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. 2014 Sep 12;289(37):25737-49.
doi: 10.1074/jbc.M114.570838. Epub 2014 Jul 29.

Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-Of-Function Capacity

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Free PMC article

Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-Of-Function Capacity

Khanh-Quynh N Nguyen et al. J Biol Chem. .
Free PMC article

Abstract

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.

Keywords: AXL; Apoptosis; Breast Cancer; Efferocytosis; Epithelial Cell; GAS6; MERTK; Phagocytosis; Protein S; Receptor-tyrosine Kinase.

Figures

FIGURE 1.
FIGURE 1.
Stable MERTK expression in non-transformed MCF10A cells. A, MCF10A cells were infected with either pMSCV empty vector (left panel) or pMSCV-MERTK (right panel) retroviral particles, and after selection with puromycin, MERTK was detected with anti-MERTK antibodies by flow cytometry. B, MCF10A-pMSCV empty vector control, MCF10A-MERTK, or highly invasive MDA-MB-231 cells were grown in soft agar in the presence of human GAS6, and colonies (where applicable) were stained with crystal violet after 21 days. C, MCF10A-pMSCV and MCF10A-MERTK stable cell lines were starved overnight in medium containing 0.5% HS and full MCF10A supplements. Cells were collected in starvation serum (0.5% HS plus full MCF10A supplements), after which 50 μl of cell suspension containing 2.5 × 103 cells was added to 100 μl of GAS6 conditioned medium or normal growth medium containing 5% HS and full MCF10A supplements. Real-time proliferation was monitored every 5 h for 160 h by xCELLigence (ACEA Biosciences). D, representative confocal imaging of acini by MCF10A (20× magnification), MCF10A-MERTK (40× magnification), and MCF10A-pMSCV (40× magnification) cells grown in Matrigel for 7 (iv) or 12 (i, ii, and iii) days. Acini were fixed and stained for actin conjugated with Alexa Fluor® 568 phalloidin (red), nuclei with DAPI (blue), and cleaved caspase-3 (green) to identify apoptotic cells (iv).
FIGURE 2.
FIGURE 2.
MERTK promotes migration and chemoprotection. A, real-time monitoring of cell migration in the MCF10A-MERTK and MCF10A-pMSCV stable cell lines was analyzed by xCELLigence. Fibronectin (10 μg/ml) was coated in the top chamber of the CIM-Plate 16, after which 5 × 104 MCF10A-MERTK or MCF10A-pMSCV cells were cultured in 3% serum and one fifth of the complete MCF10A supplements described under “Experimental Procedures.” MCF10A-MERTK and MCF10A-pMSCV cells were allowed to migrate toward either human GAS6 conditioned medium in 1% serum or control conditioned medium in 1% serum located in the bottom chamber of the CIM-Plate 16. The experiment was done in four replicates, and the representative of two independent experiments is shown (n = 2). B and C, MCF10A-MERTK and MCF10A-pMSCV (in the presence of human GAS6) were treated with 50 μm of camptothecin (B) or 20 μm of 5-FU (C) with or without PI3K/AKT inhibitor, LY-294002 (50 μm), for 24 h and stained with FITC-conjugated Annexin V and propidium iodine (PI). Total cell death (Annexin V and/or PI positive) was evaluated by flow cytometry. The experiment was done in triplicate, and the representative of two independent experiments is shown (n = 2). D, MCF10A-MERTK and MCF10A-pMSCV were starved for 6 h and treated with either human GAS6-conditioned medium or control conditioned medium for 30 min, after which cell lysates were collected, and MERTK and AKT phosphorylation were determined by immunoblotting with their respective antibodies.
FIGURE 3.
FIGURE 3.
MERTK drives efferocytosis in PMECs and when stably expressed in MCF10A cells. A, primary mammary epithelial cells (PMECs) isolated from wild type (WT) or knock-out Mertk(−/−) mice were labeled with CFDA-green. Cells were starved for 6 h and then co-cultured with PKH26-red apoptotic CEM for 5 h and efferocytosis was analyzed by flow cytometry. The experiment was done in triplicate and the representative of 2 independent experiments is shown (n = 2). B, PKH26-red MCF10A-MERTK and MCF10A-pMSCV stable cells were starved for 6 h and then co-cultured with PKH67-green apoptotic CEM in 10% FBS medium for 3 h, and efferocytosis was determined by flow cytometry. C, efferocytosis was carried out as described in B with human GAS6 conditioned medium, GAS6 (1) or (0.5), instead of 10% serum. GAS6 (0.5) was generated by mixing equal volumes of GAS6 (1) with fresh RPMI. NS = not significant. D, representative confocal imaging of PKH26-red MCF10A-MERTK or MCF10A-pMSCV phagocytes and PKH67-green apoptotic CEM co-cultures. The experiment was performed as described in B in an 8-chambered slide.
FIGURE 4.
FIGURE 4.
