Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the ...prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin‐dependent kinase inhibitor 2A) and CDKN2B (cyclin‐dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis‐acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell‐cycle, p53 signaling, phosphoinositide 3‐kinase signaling, mitogen‐activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. Conclusion: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease. (Hepatology 2013;58:706–717)
Unlike the majority of cancers, survival for lung cancer has not shown much improvement since the early 1970s and survival rates remain low. Genetically engineered mice tumor models are of high ...translational relevance as we can generate tissue specific mutations which are observed in lung cancer patients. Since these tumors cannot be detected and quantified by traditional methods, we use micro-computed tomography imaging for longitudinal evaluation and to measure response to therapy. Conventionally, we analyze microCT images of lung cancer via a manual segmentation. Manual segmentation is time-consuming and sensitive to intra- and inter-analyst variation. To overcome the limitations of manual segmentation, we set out to develop a fully-automated alternative, the Mouse Lung Automated Segmentation Tool (MLAST). MLAST locates the thoracic region of interest, thresholds and categorizes the lung field into three tissue categories: soft tissue, intermediate, and lung. An increase in the tumor burden was measured by a decrease in lung volume with a simultaneous increase in soft and intermediate tissue quantities. MLAST segmentation was validated against three methods: manual scoring, manual segmentation, and histology. MLAST was applied in an efficacy trial using a Kras/Lkb1 non-small cell lung cancer model and demonstrated adequate precision and sensitivity in quantifying tumor growth inhibition after drug treatment. Implementation of MLAST has considerably accelerated the microCT data analysis, allowing for larger study sizes and mid-study readouts. This study illustrates how automated image analysis tools for large datasets can be used in preclinical imaging to deliver high throughput and quantitative results.
The CDK4/6 inhibitor palbociclib reduces tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Palbociclib in combination ...with anti-hormonal therapy brings significant benefit to breast cancer patients. In this study, novel combination approaches and underlying molecular/cellular mechanisms for palbociclib were explored in squamous cell lung cancer (SqCLC), the second most common subtype of non-small cell lung cancer. While approximate 20% lung patients benefit from immunotherapy, most SqCLC patients who receive platinum-doublet chemotherapy as first-line treatment, which often includes a taxane, are still in need of more effective combination therapies. Our results demonstrated enhanced cytotoxicity and anti-tumor effect with palbociclib plus taxanes at clinically achievable doses in multiple SqCLC models with diverse cancer genetic backgrounds. Comprehensive gene expression analysis revealed a sustained disruption of pRB-E2F signaling by combination that was accompanied with enhanced regulation of pleiotropic biological effects. These included several novel mechanisms such as abrogation of G2/M and mitotic spindle assembly checkpoints, as well as impaired induction of hypoxia-inducible factor 1 alpha (HIF-1α). The decrease in HIF-1α modulated a couple key angiogenic and anti-angiogenic factors, resulting in an enhanced anti-angiogenic effect. This preclinical work suggests a new therapeutic opportunity for palbociclib in lung and other cancers currently treated with taxane based chemotherapy as standard of care.
Aim
This study aimed at exploring changes in YAP expression and their effect on periodontitis (PD) combined with traumatic occlusion (TO).
Materials and Methods
BALB/cJ mice were used to establish a ...PD model by local administration of Porphyromonas gingivalis (P.g, ATCC 33277) and a TO model by occlusal elevation (OE) using composite resin bonding on the bilateral maxillary molar. The mouse fibroblast cell line (L929) and pre‐osteoblast cell line (MC3T3‐E1) were subjected to cyclic tensile/compressive stress and inflammatory stimuli (lipopolysaccharide from Escherichia coli) to verify in vivo results.
Results
Severe bone resorption was observed by microCT scanning in OE with P.g group, when compared to OE only and P.g only groups. Mechanical stress caused by OE activated the Hippo‐YAP pathway in periodontal tissues and upregulated the expression of JNK/AP‐1. OE with P.g further promoted the expression of YAP and JNK/AP1, leading to the upregulation of the JNK/AP‐1 related inflammatory cytokines TNF‐α and IL6. Similar results were obtained when osteoblasts were subjected to mechanical stress in vitro.
Conclusions
Our study demonstrated that periodontitis with TO caused severe inflammation‐induced bone resorption. Activation of YAP and upregulation of JNK/AP‐1 induced by TO potentially aggravated the symptoms of PD.
Recent regulatory guidance suggests that metabolites identified in human plasma should be present at equal or greater levels in one of the animal species used in safety assessments. In this report, a ...high-performance liquid chromatography-tandem mass spectrometry method is described whereby quantitative comparisons of exposures to metabolites between species can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods. This novel method was tested using six drug-metabolite combinations. Plasma samples from animals are mixed with control plasma from humans and vice versa to remove possible differential effects of matrices. Through multiple ion monitoring-triggered enhanced product ion (EPI) scans, all metabolites were qualitatively confirmed, and daughter ions were selected for the most sensitive mass transitions to trigger EPI scans. Direct comparisons of metabolites in animal versus human plasma were achieved by calculating the peak area ratios of the metabolites versus an internal standard. Linearity of instrument responses was established by serial dilution. A statistical analysis demonstrated that experimentally measured ratios of the parent and metabolites in rat versus human correlated well with the nominal ratios of concentrations using linear regression with an average slope of 0.99 ± 0.08 (r = 0.994 ± 0.005). This analysis showed that if the experimentally determined ratio of mass spectrometer responses is ≥ 2.0, then the actual exposure ratio is unity or greater (p < 0.01). This method offers time- and resource-sparing advantages to ascertaining metabolite exposure comparisons between humans and laboratory animal species. A strategy for application of this approach within standard drug development processes is described.
