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J.L. Ramírez Serrano
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OA10 - EGFR Mutations (ID 382)
- Event: WCLC 2016
- Type: Oral Session
- Track: Biology/Pathology
- Presentations: 1
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OA10.03 - YAP-NOTCH and STAT3 Signaling Rebound as a Compensatory Response to Gefitinib or Osimertinib Treatment in EGFR Mutant Lung Cancer (ID 4144)
11:20 - 11:30 | Author(s): J.L. Ramírez Serrano
- Abstract
- Presentation
Background:
Preclinical studies provide insights to therapy mechanisms of resistance that are not feasible with clinical studies. We investigated the signaling pathways that could be involved in adaptive resistance to gefitinib and/or osimertinib in EGFR mutant cells.
Methods:
We performed several laboratory methods to examine the signaling pathways involved in EGFR mutations. Signal transduction pathway analysis was designed using the Ingenuity Pathway Analysis (IPA) software (https://www.ingenuity.com/) Figure 1
Results:
Pathways mediating EGFR mutations are: i) ERK1/2 via Ras and MEK1/2 ii) AKT via PI3K and iii) STAT3 via JAK (Figure). By Western blot analysis, phosphorylation of Tyr705 on STAT3 was noted after 2 hours of gefitinib or osimertinib treatment in PC9 and H1975 EGFR mutant cells. Unexpectedly, YAP1 phosphorylation on Tyr357 and Notch activation was detected. Co-targeting STAT3 and Src with gefitinib or osimertinib ablates activation of STAT3 and YAP1-NOTCH3 signaling pathways (Figure). In vitro and in vivo, the combinatory therapy of gefitinib or osimertinib plus TPCA-1 (a dual inhibitor of IKKs and STAT3) plus saracatinib (a SFK inhibitor) leads to significant tumor shrinkage in PC9 and H1975 cells. In tumor samples of 64 EGFR mutant NSCLC patients treated with gefitinib, the median progression free survival (PFS) was significantly shorter in those with high levels of HES1, ALDH1A1, ALDH1A3, Bmi1, AXL, CDCP1, SHP2 and ILK (Figure). However, the mRNA levels of STAT3 and YAP1 stand out in the prediction of shorter PFS with a hazard ratio of 3.02 and 2.57, respectively (P<0.001)
Conclusion:
For the first time ever, we reported gefitinib induced activation of theYAP1-NOTCH signaling pathway, in addition to activation of STAT3, in EGFR mutant cells. Secondly, co-targeting STAT3 and Src, together with EGFR, causes significant tumor growth inhibition, in comparison with gefitinib or osimertinib single therapy.
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-064 - MET-Dependent Activation of STAT3 as Mediator of Resistance to MEK Inhibitors in KRAS-Mutant Lung Cancer (ID 4240)
14:30 - 14:30 | Author(s): J.L. Ramírez Serrano
- Abstract
Background:
Targeting the MAPK pathway by MEK inhibition results in limited activity in patients with KRAS-mutant non-small cell lung cancer (NSCLC). The lack of effectiveness may be associated with activation of other effectors including STAT3, as well as MEK inhibition relief from negative feedback loops. Indeed, in KRAS-mutant colorectal cancer, MEK inhibition decreases the activity of the metalloprotease ADAM17, which normally inhibits MET signaling and STAT3 activation by promoting shedding of MET endogenous antagonist, soluble "decoy" MET. Herein, we explore the MET-dependent activation of STAT3 as a mediator of resistance to MEK inhibitors, and whether MET or STAT3 inhibitors can synergistically increase MEK-inhibitor-induced growth inhibition in KRAS-mutant NSCLC cells in vitro.
Methods:
Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with the allosteric MEK inhibitor, selumetinib, the small-molecule dual inhibitor of the MET and ALK receptor tyrosine kinases, crizotinib, and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit, that exerts an anticancer effect by inhibiting STAT3. RNA was isolated from four KRAS cell lines and the STAT3 and MET mRNA expression analysis was performed by TaqMan based qRT-PCR. Western blotting was used to assess the effect of selumetinb on ERK, AKT and STAT3 phosphorylation.
Results:
We first evaluated the efficacy of the MEK inhibitor selumetinib in our KRAS-mutant NSCLC cell line panel using an MTT cell proliferation assay. H460 cells were relatively insensitive to selumetinib. Following 48-hour treatment with selumetinib, ERK1/2 and AKT phosphorylation were suppressed but a rebound activation of STAT3 occurred in H460 cells. We next investigated whether MET expression was related to the feedback activation of STAT3 signaling following MEK inhibitor treatment. We compared gene expression profiles of the H460 cell line before and after treatment with selumetinib. Interestingly, we found significant upregulation of MET and STAT3 mRNA expression after seven days of selumetinib treatment. To further interrogate the relationship between MEK inhibition and MET-mediated STAT3 reactivation, H460 cells were treated with the combination of selumetinib and crizotinib or selumetinib and evodiamine. A 72-hour exposure to both combinations resulted in a clear cell synergism, as measured by the combination index (CI) analysis, with a CI of 0.79 and 0.78 respectively.
