Enhancement of TKI sensitivity in lung adenocarcinoma through m6A-dependent translational repression of Wnt signaling by circ-FBXW7

Abstract

Background: Tyrosine kinase inhibitors (TKIs) that specifically target mutational points in the EGFR gene have significantly reduced suffering and provided greater relief to patients with lung adenocarcinoma (LUAD). The third-generation EGFR-TKI, Osimertinib, has been successfully employed in clinical treatments to overcome resistance to both original and acquired T790M and L858R mutational points. Nevertheless, the issue of treatment failure response has emerged as an insurmountable problem.

Methods: By employing a combination of multiple and integrated approaches, we successfully identified a distinct population within the tumor group that plays a significant role in carcinogenesis, resistance, and recurrence. Our research suggests that addressing TKI resistance may involve targeting the renewal and repopulation of stem-like cells. To investigate the underlying mechanisms, we conducted RNA Microarray and m6A Epi-Transcriptomic Microarray analyses, followed by assessment of transcription factors. Additionally, we specifically designed a tag to detect the polypeptide circRNA-AA, and its expression was confirmed through m6A regulations.

Results: We initially identified unique molecular signatures present in cancer stem cells that contributed to poor therapeutic responses. Activation of the alternative Wnt pathway was found to sustain the renewal and resistant status of these cells. Through bioinformatics analysis and array studies, we observed a significant decrease in the expression of circFBXW7 in Osimertinib-resistant cell lines. Notably, the abnormal expression pattern of circFBXW7 determined the cellular response to Osimertinib. Functional investigations revealed that circFBXW7 inhibits the renewal of cancer stem cells and resensitizes both resistant LUAD cells and stem cells to Osimertinib. In terms of the underlying mechanism, we discovered that circFBXW7 can be translated into short polypeptides known as circFBXW7-185AA. These polypeptides interact with β-catenin in an m6A-dependent manner. This interaction leads to reduced stability of β-catenin by inducing subsequent ubiquitination, thereby suppressing the activation of canonical Wnt signaling. Additionally, we predicted that the m6A reader, YTHDF3, shares common binding sites with hsa-Let-7d-5p. Enforced expression of Let-7d post-transcriptionally decreases the levels of YTHDF3. The repression of Let-7d by Wnt signaling releases the stimulation of m6A modification by YTHDF3, promoting the translation of circFBXW7-185AA. This creates a positive feedback loop contributing to the cascade of cancer initiation and promotion.

Conclusions: Our bench study, in vivo experiments, and clinical validation have unequivocally shown that circFBXW7 effectively inhibits the abilities of LUAD stem cells and reverses resistance to TKIs by modulating Wnt pathway functions through the action of circFBXW7-185AA on β-catenin ubiquitination and inhibition. The regulatory role of circRNA in Osimertinib treatment has been rarely reported, and our findings reveal that this process operates under the influence of m6A modification. These results highlight the tremendous potential of this approach in enhancing therapeutic strategies and overcoming resistance to multiple TKI treatments.

Keywords: Lung adenocarcinoma; Therapy resistance; Translated circRNAs; Tyrosine kinase inhibitor; m6A modification.

Fig. 1

Signatures of Wnt signaling expressing status and their clinical relevance. A The SOX family and Snail, which are critical effectors of the Wnt signaling pathway with transcriptional and pluripotency promotion ability, exhibit significantly abnormal expression patterns. B RNA array and chip sequencing techniques were employed to analyze the expression patterns of the SOX family. C Survival analysis of Wnt signaling family factors demonstrates their negative roles in predicting overall survival and progression-free survival. D KEGG pathway evaluation and ROC-Plotter associated Dep-map calculation were performed to assess the effects of Osimertinib treatment. E The most differentially expressed functional factors and ligands were presented. F By clustering Wnt signaling genes, it was observed that abnormal activation of the Wnt pathway correlates significantly with diverse treatment responses and long-term therapeutic outcomes

Fig. 2

Stem-like cells retained the regeneration capability to re-produce lung cancer group in a Wnt activation dependent manner. Different markers referring to identifying the Stem-like cells may distinguish diverse sub-group of different signatures. A In HCC827 cell lines and H1975 cell lines, the groups of stem-like cells with CD133⁺/CD24⁻ markers was 21.4% and 17.2% relatively. B The stem cells of ALDH1A1⁺ surface marker accounted for a small group of 2.34% (HCC827) and 1.31% (H1975) respectively. C the ALDH1A1⁺ cells could renew themselves and form the spheres effectively. D The amplified stem cells population of spheres were naturally resistant to Osimertinib treatment, as was the Osimertinib resistant lung cancer cells. E–F Schematic images illustrated the different cells division manners, and the unpaired DNA segregation modes. The palette color set of rainbow mode was used to show the glow scale of each single cell at the dividing process. Wnt signaling effectors were overexpressed in lung cancer stem cells (G), and in the resistant cells (H). I-J Wnt signaling functions were verified to be effectively repressed by Capmatinib, TCF-4, SNAI1, SOX2 inhibition, and then was found to promote the differentiability-oriented symmetrical cell dividing, and decreased the asymmetrical self-renewing and the symmetrical self-renewing

