Dissecting the heterogeneity and tumorigenesis of BRCA1 deficient mammary tumors via single cell RNA sequencing.

Background: BRCA1 plays critical roles in mammary gland development and mammary tumorigenesis. And loss of BRCA1 induces mammary tumors in a stochastic manner. These tumors present great heterogeneity at both intertumor and intratumor levels. Methods: To comprehensively elucidate the heterogeneity of BRCA1 deficient mammary tumors and the underlying mechanisms for tumor initiation and progression, we conducted bulk and single cell RNA sequencing (scRNA-seq) on both mammary gland cells and mammary tumor cells isolated from Brca1 knockout mice. Results: We found the BRCA1 deficient tumors could be classified into four subtypes with distinct molecular features and different sensitivities to anti-cancer drugs at the intertumor level. Whereas within the tumors, heterogeneous subgroups were classified mainly due to the different activities of cell proliferation, DNA damage response/repair and epithelial-to-mesenchymal transition (EMT). Besides, we reconstructed the BRCA1 related mammary tumorigenesis to uncover the transcriptomes alterations during this process via pseudo-temporal analysis of the scRNA-seq data. Furthermore, from candidate markers for BRCA1 mutant tumors, we discovered and validated one oncogene Mrc2, whose loss could reduce mammary tumor growth in vitro and in vivo. Conclusion: Our study provides a useful resource for better understanding of mammary tumorigenesis induced by BRCA1 deficiency.

Detection of circulating genetically abnormal cells in peripheral blood for early diagnosis of non-small cell lung cancer.

BACKGROUND: Circulating genetically abnormal cells (CACs) with specific chromosome variations have been confirmed to be present in non-small cell lung cancer (NSCLC). However, the diagnostic performance of CAC detection remains unclear. This study aimed to evaluate the potential clinical application of the CAC test for the early diagnosis of NSCLC. METHODS: In this prospective study, a total of 339 participants (261 lung cancer patients and 78 healthy volunteers) were enrolled. An antigen-independent fluorescence in situ hybridization was used to enumerate the number of CACs in peripheral blood. RESULTS: Patients with early-stage NSCLC were found to have a significantly higher number of CACs than those of healthy participants (1.34 vs. 0.19; P < 0.001). The CAC test displayed an area under the receiver operating characteristic (ROC) curve of 0.76139 for discriminating stage I NSCLC from healthy participants with 67.2% sensitivity and 80.8% specificity, respectively. Compared with serum tumor markers, the sensitivity of CAC assays for distinguishing early-stage NSCLC was higher (67.2% vs. 48.7%, P < 0.001), especially in NSCLC patients with small nodules (65.4% vs. 36.5%, P = 0.003) and ground-glass nodules (pure GGNs: 66.7% vs. 40.9%, P = 0.003; mixed GGNs: 73.0% vs. 43.2%, P < 0.001). CONCLUSIONS: CAC detection in early stage NSCLC was feasible. Our study showed that CACs could be used as a promising noninvasive biomarker for the early diagnosis of NSCLC. KEY POINTS: What this study adds: This study aimed to evaluate the potential clinical application of the CAC test for the early diagnosis of NSCLC. Significant findings of the study: CAC detection in early stage NSCLC was feasible. Our study showed that CACs could be used as a promising noninvasive biomarker for the early diagnosis of NSCLC.

Tyrosine Kinase Inhibitors Could Be Effective Against Non-small Cell Lung Cancer Brain Metastases Harboring Uncommon EGFR Mutations.

