Rnd3 regulates cell proliferation through NICD signaling in lung adenocarcinoma cells, A549.

<p>(<b>A</b>) Rnd3 mRNA detected by qRT-PCR is down-regulated in A459 compared to HBEC. (<b>B</b>) <b>&</b> (<b>C</b>) A western blot to detect phosphorylated MYPT1, NICD in cells. (<b>D</b>) <b>&</b> (<b>E</b>) The application of compound E blocks the proliferation of A459 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (<b>F</b>) <b>&</b> (<b>G</b>) Transient overexpression of Rnd3 in A549 cells down-regulated NICD and Hes1 in a dosage dependent manner. Data represent means ± S.D. *<i>p</i><0.05 compared to control (group 0); #<i>p</i><0.05 compared to group 1; $<i>p</i><0.05 compared to group 3. Data represent means ± S.D.</p

H520-Rnd3 and H358-Rnd3 cells have a lower proliferation rate.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) The generation of an H358 cell line stably expressing GFP-tagged Rnd3. The Rnd3 expression was verified by both Rnd3 and GFP antibodies. (<b>C</b>) <b>&</b> (<b>D</b>) The generation of an H358 cell line stably expressing GFP-tagged Rnd3. The Rnd3 expression was verified by both Rnd3 and GFP antibodies. (<b>E</b>) <b>&</b> (<b>F</b>) H358-Rnd3 has a lower proliferation rate compared to H358 cells as detected by BrdU incorporation. (<b>G</b>) <b>&</b> (<b>H</b>) H520-Rnd3 has a lower proliferation rate compared to H520 cells as detected by BrdU incorporation. (<b>I</b>) <b>&</b> (<b>J</b>) Cell number was quantified at different time point. An average of three samples at each time point was presented in this figure. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D.</p

Rnd3 inhibits proliferation by promoting NICD degradation in H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) Hes1 was up-regulated in H520 and H358 cells compared to HBEC cells. (<b>C–F</b>) Hes1 expression decreased when Rnd3 was stably overexpressed in H358 and H520 cells. (<b>G</b>) <b>&</b> (<b>H</b>) Transient overexpression of Rnd3 in H358 cells down-regulated NICD and Hes1 in a dosage dependent manner. (<b>I</b>) <b>&</b> (<b>J</b>) Inhibition of proteasome activity by MG132 (final concentration of 15 µM) abolished the Rnd3 dosage dependent NICD down-regulation in H358 cells. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D. *<i>p</i><0.05 compared to control (group 0); <b>#</b><i>p</i><0.05 compared to group 1; <b>$</b><i>p</i><0.05 compared to group 3. Data represent means ± S.D.</p

Inhibition of Notch signaling prevented proliferation of H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) The application of compound E blocks the proliferation of H358 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. (<b>C</b>) <b>&</b> (<b>D</b>) The application of compound E blocks the proliferation of H520 cells as detected by a BrdU incorporation assay. The cells were treated with compound E at the final concentration of 5 nM and synchronized by serum depletion followed by growth in media for 12 hours. Then, the cells were treated with BrdU for 30 minutes before being harvested for analysis. BrdU-positive cells were quantified from 8 images taken from four slides. Data represent means ± S.D.</p

Reintroduction of Rnd3 corrects Rho and Notch signaling in H520 and H358 cells.

<p>(<b>A</b>) <b>&</b> (<b>B</b>) Decreased NICD expression in H520-Rnd3 cells compared to H520 cells. (<b>C</b>) <b>&</b> (<b>D</b>) Decreased Rho Kinase activity in H520-Rnd3 cells compared to H520 cells detected by antibodies specific for pMYPT1 and pMLC2. (<b>E</b>) <b>&</b> (<b>F</b>) Decreased NICD expression in H358-Rnd3 cells compared to H358 cells. (<b>G</b>) <b>&</b> (<b>H</b>) Decreased Rho Kinase activity in H358-Rnd3 cells compared to H358 cells detected by antibodies specific for pMYPT1 and pMLC2. Western blots were quantified from three independent experimental repeats. Data represent means ± S.D.</p

Rnd3 is down-regulated in non-small lung cancer cell lines, H520 and H358.

