Phase separation underlies signaling activation of oncogenic NTRK fusions.

NTRK (neurotrophic tyrosine receptor kinase) gene fusions that encode chimeric proteins exhibiting constitutive activity of tropomyosin receptor kinases (TRK), are oncogenic drivers in multiple cancer types. However, the underlying mechanisms in oncogenesis that involve various N-terminal fusion partners of NTRK fusions remain elusive. Here, we show that NTRK fusion proteins form liquid-like condensates driven by their N-terminal fusion partners. The kinase reactions are accelerated in these condensates where the complexes for downstream signaling activation are also concentrated. Our work demonstrates that the phase separation driven by NTRK fusions is not only critical for TRK activation, but the condensates formed through phase separation serve as organizational hubs for oncogenic signaling.

CENPF/CDK1 signaling pathway enhances the progression of adrenocortical carcinoma by regulating the G2/M-phase cell cycle.

BACKGROUND: Adrenocortical carcinoma (ACC) is an aggressive and rare malignant tumor and is prone to local invasion and metastasis. And, overexpressed Centromere Protein F (CENPF) is closely related to the oncogenesis of various neoplasms, including ACC. However, the prognosis and exact biological function of CENPF in ACC remains largely unclear. METHODS: In the present essay, the expression patterns and prognostic value of CENPF in ACC were investigated in clinical specimens and public cancer databases, including GEO and TCGA. The potential signaling mechanism of CENPF in ACC was studied based on gene-set enrichment analysis (GSEA). Furthermore, a small RNA interference experiment was conducted to probe the underlying biological function of CENPF in the human ACC cell line, SW13 cells. Lastly, two available therapeutic strategies, including immunotherapy and chemotherapy, have been further explored. RESULTS: The expression of CENPF in human ACC samples, GEO, and TCGA databases depicted that CENPF was overtly hyper-expressed in ACC patients and positively correlated with tumor stage. The aberrant expression of CENPF was significantly correlated with unfavorable overall survival (OS) in ACC patients. Then, the GSEA analysis declared that CENPF was mainly involved in the G2/M-phase mediated cell cycle and p53 signaling pathway. Further, the in vitro experiment demonstrated that the interaction between CENPF and CDK1 augmented the G2/M-phase transition of mitosis, cell proliferation and might induce p53 mediated anti-tumor effect in human ACC cell line, SW13 cells. Lastly, immune infiltration analysis highlighted that ACC patients with high CENPF expression harbored significantly different immune cell populations, and high TMB/MSI score. The gene-drug interaction network stated that CENPF inhibitors, such as Cisplatin, Sunitinib, and Etoposide, might serve as potential drugs for the therapy of ACC. CONCLUSION: The result points out that CENPF is significantly overexpressed in ACC patients. The overexpressed CENPF predicts a poor prognosis of ACC and might augment the progress of ACC. Thus, CENPF and related genes might serve as a novel prognostic biomarker or latent therapeutic target for ACC patients.

Long noncoding RNA DLX6-AS1 promotes liver cancer by increasing the expression of WEE1 via targeting miR-424-5p.

Long noncoding RNAs (lncRNAs) played an important role in tumorigenesis and development of hepatocellular carcinoma (HCC). In this study, we first demonstrated that lncRNA DLX6 antisense RNA 1 (DLX6-AS1) was upregulated in cancer tissues and cells lines compared with normal adjacent and cell line. Knock-down DLX6-AS1 by transfection with small interfering RNA (siRNA) suppressed cell proliferation, migration, and invasion of HCC cells. Cell cycle analysis showed that cells transfected with siRNA were arrested in G0/G1 phase. Then, we performed dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay to show that DLX6-AS1 could bind with miR-424-5p. And cotransfection inhibitor of miR-424-5p with siRNA of DLX6-AS1 could abolish the inhibitory effect of siRNA of DLX6-AS1 on cell proliferation, migration, and invasion. Moreover, we further demonstrated that the oncogene WEE1 G2 checkpoint kinase (WEE1) was the target of miR-424-5p and expression levels of WEE1 were positive correlation with that of DLX6-AS1. Taken together, these results suggested that upregulated DLX6-AS1 promoted cell proliferation, migration, and invasion of HCC through increasing expression of WEE1 via targeting miR-424-5p.