Fig. 3

CircRNAs participated in the Osimertinib therapy resistance. A-B The ribosomal RNA-depleted total RNAs of the resistant HCC827OR cells and H1975OR cells were adjusted to perform the RNA-seq, and the differentially expressed circRNAs were preliminary screening. The informatic analysis of cellular and signaling functions indicated the differentially expressed circRNAs were possibly participated in microRNAs functions (C), in cell division (D), in molecular functions, and in cellular components (E). F The candidate circRNAs with significant dysregulations and potential abnormal functions were laid out

Fig. 4

m6A modified circ-FBXW7 expression in resistant lung cancer cells A The m6A levels of total RNAs from resistant cells were more abundant than that of sensitive original cells. B The differentially expressed hsa-circ-0001451 (circ-FBXW7) was matched in circRNA Database and the structure was illustrated for mechanistic understanding. C circ-FBXW7 was lowly expressed in Osimertinib resistant cells and in stem-like cells. D-F The lentiviral based YTHDF3 knock-down systems inhibited its expression significantly, and LC–MS/MS tests revealed the YTHDF3 associated m6A levels. F-G YTHDF3 inhibition decreased the m6A modification and therefore promoted the stem cells renewal that identified by spheres forming assay and surface marker of ALDH1A1

Fig. 5

Translated circ-FBXW7 suppressed stem cells’ renewal in a β-catenin ubiquitylation way. A Schematic image illustrated the IRES independent translation potential of circ-FBXW7. B Designation of junction probe and Flag-tag to detect translated circ-FBXW7-AA. C The specifically designed and synthesized FLAG antibody successfully detected circ-FBXW7-AA in 293 T cells. D The reader of YTHDF3 inhibition blocked m6A quantity, and decreased circ-FBXW7-AA level. Over-expressed circ-FBXW7 inhibited the proliferation of stem cells group (E), and stimulated the differentiated-orientation cells dividing (F) The tags of “negative” and “positive” are referring to marker of circ-FBXW7 solely, and the positive-circFBXW7 was set as the positive control. G The increased differentiated-orientation cells dividing, together with the increased asymmetric cells dividing, decreased the malignant behaviors. Similarly, YTHDF3 inhibition failed to stimulate the circ-FBXW7 dependent differentiated cells division, and helped to restore the symmetric cells dividing. H The selecting cells of FSC gate was grouped based on distribution patterns of phenotypic markers of circ-FBXW7/H3K27me3/ALDH1A1. I The cells with negative label (circ-FBXW7⁻/H3K27me3⁺/ALDH1A1⁺) showed much higher ability to form the spheres when assessing in per 100 cells, and the positive label (circ-FBXW7⁺/H3K27me3⁻/ALDH1A1⁻) greatly decreased the spheres forming ability. J Enforced circ-FBXW7 stimulated symmetric differentiation dividing, and consequently resulted in spheres forming inhibition. K The overexpressed circ-FBXW7 affected the downstream Wnt/β-catenin signaling (CCND1/CMYC) to conduct the renewal inhibition effects. L Co-immunoprecipitation assay revealed the endogenous circ-FBXW7-185AA could interact with endogenous β-catenin in both HCC827 cells and H1975 cells. Circ-FBXW7-185AA negatively regulated the protein stability of β-catenin (M), the ubiquitination status of β-catenin increased with overexpressed circ-FBXW7-185AA (N). O The ubiquitination level of β-catenin gradually increased with the overexpressed circ-FBXW7-185AA, and MG132 weakened the inhibitory effect of circ-FBXW7-185AA on β-catenin expression

Fig. 6

Circ-FBXW7 did not function through Let-7 rescue, but formed negative feedback through Wnt/β-catenin signaling mediation. A The overexpression of circ-FBXW7 led to an upregulation of Let-7d expression. B In Osimertinib-resistant HCC827OR and H1975OR cells, inhibition of the Wnt signaling pathway resulted in increased Let-7d expression. Strong stimulation of Let-7d expression was observed upon β-catenin inhibition. C The effects of Wnt signaling on sphere-forming ability were assessed, and no significant differences were observed among groups treated with circ-FBXW7, si-β-catenin, or their combination, indicating their similar functions. However, the synthesized compound of Wnt signaling stimulator increased sphere-forming ability, which was partially relieved by circ-FBXW7. D Putative binding sites were identified between the m6A readers (YTHDF3) and Let-7 miRNAs. E Manipulation of Let-7b, Let-7d, or Let-7i did not significantly alter the mRNA levels of YTHDF3. F Among the Let-7 miRNAs tested, Let-7d showed the most effective decrease in YTHDF3 expression. G Let-7d inhibited the luciferase activity of wild-type YTHDF3 mRNA, but had no effect on mutant groups. H Let-7d inhibited circ-FBXW7 expression in an YTHDF3 repression-dependent manner. I Let-7d and circ-FBXW7 formed a negative feedback loop. J Enforcing circ-FBXW7, Let-7d, or inhibiting YTHDF3 all decreased the self-renewal ability of resistant H1975OR and HCC827OR cells, with Capmatinib used as a positive control. K Upregulation of circ-FBXW7 and Let-7d reduced the stem cell population, while inhibition of YTHDF3 increased stem cells expressing the ALDH1A1 surface marker