Zixuan Li

Overexpression of yes-associated protein contributes to progression and poor prognosis of non-small-cell lung cancer

Yes‐associated protein (YAP), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene. This study aimed to assess the clinical significance and biological functions of YAP in non‐small‐cell lung cancer (NSCLC). We investigated the expression of YAP in 92 cases of NSCLC tissue by immunohistochemistry and found that YAP was expressed in 66.3% (61/92) cases and predominantly presented in the nucleus. The expression of YAP in NSCLC was significantly correlated with p‐TNM stage (P = 0.0037) and lymph node metastasis (P = 0.0093). Importantly, YAP expression was associated with short overall survival. Further study in NSCLC cell lines in which YAP was either overexpressed or depleted confirmed that YAP markedly promoted cell proliferation and invasion. These results indicate that YAP plays an important role in NSCLC and might be a useful therapeutic target of NSCLC.

MicroRNA-449a Is Downregulated in Non-Small Cell Lung Cancer and Inhibits Migration and Invasion by Targeting c-Met

MicroRNA-449a is expressed at a low level in several tumors and cancer cell lines, and induces G1 arrest, apoptosis, and senescence. To identify the function of miR-449a in non-small cell lung cancer (NSCLC), we discussed the potential relevance of miR-449a to clinicopathological characteristics and prognosis in NSCLC. We also investigated the impact of miR-449a on migration and invasion in NSCLC cells. The expression of miR-449a in NSCLC tissues and cell lines was detected using RT-qPCR. In vitro, gain-of-function, loss-of-function experiments, and fluorescence assays were performed to identify the potential target of miR-449a and the function of miR-449a in NSCLC cells. MiR-449a was downregulated in both NSCLC tissues and cell lines. Moreover, a low expression level of miR-449a appeared to be correlated with lymph node metastasis and poor survival. In vitro, miR-449 regulated cell migration and invasion in NSCLC cells as a potential tumor suppressor, at least in part by targeting c-Met. Furthermore, reciprocal expression of miR-449a and c-Met was shown in NSCLC tissue samples. This study indicates that miR-449a might be associated with NSCLC progression, and suggests a crucial role for miR-449a in NSCLC.

Overexpression of TRIM24 Correlates with Tumor Progression in Non-Small Cell Lung Cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of TRIM24 in patients with non-small cell lung cancer (NSCLC). The expression profile of TRIM24 in NSCLC tissues and adjacent noncancerous lung tissues was detected by immunohistochemistry. TRIM24 was found to be overexpressed in 81 of 113 (71.7%) human lung cancer samples and correlated with p-TNM stage (p  = 0.0006), poor differentiation (p = 0.004), Ki67 index (p<0.0001), cyclin D1(p = 0.0096) and p-Rb expression (p = 0.0318). In addition, depleting TRIM24 expression by small interfering RNA inhibited growth and invasion in lung cell lines. Moreover, TRIM24 depletion induced cell cycle arrest at the G1/S boundary and induced apoptosis. Western blotting analysis revealed that knockdown of TRIM24 decreased the protein levels of Cyclin A, Cyclin B, Cyclin D1, cyclin E and p-Rb and increased P27 expression. These results indicate that TRIM24 plays an important role in NSCLC progression.

Downregulation of Mig-6 in nonsmall-cell lung cancer is associated with EGFR signaling

Downregulation of Mig‐6 expression has been implicated in several human cancers and its loss can lead to prolonged activation of EGFR and carcinogenesis. The present study aimed to investigate the clinical significance of loss of Mig‐6 expression in nonsmall‐cell lung cancer (NSCLC) and the biological functions of Mig‐6 in NSCLC cell lines. Mig‐6 expression was downregulated in 47/91 (51.6%) cases of NSCLC that were examined. Mig‐6 downregulation significantly correlated with poor differentiation (P = 0.0131), histological type (P = 0.0021), and EGFR expression (P = 0.003). In addition, knockdown of Mig‐6 expression in H1299 and BE1 cells promoted EGF‐induced tumor cell proliferation and migration. Furthermore, Mig‐6 knockdown led to a significant increase in phospho‐AKT, phospho‐ERK, phospho‐EGFR as well as MMP‐2 and MMP‐9 levels. These results indicate that downregulated Mig‐6 in NSCLC tissues may serve as a new marker that can predict the activation of EGFR signaling pathway.

CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells.

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G2/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G0/G1 and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G2/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G0/G1 and G1/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.

Overexpression of CARMA3 in Non-Small-Cell Lung Cancer Is Linked for Tumor Progression

We aimed to investigate the clinical significance of the expression of novel scaffold protein CARMA3 in non-small-cell lung cancer (NSCLC) and the biological function of CARMA3 in NSCLC cell lines. We observed moderate to high CARMA3 staining in 68.8% of 141 NSCLC specimens compared to corresponding normal tissues. The overexpression of CARMA3 was significantly correlated with TNM stage (P = 0.022) and tumor status (P = 0.013). CARMA3 upregulation also correlated with a shorter survival rate of patients of nodal status N0 (P = 0.042)as well as the expression of epidermal growth factor receptor (EGFR) (P = 0.009). In EGFR mutation positive cases, CARMA3 expression was much higher (87.5%) compared to non-mutation cases (66.1%). In addition, we observed that knockdown of CARMA3 inhibits tumor cell proliferation and invasion, and induces cell cycle arrest at the boundary between the G1 and S phase. We further demonstrated a direct link between CARMA3 and NF-κB activation. The change of biological behavior in CARMA3 knockdown cells may be NF-κB-related. Our findings demonstrated, for the first time, that CARMA3 was overexpressed in NSCLC and correlated with lung cancer progression, EGFR expression, and EGFR mutation. CARMA3 could serve as a potential companion drug target, along with NF-kB and EGFR in EGFR-mutant lung cancers.

