Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia

Abstract

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.

Keywords: breast adenocarcinoma; hypoxic tumor microenvironment; immunotherapy; innate immunity.

Fig. 1.

Hypoxia-induced autophagy decreases the susceptibility of breast cancer cells to NK-mediated lysis in vitro. (A) Cytotoxicity assays were performed in duplicate using NK cells isolated from eight healthy donors (D1 to D8) at 5/1 or 1/1 E/T (Effector/Target) ratios on normoxic (N) or hypoxic (H) MCF-7 cells. Cell death was assessed by flow cytometry using TO-PRO-3. (B, Upper) Cells were cultured under normoxia (N) or hypoxia (H) conditions for 16 h. The expression levels of hypoxia inducible factor 1α, p62, and LC3 were assessed by immunoblot. (B, Middle) Untreated (−CQ) or chloroquine (60 µM) treated (+CQ) cells cultured under normoxia (N) or hypoxia (H) were assessed by immunoblot for the expression of LC3. (B, Lower) GFP–LC3 expressing normoxic or hypoxic MCF-7 cells were analyzed by fluorescence microscopy for the autophagosome formation (dots). (Scale bar, 10 μm.) (C) MCF-7 cells were cultured under normoxia, hypoxia, or starvation [Earle's balanced salt solution (EBSS)]. Cytotoxicity assays were performed using NK cells isolated from a healthy donor (D12). The percentage of tumor cell lysis is reported as an average (±SEM) of three experiments performed. Statistically significant differences are indicated by asterisks (*P < 0.05; **P < 0.005; ***P < 0.0005). (D, Upper Left) Autophagy-competent and -defective MCF-7 cells were generated by culturing cells expressing BECN1 under the control of a tetracycline-responsive promoter (BECN1) in the absence (−) or presence (+) of doxycycline (100 ng/mL), respectively. Beclin1 expression was assessed by immunoblot. (D, Right) The formation of autophagosomes (dot-like structures) in autophagy-competent (−Dox) and autophagy-defective (+Dox) hypoxic cells was assessed using immunofluorescence microscopy with an Alexa-Fluor-488–coupled LC3 antibody. Nuclei were stained with DAPI. (Scale bar, 10 µm.) (D, Lower Left) Cytotoxicity assays were performed on autophagy competent (BECN1+) or defective (BECN1−) MCF-7 cells cultured under normoxic (N) or hypoxic (H) conditions with NK cells isolated from a healthy donor (D13). The percentage of tumor cell lysis is reported as an average (±SEM) of three experiments, and statistically significant differences are indicated by asterisks (*P < 0.05).

Fig. 2.

