Growth inhibition of KRAS‑ and EGFR‑mutant lung adenocarcinoma by cosuppression of STAT3 and the SRC/ARHGAP35 axis

  • Authors:
    • Ken Onodera
    • Akira Sakurada
    • Hirotsugu Notsuda
    • Tatsuaki Watanabe
    • Yasushi Matsuda
    • Masafumi Noda
    • Chiaki Endo
    • Yoshinori Okada
  • View Affiliations

  • Published online on: July 2, 2018
  • Pages: 1761-1768
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The need for effective treatment of KRAS‑mutant lung cancer is an emerging issue. Rho GTPase‑activating protein 35 (ARHGAP35) is reported to be a possible molecular target for lung adenocarcinoma. We investigated the effect of long‑term ARHGAP35 suppression on the proliferation, migration and molecular dynamics of lung adenocarcinomas harboring KRAS and EGFR gene mutations. Lung adenocarcinoma cell lines A549 (KRAS‑mutant) and PC9 and H1975 (EGFR‑mutants) were used, and ARHGAP35 knockdown was carried out using puromycin. Cell viability, migration and molecular dynamics were assayed 1 month after introducing small hairpin RNA. The compensatory upregulated mechanism was screened by western blotting and confirmed by a specific inhibitor. Finally, we tested the effects of cosuppression of the SRC/ARHGAP35 axis and the identified pathway in vitro. ARHGAP35 suppression was attenuated by long‑term knockdown of the target genes. Compensatory mechanisms by SRC and STAT3 caused attenuation in A549 cells. After long‑term ARHGAP35 knockdown, both A549 and PC9 cells were more sensitive to treatment with a STAT3 inhibitor. The suppressive effect of ARHGAP35 knockdown on migration was sustained, but only modest, in all cell lines. Synergistic and strong growth inhibition was observed with concomitant use of an SRC inhibitor and a STAT3 inhibitor in A549 cells. STAT3 activation compensated for ARHGAP35 knockdown in lung adenocarcinoma with the KRAS mutation. Moreover, cosuppression of the STAT3 pathway and SRC/ARHGAP35 axis may be an effective strategy for treating lung adenocarcinoma, especially in the presence of a KRAS mutation.


Lung cancer is the leading cause of mortality worldwide, being responsible for 1.59 million patient deaths every year. Although early diagnosis increases the potential for curative surgical resection, more than half of cases present with advanced disease and require treatment with chemotherapy and radiotherapy (1). A recent breakthrough, the discovery of driver mutations and the development of corresponding tyrosine kinase inhibitors (TKIs), has shown significant clinical benefits to date (2,3). However, targeting KRAS, the second major driver, is an emerging problem as its activation is different from the usual kinase-based signaling. In addition, overcoming an acquired resistance, including a mutation at exon 20 in ERBB2, (4,5) and activation of the HGF and MET signaling pathway, (6,7) has yet been a problem to resolve although the specific inhibitor targeting T790M in the EGFR gene (8,9) has been developed.

The concept of synthetic lethality leading to cell death in the presence of a combination of mutations in multiple genes was first advanced in 1945 (10). This was later highlighted in 2005 by reports indicating that breast cancer with the breast cancer susceptibility gene mutation (BRCA1, DNA repair associated) was very sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) (11,12). Thus, it seemed that simultaneous suppression of two major DNA repair genes could disrupt DNA replication in cancer cells. Since this discovery, other therapeutic approaches based on synthetic lethality have been sought in various malignancies known to have specific gene alterations (13). Notably, the KRAS mutation has been a major target for synthetic lethality, and the cosuppression of MEK with RAF1 or BCL-XL was identified as being effective at inducing synthetic lethality in cancer cells with this mutation (14,15). To establish effective clinical treatments for lung cancer with KRAS mutations, it is important to identify additional combinations that cause synthetic lethality.

