CD317 Activates EGFR by Regulating Its Association with Lipid Rafts

Abstract

EGFR regulates various fundamental cellular processes, and its constitutive activation is a common driver for cancer. Anti-EGFR therapies have shown benefit in cancer patients, yet drug resistance almost inevitably develops, emphasizing the need for a better understanding of the mechanisms that govern EGFR activation. Here we report that CD317, a surface molecule with a unique topology, activated EGFR in hepatocellular carcinoma (HCC) cells by regulating its localization on the plasma membrane. CD317 was upregulated in HCC cells, promoting cell-cycle progression and enhancing tumorigenic potential in a manner dependent on EGFR. Mechanistically, CD317 associated with lipid rafts and released EGFR from these ordered membrane domains, facilitating the activation of EGFR and the initiation of downstream signaling pathways, including the Ras-Raf-MEK-ERK and JAK-STAT pathways. Moreover, in HCC mouse models and patient samples, upregulation of CD317 correlated with EGFR activation. These results reveal a previously unrecognized mode of regulation for EGFR and suggest CD317 as an alternative target for treating EGFR-driven malignancies. SIGNIFICANCE: Activation of EGFR by CD317 in hepatocellular carcinoma cells suggests CD317 as an alternative target for treating EGFR-dependent tumors.

Fig. 1.. Upregulation of CD317 expression correlates with tumorigenesis

(A) CD317 upregulation in hepatocellular carcinoma. CD317 protein expression in tumors and normal tissues from patients diagnosed with hepatocellular carcinoma was analyzed by immunohistochemistry. CD317-positive cells are shown in brown. Quantitation of the CD317 expression in tumors (n = 38) and normal tissues (n = 10) was performed as described in Methods. Each data point represents a CD317 expression score of patients. The horizontal bars represent the medians. Scale bar: 100 μm. (B) Levels of CD317 protein in the human cell lines. (C) Immunoblot analysis of HCC cells with or without CD317 overexpression or knockdown. (D) Proliferation of HepG2, Bel7402, and Huh7 cells transiently transfected with control (MigR1) or CD317 plasmid, or with control (siCtrl) or CD317 siRNA (siCD317). * P < 0.05 and *** P < 0.001 for CD317 vs MigR1 group. ^## P < 0.005 and ^### P < 0.001 for CD317 siRNA vs control siRNA group. (E) Colony formation of HepG2 and Bel7402 cells transiently transfected with MigR1, CD317, siCD317 or control siRNA. ** P < 0.005, *** P < 0.001. (F-I) HepG2 cells transfected with control (PLVX) or CD317 plasmid were injected s.c. into nude mice. Shown are tumor incidences (F) and tumor growth (G) overtime, and typical whole-body fluorescence images (H, upper panel), tumor appearance (H, bottom panel), and weight (I, mean ± SEM) at 28 days post-injection. ** P < 0.005. (J) Total lysates from PLVX and CD317 tumors were analyzed by western blotting. (K-M) HepG2 cells transfected with control (shCtrl), CD317 shRNA-1 (sh317–1), or CD317 shRNA-2 (sh317–2) were injected s.c. into nude mice. Shown are tumor growth (K) overtime, and typical whole-body images (L, upper panel), tumor appearance (L, bottom panel), and weight (M, mean ± SEM) at 23 days post-injection. (N) Total lysates from shCtrl, sh317–1 and sh317–2 tumors were analyzed by western blotting.

Fig. 2.. CD317 accelerates cell cycle transition in vitro

(A, B) HepG2, Bel7402, and Huh7 cells were transfected with the control vector (MigR1) or either CD317 (A), or HepG2 and Bel7402 cells were transfected with control or CD317 siRNA (B). Percentage of cells in G₀/G₁, S, and G₂/M phase was quantified (means ± SEM). ** P < 0.005, *** P < 0.001 (C, D) Expression of cell cycle-related proteins in HepG2, Bel7402, and Huh7 cells transfected with MigR1 or CD317 (C), or in HepG2, Bel7402 cells transfected with control or CD317 siRNA (D). Blots in C and D were exposed for different times to better show the effects of CD317 overexpression or knockdown. (E, G) Expression of cell cycle regulatory proteins in HepG2 cells transfected with MigR1 vector, wild type CD317, or the indicated CD317 mutants (E), or with the control vector pCMV and CD317-ECD-His (G). In (G), the endogenous CD317 and CD317-ECD-His was detected by anti-CD317 antibody, while CD317-ECD-His was also detected by anti-His antibody. (F, H) Cell cycle progression of HepG2 cells transfected with MigR1 vector, wild type CD317, or the indicated CD317 mutants (F), or with the control vector pCMV and CD317-ECD-His (H). Values represent means ± SEM. ** P < 0.005, *** P <0.001. The experiments were repeated at least three times with similar results.