Hypoxia-induced autophagy impairs tumor cell susceptibility to NK-mediated lysis without affecting NK cell function. (A) Autophagy-competent (BECN1+) and -defective (BECN1−) MCF-7 cells cultured under normoxia (N) or hypoxia (H) were assessed by flow cytometry for the expression of MHC class I molecules and NK cell-activating NKG2D ligands . Fluorescence intensity is reported as an average (±SEM) of three experiments. Statistically significant differences are indicated by asterisks (*P < 0.05; **P < 0.005; ***P < 0.0005). (B) BECN1+ or BECN1− MCF-7 cells were pretreated with mouse IgM anti-pan HLA class I A6-136 mAb and incubated under normoxic (N) or hypoxic (H) conditions before presentation to NK cells at a 5/1 E/T ratio. The percentage of target cell lysis is reported. Statistically significant differences are indicated by asterisks (**P < 0.005). (C) BECN1+ or BECN1− MCF-7 cells cultured under normoxia (N) or hypoxia (H) were incubated with NK cells. The percentage of conjugate formation at indicated time was determined by flow cytometry. Results are reported as an average (±SEM) of three independent experiments. No statistically significant differences were observed. (D) NK cells as effectors (E) were cultured alone or with normoxic (N) or hypoxic (H) MCF-7 cells at a 5/1 E/T ratio. The level of CD107a (a degranulation marker) on the surface of the NK cells was assessed by flow cytometry. Fluorescence intensity is reported as an average (±SEM) of five experiments performed with NK cells from different donors. (E) Normoxic (N) or hypoxic (H) MCF-7 cells were loaded with exogenous, activated granzyme B (0.8 μg/mL) using the pore-forming protein streptolysin-O. The percentage of cell death was determined by flow cytometry. Results are reported as an average (±SEM) of three experiments. (F) PKH-26–stained normoxic or hypoxic MCF-7 cells (red) were cocultured with YT–Indy–NK cells expressing GFP–GzmB (green) at 5/1 E/T ratio. The content of NK-derived GFP–GzmB in target cells was monitored after 30 min of coculture by a Zeiss laser-scanning confocal microscope (LSM-510 Meta) with a 60× oil immersion objective. (Scale bar, 10 μm.) (G) Autophagy-competent (BECN1+) or -defective (BECN1−) MCF-7 cells were incubated under normoxia (N) or hypoxia (H) and cocultured with NK cells at 5/1 E/T ratio for 0 and 30 min. Following separation, tumor cells were lysed and subjected to immunoblot for the intracellular GzmB content. NK cell lysate was used as a control for GzmB detection. The expression of LC3 was reported as a marker for autophagy. (H) Normoxic (N) or hypoxic (H) MCF-7 cells were cultured alone (−) or with NK cells (+) at 5/1 ratio for 30 min in the presence (+) or absence (−) of chloroquine (CQ) or e64d/pepstatin. Tumor cells separated from NK were subjected to immunoblot analysis to evaluate the intracellular GzmB and perforin content.

Fig. 3.

In vitro tumor cells degrade NK cell-derived granzyme B in lysosomes via autophagy. (A) NK cells were cocultured with autophagy-competent (BECN1+) or -defective (BECN1−) normoxic or hypoxic MCF-7 cells. Cell lysates of separated tumor cells were subjected to subcellular fractionation. Fractions 1 to 12 were characterized by Western blot using the indicated antibodies. * indicates an unspecific band. (B) Chloroquine-treated hypoxic MCF-7 cells (T) were cocultured with GzmB–GFP–expressing NK cells (E). Target cells were stained with an Alexa-Fluor-568–coupled rabbit anti-LC3 antibody to visualize autophagosomes (red) and with DAPI to stain nuclei (blue). Colocalization of GzmB with LC3 in autophagosomes was visualized by confocal microscopy using a 100× oil immersion objective. An enlarged image (400×) of the overlay (box) is shown. (Scale bar, 10 µm.) (C) Chloroquine treated GFP–LC3 –expressing MCF-7 cell were cultured under hypoxia stained with anti-EEA1 Alexa-Fluor-568–coupled anti-rabbit IgG antibody (red) and DAPI for nuclei. Cells were analyzed by confocal microscopy. The overlay image shows colocalization of LC3 and EEA1 (yellow dots). (Scale bar, 10 µm.)

Fig. 4.

Targeting autophagy in vivo improves tumor elimination by NK cells. (A) Control (NK+) and NK-depleted (NK−) C57BL/6 (Left) or BALB/c (Right) mice were engrafted with B16–F10 murine melanoma cells and 4T1 mammary carcinoma cells, respectively. Tumor growth in NK+ and NK− C57BL/6 (n = 7) and BALB/c (n = 10) mice was monitored using caliper measurements on the indicated days. Statistically significant differences in tumor volume are indicated by asterisks (*P < 0.05; **P < 0.005). (B) Autophagy-competent (BECN1+) or -defective (BECN1−) B16–F10 or 4T1 cells were injected s.c. (Left) or in the mammary fat pad (Right), respectively, in control (NK+) and NK-depleted (NK−) C57BL/6 (n = 7) and BALB/c (n = 10) mice. Tumor growth was monitored using caliper measurements on the indicated days. Statistically significant differences are indicated by asterisks (*P < 0.05).