PSMG2-controlled proteasome-autophagy balance mediates the tolerance for MEK-targeted therapy in triple-negative breast cancer

Abstract

Although the MAPK pathway is aberrantly activated in triple-negative breast cancers (TNBCs), the clinical outcome of MEK-targeted therapy is still poor. Through a genome-wide CRISPR-Cas9 library screening, we find that inhibition of PSMG2 sensitizes TNBC cells BT549 and MB468 to the MEK inhibitor AZD6244. Mechanistically, PSMG2 knockdown impairs proteasome function, which in turn activates autophagy-mediated PDPK1 degradation. The PDPK1 degradation significantly enhances AZD6244-induced tumor cell growth inhibition by interrupting the negative feedback signals toward the AKT pathway. Consistently, co-targeting proteasomes and MEK with inhibitors synergistically suppresses tumor cell growth. The autophagy inhibitor chloroquine partially relieves the PDPK1 degradation and reverses the growth inhibition induced by combinatorial inhibition of MEK and proteasome. The combination regimen with the proteasome inhibitor MG132 plus AZD6244 synergistically inhibits tumor growth in a 4T1 xenograft mouse model. In summary, our study not only unravels the mechanism of MEK inhibitor resistance but also provides a combinatorial therapeutic strategy for TNBC in clinics.

Keywords: AKT pathway; CRISPR-Cas9; MAPK pathway; PDPK1; PSMG2; autophagy; proteasome; resistance; targeted therapy; triple-negative breast cancer.

Graphical abstract

Figure 1

CRISPR-Cas9 screening identified synthetic lethal genes of MEK inhibitors (A) The experimental outline of genome-wide CRISPR-Cas9 knockout library screening in BT549 cells. (B) Comparing the sgRNA data of DMSO-Day7 and AZD-Day7, 1,649 genes were significantly downregulated in the AZD-Day7 group. In the scatter diagram, the color and size of the dots indicate the number of detected sgRNAs for each gene and the down recurrence (sgRNA_down/sgRNA_total), respectively. (C) The upset diagram shows the numbers of overlapping genes that may drive MEK inhibitor resistance under different criteria. (D) The scatterplot shows that 52 final candidate genes potentially confer tolerance to MEK inhibitors in our model cells. According to the log₂FC, the top 10 genes are marked with different colored dots and gene symbols.

Figure 2

PSMG2 was the determinant of MEK inhibitor resistance in TNBC cells (A) Clonogenic assays of BT549 and MB468 cells with or without knockdown of 10 candidate genes and with or without AZD6244 (1 μM) treatment for 10–14 days. (B and C) Western blot shows the overall ubiquitination level of BT549 and MB468 cells after PSMG2 knockdown or MG132 (2 μΜ) for 24 h. Loading control, β-actin. (D) Clonogenic assays of BT549 and MB468 cells treated with AZD6244 (1 μM) alone or MG132 (0.4 μM) alone or the combination for 10–14 days. (E) The results of the clonogenic assays (D) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗∗∗p < 0.001; one-way ANOVA. (F) The relative synergy of different concentrations in drug combinations was calculated by CalcuSyn v.2.0 (expressed as log₁₀ of CI value) in BT549 and MB468 cells. (G) BT549 and MB468 cells transfected with vector (cPPT) or the overexpression vector for PSMG2 (PSMG2) were treated with AZD6244 (1 μM), MG132 (0.4 μM), or the combination for 10–14 days. (H and I) The results of the clonogenic assays (G) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗∗∗∗p < 0.0001; one-way ANOVA.

Figure 3

PSMG2 knockdown downregulated PDPK1/AKT signaling (A) The scatterplot shows the correlation between proteasome and all 50 hallmark gene sets in the two single-cell transcriptome datasets (GEO: GSE75688 and GEO: GSE11838). The x axis and y axis represent the correlation coefficients of proteasome and hallmark gene sets in the GEO: GSE11838 and GEO: GSE75688 datasets, respectively. The size and color of the points represent the −log₁₀ p value in the GEO: GSE11838 and GEO: GSE75688 datasets, respectively. The points of the top 5 signal pathways in the comprehensive correlation are marked with different colored outer circles. (B) The scatterplot shows the high correlation between mTOR signaling and proteasome in two independent single-cell transcriptomic datasets (GEO: GSE75688 and GEO: GSE11838). The pink triangles represent the GEO: GSE75688 dataset, and the blue dots represent the GEO: GSE11838 dataset. (C) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were treated with AZD6244 (1 μM) for 2 or 24 h, and then the phosphorylation of ERK in the MAPK pathway and the phosphorylation and total protein of AKT and PDPK1 in the upstream mTOR pathway were detected by western blot. Loading control, β-actin. (D) BT549 and MB468 cells were treated with or without MG132 (2 μM) for 24 h and the related proteins were detected by western blot. Loading control, β-actin. (E) After transfection with vector (pLKO.1) or PDPK1 shRNA (shPDPK1), BT549 and MB468 cells were treated with AZD6244 (1 μM) for 2 or 24 h, and then the phosphorylation of ERK and AKT and total protein levels of PDPK1 were detected by western blot. Loading control, β-actin. (F) Clonogenic assays of PDPK1-knockdown cells (BT549 and MB468) treated with or without AZD6244 (1 μM) for 10–14 days. (G) The results of the clonogenic assays (F) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗∗∗p < 0.001; one-way ANOVA. (H) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were transfected with vector (cPPT) or the overexpression vector for PDPK1 (PDPK1), then the phosphorylation of PDPK1 and AKT was analyzed by western blot. Loading control, β-actin. (I) Under PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were transfected with vector (cPPT) or the vector for PDPK1 expression (cPPT-PDPK1) and treated with or without AZD6244 (0.1 or 0.5 μM) for 10–14 days. (J and K) The results of the clonogenic assays (I) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗p < 0.01, ∗∗∗∗p < 0.0001; one-way ANOVA.

