LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Abstract

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.

Keywords: CMA; PCC; TUNEL; autophagy; doxorubicin; tissues; tumor.

Figure 1. Effect of LAMP2A expression on known CMA substrates. Proliferating T47D cells were either transfected with LAMP2A or empty pcDNA3 vector for 48 h (A) and with LAMP2A-siRNA or control siRNA for 72 h (B). The cells were then harvested, and protein lysates were fractionated on a 4–20% Tris Glycine SDS PAGE and immunoblotted with indicated antibodies. Densitometric analyses with respect to ACTB from three independent experiments are shown on the right of each panel. Error bars are SEM and asterisk represents a statistically significant difference (*p < 0.05, ANOVA; Tukey test). LAMP2A downregulated T47D cells were either treated with proteasome inhibitor, MG132 (C) or incubated with lysosomal inhibitor, bafilomycin A₁ (BAFA₁) (D) as described under methods and followed by SDS PAGE and immunoblotting with indicated antibodies. DMF and DMSO were the solvents for the inhibitors and thus serve as controls.

Figure 2. Effect of LAMP2A expression on the half-life of GAPDH. Proliferating MCF-7 cells were transfected with LAMP2A or pcDNA3 for 24 h (A), and similarly proliferating T47D cells were transfected with LAMP2A siRNA or control siRNA (C). Representative autoradiograms of [S]-Methionine labeled GAPDH at the indicated time points from MCF7 (A) and T47D (C) cells are shown following immunoprecipitation with anti-GAPDH antibody in a pulse-chase experiment (see methods). Lanes are labeled according to treatments and the duration of hours after removal of [S]-Methionine (chase period). ‘0’ represents initial labeling. Quantifications of the labeled bands from three independent experiments were performed using Image J software and are shown in (B) (MCF-7) and (D) (T47D) with error bars (SD). Linear trend lines were drawn through the time points, and the equations of the lines are shown on the graph.

Figure 3. Breast tumors express higher levels of LAMP2A. The breast cancer OncoPair INSTA-Blot^TM that contains 14 lanes of paired breast tumor, and matched normal adjacent tissue lysates from seven different patient donors was used. (A) Amido black staining of the blot. (B) Table portraying the type, grades, stages of breast tumors, sex and age of the breast cancer patients. (C) Patient tissue blot was immunoblotted with indicated antibodies. (D–I) Protein expression bands (as indicated) were quantified by densitometry, and the levels were normalized to ACTB (arbitrary units). The values are means ± SEMs of at least three independent experiments, and asterisk represents a statistically significant difference between samples (*p < 0.05).

Figure 4. LAMP2A overexpression stimulates breast cancer cell survival during oxidative stress. Proliferating MCF-7 (A and B) and T47D (C and D) cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h (A and C), as well as transfected with LAMP2A siRNA or control siRNA for 72 h (B and D). (E) demonstrates T47D cells overexpressed with LAMP1 and LAMP2B. Treatment with H₂O₂ was performed in the final 24 h of incubation and cell survival assay was performed as described under methods. Cell viability values are means + SEM of three independent experiments and are normalized to the control (vehicle only) treatment. Significant induction in survival due to LAMP2A and reduction due to LAMP2A siRNA are indicated by an asterisk (*p < 0.05, ANOVA; Tukey test).

Figure 5. Ectopic expression of LAMP2A inhibits protein carbonyl content (PCC) formation. Proliferating T47D cells were transiently transfected with LAMP2A or empty pcDNA3 vector for 48 h (A and C) as well as transfected with LAMP2A siRNA or control siRNA for 72 h (B and D). Cytosolic extracts were prepared; and 50 μg aliquots were tested for their PCC. (C and D) represent the quantitative estimation of PCC, expressed as means ± SEMs of three independent experiments. Significant changes in the PCC contents are indicated by an asterisk (*p < 0.05 ANOVA; Tukey test). Western blots showing the protein levels of LAMP2A and ACTB in different transfection conditions are provided in (E and F).

Figure 6. Knockdown of LAMP2A triggers AKT1 phosphorylation at Ser473. Proliferating T47D cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h (A, C and E) and with LAMP2A siRNA or control siRNA for 72 h (B, D and F). (A and B) showing western blots analysis of total AKT1 and phospho AKT1 (Ser 473). (C and D) demonstrate AKT1 kinase activity using AKT1 substrate GSK3 fusion protein as a measure of AKT1 activation. (E and F) demonstrate the AKT1 substrate assay under oxidative stress condition rendered by 150 nM H₂O₂. Densitometric analyses from (E and F) are given under each panel. Data are mean ± SEMs, and significant changes are indicated by an asterisk (*p < 0.05 ANOVA; Tukey test). “^#” represents the sample lanes that were treated with 1 μM Wortmannin. (G and H) Starvation does not promote AKT1 phosphorylation. Both MCF-7 and T47D cells were starved as described under methods and the cells were harvested after 48 h followed by SDS PAGE, and immunoblotted with indicated antibodies.

Figure 7. LAMP2A modulates ROS generation in breast cancer cells. Proliferating T47D cells were either transfected with LAMP2A or empty pcDNA3 vector for 48 h (A and C) and with LAMP2A siRNA or control siRNA for 72 h (B and D) in the presence of 150 nm H₂O₂ for the last 24 h. (A and B) Cells were assayed for carboxy-DCF fluorescence by flow cytometry, and the total geometric means of the carboxy-DCF tagged cells were plotted in each case. (C and D) Cellular uptakes of carboxy-DCF by the indicated cells were imaged live using confocal microscopy (Fig. S9). The green fluorescence due to carboxy DCF among different samples was analyzed using Metamorph imaging software and represented as bar diagram. Error bars are SEM and asterisk represents a statistically significant difference (*p < 0.05, ANOVA; Tukey test).

Figure 8. Downregulation of LAMP2A triggers apoptosis in breast cancer cells. Proliferating T47D cells were either transfected with LAMP2A or empty pcDNA3 vector for 48 h or transfected with LAMP2A siRNA or control siRNA for 72 h followed by incubation with 150 nm H₂O₂ for the last 24 h. The cells were then assayed using Apo-Direct apoptosis kit. Percentage of FITC positive cells from both LAMP2A overexpressed (A) and underexpressed (B) samples were represented as a bar diagram. (C and D) demonstrate immunoblotting with CASP3 and cleaved CASP3 fragments in the indicated samples. Protein expression levels of BAX and BCL2 in LAMP2A overexpressed cells, as well as LAMP2A knock down cells, are shown in (E and F). The densitometric analyses of the indicated proteins with respect to ACTB from three independent experiments are shown as bar diagram in (G and H). Values are means ± SEMs from three independent experiments. Significant changes are indicated by an asterisk (*p < 0.05 ANOVA; Tukey test).

Figure 9. Inhibition of LAMP2A sensitizes breast tumor cells to DNA damaging drug. Proliferating MCF-7 cells (A and B) and T47D cells (C and D) were transfected with LAMP2A siRNA or control siRNA for 72 h and treated/untreated with 2.5 μM doxorubicin for the last 24 h of transfections. The cells were then subjected to cell survivability assay as described under methods. – DOX indicates no doxorubicin treatments where as + DOX indicates cells treated with doxorubicin. The DOX treatments were compared with no DOX treatment in (B) for MCF-7 cells and (D) for T47D cells. Error bars representing SEM and significant changes is indicated by an asterisk (*p < 0.05 ANOVA; Tukey test).