TMEM120B strengthens breast cancer cell stemness and accelerates chemotherapy resistance via ß1-integrin/FAK-TAZ-mTOR signaling axis by binding to MYH9.

BACKGROUND: Breast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however, the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues, especially in malignant tissues; however, its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance. METHODS: Both bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung, breast, gastric, colon, and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next, colony formation assay, MTT assay, EdU assay, transwell assay, wound healing assay, flow cytometric analysis, sphere formation assay, western blotting analysis, mouse xenograft model analysis, RNA-sequencing assay, immunofluorescence assay, and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation, invasion, stemness, chemotherapy sensitivity, and integrin/FAK/TAZ/mTOR activation. Further, liquid chromatography-tandem mass spectrometry analysis, GST pull-down assay, and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B, myosin heavy chain 9 (MYH9), and CUL9. RESULTS: TMEM120B expression was elevated in lung, breast, gastric, colon, and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage, lymph node metastasis, and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation, invasion, and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9, which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore, TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely, overexpression of TMEM120B-∆CCD delayed the formation of FAs, suppressed TAZ-mTOR signaling, and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1-2) than in those with better outcomes (Miller/Payne grades 3-5). CONCLUSIONS: Our study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the ß1-integrin/FAK-TAZ-mTOR signaling axis, maintaining stemness and accelerating chemotherapy resistance.

Silencing lncRNA-DARS-AS1 suppresses nonsmall cell lung cancer progression by stimulating miR-302a-3p to inhibit ACAT1 expression.

Long noncoding RNAs (LncRNAs) have been gaining attention as potential therapeutic targets for lung cancer. In this study, we investigated the expression and biological behavior of lncRNA DARS-AS1, its predicted interacting partner miR-302a-3p, and ACAT1 in nonsmall cell lung cancer (NSCLC). The transcript level of DARS-AS1, miR-302a-3p, and ACAT1 was analyzed using qRT-PCR. Endogenous expression of ACAT1 and the expression of-and changes in-AKT/ERK pathway-related proteins were determined using western blotting. MTS, Transwell, and apoptosis experiments were used to investigate the behavior of cells. The subcellular localization of DARS-AS1 was verified using FISH, and its binding site was verified using dual-luciferase reporter experiments. The binding of DARS-AS1 to miR-302a-3p was verified using RNA co-immunoprecipitation. In vivo experiments were performed using a xenograft model to determine the effect of DARS-AS1 knockout on ACAT1 and NSCLC. lncRNA DARS-AS1 was upregulated in NSCLC cell lines and tissues and the expression of lncRNA DARS-AS1 was negatively correlated with survival of patients with NSCLC. Knockdown of DARS-AS1 inhibited the malignant behaviors of NSCLC via upregulating miR-302a-3p. miR-302a-3p induced suppression of malignancy through regulating oncogene ACAT1. This study demonstrates that the DARS-AS1-miR-302a-3p-ACAT1 pathway plays a key role in NSCLC.

BAIAP2L1 accelerates breast cancer progression and chemoresistance by activating AKT signaling through binding with ribosomal protein L3.

BAI1-associated protein 2-like 1 (BAIAP2L1), also known as insulin receptor tyrosine kinase substrate, modulates the insulin network; however, its function in breast cancer has not been explored. Immunohistochemical analysis of 140 breast cancer specimens (77 triple-negative and 63 nontriple-negative cases) indicated that BAIAP2L1 expression was higher in breast cancer tissues (56/140, 40%) than in normal breast tissues (28.3%, 15/53; p < 0.001). BAIAP2L1 expression in breast cancer was correlated with triple-negative breast cancer (p = 0.0013), advanced TNM stage (p = 0.001), lymph node metastasis (p = 0.001), and poor patient prognosis (p = 0.001). BAIAP2L1 overexpression could accelerate breast cancer proliferation, invasion, and stemness in vivo and in vitro, possibly through the activation of AKT, Snail, and cyclin D1. Treatment with the AKT inhibitor LY294002 reduced the effects of BAIAP2L1 overexpression on breast cancer cells. BAIAP2L1 may bind to the AA202-288 of ribosomal protein L3 (RPL3) within its SRC homology 3 (SH3) domain, the loss of which may abolish the transduction of the AKT signaling pathway by promoting the degradation of PIK3CA. Moreover, BAIAP2L1 overexpression may induce chemotherapy resistance, with BAIAP2L1 expression being higher in patients with advanced Miller grades than those with lower grades. Our results indicated that BAIAP2L1 promotes breast cancer progression through the AKT signaling pathway by interacting with RPL3 through its SH3 domain.

