Amino Acid-Starved Cancer Cells Utilize Macropinocytosis and Ubiquitin-Proteasome System for Nutrient Acquisition

Abstract

To grow in nutrient-deprived tumor microenvironment, cancer cells often internalize and degrade extracellular proteins to refuel intracellular amino acids. However, the nutrient acquisition routes reported by previous studies are mainly restricted in autophagy-lysosomal pathway. It remains largely unknown if other protein degradation systems also contribute to the utilization of extracellular nutrients. Herein, it is demonstrated that under amino acid starvation, extracellular protein internalization through macropinocytosis and protein degradation through ubiquitin-proteasome system are activated as a nutrient supply route, sensitizing cancer cells to proteasome inhibition. By inhibiting both macropinocytosis and ubiquitin-proteasome system, an innovative approach to intensify amino acid starvation for cancer therapy is presented. To maximize therapeutic efficacy and minimize systemic side effects, a pH-responsive polymersome nanocarrier is developed to deliver therapeutic agents specifically to tumor tissues. This nanoparticle system provides an approach to exacerbate amino acid starvation for cancer therapy, which represents a promising strategy for cancer treatment.

Keywords: amino acid starvation; cancer starvation therapy; macropinocytosis; pH-responsive polymersomes; ubiquitin-proteasome system.

Figure 1

Schematic illustration depicting the pH‐responsive polymersomes loaded with BTZ and EIPA exhausting intracellular amino acids for cancer starvation therapy. Created with BioRender.com.

Figure 2

Amino acid‐mediated starvation induces UPS activation for internalized protein degradation. A) Representative CLSM images of FITC‐BSA (0.2 mg mL⁻¹) internalized by A549 cancer cells treated with DMEM or EBSS medium for 4 h. B) Flow cytometry analysis, and C) quantified result of FITC‐BSA (0.2 mg mL⁻¹) internalization in A549 cells treated with DMEM or EBSS medium for 4 h. D,E) Western blot analysis of K48‐polyubiquitinated proteins in A549 cells treated with DMEM, EBSS, EBSS plus BTZ (10 µm) for 2 h or 24 h. F) Flow cytometry result, and G) quantified analysis of proteasome activity experiment in A549 cells receiving DMEM, EBSS, EBSS plus BTZ (10 µm) for 4 h. H) Representative CLSM images of DQ‐BSA (10 µg mL⁻¹) internalization and degradation analysis in A549 cells receiving DMEM, EBSS, EBSS plus BTZ (10 µm) for 4 h. I) Flow cytometry result, and J) quantified analysis of DQ‐BSA uptake and degradation analysis in A549 cells receiving indicated treatments for 4 h. Data are represented as mean ± SD (n = 3). ^** p <0.01, ^**** p <0.0001.

Figure 3

UPS activity in starvation‐adapted A549 cells and LUAD tumor tissues. A) Outline of the process for starvation‐adapted cell line construction. B) Top 70 DEGs of RNAseq analysis between normal A549 cells and starvation‐adapted A549 cells. C) GSEA analysis of all DEGs between normal A549 cells and starvation‐adapted A549 cells. D) Flow cytometry analysis of proteasome activity in normal A549 cells and starvation‐adapted A549 cells, treated with normal DMEM or EBSS, separately. E) Flow cytometry analysis of FITC‐BSA internalization in normal A549 cells and starvation‐adapted A549 cells, treated with normal DMEM or EBSS, separately. F) Volcano plot of UPS‐associated DEGs between lung tumor tissues and surrounding normal tissues. G) GO enrichment analysis of UPS‐associated DEGs between lung tumor tissues and surrounding normal tissues. H) Cell viability analysis of A549 cells treated with DMSO or BTZ (20 µM), in DMEM or EBSS medium for 24 h. I) Western blot analysis of p‐S6K (70 kDa), S6K (70 kDa), and mTOR (289 kDa) in A549 cells treated with normal DMEM or EBSS medium for 2 h. J) Western blot analysis of K48‐polyubiquitinated proteins in A549 cells treated with DMSO or rapamycin (100 µm) for 2 h. K) Flow cytometry and L) quantified analysis of proteasome activity probe in A549 cells treated with a series of concentrations of rapamycin for 4 h. Data are represented as mean ± SD (n = 3). ^** p <0.01, ^*** p<0.001,^**** p <0.0001, ns: not significant.