Immune signatures of the POLE mutation in endometrial carcinomas: a systematic study based on TCGA data and clinical cohort validation.

Background: POLE is a critical biomarker for endometrial cancer (ECs) prognosis and therapeutic decision. However, the immune infiltration and immunotherapy-related gene expression in the tumor microenvironment (TME) of POLE-mutated ECs remain unresolved. Methods: The TCGA database was used to characterize the TME of POLE mutants, which primarily included immune cells and co-expression genes. We used immunohistochemistry (IHC) to determine immune cell abundance and PD-L1 expression in 104 EC tissues, including 11 POLE mutants and 93 wild-type. Results: The bioinformatic study found significant differences in gene expression of the chemokine family, immune-cell markers, and lysozyme in POLE mutants, along with immune response activation. In POLE-mutated ECs, the abundance of CD4+T, CD8+T, M1 macrophages, and dendritic cells increased considerably. Furthermore, POLE mutations may enhance immune cell recruitment or activation and lymphocyte homing in ECs. POLE mutants also had increased expression of immune-checkpoint suppressor genes such as PD-L1, CTLA-4, TIM-3, and others. The tumor mutation burden (TMB) was higher in ECs with POLE mutation. In the validation cohort, we discovered that POLE mutations were related to the immune infiltration abundance of CD8+, CD4+, and Foxp3+ cells and PD-L1 expression by IHC. The prognosis of TCGA-ECs showed that the survival time of the CD8, CD4, PD-L1, or Foxp3 over-expression subgroup of the POLE mutants was significantly prolonged compared to the down-regulation subgroup or the POLE wild-type. Conclusion: The infiltration abundance of CD8+ T, CD4+ T, Foxp3+ T cells, and the expression of PD-L1 harbor crucial value for the prognosis or individualized therapy of POLE-mutated ECs.

Centromere Protein F (CENPF) Serves as a Potential Prognostic Biomarker and Target for Human Hepatocellular Carcinoma.

Overexpression of Centromere Protein F (CENPF) is associated with tumorigenesis of many human malignant tumors. But the molecular mechanism and prognostic value of CENPF in patients with hepatocellular carcinoma (HCC) are still unclear. In this essay, expression of CENPF in HCC tumors were evaluated in a series of databases, including GEO, TCGA, Oncomine, GEPIA, The Human Protein Atlas and Kaplan-Meier plotter. It was apparent that mRNA and protein expression levels of CENPF were significantly increased in patients with HCC and were manifestly associated with the tumor stage of HCC. Aberrant expressions of CENPF were significantly linked with worse overall survival (OS) and progression-free survival (PFS) in HCC patients. Then, immunohistochemistry of CENPF in human HCC samples was carried out to suggest that CENPF protein was over-expressed in HCC tissues, compared with paired adjacent non-cancerous samples. And small interfering RNAs of CENPF in the human HepG2 cells were further performed to reveal that down-regulation of CENPF significantly inhibited cell proliferation, cell migration, and cell invasion, but slightly promoted cell apoptosis in human HepG2 cells. Moreover, the gene-set enrichment analysis (GSEA) was conducted to probe the biology process and molecular signaling pathway of CENPF in HCC. The GSEA analysis pointed out that CENPF was principally enriched in cell cycle and closely related to E2F1 and CDK1 in the regulation of cell cycle, especially during G2/M transition of mitosis in HCC. Additionally, immune infiltration analysis by CIBERSORTx revealed that mutilpe immune cells, including Treg, etc., were significantly different in HCC samples with CENPFhigh, compared with CENPFlow. These results collectively demonstrated that CENPF might serve as a potential prognostic biomarker and novel therapeutic target for HCC. However, further research is needed to validate our findings and promote the clinical application of CENPF in HCC.