Rho GTPase-activating protein 35 (ARHGAP35) is a RhoGAP protein (16,17) reported to be the principal substrate of SRC and controller of RhoA activity (through the binding of RhoA) (18,19) and also known with altered names such as p190RhoGAP, GRF-1 and GRLF1. To date, however, the role of this molecule in cancer has been uncertain, with both tumor suppressor effects and oncogenic effects reported (20,21). In proteome studies, the phosphorylation status of tyrosine Y1105 in ARHGAP35 has consistently been reported to change, being dramatically suppressed by EGFR-TKI treatment for lung adenocarcinoma in the presence of the EGFR mutation (22,23). Therefore, we aimed to focus on ARHGAP35 as a possible key molecule in the proliferation and metastasis of lung adenocarcinoma. To this end, our group previously showed that ARHGAP35 messenger RNA (mRNA) was overexpressed in lung cancer cell lines compared to normal cells, and that positive protein expression was widely observed in lung cancer cells in surgically resected specimens immunohistochemically (24). It was also observed that ARHGAP35 knockdown significantly suppressed the viability, migration and invasion of lung adenocarcinoma cells, including KRAS mutants (24).

In the present study, we investigated the effect of long-term ARHGAP35 suppression in lung cancer cells. Our aim was to identify a compensatory pathway that could be both a potential mechanism of acquired resistance for SRC/ARHGAP35 inhibition and a candidate for synthetic lethality when used in combination with inhibition of the SRC/ARHGAP35 axis.

Materials and methods

Cell lines and culture

A549 and H1975 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VI, USA). PC9 was obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). All cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS) in a humidified 5% CO2 incubator at 37°C. These cells are histologically adenocarcinomas, and A549 has the wild-type (WT) EGFR and KRAS mutations (codon 12), PC-9 has the EGFR mutation (exon 19 del), and H1975 has the EGFR mutation (L858R and T790M). A549 and H1975 cells have been reported to be EGFR-TKI resistant, whereas PC9 is EGFR-TKI sensitive. To test whether there was concomitant inhibition of STAT3 and SRC, cells were treated with combinations of different concentrations of a STAT3 inhibitor (S3I-201; Santa Cruz Biotechnology, Dallas, TX, USA) and SRC inhibitor (SKI-1; Abcam, Cambridge, UK). Drug concentrations of S3I-201 were adjusted to 0, 100, 200 and 400 µM and those for SKI-1 were adjusted to 0, 2.5, 5 and 10 µM.

RNA interference (RNAi)

Lentiviral transfection with small hairpin RNA (shRNA) was conducted using MISSION® shRNA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells were transfected with shRNA directed to ARHGAP35 or negative control shRNA using hexadimethrine bromide. Transfected cells were cultured with media containing puromycin and selected clones were maintained with puromycin over 1 month.

Western blot analysis

Cell lines were washed with ice-cold PBS and lysed in ice-cold lysis buffer (pH 8.0 50 mM HEPES, 150 mM NaCl, 100 mM NaF, 10 mM Na4P2O7H2O, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol and 1% Triton X-100) which was added Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and PhosSTOP phosphatase Inhibitor Cocktail (Roche). The cell lysates were centrifuged at 15,000 × g for 20 min at 4°C to collect the supernatant. We calculated protein concentrations by Bradford method using Bio-Rad Protein assay (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Protein samples (10 µg) were separated by SDS-PAGE Mini-PROTEAN TGX Gel (GE Healthcare Life Sciences) and transferred onto a polyvinylidene difluoride membrane (PVDF; Hybond-P®; GE Healthcare Life Sciences). The PVDF membranes with proteins were blocked with 5% non-fat dry milk (NFDM) for 1 h at room temperature. The primary antibodies dissolved in 5% bovine serum albumin (BSA) were used to detect the target protein blots at 4°C overnight for incubation. Protein were incubated on the membranes with primary antibodies dissolved in 5% BSA (or 5% NFDM) at 4°C overnight and secondary antibodies labeled with horseradish peroxidase dissolved in blocking buffer for 1 h at room temperature. The proteins were visualized on ImageQuant LAS 4000 Mini (GE Healthcare Life Sciences), using the enhanced chemiluminescence western blot detection system. The primary antibodies used in the present study included: ARHGAP35 (1:1,000; cat. no. 2860), SRC (1:1,000; cat. no. 2109), AKT (1:1,000; cat. no. 4691), STAT3 (1:2,000; cat. no. 4904), MEK (1:1,000; cat. no. 9126), PRKCD (1:1,000, 5% NFDM; cat. no. 9616), PRKCZ (1:1,000; cat. no. 9368), p-SRC (1:1,000; cat. no. 2101), p-AKT (1:2,000; cat. no. 4060), p-STAT3 (1:2,000; cat. no. 9145), p-MEK (1:1,000; cat. no. 9121) and β-actin (1:1,000; cat. no. 4967) all from Cell Signaling Technology (Boston, MA, USA).