ZNF500 abolishes breast cancer proliferation and sensitizes chemotherapy by stabilizing P53 via competing with MDM2.

Zinc finger protein 500 (ZNF500) has an unknown expression pattern and biological function in human tissues. Our study revealed that the ZNF500 mRNA and protein levels were higher in breast cancer tissues than those in their normal counterparts. However, ZNF500 expression was negatively correlated with advanced TNM stage (p = 0.018), positive lymph node metastasis (p = 0.014), and a poor prognosis (p < 0.001). ZNF500 overexpression abolished in vivo and in vitro breast cancer cell proliferation by activating the p53-p21-E2F4 signaling axis and directly interacting with p53 via its C2H2 domain. This may prevent ubiquitination of p53 in a manner that is competitive to MDM2, thus stabilizing p53. When ZNF500-∆C2H2 was overexpressed, the suppressed proliferation of breast cancer cells was neutralized in vitro and in vivo. In human breast cancer tissues, ZNF500 expression was positively correlated with p53 (p = 0.022) and E2F4 (p = 0.004) expression. ZNF500 expression was significantly lower in patients with Miller/Payne Grade 1-2 than in those with Miller/Payne Grade 3-5 (p = 0.012). ZNF500 suppresses breast cancer cell proliferation and sensitizes cells to chemotherapy.

Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.

ARHGEF40 promotes non-small cell lung cancer proliferation and invasion via the AKT-Wnt axis by binding to RhoA.

Rho guanine nucleotide exchange factor 40 (ARHGEF40) is a member of the Dbl-family of guanine nucleotide factor proteins. However, its expression pattern and biological function in malignant tumors, notably in nonsmall cell lung cancer (NSCLC) are currently unknown. The present study demonstrated that ARHGEF40 was highly expressed in NSCLC specimens and that its expression was significantly associated with advanced TNM stage (p < 0.001), lymph node metastasis (p = 0.002), and poor prognosis (p = 0.0056). In addition, ARHGEF40 accelerated nuclear translocation of the key component ß-catenin and increased the expression levels of the Wnt signaling pathway targets c-myc, cyclin D1 and MMP7. Moreover, it promoted lung cancer cell proliferation and invasion in vitro and in vivo. To elucidate the underlying molecular mechanism, the current study demonstrated that ARHGEF40 could induce activation of the Wnt signaling pathway by increasing the phosphorylation levels of AKT and GSK3ß via interaction with RhoA. Moreover, the Dbl homology (DH)-pleckstrin homology (PH) domain of ARHGEF40 was responsible for this interaction. Its deletion abolished the binding, which blocked the activation of the Wnt signaling. Taken together, the data indicated that ARHGEF40 promoted the malignant phenotype of lung cancer cells by activating the AKT-Wnt axis. This was achieved by its interaction with RhoA via the DH-PH domain. ARHGEF40 may serve as a novel target for NSCLC treatment.

TMEM206 promotes the malignancy of colorectal cancer cells by interacting with AKT and extracellular signal-regulated kinase signaling pathways.

