An ATP-binding cassette transporter G2 (CgABCG2) regulates the haemocyte proliferation by modulating the G1/S phase transition of cell cycle in oyster Crassostrea gigas.

ATP-binding cassette transporter G2 (ABCG2) is a half-transporter of the G subfamily in ATP-binding cassette transporters (ABC transporter), which is involved in the regulation of multidrug-resistant, cell cycle, and cell proliferation. In the present study, a homologue of ABCG2 (named as CgABCG2) with the conserved AAA domain and ABC2 membrane domain was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgABCG2 was of 1956 bp encoding a predicted polypeptide of 652 amino acids, which shared 56.7%-65.7% sequence similarities with previously identified ABCG2s from other animals. The mRNA transcripts of CgABCG2 were detected in all the tested tissues with higher expression levels in gonad and haemocytes (19.31-fold and 11.23-fold of that in adductor muscle respectively, p < 0.05). CgABCG2 was mainly distributed on the cell membrane of the haemocytes with a partial distribution in the cytoplasm and nucleus. After Vibrio splendidus stimulation, the mRNA expression level of CgABCG2 in haemocytes was significantly up-regulated at 3 h and 6 h, which was 5.22-fold and 8.60-fold (p < 0.05) of that in control, respectively. After the expression of CgABCG2 was interfered by RNAi, the number of cells with EdU positive signals was reduced in both haemocytes and the potential hematopoietic sites. And the mRNA expression level of CgPCNA, CgGATA3, CgRunx, CgSCL and CgC-kit decreased significantly (p < 0.05), which were about 0.66-, 0.37-, 0.32-, 0.50-, and 0.50-fold of that in the negative control group, respectively. While the mRNA expression level of CgCDK2 increased significantly (1.84-fold to that in control, p < 0.05) and that of stem cell-related factor CgSOX2 did not change significantly in the si-CgABCG2 oysters. Moreover, the cell cycle of haemocytes was detected by flow cytometry, which was arrested at G0/G1 phase in the si-CgABCG2 oysters. All the results collectively suggested that CgABCG2 might involve the proliferation of haemocytes by regulating the expression of haematopoiesis related transcription factors and the G1/S phase transition of the cell cycle in oyster C. gigas.

ROS function as an inducer of autophagy to promote granulocyte proliferation in Pacific oyster Crassostrea gigas.

Hematopoiesis is the biological process to generate new blood cells in the living body and reactive oxygen species (ROS) contribute significantly to the regulation of haematopoietic cell homeostasis. In the present study, the involvement of ROS in the proliferation of haemocytes was examined in Pacific oyster Crassostrea gigas. The ROS content in haemocytes increased significantly after lipopolysaccharide (LPS) treatment, but decreased after the treatment with antioxidant N-Acetyl-L-cysteine (NAC, a scavenger of ROS). The percentage of 5-ethynyl-2'-deoxyuridine labeled (EdU+) granulocytes in total haemocytes significantly increased at 12 h (4.12-fold, p < 0.001) and 24 h (2.36-fold, p < 0.001) after LPS treatment, while decreased at 12 h (0.26-fold, p < 0.001) and 24 h (0.61-fold, p < 0.05) after NAC treatment, respectively. Meanwhile, the percentage of haemocytes with autophagosome positive signals significantly increased at 12 h (1.17-fold, p < 0.01) and 24 h (1.19-fold, p < 0.05) after LPS treatment, but significantly reduced at 12 h (0.41-fold, p < 0.001) and 24 h (0.28-fold, p < 0.001) after the NAC treatment, respectively. After ammonium chloride (NH4Cl) treatment, the percentage of haemocytes with autophagosome and EdU+ granulocytes significantly increased at 12 h, which was 1.27-fold (p < 0.01) and 1.70-fold (p < 0.01) of control group, respectively. These results collectively suggested that ROS produced after LPS treatment could act as an inducer for autophagy and involved in regulating the proliferation of some granulocytes in C. gigas.

The proliferating cell nuclear antigen (PCNA) is a potential proliferative marker in oyster Crassostrea gigas.

