acids to this conserved core. These ID regions originated independently across different animal lineages giving rise to different types of OVOL genes over the course of metazoan evolution. We illustrated the molecular evolution of metazoan OVOL genes over a period of 700 million years (MY). This study both extends our current understanding of the structure/function relationship of metazoan OVOL genes, and assembles a good platform for further characterization of OVOL genes from diverged organisms

RhoC Induces Differential Expression of Genes Involved in Invasion and Metastasis in MCF10A Breast Cells

Inflammatory breast cancer (IBC) is the most deadly form of breast cancer in humans presumably due to its ability to metastasize from its inception. In our laboratory, overexpression of RhoC GTPase was observed to be specific for IBC tumors, but not for stage-matched, non-IBC tumors. RhoC is known to contribute to an IBC-like phenotype in HPV-E6E7 immortalized breast cells. To further study the effect of RhoC overexpression on IBC metastasis, we generated stable transfectants of spontaneous immortalized mammary epithelial cells (MCF10A) overexpressing wild-type RhoC or a constitutively active RhoC mutant (G14V). Both the RhoC wild type and the G14V transfectants were highly invasive and proliferated more rapidly compared to vector-only control clones. Overexpression of RhoC led to an increase in actin stress fiber and focal adhesion contact formation. Comparative microarray analysis of these clones further revealed that RhoC overexpression upregulated 108 genes whereas seven genes were down-regulated. We have further verified by quantitative RT-PCR that genes involved in cell proliferation, invasion/adhesion, and angiogenesis were modulated by RhoC. This work suggests strong candidates for the downstream oncogenic functions of RhoC.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44221/1/10549_2004_Article_5264607.pd

Ultra‐Specific Isolation of Circulating Tumor Cells Enables Rare‐Cell RNA Profiling

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134266/1/advs147_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134266/2/advs147-sup-0001-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134266/3/advs147.pd

Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma

Pancreatic cancer is characterized by nearly universal activating mutations in KRAS. Among other somatic mutations, TP53 is mutated in more than 75% of human pancreatic tumors. Genetically engineered mice have proven instrumental in studies of the contribution of individual genes to carcinogenesis. Oncogenic Kras mutations occur early during pancreatic carcinogenesis and are considered an initiating event. In contrast, mutations in p53 occur later during tumor progression. In our model, we recapitulated the order of mutations of the human disease, with p53 mutation following expression of oncogenic Kras. Further, using an inducible and reversible expression allele for mutant p53, we inactivated its expression at different stages of carcinogenesis. Notably, the function of mutant p53 changes at different stages of carcinogenesis. Our work establishes a requirement for mutant p53 for the formation and maintenance of pancreatic cancer precursor lesions. In tumors, mutant p53 becomes dispensable for growth. However, it maintains the altered metabolism that characterizes pancreatic cancer and mediates its malignant potential. Further, mutant p53 promotes epithelial-mesenchymal transition (EMT) and cancer cell invasion. This work generates new mouse models that mimic human pancreatic cancer and expands our understanding of the role of p53 mutation, common in the majority of human malignancies

Development and Evaluation of Active Case Detection Methods to Support Visceral Leishmaniasis Elimination in India.

As India moves toward the elimination of visceral leishmaniasis (VL) as a public health problem, comprehensive timely case detection has become increasingly important, in order to reduce the period of infectivity and control outbreaks. During the 2000s, localized research studies suggested that a large percentage of VL cases were never reported in government data. However, assessments conducted from 2013 to 2015 indicated that 85% or more of confirmed cases were eventually captured and reported in surveillance data, albeit with significant delays before diagnosis. Based on methods developed during these assessments, the CARE India team evolved new strategies for active case detection (ACD), applicable at large scale while being sufficiently effective in reducing time to diagnosis. Active case searches are triggered by the report of a confirmed VL case, and comprise two major search mechanisms: 1) case identification based on the index case's knowledge of other known VL cases and searches in nearby houses (snowballing); and 2) sustained contact over time with a range of private providers, both formal and informal. Simultaneously, house-to-house searches were conducted in 142 villages of 47 blocks during this period. We analyzed data from 5030 VL patients reported in Bihar from January 2018 through July 2019. Of these 3033 were detected passively and 1997 via ACD (15 (0.8%) via house-to-house and 1982 (99.2%) by light touch ACD methods). We constructed multinomial logistic regression models comparing time intervals to diagnosis (30-59, 60-89 and ≥90 days with =90 days compared to the referent of <30 days for ACD vs PCD were 0.88, 0.56 and 0.42 respectively. These ACD strategies not only reduce time to diagnosis, and thus risk of transmission, but also ensure that there is a double check on the proportion of cases actually getting captured. Such a process can supplement passive case detection efforts that must go on, possibly perpetually, even after elimination as a public health problem is achieved

Physics Potential of the ICAL detector at the India-based Neutrino Observatory (INO)

The upcoming 50 kt magnetized iron calorimeter (ICAL) detector at the India-based Neutrino Observatory (INO) is designed to study the atmospheric neutrinos and antineutrinos separately over a wide range of energies and path lengths. The primary focus of this experiment is to explore the Earth matter effects by observing the energy and zenith angle dependence of the atmospheric neutrinos in the multi-GeV range. This study will be crucial to address some of the outstanding issues in neutrino oscillation physics, including the fundamental issue of neutrino mass hierarchy. In this document, we present the physics potential of the detector as obtained from realistic detector simulations. We describe the simulation framework, the neutrino interactions in the detector, and the expected response of the detector to particles traversing it. The ICAL detector can determine the energy and direction of the muons to a high precision, and in addition, its sensitivity to multi-GeV hadrons increases its physics reach substantially. Its charge identification capability, and hence its ability to distinguish neutrinos from antineutrinos, makes it an efficient detector for determining the neutrino mass hierarchy. In this report, we outline the analyses carried out for the determination of neutrino mass hierarchy and precision measurements of atmospheric neutrino mixing parameters at ICAL, and give the expected physics reach of the detector with 10 years of runtime. We also explore the potential of ICAL for probing new physics scenarios like CPT violation and the presence of magnetic monopoles.Comment: 139 pages, Physics White Paper of the ICAL (INO) Collaboration, Contents identical with the version published in Pramana - J. Physic

Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast

Micropapillary carcinoma ( MPC ) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations ( CNAs ) distinct from that of grade‐ and oestrogen receptor ( ER )‐matched invasive carcinomas of no special type ( IC‐NSTs ). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray‐based comparative genomic hybridization ( aCGH ) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs . Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC‐NSTs , and recurrent mutations affecting mitogen‐activated protein kinase family genes and NBPF10 . RNA ‐sequencing analysis identified 17 high‐confidence fusion genes, eight of which were validated and two of which were in‐frame. No recurrent fusions were identified in an independent series of MPCs and IC‐NSTs . Forced expression of in‐frame fusion genes ( SLC2A1–FAF1 and BCAS4–AURKA ) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out‐of‐frame rearrangements was found in one MPC and in 13% of HER2 ‐positive breast cancers, identified through a re‐analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild‐type CDK12 in a CDK12 ‐null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106752/1/path4325.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/106752/2/path4325-sup-0001-AppendixS1.pd