NSCLC cells acquire resistance to AZD9291 by reducing CAMSAP3

NSCLC cells acquire resistance to AZD9291 by reducing CAMSAP3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NSCLC cells acquire resistance to AZD9291 by reducing CAMSAP3 Huadong Liu, Fei Yang, Zhen Wang, Xiao Han, Jinjin Zhong, Zhanwu Hou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6539573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract AZD9291, also known as Osimertinib, is a highly potent and selective EGFR mutants (including exon 19 deletion, L858R/T790M) inhibitor that significantly inhibits EGFR phosphorylation signaling. However, acquired resistance to AZD9291 is inevitable in the treatment of non-small cell lung cancer (NSCLC). Microtubules, key cytoskeletal components involved in intracellular cargo transport, mediate EGFR-endosomal recycling, yet their specific role in AZD9291 resistance remains to be elucidated. In this study, we found that centrosomal microtubule formation was increased in AZD9291-resistant NSCLC cells, and calmodulin-regulated hemosiderin-associated protein 3 (CAMSAP3) was identified as the key molecule responsible for the change of microtubule morphology. Genetic modulation of CAMSAP3 expression through silencing or overexpression directly altered microtubule architecture and restored AZD9291 sensitivity. Furthermore, we demonstrated that full-length CAMSAP3 is essential for proper localization of the microtubule-dependent endosomal-lysosomal system. CAMSAP3 depletion caused EGFR translocation to the perinuclear microtubule organizing center (MTOC), thereby blocking plasma membrane recycling and promoting lysosomal degradation. These findings establish CAMSAP3 as a key regulator of EGFR signaling and AZD9291 response in NSCLC, suggesting its therapeutic potential for overcoming drug resistance in lung cancer. Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Biological sciences/Cell biology/Cytoskeleton Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION First-generation EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, and second-generation TKIs including afatinib and dacotinib, have consistently demonstrated high efficacy in treating metastatic non-small cell lung cancer with specific EGFR mutations, particularly exon 19 deletions and the L858R mutation [1, 2]. However, drug resistance develops rapidly, with the gatekeeper T790M mutation of the EGFR gene leading to drug resistance to first- and second-generation TKIs [3-5]. AZD9291, a third-generation EGFR TKI that irreversible targets EGFR-T790M mutations, is the preferred therapeutic option for EGFR-mutant NSCLC [6, 7]. However, resistance to AZD9291 is also inevitable. The mechanisms of acquired resistance to AZD9291 are highly heterogeneous, such as T790M deletion, C797S mutation, MET and PTK7 amplification [8-10]. However, the mechanism of AZD9291 resistance remains unknown in approximately 40% of patients. Wild-type EGFR is degraded within a short period of time after activation by ligands such as EGF by endocytosis, a cellular process that selectively internalizes cell surface proteins through plasma membrane invagination into endosomal vesicles for degradation [15, 16]. EGFR-T790M mutant has a propensity to heterodimerize with ErbB2 and defects in the endosome-lysosome pathway, escape CBL-mediated ubiquitylation and subsequent lysosomal degradation, resulting in activated phosphorylated EGFR being trapped in the endosome or recycling to the cell surface, amplifying downstream signals and survival pathways [17]. The recycling endosomes were transported through microtubules, along with other organelles such as the Golgi apparatus and lysosomes, which is involved in cellular processes such as tumor cell migration and invasion [11, 12]. Microtubules regulated EGFR trafficking has been reported to be associated with TKIs resistance [19]. Microtubule protein de-tyrosine promotes kinesin family member 3C (KIF3C) can enhance endosomal recycling of EGFR, leading to prolonged activation of PI3K/Akt/mTOR signaling [13]. Previous studies have shown that faster recycling of EGFR back to the cell surface leads to activation of the downstream ERK1/2 pathway and increased sensitivity to EGFR inhibitors [18]. The formation of centrosomal microtubules is contingent upon the abortive formation of microtubules (called nucleation) and the fixation of the negative end of microtubules at the MTOC (called anchoring) [14-16]. However, noncentrosomal microtubules predominate in epithelial cells. The release of microtubules (MTs) from centrosomes following nucleation appears to be pivotal in the organization of epithelial MTs [17-19]. Several microtubule-binding proteins, particularly CAMSAP3, have been reported to regulate microtubule nucleation and anchoring in epithelial cells. It was found that CAMSAP3 knockdown in NSCLC cell lines H460, A549, and H23 increased the levels of hypoxia-inducible factor-1β (HIF-1β) and its downstream targets vascular endothelial growth factor A (VEGFA), matrix metalloproteinases MMP2 and MMP9, resulting in high invasive capacity in NSCLC [20]. In this study we found CAMSAP3 is a key regulator of microtubule morphology in AZD9291 resistance cells. Depletion of CAMSAP3 altered the AZD9291 sensitivity of H1975 or HCC827 cell lines. Further studies revealed that microtubule morphology as well as the endosomal-lysosomal system exhibited similar localization in drug-resistant cells and CAMSAP3 knockdown cells, suggesting that CAMSAP3 further affects the endosomal-lysosomal system through microtubule network remodeling. In conclusion, our findings establish CAMSAP3 as a key regulator of EGFR signaling and AZD9291 response in NSCLC, suggesting its therapeutic potential for overcoming drug resistance in lung cancer. MATERIALS AND METHODS AZD9291-resistant H1975/HCC827 cell lines The H1975 and HCC827 NSCLC cell lines were purchased from the National Collection of Authenticated Cell Cultures. RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.1 mg/mL streptomycin and 100 U/mL penicillin was used for H1975/HCC827 cells’ culturing. For acquiring drug-resistance cells, H1975/HCC827 cells were cultured in RPMI 1640 medium containing different concentrations of AZD9291 for 4 months. H1975/HCC827 cells were regarded as resistance to AZD9291 when H1975/HCC827 cells could grow well in 1 µmol/L AZD9291 medium. Immunofluorescent staining For immunofluorescent staining experiments, cells were fixed with 100% ice-cold methanol for 5 min at -20°C, then washed three times with PBS, and incubated with 5% bovine serum albumin (BSA, Solarbio, Beijing, China) at room temperature for 1 h. Cells were incubated with primary antibodies for 3 h and secondary antibodies for 1 h. Images of the stained cells were captured using a Super-resolution Confocal Microscope (Leica TCS SP8 STED 3X). ImageJ was employed to analyze fluorescence colocalization. MTT assay MTT assay was performed for cell viability detection. After treatment, the supernatant of cell medium was discarded, and 100 µL of 1 mg/mL MTT solution within serum-free medium was added into each well and incubated at 37°C for 3 h. After that, the supernatant with MTT was discarded, followed by the addition of 100 µL of dimethyl sulfoxide (DMSO, Solarbio, Beijing, China). The absorbance at 490 nm was measured by a microplate reader. Crystal violet staining assay 1x10 3 cells were seeded in 12-well plate and cultured for two days. After treatment, cells were fixed by 4% paraformaldehyde for 7 min and stained by 0.1% crystal violet for 15 min, The picture was obtained using light microscope. After that, 100 µL of 10% acetic acid was used to dissolve crystal violet for cell survival. The absorbance at 570 nm was measured by a microplate reader. Quantitative real‑time PCR (qRT‑PCR) After PBS washes three times, mRNA was extracted by TRIzol Reagent (Invitrogen). 1 µg of RNA was used for cDNA reverse transcription with 5X RT Premix (#AG11706, Accurate Biology). SYBR Green Premix Pro (AG11701, Accurate Biology) was used to amplify genes in ABI 7500 (Applied Biosystems). β-actin was taken as control. The qPCR primers were shown in Supplementary Table S1 . Western blot analysis Western blot analysis After PBS washing, cells were harvested with IP lysis buffer (P0013, Beyotime Biotechnology), supplemented with PMSF and phosphorylation inhibitors. And then the cell lysate was collected into 1.5 mL tubes, followed by centrifugation at 4°C for 10 min at 12000 rpm speed. Equal amounts of protein were separated on SDS-PAGE gels and transferred onto NC membranes (Millipore). The membranes were blocked with 5% milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6) at room temperature for 1 h. After TBST washing, primary antibodies were added and incubated at 4°C overnight, and the secondary antibody was incubated for 1 h at room temperature. ECL kit (Pierce) was used to visualize by a Chem imaging system. Antibodies used are as indicated: CAMSAP3, SAB4200415, Sigma-Aldrich; Tubulin beta, M20005, Abmart; Tubulin gamma, T55405s, Abmart; Phospho-EGFR (Y1068) 3777T, Cell Signaling Technology; EGFR, sc-373746, Santa Cruz Biotechnology; Phospho-ERK1/2 (T202/Y204), TA1015, Abmart; ERK1/2, T40071, Abmart; EEA1, 3288T, Cell Signaling Technology; RAB11A, D4F5, Cell Signaling Technology; GAPDH, AC002, ABclonal; LAMP1, 9091, Cell Signaling Technology. siRNA transfection Specific siRNAs targeting genes of interest were purchased from Shanghai ShengGong (Shanghai, China). Cells were transfected with siRNAs by using INTERFERin® (Polyplus-transfection S.A, Illkirch, France) according to the manufacturer’s protocols. The sequences of siRNA used to knock down the indicated genes were listed in Supplementary Table S2 . Lentiviral shRNA constructs and infection Lentiviral shRNA against CAMSAP3 and scramble control shRNA were constructed according to previous report [ 10 ]. H1975 cells were infected with lentivirus at approximately 60–70% confluence, 10 µg/mL polybrene was added into medium as the same time and incubated for 48 h, then the medium was supplemented with 2 µg/mL puromycin for screening. The sequences of shRNA used to knock down the indicated genes were listed in Supplementary Table S3. Animal experiments To evaluate the impact of CAMSAP3 knock down on AZD9291-resistant NSCLC, we implanted 2 × 10 6 H1975 cells transfected with either control shRNA (shCtrl) or CAMSAP3 shRNA (shCAMSAP3) plasmids into the subcutaneous tissue of 5-week-old female nude mice (GemPharmatech, Chengdu, China) to generate xenograft tumors. The study comprised five mice per group (n = 5). Once the maximum tumor size reached approximately 100 mm 3 , the mice were administered either saline (vehicle control) or AZD9291 (5 mg/kg body weight, once per day, oral gavage). At predetermined time points, the length (L) and width (W) of the tumours were measured. The tumour volume was calculated using the formula: (L x W 2 )/2. Tumours were harvested at the conclusion of the final measurement for the purpose of further analysis. Plasmids transfection For transfection, 1 µg plasmid was added in 200 µL opti-MEM, and then 5 µL PEI transfection reagent was added. The mix were added into the medium without any antibiotic after 15 min of incubation. After 4–6 h, the medium was replaced by fresh complete medium. 