Proliferating cell nuclear antigen (PCNA) is a crucial eukaryotic replication accessory factor in the regulation of DNA synthesis, which is always used as a proliferation marker for haematopoiesis in vertebrates. In the present study, a homologue of PCNA (named as CgPCNA) with a conserved N-terminal PCNA domain and a C-terminal PCNA domain was identified from oyster Crassostrea gigas. The deduced amino acid sequence of CgPCNA shared 85.4% and 86.6% similarities with the PCNAs identified in Mus musculus and Homo sapiens, respectively. CgPCNA was firstly clustered with PCNAs from molluscs, and then with PCNAs from arthropods to form a group falling into the invertebrate clade in the phylogenic tree. The mRNA transcripts of CgPCNA were detected in all tested tissues with higher expression level in gonad, gills and haemolymph. They were also detected in granulocytes, semi-granulocytes and agranulocytes with no significant differences, but the protein level of CgPCNA in agranulocytes was significantly higher (3.67-fold, p < 0.05) than that in granulocytes. In the haemocytes, CgPCNA was mainly distributed in the nucleus and less in the cytoplasm of haemocytes. CgPCNA protein was observed at the tubule lumen regions of gills vessels, and especially colocalized with the EdU signals. After lipopolysaccharide (LPS) and Vibrio splendidus stimulation, the expression level of CgPCNA mRNA in haemocytes was significantly (p < 0.05) up-regulated at 6 h and 12 h, which was 13.87-fold and 3.89-fold of that in control, respectively. In the oysters treated with the recombinant protein CgAstakine (rCgAstakine), the protein abundance of CgPCNA was enhanced in agranulocytes and gills, while no significant change was observed in semi-granulocytes and granulocytes. These results collectively indicated that CgPCNA was highly expressed in the newborn agranulocytes and the potential haematopoietic sites, and it might be applied as a marker for haemocytes proliferation in oysters.

DNA binding protein CgIkaros-like regulates the proliferation of agranulocytes and granulocytes in oyster (Crassostrea gigas).

DNA-binding protein Ikaros is a major determinant of haematopoietic lineage, especially in the development, differentiation and proliferation of lymphocytes. In the present study, a Ikaros homologue (designed as CgIkaros-like) was identified and characterized as a vital determinant in the proliferation of haemocytes during haematopoiesis of Pacific oyster Crassostrea gigas. The complete coding sequence of CgIkaros-like was of 1329 bp encoding a predicted polypeptide of 442 amino acids with four ZnF regions, locating at the C-terminus and N-terminus respectively. The highest expression level of CgIkaros-like mRNA was found in gills, followed by haemocytes and gonad. The mRNA transcripts of CgIkaros-like could be detected in all the haemocytes with higher abundance in semi-granulocytes and agranulocytes. CgIkaros-like protein was localized in both of cytoplasm and nucleus with higher abundance in nucleus of oyster haemocytes. The mRNA and protein expression levels of agranulocyte marker CgCD9, granulocyte marker CgAATase, cell cycle related gene CgCDK2, Notch receptor CgNotch and Notch target gene CgHes1 all increased significantly (p < 0.05) after CgIkaros-like was interfered by siRNAs, which were about 27.33-, 2.63-, 24.34-, 4.45- and 6.08-fold of that in the siRNA-NC control group, respectively. While the transcripts of CgGATA3 and CgRunx did not change significantly after CgIkaros-like was interfered. These results demonstrated that CgIkaros-like functioned as a transcription factor combined with Notch pathway to mediate CgCDK2 and regulate the proliferation of oyster haemocytes.

Efficacy and Resistance of ALK Inhibitors in Two Inflammatory Myofibroblastic Tumor Patients with ALK Fusions Assessed by Whole Exome and RNA Sequencing.

We report two inflammatory myofibroblastic tumor (IMT) patients with ALK fusions (RRBP-ALK and TNS1-ALK, respectively). They both received tumor resection surgery and treatment with ALK inhibitors crizotinib followed by alectinib, and upon receiving each of the drugs, showed a brief response, then experienced recurrence or progression of the disease. During the treatment, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) were applied to monitor potential drug-induced gene mutation and expression changes. A novel, secondary mutation in ALK exon 23 (L1196Q) was identified in patient 1 after alectinib resistance developed. Guided by this result, a newer ALK inhibitor, ceritinib was prescribed. The patient was able to achieve a partial response (PR) and is in good condition as of the manuscript date. On the contrary, there was no secondary mutation identified in ALK in patient 2 after drug resistance. While the expression of PTCH1, a negative regulator of the sonic hedgehog (SHH) signaling pathway, was significantly reduced at the time after the treatment with crizotinib before that of alectinib. The expression of PTCH1 was also reduced after the treatment with alectinib. It was reported that ALK can exert its biological functions partially by activating SHH signaling pathway. The down-regulation of PTCH1 suggests the compensatory activation of SHH pathway may cause resistance to ALK inhibitors in IMT. Going forward, monitoring gene mutation and expression changes through DNA and RNA sequencing will be able to offer opportunities to investigate potential mechanisms of drug resistance and will help to achieve precise prescription for better treatment outcomes.

