ANXA3/JNK Signaling Promotes Self-Renewal and Tumor Growth, and Its Blockade Provides a Therapeutic Target for Hepatocellular Carcinoma - PubMed (original) (raw)

. 2015 Jul 14;5(1):45-59.

doi: 10.1016/j.stemcr.2015.05.013. Epub 2015 Jun 18.

Tsun-Ming Fung 1, Steve T Luk 1, Kai-Yu Ng 1, Terence K Lee 2, Chi-Ho Lin 3, Judy W Yam 2, Kwok Wah Chan 4, Fai Ng 5, Bo-Jian Zheng 5, Yun-Fei Yuan 6, Dan Xie 6, Chung-Mau Lo 7, Kwan Man 7, Xin-Yuan Guan 8, Stephanie Ma 9

Affiliations

ANXA3/JNK Signaling Promotes Self-Renewal and Tumor Growth, and Its Blockade Provides a Therapeutic Target for Hepatocellular Carcinoma

Man Tong et al. Stem Cell Reports. 2015.

Abstract

Frequent tumor relapse in hepatocellular carcinoma (HCC) has been commonly attributed to the presence of residual cancer stem cells (CSCs) after conventional treatments. We have previously identified and characterized CD133 to mark a specific CSC subset in HCC. In the present study, we found endogenous and secretory annexin A3 (ANXA3) to play pivotal roles in promoting cancer and stem cell-like features in CD133+ liver CSCs through a dysregulated JNK pathway. Blockade of ANXA3 with an anti-ANXA3 monoclonal antibody in vitro as well as in human HCC xenograft models resulted in a significant reduction in tumor growth and self-renewal. Clinically, ANXA3 expression in HCC patient sera closely associated with aggressive clinical features. Our results suggest that ANXA3 can serve as a novel diagnostic biomarker and that the inhibition of ANXA3 may be a viable therapeutic option for the treatment of CD133+ liver-CSC-driven HCC.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract

Figure 1

Figure 1

Endogenous and Secretory Overexpression of ANXA3 Is Strongly Associated with HCC Pathogenesis (A) Relative ANXA3 expression in sorted CD133 subsets isolated from HCC cells and clinical samples by qPCR. Results represent mean ± SD of duplicate wells in three independent experiments. (B) Western blot showing expression of ANXA3 in sorted CD133 subsets isolated from HCC cells. (C) Secretory ANXA3 expression levels in sorted CD133 subsets isolated from HCC cells as detected by ELISA. (D) Dual-color IF images of CD133 (red) and ANXA3 (green) in Huh7. Nuclei stained with DAPI (blue). (E) Western blots showing expression of CD133 and ANXA3 in a panel of liver cancer cell lines. (F) Western blot showing expression of ANXA3 in HCC (T) and matched non-tumor liver (N) specimens from six individual patients. Association between tumor stage and ANXA3 overexpression in HCC. ∗p < 0.05. Results represent mean ± SD from three independent experiments. (G) Boxplots showing expression of ANXA3 in the sera collected from healthy individuals (normal), HBV carriers (HBV+), patients with liver cirrhosis, and patients with either early HCC (I & II) or advanced HCC (III & IV). Results represent mean ± SD of duplicate wells in three independent experiments. (H) ROC curve analysis of the sensitivity and specificity of using AFP, ANXA3, or a combination of both for HCC diagnosis. Summary of ROC curve analysis with area under the curve (AUC) and 95% CI values. See also Figure S1.

Figure 2

Figure 2

Endogenous ANXA3 Confers Enhanced Cancer and Stem Cell-like Properties in HCC (A) Quantification of hepatospheres in HCC cells with ANXA3 stably expressed (MIHA EV and ANXA3 O/E) or repressed (Huh7 NTC and ANXA3 knockdown clones 244 and 246). ∗p < 0.05. EV, empty vector; O/E, overexpression; NTC, non-target control. Scale bar, 100 μm. Results represent mean ± SD of 12 replicates in three independent experiments. (B) Percentage of Annexin-V-positive cells in MIHA and Huh7 with ANXA3 stably expressed and repressed, respectively, following STS treatment. Results represent mean ± SD of three independent experiments. (C) Western blot showing expression of total and cleaved caspase-3 and PARP after STS treatment. (D) Percentage of Annexin-V-positive cells in MIHA and Huh7 with ANXA3 stably expressed and repressed, respectively, following 5-FU and cisplatin treatment. Results represent mean ± SD of three independent experiments. (E) Western blot showing expression of ANXA3 in Huh7 following treatment with increasing concentrations of 5-FU and cisplatin. (F) Quantification of capillary tubes formed by human umbilical vein endothelial cells (HUVECs) following treatment with supernatant collected from MIHA and Huh7 with ANXA3 stably expressed and repressed, respectively. ∗p < 0.05. Scale bar, 100 μm. Results represent mean ± SD of duplicate wells in three independent experiments. See also Figures S2 and S3.