48–72 hours later, cells were harvested for analysis. The sequences of the constructed plasmids were shown in Supplementary Table S4. Lysosomal pH assay LysoSensor Green DND-189 (40767ES50, Yeason, China) was used to detect lysosomal pH. Cells were stained with 1 µM Lysotracker Green DND-189 diluted in RPMI-1640 at room temperature for 10 min, washed twice with PBS and added fresh medium. Hoechst 33342 (HY-15559, MCE, China) was used to stain nucleus. Images of the marked cells were captured using a Super-resolution Confocal Microscope (Leica TCS SP8 STED 3X) and FlowJo software were performed to detect and analyze the fluorescence intensity. Statistical analysis Excel was used for statistical analysis. The between-group variance was analyzed by unpaired two-tailed Student’s t test. Data are represented as the mean ± SD from at least three independent experiments. There were statistically significant differences between two groups when p < 0.05. RESULTS Microtubule remodeling accompanies AZD9291 resistance in NSCLC cells Microtubules and their post-translational modifications have been implicated in EGFR transport and chemotherapy resistance in lung squamous cell carcinoma. However, alterations in microtubules and their role in NSCLC resistance to AZD9291 remain unreported. To explore the biological function of microtubules in NSCLC resistance, the microtubule morphology in AZD9291-sensitive (H1975) and resistant (H1975AR) cells were analyzed using β-tubulin immunofluorescence staining. The results revealed that significant morphological differences, including distinct perinuclear microtubule clusters uniquely were observed in H1975AR cells (Fig. 1 A). To test whether this morphological change was cell line dependent, we established another AZD9291 resistance cells (HCC827AR) and similar phenomenon was observed (Fig. S1 A). In contrast, F-actin staining of microfilaments showed no structural differences between the resistant and sensitive cells (Fig. S1 B). To investigate whether the morphological change of in the resistant cells was on account of microtubule aggregation, H1975 and H1975AR cells were treated with nocodazole (a microtubule-depolymerizing agent) to assess post-washout microtubule regeneration. The result showed that microtubules depolymerized in both cell lines upon nocodazole treatment, and nucleation resumed within 5 minutes of nocodazole washout (Fig. 1 A). However, after 60 minutes, microtubules in H1975 sensitive cells dispersed into the cytoplasm, whereas those in resistant cells remained anchored at perinuclear microtubule-organizing centers (MTOCs) (Fig. 1 A). Co-staining with β-tubulin and γ-tubulin confirmed these clusters as centrosomal microtubules, indicating a marked increase in centrosomal microtubule proportion in resistant cells, particularly in H1975AR (Fig. 1 B). To verify the effect of microtubule aggregation in the morphological differences between AZD9291-resistant and sensitive cells, depolymerizing agent nocodazole and stabilizing agent paclitaxel were used for cell disposing. The result showed that short-term (1 hour) paclitaxel or nocodazole exposure disrupted microtubules in both H1975 and H1975AR cells (Fig. S1 C). Prolonged treatment (48 hours) resulted in a more pronounced reduction in cell viability and survival in H1975AR and HCC827AR cells compared to their sensitive counterparts (Fig. 1 C, 1 D), suggesting heightened sensitivity of resistant cells to microtubule-targeting agents. To investigate the targets that give rise to the morphological difference in AZD9291-resistant cell, we analyzed transcriptional levels of microtubule associated genes including CAMSAP1/2/3 , NINEIN , TPX2 , etc., microtubule dynamics genes including SPASTIN , KIF3A , DCTN1 , etc., and centrosome function genes including TUBG1 , NEDD1 , NUMA1 , etc (Fig. 2 A). The result showed that the mRNA level of CAMSAP3 decreased nearly 60% in AZD9291-resistant cells than in sensitive cells, indicating that CAMSAP3 might play important role in centrosomal/non-centrosomal microtubule regulation. CAMSAP3 contributes to AZD9291 resistance in NSCLC In order to further validate the role of CAMSAP3 in NSCLC cell resistance to AZD9291, the protein expression of CAMSAP3 in H1975AR was further assessed via western blot and immunofluorescence staining. Consistent with RT-qPCR results, the protein level of CAMSAP3 markedly reduced in H1975AR cell (Fig. 2 B). Moreover, Kaplan-Meier survival analysis revealed shorter overall survival in lung adenocarcinoma patients with low CAMSAP3 levels, implicating the prognostic relevance between CAMSAP3 expression and lung cancer prograssion (Fig. 2 C). Next, the localization of CAMSAP3 in NSCLC cells was assessed. As shown in Fig. 2 D, co-immunofluorescence staining of CAMSAP3 and β-tubulin demonstrated uniform perinuclear CAMSAP3 distribution with predominant centrosomal microtubule organization in H1975 sensitive cells. In contrast, AZD9291-resistant cells displayed reduced CAMSAP3 expression and retained centrosomal microtubule clustering, a pattern replicated in HCC827AR cells (Fig. S2 A-C). These findings suggested that the centrosomal microtubule aggreation regulater CAMSAP3 was dromatically dedreased in AZD9291 resistance NSCLC, which might effect the lung cancer prograssion. CAMSAP3 modulates AZD9291 sensitivity and microtubule morphology After clear the role of CAMSAP3 in microtubule aggregation, the biological function of CAMSAP3 in NSCLC drug resistance was then assessed. CAMSAP3 was knocked down by siRNA in H1975 and HCC827 cells, as shown in Fig. 3 A and Fig. S3A, CAMSAP3 knockdown efficiency were detected in protein level and mRNA level, respectively. For microtubule morphology, co-immunofluorescence staining of CAMSAP3 and β-tubulin revealed centrosomal microtubule clustering in CAMSAP3-depleted H1975 (Fig. 3 ) and HCC827 cells (Fig. S3B), which was consistent with our preceding hypothesis. Then MTT and crystal violet assay demonstrated increased cell viability and staining intensity in CAMSAP3-knockdown H1975 (Fig. 3 C, 3 D) and HCC827 cells (Fig. S3C, S3D) treated with AZD9291, indicating that CAMSAP3 silencing accelerated AZD9291 resistance. In order to validate the obove findings in vivo , H1975 cells stably expressing control shRNA (shCtrl) or shCAMSAP3 were used for xenograft assay (Fig. S3E). β-tubulin staining confirmed enhanced microtubule clustering in CAMSAP3 stable silencing H1975 cell line (Fig. S3F). Nude mice were treated with 5 mg/kg AZD9291 when subcutaneous tumors volumes reaching ~ 100 mm³ (day 0). Tumor volumes were monitored day 17, until tissues were harvested post-measurement (Fig. 3 E). As shown in Fig. 3 F and Fig. 3 G, AZD9291 reduced tumor volumes in both groups, while the final tumor volumes inhibition ratio revealed that CAMSAP3 knockdown significantly increased the tumor resistance to AZD9291 compared with the control group (Fig. 3 G), which confirming the critical role of CAMSAP3 in AZD9291 resistance modulation. CAMSAP3 was composed of various functional domains, to assess the which component of CAMSAP3 was responsible for the microtubule remodeling and cell resistance modulation, different truncated CAMSAP3, including FLAG-tagged plasmids encoding full-length CAMSAP3 (CAMSAP3-FL), truncations lacking the CH (CAMSAP3-ΔCH) or HCKK domains (CAMSAP3-ΔHCKK), and an HCKK-only construct (CAMSAP3-HCKK) were overexpressed in AZD9291-resistant cells (Fig. 4 A). The cell microtubule morphology staining showed that only CAMSAP3-FL partially restored non-centrosomal microtubule distribution in H1975AR cells (Fig. 4 B, 4 C), though cytoplasmic parallel microtubule organization (as in sensitive cells; Fig. 1 A) was not fully recapitulated. CAMSAP3-ΔHCKK localized aberrantly to microtubule-organizing centers (MTOCs), highlighting the HCKK domain’s necessity for proper CAMSAP3 localization (Fig. 4 C). The function of different truncated CAMSAP3 in AZD9291 sensitivity was then assessed. MTT assay was performed to examine the effect of truncated CAMSAP3s to cell viability. As shown in Fig. 4 D, CAMSAP3-FL overexpression restored AZD9291 sensitivity in resistant cells compared to truncated variants. Notably, transient CAMSAP3-FL transfection induced significant higher cell death ratio, so stable CAMSAP3-FL overexpression cell line could not be established. The above findings suggested that the microtubule networks disruption by forced CAMSAP3 overexpression was critical for AZD9291-resistant cells’ survival. CAMSAP3 disrupts lysosomal and EGFR localization EGFR degradation and downregulation is one of the most important factors contribute to AZD9291 resistance, and the lysosomal pathway is critical for EGFR degradation and downregulation. But the relationship between microtubule-driven lysosomal alterations and AZD9291 resistance hadn’t been thrashed out. Due to the early endosomes (EEA1), lysosomes (LAMP1), and recycling endosomes (RAB11A) were the mainly regulators of the lysosomal alterations, they were stained in H1975 and H1975AR cells, respectively. As shown in Fig. 5 A, EEA1, LAMP1, and RAB11A were well-distributed in cytoplasmic in sensitive H1975 cells, whereas all three organelles were clustered at perinuclear microtubule-organizing centers (MTOCs) in H1975AR and CAMSAP3-knockdown cells. Western blotting showed reduced RAB11A level and elevated LAMP1 levels in H1975AR and CAMSAP3-knockdown cells (except H1975 siCAMSAP3) (Fig. 5 B), indicating that CAMSAP3 deficiency alters endo-lysosomal trafficking and lysosomal protein expression. Mutant EGFRs (exon 19 deletions, T790M/L858R) are typically resistant to degradation due to ubiquitination/lysosomal defects. Surprisingly, EGFR and pEGFR (Y1068) levels greatly reduced both in AZD9291-resistant and CAMSAP3-knockdown H1975 cells (Fig. 6 B). Meanwhile, co-immunofluorescence staining showed that of CAMSAP3 knockdown enhanced the colocalization of pEGFR (Y1068) and LAMP1 both in H1975 (Fig. 5 C) and HCC827 cells (Fig. S4A), demonstrating that CAMSAP3 was associated with EGFR localization in lysosomal. Lysosomal acidification is an important factor of lysosomal activity during protein degradation. LysoSensor Green DND-189 staining (a pH-sensitive indicator) showed a heightened lysosomal acidification in H1975AR and CAMSAP3-knockdown cells (Fig. 5 D), verifying the role of CAMSAP3 in lysosome degradation activity. Next, hydroxychloroquine (CQ) was added for lysosomal activity inhibition, western blotting showed that cells treated with combined CAMSAP3 siRNA and CQ exhibited significantly higher EGFR degradation efficiency than the cells treated with CQ singly (Fig. 5 E, S4 B), confirming that loss of CAMSAP3 promoted lysosomal degradation of mutant EGFR. Since EGFR trafficking was regulated by microtubules, so the localization of EGFR/pEGFR (Y1068) in AZD9291-resistant and sensitive cells were examined next. As shown in Fig. 6 A and Fig. S5A, EGFR/pEGFR predominantly localized to the plasma membrane in sensitive cells. Whereas in resistant cells, EGFR/pEGFR showed perinuclear MTOC accumulation, accompanied by a reduced EGFR/pEGFR level. Afterwards, continuous processing by AZD9291 lead to pEGFR detection in resistant cells, and CAMSAP3 knockdown similarly shifted EGFR/pEGFR to perinuclear MTOCs both in H1975 and HCC827 cells (Fig. 6 A, S5 A). Western blotting confirmed reduced expression or levels of EGFR, pEGFR, and pERK1/2 (T202/Y204) in AZD9291-resistant and CAMSAP3 knockdown cells compared with the sensitive cells (Fig. 6 B, S5 B), affirm the role of CAMSAP3 in EGFR degradation. However, the transcriptional level of EGFR remained unchanged in AZD9291-resistant and CAMSAP3 knockdown cell (Fig. S5C and S5D), suggesting that loss of EGFR was only regulated by post-translational degradation pathway. DISCUSSION AZD9291 has become the first-line treatment choice for EGFR-mutant NSCLC, with greater efficacy and improved overall survival compared to the previous generation of EGFR TKIs [ 8 , 21 ]. The interaction of the microtubule system with tyrosine kinases (TKs) signaling pathways plays a key role in tumor drug resistance [ 22 ]. Tubulin-binding agents (e.g., paclitaxel, docetaxel) inhibit microtubule dynamics by targeting β-tubulin in α/β-tubulin heterodimers, inducing mitotic arrest and apoptosis. These agents, particularly paclitaxel, form the backbone of first-line NSCLC therapy, administered either alone or combined with platinum-based chemotherapy (cisplatin/carboplatin) [ 23 , 24 ]. Preclinical evidences demonstrate that chronic EGFR inhibition triggers non-genetic resistance through TPX2-mediated Aurora kinase A (AURKA) activation, thus combining an AURKA inhibitor with third-generation EGFR-TKI AZD9291 can overcome acquired resistance in NSCLC [ 25 ]. TPX2 and AURKA were involving microtubule nucleation/mitotic spindle assembly [ 26 , 27 ], and our study revealed distinct microtubule localization patterns between drug-sensitive and resistant cells. In sensitive cells, microtubules were released into the cytoplasm following nucleation, whereas in AZD9291-resistant cells, microtubules remained anchored in the perinuclear region post-nucleation. These findings suggest that altered microtubule dynamics may contribute to TKI resistance. Studying microtubule dynamics in drug-resistant cells is an important approach to understanding and overcoming drug resistance. The EGFR signaling pathway is a central driver of tumor growth, but inhibitors like Osimertinib often face resistance [ 28 ]. NSCLC EGFR TKIs resistance was classically linked to secondary EGFR mutations (e.g., T790M/C797S), > 40% of EGFR TKIs resistance cases now originate from tumor cells abandoning EGFR dependency through: 1) ‌Bypass activation‌, including MET amplification-mediated ERBB3-PI3K/AKT signaling, HER2/HER3 heterodimer-driven MAPK activation and AXL-RTK-mediated EMT [2 9 – 31 ]; 2) ‌Downstream mutations‌ (e.g., KRAS G12C/BRAF V600E) that bypass EGFR regulation [32]; or 3) ‌Phenotypic plasticity‌ through tumor stem cell maintenance via WNT/Notch pathways or metabolic reprogramming (e.g., glutamine dependency) [33, 34]. These mechanisms illustrate that EGFR TKIs resistance often emerged when cells survive on EGFR independent manner. Previous studies have demonstrated that AZD9291 promotes EGFR degradation [35]. Consistent with these findings, we observed EGFR degradation in AZD9291-resistant cells, suggesting that EGFR may become dispensable for resistant cell survival. However, the precise molecular mechanisms regulating this degradation process remain incompletely understood. Therefore, further investigation is warranted to elucidate the mechanisms underlying EGFR degradation in drug-resistant cells. EGFR, a transmembrane receptor tyrosine kinase, undergoes EEA1-mediated endocytosis followed by lysosomal degradation or plasma membrane recycling [ 36 , 37 ]. Mutant EGFR evades CBL-mediated ubiquitination and lysosomal degradation, leading to persistent activation of EGFR signaling [ 38 ]. Single-cell sequencing further reveals that EGFR-independent clones in the tumor microenvironment propagate resistance via exosomal miRNAs, underscoring the need to target the tumor ecosystem in TKIs resistant cells [ 39 , 40 ]. In this study, we observed perinuclear accumulation of EGFR at microtubule organizing centers (MTOCs) in AZD9291-resistant NSCLC cells. Mechanistic investigations revealed that CAMSAP3, a microtubule-remodeling protein, promotes EGFR depletion through lysosomal degradation. Specifically, CAMSAP3 knockdown induced aberrant non-centrosomal microtubule assembly, which disrupted normal EGFR trafficking in sensitive cells. This disruption diverted EGFR/pEGFR from the plasma membrane recycling pathway to lysosomal degradation (Fig. 7 ). These findings demonstrated that loss of CAMSAP3 induces NSCLC cell grow on EGFR independent manner, which lead to the acquired resistance to AZD9291. In conclusion, we hypothesized that loss of CAMSAP3 prompts NSCLC cellular resistance to AZD9291 through EGFR independent pathways. The precise molecular mechanisms and regulatory networks underlying CAMSAP3-mediated acquired resistance require further exploration using omics approaches. Declarations AUTHOR INFORMATION These authors contributed equally: Fei Yang, Zhen Wang. Authors and Affiliations Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science, Xi’an Jiaotong University, Xi’an 710049, PR China. Jiangang Long, Fei Yang, Jinjin Zhong, Zhanwu Hou & Shuying Bian School of Life Sciences and Health, University of Health and Rehabilitation Sciences, Qingdao 266113, China. Huadong Liu, Zhen Wang Department of Pathogen Biology, School of Basic Medical Science, Xi'an Medical University, Xi'an 710021, China Xiao Han Contributions Huadong Liu and Jiangang Long conceived and supervised the project. Fei Yang, Zhen Wang acquired and interpreted data, and drafted the manuscript. Xiao Han, Jinjin Zhong, Zhanwu Hou and Shuying Bian conducted the experiments. All authors read and approved the final manuscript version. Corresponding authors Correspondence to Huadong Liu or Jiangang Long. FUNDING This work was supported by research grant from the National Key R&D Program of China (2023YFA1801200), the Natural Science Foundation of Shandong (No. ZR2022LSW003, H.L.). The authors also thank Ruoyuan Liu and Ying Hao at the Instrument Analysis Center of XJTU for their assistance in capturing images of the stained cells. COMPETING INTERESTS The authors declare no competing interests. ETHICS APPROVAL All animal protocols were approved by the Animal Care and Use Committee of the School of Life Science and Technology, Xi’an Jiaotong University (Approval ID: AE-2025-2589). References A. J. Barker, K. H. Gibson, W. Grundy, A. A. Godfrey, J. J. Barlow, M. P. Healy et al. , Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg Med Chem Lett 11 , 1911-1914 (2001). J. D. Moyer, E. 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Wang et al. , TRIB3-EGFR interaction promotes lung cancer progression and defines a therapeutic target. Nat Commun 11 , 3660 (2020). X. Zhang, C. Chen, C. Ling, S. Luo, Z. Xiong, X. Liu et al. , EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis. Cell Death Dis 13 , 934 (2022). D. Palmieri, A. Bouadis, R. Ronchetti, M. J. Merino, P. S. Steeg, Rab11a differentially modulates epidermal growth factor-induced proliferation and motility in immortal breast cells. Breast Cancer Res Treat 100 , 127-137 (2006). K. Shtiegman, B. S. Kochupurakkal, Y. Zwang, G. Pines, A. Starr, A. Vexler et al. , Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling. Oncogene 26 , 6968-6978 (2007). L. V. Sequist, J. von Pawel, E. G. Garmey, W. L. Akerley, W. Brugger, D. Ferrari et al. , Randomized phase II study of erlotinib plus tivantinib versus erlotinib plus placebo in previously treated non-small-cell lung cancer. J Clin Oncol 29 , 3307-3315 (2011). A. N. Hata, M. J. Niederst, H. L. Archibald, M. Gomez-Caraballo, F. M. Siddiqui, H. E. Mulvey et al. , Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat Med 22 , 262-269 (2016). Additional Declarations (Not answered) Supplementary Files Supplementarytables.docx Supplementary tables Originalwesternblots.docx Original western blots FigS1.tif Fig. S1 Representative immunofluorescence staining in H1975/H1975AR and HCC827/HCC827AR cells. (A) Immunofluorescence staining for β-tubulin and γ-tubulin in HCC827/HCC827AR cells. Centrosomes were stained with anti-γ-tubulin antibodies. The arrowheads indicate centrosomal positions. DAPI stains nucleus (Scale bars, 10 μm). (B) Immunofluorescence staining for F-actin in H1975/H1975AR cells. DAPI stains nucleus (Scale bars, 10 μm). (C) Immunofluorescence staining for β-tubulin in H1975 and H1975AR cells treated by nocodazole (1 μg/μL) and pclitaxel (1 μg/μL) for 1 h. DAPI stains nucleus (Scale bars, 10 μm). FigS2.tif Fig. S2 Identification of CAMSAP3 expression by RT-qPCR, western blot and immunofluorescence staining in HCC827/HCC827AR cells. (A) RT-qPCR analysis of CAMSAP1/2/3 mRNA levels in HCC827 and HCC827AR cells (x ± s, n = 3), * p < 0.05, ** p < 0.01. (B) Western blot analysis of CAMSAP3 protein levels in HCC827 and HCC827AR cells (x ± s, n = 3). (C) Co-immunofluorescence staining for β-tubulin and CAMSAP3 in HCC827/HCC827AR cells. The arrowheads indicate centrosomal positions. DAPI stains nucleus (Scale bars, 10 μm). FigS3.tif Fig. S3 Identification of CAMSAP3 knockdown efficiency in HCC827/HCC827AR cells. (A) CAMSAP3 knockdown efficiency in HCC827 cells with the indicated siRNAs (x ± s, n = 3), ** p < 0.01. (B) Co-immunofluorescence staining for β-tubulin and CAMSAP3 in HCC827 cells transfected with the indicated siRNAs. The arrowheads indicate centrosomal positions. DAPI stains nucleus (Scale bars, 10 μm). The number of cells with centrosomal microtubules in Fig. S3B was quantified, in which 100 cells were analyzed per experiment, * p < 0.05. (C) After silencing CAMSAP3 with the specific siRNA for 24 h in HCC827 cells, followed by treatment with 0.1 μM AZD9291 for 48 h, the cell viabilities were detected by MTT (x ± s, n = 6), *** p < 0.001. (D) After silencing CAMSAP3 with the specific siRNA for 24 h in HCC827 cells, followed by treatment with 0.1 μM AZD9291 for 48 h, the cell survival was detected by Crystal Violet Staining (x ± s, n = 3), *** p < 0.001. (E) Stable CAMSAP3 knockdown efficiency with the indicated shRNAs in H1975 cells. (F) Immunostaining for β-tubulin in H1975_shCtrl and H1975_shCAMSAP3 cells. The arrowheads indicate centrosomal positions (Scale bars, 10 μm). FigS4.tif Fig. S4 Identification of the localization