EMS1/DLL4-Notch Signaling Axis Augments Cell Cycle-Mediated Tumorigenesis and Progress in Human Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant neoplasm that is prone to local invasion and metastasis. Meanwhile, overexpressed endothelial cell-specific molecule 1 (ESM1) is closely related to tumorigenesis of multitudinous tumors. However, the prognosis value and biological function of ESM1 in ACC remains undefined. In the current essay, the assessment in human ACC samples and multiple public cancer databases suggested that ESM1 was significantly overexpressed in ACC patients. The abnormal expression of ESM1 was evidently correlated with dismal overall survival (OS) in ACC patients. Then, the gene-set enrichment analysis (GSEA) was applied to unravel that ESM1 was mostly involved in cell cycle and Notch4 signaling pathway. Furthermore, in vitro experiment, RNA interference of ESM1 was carried out to state that ESM1 augments CDK1 and p21-mediated G2/M-phase transition of mitosis, cell proliferation via DLL4-Notch signaling pathway in human ACC cell line, SW13 cells. Additionally, two possible or available therapeutic strategies, including immunotherapy and chemotherapy, have been further explored. Immune infiltration analysis highlighted that no significant difference was found in ACC patients between EMS1high and EMS1low group for immune checkpoint-related genes. In addition, the overexpression of ESM1 might trigger the accumulation of tumor mutation burden (TMB) during the cell cycle of DNA replication in ACC. The gene-drug interaction network then indicated that ESM1 inhibitors, such as cisplatin, might serve as potential drugs for the therapy of ACC. Collectively, the results asserted that ESM1 and related regulators might act as underlying prognostic biomarkers or novel therapeutic targets for ACC.

Identification of potential target genes and crucial pathways in small cell lung cancer based on bioinformatic strategy and human samples.

Small cell lung cancer (SCLC) is a carcinoma of the lungs with strong invasion, poor prognosis and resistant to multiple chemotherapeutic drugs. It has posed severe challenges for the effective treatment of lung cancer. Therefore, searching for genes related to the development and prognosis of SCLC and uncovering their underlying molecular mechanisms are urgent problems to be resolved. This study is aimed at exploring the potential pathogenic and prognostic crucial genes and key pathways of SCLC via bioinformatic analysis of public datasets. Firstly, 117 SCLC samples and 51 normal lung samples were collected and analyzed from three gene expression datasets. Then, 102 up-regulated and 106 down-regulated differentially expressed genes (DEGs) were observed. And then, functional annotation and pathway enrichment analyzes of DEGs was performed utilizing the FunRich. The protein-protein interaction (PPI) network of the DEGs was constructed through the STRING website, visualized by Cytoscape. Finally, the expression levels of eight hub genes were confirmed in Oncomine database and human samples from SCLC patients. It showed that CDC20, BUB1, TOP2A, RRM2, CCNA2, UBE2C, MAD2L1, and BUB1B were upregulated in SCLC tissues compared to paired adjacent non-cancerous tissues. These suggested that eight hub genes might be viewed as new biomarkers for prognosis of SCLC or to guide individualized medication for the therapy of SCLC.

Rare variants of solitary fibrous tumor.

OBJECTIVE: Some cases of solitary fibrous tumor (SFT) exhibit unusual histologic features that may cause diagnostic difficulty, such as fascicular monotonous spindle cells accompanied by hyalinized blood vessels and numerous evenly distributed mast cells, and features mimicking myxoid liposarcoma. Awareness of these features is important for reaching correct diagnosis of similar cases. METHODS: Three cases of SFT with the above unusual features were retrieved from our consult files for review, including H&E slides and immunohistochemical stains. In addition, FISH analysis for SS18-SSX (SYT), DDIT3 and MDM2 were performed. Furthermore, formalin-fixed paraffin-embedded (FFPE) tissue sections were tested for 8 fusion variants of NAB2-STAT6 by qualitative endpoint reverse-transcriptase (RT)-PCR. RESULTS: Neoplastic cells from all 3 cases are positive for CD34, CD99, and STAT6 immunohistochemically. In addition, the tumors are positive for NAB2-STAT6 fusion gene. Mast cells from the first case possess nonneoplastic phenotype and are positive for CD117 and tryptase staining but negative for CD25. CONCLUSIONS: The three cases studied here represent rare types of SFT, which differ from classical "pattern-less" pattern of SFT. Correct diagnosis required a combination of CD34 and STAT6 immunostaining and NAB2-STAT6 fusion gene analysis.