Quantitative assay for Rho kinase activity

Measurement of Rho kinase (ROCK) activity was performed using a ROCK Activity Immunoblot kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's instructions.


Total RNA isolation from cell lines and complementary DNA synthesis was performed using TaqMan® Gene Expression Cells-to-Ct™ Kits (Thermo Fisher Scientific, Inc.). All mRNA was measured by qRT-PCR using an ABI PRISM 7000 Sequence Detection System (Thermo Fisher Scientific) with primers (Thermo Fisher Scientific; ARHGAP35: Hs00534180_m1, SRC: Hs01082246_m1, STAT3: Hs00374280_m1, 18S rRNA: Hs99999901_s1). SRC, STAT3 and 18S ribosomal RNA primer (Thermo Fisher Scientific). PCR reaction conditions were performed as follows: 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 60 sec.

Cell viability assay

Measurement of cell viability was performed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Cells were seeded onto a 96-well plate at a concentration of 3,000 cells/well and were incubated at 37°C. At 72 h, the optical density was measured at 490 nm using a microtiter plate reader, and the rate of cell survival was expressed as the absorbance. To assess the effect of an inhibitor, cell viability was evaluated 48 h after treatment.

Cell migration assay

Cell migration was evaluated by scratch assay, as described in a previous report (25). Cells were seeded onto a 24-well plate at a concentration of 150,000 cells/well and were incubated at 37°C. At 24 h, the cell monolayer was scratched in a straight line to create a scratch with Cell Scratcher (AGC, Tokyo, Japan). Twenty-four and 48 h later, we measured the width of the scratch using an optical microscope without stain, and calculated the rate of cell migration.

Statistical analysis

All experiments were performed in triplicate and analyzed using JMP Pro 11 (SAS Institute, Inc., Cary, NC, USA). Unpaired Student's t-tests were used for comparisons between two groups. P<0.05 were considered to indicate a statistically significant result. The Chou-Talalay method was used to evaluate synergistic effect of drug combination as described in a previous report (26). Combination index (CI) was calculated based on the effect ratio of cell viability under various drug concentrations using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). CI <0.9, 0.9–1.1 and >1.1 were regarded as synergism, additive effect and antagonism, respectively (27).


Suppressive effects of cell viability are attenuated by long-term ARHGAP35 knockdown in lung adenocarcinoma cell lines

We established lung adenocarcinoma cell clones by continuous exposure to puromycin for a month after introducing shRNA against ARHGAP35 into cells. qRT-PCR (Fig. 1A) and western blot (Fig. 1B) analyses showed that ARHGAP35 was significantly suppressed in these cells. In the MTS assay, cell viability with long-term ARHGAP35 knockdown was comparable to the cell viability without knockdown in all cell lines (Fig. 1C). This suggested that the suppressive effects of cell viability were attenuated by long-term knockdown.

Molecular dynamics in EGFR signaling pathway changes after long-term ARHGAP35 knockdown

We also evaluated the molecular dynamics of the RAS/RAF1/MAPK, STAT, PI3K, MET and ERBB2 pathways (Figs. 2 and 3). Western blots showed increases in SRC and STAT3 total protein, and increased levels of phosphorylated STAT3 in A549 cells after long-term ARHGAP35 knockdown (Fig. 2). PRKCZ, MET and ERBB2 levels were also increased with ARHGAP35 knockdown in the A549 cells (Fig. 2 and data not shown). By contrast, PRKCD, PRKCZ, MET and ERBB2 levels were decreased after long-term ARHGAP35 knockdown in the H1975 cells (Fig. 2 and data not shown). SRC, MEK, STAT3, AKT, PRKCD, PRKCZ and MET levels were decreased with long-term ARHGAP35 knockdown in the PC9 cells (Fig. 2 and data not shown). qRT-PCR showed increased SRC mRNA in A549 cells and decreased SRC (Fig. 3A) and STAT3 (Fig. 3B) mRNA in the H1975 cells. ROCK activity, which is associated with cell migration, was within normal limits in all cell lines with ARHGAP35 knockdown (Fig. 4).

