Intervention of AXL in EGFR Signaling via Phosphorylation and Stabilization of MIG6 in Non-Small Cell Lung Cancer.

About 80% of lung cancer patients are diagnosed with non-small cell lung cancer (NSCLC). EGFR mutation and overexpression are common in NSCLC, thus making EGFR signaling a key target for therapy. While EGFR kinase inhibitors (EGFR-TKIs) are widely used and efficacious in treatment, increases in resistance and tumor recurrence with alternative survival pathway activation, such as that of AXL and MET, occur frequently. AXL is one of the EMT (epithelial-mesenchymal transition) signature genes, and EMT morphological changes are also responsible for EGFR-TKI resistance. MIG6 is a negative regulator of ERBB signaling and has been reported to be positively correlated with EGFR-TKI resistance, and downregulation of MIG6 by miR-200 enhances EMT transition. While MIG6 and AXL are both correlated with EMT and EGFR signaling pathways, how AXL, MIG6 and EGFR interplay in lung cancer remains elusive. Correlations between AXL and MIG6 expression were analyzed using Oncomine or the CCLE. A luciferase reporter assay was used for determining MIG6 promoter activity. Ectopic overexpression, RNA interference, Western blot analysis, qRT-PCR, a proximity ligation assay and a coimmunoprecipitation assay were performed to analyze the effects of certain gene expressions on protein-protein interaction and to explore the underlying mechanisms. An in vitro kinase assay and LC-MS/MS were utilized to determine the phosphorylation sites of AXL. In this study, we demonstrate that MIG6 is a novel substrate of AXL and is stabilized upon phosphorylation at Y310 and Y394/395 by AXL. This study reveals a connection between MIG6 and AXL in lung cancer. AXL phosphorylates and stabilizes MIG6 protein, and in this way EGFR signaling may be modulated. This study may provide new insights into the EGFR regulatory network and may help to advance cancer treatment.

Generating dual-wavelength VECSEL by selecting birefringence filter material and the application toward mid-infrared region via intracavity OPO.

A method for realizing a synchronized watt-level dual-wavelength vertical-external-cavity surface-emitting-laser (VECSEL) by using a tilted birefringence filter (BRF) is demonstrated. It is verified that by selecting suitable BRF material with different refractive index differences between extraordinary wave and ordinary wave, the dual-wavelength emission with a free spectrum range from sub-THz to tens of THz can be achieved. The output characteristics of such a dual-wavelength VECSEL are thoroughly investigated including its wavelength tunability and power difference. Finally, the intracavity optical parametric oscillator is applied to efficiently convert the dual-wavelength laser toward the mid-infrared region. The gain competition and longitudinal mode hopping performances for the multi-wavelength mid-infrared output are explored.

Low-threshold dual-wavelength CW mid-IR laser from shared intracavity quasi-phase-matched OPO.

A low-threshold continuous wave dual-wavelength mid-infrared laser is demonstrated by using an intracavity optical parametric oscillator (OPO) with a dual-wavelength pump wave. To realize a high-quality dual-wavelength pump wave, a composite Nd:YVO4/Nd:GdVO4 gain medium is applied for a linear polarized state and synchronization output. With the quasi-phase-matching OPO process, it is found that the dual-wavelength pump wave shares equal signal wave oscillation and leads to a lower OPO threshold. Finally, a diode threshold pumped power of barely 2 W can be achieved for the balanced intensity dual-wavelength watt-level mid-IR laser.

Exploring the influence of pump beam quality on designing millijoule diode-end-pumped passively Q-switched lasers.

To practically predict the design criteria of diode-end-pumped passively Q-switched (PQS) lasers with energy scaling to millijoule region, an analytical model with longitudinally spatial dependence is derived to investigate the influence of pump beam quality. In comparison with PQS theory that considers transverse spatial dependence only, it is found that the threshold pump power can be up to 5 times larger when the beam quality factor was 80. This result indicates the importance of considering pump beam quality when designing PQS lasers especially for operation at high pump power level. The theoretical results are verified by a series of PQS experiments. The influence of thermal lensing effect on cavity design is further discussed to obtain good laser quality. Finally, an end-pumped millijoule PQS laser is successfully realized based on the theoretical analysis and the resonator design.

MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/ß-catenin pathway.

BACKGROUND: The development of drug resistance in oral squamous cell carcinoma (OSCC) that frequently leads to recurrence and metastasis after initial treatment remains an unresolved challenge. Presence of cancer stem cells (CSCs) has been increasingly reported to be a critical contributing factor in drug resistance, tumor recurrence and metastasis. Thus, unveiling of mechanisms regulating CSCs and potential targets for developing their inhibitors will be instrumental for improving OSCC therapy. METHODS: siRNA, shRNA and miRNA that specifically target keratin 17 (KRT17) were used for modulation of gene expression and functional analyses. Sphere-formation and invasion/migration assays were utilized to assess cancer cell stemness and epithelial mesenchymal transition (EMT) properties, respectively. Duolink proximity ligation assay (PLA) was used to examine molecular proximity between KRT17 and plectin, which is a large protein that binds cytoskeleton components. Cell proliferation assay was employed to evaluate growth rates and viability of oral cancer cells treated with cisplatin, carboplatin or dasatinib. Xenograft mouse tumor model was used to evaluate the effect of KRT17- knockdown in OSCC cells on tumor growth and drug sensitization. RESULTS: Significantly elevated expression of KRT17 in highly invasive OSCC cell lines and advanced tumor specimens were observed and high KRT17 expression was correlated with poor overall survival. KRT17 gene silencing in OSCC cells attenuated their stemness properties including markedly reduced sphere forming ability and expression of stemness and EMT markers. We identified a novel signaling cascade orchestrated by KRT17 where its association with plectin resulted in activation of integrin ß4/α6, increased phosphorylation of FAK, Src and ERK, as well as stabilization and nuclear translocation of ß-catenin. The activation of this signaling cascade was correlated with enhanced OSCC cancer stemness and elevated expression of CD44 and epidermal growth factor receptor (EGFR). We identified and demonstrated KRT17 to be a direct target of miRNA-485-5p. Ectopic expression of miRNA-485-5p inhibited OSCC sphere formation and caused sensitization of cancer cells towards cisplatin and carboplatin, which could be significantly rescued by KRT17 overexpression. Dasatinib treatment that inhibited KRT17-mediated Src activation also resulted in OSCC drug sensitization. In OSCC xenograft mouse model, KRT17 knockdown significantly inhibited tumor growth, and combinatorial treatment with cisplatin elicited a greater tumor inhibitory effect. Consistently, markedly reduced levels of integrin ß4, active ß-catenin, CD44 and EGFR were observed in the tumors induced by KRT17 knockdown OSCC cells. CONCLUSIONS: A novel miRNA-485-5p/KRT17/integrin/FAK/Src/ERK/ß-catenin signaling pathway is unveiled to modulate OSCC cancer stemness and drug resistance to the common first-line chemotherapeutics. This provides a potential new therapeutic strategy to inhibit OSCC stem cells and counter chemoresistance.

Power scaling of continuous-wave second harmonic generation in a MgO:PPLN ridge waveguide and the application to a compact wavelength conversion module.

The power scaling of continuous-wave (CW) second harmonic generation (SHG) in a MgO:periodically poled lithium niobite (PPLN) ridge waveguide is investigated. The nonlinear coefficient and propagation loss factors of the MgO:PPLN waveguide are verified for future reference. The MgO:PPLN waveguide structure is determined according to a practical fiber coupling configuration, as well as the theoretical model of output power characteristics. Utilizing the designed MgO:PPLN waveguide, the 775 nm SHG power successfully reaches up to 4.02 W at an incident power of 7 W with overall conversion efficiency of 58%. Finally, a compact all-fiber waveguide conversion module is demonstrated for verifying the feasibility of commercial applications.

Investigation of anisotropic thermal lens effect in a dual-polarized Nd:YLF laser.