and expression of LAMP1 in HCC827/HCC827AR cells. (A) Co-immunofluorescence staining for LAMP1 and pEGFR (Y1068) in HCC827 cells transfected with the indicated siRNAs. DAPI stains nucleus (Scale bars, 20 μm). Co-localizations of LAMP1 and pEGFR (Y1068) were quantified as Manders colocalization coefficients, * p < 0.05. (B) Western blot analysis of for EGFR and pEGFR (Y1068) levels in HCC827 cells transfected with the indicated siRNAs with or without 25 μM CQ treatment for 48 h (x ± s, n = 3). Band intensities were measured and normalized using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bands, * p < 0.05. FigS5.tif Fig. S5 Identification of the mRNA level and protein level of EGFR in HCC827/HCC827AR cells. (A) Double immunostaining for EGFR and pEGFR (Y1068) in HCC827/HCC827AR and HCC827 cells transfected with the indicated siRNA. DAPI stains nucleus (Scale bars, 20 μm). The number of cells with internalization of EGFR into MTOC in Fig. S4C was quantified, in which 50 cells were analyzed per experiment, p < 0.05, ** p < 0.001. (B) Western blot analysis of EGFR and pEGFR (Y1068), ERK1/2 and pERK1/2 (T202/Y204) levels in HCC827/HCC827AR and HCC827 cells transfected with the indicated siRNA (x ± s, n = 3). Band intensities were measured and normalized using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bands, * p < 0.05, p < 0.01, * p < 0.001. (C) RT-qPCR analysis of EGFR mRNA levels in H1975/H1975AR and H1975 cells transfected with the indicated siRNA (x ± s, n = 3). (D) RT-qPCR analysis of EGFR mRNA levels in HCC827/HCC827AR and HCC827 cells transfected with the indicated siRNA (x ± s, n = 3). Cite Share Download PDF Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 10 Jul, 2025 Review # 2 received at journal 30 Jun, 2025 Review # 1 received at journal 16 Jun, 2025 Reviewer # 2 agreed at journal 13 Jun, 2025 Reviewer # 1 agreed at journal 28 May, 2025 Reviewers invited by journal 16 May, 2025 Submission checks completed at journal 28 Apr, 2025 First submitted to journal 27 Apr, 2025 Editor assigned by journal 27 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6539573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":457453949,"identity":"62833288-c24e-47bb-a64d-bc53b2b02db5","order_by":0,"name":"Huadong Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACAyCWAGIZNgbmA0DagoGBh0gtPGwMbAkMDAkSJGgBIgPitJhLJD+88XFHLQ+fRM7Hx7w/JOT4eQ4wfviYg1uL5Yw0Y8uZZ47zsEnkbjbmSZAwluxtYJacuQ2Pw24kmEnzth0DadkmDdSSuOE8AxszL14t6d+gWnKeEaslB2RLDUgLG0TL2QYCWs68Kbac2XaAh43nmbHhnDSgX3oONuP3y/H0jTc+ttXJybcnP3zwxsYGGGLJBz98xKMFCg4jcxgbCKoHgjpiFI2CUTAKRsFIBQBjnUifXVzTRgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Health and Rehabilitation Sciences","correspondingAuthor":true,"prefix":"","firstName":"Huadong","middleName":"","lastName":"Liu","suffix":""},{"id":457453950,"identity":"80e4a000-a964-4463-b1eb-3cb8415f3072","order_by":1,"name":"Fei Yang","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Yang","suffix":""},{"id":457453951,"identity":"013d9db0-7f25-4a67-8932-1c4934d5e6ec","order_by":2,"name":"Zhen Wang","email":"","orcid":"","institution":"University of Health and Rehabilitation Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Wang","suffix":""},{"id":457453952,"identity":"2a4989e6-7914-4b61-b20b-4a248ebafe27","order_by":3,"name":"Xiao Han","email":"","orcid":"","institution":"Xi'an Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Han","suffix":""},{"id":457453953,"identity":"83885d33-4f37-4120-b7c5-f00588e80073","order_by":4,"name":"Jinjin Zhong","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jinjin","middleName":"","lastName":"Zhong","suffix":""},{"id":457453954,"identity":"6ba3b95d-e72e-4b08-86b6-047ecb2f8d19","order_by":5,"name":"Zhanwu Hou","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Zhanwu","middleName":"","lastName":"Hou","suffix":""},{"id":457453955,"identity":"bbb02dcb-e0ec-4dc0-87b4-e429112b5e50","order_by":6,"name":"Shuying Bian","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Shuying","middleName":"","lastName":"Bian","suffix":""},{"id":457453956,"identity":"2498b486-2b0b-4ef5-bf36-9df08ff0a847","order_by":7,"name":"Jiangang Long","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jiangang","middleName":"","lastName":"Long","suffix":""}],"badges":[],"createdAt":"2025-04-27 09:45:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6539573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6539573/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-08299-0","type":"published","date":"2025-12-11T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83214250,"identity":"af9e9f60-9e26-4c46-b195-5c9d509ff23f","added_by":"auto","created_at":"2025-05-21 08:51:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1685754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrotubule dynamics were altered in AZD9291-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence staining for β-tubulin in H1975/H1975AR cells treated by nocodazole (1 μg/μL) for 1 h and washout after 0,5,60 min, respectively. The arrowheads indicate nucleation positions. DAPI stains nucleus (Scale bars, 20 μm). (B) Co-immunofluorescence staining for β-tubulin and γ-tubulin in H1975/H1975AR cells. Centrosomes were stained with anti-γ-tubulin antibodies. The arrowheads indicate the positions of centrosomes. DAPI stains nucleus (Scale bars, 10 μM). The number of cells with a cluster of microtubules in Fig. 1B was quantified, in which 100 cells were analyzed per experiment, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (C) The cell viability of H1975/H1975AR and HCC827/HCC827AR cells after treated by AZD9291, nocodazole and pclitaxel for 48 h was measured by MTT assays (x ± s, n = 6), **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (D) Inhibition of the cell survival of H1975/H1975AR and HCC827/HCC827AR cells by AZD9291 (1 μM for H1975/H1975AR; 100 nM for HCC827/HCC827AR), nocodazole (100 ng/μL) and pclitaxel (10 ng/μL) for 48 h was measured by Crystal violet staining assay (x ± s, n = 3), **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/1481720b73019920336c8b19.jpg"},{"id":83213105,"identity":"f532937f-75f9-4a75-9c91-027cf9c77c05","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1174895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAMSAP3 was downregulated in both H1975AR cells and patients with poor prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(A) RT-qPCR analysis of the expression of microtubule-related genes in H1975 and H1975AR cells (x ± s, n = 3), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (B) Western blot analysis to detect the CAMSAP3 protein levels in H1975 and H1975AR cells (x ± s, n = 3). (C) Kaplan-Meier survival analysis of lung cancer patients with high and low CAMSAP3 expression from GEPIA database. (D) Co-immunofluorescence staining for β-tubulin and CAMSAP3 in H1975/H1975AR cells, the arrowheads indicated the positions of centrosomes. DAPI stains nucleus (Scale bars, 10 μm). \u003csup\u003e** \u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.01,\u003csup\u003e *** \u003c/sup\u003e\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.01.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/652995e55ed2777747e37300.jpg"},{"id":83213107,"identity":"fb1633a1-087d-4a6c-8753-4f13d203e2b3","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1261640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAMSAP3 knockdown recapitulated the altered microtubule formation and drug sensitivity seen in AZD9291-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) CAMSAP3 knockdown efficiency in H1975 cells with the indicated siRNAs (x ± s, n = 3), * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. (B) Co-immunofluorescence staining of β-tubulin and CAMSAP3 in H1975 cells transfected with the indicated siRNAs. The arrowheads indicate the position of centrosomes. DAPI stains nucleus (Scale bars, 10 μm). The number of cells with centrosomal microtubules in Fig. 3B was quantified, in which 100 cells were analyzed per experiment, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (C) The cell viabilities of CAMSAP3 silencing H1975 and HCCC827 cells were detected by MTT assay after treated with AZD9291 for 48 h, (x ± s, n = 6), ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (D) The Crystal violet staining assay were performed for CAMSAP3 silencing H1975 and HCCC827 cells after treated with AZD9291 for 48 h (x ± s, n = 3), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (E) Diagram of a mouse xenograft treated with AZD9291. (F) Tumor volume was measured on days 6, 10, 12, 14, and 17 after implantation. (G) The tumors were dissected and pictured at the end of the experiment. The rate of tumor volume inhibition of last tumor volume in Fig. 3G was quantified (x ± s, n = 5), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/160ada0d95179219618dd521.jpg"},{"id":83213109,"identity":"6ceefbae-00c3-41bd-908e-74704130f110","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1303368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of CAMSAP3 in AZD9291-resistant cells rescued the altered microtubule formation and enhanced the drug sensitivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Structures of the full-length CAMSAP3 (CAMSAP3-FL) and its three truncated forms (CAMSAP3-ΔCH; CAMSAP3-ΔHCKK; CAMSAP3-HCKK), tagged with FLAG. The region used as the antigen for generating anti-CAMSAP3 antibodies. (B) Western blots results of CAMSAP3 (CAMSAP3-FL) and its three truncated forms expressed in H1975AR cells (x ± s, n = 3). The arrowheads indicate correct protein sizes positions. (C) Co-immunofluorescence staining of FLAG and β-tubulin in H1975AR cells transfected with FLAG-tagged CAMSAP3 and its three truncated form plasmids. DAPI stains nucleus (Scale bars, 20 μm). (D) The cell viability of CAMSAP3 and its three truncated forms overexpressed H1975AR cells after treated with 1 μΜ AZD9291 for 48 h was measured by MTT assays (x ± s, n = 6), ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/f16715d170ff711586136adc.jpg"},{"id":83215269,"identity":"ea3d6ff2-9c5c-46d0-b2e5-86853dbe8775","added_by":"auto","created_at":"2025-05-21 08:59:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1399163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAMSAP3 knockdown recapitulated the endosome-lysosome dislocation seen in AZD9291-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescencestaining for EEA1, RAB11A and LAMP1 in H1975/H1975AR cells transfected with the indicated siRNA. DAPI stains nucleus (Scale bars, 20 μm). (B) Western blot analysis of for EEA1, RAB11A and LAMP1 protein levels in H1975/H1975AR cells transfected with the indicated siRNA (x ± s, n = 3). (C) Co-immunofluorescence staining for LAMP1 and pEGFR (Y1068) in H1975 cells transfected with the indicated siRNAs. DAPI stains nucleus (Scale bars, 20 μm). Colocalization of LAMP1 and pEGFR (Y1068) were quantified as Manders colocalization