Serine-arginine protein kinase 1 (SRPK1) promotes EGFR-TKI resistance by enhancing GSK3ß Ser9 autophosphorylation independent of its kinase activity in non-small-cell lung cancer.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a major challenge for clinicians and patients with non-small cell lung cancer (NSCLC). Serine-arginine protein kinase 1 (SRPK1) is a key oncoprotein in the EGFR/AKT pathway that participates in tumorigenesis. We found that high SRPK1 expression was significantly associated with poor progression-free survival (PFS) in patients with advanced NSCLC undergoing gefitinib treatment. Both in vitro and in vivo assays suggested that SRPK1 reduced the ability of gefitinib to induce apoptosis in sensitive NSCLC cells independently of its kinase activity. Moreover, SRPK1 facilitated binding between LEF1, ß-catenin and the EGFR promoter region to increase EGFR expression and promote the accumulation and phosphorylation of membrane EGFR. Furthermore, we verified that the SRPK1 spacer domain bound to GSK3ß and enhanced its autophosphorylation at Ser9 to activate the Wnt pathway, thereby promoting the expression of Wnt target genes such as Bcl-X. The correlation between SRPK1 and EGFR expression was confirmed in patients. In brief, our research suggested that the SRPK1/GSK3ß axis promotes gefitinib resistance by activating the Wnt pathway and may serve as a potential therapeutic target for overcoming gefitinib resistance in NSCLC.

GSK3ß/ITCH/c-FLIP Axis Counteracts TRAIL-induced Apoptosis in Human Lung Adenocarcinoma Cells.

AIMS: Further investigation on the mechanism of action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in NSCLC would shed light on the understanding of TRAIL resistance and provide new clues for the counter-strategy. BACKGROUND: Cellular FLICE-inhibitory protein (c-FLIP) is a critical inhibitor of TRAIL-induced apoptosis. Our previous study suggested that glycogen synthase kinase 3ß (GSK3ß) positively regulated c-FLIP expression in human lung adenocarcinoma cells. Meanwhile, other studies reported that c-FLIP was degraded by HECT-type E3 ligase ITCH (Itchy E3 Ubiquitin Protein Ligase) via the proteasome pathway. OBJECTIVE: We will explore whether ITCH is involved in the expression regulation of c-FLIP positively controlled by GSK3ß during the treatment of TRAIL. METHODS: Human lung adenocarcinoma cells were used to stably overexpress and knockdown GSK3ß. Quantitative real-time PCR (qRT-PCR) assay was used to test the expressional level of mRNA of genes. Western blot analysis was employed to detect the expression of proteins at the protein level. siRNA of ITCH was used to knock down its expression. TRAIL treatment was used to cause apoptosis. RESULTS: In the present study, we have confirmed the degradation of c-FLIP by ITCH protein and the downregulation of ITCH expression by GSK3ß in lung adenocarcinoma cells. Moreover, ITCH silencing reversed the downregulation of c-FLIP protein caused by GSK3ß-knockdown in the cells. Accordingly, TRAIL-induced apoptosis facilitated by GSK3ß knockdown was blocked by the combined interference of ITCH. CONCLUSION: These results suggested that GSK3ß/ITCH axis regulated the stability of c-FLIP and influenced TRAIL-induced apoptosis. Taken together, our study revealed a GSK3ß/ITCH/c-FLIP axis, which counteracts TRAIL-induced apoptosis in human lung adenocarcinoma cells.

ZSWIM1 Promotes the Proliferation and Metastasis of Lung Adenocarcinoma Cells through the STK38/MEKK2/ERK1/2 Axis.

Investigating the functions of the proteins with no or less functional annotations is an important goal of the HPP (Human Proteome Project) Grand Project. In this study, we investigated the function of such a protein, ZSWIM1 (C20orf162), its gene located on chromosome 20. Its expression is upregulated in lung adenocarcinoma compared with the adjacent normal tissues and negatively correlated with the overall survival. Overexpressing ZSWIM1 markedly promotes the proliferation, migration, invasion as well as epithelial-to-mesenchymal transition in lung adenocarcinoma cells, while knocking down ZSWIM1 functions oppositely. The interactome of ZSWIM1 was identified by immunoprecipitation-mass spectrometry, and we verified the interaction of ZSWIM1 with the potential partner, STK38. ZSWIM1 antagonized the function of STK38. Mechanically, ZSWIM1 promoted the activation of MEKK2/ERK1/2 pathway through interacting with STK38, leading to the release of MEKK2. Taken together, ZSWIM1 can be annotated as an oncogene in lung adenocarcinoma, and the STK38/MEKK2/ERK1/2 axis mediates its promoting role in lung adenocarcinoma.

