Prior acquired resistance to paclitaxel relays diverse EGFR-targeted therapy persistence mechanisms

Abstract

Secondary drug resistance stems from dynamic clonal evolution during the development of a prior primary resistance. This collateral type of resistance is often a characteristic of cancer recurrence. Yet, mechanisms that drive this collateral resistance and their drug-specific trajectories are still poorly understood. Using resistance selection and small-scale pharmacological screens, we find that cancer cells with primary acquired resistance to the microtubule-stabilizing drug paclitaxel often develop tolerance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), leading to formation of more stable resistant cell populations. We show that paclitaxel-resistant cancer cells follow distinct selection paths under EGFR-TKIs by enriching the stemness program, developing a highly glycolytic adaptive stress response, and rewiring an apoptosis control pathway. Collectively, our work demonstrates the alterations in cellular state stemming from paclitaxel failure that result in collateral resistance to EGFR-TKIs and points to new exploitable vulnerabilities during resistance evolution in the second-line treatment setting.

Fig. 1. Paclitaxel resistance primes selection trajectories of cancer cells to EGFR-targeted therapies.

(A) Circos plot visualization of coresistance signatures of 265 drugs screened in 1001 human cancer cell lines processed from the publicly available database of the GDSC. Drugs were classified according to their main target mode of action. Note that coresistance is viewed as either co-occurring bidirectional or one-directional event as identified in the preprocessed data files of binarized response of cell lines to drugs in the GDSC. RTK, receptor tyrosine kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; IGFR, insulin-like growth factor receptor. (B) Radial histogram plot visualization of correlated drugs ranked on the basis of their coresistance classification with paclitaxel resistance derived from their sensitivity response in the panel of cell lines tested in the GDSC datase (left). Discretization threshold of binarized drug response (sensitive or resistant) of paclitaxel and EGFR-TKIs gefitinib, erlotinib, afatinib, lapatinib, and cetuximab (right). (C) Cellular models of paclitaxel resistance used in this study. (D) Characterization of acquired primary paclitaxel and collateral docetaxel resistance in a panel of PTXP (6 cell lines), PTXR (10 cell lines), and their parental lines derived from human and mouse lung and human pancreatic cancers. Stability of resistance was assayed using 21- and 35-day drug holidays. Cells were treated with or without drugs for 72 hours with a concentration dilution series and were assayed for sulforhodamine B (SRB). Representative of three independent experiments. (E) Validation screening of collateral resistance evaluated in a panel of PTXR cell lines with selected drugs representing chemically different classes (selected from a pool of drugs with >80% coresistance classification with paclitaxel) in two PTXR cell lines with the highest resistance (A549-PTXR and PC-3–PTXR). Resistance was assessed on the basis of IC₅₀ fold change relative to parental cell lines assayed as in (D). (F) Characterization of collateral EGFR-TKI (gefitinib, erlotinib, and afatinib) resistance in the same panel of cell lines and assay as in (D). Representative of three independent experiments.

Fig. 2. Experimental model of drug persistence reveals varying resistance stabilities to EGFR-TKIs.

(A) Generation and characterization of reversible drug-tolerant, slow-growing persister A549-, H1993-, and PC-3–derived cells in response to indicated EGFR-TKI–induced selection, expansion, and drug holiday schedules in culture (assayed by SRB). Stability and reversibility of resistance were evaluated using long-term drug holiday schedules (right, bar graph; means ± SD of three biological replicates; *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test). See also Materials and Methods. (B) Characterization of collateral persistence to afatinib and lapatinib in A549-, H1993-, and PC-3–derived gefitinib or erlotinib persisters. Cells were treated with or without drugs for 72 hours with a concentration dilution series and were assayed for SRB. Representative of two independent experiments. (C) Evolution of established A549-, H1993-, and PC-3–derived persisters to gefitinib during a long-term drug holiday. Cells were grown in drug-free media and periodically retested over ~12 weeks for sensitization to EGFR-TKIs (retesting regime: 8 μM gefitinib, 72 hours, assayed by SRB). Representative of two independent experiments. (D and E) Long-term growth of indicated GPs after over ~2 months of stepwise selection to gefitinib to stabilize resistance. Cells were then retested upon treatment in 8 μM gefitinib at indicated times and were assayed by SRB. Values are relative to nontreated. Representative of two independent experiments. (F) Resistance status to both paclitaxel and gefitinib of A549-, H1993-, and PC-3–derived persister pools generated as in (D) and expanded under increasing concentrations of gefitinib. Cells were treated with or indicated concentration of drugs for 72 hours and were assayed by SRB.