Transient expression of MERTK accelerates efferocytosis. A, MCF10A was infected with either control pMSCV or human MERTK retroviral vectors and labeled with PKH26-red. 72 h post-infection, efferocytosis was carried out as described in Fig. 3B and analyzed by flow cytometry. B, immortalized mouse primary mammary epithelial cells 21 or C, HeLa cells were transfected with either bi-cistronic plasmids pIRES-EGFP-Mertk (mouse) or control pIRES2-EGFP. 48 h post transfection, these phagocytes were co-cultured with PKH26-labeled (red) apoptotic CEM cells. Efferocytosis was assayed 5 h (21 cells) or 2 h (HeLa) post co-culture, whereby double positive cells (red + green) were scored from GFP-expressing cells and analyzed by flow cytometry.
FIGURE 5.
FIGURE 5.
MERTK overexpression in human breast cancer. A, representatives of 34 breast tumor tissues and 12 normal (tumor adjacent) tissues were immunohistochemical stained with MERTK antibodies. Brown color denotes positive staining. Scale bar is 10 μm. Magnification 200×. All normal tissues were below detection for MERTK, while 24 out of 34 breast cancer tissues (70%) were positive for MERTK. B, human non-transformed breast cells MCF10A and breast cancer cells MDA-MB 231 and MCF-7 were characterized for MERTK expression by immunoblotting. Macrophage THP-1 cells were used as a positive control for MERTK. C, efferocytosis of PKH67-green labeled apoptotic CEM by PKH26-red labeled MCF10A, MCF-7, MDA-MB 231, and THP-1 macrophages was determined by flow cytometry after co-incubation for 3 h. D, MDA-MB 231 cells were infected with MERTK shRNA or scramble shRNA control. MERTK knockdown was verified by immunoblotting. 72 h post-infection, efferocytosis (for 3 h) of apoptotic CEM (PKH67 green) by MDA-MB 231 cells (PKH26 red) was analyzed by flow cytometry.
FIGURE 6.
FIGURE 6.
AXL soluble receptors block MERTK activation. A, Coomassie Blue staining of 6×-His-tagged human soluble receptors sAXL, sMERTK, and sTYRO3 used in this study. B, human GAS6 conditioned medium was incubated with 200 nm of each individual human soluble receptor at 4 °C overnight. Complexes were then “pulled down” using Ni-NTA-agarose and the GAS6 alone without any soluble receptor was used as a control (mock). The pulled down complexes were resolved in SDS-PAGE and immunoblotted with GAS6 or His-Tag antibodies. 1× = 10 μl and 2× = 20 μl of eluted protein complex loaded onto SDS gel. C, an experiment was performed similarly to B with 100 nm of human Protein S (PROS1) instead of GAS6. D, MCF10A-MERTK and MCF10A-pMSCV were starved for 6 h and treated with either GAS6 conditioned medium or control conditioned medium for 15 min. Soluble receptors sAXL (0.1 μm) or sMERTK (0.1 μm) was used as indicated, and MERTK phosphorylation was verified by immunoblotting.
FIGURE 7.
FIGURE 7.
Apoptotic cells enhance MERTK activation by GAS6 or PROS1. MCF10A-MERTK and MCF10A-pMSCV were starved for 6 h and treated with either (A) human GAS6-conditioned medium or control conditioned medium or (B) 0.5 μm human PROS1 for 15 min in the presence or absence of MCF10A apoptotic cells (ACs). Human sTYRO3 (1 μm), sAXL (1 μm), or sMERTK (5 μm) was pre-incubated in lanes 7–9 (A) or lanes 3 and 4 (B). MERTK and AXL phosphorylation was verified by immunoblotting. Densitometry of immunoblots was quantified by ImageJ where auto-phosphorylation status of MERTK or AXL was normalized by total MERTK or AXL expression level, respectively.
FIGURE 8.
FIGURE 8.
AXL soluble receptors inhibit MERTK-driven efferocytosis. A, MCF10A-MERTK and MCF10A-pMSCV labeled with PKH26-red were co-cultured with PKH67-green apoptotic CEM for 3 h in the presence or absence of 1 μm human sAXL and efferocytosis was analyzed by flow cytometry. B, a similar experiment was performed as described in A with MDA-MB 231 instead of MCF10A phagocytes. C, efferocytosis was carried as in panel A for 4 h and analyzed by Amnis Imagestream. However, instead of apoptotic CEM cells, apoptotic MCF10A cells were used in this experiment.
FIGURE 9.
FIGURE 9.
MERTK activation induces PD-L1 and PD-L2 expression. 293TN cells were transfected with pEF2-FL-Fc control or pEF2-FL-Fc-Mertk plasmids. A, 12 and 24 h post-transfection, mRNA transcripts of PD-L1 and PD-L2 in transfected cells were analyzed by real time PCR. B, 48 h post transfection, cells were collected and stained with FITC-conjugated anti-FLAG and PE-conjugated anti-PD-L1 or APC-conjugated anti-PD-L2 and analyzed by flow cytometry. Cells were gated on FLAG positive expression and then analyzed for percent cells that expressed PD-L1 or PD-L2. C, MDA-MB 231cells were transfected with either scramble shRNAs or MERTK targeting shRNAs. 30 h post-transfection, cells were starved for 5 h in serum-free medium and then treated with or without apoptotic MCF10A plus GAS6 conditioned medium. Six hours later, apoptotic MCF10A cells were washed away, and MDA-MB 231 cells were incubated in growth medium with 0.5% serum for another 30 h. Cells were then collected, stained with PE-conjugated anti PD-L1, and analyzed by flow cytometry.

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