Lenvatinib-resistant hepatocellular carcinoma promotes malignant potential of tumor-associated macrophages via exosomal miR-301a-3p

Abstract

Background: The interactions between cancer cells and tumor-associated macrophages (TAMs) via microRNAs (miRNAs) play crucial roles in malignant potential and drug resistance. However, it remains unclear how lenvatinib-resistant hepatocellular carcinoma (LR HCC) promotes TAM tumor biology. Here we investigated the crosstalk between LR HCC cells and TAMs for cancer progression and lenvatinib resistance, focusing on an exosomal miRNA.

Methods: We used two bioinformatics software programs to identify miRNAs that target PTEN in gastrointestinal cancers, then investigated exosomal miRNA expression in LR HCC conditioned medium (CM). After modifying TAMs with LR HCC CM (LR TAM), macrophage phenotype and PTEN-Nrf2 signaling pathway component expression were analyzed in LR TAMs. The malignant potential and drug resistance were investigated in naïve HCC cells cultured with LR TAM CM.

Results: LR HCC cells highly induced M2-like properties in macrophages compared with naïve HCC cells. Exosomal miR-301a-3p expression was increased in LR HCC CM, with higher activation of the PTEN/PI3K/GSK3β/Nrf2 signaling pathway in LR TAMs. Naïve HCC cells were educated with LR TAM CM to promote malignant potential and lenvatinib resistance. Inhibition of exosomal miR-301a-3p prevented the malignant potential of LR TAMs. Activation of Nrf2 signaling by LR HCC cell-derived exosomal miR-301a-3p skewed the transformation of macrophages to the M2 phenotype.

Conclusion: Our study provides new findings on the role of miR-301a-3p, suggesting it is a promising therapeutic target to improve HCC lenvatinib resistance.

Keywords: PTEN; drug resistance; exosome; hepatocellular carcinoma; lenvatinib.

FIGURE 1

Characteristics of LR HCC cells cocultured with TAMs. (A) The cell viabilities of HCC and LR HCC cells were examined by CCK‐8 assays, then cultured with complete growth medium with increasing doses of lenvatinib. (B) TAMs were stimulated with the CM of naïve HCC or LR HCC cells. (C) Morphology of TAMs cultured with the CM of M0, HCC, or LR HCC cells. (D) CD163 and CD206 expression levels were evaluated by qRT‐PCR in each TAM group. (E) PTEN and Nrf2 expression levels in TAMs were detected by qRT‐PCR after being stimulated with the CM of M0, HCC, or LR HCC cells. (F) Intranuclear Nrf2 protein expression levels in TAMs were detected by western blot analysis (W; whole, C; cytoplasm, N; nuclear). (G) TAMs stimulated with the CM of M0, Huh‐, or Huh^LR cells were stained with Nrf2 (green) and DAPI (blue). White arrowheads show the enhanced intranuclear Nrf2 signals. (H) Protein expression levels of signaling pathway components downstream of PTEN were detected in TAMs by western blot analysis after being stimulated with the CM of M0, Huh, or Huh^LR cells. CM, conditioned medium; HCC, hepatocellular carcinoma; LR, lenvatinib‐resistant; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; TAM, tumor‐associated macrophage. The data are shown as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001. Bar = 100 μm.

FIGURE 2

Enhancement of the malignant potential and drug resistance of naïve HCC cells cocultured with LR TAM CM. (A–C) The proliferation and migration capabilities of naïve HCC cells cultured with the CM of M0, HCC, or LR HCC cells were detected. (D) Cell viability was detected by CCK‐8 assays in naïve HCC cells cultured with the CM of TAM (Huh) or TAM (Huh^LR) cells for 24 h. (E) VEGF concentration was higher in LR TAM CM than in TAM CM and CM of M0 macrophages. (F) E‐cadherin, N‐cadherin, Snail, and vimentin mRNA expression levels in naïve HCC cells were detected by qRT‐PCR after being stimulated with the CM of M0, TAM (Huh), or TAM (Huh^LR) cells for 24 h. (G) E‐cadherin, N‐cadherin, and vimentin protein expression levels were detected by western blot analysis in naïve HCC cells cultured with the CM of M0, TAM (Huh), or TAM (Huh^LR) cells for 24 h. CM, conditioned medium; HCC, hepatocellular carcinoma; LR, lenvatinib‐resistant; TAM, tumor‐associated macrophage. The data are shown as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001. Bar = 100 μm.

FIGURE 3

The exosomal miR‐301a‐3p expression levels in HCC. (A) The expression patterns of miR‐301a‐3p in the HCC tissues and nontumoral tissues in microRNA expression data from the Genome Data Commons the Cancer Genome Atlas Liver Cancer. (B) High miR‐301a‐3p expression levels were significantly associated with poor survival in HCC patients from the GDC TCGA Liver Cancer dataset (P = 0.002). (C) Nanosight particle tracking was used to analyze the size and particle concentration secreted by HCC cells. (D) CD63 protein expression in HCC‐derived exosomes was detected by western blot analysis. (E) Quantification of exosomal miR‐301a‐3p expression in HCC and LR HCC cells. HCC, hepatocellular carcinoma; LR, lenvatinib‐resistant. The data are shown as mean ± standard deviation (SD). *P < 0.05. **P < 0.01. ***P < 0.001.

FIGURE 4

Decreased malignant potential by downregulating M2 polarization of TAMs through inhibition of Huh^LR‐derived exosomal miR‐301a‐3p. (A) Efficiency of miR‐301a‐3p inhibitor transfection into Huh^LR cells was assessed using qRT‐PCR for exosomal miR‐301a‐3p expression. (B) The mRNA expression levels of CD163, CD206, PTEN, and Nrf2 were evaluated by qRT‐PCR in the M0, Huh^LR, INC Huh^LR, and inhibited Huh^LR cocultured TAMs. (C,D) PTEN and Nrf2 protein expression levels in the M0, Huh^LR, INC Huh^LR, and inhibited Huh^LR cocultured TAMs were detected by western blot analysis. (E,F,G) The proliferation and migration capabilities of naïve HCC cells stimulated with the CM of M0, TAM (Huh^LR), INC TAM (Huh^LR), or inhibited TAM (Huh^LR) cells. (H) The IC50 values for lenvatinb of naive Huh cell lines stimulated with the CMs of TAM (Huh^LR), INC TAM (Huh^LR) or inhibited TAM (Huh^LR) for 24h". HCC, hepatocellular carcinoma; INC, inhibited negative control; LR, lenvatinib‐resistant; TAM, tumor‐associated macrophage. The data are shown as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001. Bar = 100 μm.

FIGURE 5

Schematic representation of the proposed interactions between LR HCC cells and TAMs. TAM, tumor‐associated macrophage; HCC, hepatocellular carcinoma; LR, lenvatinib‐resistant.