STAT3 inhibitor is more effective after long-term ARHGAP35 knockdown in A549 and PC9 cells

Using MTS assay, we measured cell viability after long-term ARHGAP35 knockdown in cells treated with a STAT3 inhibitor (Fig. 5). A549 and PC9 cells were more sensitive to the STAT3 inhibitor after long-term ARHGAP35 knockdown than when there was no knockdown, but the opposite was noted in the H1975 cells.

Suppressive effect of ARHGAP35 knockdown on migration is maintained for a long time

Using scratch assays, we measured cell migration after long-term ARHGAP35 knockdown in the cell lines (Fig. 6A and B). This demonstrated that cell migratory ability was reduced in cancers with ARHGAP35 knockdown when compared with cancers without knockdown. This was the case for all cell lines, and suggested that the suppressive effect of ARHGAP35 knockdown on migration was maintained for a long time.

SRC inhibitor and STAT3 inhibitor synergistically suppress cell growth in lung adenocarcinoma cell lines

Finally, we measured cell viability under combination treatment with an SRC inhibitor and a STAT3 inhibitor in each cell line (Fig. 7). CIs of SRC inhibitor and STAT3 inhibitor were 0.61, 0.08 and 0.17 for A549, H1975 and PC9 cell lines, respectively; suggesting that the two inhibitors synergistically suppressed cell growth.


In our previous study, we showed that ARHGAP35 knockdown by means of chemically modulated small interfering RNA suppressed the proliferation of lung adenocarcinoma cell lines with EGFR or KRAS mutations (24). In the present study, we obtained clones in which ARHGAP35 was stably knocked down, and we hypothesized that attenuated viability would be seen as a compensatory mechanism for survival in those cells.

RAS activates the RAF1/MAPK pathway and EGFR activates several downstream pathways, including the RAS/RAF1/MAPK, PI3K/AKT and STAT pathways, and these play important roles when regulating proliferation, invasion and migration (28). Among these pathways, ARHGAP35 has been reported to be inactivated by the RAS/RAF1/MAPK pathway and to regulate RhoA (24,29,30). Importantly, ARHGAP35 is also activated by SRC, which is a potential signal mediator of the EGFR pathway (19). We assumed that the RAS/RAF1/MAPK and SRC pathways were compensatory mechanisms. In addition, based on acquired resistance for EGFR-TKIs, we decided to screen MET, ERBB2, PKC, AKT and STAT3 (3133).

Western blots showed increased SRC, STAT3, PRKCZ, MET and ERBB2 levels in A549 cells after long-term ARHGAP35 knockdown. A possible explanation for this is that increased SRC, MET and ERBB2 might have accelerated STAT3 and PRKCZ downstream, ultimately attenuating ARHGAP35 knockdown. This was supported by the RT-qPCR findings. In contrast to this, the protein and mRNA levels of the molecules associated with the EGFR pathway were decreased in both the H1975 and PC9 cell lines after long-term ARHGAP35 knockdown. We therefore assume the involvement of another pathway.

Levels of STAT3 and phosphorylated STAT3 increased in the A549 cell line after long-term ARHGAP35 knockdown. STAT3 is a 92-kDa protein, encoded by STAT3 on 17q21, and is a member of the STAT family of transcriptional activators (3436). This protein is associated with cell proliferation, differentiation, invasion and apoptosis in the EGFR signaling pathway (3638). In lung cancer, STAT3 promotes cell proliferation and invasion, while its inhibition leads to the suppression of tumor proliferation in vitro and in vivo (3941).

After long-term ARHGAP35 knockdown, MTS assay indicated that A549 cells were more sensitive to the STAT3 inhibitor. This was consistent with the hypothesis that increased STAT3 levels in A549 cells after long-term ARHGAP35 knockdown would be a critical factor for cell survival. Similar but modest results were obtained in the MTS assay for the PC9 cell line, although STAT3 was not increased after long-term ARHGAP35 knockdown. A possible explanation for the different impact of the inhibitor in these two cell lines may be the different dependency on the STAT3 pathway for proliferation. We speculate that A549 cells depended heavily on the STAT3 pathway as a compensatory mechanism for viability. This presented the possibility of tumor suppression by combining STAT3 inhibition and ARHGAP35 inhibition for lung cancer with the KRAS mutation.