Fig. 3. Paclitaxel resistance–induced EMT enriches and facilitates stemness to consequentially drive EGFR-TKI persistence.

(A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of SNAI1 expression in our panel of 6 PTXP and 10 PTXR cell lines and expression of other indicated EMT and stemness markers in indicated A549-, H1993-, and PC-3–derived cells. Values are relative to parental and were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) levels (means ± SD of three biological replicates). Representative of three independent experiments. (B) Schematic of cancer stemness characterization of GPs derived from parental, PTXP, and PTXR lines. (C) Western blot analysis of expression of indicated stemness markers in A549-derived parental, PTXP, and PTXR cells upon NANOG RNA interference (RNAi) for 48 hours. Actin was used as a loading control. Representative of two independent experiments. (D) Schematic of 3D spheroid-like cell formation (herein referred to as “3D cell spheres”) in our in-house built polymeric thin-film 3D culture plates [on the surface of functionalized poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethl cyclotetrasiloxane) (pV4D4)]. 3D sphere formation is characterized with higher stemness as shown in RT-PCR analysis of gene transcripts in A549 cells. (E) Schematic of GP development in A549-derived 3D cell spheres used in (F). GEF, gefitinib; ERL, erlotinib. (F) 3D sphere diameter measurements of indicated A549-derived GPs, grown for 4 days before phase-contrast imaging (top). qRT-PCR analysis of Nanog and Lin28 expressions in the same panel of 3D cell spheres as in the top panel (middle and bottom, respectively). Values are relative to parental control (means ± SD of a representative experiment performed in triplicate; *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test). (G) Schematic of Nanog RNAi and EMT inhibition in 3D cell spheres of A549 rederived GPs used in (H). (H) 3D cell sphere diameter measurements of indicated A549 rederived GP spheres upon Nanog RNAi or EMT inhibition for 3 days (left) or treatment with or without 10 μM LY364947 for 12 hours (right). Values are relative to RNAi or dimethyl sulfoxide control (means ± SD of a representative experiment performed in triplicate; P values by Student’s t test).

Fig. 4. Stemness is functionally associated with a glycolytic metabolism in EGFR-TKI persisters derived from paclitaxel-resistant cancer cells.