Approximately 7% of non–small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK , ROS1 , and RET . Although tumors harboring ALK fusions are highly sensitive to crizotinib, ...emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3′ overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with fluorescence in situ hybridization (FISH) ( n = 52) and 97.8% concordant with IHC ( n = 179) sensitivity, 96.8% (95% CI 91.0%–98.9%); specificity, 98.8% (95% CI 93.6%–99.8%). For ROS1 and RET , we also observed 100% concordance with FISH ( n = 46 and n = 15, respectively). We identified seven ROS1 and 14 RET fusion–positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene ( CUX1 ), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET / EGFR/KRAS alterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.
Characteristic patterns of gene expression measured by DNA microarrays have been used to classify tumors into clinically relevant sub-groups. In this study, we have refined the previously defined ...sub-types of breast tumors that could be distinguished by their distinct patterns of gene expression. A total of 115 malignant breast tumors were analyzed by hierarchical clustering based on patterns of expression of 534 "intrinsic" genes and shown to subdivide into one basal-like, one ERBB2-overexpressing, two luminal-like, and one normal breast tissue-like subgroup. The genes used for classification were selected based on their similar expression levels between pairs of consecutive samples taken from the same tumor separated by 15 weeks of neoadjuvant treatment. Similar cluster analyses of two published, independent data sets representing different patient cohorts from different laboratories, uncovered some of the same breast cancer subtypes. In the one data set that included information on time to development of distant metastasis, subtypes were associated with significant differences in this clinical feature. By including a group of tumors from BRCA1 carriers in the analysis, we found that this genotype predisposes to the basal tumor subtype. Our results strongly support the idea that many of these breast tumor subtypes represent biologically distinct disease entities.
The dysfunction and apoptosis of vascular endothelial cells are the initiating links in the formation of atherosclerosis. N6-methyladenosine (m6A) is an extremely extensive RNA methylation ...modification and its abnormality leads to the occurrence of various human diseases. In this study, we explored the effects of demethylase
-ketoglutarate-dependent dioxygenase ALKB homolog 5 (ALKBH5) on TNF-α-induced apoptosis of human umbilical vein endothelial cells (HUVECs). In TNF-α-treated HUVECs, the expression of ALKBH5 was significantly decreased. ALKBH5 overexpression promoted the proliferation and inhibited the apoptosis in TNF-α-treated HUVECs, suggesting that ALKBH5 had a protective effect on cell damage induced by TNF-α. Importantly, ALKBH5 promoted the expression of Bcl-2 in HUVECs. Bcl2 overexpression reduced the expression of Gadd45, Bax, and p21, which are transcriptionally activated by p53. But the expression of p53 has not been significantly affected, indicating that Bcl2 might regulate the apoptosis by inhibiting p53 downstream targets. In addition, ALKBH5 overexpression significantly increased the level of pri-miR-7 and decreased the level of miR-7. In conclusion, ALKBH5 attenuated the TNF-α-induced cell injury via promoting Bcl2 expression. Our research expands the understanding of the progression mechanism of atherosclerosis and provides a potential strategy for the protection of vascular endothelial injury.
Colorectal cancer (CRC) patients have poor prognosis after formation of distant metastasis. Understanding the molecular mechanisms by which genetic changes facilitate metastasis is critical for the ...development of targeted therapeutic strategies aimed at controlling disease progression while minimizing toxic side effects. A comprehensive portrait of somatic alterations in CRC and the changes between primary and metastatic tumors has yet to be developed. We performed whole genome sequencing of two primary CRC tumors and their matched liver metastases. By comparing to matched germline DNA, we catalogued somatic alterations at multiple scales, including single nucleotide variations, small insertions and deletions, copy number aberrations and structural variations in both the primary and matched metastasis. We found that the majority of these somatic alterations are present in both sites. Despite the overall similarity, several de novo alterations in the metastases were predicted to be deleterious, in genes including FBXW7, DCLK1 and FAT2, which might contribute to the initiation and progression of distant metastasis. Through careful examination of the mutation prevalence among tumor cells at each site, we also proposed distinct clonal evolution patterns between primary and metastatic tumors in the two cases. These results suggest that somatic alterations may play an important role in driving the development of colorectal cancer metastasis and present challenges and opportunities when considering the choice of treatment.
•We did whole genome sequencing of 2 primary colorectal tumors and their metastases.•We catalogued somatic alterations in both sites.•Majority of these somatic alterations were present in both sites.•Metastasis specific mutations which might be functional were identified.•Distinct clonal evolution patterns were found in the two cases.
Ultra-deep next-generation sequencing of circulating tumor DNA (ctDNA) holds great promise as a tool for the early detection of cancer and for monitoring disease progression and therapeutic ...responses. However, the low abundance of ctDNA in the bloodstream coupled with technical errors introduced during library construction and sequencing complicates mutation detection.
To achieve high accuracy of variant calling via better distinguishing low-frequency ctDNA mutations from background errors, we introduce TNER (Tri-Nucleotide Error Reducer), a novel background error suppression method that provides a robust estimation of background noise to reduce sequencing errors. The results on both simulated data and real data from healthy subjects demonstrate that the proposed algorithm consistently outperforms a current, state-of-the-art, position-specific error polishing model, particularly when the sample size of healthy subjects is small.
TNER significantly enhances the specificity of downstream ctDNA mutation detection without sacrificing sensitivity. The tool is publicly available at https://github.com/ctDNA/TNER .
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