In contrast to A549 cells, however, opposite results were obtained in H1975 cells after STAT3 inhibition. Although we cannot explain these phenomena, we speculate that: i) Any specific underlying molecular mechanisms may cause unexpected results after STAT3 inhibition in lung cancer with acquired resistance for the EGFR mutation or ii) sh-Neg treatment may become a stress for H1975 since elevated protein levels of protein kinase C delta (PRKCD) and zeta (PRKCZ) in sh-Neg treated H1975 cells were observed (Fig. 2). The former could be important because STAT3 inhibitors have been studied in clinical trials for both hematological and solid malignancies (42,43). For the latter, PKCs have been reported to be upregulated by Toll-like receptors and positively regulate STAT3 in response to stress (44,45). Accordingly, sh-Neg-treated H1975 cells may have relatively strong dependence to STAT3 and respond strongly to STAT3 inhibitor. Our results that the ARHGAP35 protein level was slightly decreased and the STAT3 protein level was increased by sh-Neg treatment in H1975 cells (Figs. 1 and 2) and discrepancy between mRNA expression and protein translation for SRC and STAT3 (Figs. 1 and 3) may be affected by same reason. However, our important finding is that STAT3 inhibitor and SRC inhibitor synergistically suppressed cell growth even in EGFR-mutant cells (Fig. 7).

ARHGAP35 is poor therapeutic target because it lacks a kinase domain. A natural alternative strategy is therefore to target an upstream mediator, such as SRC, that can suppress ARHGAP35 activity. Unfortunately, a phase II clinical trial of a single-use SRC inhibitor for lung cancer has presented only modest clinical benefit (46). Based on the concept of synthetic lethality, we therefore, tested the effects of cosuppression of the SRC/ARHGAP35 axis with both a STAT3 inhibitor and an SRC inhibitor in vitro. Concomitant treatment caused a synergistic and strong effect on growth inhibition in the KRAS- and EGFR-mutant cell lines, especially in the KRAS-mutant cell line. Thus, cosuppression of the STAT3 pathway and SRC/ARHGAP35 axis may be an effective strategy for treating KRAS and EGFR mutant lung adenocarcinoma. A further study is needed to confirm whether this effect can be generalized to other cell lines.

Notably, a synergistic effect was observed for the SRC and STAT3 inhibitors in both EGFR-mutant cell lines, despite the fact that the H1975 cell line is usually resistant to STAT3 inhibitors and the PC9 cell line is relatively resistant to SRC inhibitors. Unfortunately, we cannot illuminate the mechanism underlying this synergistic effect, but based on previous reports of synthetic lethality, it is possible that mitosis regulation or DNA duplication systems could be involved (11,12,14). In a previous research, ARHGAP35 has been shown to regulate mitosis by controlling RhoA (47). In addition, a recent report showed that STAT3 upregulated TPX2, a microtubule-associated protein known to be involved in mitosis, by binding the 5′-flanking sequence of the TPX2 gene (48). Further study is warranted on this topic as STAT3 and SRC have many potential targets.

In our previous study, short-term ARHGAP35 knockdown was also shown to suppress migration in lung cancer (24). In the present study, cell migration ability was reduced in all three cell lines after ARHGAP35 knockdown, suggesting that the suppressive effect on migration may be maintained in the long term. ROCK activity was within normal limits, however, so this conflicting result will need to be resolved in future research.

In conclusion, cosuppression of STAT3 and the SRC/ARHGAP35 axis may be an effective strategy for treating KRAS- and EGFR-mutant lung adenocarcinoma.


Not applicable.


No funding was received.

Availability of data and materials

The analysed datasets generated during the present study are available from the corresponding author on reasonable request.

Authors' contributions

KO and HN performed the experiments. KO wrote the manuscript. AS and CE made substantial contributions to conception, design and intellectual content of the studies. TW, YM and MN made key contributions to the analysis and interpretation of the data. AS and YO reviewed and edited the manuscript. YO also contributed to the planning of the research. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



Committee for Scientific Affairs, The Japanese Association for Thoracic Surgery, ; Masuda M, Kuwano H, Okumura M, Amano J, Arai H, Endo S, Doki Y, Kobayashi J, Motomura N, Nishida H, et al: Thoracic and cardiovascular surgery in Japan during 2012: Annual report by The Japanese Association for thoracic surgery. Gen Thorac Cardiovasc Surg. 62:734–764. 2014. View Article : Google Scholar : PubMed/NCBI