Figure 4

PSMG2 knockdown activated autophagy in TNBC cells (A and B) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were treated with or without AZD6244 (1 μM) for 24 h, and the relative mRNA or ribosome-bound mRNA expression level of PDPK1 was detected by qRT-PCR. Error bars represent the mean ± SD, n = 3 technical replicates. (C) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, the autophagy-related proteins were detected by western blot in BT549 and MB468 cells. Loading control, β-actin. (D and E) BT549 and MB468 cells were treated with or without MG132 (2 μM) for 24 h or bortezomib (100 nM) for 36 h, and then the autophagy-related proteins were detected by western blot. Loading control, β-actin. (F) BT549 cells, transfected with vector (pLKO.1) or PSMG2 shRNA (shPSMG2), were imaged by transmission electron microscopy. Two magnified views of the electron photomicrograph show a characteristic autophagosome. N, nucleus; ER, endoplasmic reticulum; M, mitochondria; A, autophagosome. The scale bar represents 2 μm and 1 μm, respectively. (G) After PSMG2 knockdown, ER stress-related proteins were detected by western blot in BT549 and MB468 cells. Loading control, β-actin.

Figure 5

PDPK1 was degraded by autophagy in TNBC cells (A) BT549 and MB468 cells were treated with different concentrations of autophagy activator (rapamycin) for 24 h, and then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, β-actin. (B and C) Clonogenic assays of BT549 and MB468 cells treated with different concentrations of AZD6244 and LiCl (6 mM) for 10–14 days. (D) After vector (pLKO.1) or PSMG2 shRNA (shPSMG2) transfection, BT549 and MB468 cells were treated with CQ (40 μM) for 8 or 12 h, and then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, β-actin. (E) After treatment with or without MG132 (2 μM) for 24 h, BT549 and MB468 cells were treated with CQ (40 μM) for 8 or 12 h, then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, β-actin. (F) Confocal microscopy images show the merged subcellular co-localization images of PDPK1 and LAMP1 of BT549 and MB468 cells. BT549 and MB468 cells treated with MG132 (2 μM) or rapamycin (1 μM) for 24 h or transfected with PSMG2 shRNA (shPSMG2) and treated with or without CQ (40 μM) for 12 h. Nucleus and LAMP1 were stained with DAPI (blue) and Alexa Fluor 594-conjugated wheat germ agglutinin (red), respectively. PDPK1 was stained with Alexa Fluor 488 combined with wheat germ agglutinin (green). White arrows indicated the co-localization between PDPK1 and LAMP1. The scale bar represents 40 μm. (G) Clonogenic assays of BT549 and MB468 cells treated with or without different concentrations of AZD6244 and CQ (0.5 μM) for 10–14 days after PSMG2 shRNA (shPSMG2) transfection. (H and I) The results of the clonogenic assays (G) were quantified by ImageJ. The quantitative results are presented as the mean ± SD (n = 4 technical replicates); ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA.

Figure 6

MEK inhibitors and proteasome inhibitors synergistically inhibited tumor progression in vivo (A) Clonogenic assays of 4T1 cells treated with different concentrations of AZD6244 or MG132 for 10–14 days. (B) The relative synergy of different concentrations of drug combinations (expressed as log₁₀ of CI value) in 4T1 cells. (C) 4T1 cells were treated with or without MG132 (2 μM) for 24 h and the related proteins were detected by western blot. Loading control, β-actin. (D) 4T1 cells were treated with or without MG132 (2 μM) for 24 h and the autophagy-related proteins were detected by western blot. Loading control, β-actin. (E) 4T1 cells were treated with different concentrations of rapamycin for 24 h, and then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, β-actin. (F) After treatment with or without MG132 (2 μM) for 24 h, 4T1 cells were treated with CQ (40 μM) for 8 or 12 h, then PDPK1 and autophagy-related proteins were detected by western blot. Loading control, β-actin. (G) BALB/c mice were subcutaneously inoculated with 4 × 10⁵ 4T1 cells on day 0. Tumor-bearing mice (n = 8 mice) were treated with various drugs when tumor volume achieved ≈100 mm³. Details of treatment are provided in the STAR Methods. (H) The average tumor growth curves for mice treated with PBS (control), AZD6244, MG132, and AZD6244 plus MG132. The quantitative results are presented as the mean ± SD (n = 8 mice); ∗p < 0.05; one-way ANOVA. (I) Tumors were weighed after the animals were euthanized on day 24. The quantitative results are presented as the mean ± SD (n = 8 mice); ∗∗∗∗p < 0.0001; one-way ANOVA. (J) Degradation of PDPK1 and activation of autophagy were visualized by PDPK1 and LC3 IHC staining. The scale bar represents 100 μm. (K) The results of IHC staining were quantified by Image Pro Plus. The quantitative results are presented as the mean ± SD (n = 4 fields); ∗∗p < 0.01, ∗∗∗∗p < 0.0001; one-way ANOVA.

Figure 7

Schematic diagram of MEK-inhibitor-resistance mechanism in TNBC cells (A and B) Activation of the AKT pathway by MEK inhibition contributes to drug resistance in MEKi-sensitive TNBC cells. (C and D) The proteasome-autophagy balance mediates drug resistance in MEKi-resistant TNBC cells.