BACKGROUND: The roles of TMEM206, a new transmembrane protein, in cancer, including colorectal cancer (CRC), are unknown. Related family members, including TMEM16A, TMEM132A, and TMEM176B, have been shown to be involved in various biological behaviors. In addition, TMEM88 has been reported to promote non-small-cell lung cancer. In this study, we examined the roles of TMEM206 in CRC. METHOD: Real-time reverse transcription polymerase chain reaction was used to measure TMEM206 messenger RNA (mRNA) levels in clinical specimens and transfected cell lines. Immunohistochemistry was used to determine the relationship between TMEM206 expression levels and clinical data. Plasmids and small interfering RNA were used to upregulate and silence TMEM206, respectively. Protein expression levels and signaling pathway modulation were validated through western blot analysis. Colony formation, MTT, cell migration and invasion assays, and flow cytometry analyses were used to test the potential roles of TMEM206 in CRC. Co-immunoprecipitation was used to evaluate the interaction between TMEM206 and AKT. RESULTS: Investigation of the clinical significance of TMEM206 expression in CRC tissues revealed that TMEM206 mRNA and protein levels were higher in CRC tissues than in paired normal adjacent tissues (p < 0.05). TMEM206 overexpression was positively associated with T stage of cancer and UICC stage ( p < 0.05) and negatively related to differentiation of CRC ( p = 0.015). Upregulation or silencing of TMEM206 promoted or inhibited the proliferation of CRC cells and positively or negatively regulated the levels of phospho-AKT and downstream signaling pathway components (phospho-glycogen synthase kinase 3ß and cyclin D1), respectively. Moreover, silencing of TMEM206 in cell lines arrested CRC cells in the G1 stage of the cell-cycle. In addition, upregulating or silencing TMEM206 increased or decreased cell invasion and migration in vitro and positively or negatively altered levels of the phospho-extracellular signal-regulated kinase (ERK) and phospho-focal adhesion kinase 397, respectively. Co-immunoprecipitation demonstrated that AKT and TMEM206 proteins interacted. Furthermore, TMEM206 promoted the development and progression of CRC by enhancing the interactions between the AKT and ERK signaling pathways. CONCLUSION: TMEM206 controlled the progression of CRC by accelerating CRC cell proliferation and promoting CRC cell migration and invasion. The target of TMEM206 may be AKT, which is known to be involved in modulating the biological behaviors of various cancers.

ZC3H13 suppresses colorectal cancer proliferation and invasion via inactivating Ras-ERK signaling.

ZC3H13 is a canonical CCCH zinc finger protein, which harbors a somatic frame-shift mutation in colorectal cancer (CRC). However, its expression and biological function were still uncertain. In the current study, we found that ZC3H13 was served as a tumor suppressor in CRC cells, which decreased the expression of Snail, Cyclin D1, and Cyclin E1, and increased the expression of Occludin and Zo-1 through inactivating Ras-ERK signaling pathway. Furthermore, reduction of ZC3H13 associated with advanced TNM stage (p = 0.02), positive regional lymph node metastasis ( p = 0.01). Taken together, the current study indicated that ZC3H13 may be an upstream regulator of Ras-ERK signaling pathway and suppressed invasion and proliferation of CRC.

ZNF326 promotes proliferation of non-small cell lung cancer cells by regulating ERCC1 expression.

The roles and downstream target genes of the transcription factor ZNF326 in malignant tumors are unclear. Out of 146 lung cancer tissue samples, we found that high expression of ZNF326 in 82 samples was closely related to low differentiation and a high pTNM stage of non-small cell lung cancer (NSCLC) cells. In vitro and in vivo analyses showed that ZNF326 significantly promoted cell cycle progression, colony formation, and proliferation as well as the growth of NSCLC transplanted tumors. Chromatin immunoprecipitation sequencing, dual-luciferase assay, and electrophoretic mobility shift assay confirmed that the C2H2 structure of ZNF326 binds to the -833 to -875 bp region of the ERCC1 promoter to initiate transcriptional activity. This binding promoted CyclinB1 synthesis and cell cycle progression. These results show that the ZNF326 transcription factor is highly expressed in lung cancer and promotes the proliferation of NSCLC cells by regulating the expression of ERCC1.

CCDC85B promotes non-small cell lung cancer cell proliferation and invasion.