(A and B) Characterization of glycolysis and glutaminolysis dependency of indicated PTXP and PTXR cell lines upon treatment with or without 2DG or BPTES with a concentration dilution series and assayed for SRB (means ± SD of three biological replicates). (C) Colony formation assay of parental, PTXP, and PTXR derived from A549 and PC-3 cells following indicated drug treatment regimes (10 mM 2DG, 10 μM BPTES, or 8 μM GNE-140 for 24 hours, followed by 5 μM gefitinib for 72 hours and then equally reseeded in drug-free media following 8 days of culture). Representative of two independent experiments. (D) Representative images of immunofluorescence of CDK8 (green) and GLUT1 (magenta) in indicated A549-derived control and GPs. Nuclei are shown in blue [4′,6-diamidino-2-phenylindole (DAPI)]. Scale bars, 25 μm. Representative of two independent experiments. (E) Glucose titration curves for indicated A549-derived GPs. Values were normalized to growth in 10 mM glucose-containing media (0 mM glucose represents glucose-deprived media; means ± SD of two biological replicates). (F) Western blot analysis of indicated proteins in the mTOR/AMPK pathway in A549-derived GPs upon treatment with or without 10 mM 2DG for indicated times. Tubulin was used as a loading control. Representative of two independent experiments. (G) Western blot analysis of phospho-CAD (Ser¹⁸⁵⁹) [p-CAD^(Ser1859)] and AXL expressions in indicated A549-derived GPs upon treatment with or without LDHA inhibitors GNE-140 (8 μM) or oxamate (40 mM) for 24 hours, followed by treatment with or without 5 μM gefitinib for 24 hours. Tubulin was used as a loading control. Representative of two independent experiments. (H) Sensitivity characterization of indicated A549- and PC-3–derived GPs upon treatment with or without 8 μM GNE-140 or 40 mM oxamate, followed by treatment with gefitinib for 72 hours with a concentration dilution series and assayed for SRB (means ± SD of three biological replicates). (I and J) Western blot analysis of indicated stemness and glycolysis marker expressions in indicated A549-derived GPs upon treatment with or without 10 mM 2DG for 24 hours. A549-PTXR GPs underwent NANOG RNAi for 48 hours, followed by the same 2DG treatment. Tubulin and GAPDH were used as loading controls. Representative of two independent experiments. (K) Phase-contrast images of indicated A549-derived GP 3D cell spheres upon NANOG RNAi for 48 hours, followed by indicated glucose modifications for 12 hours (rescue indicates culture in 10 mM glucose–containing media). Representative of two independent experiments. Scale bars, 70 μm. (L) Glucose uptake, lactate secretion, and glutamate consumption measurements in indicated A549-derived GPs upon NANOG RNAi for 48 hours (RLU, relative luciferase units; means ± SD of three biological replicates; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test).

Fig. 5. An adaptive-like senescence phenotype is a stemness-directed metabolic stress response of EGFR-TKI persisters derived from paclitaxel-resistant cancer cells.

(A) SA-β-gal staining of indicated A549-derived GPs under indicated culture conditions and glucose modification at indicated times, followed by 3-day high-glucose culture for PTXR GPs (bottom). Cells were passaged over ~36 times before experiment. Representative images are shown (left) along with quantified data as percentage (right; means ± SD of three biological replicates; *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test). (B) Representative images of SA-β-gal (green) immunofluorescent labeling using a C12FDG substrate in indicated A549-derived GPs under the indicated culture conditions. Nuclei are shown in blue (DAPI). Scale bars, 20 μm. Representative of two independent experiments. (C) Colony counts of indicated of early serial passages of A549-derived GPs under indicated culture conditions (means ± SD of three biological replicates; *P < 0.05, **P < 0.01, Student’s t test). (D) qRT-PCR analysis of expression of p16^INK4a and other indicated SASP components in indicated A549-derived GPs upon indicated culture conditioning. Values were analyzed relative to actin levels to control for complementary DNA (cDNA) quantity (means ± SD of three biological replicates; *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test). AU, arbitrary units. (E) Western blot analysis of indicated stemness marker and Wnt5a pathway marker expressions in A549-PTXR GPs upon indicated culture conditioning. Tubulin was used as a loading control. Representative of two independent experiments. (F) Caspase 3/7 DEVDase and caspase 9 activities of indicated A549-derived GPs upon indicated culture conditioning following NANOG RNAi for 48 hours (means ± SD of three biological replicates; *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test). OD, optical density. (G) qRT-PCR analysis of interleukin-1α (IL1α) and CXCL1 expressions in A549-PTXR GPs under indicated culture condition upon treatment with or without GSK3βi inhibitor (1 μM) for 48 hours following NANOG RNAi for 48 hours (means ± SD of three biological replicates; *P < 0.05, **P < 0.01, Student’s t test). (H) Transwell Matrigel invasion assay of A549-PTXR GPs under the same condition, treatment, and RNAi as in (G) (means ± SD of three biological replicates; **P < 0.01, ***P < 0.005, Student’s t test). DMSO, dimethyl sulfoxide. (I) Representative images of active β-catenin (gray) in indicated A549-derived GPs under indicated culture conditions upon treatment with or without CHIR99021 (low, 1 μM; high, 5 μM) for 48 hours following NANOG RNAi for 48 hours. Nuclei are shown in blue (DAPI). Scale bars, 25 μm. Representative of two independent experiments.

