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  • 1806553 Statistical Analysis br The primary end point of the

    2020-08-28

    Statistical Analysis
    The primary end point of the clinical part of the study was to examine the potential effects of gene messenger RNA (mRNA) expression levels on survival. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by a nonparametric log-rank test. In addition to analyzing gene expression as a continuous variable, expression levels were divided into 2 groups according to the median relative expression. A multivariate Cox proportional hazard model was applied with treatment and potential risk factors as covariates, obtaining hazard ratios (HRs) and their 95% confidence interval  Results
    Effect of Single EGFR Inhibition on CSC Markers in EGFR-MutationePositive Cells
    We initially extended our previously published findings in which STAT3 is activated on treatment with EGFR TKIs in EGFR-mutationepositive cells,9-11 with a time-response Western blot experiment. As expected, gefitinib suppressed EGFR, AKT, and ERK1/2 phosphorylation in PC9 cells, but at 6 hours, and more clearly at 24 and 48 hours, it stimulated STAT3 phosphorylation on the critical tyrosine residue 705 (Figure 1A). Similar results were obtained when PC9 1806553 were treated with osimertinib (Figure 1B).
    Cancer Stem Cell Biomarkers
    Figure 3 Continued
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    NOTCH3 cleaved
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    Western blot experiments of protein extracts of H1975 cells treated with osimertinib showed that the drug inhibits EGFR, AKT, and ERK1/2 and increases STAT3 phosphorylation (Figure 1C).
    After confirming our previous findings, and knowing that STAT3 activation increases the ALDHþ cell subpopulation in NSCLC,14 we then explored the effect of EGFR TKIs on CSC marker expression. We reported that afatinib increases the ALDHþ subpopulation in PC9 cells.10 The same occurred when we applied the Aldefluor assay in PC9 and H1975 cells treated with gefitinib or osimertinib. Gefitinib and osimertinib in PC9 and osimertinib in H1975 cells clearly increased the ALDHþ subpopulation (Figure 2A). Specifically, in the untreated PC9 cell line existed 0.8% to 2% of ALDHþ cells, compared to 6% and 40.8% in the gefi-tinib- and osimertinib-treated cells, respectively. Similarly, in the untreated H1975 cell line existed 1.2% of ALDHþ cells, compared to 2.2% in the osimertinib-treated cell line (Figure 2A).
    Next, we analyzed the effect of gefitinib, afatinib, and osimertinib on the expression of several CSC- and EMT-related markers in both the PC9 and H1975 cell lines. The IC50 of gefitinib, afatinib, and osimertinib in PC9 and H1975 cells has previously been re-ported.10,11 Western blot time-response experiments revealed an 
    increase in the expression of most of the CSC and EMT markers analyzed, such as NOTCH3, CD44, Bmi-1, and HES1. CD44 expression was decreased in PC9 cells treated with osimertinib (Figure 2B). Our results indicate that first-, second-, and third-generation EGFR TKIs activate resistance pathways, such as STAT3, and select for a CSC-enriched cell population.
    Effect of Combined EGFR, STAT3, and Src-YAP1 Inhibition on CSC Markers in EGFR-MutationePositive Cells
    Our previous work clearly demonstrated that single EGFR in-hibition in EGFR-mutationepositive cells causes an imbalance in parallel and downstream signaling pathways that ultimately bypass the EGFR blockade.9-11 Cotargeting EGFR, STAT3, and Src-YAP1 causes significant tumor growth inhibition compared to single gefitinib, afatinib, or osimertinib.9-11 Here we tried to see the effect of this combination on CSCs by both Aldefluor assay and Western blot analysis. In the PC9 cell line, gefitinib increased the ALDHþ subpopulation, but we were not able to reverse this phenomenon with the double combination of gefitinib and a STAT3 (TPCA-1) or Src inhibitor (saracatinib or AZD05030). To our surprise, the
    Jordi Codony-Servat et al
    Table 1 Cox Regression Model of PFS Univariate Analysis With HES1, Bmi-1, ALDH1A1, and ALDH1A3 mRNA Expression as Continuous Variables
    Survival Biomarker N Contrast P HR (95% CI)
    Abbreviations: ALDH ¼ aldehyde dehydrogenase; Bmi-1 ¼ B-cellespecific Moloney murine leukemia virus integration site 1; CI ¼ confidence interval; HES1 ¼ target hairy and enhancer of split 1; HR ¼ hazard ratio; mRNA ¼ messenger RNA; OS ¼ overall survival; PFS ¼ progression-free survival.
    triple combination of gefitinib, TPCA-1, and Src inhibitor further increased the ALDHþ subpopulation (Figure 3A). In contrast, in the H1975 cell line, we observed a decrease of the ALDHþ sub-population when osimertinib was combined with TPCA-1 or sar-acatinib. This decrease was even clearer when we treated H1975 cells with the triple combination (Figure 3A). Similarly, by Western blot analysis, we observed decreased levels of cleaved intracellular NOTCH3, Bmi-1, and HES1 with the triple combination in the H1975 cell line, but not in the PC9 cell line (with the exception of cleaved intracellular NOTCH3) (Figure 3B). Thus, combined EGFR, STAT3, and Src-YAP1 inhibition increased the ALDHþ subpopulation in PC9 cells, while a significant decrease was observed when using the triple-drug combination in H1975 cells. Furthermore, CSC marker protein levels were decreased after treatment with the triple-drug combination in H1975 cells but not in PC9 cells. These results indicate the heterogeneity of resistance mechanisms in different tumor types. Finally, to delineate the CSC phenotype of cells with acquired resistance to EGFR TKIs, we investigated by Western blot analysis the expression levels of CSC markers in osimertinib-resistant PC9 cells and compared them to the parental PC9. HES1 up-regulation was the most common molecular event, observed in 4 of the 5 osimertinib-resistant clones, while NOTCH3 and ALDH1A1 were also found in some of the resistant cell lines (Figure 3C).