Coiled-coil domain containing 85 B (CCDC85B) is involved in diverse biological processes; however, its expression patterns and functions in human cancers are yet unknown. The present study demonstrated that the expression of CCDC85B in the cytoplasm of the non-small cell lung cancer (NSCLC) tumor cells was significantly higher compared to adjacent normal lung tissues (P < 0.05). Furthermore, CCDC85B expression correlated with advanced TNM stage (P = 0.004) and positive regional lymph node metastasis (P = 0.009) of NSCLC. In addition, in A549 and H1299 lung cancer cell lines, the overexpression of CCDC85B promoted cell proliferation and invasion, while siRNA-mediated CCDC85B knockdown exhibited opposite effects. CCDC85B promoted AKT and GSK3ß phosphorylation and upregulated the levels of active ß-catenin, Wnt targets c-myc, cyclin D1, and MMP7. Besides, the CCDC85B-induced upregulation of phosphorylated GSK3ß and active ß-catenin was rescued following the treatment with PI3 K inhibitor, LY294002. In conclusion, CCDC85B was associated with NSCLC progression as it promoted the proliferation and invasion of lung cancer cells through activated AKT/GSK3ß/ß-catenin oncogenic signaling pathway. Therefore, CCDC85B might serve as a novel target for NSCLC treatment.

TIMM50 promotes tumor progression via ERK signaling and predicts poor prognosis of non-small cell lung cancer patients.

TIMM50 (Translocase of the inner mitochondrial membrane 50), also called TIM50, plays an essential role in mitochondrial membrane transportation. The existing literature suggests that TIMM50 may perform as an oncogenetic protein in breast cancer. However, the molecular mechanism, especially in human non-small cell lung cancer (NSCLC), is uncertain to date. In the present study, using immunohistochemistry, we found that TIMM50 expression significantly correlated with larger tumor size (P = 0.049), advanced TNM stage (P = 0.001), positive regional lymph node metastasis (P = 0.007), and poor overall survival (P = 0.001). Proliferation and invasion assay showed that TIMM50 dramatically promoted the ability of proliferation and invasion of NSCLC cells. Subsequent Western blotting results revealed that TIMM50 enhanced the expression of Cyclin D1 and Snail, and inhibited the expression of E-cadherin. Moreover, TIMM50 facilitated the expression of phosphorylated ERK and P90RSK. Incorporation of ERK inhibitor counteracted the upregulating expression of CyclinD1, and Snail, and downregulating expression of E-cadherin expression induced by TIMM50 overexpression. In conclusion, our data indicated that TIMM50 facilitated tumor proliferation and invasion of NSCLC through enhancing phosphorylation of its downstream ERK/P90RSK signaling pathway. We speculated that TIMM50 might be a useful prognosis marker of NSCLC patients.

Correction to: Axin gene methylation status correlates with radiosensitivity of lung cancer cells.

Following publication of the original article.

ARHGEF39 promotes tumor progression via activation of Rac1/P38 MAPK/ATF2 signaling and predicts poor prognosis in non-small cell lung cancer patients.