The anisotropic thermal lens effect of a dual-polarization Nd:YLF laser is experimentally investigated by measuring the transverse beat frequency between TEM0,0 and TEM1,0 modes in the self-pulsing operation, and focal lengths of thermal lensing for both polarizations can be accurately determined. The focal length of the thermal lens for π polarization was observed to be negative and varies from -1.1 to -0.5m for the absorbed pump power increasing from 1.7 to 3.8 W. For σ polarization, the focal length of the thermal lens was determined to be positive and varies from 1.2 to 0.9 m for the absorbed pump power increasing from 8.4 to 10.9 W. The sensitivity factors of the thermal lens for both polarizations were evaluated to be Mπ=-0.54m-1/W and Mσ=0.1m-1/W, respectively.

PTBP1-mediated regulation of AXL mRNA stability plays a role in lung tumorigenesis.

AXL is expressed in many types of cancer and promotes cancer cell survival, metastasis and drug resistance. Here, we focus on identifying modulators that regulate AXL at the mRNA level. We have previously observed that the AXL promoter activity is inversely correlated with the AXL expression levels, suggesting that post-transcriptional mechanisms exist that down-regulate the expression of AXL mRNA. Here we show that the RNA binding protein PTBP1 (polypyrimidine tract-binding protein) directly targets the 5'-UTR of AXL mRNA in vitro and in vivo. Moreover, we also demonstrate that PTBP1, but not PTBP2, inhibits the expression of AXL mRNA and the RNA recognition motif 1 (RRM1) of PTBP1 is crucial for this interaction. To clarify how PTBP1 regulates AXL expression at the mRNA level, we found that, while the transcription rate of AXL was not significantly different, PTBP1 decreased the stability of AXL mRNA. In addition, over-expression of AXL may counteract the PTBP1-mediated apoptosis. Knock-down of PTBP1 expression could enhance tumor growth in animal models. Finally, PTBP1 was found to be negatively correlated with AXL expression in lung tumor tissues in Oncomine datasets and in tissue micro-array (TMA) analysis. In conclusion, we have identified a molecular mechanism of AXL expression regulation by PTBP1 through controlling the AXL mRNA stability. These findings may represent new thoughts alternative to current approaches that directly inhibit AXL signaling and may eventually help to develop novel therapeutics to avoid cancer metastasis and drug resistance.

Energy scale-up and mode-quality enhancement of the LED-pumped Nd:YAG Q-switched laser achieving a millijoule green pulse.

An efficient LED pumping module is explored for the demonstration of energy scaling of passively Q-switched output to millijoule high-pulse-energy level. For the free-running operation at fundamental wavelength, the highest optical conversion efficiency of 23.7% is reached. By using a Cr4+:YAG crystal as the saturable absorber, the passively Q-switched output energy is up to 14.5 mJ in a compact setup. The laser resonator is further optimized to shorten the output pulse width and to obtain better mode quality. The pulse width of the Q-switched emission is as short as 25 ns, and the peak power is up to 0.7 MW. With such a highly efficient configuration, the extracavity second-harmonic generation at 532 nm can be achieved with 4.5 mJ pulse energy.

Protective Effect of Antrodia cinnamomea Extract against Irradiation-Induced Acute Hepatitis.

Radiotherapy for treatment of hepatocellular carcinoma causes severe side effects, including acute hepatitis and chronic fibrosis. Complementary and alternative medicine (CAM) has emerged as an important part of integrative medicine in the management of diseases. Antrodia cinnamomea (AC), a valuable medicinal fungus originally found only in Taiwan, has been shown to possess anti-oxidation, vaso-relaxtation, anti-inflammation, anti-hepatitis, and anti-cancer effects. In this paper we evaluate the protective effects of ethanol extract of Antrodia cinnamomea (ACE) against radiotoxicity both in normal liver cell line CL48 and in tumor-bearing mice. In CL48, ACE protects cells by eliminating irradiation-induced reactive oxygen species (ROS) through the induction of Nrf2 and the downstream redox system enzymes. The protective effect of ACE was also demonstrated in tumor-bearing mice by alleviating irradiation-induced acute hepatitis. ACE could also protect mice from CCl4-induced hepatitis. Since both radiation and CCl4 cause free radicals, these results indicate that ACE likely contains active components that protect normal liver cells from free radical attack and can potentially benefit hepatocellular carcinoma (HCC) patients during radiotherapy.