Clinicopathological characterization of ten patients with primary malignant melanoma of the esophagus and literature review.

BACKGROUND: Primary malignant melanoma of the esophagus (PMME) is a rare malignant disease and has not been well characterized in terms of clinicopathology and survival. AIM: To investigate the clinical features and survival factors in Chinese patients with PMME. METHODS: The clinicopathological findings of ten cases with PMME treated at Henan Provincial People's Hospital were summarized. Moreover, the English- and Chinese-language literature that focused on Chinese patients with PMME from 1980 to September 2021 was reviewed and analyzed. Univariate and multivariate analyses were employed to investigate the clinicopathologic factors that might be associated with survival. RESULTS: A total of 290 Chinese patients with PMME, including ten from our hospital and 280 from the literature were enrolled in the present study. Only about half of the patients (55.8%) were accurately diagnosed before surgery. Additionally, 91.1% of the patients received esophagectomy, and 88 patients (36.5%) received adjuvant therapy after surgery. The frequency of lymph node metastasis (LNM) was 51.2% (107/209), and LNM had a positive rate of 45.3% even when the tumor was confined to the submucosal layer. The risk of LNM increased significantly with the pT stage [P < 0.001, odds ratio (OR): 2.47, 95% confidence interval (CI): 1.72-3.56] and larger tumor size (P = 0.006, OR: 1.21, 95%CI: 1.05-1.38). The median overall survival (OS) was 11.0 mo (range: 1-204 mo). The multivariate Cox analysis showed both the pT stage [P = 0.005, hazard ratio (HR): 1.70, 95%CI: 1.17-2.47] and LNM (P = 0.009, HR: 1.78, 95%CI: 1.15-2.74) were independent prognostic factors for OS. The median disease-free survival (DFS) was 5.3 mo (range: 0.8-114.1 mo). The multivariate analysis indicated that only the advanced pT stage (P = 0.02, HR: 1.93, 95%CI: 1.09-3.42) was a significant independent indicator of poor RFS in patients with PMME. CONCLUSION: The correct diagnosis of PMME before surgery is low, and physicians should pay more attention to avoid a misdiagnosis or missed diagnosis. Extended lymph node dissection should be emphasized in surgery for PMME even though the tumor is confined to the submucosal layer. Both the LNM and pT stage are independent prognosis factors for OS, and the pT stage is the prognosis factor for DFS in patients with PMME.

SRPK1/AKT axis promotes oxaliplatin-induced anti-apoptosis via NF-κB activation in colon cancer.

BACKGROUND: Colorectal cancer is the third most common diagnosis. Oxaliplatin is used as first-line treatment of colon cancer. However, oxaliplatin resistance greatly reduces its therapeutic effect. SRPK1 involves in pre-mRNA splicing and tumorigenesis. How SRPK1 mediates drug resistance in colon cancer is unknown. METHODS: The expression of SRPK1 was analyzed in the TCGA and the CPTAC pan-cancer samples and detected in colon cancer cell lines and tissues by IHC and western blot. The MTT and TUNEL assay were used to verify the anti-apoptosis ability of colon cancer cell. The activation of NF-κB was determined by luciferase assay and qRT-PCR. AKT, IKK, IκB and their phosphorylation level were verified by western blot. RESULTS: We found that SRPK1 expression was the second highest in TCGA and the CPTAC pan-cancer samples. The mRNA and protein levels of SRPK1 were increased in tissues from patients with colon cancer. SRPK1 was associated with clinical stage and TNM classifications in 148 cases of colon cancer patients. High SRPK1 levels correlated with poor prognosis (p < 0.001). SRPK1 overexpression enhanced the anti-apoptosis ability of colon cancer cells, whereas SRPK1 silencing had the opposite effect under oxaliplatin treatment. Mechanistically, SRPK1 enhances IKK kinase and IκB phosphorylation to promote NF-κB nuclear translocation to confer oxaliplatin resistance. CONCLUSIONS: Our findings suggest that SRPK1 participates in colon cancer progression and enhances the anti-apoptosis capacity to induce drug resistance in colon cancer cells via NF-κB pathway activation, and thus might be a potential pharmaceutically target for colon cancer treatment.

TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.

[Temporal and spatial heterogeneity analysis of optimal value of sensitive parameters in ecological process model: The BIOME-BGC model as an example.] / 生态过程模型敏感参数最优取值的时空异质性分析­­ä»¥BIOME-BGC模型为例.