coefficients, *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05. (D) Lysosomal pH was marked by LysoSensor Green DND-189 in H1975/H1975AR and H1975 cells transfected with the indicated siRNA. Hoechst 33342 was used to stain nucleus. (Scale bars, 25 μm). Relative fluorescence intensity in Fig. 6D was quantified, in which 50 cells were analyzed per experiment, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (E) Western blot analysis of for EGFR and pEGFR (Y1068) levels in H1975 cells transfected with the indicated siRNAs with or without 25 μM CQ treatment for 48 h (x ± s, n = 3). Band intensities were normalized by GAPDH, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/e45705526fda7911ed0fed5e.jpg"},{"id":83213112,"identity":"3a34c75e-9bc8-407d-892b-9e47d73b6bc0","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1613421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAMSAP3 knockdown recapitulated the EGFR dislocation seen in AZD9291-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunofluorescence staining of EGFR and pEGFR (Y1068) in H1975 and H1975AR cells transfected with the indicated siRNA, DAPI stains nucleus (Scale bars, 20 μm). (B) Western blot analysis the levels of EGFR and pEGFR (Y1068), ERK1/2 and pERK1/2 (T202/Y204) in H1975/H1975AR cells transfected with the indicated siRNA (x ± s, n = 3). Bands intensities were measured and normalized by the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (C) Co-immunofluorescence staining of EGFR and β-tubulin in H1975/H1975AR cells transfected with the indicated siRNA. DAPI stains nucleus (Scale bars, 10 μm). The number of cells with internalization of EGFR into MTOC in Fig. 5C was quantified, in which 50 cells were analyzed per experiment, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/069c84fe1a675676f3022492.jpg"},{"id":83213120,"identity":"2e6e4304-da62-4c1a-9f74-5fcb68e9ac49","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":380207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the CAMSAP3-regulated the different distribution of microtubules as well as endosome-lysosomes in AZD9291-sensitive and resistant NSCLC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCAMSAP3 knockdown in AZD9291-sensitive cells resulted in a significant increase in the proportion of non-centrosomal microtubules, which in turn altered the internalization/degradation pathway of EGFR/pEGFR, preventing EGFR/pEGFR from recycling to the cytoplasmic membrane, similar to AZD9291-resistant cells.\u003c/p\u003e","description":"","filename":"Fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/1c5029561957c7d6c7a78880.jpg"},{"id":101032195,"identity":"4c174036-7241-4f3a-b435-8e8ee8f64ee5","added_by":"auto","created_at":"2026-01-24 08:07:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10076000,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/eee07c06-78be-4853-81a5-d4acf0bc2510.pdf"},{"id":83215267,"identity":"99c34a0d-3560-4504-931b-2ee687379088","added_by":"auto","created_at":"2025-05-21 08:59:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27398,"visible":true,"origin":"","legend":"Supplementary tables","description":"","filename":"Supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/84c6f1a74cd6abea03d7e110.docx"},{"id":83214252,"identity":"031a6653-9674-4d3d-9e44-74f9cc035410","added_by":"auto","created_at":"2025-05-21 08:51:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1528141,"visible":true,"origin":"","legend":"Original western blots","description":"","filename":"Originalwesternblots.docx","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/10bc90d4e7d68d686039b3fa.docx"},{"id":83214255,"identity":"67f29efd-e272-4585-9a6e-b68a9b3f8ce3","added_by":"auto","created_at":"2025-05-21 08:51:06","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4307760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Representative immunofluorescence staining in H1975/H1975AR and HCC827/HCC827AR cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence staining for β-tubulin and γ-tubulin in HCC827/HCC827AR cells. Centrosomes were stained with anti-γ-tubulin antibodies. The arrowheads indicate centrosomal positions. DAPI stains nucleus (Scale bars, 10 μm). (B) Immunofluorescence staining for F-actin in H1975/H1975AR cells. DAPI stains nucleus (Scale bars, 10 μm). (C) Immunofluorescence staining for β-tubulin in H1975 and H1975AR cells treated by nocodazole (1 μg/μL) and pclitaxel (1 μg/μL) for 1 h. DAPI stains nucleus (Scale bars, 10 μm).\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/8a6655cb9152613b54ae9164.tif"},{"id":83213113,"identity":"9571c5f0-ae10-4b82-b8ef-bc6983c11572","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2042636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 Identification of CAMSAP3 expression by RT-qPCR, western blot and immunofluorescence staining in HCC827/HCC827AR cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR analysis of \u003cem\u003eCAMSAP1/2/3\u003c/em\u003e mRNA levels in HCC827 and HCC827AR cells (x ± s, n = 3), *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (B) Western blot analysis of CAMSAP3 protein levels in HCC827 and HCC827AR cells (x ± s, n = 3). (C) Co-immunofluorescence staining for β-tubulin and CAMSAP3 in HCC827/HCC827AR cells. The arrowheads indicate centrosomal positions. DAPI stains nucleus (Scale bars, 10 μm).\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/b2b531b44f79bd5af0092c60.tif"},{"id":83213129,"identity":"c54867cf-c4fc-4f53-a2b4-f2cfac170168","added_by":"auto","created_at":"2025-05-21 08:43:06","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2860586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3 Identification of CAMSAP3 knockdown efficiency in HCC827/HCC827AR cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) CAMSAP3 knockdown efficiency in HCC827 cells with the indicated siRNAs (x ± s, n = 3), **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (B) Co-immunofluorescence staining for β-tubulin and CAMSAP3 in HCC827 cells transfected with the indicated siRNAs. The arrowheads indicate centrosomal positions. DAPI stains nucleus (Scale bars, 10 μm). The number of cells with centrosomal microtubules in Fig. S3B was quantified, in which 100 cells were analyzed per experiment, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. (C) After silencing CAMSAP3 with the specific siRNA for 24 h in HCC827 cells, followed by treatment with 0.1 μM AZD9291 for 48 h, the cell viabilities were detected by MTT (x ± s, n = 6), ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (D) After silencing CAMSAP3 with the specific siRNA for 24 h in HCC827 cells, followed by treatment with 0.1 μM AZD9291 for 48 h, the cell survival was detected by Crystal Violet Staining (x ± s, n = 3), ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (E) Stable CAMSAP3 knockdown efficiency with the indicated shRNAs in H1975 cells. (F) Immunostaining for β-tubulin in H1975_shCtrl and H1975_shCAMSAP3 cells. The arrowheads indicate centrosomal positions (Scale bars, 10 μm).\u003c/p\u003e","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/927cb7852d898afc9a20934d.tif"},{"id":83215272,"identity":"72d01e6a-bb41-4f36-b42f-73a394736235","added_by":"auto","created_at":"2025-05-21 08:59:06","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1897502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4 Identification of the localization and expression of LAMP1 in HCC827/HCC827AR cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Co-immunofluorescence staining for LAMP1 and pEGFR (Y1068) in HCC827 cells transfected with the indicated siRNAs. DAPI stains nucleus (Scale bars, 20 μm). Co-localizations of LAMP1 and pEGFR (Y1068) were quantified as Manders colocalization coefficients, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (B) Western blot analysis of for EGFR and pEGFR (Y1068) levels in HCC827 cells transfected with the indicated siRNAs with or without 25 μM CQ treatment for 48 h (x ± s, n = 3). Band intensities were measured and normalized using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bands, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/2ef6a087ba08cb1c1434f56c.tif"},{"id":83214266,"identity":"bd0ef2fd-a011-464f-b750-ea30bd81d97a","added_by":"auto","created_at":"2025-05-21 08:51:07","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2941406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S5 Identification of the mRNA level and protein level of EGFR in HCC827/HCC827AR cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(A) Double immunostaining for EGFR and pEGFR (Y1068) in HCC827/HCC827AR and HCC827 cells transfected with the indicated siRNA. DAPI stains nucleus (Scale bars, 20 μm). The number of cells with internalization of EGFR into MTOC in Fig. S4C was quantified, in which 50 cells were analyzed per experiment, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (B) Western blot analysis of EGFR and pEGFR (Y1068), ERK1/2 and pERK1/2 (T202/Y204) levels in HCC827/HCC827AR and HCC827 cells transfected with the indicated siRNA (x ± s, n = 3). Band intensities were measured and normalized using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bands,\u003csup\u003e * \u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (C) RT-qPCR analysis of \u003cem\u003eEGFR\u003c/em\u003e mRNA levels in H1975/H1975AR and H1975 cells transfected with the indicated siRNA (x ± s, n = 3). (D) RT-qPCR analysis of \u003cem\u003eEGFR\u003c/em\u003e mRNA levels in HCC827/HCC827AR and HCC827 cells transfected with the indicated siRNA (x ± s, n = 3).\u003c/p\u003e","description":"","filename":"FigS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6539573/v1/d0dd5725dc0fc58e80303b63.tif"}],"financialInterests":"(Not answered)","formattedTitle":"NSCLC cells acquire resistance to AZD9291 by reducing CAMSAP3","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFirst-generation EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, and second-generation TKIs including afatinib and dacotinib, have consistently demonstrated high efficacy in treating metastatic non-small cell lung cancer with specific EGFR mutations, particularly exon 19 deletions and the L858R mutation [1, 2]. However, drug resistance develops rapidly, with the gatekeeper T790M mutation of the EGFR gene leading to drug resistance to first- and second-generation TKIs [3-5]. AZD9291, a third-generation EGFR TKI that irreversible targets EGFR-T790M mutations, is the preferred therapeutic option for EGFR-mutant NSCLC [6, 7]. However, resistance to AZD9291 is also inevitable. The mechanisms of acquired resistance to AZD9291 are highly heterogeneous, such as T790M deletion, C797S mutation, MET and PTK7 amplification [8-10]. However, the mechanism of AZD9291 resistance remains unknown in approximately 40% of patients.