Exploring the DBR superlattice effect on the thermal performance of a VECSEL with the finite element method.

The design criterion of thermal conductivity for the GaAs/AlAs distributed Bragg reflector (DBR) superlattice structure was thoroughly investigated to precisely analyze the thermal behaviors of the optically pumped vertically external cavity surface-emitting laser (VECSEL). A finite element model with detailed configuration of a VECSEL gain chip was constructed to fulfill the analysis. A 1060 nm VECSEL with different pump conditions was further demonstrated to verify the finite element analysis. At the VECSEL thermal rollover point, the analysis results show that the model with the superlattice property predicts more precise temperature values than that using a bulk composite property. It reveals that the accurate determination of the thermal conductivity of the DBR superlattice is significantly important for the VECSEL thermal analysis.

AXL phosphorylates and up-regulates TNS2 and its implications in IRS-1-associated metabolism in cancer cells.

BACKGROUND: TNS2 is a focal adhesions protein and a binding partner for many proteins, including the receptor tyrosine kinase Axl. Although TNS2 can bind with Axl, the details of their interactions have not been elucidated. TNS2 is involved in IRS-1 signaling pathway. In this study, we confirmed the relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. METHODS: The expression levels of TNS2, Axl, IRS-1, PDK1 and Glut4 in human cancer cells were measured by Western blot and/or IP-Western blot assays. Paired samples of pancreatic cancer and non-cancer tissues were obtained from 33 patients and were used to construct tissue microarrays. The expression levels of these markers in the tissue microarrays were measured by enzyme-linked Immunohistochemistry assay, and the relationships were analyzed by Pearson's chi-square test and two-tailed t-test analysis. RESULTS: We demonstrated for the first time that TNS2 is a phosphorylation substrate of Axl. Moreover, we found a positive relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. Based on these results, we suggest that Axl modulates glucose metabolism potentially through TNS2 and IRS-1. We hypothesize that there exists a novel mechanism whereby Axl binds to and phosphorylates TNS2, releasing TNS2 from interaction with IRS-1 and resulting in increased stability of IRS-1. The two key enzymes of aerobic glycolysis (Glut4 and PDK1) were found to be up-regulated by Axl/TNS2/IRS-1 cross-talk and may play a critical role in glucose metabolism of cancer cells. CONCLUSIONS: Our results revealed for the first time that Axl binds to and phosphorylates TNS2 and that Axl/TNS2/IRS-1 cross-talk may potentially play a critical role in glucose metabolism of cancer cells.

Exploring the self-mode locking of the 2 µm Tm:YAG laser with suppression of the self-pulsing dynamic.

A continuous-wave (cw) self-mode-locked Tm:YAG laser at 2015 nm is successfully demonstrated by suppressing the self-pulsing behavior. By using rate equations to simulate the laser temporal dynamic, the theoretical analysis indicates that the reabsorption-induced self-pulsing for the Tm:YAG laser can be suppressed with sufficient pump power. It is experimentally confirmed that at absorbed powers higher than 6 W, the self-pulsing can be eliminated and a cw self-mode-locked pulse is generated instead of the Q-switched mode locking. At an absorbed power of 6.8 W, the output power of the self-mode-locked Tm:YAG laser reaches 1.22 W with corresponding pulse duration of 3 ps and a repetition rate of 3.376 GHz.

Compact coupling scheme to achieve the synchronously dual self-mode-locked operation with a 35.2 THz optical beating.

A synchronously dual self-mode-locked (SML) operation at 946 and 1064 nm is experimentally accomplished by using a compact coupling scheme to obtain the optical beating frequency up to 35.2 THz. The SML emissions at 946 and 1064 nm are established by a monolithic Nd:YAG cavity and a Nd:YVO4 crystal in a flat-flat cavity, respectively. Two gain media are butt adjoined to be longitudinally pumped by a single laser diode. The monolithic Nd:YAG crystal is used not only as the 946 nm laser cavity, but also as the output coupler of the Nd:YVO4 1064 nm laser. More importantly, the output surface of the monolithic Nd:YAG laser is coated to generate the optical feedback for the synchronization of the dual SML operation. At a pump power of 10.7 W, the output powers at 1064 and 946 nm can simultaneously reach 1.5 W by controlling the focal position of the pump waist.