Herbst RS and Shin DM: Monoclonal antibodies to target epidermal growth factor receptor-positive tumors: A new paradigm for cancer therapy. Cancer. 94:1593–1611. 2002. View Article : Google Scholar : PubMed/NCBI


Morita S, Okamoto I, Kobayashi K, Yamazaki K, Asahina H, Inoue A, Hagiwara K, Sunaga N, Yanagitani N, Hida T, et al: Combined survival analysis of prospective clinical trials of gefitinib for non-small cell lung cancer with EGFR mutations. Clin Cancer Res. 15:4493–4498. 2009. View Article : Google Scholar : PubMed/NCBI


Mazières J, Peters S, Lepage B, Cortot AB, Barlesi F, Beau-Faller M, Besse B, Blons H, Mansuet-Lupo A, Urban T, et al: Lung cancer that harbors an HER2 mutation: Epidemiologic characteristics and therapeutic perspectives. J Clin Oncol. 31:1997–2003. 2013. View Article : Google Scholar : PubMed/NCBI


Wang SE, Narasanna A, Perez-Torres M, Xiang B, Wu FY, Yang S, Carpenter G, Gazdar AF, Muthuswamy SK and Arteaga CL: HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell. 10:25–38. 2006. View Article : Google Scholar : PubMed/NCBI


Onitsuka T, Uramoto H, Nose N, Takenoyama M, Hanagiri T, Sugio K and Yasumoto K: Acquired resistance to gefitinib: The contribution of mechanisms other than the T790M, MET, and HGF status. Lung Cancer. 68:198–203. 2010. View Article : Google Scholar : PubMed/NCBI


Yano S, Wang W, Li Q, Matsumoto K, Sakurama H, Nakamura T, Ogino H, Kakiuchi S, Hanibuchi M, Nishioka Y, et al: Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res. 68:9479–9487. 2008. View Article : Google Scholar : PubMed/NCBI


Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG and Halmos B: EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 352:786–792. 2005. View Article : Google Scholar : PubMed/NCBI


Yun CH, Mengwasser KE, Toms AV, Woo MS, Greulich H, Wong KK, Meyerson M and Eck MJ: The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA. 105:2070–2075. 2008. View Article : Google Scholar : PubMed/NCBI


Wright S and Ddbzhansky T: Genetics of natural populations Xii. Experimental reproduction of some of the changes caused by natural selection in certain populations of drosophila-pseudoobscura. Genetics. 31:125–156. 1945.


Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ and Helleday T: Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature. 434:913–917. 2005. View Article : Google Scholar : PubMed/NCBI


Farmer H, McCabe N, Lord CJ, Tutt ANJ, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, et al: : Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 434:917–921. 2005. View Article : Google Scholar : PubMed/NCBI


McLornan DP, List A and Mufti GJ: Applying synthetic lethality for the selective targeting of cancer. N Engl J Med. 371:1725–1735. 2014. View Article : Google Scholar : PubMed/NCBI


Corcoran RB, Cheng KA, Hata AN, Faber AC, Ebi H, Coffee EM, Greninger P, Brown RD, Godfrey JT, Cohoon TJ, et al: Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models. Cancer Cell. 23:121–128. 2013. View Article : Google Scholar : PubMed/NCBI


Lamba S, Russo M, Sun C, Lazzari L, Cancelliere C, Grernrum W, Lieftink C, Bernards R, Di Nicolantonio F and Bardelli A: RAF suppression synergizes with MEK inhibition in KRAS mutant cancer cells. Cell Rep. 8:1475–1483. 2014. View Article : Google Scholar : PubMed/NCBI


Ellis C, Moran M, McCormick F and Pawson T: Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 343:377–381. 1990. View Article : Google Scholar : PubMed/NCBI


Settleman J, Narasimhan V, Foster LC and Weinberg RA: Molecular cloning of cDNAs encoding the GAP-associated protein p190: Implications for a signaling pathway from ras to the nucleus. Cell. 69:539–549. 1992. View Article : Google Scholar : PubMed/NCBI


Brouns MR, Matheson SF and Settleman J: p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation. Nat Cell Biol. 3:361–367. 2001. View Article : Google Scholar : PubMed/NCBI


Chang JH, Gill S, Settleman J and Parsons SJ: c-Src regulates the simultaneous rearrangement of actin cytoskeleton, pl90RhoGAP, and pl20RasGAP following epidermal growth factor stimulation. J Cell Biol. 130:355–368. 1995. View Article : Google Scholar : PubMed/NCBI