[Expressions of CXCL13, CD10 and bcl-6 in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified].

OBJECTIVE: To study the value of immunomarkers CXCL13, CD10, bcl-6 in pathologic diagnosis of angioimmunoblastic T-cell lymphoma (AITL). METHODS: One hundred and fifteen cases of AITL, 30 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) and 30 cases of reactive lymph nodes with paracortical hyperplasia (RH) encountered during the period from January, 1990 to January, 2008 were retrieved from the archival files of the Department of Pathology, West China Hospital of Sichuan University, China. The morphologic features were reviewed and compared. Immunohistochemical study was performed by SP method for CXCL13, CD10, bcl-6, CD21, CD3epsilon, CD3, CD45RO, CD20 and Ki-67. TCR-gamma gene rearrangement study was also carried out. RESULTS: Regressed follicles were evident in 7.8% (9/115) of AITL cases, 6.7% (2/30) of PTCL, NOS cases and 83.3% (25/30) of RH cases, respectively. A marked increase of number of arborizing venules was shown in 98.3% (113/115) of AITL cases, 63.3% (19/30) of PTCL, NOS cases and 76.7% (23/30) of RH cases, respectively. In lymph nodes with paracortical hyperplasia, the expression of CXCL13, CD10 and bcl-6 were restricted to the germinal centers. In AITL, 96.5% (111/115) of cases showed CXCL13 expression, in contrast to 26.7% (8/30) of PTCL, NOS. Expression of CD10 and bcl-6 were found in the neoplastic cells in 50.4% (58/115) and 78.3% (90/115) of AITL, and 3.3% (1/30) and 3.3% (1/30) of PTCL, NOS, respectively. Irregular meshworks of CD21-positive follicular dendritic cells were found in all the AITL cases. Clonal TCR-gamma rearrangement was detected in 83% (83/100) of the AITL cases. CONCLUSIONS: AITL is a type of lymphoma originated from the follicular helper T cells. Detailed morphologic assessment and use of immunohistochemical markers are essential for accurate diagnosis.

Aberrant expression of thyroid transcription factor-1 in schwannomas.

Aberrant expression of thyroid transcription factor-1 (TTF-1) has been observed in tumors arising in locations other than thyroid gland, lung and ventral forebrain. However, TTF-1 expression in schwannomas has not yet been studied. Meanwhile, a few inconsistent changes in protein expression have been identified between schwannomas and other peripheral nerve sheath tumors. We evaluated TTF-1 expression in 161 schwannomas and 43 other peripheral nervous system lesions, including ganglioneuromas (n = 8), malignant peripheral nerve sheath tumors (MPNSTs) (n = 11), neurofibromas (n = 24), and traumatic neuromas (n = 9), using immunohistochemistry and verified it using quantitative real-time reverse-transcription polymerase chain reaction (qPCR) to explore TTF-1 expression in peripheral nervous system lesions. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained for both analyses. In this study, we observed nuclear TTF-1 staining in 109 (67.7%) schwannomas, including 102 of 131 (77.9%) conventional, 1 of 20 (5.0%) cellular and 6 of 10 (60.0%) plexiform schwannomas. Nuclear staining was not observed in normal peripheral nerves and non-schwannoma lesions. qPCR verified the aberrant expression and revealed a correlation between TTF-1 protein and mRNA levels (r = 0.633, P = .003). In conclusion, the data from our study show that TTF-1 is selectively expressed in the majority of schwannomas, particularly the conventional variants. Based on this observation, the TTF-1 protein and mRNA are specifically expressed in schwannomas. This highly aberrant expression of varying amounts of TTF-1 may provide new clues to reveal the pathogenesis of schwannoma.