Patient specific circulating tumor DNA fingerprints to monitor treatment response across multiple tumors.

BACKGROUND: Circulating tumor DNA (ctDNA) offers a convenient way to monitor tumor progression and treatment response. Because tumor mutational profiles are highly variable from person to person, a fixed content panel may be insufficient to track treatment response in all patients. METHODS: We design ctDNA fingerprint panels specific to individual patients which are based on whole exome sequencing and target to high frequency clonal population clusters in patients. We test the fingerprint panels in 313 patients who together have eight tumor types (colorectal, hepatocellular, gastric, breast, pancreatic, and esophageal carcinomas and lung cancer and cholangiocarcinoma) and exposed to multiple treatment methods (surgery, chemotherapy, radiotherapy, targeted-drug therapy, immunotherapy, and combinations of them). We also monitor drug-related mutations in the patients using a pre-designed panel with eight hotspot genes. RESULTS: 291 (93.0%) designed fingerprint panels harbor less than ten previously known tumor genes. We detected 7475 ctDNA mutations in 238 (76%) patients and 6196 (96.0%) of the mutations are detected in only one test. Both the level of ctDNA content fraction (CCF) and fold change of CCF (between the definitive and proceeding tests) are highly correlated with clinical outcomes (p-values 1.36e-6 for level and 5.64e-10 for fold change, Kruskal-Wallis test). The CCFs of PD patients are an order of magnitude higher than the CCFs of SD and OR patients (median/mean 2.22%/8.96% for SD, 0.18/0.21% for PD, and 0.31/0.54% for OR; pairwise p-values 7.8e-6 for SD ~ PD, 2.7e-4 for OR ~ PD, and 7.0e-3 for SD ~ OR, Wilcoxon rank sum test). The fold change of CCF distinguishes the patient groups even better, which increases for PD, remains stable for SD, and decreases for OR patients (p-values 0.002, ~ 1, and 0.0001 respectively, Wilcoxon signed-rank test). Eleven drug-related mutations are identified from nine out of the 313 patients. CONCLUSIONS: The ctDNA fingerprint method improves both specificity and sensitivity of monitoring treatment response across several tumor types. It can identify tumor relapse/recurrence potentially earlier than imaging-based diagnosis. When augmented with tumor hotspot genes, it can track acquired drug-related mutations in patients.

Comprehensive analysis of potential immunotherapy genomic biomarkers in 1000 Chinese patients with cancer.

BACKGROUND: Tumor mutation burden (TMB), DNA mismatch repair deficiency (dMMR), microsatellite instability (MSI), and PD-L1 amplification (PD-L1 AMP) may predict the efficacy of the PD-1/PD-L1 blockade. With the broadening landscape of immunotherapy use, it is important to identify patients who are likely to benefit from the therapy. This study aimed to characterize the distributions of these biomarkers and explore the relationships among these biomarkers for Chinese patients with cancer. METHODS: In this study, we examined the aforementioned biomarkers in more than 1000 Chinese patients with cancer. These biomarkers were determined based on whole-exome sequencing (WES) of tumor/blood samples. RESULTS: Of the 953 samples from Chinese cancer patients assessed in this study, 35% exhibited high TMB (TMB-H), 4% were positive for high MSI (MSI-H), dMMR occurred in 0.53%, and PD-L1 AMP was positive in 3.79%. We found higher rates of TMB-H among hepatocellular carcinoma, breast cancer, and esophageal cancer patients than was reported for The Cancer Genome Atlas (TCGA) data. Lung cancer patients with EGFR mutations had significantly lower TMB values than those with wild-type EGFR, and increased TMB was significantly associated with dMMR in colorectal cancer (CRC). The frequency of tumors with MSI-H was the highest in CRC and gastric cancer. PD-L1 AMP occurred most frequently in lung squamous cell carcinoma and HER2-positive breast cancer. While MSI and dMMR are associated with higher mutational loads, correlations between TMB-H and other biomarkers, between MSI-H and dMMR, and between PD-L1 AMP and other biomarkers were low, indicating different underlying causes of the four biomarkers. CONCLUSION: The results reveal the frequency of these biomarkers in different malignancies, with potential implications for PD-1/PD-L1 blockade use for Chinese patients with cancer.