Rho guanine nucleotide exchange factor 39 (ARHGEF39), also called C9orf100, is a new member of the Dbl-family of guanine nucleotide exchange factors. Although ARHGEF39 has been proven to regulate tumor progression in hepatocellular carcinoma, the downstream signaling pathway of ARHGEF39 and its clinical associations in non-small cell lung cancer (NSCLC) are currently unknown. In the present study, using MTT, colony formation, flow cytometry, mice xenografts, wound healing, and transwell assays, we showed that ARHGEF39 promoted tumor proliferation, migration, and invasion. Furthermore, ARHGEF39 promoted the expression of Cyclin A2, Cyclin D1, and MMP2 by activating Rac1, leading to increased phosphorylation of P38 and ATF2. Treatment with a P38 inhibitor counteracted the effect of ARHGEF39 overexpression on the increase in Cyclin A2, Cyclin D1, and MMP2 expression. Moreover, the elevated levels of p-P38 and p-ATF2 caused by ARHGEF39 overexpression could be inhibited by expression of a dominant negative Rac1 mutant (T17N). In addition, the inhibition of the expression of p-P38 and p-ATF2 by ARHGEF39 RNAi could be restored by the expression of a constitutively active Rac1 mutant (Q61L). A similar impact on cell growth and invasion was observed after ARHGEF39 overexpression combined with the P38 inhibitor, Rac1 T17N, or Rac1 Q61L. Using immunohistochemistry, ARHGEF39 expression was observed to correlate positively with larger tumor size in clinical samples from 109 cases of NSCLC (P = 0.008). The Kaplan-Meier test revealed that ARHGEF39 expression significantly affected the overall survival of patients with NSCLC (52.55 ± 6.40 months vs. 64.30 ± 5.40 months, P = 0.017). In conclusion, we identified that ARHGEF39 promotes tumor growth and invasion by activating the Rac1-P38-ATF2 signaling pathway, as well as increasing the expression of Cyclin A2, Cyclin D1, and MMP2 in NSCLC cells. ARHGEF39 may be a useful marker to predict poor prognosis of patients with NSCLC.

Odd-skipped related 1 inhibits lung cancer proliferation and invasion by reducing Wnt signaling through the suppression of SOX9 and ß-catenin.

The odd-skipped related 1 (OSR1) gene encodes a zinc-finger transcription factor. The expression and significance of OSR1 in human tumors remains unclear. We found that OSR1 was downregulated in lung cancers, and its expression was correlated with poor differentiation. Overexpression of OSR1 by OSR1 gene transfection into H1299 cells (H1299-OSR1) inhibited the proliferation and invasion of lung cancer cells. Knockdown of OSR1 with small interfering (si)RNA against OSR1 in A549 cells (A549-siOSR1) enhanced the proliferation and invasion of lung cancer cells. Western blot analysis showed that the expression level of GSK3ß increased, while that of p-GSK3ß, nuclear ß-catenin, cyclin D1, c-Myc and matrix metallopeptidase 7 significantly decreased in the H1299-OSR1 cells, and this pattern was reversed in the A549-siOSR1 cells compared to that in the control cells. Furthermore, upregulation of sex-determining region Y-box 9 (SOX9) by SOX9 gene transfection increased the expression of ß-catenin, which was inhibited by OSR1. The mRNA and protein expression levels of SOX9 and ß-catenin were reduced in H1299-OSR1 cells and increased in A549-siOSR1 cells. In conclusion, the expression of OSR1 was more reduced in lung cancer tissues than in normal lung tissues, and was correlated with poor differentiation. OSR1 downregulated the activity of the Wnt signaling pathway by suppressing the expression of SOX9 and ß-catenin.

ZNF326 promotes a malignant phenotype of breast cancer by interacting with DBC1.

The biological role and underlying mechanism of action of zinc-finger protein 326 (ZNF326) in malignant tumors, including breast cancer, are still not clear. In this study, we detected high expression of ZNF326 in breast cancer specimens (60/111, 54.1%) and breast cancer cell lines (7/7); the expression level of ZNF326 was inversely associated with advanced pTNM stage (P = 0.002), positive lymph node metastasis (P = 0.004), poor prognosis in patients with breast cancer (P = 0.0097), and ER/PR/Her2 status (P = 0.013). Meanwhile, the ectopic expression of ZNF326 significantly upregulated MMP7, EMT-related proteins (Snail and Slug), and cell cycle-related proteins (cyclinA2 and cyclinB1); downregulated E-cadherin expression; and promoted the proliferation and invasiveness of breast cancer cells both in vivo and in vitro. Mechanistically, co-immunoprecipitation and immunofluorescence assays both demonstrated that ZNF326 interacted with deleted in breast cancer-1 (DBC1) in breast cancer cells. Additionally, DBC1 knockdown eliminated the up-regulation of MMP7, EMT-related proteins, and cell cycle-related proteins as well as the enhanced proliferation and invasiveness induced by ZNF326. Therefore, we concluded that ZNF326 is highly expressed in breast cancer, is associated with poor prognosis, and plays a vital role in promoting the malignant phenotype of breast cancer cells by interacting with DBC1.