Background: The significance of uncommon epidermal growth factor receptor (EGFR) mutations in patients with non-small cell lung cancer (NSCLC) and brain metastasis (BM) remains unclear. Cerebrospinal fluid (CSF) liquid biopsy is a novel tool for assessing EGFR mutations in BM. This study aimed to evaluate the EGFR mutations in patients with NSCLC and newly diagnosed BM and to examine the effect of EGFR tyrosine kinase inhibitors (TKI) on BM harboring CSF-tested uncommon EGFR mutations. Methods: This was a prospective study of 21 patients with NSCLC and BM diagnosed between 04/2018 and 01/2019. CSF was obtained to detect the BM EGFR mutations by next-generation sequencing. BM characteristics at magnetic resonance imaging (MRI) and EGFR-TKI response were examined. Results: Of 21 patients with NSCLC, 10 (47.6%) had leptomeningeal metastasis (LM), while 11 (52.4%) had brain parenchymal metastasis (BPM); 13 (61.9%) had confirmed EGFR mutation-positive primary tumors. The uncommon mutation rate in CSF ctDNA was 33.3% (7/21). Among those with EGFR mutation-positive primary tumors, the rate of uncommon EGFR mutations in CSF was 53.8% (7/13). Uncommon EGFR mutations were more common in patients with LM than in patients with PBM (6/11, 54.5% vs. 1/10, 10%), and included G719A, L861Q, L703P, and G575R. TKI was effective for four patients with BMs harboring uncommon EGFR mutations. Conclusion: In patients with NSCLC and LM, the rate of uncommon EGFR mutation was high. The BMs with uncommon EGFR mutations seem to respond to EGFR-TKI treatment. CSF liquid biopsy could reveal the EGFR genetic profile of the BM and help guide treatment using small-molecule TKI.

Corrigendum: Afatinib for Advanced Non-small Cell Lung Cancer in a Case With an Uncommon Epidermal Growth Factor Receptor Mutation (G719A) Identified in the Cerebrospinal Fluid.

[This corrects the article DOI: 10.3389/fonc.2019.00628.].

Afatinib for Advanced Non-small Cell Lung Cancer in a Case With an Uncommon Epidermal Growth Factor Receptor Mutation (G719A) Identified in the Cerebrospinal Fluid.

Few previous studies of patients with non-small cell lung cancer (NSCLC) and leptomeningeal metastases have used liquid biopsy of cerebrospinal fluid (CSF) to identify epidermal growth factor receptor (EGFR) mutations and guide therapy. A 34-year-old male patient with NSCLC and leptomeningeal metastases was admitted to the Interventional Radiology Department, Tianjin Huanhu Hospital on 18th April 2018 after showing no response to chemoradiotherapy. On admission, the patient was in critical condition with an estimated survival <1 month. A ventriculoperitoneal shunt was placed in the right lateral ventricle. The CSF level of carcinoembryonic antigen (CEA) was 9,869 ng/mL. Next-generation sequencing (NGS) of the CSF revealed an EGFR G719A mutation (frequency: 55.63%), whereas sequencing of circulating tumor DNA or cells in the peripheral blood identified no clinically significant mutations. Afatinib therapy was initiated based on the NGS results. During follow-up, the patient's symptoms improved, ventricular dilatation lessened, and pulmonary lesions decreased in size. At the last follow-up (7 months), the patient continued to show a good response to afatinib therapy with minimal adverse effects. This is the first clinical study to report the use of simultaneous genetic testing of CSF and peripheral blood to guide the successful implementation of afatinib therapy in a patient with NSCLC and leptomeningeal metastases. Notably, NGS of CSF was superior to genetic testing of peripheral blood at identifying an uncommon EGFR mutation (G719A) in a patient with NSCLC and leptomeningeal metastases.

Detection of HER2-positive Circulating Tumor Cells Using the LiquidBiopsy System in Breast Cancer.