\u003c/p\u003e\n\u003cp\u003eWild-type EGFR is degraded within a short period of time after activation by ligands such as EGF by endocytosis, a cellular process that selectively internalizes cell surface proteins through plasma membrane invagination into endosomal vesicles for degradation [15, 16]. EGFR-T790M mutant has a propensity to heterodimerize with ErbB2 and defects in the endosome-lysosome pathway, escape CBL-mediated ubiquitylation and subsequent lysosomal degradation, resulting in activated phosphorylated EGFR being trapped in the endosome or recycling to the cell surface, amplifying downstream signals and survival pathways [17]. The recycling endosomes were transported through microtubules, along with other organelles such as the Golgi apparatus and lysosomes, which is involved in cellular processes such as tumor cell migration and invasion [11, 12]. Microtubules regulated EGFR trafficking has been reported to be associated with TKIs resistance [19]. Microtubule protein de-tyrosine promotes kinesin family member 3C (KIF3C) can enhance endosomal recycling of EGFR, leading to prolonged activation of PI3K/Akt/mTOR signaling [13]. Previous studies have shown that faster recycling of EGFR back to the cell surface leads to activation of the downstream ERK1/2 pathway and increased sensitivity to EGFR inhibitors [18].\u003c/p\u003e\n\u003cp\u003eThe formation of centrosomal microtubules is contingent upon the abortive formation of microtubules (called nucleation) and the fixation of the negative end of microtubules at the MTOC (called anchoring) [14-16]. However, noncentrosomal microtubules predominate in epithelial cells. The release of microtubules (MTs) from centrosomes following nucleation appears to be pivotal in the organization of epithelial MTs [17-19]. Several microtubule-binding proteins, particularly CAMSAP3, have been reported to regulate microtubule nucleation and anchoring in epithelial cells. It was found that CAMSAP3 knockdown in NSCLC cell lines H460, A549, and H23 increased the levels of hypoxia-inducible factor-1\u0026beta; (HIF-1\u0026beta;) and its downstream targets vascular endothelial growth factor A (VEGFA), matrix metalloproteinases MMP2 and MMP9, resulting in high invasive capacity in NSCLC [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study we found CAMSAP3 is a key regulator of microtubule morphology in AZD9291 resistance cells. Depletion of CAMSAP3 altered the AZD9291 sensitivity of H1975 or HCC827 cell lines. Further studies revealed that microtubule morphology as well as the endosomal-lysosomal system exhibited similar localization in drug-resistant cells and CAMSAP3 knockdown cells, suggesting that CAMSAP3 further affects the endosomal-lysosomal system through microtubule network remodeling. In conclusion, our findings establish CAMSAP3 as a key regulator of EGFR signaling and AZD9291 response in NSCLC, suggesting its therapeutic potential for overcoming drug resistance in lung cancer.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eAZD9291-resistant H1975/HCC827 cell lines\u003c/h2\u003e \u003cp\u003eThe H1975 and HCC827 NSCLC cell lines were purchased from the National Collection of Authenticated Cell Cultures. RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.1 mg/mL streptomycin and 100 U/mL penicillin was used for H1975/HCC827 cells\u0026rsquo; culturing. For acquiring drug-resistance cells, H1975/HCC827 cells were cultured in RPMI 1640 medium containing different concentrations of AZD9291 for 4 months. H1975/HCC827 cells were regarded as resistance to AZD9291 when H1975/HCC827 cells could grow well in 1 \u0026micro;mol/L AZD9291 medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e \u003cp\u003eFor immunofluorescent staining experiments, cells were fixed with 100% ice-cold methanol for 5 min at -20\u0026deg;C, then washed three times with PBS, and incubated with 5% bovine serum albumin (BSA, Solarbio, Beijing, China) at room temperature for 1 h. Cells were incubated with primary antibodies for 3 h and secondary antibodies for 1 h. Images of the stained cells were captured using a Super-resolution Confocal Microscope (Leica TCS SP8 STED 3X). ImageJ was employed to analyze fluorescence colocalization.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMTT assay\u003c/h3\u003e\n\u003cp\u003eMTT assay was performed for cell viability detection. After treatment, the supernatant of cell medium was discarded, and 100 \u0026micro;L of 1 mg/mL MTT solution within serum-free medium was added into each well and incubated at 37\u0026deg;C for 3 h. After that, the supernatant with MTT was discarded, followed by the addition of 100 \u0026micro;L of dimethyl sulfoxide (DMSO, Solarbio, Beijing, China). The absorbance at 490 nm was measured by a microplate reader.\u003c/p\u003e\n\u003ch3\u003eCrystal violet staining assay\u003c/h3\u003e\n\u003cp\u003e1x10\u003csup\u003e3\u003c/sup\u003e cells were seeded in 12-well plate and cultured for two days. After treatment, cells were fixed by 4% paraformaldehyde for 7 min and stained by 0.1% crystal violet for 15 min, The picture was obtained using light microscope. After that, 100 \u0026micro;L of 10% acetic acid was used to dissolve crystal violet for cell survival. The absorbance at 570 nm was measured by a microplate reader.\u003c/p\u003e\n\u003ch3\u003eQuantitative real‑time PCR (qRT‑PCR)\u003c/h3\u003e\n\u003cp\u003eAfter PBS washes three times, mRNA was extracted by TRIzol Reagent (Invitrogen). 1 \u0026micro;g of RNA was used for cDNA reverse transcription with 5X RT Premix (#AG11706, Accurate Biology). SYBR Green Premix Pro (AG11701, Accurate Biology) was used to amplify genes in ABI 7500 (Applied Biosystems). β-actin was taken as control. The qPCR primers were shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis\u003c/div\u003e \u003cp\u003eAfter PBS washing, cells were harvested with IP lysis buffer (P0013, Beyotime Biotechnology), supplemented with PMSF and phosphorylation inhibitors. And then the cell lysate was collected into 1.5 mL tubes, followed by centrifugation at 4\u0026deg;C for 10 min at 12000 rpm speed. Equal amounts of protein were separated on SDS-PAGE gels and transferred onto NC membranes (Millipore). The membranes were blocked with 5% milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6) at room temperature for 1 h. After TBST washing, primary antibodies were added and incubated at 4\u0026deg;C overnight, and the secondary antibody was incubated for 1 h at room temperature. ECL kit (Pierce) was used to visualize by a Chem imaging system. Antibodies used are as indicated: CAMSAP3, SAB4200415, Sigma-Aldrich; Tubulin beta, M20005, Abmart; Tubulin gamma, T55405s, Abmart; Phospho-EGFR (Y1068) 3777T, Cell Signaling Technology; EGFR, sc-373746, Santa Cruz Biotechnology; Phospho-ERK1/2 (T202/Y204), TA1015, Abmart; ERK1/2, T40071, Abmart; EEA1, 3288T, Cell Signaling Technology; RAB11A, D4F5, Cell Signaling Technology; GAPDH, AC002, ABclonal; LAMP1, 9091, Cell Signaling Technology.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003esiRNA transfection\u003c/h2\u003e \u003cp\u003eSpecific siRNAs targeting genes of interest were purchased from Shanghai ShengGong (Shanghai, China). Cells were transfected with siRNAs by using INTERFERin\u0026reg; (Polyplus-transfection S.A, Illkirch, France) according to the manufacturer\u0026rsquo;s protocols. The sequences of siRNA used to knock down the indicated genes were listed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLentiviral shRNA constructs and infection\u003c/h3\u003e\n\u003cp\u003eLentiviral shRNA against CAMSAP3 and scramble control shRNA were constructed according to previous report [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. H1975 cells were infected with lentivirus at approximately 60\u0026ndash;70% confluence, 10 \u0026micro;g/mL polybrene was added into medium as the same time and incubated for 48 h, then the medium was supplemented with 2 \u0026micro;g/mL puromycin for screening. The sequences of shRNA used to knock down the indicated genes were listed in Supplementary Table S3.\u003c/p\u003e\n\u003ch3\u003eAnimal experiments\u003c/h3\u003e\n\u003cp\u003eTo evaluate the impact of CAMSAP3 knock down on AZD9291-resistant NSCLC, we implanted 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e H1975 cells transfected with either control shRNA (shCtrl) or CAMSAP3 shRNA (shCAMSAP3) plasmids into the subcutaneous tissue of 5-week-old female nude mice (GemPharmatech, Chengdu, China) to generate xenograft tumors. The study comprised five mice per group (n\u0026thinsp;=\u0026thinsp;5). Once the maximum tumor size reached approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were administered either saline (vehicle control) or AZD9291 (5 mg/kg body weight, once per day, oral gavage). At predetermined time points, the length (L) and width (W) of the tumours were measured. The tumour volume was calculated using the formula: (L x W\u003csup\u003e2\u003c/sup\u003e)/2. Tumours were harvested at the conclusion of the final measurement for the purpose of further analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids transfection\u003c/h2\u003e \u003cp\u003eFor transfection, 1 \u0026micro;g plasmid was added in 200 \u0026micro;L opti-MEM, and then 5 \u0026micro;L PEI transfection reagent was added. The mix were added into the medium without any antibiotic after 15 min of incubation. After 4\u0026ndash;6 h, the medium was replaced by fresh complete medium. 48\u0026ndash;72 hours later, cells were harvested for analysis. The sequences of the constructed plasmids were shown in Supplementary Table S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLysosomal pH assay\u003c/h2\u003e \u003cp\u003eLysoSensor Green DND-189 (40767ES50, Yeason, China) was used to detect lysosomal pH. Cells were stained with 1 \u0026micro;M Lysotracker Green DND-189 diluted in RPMI-1640 at room temperature for 10 min, washed twice with PBS and added fresh medium. Hoechst 33342 (HY-15559, MCE, China) was used to stain nucleus. Images of the marked cells were captured using a Super-resolution Confocal Microscope (Leica TCS SP8 STED 3X) and FlowJo software were performed to detect and analyze the fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExcel was used for statistical analysis. The between-group variance was analyzed by unpaired two-tailed Student\u0026rsquo;s t test. Data are represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from at least three independent experiments. There were statistically significant differences between two groups when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicrotubule remodeling accompanies AZD9291 resistance in NSCLC cells\u003c/h2\u003e \u003cp\u003eMicrotubules and their post-translational modifications have been implicated in EGFR transport and chemotherapy resistance in lung squamous cell carcinoma. However, alterations in microtubules and their role in NSCLC resistance to AZD9291 remain unreported. To explore the biological function of microtubules in NSCLC resistance, the microtubule morphology in AZD9291-sensitive (H1975) and resistant (H1975AR) cells were analyzed using β-tubulin immunofluorescence