LED-side-pumped Nd:YAG laser with >20% optical efficiency and the demonstration of an efficient passively Q-switched LED-pumped solid-state laser.

A LED-array-pumped Nd:YAG laser with optical conversion efficiency up to 20.2% is demonstrated by direct side-pumping without a coupling lens. The 810-nm LED array with a full-width-half-maximum of 30 nm and an area fill factor of 50% is designed to attain high spectral absorption efficiency with a pump density of 151.8 W/cm2. Furthermore, higher than 45% overall coupling efficiency is achieved by placing the LED array as close as possible to the side of the gain medium for overcoming the large pumped divergence. More importantly, by using the efficient pump scheme, the demonstration of a passively Q-switched LED-pumped laser is successfully realized with a pulsed energy of 1.42 mJ.

Negative feedback regulation of AXL by miR-34a modulates apoptosis in lung cancer cells.

The AXL receptor tyrosine kinase is frequently overexpressed in cancers and is important in cancer invasion/metastasis and chemoresistance. Here, we demonstrate a regulatory feedback loop between AXL and microRNA (miRNA) at the post-transcriptional level. Both the GAS6-binding domain and the kinase domain of AXL, particularly the Y779 tyrosine phosphorylation site, are shown to be crucial for this autoregulation. To clarify the role of miRNAs in this regulation loop, approaches using bioinformatics and molecular techniques were applied, revealing that miR-34a may target the 3' UTR of AXL mRNA to inhibit AXL expression. Interestingly and importantly, AXL overexpression may induce miR-34a expression by activating the transcription factor ELK1 via the JNK signaling pathway. In addition, ectopic overexpression of ELK1 promotes apoptosis through, in part, down-regulation of AXL. Therefore, we propose that AXL is autoregulated by miR-34a in a feedback loop; this may provide a novel opportunity for developing AXL-targeted anticancer therapies.

Oxidative stress enhances Axl-mediated cell migration through an Akt1/Rac1-dependent mechanism.

Persistent oxidative stress is common in cancer cells because of abnormal generation of reactive oxygen species (ROS) and has been associated with malignant phenotypes, such as chemotherapy resistance and metastasis. Both overexpression of Axl and abnormal ROS elevation have been linked to cell transformation and increased cell migration. However, the relationship between Axl and ROS in malignant cell migration has not been previously evaluated. Using an in vitro human lung cancer model, we examined the redox state of lung adenocarcinoma cell lines of low metastatic (CL1-0) and high metastatic (CL1-5) potentials. Here we report that Axl activation elicits ROS accumulation through the oxidase-coupled small GTPase Rac1. We also observed that oxidative stress could activate Axl phosphorylation to synergistically enhance cell migration. Further, Axl signaling activated by H2O2 treatment results in enhancement of cell migration via a PI3K/Akt-dependent pathway. The kinase activity of Axl is required for the Axl-mediated cell migration and prolongs the half-life of phospho-Akt under oxidative stress. Finally, downregulation of Akt1, but not Akt2, by RNAi in Axl-overexpressing cells inhibits the amount of activated Rac1 and the ability to migrate induced by H2O2 treatment. Together, these results show that a novel Axl-signaling cascade induced by H2O2 treatment triggers cell migration through the PI3K/Akt1/Rac1 pathway. Elucidation of redox regulation in Axl-related malignant migration may provide new molecular insights into the mechanisms underlying tumor progression.

Epigenetic inactivation of the metastasis suppressor RECK enhances invasion of human colon cancer cells.