CCL21/CCR7 Prevents Apoptosis via the ERK Pathway in Human Non-Small Cell Lung Cancer Cells

Previously, we confirmed that C-C chemokine receptor 7 (CCR7) promotes cell proliferation via the extracellular signal-regulated kinase (ERK) pathway, but its role in apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. A549 and H460 cells of NSCLC were used to examine the effect of CCL21/CCR7 on apoptosis using flow cytometry. The results showed that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant decline in the percent of apoptosis. Western blot and real-time PCR assays indicated that activation of CCR7 significantly caused upregulation of anti-apoptotic bcl-2 and downregulation of pro-apoptotic bax and caspase-3, but not p53, at both protein and mRNA levels. CCR7 small interfering RNA significantly attenuated these effects of exogenous CCL21. Besides, PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished these effects of CCL21/CCR7. Coimmunoprecipitation further confirmed that there was an interaction between p-ERK and bcl-2, bax, or caspase-3, particularly in the presence of CCL21. These results strongly suggest that CCL21/CCR7 prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax and caspase-3 potentially via the ERK pathway in A549 and H460 cells of NSCLC.

Low expression of Mig-6 is associated with poor survival outcome in NSCLC and inhibits cell apoptosis via ERK-mediated upregulation of Bcl-2

Mitogen-inducible gene-6 (Mig-6), an immediate early response gene, is a specific negative regulator of epidermal growth factor receptor (EGFR). Ablation of Mig-6 has been shown to induce tumor formation in various tissues, supporting the tumor suppressor function of Mig-6. However, little is known about the role of Mig-6 in non-small cell lung cancer (NSCLC) apoptosis, nor has the contribution of upregulated Mig-6 on biological behaviors of A549 and H157 cells previously been reported. The aim of the present study was to investigate the effects of exogenously transfected Mig-6 on proliferation, invasion and apoptosis of A549 and H157 cells and to identify novel underlying mechanisms of Mig-6-induced apoptosis. We used immunohistochemical staining to examine the expression of Mig-6 protein in NSCLC tissues. For evaluation of the prognostic value of Mig-6 expression to each clinicopathologic factor, Kaplan-Meier method and Coxs proportional hazards model were employed. Mig-6 low expression was correlated with a poor prognosis in patients with lung cancer. Patients with high expression of Mig-6 had a statistically significantly longer survival than those with low expression of Mig-6. Coxs regression analysis indicated that loss of Mig-6 expression was an independent, unfavorable prognostic factors. We utilized siRNA-targeting Mig-6 and Mig-6 overexpression plasmid to determine the effect of Mig-6 on lung cancer cells. Flow cytometry studies revealed Mig-6 overexpression promoted apoptosis in NSCLC cell lines. siRNA-mediated Mig-6 knockdown inhibited apoptosis of cancer cells, but this anti-apoptotic effect was abolished by inhibition of ERK. Upregulation of Mig-6 decreased the proliferation and invasive potential of transfected cells. Moreover, upregulation of Mig-6 inhibited proliferation and invasion of A549 and H157 cells. Collectively, our results showed that Mig-6 is a potential biomarker for evaluation of tumor prognosis of lung cancer. Mig-6 promotes apoptosis in lung cancer cells via the ERK pathway.

CARMA3 regulates the invasion, migration, and apoptosis of non-small cell lung cancer cells by activating NF-кB and suppressing the P38 MAPK signaling pathway.

In our previous study, CARMA3 overexpression in lung cancer cells promoted cell proliferation and invasion; however, the mechanism underlying the role of CARMA3 in cancer cell invasion remained unclear. In the present study, knockdown of CARMA3 in A549 and H1299 cells suppressed cell invasion and migration, and downregulated matrix metalloprotease 9 expression at the protein and mRNA levels, as shown by Western blotting and real-time PCR. CARMA3 knockdown increased cell apoptosis, as shown by flow cytometry, increased the mRNA and protein expression levels of Bax and Caspase3, and downregulated Bcl-2 in A549 and H1299 cells. Phosphorylated P38 levels increased and NF-кB activation decreased following knockdown of CARMA3. SB203580, a P38 MAPK inhibitor, activated NF-кB, increased cell migration, and inhibited cell apoptosis after knockdown of CARMA3 compared to knockdown of CARMA3 without SB203580. These findings indicate that CARMA3 may suppress the activation of the P38 MAPK signaling pathway to regulate invasion, migration and apoptosis of lung cancer cells by activating NF-кB (P65) in the nucleus.

Malignant phosphaturic mesenchymal tumor with pulmonary metastasis: A case report

Rationale: Phosphaturic mesenchymal tumor (PMT) is a new tumor entity of soft tissue and bone tumor recently accepted by the World Health Organization, which typically causes the paraneoplastic syndrome of tumor-induced osteomalacia (TIO). The majority of PMTs follow a benign clinical course and local recurrence occurs in < 10% of cases, malignant PMTs with distant organ metastasis are extremely uncommon. Patient concerns: We reported a 41-year-old woman who was diagnosed with PMT 10 years ago with a repeated recurrence and pulmonary metastasis. Diagnoses: Based on clinical manifestations, MRI scan, serum biochemical indicators evaluation, followed by histopathological examination, the patient was diagnosed as malignant PMT with pulmonary metastasis. Interventions: The patient was treated with calcium, phosphorus, and vitamin D after surgical resection and measured the serum ion concentrations every 3 months. Outcomes: The patient had a favorable outcome for 10 months without recurrence. Lessons: PMTs lack of characteristic histological morphology, some recurrence cases may appear benign morphologically; the malignant PMTs are easily overlooked. Patients with PMT should be carefully evaluated and monitored, in order to early identify its malignant potential.