Our understanding of metastatic prostate cancer (mPrCa) has dramatically advanced during the genomics era. Nonetheless, many aspects of the disease may still be uncovered through reanalysis of public ...datasets. We integrated the expression datasets for 209 PrCa tissues (metastasis, primary, normal) with expression, gene dependency (GD) (from CRISPR/cas9 screen), and drug viability data for hundreds of cancer lines (including PrCa). Comparative statistical and pathways analyses and functional annotations (available inhibitors, protein localization) revealed relevant pathways and potential (and previously reported) protein markers for minimally invasive mPrCa diagnostics. The transition from localized to mPrCa involved the upregulation of DNA replication, mitosis, and PLK1-mediated events. Genes highly upregulated in mPrCa and with very high average GD (~1) are potential therapeutic targets. We showed that fostamatinib (which can target PLK1 and other over-expressed serine/threonine kinases such as AURKA, MELK, NEK2, and TTK) is more active against cancer lines with more pronounced signatures of invasion (e.g., extracellular matrix organization/degradation). Furthermore, we identified surface-bound (e.g., ADAM15, CD276, ABCC5, CD36, NRP1, SCARB1) and likely secreted proteins (e.g., APLN, ANGPT2, CTHRC1, ADAM12) that are potential mPrCa diagnostic markers. Overall, we demonstrated that comprehensive analyses of public genomics data could reveal potentially clinically relevant information regarding mPrCa.
If properly translated to clinical use, our knowledge about biomarkers may lead to a more effective way of combating colorectal cancer (CRC). Biomarkers are biomolecular, genetic, or cytogenetic ...attributes indicative of the disease’s progression, predisposition, prognosis, or therapeutic options. For CRC, these include chromosomal instability, mutations in
KRAS
and
TP53
, loss of 18q, and elevated level of carcinoembryonic antigen (CEA), which are all associated with poor prognosis. The prognostic significance of 18q loss can be attributed to reduced expression of
SMAD4
, or
DCC
, although the chromosomal arm is actually heavily populated by genes whose downregulation correlate to worse survival. Potentially, identification of prognostic biomarkers can help the oncologist decide whether adjuvant chemotherapy is necessary after surgery. Testing for therapeutic biomarkers can be necessary if targeted therapeutics are being considered. The identification of highly penetrant predisposition markers (such as mutations in
APC
and
MLH1
) can be a lifesaver for carrier individuals, who would then have to undergo colonoscopy at an earlier age. Even sporadic CRCs may have some hereditary components, according to recent studies. Genome-wide association studies (using SNP arrays) showed that polymorphisms of certain genes can have subtle influence on CRC predisposition. Our own SNP array-based analysis suggested that long stretches of germline homozygosity (autozygosity), indicative of consanguinity, may also factor in CRC predisposition.
During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study ...this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1-4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA, MYC, MET, FGF6, FGF23, LYN, MMP9, MYBL2, AURKA, UBE2C, and PTEN. Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68, CSMD1, POLR1D, and PMEPA1. From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.
A circulating tumor cell (CTC) selection microfluidic device was integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for the detection of ...mutations contained within the genomic DNA of the CTCs. Molecular profiling of CTCs can provide important clinical information that cannot be garnered simply by enumerating the selected CTCs. We evaluated our approach using SW620 and HT29 cells (colorectal cancer cell lines) seeded into whole blood as a model system. Because SW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using a microfluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device was operated at an optimized flow rate of 2 mm s−1, which allowed for the ability to process 1 mL of whole blood in <40 min. The selected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through a pair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% ± 4%. Following enumeration, the CTCs were hydrodynamically transported at a flow rate of 1 μL min−1 to an on-chip electromanipulation unit, where they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using an electric field of 100 V cm−1, the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 μL, providing an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillary electrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with the amplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 of the KRAS gene were successfully detected in the SW620 CTCs for samples containing <10 CTCs in 1 mL of whole blood. However, the HT29 cells did not contain these mutations, consistent with their known genotype.
Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the ...markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR.
In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood.
Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27).
This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary ...polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-
ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7
min for PCR, 8.1
min for LDR, 5
min for hybridization, 10
min for washing and 2.6
min for fluorescence scanning (total processing time
=
ca. 50
min) with an order of magnitude reduction in reagents compared to bench-top formats.
L1-CAM, a neuronal cell adhesion receptor, is also expressed in a variety of cancer cells. Recent studies identified L1-CAM as a target gene of beta-catenin-T-cell factor (TCF) signaling expressed at ...the invasive front of human colon cancer tissue. We found that L1-CAM expression in colon cancer cells lacking L1-CAM confers metastatic capacity, and mice injected in their spleen with such cells form liver metastases. We identified ADAM10, a metalloproteinase that cleaves the L1-CAM extracellular domain, as a novel target gene of beta-catenin-TCF signaling. ADAM10 overexpression in colon cancer cells displaying endogenous L1-CAM enhanced L1-CAM cleavage and induced liver metastasis, and ADAM10 also enhanced metastasis in colon cancer cells stably transfected with L1-CAM. DNA microarray analysis of genes induced by L1-CAM in colon cancer cells identified a cluster of genes also elevated in a large set of human colon carcinoma tissue samples. Expression of these genes in normal colon epithelium was low. These results indicate that there is a gene program induced by L1-CAM in colon cancer cells that is also present in colorectal cancer tissue and suggest that L1-CAM can serve as target for colon cancer therapy.
Several studies have verified the existence of multiple chromosomal abnormalities in colon cancer. However, the relationships between DNA copy number and gene expression have not been adequately ...explored nor globally monitored during the progression of the disease. In this work, three types of array-generated data (expression, single nucleotide polymorphism, and comparative genomic hybridization) were collected from a large set of colon cancer patients at various stages of the disease. Probes were annotated to specific chromosomal locations and coordinated alterations in DNA copy number and transcription levels were revealed at specific positions. We show that across many large regions of the genome, changes in expression level are correlated with alterations in DNA content. Often, large chromosomal segments, containing multiple genes, are transcriptionally affected in a coordinated way, and we show that the underlying mechanism is a corresponding change in DNA content. This implies that whereas specific chromosomal abnormalities may arise stochastically, the associated changes in expression of some or all of the affected genes are responsible for selecting cells bearing these abnormalities for clonal expansion. Indeed, particular chromosomal regions are frequently gained and overexpressed (e.g., 7p, 8q, 13q, and 20q) or lost and underexpressed (e.g., 1p, 4, 5q, 8p, 14q, 15q, and 18) in primary colon tumors, making it likely that these changes favor tumorigenicity. Furthermore, we show that these aberrations are absent in normal colon mucosa, appear in benign adenomas (albeit only in a small fraction of the samples), become more frequent as disease advances, and are found in the majority of metastatic samples.
The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most ...commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR–ligase detection reaction–real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient–derived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.
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