Conclusion:
Collectively our results showed that the feedback STAT3 activation induced by MET, mitigates the effect of MEK inhibition, and provides rationale for further assessment of combined MEK and MET or STAT3 inhibition in KRAS-mutant NSCLC.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-038 - STAT3 and Src-YAP1 Inhibition Results in Greater Necitumumab Sensitivity in Lung Squamous Cell Carcinoma (ID 4242)
14:30 - 14:30 | Author(s): J.L. Ramírez Serrano
- Abstract
Background:
The anti-EGFR monoclonal antibody (mAb), necitumumab, has been recently approved in combination with chemotherapy, as 1st-line treatment for advanced lung squamous cell carcinoma (LSCC) patients, but with minimal survival benefit. Evidence continues to accumulate that signal transducer and activator of transcription 3 (STAT3) is a promising molecular target for cancer therapies. STAT3 is activated by tyrosine phosphorylation in response to EGF and interleukin-6 (IL-6). In addition to STAT3, IL-6 activates the Src family kinases, and subsequently YES-associated protein 1 (YAP1). STAT3 and Src-YAP1 activation contributes to EGFR inhibitor resistance and concomitant targeting of EGFR and STAT3-Src may represent an effective treatment strategy for LSCC.
Methods:
RNA was isolated from six LSCC cell lines and the mRNA expression analysis of EGFR, STAT3, Src and YAP1 was performed by TaqMan based qRT-PCR. Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with necitumumab and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit that exerts an anticancer effect by inhibiting STAT3 and Src. Western blotting was performed to assess the effect of necitumumab on EGFR downstream signaling pathways.
Results:
We first evaluated the expression of EGFR in our panel of LSCC cell lines. We found that almost all of them homogeneously express high levels of EGFR. We then assessed the effect of necitumumab on EGFR downstream signaling in the SK-MES1 cell line. Treatment of SK-MES1 cells with 25ug/ml of necitumumab for seven days was unable to ablate STAT3, Src or YAP1 mRNA expression. Consistent with this, we found that necitumumab suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation on the critical tyrosine residue 705 in a time and dose-dependent manner. We examined the growth inhibitory effect of the necitumumab and evodiamine combination. We performed an MTT cell proliferation assay on SK-MES1 cells and we used a constant ratio drug combination method to determine synergy, additivity, or antagonism. The combination of necitumumab and evodiamine resulted in a clear synergism in SK-MES1 cells as measured by the combination index (CI) analysis, with a CI of 0.74. Experiments in the rest of our LSCC cell lines are ongoing.
Conclusion:
Herein we have examined the role of STAT3 and Src-YAP1 in the context of treatment with the FDA-approved EGFR mAb, necitumumab. Our data provide initial evidence that co-activation of STAT3 and Src-YAP1 may limit the cellular response to EGFR inhibition in LSCC.
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P3.02b - Poster Session with Presenters Present (ID 494)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.02b-047 - Co-Activation of STAT3 and YAP1 Signaling Pathways Limits EGFR Inhibitor Response in Lung Cancer (ID 4168)
14:30 - 14:30 | Author(s): J.L. Ramírez Serrano
- Abstract
Background:
EGFR tyrosine kinase inhibitors (TKIs) induce early activation of several signaling pathways. Interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) hyper-activation occur following EGFR TKI therapy in EGFR-mutant NSCLC cells. We explored the relevance of co-targeting EGFR, STAT3 and Src-YES-associated protein 1 (YAP1) signaling in EGFR-mutant NSCLC.
Methods:
We combined in vitro and in vivo approaches to explore whether concomitant activation of STAT3 and Src-YAP1 can limit the effectiveness of EGFR TKIs in EGFR-mutant NSCLC cells and xenograft models. In two cohorts of EGFR-mutant NSCLC patients, we examined messenger RNA (mRNA) gene expression within signaling pathways, leading to EGFR TKI resistance.
Results:
Gefitinib suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation (pSTAT3-Tyr705). In EGFR mutant cells, gefitinib plus TPCA-1 (STAT3 inhibitor) abolished pSTAT3-Tyr705 but not the YAP1 phosphorylation on tyrosine 357 by Src family kinases (SFKs). The triple combination of gefitinib, TPCA-1 and AZD0530 (SFK inhibitor) ablated both STAT3 and YAP1 phosphorylation and was highly synergistic, according to the combination index. In two EGFR mutant xenograft mouse models, the triple combination of gefitinib, TPCA-1 and AZD0530 markedly and safely suppressed tumor growth. High levels of STAT3 or YAP1 mRNA expression were associated with worse outcome to EGFR TKI in 64 EGFR-mutant NSCLC patients. Median progression-free survival (PFS) was 9.6 (95%CI, 5.9-14.1) and 18.4 months (95%CI, 8.8-30.2) for patients with high and low STAT3 mRNA, respectively (p<0.001), (HR for disease progression, 3.02; 95% CI, 1.54-5.93; p=0.0013). Median PFS was 9.6 (95%CI, 7.7-15.2) and 23.4 months (95%CI, 13.0-28.1) for patients with high and low YAP1 mRNA, respectively (p=0.005), (HR for disease progression, 2.57; 95%CI, 1.30-5.09; p=0.0067). The results were similar in the validation cohort of 55 EGFR-mutant NSCLC patients treated with first-line EGFR TKI in the Department of Oncology of Shanghai Pulmonary Hospital.
Conclusion:
Our study reveals that STAT3 and Src-YAP1 signaling activation occurs following single EGFR TKI in EGFR-mutant NSCLC. STAT3 and YAP1 mRNA levels were significantly predictive of progression-free survival in the original as well as in the validation cohort of EGFR-mutant NSCLC patients. Co-targeting STAT3 and Src in combination with EGFR TKI could substantially improve survival.