Figure 4

Synergistic anti‐cancer effect of EIPA and BTZ. A) Representative CLSM images of TMR‐Dextran internalization in A549 cells treated with DMEM, EBSS, or EBSS plus EIPA (50 µm, pretreated 1 h before) for 1 h. B) Flow cytometry and quantified analysis of TMR‐Dextran internalization in A549 cells treated with DMEM, EBSS, or EBSS plus EIPA (50 µm, pretreated 1 h before) for 1 h. C) Representative CLSM images of FITC‐BSA internalization in A549 cells treated with DMEM, EBSS, or EBSS plus EIPA (50 µM, pretreated 1 h before) for 1 h. D) Flow cytometry and quantified analysis of FITC‐BSA internalization in A549 cells treated with DMEM, EBSS, or EBSS plus EIPA (50 µM, pretreated 1 h before) for 1 h. E) Representative co‐localization analysis of FITC‐BSA and TMR‐Dextran in A549 cells using CLSM. F) Cell viability analysis of A549 cells treated with different concentrations of EIPA and BTZ for 24 h in EBSS medium. G) Flow cytometric apoptosis analysis, and H) quantified result of A549 cells treated with EIPA (50 µM), BTZ (20 µm), or their combination in EBSS medium for 24 h. I) Flow cytometry, and J) quantified analysis of DQ‐BSA fluorescence intensity in A549 cells treated with EIPA (50 µM), BTZ (20 µM), or their combination in DMEM or EBSS medium for 24 h. K) The level of GSH in A549 cells treated with EIPA (50 µM), BTZ (10 µm), or their combination in DMEM or EBSS (3% BSA) medium for 24 h. L) DCFH‐DA flow cytometric analysis in A549 cells treated with EIPA (50 µm), BTZ (10 µm), or their combination in indicated medium for 24 h. Data are represented as mean ± SD (n = 3). ^**p <0.01, ^*** p <0.001, ^**** p <0.0001.

Figure 5

Combination of EIPA and BTZ circumvents compensatory protein catabolism. A) Scheme depicting the influence of EIPA on intracellular protein degradation. Created with BioRender.com. B) Flow cytometry and C) quantified analysis of cells pretreated with DQ‐BSA for 1 h, then cultured in DMEM, EBSS plus BSA, EBSS plus BSA and EIPA for another 3 h. D) Scheme depicting the effect of BTZ on extracellular protein internalization and lysosome‐dependent degradation. Created with BioRender.com. E) Floy cytometry and F) quantified analysis of FITC‐BSA internalization in A549 cells receiving DMEM, EBSS, or EBSS plus BTZ for 4 h. G) Representative CLSM images and H,I) quantified analysis of mCherry‐GFP‐LC3 reporter system and BSA‐AF647 internalization in A549 cells treated with DMEM, EBSS, and EBSS plus BTZ for 4 h using FIJI‐ImageJ software with ComDet plugin. J) Western blot analysis of SQSTM1 (62 kDa) in A549 cells treated with indicated drugs in DMEM or EBSS plus BSA medium for 4 h. Data are represented as mean ± SD (n = 3). ^* p <0.05, ^** p <0.01, ^**** p <0.0001.

Figure 6

Characterization of polymersomes encapsulating EIPA and BTZ. A) Scheme of the polymersomes encapsulating EIPA and BTZ, which can undergo pH‐responsive cleavage. Created with BioRender.com. B) Average sizes of ENPs, BNPs, and EBNPs using DLS. C) TEM image of EBNPs. D) Stability test of ENPs, BNPs, and EBNPs in DMEM with 10% FBS at 37 °C for 5 days. E) Drug release profiles of EBNPs for 24 h at pH 7.4 and pH 5.0, separately. F) Cell viability of A549 cells treated with ENPs, BNPs, and EBNPs for 24 h. Concentrations of EIPA was 25, 50, or 100 µm. Concentrations of BTZ was 14, 28, or 56 µm. Data are represented as mean ± SD (n = 3). ^** p <0.01, ^*** p <0.001, ^**** p <0.0001.

Figure 7

Anti‐tumor effect of polymersome nanoparticles in xenograft tumor mouse model. A) Schematic illustration of the schedule for xenograft tumor model implantation and synergistic therapy. B) Images of xenograft tumor tissues excised from tumor‐bearing mice at the endpoint of treatments (n = 4). C) Tumor volume profile of tumor‐bearing mice receiving different treatments. D) Tumor weight of xenograft tumor tissues excised from tumor‐bearing mice at the endpoint of treatments (n = 4). E) Representative H&E staining analysis of tumor tissues excised from tumor‐bearing mice at the endpoint of treatments. F) Representative TUNEL assay of tumor tissues excised from tumor‐bearing mice at the endpoint of treatments.^* p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001.