WWC3 regulates the Wnt and Hippo pathways via Dishevelled proteins and large tumour suppressor 1, to suppress lung cancer invasion and metastasis.

The scaffolding protein WWC (WW and C2-domain containing) family is known to regulate cell proliferation and organ size via the Hippo signalling pathway. However, the expression level of WWC3 in human tumours and the mechanisms underlying its role in cellular signal transduction have not yet been reported. Herein, we explored the potential roles of WWC3 in lung cancer cells and the corresponding molecular mechanisms. We found low WWC3 expression in both lung cancer cell lines and lung cancer specimens, which was associated with low differentiation, advanced pTNM stage, positive lymph node metastasis, and poor prognosis in patients with lung cancer. Moreover, the overexpression of WWC3 inhibited the proliferation and invasiveness of lung cancer cells. These effects were mediated by the inhibition and stimulation of the Wnt and Hippo pathways, respectively, in vitro and in vivo. Specifically, WWC3 interacts with Dishevelled (Dvl) proteins, prevents casein kinase 1ϵ from phosphorylating Dvls, and inhibits ß-catenin nuclear translocation to inhibit the Wnt pathway. Deleting the WW and C-terminal PDZ-binding domains of WWC3 abrogated these effects. Moreover, the interaction of WWC3 with Dvls reduced the interaction between WWC3 and large tumour suppressor 1 (LATS1), as well as decreasing LATS1 phosphorylation to increase the nuclear importation of yes-associated protein (YAP) and attenuate the Hippo pathway. Deleting the WW domain of WWC3 abrogated this effect. These findings demonstrate the molecular interplay between WWC3, Dvls, and LATS1, and reveal a link between the Wnt and Hippo pathways, which provides a potential target for clinical intervention in lung cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Kctd20 promotes the development of non-small cell lung cancer through activating Fak/AKT pathway and predicts poor overall survival of patients.

Kctd20 (potassium channel tetramerization protein domain containing 20) is a positive regulator of Akt signaling. However, the role of Kctd20 during the course of tumorigenesis and development is unclear. Using immunohistochemistry, we demonstrated that, in non-small cell lung cancer (NSCLC) patients, Kctd20 protein expression significantly correlates with advanced TNM stage (P < 0.001), positive status for regional lymph node metastasis (P = 0.019), and poor overall survival (P = 0.013). Proliferation and invasion assays showed that Kctd20 dramatically promotes the proliferation and invasion of NSCLC cells (P = 0.007 and P < 0.001, respectively). Subsequent Western Blot and qPCR experiments revealed an upregulation of Cyclin D1 and downregulation of E-cadherin in Kctd20-overexpressing cells. After depleting Kctd20, downregulaton of Cyclin D1, and upegulation of E-cadherin was observed. After overexpressing Kctd20, the levels of phosphorylated Fak (Tyr397) and Akt (Thr308) increased, while after transfection with Kctd20-siRNA these phosporylated proteins were downregulated. Moreover, in Kctd20-overexpressing cells, treatment with an Akt inhibitor reduced expression of p-Akt and Cyclin D1, enhanced E-cadherin expression, and did not impact p-Fak levels. When Kctd20-overexpressing cells were treated with a Fak inhibitor, the same effects were seen, and the level of p-Akt was reduced. Our results suggest that Kctd20 impacts proliferation and invasion of NSCLC through enhancing Fak (Tyr397) and Akt (Thr 308) phosphorylation. Kctd20 may predict prognosis and be targeted therapeutically in NSCLC.

Lasp2 enhances tumor invasion via facilitating phosphorylation of FAK and predicts poor overall survival of non-small cell lung cancer patients.