<p>(<b>A</b>) Rnd3 mRNA detected by qRT-PCR is down-regulated in H358 and H520 compared to HBEC. (<b>B</b>) Rnd3 protein expression levels in cells by western blot. (<b>C</b>) Densitometry quantification of western band intensity in B. (<b>D</b>), (<b>F</b>), (<b>H</b>) <b>&</b> (<b>I</b>) A western blot to detect phosphorylated MYPT1, phosphorylated MLC2, ROCK1 and NICD in cells. (<b>E</b>), (<b>G</b>) <b>&</b> (<b>J</b>) Densitometry quantification of western band intensity showed up-regulation of Rho Kinase activity and NICD expression in H358 and H520 cells compared to HBEC. Western blots were quantified from three independent experimental repeats. BrdU-positive cells were quantified from 8 images taken from four slides. Data represent means ± S.D.</p

DataSheet1_Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer.docx

Profilin 1 (PFN1), an actin-binding protein, plays contrasting roles in the metastasis of several cancers; however, its role in non-small cell lung cancer (NSCLC) metastasis remains unclear. Here, PFN1 expression was upregulated in metastatic NSCLC tissues. PFN1 overexpression significantly promotes NSCLC metastasis in vitro and in vivo. Proteomics analysis revealed PFN1 involvment in microvesicles (MVs) secretion. In vitro experiments confirmed that PFN1 overexpression increased secretion of MVs. MVs are important mediators of metastasis. Here, we show an increased abundance of MVs in the sera of patients with metastatic NSCLC compared to that in the sera of patients with non-metastatic NSCLC. Both in vitro and in vivo experiments revealed that PFN1 could increase MV secretion, and MVs derived from PFN1-overexpressing cells markedly promoted NSCLC metastasis. We then elucidated the mechanisms underlying PFN1-mediated regulation of MVs and found that PFN1 could interact with ROCK1 and enhance its kinase activity to promote myosin light chain (MLC) phosphorylation for MV secretion. Inhibition of ROCK1 decreased MV secretion and partially reversed the PFN1-induced promotion of NSCLC metastasis. Collectively, these findings show that PFN1 regulates MV secretion to promote NSCLC metastasis. PFN1 and MVs represent potential predictors or therapeutic targets for NSCLC metastasis.</p

Table1_Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer.docx

Profilin 1 (PFN1), an actin-binding protein, plays contrasting roles in the metastasis of several cancers; however, its role in non-small cell lung cancer (NSCLC) metastasis remains unclear. Here, PFN1 expression was upregulated in metastatic NSCLC tissues. PFN1 overexpression significantly promotes NSCLC metastasis in vitro and in vivo. Proteomics analysis revealed PFN1 involvment in microvesicles (MVs) secretion. In vitro experiments confirmed that PFN1 overexpression increased secretion of MVs. MVs are important mediators of metastasis. Here, we show an increased abundance of MVs in the sera of patients with metastatic NSCLC compared to that in the sera of patients with non-metastatic NSCLC. Both in vitro and in vivo experiments revealed that PFN1 could increase MV secretion, and MVs derived from PFN1-overexpressing cells markedly promoted NSCLC metastasis. We then elucidated the mechanisms underlying PFN1-mediated regulation of MVs and found that PFN1 could interact with ROCK1 and enhance its kinase activity to promote myosin light chain (MLC) phosphorylation for MV secretion. Inhibition of ROCK1 decreased MV secretion and partially reversed the PFN1-induced promotion of NSCLC metastasis. Collectively, these findings show that PFN1 regulates MV secretion to promote NSCLC metastasis. PFN1 and MVs represent potential predictors or therapeutic targets for NSCLC metastasis.</p

Table_3_The Predictive Values of Advanced Non-Small Cell Lung Cancer Patients Harboring Uncommon EGFR Mutations—The Mutation Patterns, Use of Different Generations of EGFR-TKIs, and Concurrent Genetic Alterations.xlsx