The ecological process models are powerful tools for studying terrestrial ecosystem water and carbon cycle at present. However, there are many parameters for these models, and weather the reasonable values of these parameters were taken, have important impact on the models simulation results. In the past, the sensitivity and the optimization of model parameters were analyzed and discussed in many researches. But the temporal and spatial heterogeneity of the optimal parameters is less concerned. In this paper, the BIOME-BGC model was used as an example. In the evergreen broad-leaved forest, deciduous broad-leaved forest and C3 grassland, the sensitive parameters of the model were selected by constructing the sensitivity judgment index with two experimental sites selected under each vegetation type. The objective function was constructed by using the simulated annealing algorithm combined with the flux data to obtain the monthly optimal values of the sensitive parameters at each site. Then we constructed the temporal heterogeneity judgment index, the spatial heterogeneity judgment index and the temporal and spatial heterogeneity judgment index to quantitatively analyze the temporal and spatial heterogeneity of the optimal values of the model sensitive parameters. The results showed that the sensitivity of BIOME-BGC model parameters was different under different vegetation types, but the selected sensitive parameters were mostly consistent. The optimal values of the sensitive parameters of BIOME-BGC model mostly presented time-space heterogeneity to different degrees which varied with vegetation types. The sensitive parameters related to vegetation physiology and ecology had relatively little temporal and spatial heterogeneity while those related to environment and phenology had generally larger temporal and spatial heterogeneity. In addition, the temporal heterogeneity of the optimal values of the model sensitive parameters showed a significant linear correlation with the spatial heterogeneity under the three vegetation types. According to the temporal and spatial heterogeneity of the optimal values, the parameters of the BIOME-BGC model could be classified in order to adopt different parameter strategies in practical application. The conclusion could help to deeply understand the parameters and the optimal values of the ecological process models, and provide a way or reference for obtaining the reasonable values of parameters in models application.

Pingyangmycin Pretreatment Influences the Biological Behavior of Ocular Venous Malformation and Relates with Galectin-3 Expression.

BACKGROUND: Galectin-3 (Gal-3) plays a role in the mechanisms underlying ocular venous malformation. We conducted this study to investigate the effect of pingyangmycin pretreatment on the Gal-3 expressions and biological behavior of ocular venous malformation. METHODS: Tissue samples were collected from 136 patients with ocular venous malformation. Patients were randomly divided into pingyangmycin (n = 69) and nonpingyangmycin group (n = 67). Patients in the pingyangmycin group received a local injection of 0.02% pingyangmycin once every 2 days for 2 weeks (7 doses) before removal surgery, whereas patients in the nonpingyangmycin group underwent removal surgery without local injection. The protein and messenger RNA (mRNA) expression of Gal-3 were detected by using immunohistochemistry and in situ hybridization. RESULTS: Gal-3 protein was expressed in 35 (52%) of 67 samples in the nonpingyangmycin group and in 19 (28%) of 69 samples in the pingyangmycin group (P < 0.05). Gal-3 mRNA expression was detected in 39 (58%) of 67 samples in the nonpingyangmycin group and 22 (32%) of 69 samples in the pingyangmycin group (P < 0.05). The higher Gal-3 expressions were detected in samples with deeper invasiveness than those with superficial invasiveness before (χ2 = 12.720 and 13.369, respectively, both P < 0.05) and after pingyangmycin treatment (χ2 = 8.429 and 4.590, respectively, both P < 0.05). It was more frequently detected in mesh-like lesions with unclear boundary than round lesions with clear boundary before (χ2 = 30.291 and 41.466, respectively, both P < 0.05) and after pingyangmycin treatment (χ2 = 14.619 and 15.130, respectively, both P < 0.05). Pingyangmycin treatment led to a significant difference in Gal-3 expressions at both protein and mRNA levels (χ2 = 8.664 and 9.524, respectively, both P < 0.05). CONCLUSIONS: Gal-3 expression may be involved in the development and invasiveness of ocular venous malformation, and pingyangmycin can inhibit Gal-3 expression, indicating a role of pingyangmycin treatment before the removal of ocular venous malformation.

Primary extragastrointestinal stromal tumor arising in the vaginal wall: Significant clinicopathological characteristics of a rare aggressive soft tissue neoplasm.