Fig. 6. Apoptosis evasion by FOXO3a is crucial to initiate and maintain phenotypic transitions to a stable secondary resistance.

(A) Kaplan-Meier plots of RFS of patients with breast cancer, categorized in cohort groups of systemically untreated, chemotherapy-treated, and adjuvant chemotherapy–treated patients (top) and overall and first progression survival of patients with lung cancer (bottom). Patient survival data were stratified by FOXO3a expression (low or high) in their primary tumors. P values were calculated using a log-rank test. (B) qRT-PCR analysis of FOXO3a expression in FFPE tumor tissue sections from patients with breast cancer who underwent sequential multidrug chemotherapy. Log-transformed gene expression values are relative to the sample with the lowest FOXO3a expression and were normalized to GAPDH levels (means ± SD of two biological replicates). (C) Immunohistochemical analysis of indicated FFPE tumor tissue sections used in (B). Sections were blocked and probed with FOXO3a antibody and detected using a 3,3’-Diaminobenzidine (DAB) chromagen kit (right). Replicate tissue sections were stained with hematoxylin and eosin (H&E) (left). All sections were photographed with an inverted phase-contrast microscope (original magnification, ×200). Scale bars, 150 μm. Representative of two independent experiments. (D) Western blot analysis of indicated apoptotic signal expressions in A549-derived parental, PTXP, PTXR, and indicated GPs. Treatment with 20 nM paclitaxel for 24 hours in parental cells served as a positive control. GAPDH was used as a loading control. Representative of two independent experiments. PARP, poly(adenosine 5´-diphosphate–ribose) polymerase. (E) FOXO reporter activity induced by FOXO3 in the same cells and conditions as in (D). Values represent ratio fold induction normalized reporter activity in the presence of FOXO3 to that in the presence of the negative control expression vector (transfected for 48 hours); all relative to parental control. Reporter activity was measured upon treatment with indicated concentration dilution series of gefitinib for 72 hours in indicated A549-PTXR GPs before plasmid transfections for 48 hours (right; means ± SD of three biological replicates). LU, luciferase units. (F) Western blot analysis of phospho-FOXO3a (T32) expression in indicated A549-derived persisters upon treatment with 8 μM gefitinib for 72 hours. Representative of two independent experiments. SE, short exposure; LE, long exposure. (G) qRT-PCR analysis of expression of FOXO3a target genes IRS2 and TNFSF10 in the same cells and treatment as in (E). Values are relative to parental and were normalized to GAPDH levels (means ± SD of three biological replicates). (H) Western blot analysis of FOXO3a expression in indicated A549-derived GPs upon FOXO3a RNAi for 48 hours. Tubulin was used as a loading control. Representative of two independent experiments. (I) Western blot analysis of active FOXO3a expression in A549-PTXR GPs upon transfection with indicated plasmids for 36 hours to induce transcription of an active mutant form of FOXO3a [hemagglutinin (HA)–tagged FOXO3a.TM]. Tubulin was used as a loading control. Representative of two independent experiments. (J and K) Characterization of FOXO3a translocation in indicated A549-derived GPs by Western blotting (J) (α-tubulin and histone were used as loading controls for nuclear and cytoplasmic lysates, respectively) and immunofluorescence (K) (cells were transfected with or without HA-tagged FOXO3a.TM for 36 hours). Representative of two independent experiments. (L) Gefitinib resistance characterization of indicated A549-derived GPs upon FOXO3a RNAi for 48 hours or transfection with control or HA-tagged FOXO3a.TM plasmid for 36 hours following gefitinib treatment for 72 hours with a concentration dilution series and were assayed for SRB. Values are relative to parental control (means ± SD of four biological replicates).

Fig. 7. Schematic overview of our study.