staining. The results revealed that significant morphological differences, including distinct perinuclear microtubule clusters uniquely were observed in H1975AR cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To test whether this morphological change was cell line dependent, we established another AZD9291 resistance cells (HCC827AR) and similar phenomenon was observed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). In contrast, F-actin staining of microfilaments showed no structural differences between the resistant and sensitive cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether the morphological change of in the resistant cells was on account of microtubule aggregation, H1975 and H1975AR cells were treated with nocodazole (a microtubule-depolymerizing agent) to assess post-washout microtubule regeneration. The result showed that microtubules depolymerized in both cell lines upon nocodazole treatment, and nucleation resumed within 5 minutes of nocodazole washout (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, after 60 minutes, microtubules in H1975 sensitive cells dispersed into the cytoplasm, whereas those in resistant cells remained anchored at perinuclear microtubule-organizing centers (MTOCs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Co-staining with β-tubulin and γ-tubulin confirmed these clusters as centrosomal microtubules, indicating a marked increase in centrosomal microtubule proportion in resistant cells, particularly in H1975AR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo verify the effect of microtubule aggregation in the morphological differences between AZD9291-resistant and sensitive cells, depolymerizing agent nocodazole and stabilizing agent paclitaxel were used for cell disposing. The result showed that short-term (1 hour) paclitaxel or nocodazole exposure disrupted microtubules in both H1975 and H1975AR cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Prolonged treatment (48 hours) resulted in a more pronounced reduction in cell viability and survival in H1975AR and HCC827AR cells compared to their sensitive counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), suggesting heightened sensitivity of resistant cells to microtubule-targeting agents. To investigate the targets that give rise to the morphological difference in AZD9291-resistant cell, we analyzed transcriptional levels of microtubule associated genes including \u003cem\u003eCAMSAP1/2/3\u003c/em\u003e, \u003cem\u003eNINEIN\u003c/em\u003e, \u003cem\u003eTPX2\u003c/em\u003e, etc., microtubule dynamics genes including \u003cem\u003eSPASTIN\u003c/em\u003e, \u003cem\u003eKIF3A\u003c/em\u003e, \u003cem\u003eDCTN1\u003c/em\u003e, etc., and centrosome function genes including \u003cem\u003eTUBG1\u003c/em\u003e, \u003cem\u003eNEDD1\u003c/em\u003e, \u003cem\u003eNUMA1\u003c/em\u003e, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The result showed that the mRNA level of \u003cem\u003eCAMSAP3\u003c/em\u003e decreased nearly 60% in AZD9291-resistant cells than in sensitive cells, indicating that CAMSAP3 might play important role in centrosomal/non-centrosomal microtubule regulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCAMSAP3 contributes to AZD9291 resistance in NSCLC\u003c/h2\u003e \u003cp\u003eIn order to further validate the role of CAMSAP3 in NSCLC cell resistance to AZD9291, the protein expression of CAMSAP3 in H1975AR was further assessed via western blot and immunofluorescence staining. Consistent with RT-qPCR results, the protein level of CAMSAP3 markedly reduced in H1975AR cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, Kaplan-Meier survival analysis revealed shorter overall survival in lung adenocarcinoma patients with low CAMSAP3 levels, implicating the prognostic relevance between CAMSAP3 expression and lung cancer prograssion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Next, the localization of CAMSAP3 in NSCLC cells was assessed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, co-immunofluorescence staining of CAMSAP3 and β-tubulin demonstrated uniform perinuclear CAMSAP3 distribution with predominant centrosomal microtubule organization in H1975 sensitive cells. In contrast, AZD9291-resistant cells displayed reduced CAMSAP3 expression and retained centrosomal microtubule clustering, a pattern replicated in HCC827AR cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-C). These findings suggested that the centrosomal microtubule aggreation regulater CAMSAP3 was dromatically dedreased in AZD9291 resistance NSCLC, which might effect the lung cancer prograssion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCAMSAP3 modulates AZD9291 sensitivity and microtubule morphology\u003c/h2\u003e \u003cp\u003eAfter clear the role of CAMSAP3 in microtubule aggregation, the biological function of CAMSAP3 in NSCLC drug resistance was then assessed. CAMSAP3 was knocked down by siRNA in H1975 and HCC827 cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig. S3A, CAMSAP3 knockdown efficiency were detected in protein level and mRNA level, respectively. For microtubule morphology, co-immunofluorescence staining of CAMSAP3 and β-tubulin revealed centrosomal microtubule clustering in CAMSAP3-depleted H1975 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and HCC827 cells (Fig. S3B), which was consistent with our preceding hypothesis. Then MTT and crystal violet assay demonstrated increased cell viability and staining intensity in CAMSAP3-knockdown H1975 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and HCC827 cells (Fig. S3C, S3D) treated with AZD9291, indicating that CAMSAP3 silencing accelerated AZD9291 resistance. In order to validate the obove findings \u003cem\u003ein vivo\u003c/em\u003e, H1975 cells stably expressing control shRNA (shCtrl) or shCAMSAP3 were used for xenograft assay (Fig. S3E). β-tubulin staining confirmed enhanced microtubule clustering in CAMSAP3 stable silencing H1975 cell line (Fig. S3F). Nude mice were treated with 5 mg/kg AZD9291 when subcutaneous tumors volumes reaching\u0026thinsp;~\u0026thinsp;100 mm\u0026sup3; (day 0). Tumor volumes were monitored day 17, until tissues were harvested post-measurement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, AZD9291 reduced tumor volumes in both groups, while the final tumor volumes inhibition ratio revealed that CAMSAP3 knockdown significantly increased the tumor resistance to AZD9291 compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), which confirming the critical role of CAMSAP3 in AZD9291 resistance modulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCAMSAP3 was composed of various functional domains, to assess the which component of CAMSAP3 was responsible for the microtubule remodeling and cell resistance modulation, different truncated CAMSAP3, including FLAG-tagged plasmids encoding full-length CAMSAP3 (CAMSAP3-FL), truncations lacking the CH (CAMSAP3-ΔCH) or HCKK domains (CAMSAP3-ΔHCKK), and an HCKK-only construct (CAMSAP3-HCKK) were overexpressed in AZD9291-resistant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The cell microtubule morphology staining showed that only CAMSAP3-FL partially restored non-centrosomal microtubule distribution in H1975AR cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), though cytoplasmic parallel microtubule organization (as in sensitive cells; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was not fully recapitulated. CAMSAP3-ΔHCKK localized aberrantly to microtubule-organizing centers (MTOCs), highlighting the HCKK domain\u0026rsquo;s necessity for proper CAMSAP3 localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The function of different truncated CAMSAP3 in AZD9291 sensitivity was then assessed. MTT assay was performed to examine the effect of truncated CAMSAP3s to cell viability. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, CAMSAP3-FL overexpression restored AZD9291 sensitivity in resistant cells compared to truncated variants. Notably, transient CAMSAP3-FL transfection induced significant higher cell death ratio, so stable CAMSAP3-FL overexpression cell line could not be established. The above findings suggested that the microtubule networks disruption by forced CAMSAP3 overexpression was critical for AZD9291-resistant cells\u0026rsquo; survival.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCAMSAP3 disrupts lysosomal and EGFR localization\u003c/h2\u003e \u003cp\u003eEGFR degradation and downregulation is one of the most important factors contribute to AZD9291 resistance, and the lysosomal pathway is critical for EGFR degradation and downregulation. But the relationship between microtubule-driven lysosomal alterations and AZD9291 resistance hadn\u0026rsquo;t been thrashed out. Due to the early endosomes (EEA1), lysosomes (LAMP1), and recycling endosomes (RAB11A) were the mainly regulators of the lysosomal alterations, they were stained in H1975 and H1975AR cells, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, EEA1, LAMP1, and RAB11A were well-distributed in cytoplasmic in sensitive H1975 cells, whereas all three organelles were clustered at perinuclear microtubule-organizing centers (MTOCs) in H1975AR and CAMSAP3-knockdown cells. Western blotting showed reduced RAB11A level and elevated LAMP1 levels in H1975AR and CAMSAP3-knockdown cells (except H1975 siCAMSAP3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that CAMSAP3 deficiency alters endo-lysosomal trafficking and lysosomal protein expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMutant EGFRs (exon 19 deletions, T790M/L858R) are typically resistant to degradation due to ubiquitination/lysosomal defects. Surprisingly, EGFR and pEGFR (Y1068) levels greatly reduced both in AZD9291-resistant and CAMSAP3-knockdown H1975 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Meanwhile, co-immunofluorescence staining showed that of CAMSAP3 knockdown enhanced the colocalization of pEGFR (Y1068) and LAMP1 both in H1975 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and HCC827 cells (Fig. S4A), demonstrating that CAMSAP3 was associated with EGFR localization in lysosomal. Lysosomal acidification is an important factor of lysosomal activity during protein degradation. LysoSensor Green DND-189 staining (a pH-sensitive indicator) showed a heightened lysosomal acidification in H1975AR and CAMSAP3-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), verifying the role of CAMSAP3 in lysosome degradation activity. Next, hydroxychloroquine (CQ) was added for lysosomal activity inhibition, western blotting showed that cells treated with combined CAMSAP3 siRNA and CQ exhibited significantly higher EGFR degradation efficiency than the cells treated with CQ singly (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB), confirming that loss of CAMSAP3 promoted lysosomal degradation of mutant EGFR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince EGFR trafficking was regulated by microtubules, so the localization of EGFR/pEGFR (Y1068) in AZD9291-resistant and sensitive cells were examined next. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Fig. S5A, EGFR/pEGFR predominantly localized to the plasma membrane in sensitive cells. Whereas in resistant cells, EGFR/pEGFR showed perinuclear MTOC accumulation, accompanied by a reduced EGFR/pEGFR level. Afterwards, continuous processing by AZD9291 lead to pEGFR detection in resistant cells, and CAMSAP3 knockdown similarly shifted EGFR/pEGFR to perinuclear MTOCs both in H1975 and HCC827 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA). Western blotting confirmed reduced expression or levels of EGFR, pEGFR, and pERK1/2 (T202/Y204) in AZD9291-resistant and CAMSAP3 knockdown cells compared with the sensitive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB), affirm the role of CAMSAP3 in EGFR degradation. However, the transcriptional level of EGFR remained unchanged in AZD9291-resistant and CAMSAP3 knockdown cell (Fig. S5C and S5D), suggesting that loss of EGFR was only regulated by post-translational degradation pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAZD9291 has become the first-line treatment choice for EGFR-mutant NSCLC, with greater efficacy and improved overall survival compared to the previous generation of EGFR TKIs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The interaction of the microtubule system with tyrosine kinases (TKs) signaling pathways plays a key role in tumor drug resistance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Tubulin-binding agents (e.g., paclitaxel, docetaxel) inhibit microtubule dynamics by targeting β-tubulin in α/β-tubulin heterodimers, inducing mitotic arrest and apoptosis. These agents, particularly paclitaxel, form the backbone of first-line NSCLC therapy, administered either alone or combined with platinum-based chemotherapy (cisplatin/carboplatin) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Preclinical evidences demonstrate that chronic EGFR inhibition triggers non-genetic resistance through TPX2-mediated Aurora kinase A (AURKA) activation, thus combining an AURKA inhibitor with third-generation EGFR-TKI AZD9291 can overcome acquired resistance in NSCLC [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. TPX2 and AURKA were involving microtubule nucleation/mitotic spindle assembly [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and our study revealed distinct microtubule localization patterns between drug-sensitive and resistant cells. In sensitive cells, microtubules were released into the cytoplasm following nucleation, whereas in AZD9291-resistant cells, microtubules remained anchored in the perinuclear region post-nucleation. These findings suggest that altered microtubule dynamics may contribute to TKI resistance. Studying microtubule dynamics in drug-resistant cells is an important approach to understanding and overcoming drug resistance.\u003c/p\u003e \u003cp\u003eThe EGFR signaling pathway is a central driver of tumor growth, but inhibitors like Osimertinib often face resistance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. NSCLC EGFR TKIs resistance was classically linked to secondary EGFR mutations (e.g., T790M/C797S), \u0026gt;\u0026thinsp;40% of EGFR TKIs resistance cases now originate from tumor cells abandoning EGFR dependency through: 1) \u0026zwnj;Bypass activation\u0026zwnj;, including MET amplification-mediated ERBB3-PI3K/AKT signaling, HER2/HER3 heterodimer-driven MAPK activation and AXL-RTK-mediated EMT [2\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]; 2) \u0026zwnj;Downstream mutations\u0026zwnj; (e.g., KRAS G12C/BRAF V600E) that bypass EGFR regulation [32]; or 3) \u0026zwnj;Phenotypic plasticity\u0026zwnj; through tumor stem cell maintenance via WNT/Notch pathways or metabolic reprogramming (e.g., glutamine dependency) [33, 34]. These mechanisms illustrate that EGFR TKIs resistance often emerged when cells survive on EGFR independent manner. Previous studies have demonstrated that AZD9291 promotes EGFR degradation [35]. Consistent with these findings, we observed EGFR degradation in AZD9291-resistant cells, suggesting that EGFR may become dispensable for resistant cell survival. However, the precise molecular mechanisms regulating this degradation process remain incompletely understood. Therefore, further investigation is warranted to elucidate the mechanisms underlying EGFR degradation in drug-resistant cells.\u003c/p\u003e \u003cp\u003eEGFR, a transmembrane receptor tyrosine kinase, undergoes EEA1-mediated endocytosis followed by lysosomal degradation or plasma membrane recycling [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Mutant EGFR evades CBL-mediated ubiquitination and lysosomal degradation, leading to persistent activation of EGFR signaling [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Single-cell sequencing further reveals that EGFR-independent clones in the tumor microenvironment propagate resistance via exosomal miRNAs, underscoring the need to target the tumor ecosystem in TKIs resistant cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, we observed perinuclear accumulation of EGFR at microtubule organizing centers (MTOCs) in AZD9291-resistant NSCLC cells. Mechanistic investigations revealed that CAMSAP3, a microtubule-remodeling protein, promotes EGFR depletion through lysosomal degradation. Specifically, CAMSAP3 knockdown induced aberrant non-centrosomal microtubule assembly, which disrupted normal EGFR trafficking in sensitive cells. This disruption diverted EGFR/pEGFR from the plasma membrane recycling pathway to lysosomal degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings demonstrated that loss of CAMSAP3 induces NSCLC cell grow on EGFR independent manner, which lead to the acquired resistance to AZD9291.\u003c/p\u003e \u003cp\u003eIn conclusion, we hypothesized that loss of CAMSAP3 prompts NSCLC cellular resistance to AZD9291 through EGFR independent pathways. The precise molecular mechanisms and regulatory networks underlying CAMSAP3-mediated acquired resistance require further exploration using omics approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally: Fei Yang, Zhen Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCenter for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science, Xi’an Jiaotong University, Xi’an 710049, PR China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiangang Long, Fei Yang, Jinjin Zhong, Zhanwu Hou \u0026amp; Shuying Bian\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchool of Life Sciences and Health, University of Health and Rehabilitation Sciences, Qingdao 266113, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuadong Liu, Zhen Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Pathogen Biology, School of Basic Medical Science, Xi'an Medical University, Xi'an 710021, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiao Han\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuadong Liu and Jiangang Long conceived and supervised the project. Fei Yang, Zhen Wang acquired and interpreted data, and drafted the manuscript. Xiao Han, Jinjin Zhong, Zhanwu Hou and Shuying Bian conducted the experiments. All authors read and approved the final manuscript version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Huadong Liu or Jiangang Long.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by research grant from the National Key R\u0026amp;D Program of China (2023YFA1801200), the Natural Science Foundation of Shandong (No. ZR2022LSW003, H.L.). The authors also thank Ruoyuan Liu and Ying Hao at the Instrument Analysis Center of XJTU for their assistance in capturing images of the stained cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal protocols were approved by the Animal Care and Use Committee of the School of Life Science and Technology, Xi’an Jiaotong University (Approval ID: AE-2025-2589).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. J. Barker, K. H. Gibson, W. Grundy, A. A. Godfrey, J. J. Barlow, M. P. 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However, acquired resistance to AZD9291 is inevitable in the treatment of non-small cell lung cancer (NSCLC). Microtubules, key cytoskeletal components involved in intracellular cargo transport, mediate EGFR-endosomal recycling, yet their specific role in AZD9291 resistance remains to be elucidated. In this study, we found that centrosomal microtubule formation was increased in AZD9291-resistant NSCLC cells, and calmodulin-regulated hemosiderin-associated protein 3 (CAMSAP3) was identified as the key molecule responsible for the change of microtubule morphology. Genetic modulation of CAMSAP3 expression through silencing or overexpression directly altered microtubule architecture and restored AZD9291 sensitivity. Furthermore, we demonstrated that full-length CAMSAP3 is essential for proper localization of the microtubule-dependent endosomal-lysosomal system. CAMSAP3 depletion caused EGFR translocation to the perinuclear microtubule organizing center (MTOC), thereby blocking plasma membrane recycling and promoting lysosomal degradation. These findings establish CAMSAP3 as a key regulator of EGFR signaling and AZD9291 response in NSCLC, suggesting its therapeutic potential for overcoming drug resistance in lung cancer.\u003c/p\u003e","manuscriptTitle":"NSCLC cells acquire resistance to AZD9291 by reducing CAMSAP3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-21 08:43:01","doi":"10.21203/rs.3.rs-6539573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-10T15:13:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-30T16:41:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-16T10:53:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-13T13:31:49+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-28T15:43:00+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-16T07:12:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T13:26:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-04-27T09:42:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-27T09:42:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cebe495b-02d2-4ea1-8aeb-b107c93abe21","owner":[],"postedDate":"May 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48618202,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"},{"id":48618203,"name":"Biological sciences/Cell biology/Cytoskeleton"}],"tags":[],"updatedAt":"2026-01-24T08:06:57+00:00","versionOfRecord":{"articleIdentity":"rs-6539573","link":"https://doi.org/10.1038/s41419-025-08299-0","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-12-11 05:00:00","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-05-21 08:43:01","video":"","vorDoi":"10.1038/s41419-025-08299-0","vorDoiUrl":"https://doi.org/10.1038/s41419-025-08299-0","workflowStages":[]},"version":"v1","identity":"rs-6539573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6539573","identity":"rs-6539573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}