Kusama T, Mukai M, Endo H, Ishikawa O, Tatsuta M, Nakamura H and Inoue M: Inactivation of Rho GTPases by p190 RhoGAP reduces human pancreatic cancer cell invasion and metastasis. Cancer Sci. 97:848–853. 2006. View Article : Google Scholar : PubMed/NCBI


Shen CH, Chen HY, Lin MS, Li FY, Chang CC, Kuo ML, Settleman J and Chen RH: Breast tumor kinase phosphorylates p190RhoGAP to regulate rho and ras and promote breast carcinoma growth, migration, and invasion. Cancer Res. 68:7779–7787. 2008. View Article : Google Scholar : PubMed/NCBI


Guo A, Villén J, Kornhauser J, Lee KA, Stokes MP, Rikova K, Possemato A, Nardone J, Innocenti G, Wetzel R, et al: Signaling networks assembled by oncogenic EGFR and c-Met. Proc Natl Acad Sci USA. 105:692–697. 2008. View Article : Google Scholar : PubMed/NCBI


Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, et al: Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 131:1190–1203. 2007. View Article : Google Scholar : PubMed/NCBI


Notsuda H, Sakurada A, Endo C, Okada Y, Horii A, Shima H and Kondo T: p190A RhoGAP is involved in EGFR pathways and promotes proliferation, invasion and migration in lung adenocarcinoma cells. Int J Oncol. 43:1569–1577. 2013. View Article : Google Scholar : PubMed/NCBI


Liang CC, Park AY and Guan JL: In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2:329–333. 2007. View Article : Google Scholar : PubMed/NCBI


Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 22:27–55. 1984. View Article : Google Scholar : PubMed/NCBI


Zhu H, Ding WJ, Wu R, Weng QJ, Lou JS, Jin RJ, Lu W, Yang B and He QJ: Synergistic anti-cancer activity by the combination of TRAIL/APO-2L and celastrol. Cancer Invest. 28:23–32. 2010. View Article : Google Scholar : PubMed/NCBI


Scaltriti M and Baselga J: The epidermal growth factor receptor pathway: A model for targeted therapy. Clin. Cancer Res. 12:5268–5272. 2006.


Chen JC, Zhuang S, Nguyen TH, Boss GR and Pilz RB: Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity. J Biol Chem. 278:2807–2818. 2003. View Article : Google Scholar : PubMed/NCBI


Pullikuth AK and Catling AD: Extracellular signal-regulated kinase promotes Rho-dependent focal adhesion formation by suppressing p190A RhoGAP. Mol Cell Biol. 30:3233–3248. 2010. View Article : Google Scholar : PubMed/NCBI


Appleman LJ: MET signaling pathway: A rational target for cancer therapy. J Clin Oncol. 29:4837–4838. 2011. View Article : Google Scholar : PubMed/NCBI


Moasser MM: The oncogene HER2: Its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene. 26:6469–6487. 2007. View Article : Google Scholar : PubMed/NCBI


Nakamura T, Sakai K, Nakamura T and Matsumoto K: Hepatocyte growth factor twenty years on: Much more than a growth factor. J Gastroenterol Hepatol. 26 Suppl 1:S188–S202. 2011. View Article : Google Scholar


Aggarwal BB, Kunnumakkara AB, Harikumar KB, Gupta SR, Tharakan ST, Koca C, Dey S and Sung B: Signal transducer and activator of transcription-3, inflammation, and cancer: How intimate is the relationship? Ann NY Acad Sci. 1171:59–76. 2009. View Article : Google Scholar : PubMed/NCBI


Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M and Kishimoto T: Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell. 77:63–71. 1994. View Article : Google Scholar : PubMed/NCBI


Zhong Z, Wen Z and Darnell JE Jr: Stat3: A STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science. 264:95–98. 1994. View Article : Google Scholar : PubMed/NCBI


Dauer DJ, Ferraro B, Song L, Yu B, Mora L, Buettner R, Enkemann S, Jove R and Haura EB: Stat3 regulates genes common to both wound healing and cancer. Oncogene. 24:3397–3408. 2005. View Article : Google Scholar : PubMed/NCBI


Turkson J and Jove R: STAT proteins: Novel molecular targets for cancer drug discovery. Oncogene. 19:6613–6626. 2000. View Article : Google Scholar : PubMed/NCBI