Fig. 3.. CD317 function is dependent on EGFR-STAT3/ERK axis

(A) STAT3 and ERK½ phosphorylation levels in HepG2, Bel7402 and Huh7 cells transiently transfected with MigR1 or CD317 (left), or with control siRNA or CD317 siRNA (right). The levels of CD317, pSTAT3, total STAT3, pERK½, and total ERK½ were determined by Western blot. (B) HepG2 cells were transfected with control MigR1 vector, CD317, or CD317 N65/92D (left), Huh7 cells were transfected with control MigR1 vector or CD317 (centered), and Bel7402 cells were transfected with control or CD317 siRNA (right). The levels of CD317, pY845 EGFR, pY1068 EGFR, and total EGFR were analyzed by Western blot. (C) Representative FACS graphs (left) and statistical analysis (right) of EGFR phosphorylation levels in HepG2 cells transiently transfected with control MigR1 vector or CD317. Mean fluorescence intensity (MFI) values of GFP-negative and -positive cells was analyzed as indicated. * P < 0.05, ** P < 0.005, *** P < 0.001. (D) HepG2 cells transfected with MigR1 or CD317 were treated with or without Erlotinib (50 μM). Cells were analyzed for the activation of EGFR, STAT3, and ERK½ (top), expression of cell cycle-related proteins (bottom). (E, F) HepG2 cells stably infected with control (PLVX) or CD317-expressing lentiviruses, and Huh7 cells transfected with control (MigR1) or CD317 vectors were treated with or without Erlotinib (50 μM). Cell cycle distribution (E) and cell proliferation (F) were analyzed. ** P < 0.005, *** P < 0.001 for CD317 vs control group. (G) Huh7 cells transfected with control (MigR1) or CD317 vectors were treated with control or EGFR siRNA (siEGFR). Activation of STAT3 and ERK½ were analyzed. (H-J) HepG2 cells stably infected with PLVX or CD317 lentiviruses were treated with control or EGFR siRNA. Activation of EGFR, STAT3, and ERK½ (H, left), expression of cell cycle-related proteins (H, right), cell cycle distribution (I), and cell proliferation (J) were analyzed. *** P < 0.001 for CD317 vs PLVX group.

Fig. 4.. CD317 regulates EGFR in a lipid raft-dependent manner

(A, B) HepG2 cells were transfected with MigR1 or CD317 plasmid (A), and Bel7402 cells were transfected with control or CD317 siRNA (B). 48 h later, mRNA levels of CD317, EGFR, EGF, TGF-α and AREG were determined by real-time PCR (left), the protein levels of TGF-α and AREG in culture supernatants were detected by ELISA (right). Values represent means ± SEM, ** P < 0.005. The experiments were repeated at least three times with similar results. (C) Lysates of HepG2 cells were immunoprecipitated with anti-CD317 or anti-EGFR antibodies as indicated. Cell lysates (input) and immunoprecipitates were analyzed by Western blotting. (D) HepG2 cells transfected with control or CD317 siRNA were immunostained for EGFR (red), lipid rafts (green), and DNA (DAPI, blue). Shown are representative fluorescence images and EGFR-lipid rafts co-localization plot (left), and Pearson’s correlation coefficient (R) (right; mean ± SEM for at least 30 cells). *** P < 0.001. Scale bar: 20 μm. (E) HepG2 cells were transfected with control or CD317 siRNA, along with control (pLVX) or siRNAs-resistant (SR) CD317, delCT, and delGPI plasmids. EGFR in non-lipid raft (N) and lipid raft fractions were analyzed by Western blot. Transferrin receptor (TfR) and Caveolin-1 serve as loading controls for non-raft proteins and lipid raft proteins, respectively. (F) EGFR activation in CD317-knockdown HepG2 cells expressing the indicated siRNAs-resistant CD317 plasmids or the PLVX control vector. (G, H) HepG2 cells expressing MigR1, CD317, HA-delCT-CD317 (delCT), and delGPI-CD317-HA (delGPI) (H, right) were analyzed for cell cycle progression (G) and EGFR activation (H, left). ** P < 0.005, *** P < 0.001 for CD317 or delCT vs PLVX group. (I) HepG2 and Huh7 cells were transfected with MigR1 or CD317 plasmid, and Bel7402 cells were transfected with Ctrl or CD317 siRNA and treated with or without 10 mM Methyl-β-cyclodextrin (MβCD) for 30 min. Thereafter, cholesterol was reloaded for 90 min using cholesterol-saturated MβCD (2:10 chol-MβCD, 2 mM cholesterol, and 10 mM MβCD) in Huh7 cells. The levels of CD317, pY845-EGFR, pY1068-EGFR, and total EGFR were determined by Western blot.

Fig. 5.. Upregulation of CD317 correlates with the activation of EGFR in HCC

(A) Immunohistological staining of CD317, pY845-EGFR, and PCNA in HCC sections. Scale bars in black and white indicate 50 and 20 μm, respectively. (B) Statistical results of data shown in (a). Shown are correlation co-efficiency between the phosphor-Y845 EGFR and the CD317 staining levels in tissue sections (n = 110) (upper), and nuclear PCNA expression in CD317^high (score ≥ 6, n = 56) and CD317^low (score < 6, n = 54) patients (bottom). Each data point represents a patient. The horizontal bars represent the medians. (C) Proposed role of CD317 in HCC development. CD317 expression is markedly up-regulated in HCC, leading to over-activation of EGFR and the downstream molecules, such as STAT3 and ERK½, by modulating lipid raft dynamic. STAT3 collaborated with ERK½ signaling to activate the expression of Cyclin D1 and PCNA, inhibits p16 expression meanwhile, which consequently accelerates G₁/S phase transition and uncontrolled proliferation. At last, CD317 promotes HCC development.