Gastrointestinal (GI) stromal tumor is the most common mesenchymal neoplasm of the GI tract but also occurs with a lower frequency in extragastrointestinal regions and is called extragastrointestinal stromal tumor (EGIST). We report an unusual case of EGIST presenting as a vaginal mass. A 41-year-old woman presented with a gradually enlarging vaginal mass for the last 2 years. Physical examination revealed an elliptical, non-tender mass about 7.5 cm × 7 cm in size in the posterior vaginal wall and was resected completely. Under histological examination, the tumor showed a spindle cell type with coagulation necrosis, hemorrhage and high mitotic count. Immunohistochemical analysis revealed tumor cells were positive for DOG1, CD117, CD34 and p53 protein. Ki-67 labeling was 8%. Genetic analysis showed a deletion of exon 11 of the c-kit gene at codons 557-558. EGISTs should be kept in mind in the differential diagnosis in patients presenting with solid mass of the vaginal wall.

[Correlation of Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) to fascin and phosphorylated Stat3 in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: Latent membrane protein 1 (LMP1) of Epstein-Barr (EB) virus is an important oncogene. Fascin is an actin cross-linking protein involved in cell migration and adhesion. Phosphorylated signal transducer and activator of transcription 3 (pStat3) is a member of STATs family, which is closely related to tumorigenesis. This study was to investigate expressions of LMP1, Fascin and pStat3 in nasopharyngeal carcinoma (NPC) tissues and lymph node metastases of NPC, thus to explore their correlations to the initiation and progression of NPC. METHODS: Expressions of EBV-encoded small RNA (EBER), LMP1, Fascin, pStat3, p53, Ki-67 and Bcl-2 were detected in 43 NPC tissues (21 with and 22 without lymph node metastases) and 21 corresponding lymph node metastases using in situ hybridization or immunohistochemistry (IHC). Data were statistically analyzed. Expressions of pStat3 and Fascin were measured in the NPC cell line CNE1 transfected with LMP1-expressing plasmid using Western blot. RESULTS: Positive EBER expression was detected in all 43 NPC tissues and 21 lymph node metastases in NPC. The expression rates of LMP1, Fascin, pStat3, p53, Ki-67, and Bcl-2 were 69.8% (30/43), 93.0% (40/43), 72.1% (31/43), 90.7% (39/43), 88.4% (38/43) and 88.4% (38/43)in 43 NPC tissues, respectively. LMP1 expression was positively correlated with the expression level of Fascin, pStat3, p53, Ki-67 and Bcl-2 in 43 NPC cases(P < 0.05). LMP1 was found in 10 out of 21 (46.7%) lymph node metastases in NPC. In addition, LMP1 expression dramatically increased pStat3 and Fascin in CNE1 cells. CONCLUSIONS: LMP1 is expressed in lymph node metastatases in NPC. The expression of LMP1 is positively correlated with Fascin, pStat3 and the proliferation index of tumor cells. Moreover, LMP1 up-regulates pStat3 and Fascin in NPC cells.

[Analysis of Epstein-Barr virus BamH I "f" variant in nodal metastasis of nasopharyngeal carcinoma].

OBJECTIVE: To investigate the Epstein-Barr virus (EBV) BamH I "f" variant in primary nasopharyngeal carcinoma (NPC) and its metastases in lymph nodes (LN). METHODS: In situ hybridization was used to detect EBV-encoded small RNA (EBER) expression in 21 paired paraffin-embedded tissue from primary NPC and their lymph node metastases and 22 primary NPC without lymph node metastasis. PCR and restriction fragment length polymorphism (RFLP) assay were used to detect EBV BamH I "f" variant in all cases of NPCs, lymph node metastases and 50 cases of chronic inflammation of nasopharynx from Canton. RESULTS: All cases of NPCs and their lymph node metastases showed EBER expression, indicating a high EBV-positive rate in Cantonese NPC patients. EBV BamH I "f" variant was found in 11 cases (52.4%, 11/21) of primary NPCs with LN metastasis, 12 cases (57.1%, 12/21) of the LN metastases, and 18 cases (81.8%, 18/22) of primary NPCs without LN metastasis. However, of the 50 cases of chronic inflammation of nasopharynx, only one case (2.1%, 1/47) demonstrated BamH I "f" variant. The frequency of BamH I "f" variant in NPC was therefore dramatically higher than that in chronic inflammation of nasopharynx. It is of note that atypical hyperplasia was observed in a few epithelial cells from the case of chronic inflammation of nasopharynx expressing BamH I "f" variant. CONCLUSIONS: The frequency of EBV BamH I "f" variant in NPC is significantly higher than that in chronic inflammation of nasopharynx. It is the first demonstration that the BamH I "f" variant is also present in the LN metastases of NPC. The frequency of BamH I "f" variant in metastatic NPC of the lymph node is almost equal to that of primary NPCs.