IntroductionEpidermal growth factor receptor (EGFR) 19del and L858R mutation are known as “common mutations” in non-small cell lung cancer (NSCLC) and predict sensitivities to EGFR tyrosine kinase inhibitors (TKIs), whereas 20ins and T790M mutations confer drug-resistance to EGFR-TKIs. The role of the remaining uncommon EGFR mutations remains elusive.MethodsWe retrospectively screened a group of NSCLC patients with uncommon EGFR mutations other than 20ins and T790M. The mutation patterns, use of different generations of EGFR-TKIs, and concurrent genetic alterations were analyzed. Meanwhile, a cohort of patients with single 19del or L858R were included for comparison.ResultsA total of 180/1,300 (13.8%) patients were identified. There were 102 patients with advanced or recurrent NSCLC that received first-line therapy of gefitinib/erlotinib/icotinib and afatinib and were eligible for analysis. The therapeutic outcomes among patients with common mutations (EGFRcm, n = 97), uncommon mutation plus common mutations (EGFRum+EGFRcm, n = 52), complex uncommon mutations (complex EGFRum, n = 22), and single uncommon mutations (single EGFRum, n = 28) were significantly different (ORRs: 76.3%, 61.5%, 54.5%, and 50.0%, respectively, p = 0.023; and mPFS: 13.3, 14.7, 8.1, and 6.0 months, respectively, p = 0.004). Afatinib showed superior efficacy over gefitinib/erlotinib/icotinib in EGFRcm (ORR: 81.0% vs. 75.0%, p = 0.773; mPFS: 19.1 vs. 12.0m, p = 0.036), EGFRum+EGFRcm (ORR: 100% vs. 54.5%, p = 0.017; mPFS: NE vs. 13.6m, p = 0.032), and single EGFRum (ORR: 78.6% vs. 21.4%, p = 0.007; mPFS: 10.1 vs. 3.0m, p = 0.025) groups. Comprehensive genomic profiling by Next Generation Sequencing encompassing multiple cancer-related genes was performed on 51/102 patients; the mPFS of patients without co-mutation (n = 16) and with co-mutations of tumor-suppressor genes (n = 31) and driver oncogenes (n = 4) were 31.1, 9.2, and 12.4 months, respectively (p = 0.046). TP53 mutation was the most common co-alteration and showed significantly shorter mPFS than TP53 wild-type patients (7.0 vs. 31.1m, p ConclusionsUncommon EGFR mutations constitute a highly heterogeneous subgroup of NSCLC that confer different sensitivities to EGFR-TKIs with regard to the mutation patterns. Afatinib may be a better choice for most uncommon EGFR mutations. Concurrent 19del/L858R and tumor-suppressor gene alterations, especially TP53, can be established as prognostic biomarkers.</p

Table_2_The Predictive Values of Advanced Non-Small Cell Lung Cancer Patients Harboring Uncommon EGFR Mutations—The Mutation Patterns, Use of Different Generations of EGFR-TKIs, and Concurrent Genetic Alterations.xlsx

IntroductionEpidermal growth factor receptor (EGFR) 19del and L858R mutation are known as “common mutations” in non-small cell lung cancer (NSCLC) and predict sensitivities to EGFR tyrosine kinase inhibitors (TKIs), whereas 20ins and T790M mutations confer drug-resistance to EGFR-TKIs. The role of the remaining uncommon EGFR mutations remains elusive.MethodsWe retrospectively screened a group of NSCLC patients with uncommon EGFR mutations other than 20ins and T790M. The mutation patterns, use of different generations of EGFR-TKIs, and concurrent genetic alterations were analyzed. Meanwhile, a cohort of patients with single 19del or L858R were included for comparison.ResultsA total of 180/1,300 (13.8%) patients were identified. There were 102 patients with advanced or recurrent NSCLC that received first-line therapy of gefitinib/erlotinib/icotinib and afatinib and were eligible for analysis. The therapeutic outcomes among patients with common mutations (EGFRcm, n = 97), uncommon mutation plus common mutations (EGFRum+EGFRcm, n = 52), complex uncommon mutations (complex EGFRum, n = 22), and single uncommon mutations (single EGFRum, n = 28) were significantly different (ORRs: 76.3%, 61.5%, 54.5%, and 50.0%, respectively, p = 0.023; and mPFS: 13.3, 14.7, 8.1, and 6.0 months, respectively, p = 0.004). Afatinib showed superior efficacy over gefitinib/erlotinib/icotinib in EGFRcm (ORR: 81.0% vs. 75.0%, p = 0.773; mPFS: 19.1 vs. 12.0m, p = 0.036), EGFRum+EGFRcm (ORR: 100% vs. 54.5%, p = 0.017; mPFS: NE vs. 13.6m, p = 0.032), and single EGFRum (ORR: 78.6% vs. 21.4%, p = 0.007; mPFS: 10.1 vs. 3.0m, p = 0.025) groups. Comprehensive genomic profiling by Next Generation Sequencing encompassing multiple cancer-related genes was performed on 51/102 patients; the mPFS of patients without co-mutation (n = 16) and with co-mutations of tumor-suppressor genes (n = 31) and driver oncogenes (n = 4) were 31.1, 9.2, and 12.4 months, respectively (p = 0.046). TP53 mutation was the most common co-alteration and showed significantly shorter mPFS than TP53 wild-type patients (7.0 vs. 31.1m, p ConclusionsUncommon EGFR mutations constitute a highly heterogeneous subgroup of NSCLC that confer different sensitivities to EGFR-TKIs with regard to the mutation patterns. Afatinib may be a better choice for most uncommon EGFR mutations. Concurrent 19del/L858R and tumor-suppressor gene alterations, especially TP53, can be established as prognostic biomarkers.</p