Syk Kinase ; Tyrosine/metabolism* ; Phosphorylation

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.
Humans ; Animals ; Mice ; c-Mer Tyrosine Kinase/metabolism* ; Receptor Protein-Tyrosine Kinases/genetics* ; Axl Receptor Tyrosine Kinase ; Proto-Oncogene Proteins/metabolism* ; B7-H1 Antigen/genetics* ; Triple Negative Breast Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Biomarkers ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9

The characteristic genetic abnormality of neuroendocrine neoplasms (NENs), a heterogeneous group of tumors found in various organs, remains to be identified. Here, based on the analysis of the splicing variants of an oncogene Focal Adhesion Kinase (FAK) in The Cancer Genome Atlas datasets that contain 9193 patients of 33 cancer subtypes, we found that Box 6/Box 7-containing FAK variants (FAK^6/7) were observed in 7 (87.5%) of 8 pancreatic neuroendocrine carcinomas and 20 (11.76%) of 170 pancreatic ductal adenocarcinomas (PDACs). We tested FAK variants in 157 tumor samples collected from Chinese patients with pancreatic tumors, and found that FAK^6/7 was positive in 34 (75.6%) of 45 pancreatic NENs, 19 (47.5%) of 40 pancreatic solid pseudopapillary neoplasms, and 2 (2.9%) of 69 PDACs. We further tested FAK splicing variants in breast neuroendocrine carcinoma (BrNECs), and found that FAK^6/7 was positive in 14 (93.3%) of 15 BrNECs but 0 in 23 non-NEC breast cancers. We explored the underlying mechanisms and found that a splicing factor serine/arginine repetitive matrix protein 4 (SRRM4) was overexpressed in FAK^6/7-positive pancreatic tumors and breast tumors, which promoted the formation of FAK^6/7 in cells. These results suggested that FAK^6/7 could be a biomarker of NENs and represent a potential therapeutic target for these orphan diseases.
Female ; Humans ; Alternative Splicing ; Breast Neoplasms/metabolism* ; Carcinoma, Pancreatic Ductal/pathology* ; Focal Adhesion Protein-Tyrosine Kinases/therapeutic use* ; Nerve Tissue Proteins/genetics* ; Neuroendocrine Tumors/genetics* ; Oncogenes ; Pancreatic Neoplasms/metabolism*

Objective: To investigate the clinicopathological features, treatment and prognosis of patients with RET fusion positive non-small cell lung cancer (NSCLC). Methods: A total of 1 089 NSCLCs were retrieved at Affiliated Hospital of Jiangnan University from August 2018 to April 2020. In all cases, multiple gene fusion detection kits (fluorescent PCR method) were used to detect the gene status of RET, EGFR, ALK, ROS1, KRAS, BRAF and HER2; and immunohistochemical method was used to detect the expression of PD-L1 and mismatch repair related proteins. The correlation between RET-fusion and patients' age, gender, smoking history, tumor stage, grade, pathologic type, and PD-L1, mismatch repair related protein expression was analyzed. Results: There were 22 cases (2.02%) detected with RET fusion-positive in 1 089 NSCLC patients, in which 11 males and 11 females; and the median age was 63.5 years. There were 20 adenocarcinomas, including 11 acinar predominant adenocarcinoma (APA), five solid predominant adenocarcinoma (SPA) and four lepidic predominant adenocarcinoma (LPA); There were one case each of squamous cell carcinoma (non-keratinizing type) and sarcomatoid carcinoma (pleomorphic carcinoma). There were 6 and 16 patients with RET fusion-positive who were in stage Ⅰ-Ⅱ and Ⅲ-Ⅳ respectively, and 16 cases with lymph node metastasis, 11 cases with distant metastasis. Among RET fusion-positive cases, one was detected with HER2 co-mutation. The tumor proportion score of PD-L1≥1% in patients with RET fusion positive lung cancer was 54.5% (12/22). Defects in mismatch repair protein expression were not found in patients with RET fusion positive NSCLC. Four patients with RET fusions positive (two cases of APA and two cases of SPA) received pratinib-targeted therapy, and two showed benefits from this targeted therapy. Conclusions: The histological subtypes of RET fusions positive NSCLC are more likely to be APA or SPA. RET fusion-positive NSCLC patients are associated with advanced clinical stage, lymph node metastases, and they may benefit from targeted therapy with RET-specific inhibitors.
Male ; Female ; Humans ; Middle Aged ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; B7-H1 Antigen/genetics* ; Protein-Tyrosine Kinases/genetics* ; Proto-Oncogene Proteins c-ret/metabolism* ; Proto-Oncogene Proteins/genetics* ; Adenocarcinoma/pathology* ; Carcinoma, Squamous Cell/genetics* ; Mutation

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by Transwell^TM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.
Animals ; Mice ; Focal Adhesion Protein-Tyrosine Kinases/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Extracellular Traps/metabolism* ; Cell Movement ; Cell Proliferation ; RNA, Small Interfering/genetics* ; Colorectal Neoplasms/genetics* ; Cell Line, Tumor

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.
Humans ; Animals ; Mice ; c-Mer Tyrosine Kinase/metabolism* ; Receptor Protein-Tyrosine Kinases/genetics* ; Axl Receptor Tyrosine Kinase ; Proto-Oncogene Proteins/metabolism* ; B7-H1 Antigen/genetics* ; Triple Negative Breast Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Biomarkers ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9

Animals ; Cell Proliferation ; Hepatic Stellate Cells ; Liver Cirrhosis/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; Rats

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.
Benzyl Alcohols ; Escherichia coli/metabolism* ; Gastrodia/chemistry* ; Metabolic Engineering ; Tyrosine/metabolism*

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.
Branchio-Oto-Renal Syndrome/pathology* ; Chromosome Deletion ; Comparative Genomic Hybridization ; Genetic Research ; Homeodomain Proteins/genetics* ; Humans ; Intracellular Signaling Peptides and Proteins ; Nuclear Proteins/metabolism* ; Pedigree ; Protein Tyrosine Phosphatases/metabolism*

OBJECTIVE: To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models. METHODS: Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot. RESULTS: After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05). CONCLUSION In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.
Animals ; Connexin 43/pharmacology* ; Disease Models, Animal ; Dopaminergic Neurons/metabolism* ; Mice ; Mice, Inbred C57BL ; Oxidopamine/metabolism* ; Parkinson Disease/metabolism* ; Peptides/pharmacology* ; Tyrosine 3-Monooxygenase/pharmacology*