BACKGROUND: Most previous studies of circulating tumor cells (CTCs) are based on the CellSearch platform, but CellSearch has a number of limitations. This study aimed to use the LiquidBiopsy system and immunofluorescence to test the human epidermal growth factor receptor 2 (HER2) status of CTCs in patients with breast cancer. MATERIALS AND METHODS: The LiquidBiopsy system was used to detect HER2-positive (HER2+) cells in whole blood by microfluidic immunomagnetic bead screening and immunofluorescence assay, according to the manufacturer;s instructions. HER2 expression on CTCs was assessed using the Ariol system, calibrated through spiking experiments of 100 cells (BT474, SKBR3, A431, and MDA-MB-231) and 2.5 × 107 white blood cells/mL from healthy donors. Seventy-one patients with breast cancer and 107 non-cancer donors consented to provide blood. RESULTS: Based on breast cancer cell lines experiments, HER2+ CTCs were defined as CTCs with HER2 immunofluorescence intensity ≥ 3.5 times higher than the CD45 immunofluorescence intensity (100% sensitivity and 99.9% specificity). Among the 71 patients with breast cancer, 31 (43.7%) had HER2+ tumor. Among the HER2+ patients, 41.9% (13/31) were found to be HER2+ based on CTC ≥ 1, and 25.8% (8/31) were positive based on CTC ≥ 3. In HER2-negative patients by pathologic examination, 1 (2.5%) patient was found to have ≥ 3 HER2+ CTCs, whereas 15 (37.5%) patients had ≥ 1 HER2+ CTC. HER2+ CTCs were detected at all stages, even in early breast cancer, but the detection rate was higher in metastatic breast cancer. CONCLUSION: This proof-of-concept study strongly suggests that HER2+ CTCs can be detected using the LiquidBiopsy system.

A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles.

BACKGROUND: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. METHODOLOGY/FINDINGS: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. CONCLUSIONS: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses.

Overexpression of eukaryotic initiation factor 5A2 enhances cell motility and promotes tumor metastasis in hepatocellular carcinoma.

UNLABELLED: A high incidence of tumor recurrence and metastasis has been reported in hepatocellular carcinoma (HCC) patients; however, the underlying molecular mechanisms are largely unknown. In the present study a novel metastasis-related gene, eukaryotic initiation factor 5A2 (EIF5A2), was characterized for its role in HCC metastasis and underlying molecular mechanisms. Overexpression of EIF5A2 messenger RNA (mRNA) was detected in 50/81 (61.7%) of HCCs, which was significantly higher than those in nontumorous liver tissues. Compared with matched primary HCC, higher expression of EIF5A2 protein was observed in 25/47 (53.2%) of metastatic tumors. Functional studies found that ectopic expression of EIF5A2 could enhance cancer cell migration and invasion in vitro and tumor metastasis in vivo in an experimental mouse model. Moreover, inhibition of EIF5A by small interfering RNA (siRNA) or deoxyhypusine synthase (DHPS) inhibitor GC7, which inhibits EIF5A2 maturation, could effectively decrease cell motility. Further study found that EIF5A2 was able to induce epithelial-mesenchymal transition (EMT), a key event in tumor invasion and metastasis, characterized by down-regulation of epithelial markers (E-cadherin and beta-catenin) and up-regulation of mesenchymal markers (fibronectin, N-cadherin, alpha-SMA, and vimentin). In addition, EIF5A2 could also activate RhoA/Rac1 to stimulate the formation of stress fiber and lamellipodia. CONCLUSION: EIF5A2 plays an important role in HCC invasion and metastasis by inducing EMT, as well as stimulating cytoskeleton rearrangement through activation of RhoA and Rac1.

Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

UNLABELLED: Amplification of 1q21 is the most frequent genetic alteration in human hepatocellular carcinoma (HCC), being detected in 58%-78% of primary HCC cases by comparative genomic hybridization. Recently, we isolated a candidate oncogene, Amplified in Liver Cancer 1 (ALC1), from 1q21 by hybrid selection. Here we demonstrate that ALC1 was frequently amplified and overexpressed in HCC. ALC1-transfected cells possessed a strong oncogenic ability, increasing the colony formation in soft agar and increasing the tumorigenicity in nude mice, which could be effectively suppressed by small interfering RNA against ALC1. Functional studies showed that overexpression of ALC1 could promote G1/S phase transition and inhibit apoptosis. Molecular studies revealed that the oncogenic function of ALC1 might be associated with its roles in promoting cell proliferation by down-regulating p53 expression. CONCLUSION: These results suggest that ALC1 is the target oncogene within the 1q21 amplicon and plays a pivotal role in HCC pathogenesis.

Simvastatin potentiates tumor necrosis factor alpha-mediated apoptosis of human vascular endothelial cells via the inhibition of the geranylgeranylation of RhoA.