Figure 3

Figure 3

Endogenous ANXA3 Contributes to Augmented Tumor-Initiating, Self-Renewal, and Metastatic Potential In Vivo (A) Top: representative xenograft tumors derived from MHCC97L-EV and -ANXA3 O/E or Huh7-NTC and -ANXA3 knockdown clones 244 and 246 cells 4 weeks after subcutaneous injection (n = 5–6). Scale bar, 1 cm. Bottom: H&E- images of tumors derived from MHCC97L-EV, -ANXA3 O/E, and Huh7-NTC cells. Scale bar, 200 μm. (B) IHC staining for expression of ANXA3 and PCNA in the resected xenograft tumors derived from MHCC97L-EV and -ANXA3 O/E cells. Scale bar, 200 μm. (C) Ex vivo imaging of lungs harvested from mice that received orthotopic injections of MHCC97L-EV or -ANXA3 O/E cells in the liver. Luciferase signals shown as dot plot. H&E images of lung tissue harvested. Scale bars represent 1 cm (top) and 100 μm (bottom). Results represent mean ± SD of six mice from one independent experiment. (D) Top: images of tumors (black arrows) formed in NOD/SCID mice injected subcutaneously with CD133+ANXA3+, CD133+ANXA3−, CD133−ANXA3+, and CD133−ANXA3− cells isolated from Huh7 (picture representative of a 5,000-cell injection) (n = 8 for primary and n = 4 for secondary implantations). Scale bar, 1 cm. Bottom: engraftment rates of CD133+ and CD133− subsets with or without ANXA3 repressed in Huh7. See also Figures S2 and S3.

Figure 4

Figure 4

Secretory ANXA3 Confers Enhanced Cancer and Stem Cell-like Properties in HCC In Vitro and In Vivo (A) Secretory ANXA3 overexpression and recombinant ANXA3 model systems. (B) Quantification of number of MIHA and MHCC97L cells that migrated or invaded following co-culture with empty vector control medium (EV CM) or ANXA3-containing conditioned medium (ANXA3 O/E CM). ∗p < 0.05. Scale bar, 200 μm. Results represent mean ± SD from three independent experiments. (C) Quantification of hepatospheres formed following co-culture with EV CM or ANXA3 O/E CM. ∗∗∗p < 0.001. Scale bar, 100 μm. Results represent mean ± SD of 12 replicates in three independent experiments. (D) Percentage of Annexin-V-positive cells in MIHA and MHCC97L co-cultured with EV CM or ANXA3 O/E CM, following treatment with STS, 5-FU, or cisplatin. Results represent mean ± SD of three independent experiments. (E) Top and bottom: quantification of number of MIHA that migrated and formed hepatospheres following treatment with recombinant ANXA3 protein (rANXA3). ∗p < 0.05 and ∗∗p < 0.01. Results represent mean ± SD from three independent experiments. Middle: quantification of capillary tubes formed by HUVECs following treatment with rANXA3. ∗∗∗p < 0.001. Scale bars represent 200 μm (top) and 100 μm (middle and bottom). Results represent mean ± SD of duplicate wells in three independent experiments. (F) Representative xenograft tumors derived from MHCC97L cells co-cultured with EV CM or ANXA3 O/E CM 4 weeks after subcutaneous injection (n = 8). Dot plot shows the tumor weight of each xenograft. Results represent mean ± SD of eight mice from one independent experiment.

Figure 5

Figure 5

Exogenous ANXA3 Is Internalized through Caveolin-1-Dependent Endocytosis (A) Representative 2D and 3D confocal microscopy images of CFDA-stained PLC8024 following co-culture with SNAP-tagged ANXA3 proteins (SNAP-ANXA3). (B) Coomassie-blue-stained SDS-PAGE gel showing cytoplasmic and membrane sub-fractions of PLC8024 treated with 45 or 90 μg of recombinant FLAG-ANXA3 protein (rANXA3). Arrow depicts 35-kDa band, which corresponds to rANXA3. (C) Western blot showing expression of ANXA3 in cytoplasmic and membrane sub-factions of PLC8024 treated with 90 μg of rANXA3 and immunoprecipitated by FLAG antibody. (D) Western blot showing expression of FLAG, ANXA3, and CAV1 in PLC8024 and MHCC97L with or without CAV1 stably repressed and with or without rANXA3 treatment. (E) Western blot showing expression of FLAG, ANXA3, and CAV1 in PLC8024 with or without Myc-CAV1 stably overexpressed and with or without rANXA3 treatment. (F) Western blot showing expression of FLAG and ANXA3 in PLC8024, MIHA, and MHCC97L co-treated with rANXA3, heparinase I, heparinase II, or their combination. Representative images from three independent experiments are shown for all experiments.