Li CJ, Li YC, Zhang DR and Pan JH: Signal transducers and activators of transcription 3 function in lung cancer. J Cancer Res Ther. 9 Suppl 2:S67–S73. 2013. View Article : Google Scholar : PubMed/NCBI


Siveen KS, Sikka S, Surana R, Dai X, Zhang J, Kumar AP, Tan BK, Sethi G and Bishayee A: Targeting the STAT3 signaling pathway in cancer: Role of synthetic and natural inhibitors. Biochim Biophys Acta. 1845:136–154. 2014.PubMed/NCBI


Song L, Rawal B, Nemeth JA and Haura EB: JAK1 activates STAT3 activity in non-small-cell lung cancer cells and IL-6 neutralizing antibodies can suppress JAK1-STAT3 signaling. Mol Cancer Ther. 10:481–494. 2011. View Article : Google Scholar : PubMed/NCBI


Bendell JC, Hong DS, Burris HA III, Naing A, Jones SF, Falchook G, Bricmont P, Elekes A, Rock EP and Kurzrock R: Phase 1, open-label, dose-escalation, and pharmacokinetic study of STAT3 inhibitor OPB-31121 in subjects with advanced solid tumors. Cancer Chemother Pharmacol. 74:125–130. 2014. View Article : Google Scholar : PubMed/NCBI


Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF, Catalano JV, Deininger M, Miller C, Silver RT, et al: A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med. 366:799–807. 2012. View Article : Google Scholar : PubMed/NCBI


Jain N, Zhang T, Kee WH, Li W and Cao X: Protein kinase C δ associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J Biol Chem. 274:24392–24400. 1999. View Article : Google Scholar : PubMed/NCBI


Olejniczak M, Galka P and Krzyzosiak WJ: Sequence-non-specific effects of RNA interference triggers and microRNA regulators. Nucleic Acids Res. 38:1–16. 2010. View Article : Google Scholar : PubMed/NCBI


Johnson FM, Bekele BN, Feng L, Wistuba I, Tang XM, Tran HT, Erasmus JJ, Hwang LL, Takebe N, Blumenschein GR, et al: Phase II study of dasatinib in patients with advanced non-small-cell lung cancer. J Clin Oncol. 28:4609–4615. 2010. View Article : Google Scholar : PubMed/NCBI


Chircop M: Rho GTPases as regulators of mitosis and cytokinesis in mammalian cells. Small GTPases. 5:e297702014. View Article : Google Scholar : PubMed/NCBI


Cocchiola R, Grillo C, Altieri F, Chichiarelli S, Turano C and Eufemi M: Upregulation of TPX2 by STAT3: Identification of a novel STAT3 binding site. PLoS One. 9:e1130962014. View Article : Google Scholar : PubMed/NCBI

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Volume 40 Issue 3

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Online ISSN:1791-2431

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Onodera K, Sakurada A, Notsuda H, Watanabe T, Matsuda Y, Noda M, Endo C and Okada Y: Growth inhibition of KRAS‑ and EGFR‑mutant lung adenocarcinoma by cosuppression of STAT3 and the SRC/ARHGAP35 axis. Oncol Rep 40: 1761-1768, 2018
Onodera, K., Sakurada, A., Notsuda, H., Watanabe, T., Matsuda, Y., Noda, M. ... Okada, Y. (2018). Growth inhibition of KRAS‑ and EGFR‑mutant lung adenocarcinoma by cosuppression of STAT3 and the SRC/ARHGAP35 axis. Oncology Reports, 40, 1761-1768.
Onodera, K., Sakurada, A., Notsuda, H., Watanabe, T., Matsuda, Y., Noda, M., Endo, C., Okada, Y."Growth inhibition of KRAS‑ and EGFR‑mutant lung adenocarcinoma by cosuppression of STAT3 and the SRC/ARHGAP35 axis". Oncology Reports 40.3 (2018): 1761-1768.
Onodera, K., Sakurada, A., Notsuda, H., Watanabe, T., Matsuda, Y., Noda, M., Endo, C., Okada, Y."Growth inhibition of KRAS‑ and EGFR‑mutant lung adenocarcinoma by cosuppression of STAT3 and the SRC/ARHGAP35 axis". Oncology Reports 40, no. 3 (2018): 1761-1768.