HMG-CoA reductase inhibitors (statins) are widely used in the treatment and prevention of atherosclerosis. Here we demonstrate that the HMG-CoA reductase inhibitor simvastatin potentiates TNFalpha-mediated apoptosis and TNFalpha signaling in human umbilical vein endothelial cells (HUVECs). While 2.5 microM simvastatin or 40 ng/ml TNFalpha alone had only a small effect on apoptosis in HUVECs, co-incubation with simvastatin and TNFalpha markedly increased apoptosis in a time- and dose-dependent manner as measured by FACS analysis of propidium iodide-stained cells. Geranylgeraniol, which serves as a substrate for the geranylgeranylation of small GTP binding proteins such as RhoA, which is required for the function and membrane localization of Rho, reversed the effect of simvastatin on apoptosis. GGTI, an inhibitor of protein geranylgeranylation, mimicked the effect of simvastatin on apoptosis and interfered with the membrane localization of RhoA. Furthermore, simvastatin increased the expression of the TNFalpha type I receptor (TNFalphaRI) with a dose dependence and a dependence on geranylgeranylation similar to that demonstrated for the potentiation of TNFalpha-mediated apoptosis. Adenoviral expression of a dominant-negative RhoA mimicked the effect of simvastatin on the expression of TNFalphaRI, while adenoviral expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on the expression of TNFalphaRI. Simvastatin also potentiated TNFalpha signaling as determined by increased TNFalpha-mediated E-selectin expression. These data support the conclusion that TNFalpha signaling is under the negative control of RhoA and that statins potentiate TNFalpha signaling at least in part via interference with RhoA inhibition of TNFalpha type I receptor expression.

Oncogenic transformation by SEI-1 is associated with chromosomal instability.

Amplification of SEI-1, a cell cycle regulatory gene at 19q13.1, is commonly detected in ovarian cancer, suggesting a role in the pathogenesis of ovarian cancer. In the present study, the oncogenic potential of SEI-1 was shown by anchorage-independent growth and tumor formation in nude mice with SEI-1-transfected NIH 3T3 mouse fibroblast cells. Silencing of SEI-1 gene expression by small interfering RNAs in ovarian cancer cell line SKOV3 could inhibit cell growth as well as colony formation on soft agar. Chromosomal alterations including the formation of double minutes were observed in tumor cells derived from SEI-1-transformed NIH 3T3 cells. Micronulei formation, which is an indicator of nuclear abnormality and genomic instability, was markedly increased in SEI-1-transfected cells. These data suggest that the oncogenic role of SEI-1 might be mediated at least in part via an effect on genomic instability. Furthermore, overexpression of SEI-1 was associated with higher tumor grades and late Fesddration Internationale des Gynaecologistes et Obstetristes (FIGO) stages in ovarian carcinomas. These data strongly suggest that SEI-1 plays an important role in the development and progression of ovarian cancer.

TGFbeta regulates the expression of G alpha(i2) via an effect on the localization of ras.

The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M(2) muscarinic receptors, Galpha(i2) and the G-protein coupled inward rectifying K(+) channel, GIRK1. Here TGFbeta(1) is shown to decrease the expression of Galpha(i2) in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95+/-2% (+/-SEM, n=8) inhibition of beat rate in control cells to 18+/-2% (+/-SEM,n =8) in TGFbeta(1) treated cells. Data support the conclusion that TGFbeta regulation of Galpha(i2) expression was mediated via an effect on Ras. TGFbeta(1) inhibited Galpha(i2) promoter activity by 56+/-6% (+/-SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFbeta on Galpha(i2) expression and stimulated Galpha(i2) promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFbeta(1) on Galpha(i2) promoter activity. TGFbeta had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFbeta(1) inhibition of Ras localization to the membrane, but also reversed TGFbeta(1) inhibition of Galpha(i2)promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFbeta(1) on Ras localization and Galpha(i2) promoter activity. These data suggest a novel relationship between TGFbeta signaling, regulation of Ras function and the autonomic response of the heart.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA.

Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor-stimulated chick chorioallantoic membranes and basic fibroblast growth factor-stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.