Using clinical genomic sequencing to guide personalized cancer therapy in China.

Aim: To evaluate whether clinical genomic sequencing may benefit Chinese patients with stage IV cancer. Patients & methods: Chinese patients with cancer and their oncologists were provided with genomic sequencing results and corresponding clinical treatment recommendations based on evidence-based medicine, defined as CWES (clinical whole-exome sequencing) analysis. Chinese patients with stage IV cancer who failed the previous treatment upon receiving the CWES reports were included for analyzing the impact of CWES on clinical outcomes in 1-year follow-ups. Results: A total of 88.6% of 953 Chinese patients with cancer had clinically actionable somatic genomic alterations. Eleven patients followed the CWES reports, and 11 patients did not follow the CWES suggestions. The median progression-free survival of two groups were 12 and 4 months, and 45 and 91% of patients failed this round of therapy, respectively. Conclusion: The current study suggested that CWES has the potential to increase clinical benefits for Chinese patients with stage IV cancer.

Comprehensive analysis of age-related somatic mutation profiles in Chinese young lung adenocarcinoma patients.

BACKGROUND: Lung adenocarcinoma in young adults is a rare entity with the oncogenic genetic alterations associated being poorly understood. In the present study, the effect of genetic alterations in lung adenocarcinoma patients diagnosed in young patients is reported. METHODS: Twenty young lung adenocarcinoma patients (age years: median: 33.5, range: 24-36) were enrolled in the current study and 24 patients who were at common age of the disease onset (age years: median: 61.5, range: 52-79) were selected for comparison. Paraffin sections of lung adenocarcinoma were analyzed using the whole-exome sequencing platform. RESULTS: Similar number of somatic mutations per tumor were found in the young patients and their older counterparts. Although no age-related differences were detected in the numbers of lung adenocarcinoma patients harboring well-known gene variants, mutations in FRG1 and KMT2C were associated with a younger age especially after correcting for tobacco smoking and sex (FRG1: P = 0.027, KMT2C: P = 0.046). Five genetic variants showed higher alteration frequencies in young patients compared to the unclassified East Asian population, suggesting these mutations as disease-related hereditary germline variants. CONCLUSIONS: These results suggest different characteristics of lung adenocarcinoma between the young and the patients at common age of onset. Young patients with lung adenocarcinoma have a distinctly unique prevalence of oncogenic genetic alterations.

Structure and molecular mechanism of an anion-selective mechanosensitive channel of small conductance.

Mechanosensitive (MS) channels are universal cellular membrane pores. Bacterial MS channels, as typified by MS channel of small conductance (MscS) from Escherichia coli (EcMscS), release osmolytes under hypoosmotic conditions. MS channels are known to be ion selective to different extents, but the underlying mechanism remains poorly understood. Here we identify an anion-selective MscS channel from Thermoanaerobacter tengcongensis (TtMscS). The structure of TtMscS closely resembles that of EcMscS, but it lacks the large cytoplasmic equatorial portals found in EcMscS. In contrast, the cytoplasmic pore formed by the C-terminal ß-barrel of TtMscS is larger than that of EcMscS and has a strikingly different pattern of electrostatic surface potential. Swapping the ß-barrel region between TtMscS and EcMscS partially switches the ion selectivity. Our study defines the role of the ß-barrel in the ion selection of an anion-selective MscS channel and provides a structural basis for understanding the ion selectivity of MscS channels.

Spermidine oxidase-derived H2O2 regulates pollen plasma membrane hyperpolarization-activated Ca(2+) -permeable channels and pollen tube growth.

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca(2+) concentration that accompanies pollen tube growth. Using the whole-cell patch clamp and outside-out single-channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization-activated Ca(2+) current: the addition of Spd cannot induce the channel open probability increase in excised outside-out patches, indicating that the effect of Spd in the induction of Ca(2+) currents is exerted via a second messenger. This messenger is hydrogen peroxide (H2O2), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization-activated Ca(2+) -permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca(2+) -permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal-encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca(2+) -permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca(2+) influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd-derived H2O2 signals Ca(2+) influx, thereby regulating pollen tube growth.