Lasp2, as well as Lasp1, is a member of the LIM-protein subfamily of the nebulin group characterized by the combined presence of LIM and SH3 domains. Lasp1 and Lasp2 are highly conserved in their LIM, nebulin-like, and SH3 domains but differ significantly at their linker regions. Lasp1 had been described as an oncogenic protein that was highly expressed in diverse cancer types and facilitated tumor proliferation, invasion, and metastasis process. However, unlike Lasp1, little is known about the functions of Lasp2. In the present study, using immunohistochemistry, we found that Lasp2 expression was significantly correlated with histological type (P = 0.012), advanced TNM stage (P = 0.024), positive regional lymph node metastasis (P = 0.035), and poor overall survival (P = 0.001). Would healing assay and transwell assay results indicated that Lasp2 promoted tumor migration and invasion in NSCLC cells. Furthermore, Lasp2 facilitated Snail expression and inhibited Zo-1. The levels of phosphorylated FAK (Tyr397 and Tyr925) were obviously increased after overexpressing Lasp2 and were downregulated by transfecting Lasp2-siRNA. FAK inhibitor counteracted upregulating Snail expression and downregulating of Zo-1 expression induced by Lasp2 overexpression. Taken together, Lasp2 may facilitate tumor migration and invasion of NSCLCs through FAK-Snail/Zo-1 signaling pathway. Lasp2 may be a potential prognostic predictor of NSCLC patients.

Alex3 suppresses non-small cell lung cancer invasion via AKT/Slug/E-cadherin pathway.

Alex3, is a newly identified mitochondrial protein, regulates mitochondrial dynamics and is involved in neural development. However, its expression pattern and clinicopathological relevance in human tumors are still unclear. In this study, Immunohistochemistry assay was performed in 109 cases of lung cancer samples and found that Alex 3 expression in lung cancer tissues was significantly lower than adjacent normal lung tissues (28.4% vs 52.6%, p < 0.001). Sequent statistical analysis indicated that negative Alex3 expression was significantly associated with advanced tumor-node-metastasis stages (p = 0.001), positive lymph node metastasis (p = 0.005), and poor prognosis (p = 0.008). After overexpression of Alex3, levels of p-AKT and Slug were downregulated, while level of E-cadherin was upregulated, which results in the inhibition of invasion and migration ability of lung cancer cells. In conclusion, reduction of Alex3 correlates with the development of non-small cell lung cancer and predicts adverse clinical outcome of non-small cell lung cancer patients. The effect of Alex3 on inhibiting invasion and migration may attribute to upregulation of E-cadherin expression through AKT-Slug pathway inactivation.

Inversin correlates with the malignant phenotype of non-small cell lung cancer and promotes the invasiveness of lung cancer cells.

Inversin, encoded by NPHP2, is one of the 10 NPHP proteins known to be involved in nephronophthisis (an autosomal recessive cystic kidney). Although the previous reports showed that inversin played an important role in embryonic development and renal diseases, its function in cancer was not revealed clearly so far. As measured by immunohistochemical staining, inversin was highly expressed in the cytoplasm of lung cancer samples (63.4%, 161/254) compared with adjacent normal lung tissues (22.0%, 11/50, p < 0.01). Moreover, its expression was positively correlated with differentiation ( p = 0.014), tumor node metastasis staging ( p = 0.007), and lymph node metastasis ( p = 0.020). The overall survival of non-small cell lung cancer patients with inversin positive expression (45.41 ± 1.800 months) was significantly reduced compared with those with inversin negative expression (51.046 ± 2.238 months, p = 0.042). Consistently, we found that the invasion capacity of A549 cells transfected with inversin was significantly stronger than that of control cells ( p < 0.05), while inversin siRNA-treatment significantly reduced cell invasion in H1299 cells ( p < 0.05). Additionally, we demonstrated that inversin could upregulate the expression of N-cadherin, Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-9. Collectively, these results indicated that inversin might promote the tumorigenicity of lung cancer cells and serve as a novel therapeutic target of non-small cell lung cancer.