Korean J Physiol Pharmacol.  2023 Mar;27(2):157-165. 10.4196/kjpp.2023.27.2.157.

The expression of Rab5 and its effect on invasion, migration and exosome secretion in triple negative breast cancer

• Qiao L
• Dong C
• Zhang J
• Sun G

• KMID: 2539570
• DOI: http://doi.org/10.4196/kjpp.2023.27.2.157

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and current therapeutic strategies are limited in their effectiveness. The expressions of Rab5 and the M2 tumor-associated macrophage marker CD163 in tissues were detected by Western blot. The migration and invasion of cells were determined using a Transwell assay. The expressions of the exosome markers were evaluated by Western blot. The polarization of human macrophages (THP-1) was determined by incubation of THP-1 cells with conditioned medium or exosomes collected from MDA-MB-231 cells with indicated transfections or by a coculture system of THP-1 and MDA-MB-231 cells. The M1 and M2 macrophage markers were evaluated by qRT-PCR. The expression of Rab5 in TNBC was significantly higher than that in normal breast tissue. Rab5 expressions in triple-negative and luminal A breast cancer were higher than those in other molecular subtypes. Higher CD163 expression was observed in triple-negative breast cancer and in triple-negative and luminal B subtypes. Rab5 knockdown suppressed but Rab5 overexpression promoted the migration and invasion capacity of MDA-MB-231 cells. The levels of CD63 and CD9 in the medium of Rab5 knockdown cells were lower than those in control cells, whereas higher levels of CD63 and CD9 were observed in Rab5 overexpression cells. Rab5 knockdown decreased the excretion but did not alter the diameter of the exosomes. Knockdown of Rab5 facilitated the anti-tumor polarization of macrophages, which was partially reversed by Rab5 overexpression. Therefore, Rab5 is expected to be a potential therapeutic target for triple-negative breast cancer.

Keyword

Exosomes; Rab5 GTP-binding proteins; Triple-negative breast cancer; Tumor-associated macrophages

Figure

• 

  Fig. 1 The protein level of Rab5 in normal breast tissue (n = 3) and luminal A (n = 4), luminal B (n = 4), Her2+ (n = 4) and triple-negative breast cancer (TNBC) tissues (n = 4). Data was represented as mean ± SD. *p < 0.05 vs. normal tissue.

• 

  Fig. 2 Knockdown of Rab5 in MDA-MB-231 cells by small interfering RNA. (A) Effect of siRNA concentration and incubation time on transfected with siRNAs (n = 5), cells were stained with Block-iT Alexa Fluor Red, magnification 100×. (B) mRNA level of Rab5 in MDA-MB-231 cells transfected with siRNAs (n = 5). (C) Protein level of Rab5 in MDA-MB-231 cells transfected with siRNAs (n = 3). (D) mRNA and (E) protein levels of Rab5 in MDA-MB-231 cells transfected with empty or Rab5-containing vectors. Data was represented as mean ± SD. *p < 0.05 vs. control.

• 

  Fig. 3 Migration and invasion capacity of MDA-MB-231 cells transfected with siRab5 (A) or vector containing Rab5 (B), n = 5. Cells were stained with Giemsa in migration and invasion assays, magnification 100×. Data was represented as mean ± SD. *p < 0.05 vs. control.

• 

  Fig. 4 The protein levels of exosome hallmarks CD63 and CD9 in the supernatant of MDA-MB-231 cells transfected with siRab5 (A) or vector containing Rab5 (B), n = 3. Data was represented as mean ± SD. *p < 0.05 vs. control.

• 

  Fig. 5 The implication of Rab5 in the polarization of macrophages. (A) Human THP-1 macrophages were treated with conditioned medium, exosomes collected from MDA-MB-231 cells, or were cocultured with MDA-MB-231 cells. The M2 (CD206, IL-10, and CD163) and M1 (iNOS, IL-1β, and TNF-α) markers in THP-1 cells treated with conditioned medium (B) or exosomes (C) or cocultured with MDA-MB-231 cells (D) were determined by qRT-PCR. (E) The expression of M1/M2 macrophage markers in THP-1 cells cocultured with MDA-MB-231 cells with Rab5 overexpression. The mock treatment represents THP-1 cells treated with normal medium alone. Data was represented as mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001 vs. mock treatment.

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