Effect of lncRNA PCGEM1 on malignant biological behavior of lung cancer A549 cells and its mechanism / 中国肿瘤生物治疗杂志

@#[Abstract] Objective ： ：To investigate the long-chain noncoding RNA (Lnc RNA) PCGEM1 regulating the lung cancer (LC) cell invasion and metastasis through the TGF-β/Smad signaling pathways. Methods： ：From March 2016 to May 2018, total 62 cases of LC patients receiving surgical treatment in our hospital were collected, including cancer tissues and normal tissues more than 2 cm away from the cancer tissues. qRT-PCR was used to detect the expression of lncRNA PCGEM1 and miR-148a in LC, corresponding para-cancer tissues and different LC cell strains. LncRNA PCGEM1 silenced cell line A549-siPCGEM1 and negative control A549-NC were constructed, and A549 was used as blank control. MTT and plate cloning assay were used to detect the effect of PCGEM1 on the proliferation of A549 cells. Transwell and scratch assay were used to detect the effect of PCGEM1 on the invasion and migration of A549 cells. The bioinformatics website StarBase was used to predict the complementary binding miRNAof PCGEM1. Furthermore, according to the website Targetscan, the genes that the corresponding miRNAs could target and bind were predicted. Results： ：qRT-PCR results showed that the expression of PCGEM1 in LC tissues and lung cancer cell lines was higher than that in normal tissues, and the expression level of miR-148a was lower than that in normal tissues (all P<0.05). The expression level of PCGEM1 in A549 cells was the highest, and the difference was statistically significant compared with other cell lines (P<0.05). After successful construction of PCGEM1 silenced cells, compared with the blank control group and A549-NC group, the cell OD492nm value of A549-siPCGEM1 group was significantly decreased, the number of cell clones and the number of matrigel matrix gels was significantly reduced, the cell migration rate was significantly reduced, the differences were statistically significant (P<0.05). According to the prediction results of StarBase website, PCGEM1 could be complementary to miR-148a, and the prediction analysis on microRNA.org website shows that miR-148a had a targeted binding site with TGF-β2. qRT-PCR and Western blotting results showed that the expression of miR-148a was significantly increased in the A549-siPCGEM1 group compared with the blank control group and A549-NC group, and the expression of TGF-β2 and p-Smad 2 was significantly decreased (P<0.05), while the expression of the above indicators in the blank control group and A549-NC group was not statistically significant (P>0.05). Conclusion： ：Lnc RNA PCGEM1 is highly expressed in lung cancer. High expression of PCGEM1 may enhance the TGF-β2/Smad2 signaling pathway by downregulation of miR-148a, thus promoting the development of LC and the malignant biological behavior.

[Study on significance of single nucleotide polymorphisms in the redox domain of aprimidinic/apurinic endonuclease/redox factor-1 gene in Chinese sporadic colorectal cancer].

OBJECTIVE: To investigate the relationship between the single nucleotide polymorphisms(SNPs) in the redox domain of aprimidinic/apurinic endonuclease/redox factor-1(APEX) gene and the development of sporadic colorectal cancer. METHODS: One hundred and fifty cases of sporadic colorectal cancers and 143 peripheral blood samples from healthy population were screened for genetic polymorphisms or mutations in the redox domain by denaturing gradient gel electrophoresis followed by DNA sequencing. RESULTS: There were two SNPs identified in the redox domain of APEX gene, namely, 453G to T and 1247A to G. The gene frequencies of 453T and 1247G were 1.3% and 5.7%, respectively, in patient group, while 1.05% and 4.55%, respectively, in healthy population. The genotype distribution at the two sites in healthy population was consistent with Hardy-Weinberg equilibrium. There was no difference in gene frequencies at the two sites between cancer patients and healthy population. CONCLUSION: The polymorphisms in the redox domain of APEX gene are irrelevant to the development of sporadic colorectal cancer, but their distribution may vary greatly among tribes.