Stimulation of Let-7 Maturation by Metformin Improved the Response to Tyrosine Kinase Inhibitor Therapy in an m6A Dependent Manner

Abstract

The molecular mechanism of the tyrosine kinase inhibitor (TKI) resistant lung adenocarcinoma is currently unclear, and the role of methylated adenosine at the N6 position in the resistance of cancer stem cells (CSCs) therapy is unknown. This study identified a novel and effective strategy to enhance TKIs therapy response. We first confirmed the sensitization of Metformin enforcing on Osimertinib treatment and revealed the mature miRNAs signatures of the Osimertinib resistant H1975 and HCC827 cells. Let-7b expression was stimulated when adding Metformin and then increasing the therapy sensitivity by decreasing the stem cell groups expanding. Methyltransferase-like 3 (METTL3) increased the pri-Let-7b, decreased both the pre-Let-7b and mature Let-7b, attenuating the Let-7b controlling of stem cell renewal. The addition of Metformin increased the bindings of DNA methyltransferase-3a/b (DNMT3a/b) to the METTL3 promoter. With the help of the readers of NKAP and HNRNPA2B1, the cluster mediated m6A formation on pri-Let-7b processing increased the mature Let-7b, the key player in suppressing Notch signaling and re-captivating Osimertinib treatment. We revealed that the maturation processing signaling stimulated the methylation regulation of the miRNAs, and may determine the stemness control of the therapy resistance. Our findings may open up future drug development, targeting this pathway for lung cancer patients.

Keywords: cancer stem-like cells; miRNAs maturation; n6-methyladenosine; therapy resistance; tyrosine kinase inhibitor.

Figure 1

Notch signaling signatures and Let-7b expression deviations in Osimertinib resistant cells. (A) Proliferation inhibitive ratios were detected for defining the Osimertinib resistant H1975OR cells and HCC827OR cells. (B) The samples of lung Adenocarcinoma from the TCGA database (Pan-Cancer Atlas) were analyzed with mutation and CNA data, and Notch signaling participants were universally activated. (C, D) Key functional factors of Notch signaling were primarily screened and confirmed by western blotting, and Notch signaling factors were overexpressed in resistant cells (The grouping of gels/blots were cropped from different parts, and the full-length gels could be referred to in the supplemental data). (E) The inverse relationship between Let-7b and key Notch signaling activators was defining with using data from Star-Base Project. (F) Let-7b decreased significantly in Osimertinib resistant H1975OR and HCC827OR cells ( Figure 2F ).

Figure 2

Notch signaling activation dependent stem cells renewal was responsible for Osimertinib resistance. (A, B) The ratio of ALDH1A1 (ALDH, ALDH1) positive cells accounted for less than 3% in H1975 and HCC827 cells, and Osimertinib decreased the ALDH1 positive stem cells ratio of H1975 and HCC827 effectively, however in H1975OR and HCC827OR cells, the ratios increased incredibly, and the stem cells ratio did not react to Osimertinib treatment. (C) Proliferation inhibition ratio was essentially the same between H1975 cells and H1975OR cells, between HCC827 cells and HCC827OR cells. (D) Let-7b decreased significantly in the spheres of H1975 cells and HCC827 cells, and Osimertinib did not change the Let-7b expression compared to the negative control. (E, F) Stem cells were identified with overexpressed stem associated markers, and the key notch signaling actors were excessively activated in spheres of stem cells (The grouping of gels/blots were cropped from different parts, and the full-length gels could be referred to in the Supplemental Data ). *P < 0.05

Figure 3

Metformin decreased the stem cells ratio and sensitized the resistant lung cancer cells to Osimertinib. (A) The proliferation ratio of H1975OR cells and HCC827OR cells decreased more greatly than the control groups when culturing with Metformin. (B) The proliferative index of cancer cells from the spheres of H1975 and HCC827 cells decreased significantly when co-cultured with Metformin. (C) Metformin decreased the ratios of ALDH1A1 positive cells in the group receiving Osimertinib treatment, however, the stem cells ratio did not change greatly when using Metformin alone. *P < 0.05.

Figure 4

Metformin increased the mature Let-7b through stimulating METTL3 expression and re-sensitized the resistant lung cancer cells. (A) Let-7b increased significantly in the resistant cells group when receiving Metformin and Osimertinib. (B) Metformin caused the inconsistent levels of pri-let-7b and pre-let-7b, stimulating the miRNA maturation. (C) METTL3, METTL14, and Wilms tumor 1-associated protein (WTAP) are three components of the writer protein complex in m6A process, and Metformin only increased the METTL3 expression greatly. (D) METTL3 alone remarkably decreased pri-Let-7b, and increased both pre-Let-7b and Let-7b-5p (the grouping of gels/blots were cropped from different parts, and the full-length gels could be referred to in the supplemental data). Knocking down of METTL3 abolished the Metformin effects of induction of Let-7b-5p overexpression in H1975OR cells (E, above) and HCC827OR cells (E, below).

Figure 5

Metformin inhibited DNMT3 promoter activity and facilitated the METTL3 mediated Let-7b maturation. (A) The possible CpG island upstream of the METTL3 locus referring to Let-7b was generated and exhibited. (B) Cells treated with Metformin had significantly lower methylation within METTL3 CpG island in comparison to cells without the exposure. (C) Metformin effectively reduced the bindings of DNMT3a and DNMT3b to the METTL3 promoter. (D) m6A-specific RNA immunoprecipitation (RIP) coupled qRT-PCR analysis referring to METTL3-mediated m6A on the pri-Let-7b maturation revealed that m6A-pri-Let-7b decreased significantly when the Osimertinib resistant cells received the Metformin. (E) Overexpressing either DNMT3a or DNMT3b into the H293T cells could reduce the m6A-pri-Let-7b levels, and knocking down METTL3 stimulated the m6A-pri-Let-7b expression. (F) RIP-coupled RT-PCR and western blotting in m6A-Let-7b and DGCR8 groups under Metformin treatment showed that both NKAP and HNRNPA2B1 bound with DGCR8 and m6A-Let-7b significantly, and m6A-Let-7b existed with DGCR8 and DROSHA to perform maturation functions (The grouping of gels/blots were cropped from different parts, and the full-length gels could be referred to in the Supplemental Data ). RIP-coupled qRT-PCR indicated the bindings between NKAP/HNRNPA2B1 and m6A-Let-7b in H1975OR (G, left) cells and HCC827OR cell (G, right).

Figure 6

Let-7b was critical for sensitizing the Osimertinib treatment. Let-7b inhibition greatly abolished the Metformin effects in resistant cells (A) and in stem cells (B) when receiving Osimertinib, with Notch signaling re-activation (C, the grouping of gels/blots were cropped from different parts, and the full-length gels could be referred to in the Supplemental Data ). (D, E) Let-7b inhibition specifically diminished the Metformin decreasing of stem cells group. (F) The regulative connections and the mechanistic m6A regulations of Let-7 were generated and drawn by PowerPoint and were illustrated as one image. *P < 0.05.