Down-regulation of RECK, an important metastasis suppressor gene, has been found in human colon cancer. However, the molecular mechanism for this down- regulation and its biological significance are still unclear. In the present study, we investigated whether down-regulation of RECK is caused by epigenetic inactivation via promoter methylation and tested the effect of DNA methyltransferase (DNMT) inhibitor on RECK expression and cell invasion. The mRNA and protein levels of RECK in colon tumor tissues and their normal counterparts were compared. We found that down-regulation of RECK was found in 48% of the twenty five tumors analyzed. MSP analysis demonstrated that methylation of RECK promoter was detected in 44% (11/25) of the tumor tissues and a strong correlation between down-regulation and promoter methylation was found (P = 0.028). Promoter methylation was also found in SW480 and SW620 human colon cancer cell lines. DNA methyltransferase (DNMT) inhibitor 5'-azacytidine reversed promoter methylation, restored RECK expression and suppressed invasion by these two cell lines. Restoration of RECK is critical for 5'-azacytidine-mediated suppression of cell invasion because inhibition of RECK by a specific antibody significantly attenuated the anti-invasive ability of 5'-azacytidine. Taken together, our results suggest that down-regulation of the metastasis suppressor RECK in colon cancer is associated with promoter methylation and that a DNMT inhibitor may restore RECK expression to inhibit cell invasion.

Downregulation of RECK by promoter methylation correlates with lymph node metastasis in non-small cell lung cancer.

In the present study, we addressed the molecular mechanism of the downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), a critical tumor suppressor that can potently inhibit angiogenesis and metastasis, in non-small cell lung cancer and its clinical significance. The methylation status of the RECK gene promoter was studied by methylation-specific polymerase chain reaction. RECK mRNA and protein levels were investigated by reverse transcription-polymerase chain reaction and western blot analysis. Downregulation of RECK was observed in 60% of the 55 tumors analyzed. Using methylation-specific polymerase chain reaction analysis methylation of the RECK promoter was detected in 63.6% (35/55) of the tumor tissues. A strong correlation between downregulation and promoter methylation was found in these tumors (P = 0.000005). More importantly, downregulation of RECK significantly correlated with lymph node metastasis (P = 0.038). Mutation of codon 12 of the K-ras gene was detected in 25.5% (14/55) of lung tumor tissues. Statistical analysis indicated that K-ras mutation was linked with RECK promoter methylation (P = 0.047) and downregulation (P = 0.023). Promoter methylation was also detected in human lung cancer cell lines, and the DNA methyltransferase inhibitor 5'-azacytidine reversed the expression of RECK and reduced the invasive ability of these cell lines. Collectively, our results suggest that downregulation of the metastasis suppressor RECK is caused by promoter methylation in non-small cell lung cancer and is associated with K-ras mutation and lymph node metastasis.

Silencing of the metastasis suppressor RECK by RAS oncogene is mediated by DNA methyltransferase 3b-induced promoter methylation.

RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase activity to suppress tumor invasion and metastasis. Our previous study indicated that oncogenic RAS inhibited RECK expression via a histone deacetylation mechanism. In this study, we address whether DNA methyltransferases (DNMT) participate in the inhibition of RECK by RAS. Induction of Ha-RAS(Val12) oncogene increased DNMT3b, but not DNMT1 and DNMT3a, expression in 2-12 cells. In addition, induction of DNMT3b by RAS was through the extracellular signal-regulated kinase signaling pathway. Oncogenic RAS increased the binding of DNMT3b to the promoter of RECK gene and this binding induced promoter methylation, which could be reversed by 5'-azacytidine and DNMT3b small interfering RNA (siRNA). The MEK inhibitor U0126 also reversed RAS-induced DNMT3b binding and RECK promoter methylation. Treatment of 5'-azacytidine and DNMT3b siRNA restored RECK expression in 2-12 cells and potently suppressed RAS-stimulated cell invasion. In addition, the inhibitory effect of 5'-azacytidine on RAS-induced cell invasion was attenuated after knockdown of RECK by siRNA. Interestingly, human lung cancer cells harboring constitutively activated RAS exhibited lower RECK expression and higher promoter methylation of RECK gene. 5'-Azacytidine and DNMT3b siRNA restored RECK expression in these cells and effectively suppressed invasiveness. Collectively, our results suggest that RAS oncogene induces RECK gene silencing through DNMT3b-mediated promoter methylation, and DNMT inhibitors may be useful for the treatment of RAS-induced metastasis.