Figure 6

Figure 6

Increased ANXA3 Expression Enhances JNK Signaling in CD133+ Liver CSCs (A) Gene expression profiling coupled with GeneGo Metacore analysis in PLC8024 with or without ANXA3 repressed identified a deregulated JNK/AP-1 signaling network. (B) Western blot showing expression of ANXA3, a key player in the JNK pathway (p-MKK4, JNK kinase activity, total JNK), and its downstream targets (c-MYC and p21) in Huh7, PLC8024, and MIHA with or without ANXA3 stably repressed or overexpressed. (C) JNK kinase assay and western blot analysis of activity and expression of JNK-related proteins in MIHA co-cultured with ANXA3 O/E CM, rANXA3, or their controls. (D) Luciferase reporter assay for activity of AP-1 transcription factor in Huh7 with or without ANXA3 stably repressed. ∗p < 0.05. Results represent mean ± SD from triplicate wells in three independent experiments. (E) Dual-color IF images of ANXA3 (green) and p-JNK (red) in Huh7. Nuclei stained with DAPI (blue). (F) Quantification of number of colonies formed in the indicated stable cell lines with or without JNK inhibitor. ∗∗∗p < 0.001. Scale bar, 5 mm. Results represent mean ± SD from triplicate wells in three independent experiments. (G) Quantification of number of cells that migrated or invaded following co-culture with or without JNK inhibitor. ∗p < 0.05 and ∗p < 0.01. Scale bar, 100 μm. Results represent mean ± SD from three independent experiments. (H) Quantification of capillary tubes formed by HUVECs following treatment with supernatant collected from the indicated cell lines with or without JNK inhibitor. ∗p < 0.05. Scale bar, 100 μm. Results represent mean ± SD of duplicate wells in three independent experiments. (I) Quantification of hepatospheres with or without JNK inhibitor. Scale bar, 100 μm. Results represent mean ± SD of 12 replicates in three independent experiments. (J) Percentage of Annexin-V-positive cells in the indicated stable cell lines with or without JNK inhibitor, following treatment with STS, 5-FU, or cisplatin. Results represent mean ± SD of three independent experiments. (K and L) Western blot showing expression of ANXA3, c-MYC, and p21 in ANXA3-overexpressing MIHA (K) and parental Huh7 and PLC8024 (L) treated with 25 or 50 μM JNK inhibitor (SP600125). See also Figure S4.

Figure 7

Figure 7

ANXA3 Ablation Reduces Cancer and Stem Cell-like Properties in HCC In Vitro, Attenuates Tumor Formation In Vivo, and Decreases Liver CSC Subpopulations (A) Western blot showing specificity of the ANXA3 monoclonal antibody in a panel of liver cell lines. (B) XTT assay showing growth rates of Huh7 and MIHA in the absence or presence of ANXA3 mAb. ∗p < 0.05. Results represent mean ± SD of triplicate wells in three independent experiments. (C) Percentage of Annexin-V-positive cells in Huh7 following treatment with 25 μg/ml IgG control or ANXA3 mAb. Results represent mean ± SD from three independent experiments. (D) Quantification of cells that migrated or invaded following treatment with IgG control or ANXA3 mAb. ∗p < 0.05 and ∗∗∗p < 0.001. Scale bar, 100 μm. Results represent mean ± SD from three independent experiments. (E) Quantification of capillary tubes formed by HUVECs following treatment with supernatant collected from HCC cells treated with IgG control or ANXA3 mAb. ∗p < 0.05. Scale bar, 100 μm. Results represent mean ± SD of duplicate wells in three independent experiments. (F) Quantification of hepatospheres in Huh7 and clinical samples following treatment with IgG control or ANXA3 mAb. ∗p < 0.05 and ∗∗∗p < 0.01. Scale bar, 100 μm. Results represent mean ± SD of 12 replicates in three independent experiments. (G) XTT assay showing percentage growth inhibition in Huh7 following treatment with ANXA3 mAb, cisplatin, their combination, or their controls. ∗p < 0.05. Results represent mean ± SD of triplicate wells in three independent experiments. (H) Representative xenograft tumors resected from mice treated with PBS, IgG control, ANXA3 mAb, cisplatin, or a combination of ANXA3 mAb and cisplatin (n = 5). Graph of average tumor volumes of mice along treatment course. Red arrows indicate the days when treatment was administered. Scale bar, 1 cm. Results represent mean ± SD of five mice from one independent experiment. (I) Representative images of secondary tumors (black arrows) formed in NOD/SCID mice injected subcutaneously with cells harvested from residual primary tumors shown in (A). (J) Flow cytometry for CD133 in residual xenografts of the indicated treatment groups. (K) H&E and IHC staining for expression of ANXA3 and PCNA in the resected xenograft tumors. Scale bar, 200 μm. (L) JNK kinase assay and western blot analysis of activity and expression of JNK-related proteins in Huh7 or HCC xenografts resected from mice treated with IgG control or ANXA3 mAb. Images shown of data gathered from n = 5 mice for in vivo studies shown in (H)–(K). See also Figure S5.

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