Blockade of CD47-mediated cathepsin S/protease-activated receptor 2 signaling provides a therapeutic target for hepatocellular carcinoma

Authors

  • Terence Kin-Wah Lee,

    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
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    • These authors contributed equally to this work.

  • Vincent Chi-Ho Cheung,

    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
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    • These authors contributed equally to this work.

  • Ping Lu,

    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
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  • Eunice Yuen Ting Lau,

    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
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  • Stephanie Ma,

    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
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  • Kwan Ho Tang,

    1. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
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  • Man Tong,

    1. Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
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  • Jessica Lo,

    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
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  • Irene Oi Lin Ng

    Corresponding author
    1. State Key Laboratory for Liver Research, The University of Hong Kong, Pokfulam, Hong Kong
    2. Departments of Pathology, The University of Hong Kong, Pokfulam, Hong Kong
    • Address reprint requests to: Irene O.L. Ng, M.D., Ph.D., Room 127B, University Pathology Building, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong. E-mail: iolng@hku.hk; fax: +852-2872-5197 or Terrence K.W. Lee, Ph.D., Room 704, 7/F, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong. E-mail: tkwlee@hkucc.hku.hk; fax: +852-2819-5375.

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  • Potential conflict of interest: Nothing to report.

  • The study was supported by the Hong Kong Research Grants Council Collaborative Research Fund (HKU 1/06C and HKU 7/CRF/09), Hong Kong Research Grants Council General Research Fund (HKU 771213M; to T.K.W.L.), and the National Natural Science Foundation of China (project no.: 81272440; to T.K.W.L.). I.O.L.N. is Loke Yew Professor in Pathology.

Abstract

Identification of therapeutic targets against tumor-initiating cells (TICs) is a priority in the development of new therapeutic paradigms against cancer. We enriched a TIC population capable of tumor initiation and self-renewal by serial passages of hepatospheres with chemotherapeutic agents. In chemoresistant hepatospheres, CD47 was found to be up-regulated, when compared with differentiated progenies. CD47 is preferentially expressed in liver TICs, which contributed to tumor initiation, self-renewal, and metastasis and significantly affected patients' clinical outcome. Knockdown of CD47 suppressed stem/progenitor cell characteristics. CD47+ hepatocellular carcinoma (HCC) cells preferentially secreted cathepsin S (CTSS), which regulates liver TICs through the CTSS/protease-activated receptor 2 (PAR2) loop. Suppression of CD47 by morpholino approach suppressed growth of HCC in vivo and exerted a chemosensitization effect through blockade of CTSS/PAR2 signaling. Conclusion: These data suggest that CD47 may be an attractive therapeutic target for HCC therapy. (Hepatology 2014;60:179–191)

Abbreviations
5MM

5-mispair morpholino

Ab

antibody

cDNA

complementary DNA

CIS

cisplatin

CTSS

cathepsin S

DFS

disease-free survival

DOX

doxorubicin

ELISA

enzyme-linked immunosorbent assay

EpCAM

epithelial cell adhesion molecule

FCM

flow cytometry

GEO

Gene Expression Omnibus

HCC

hepatocellular carcinoma

HUVECs

human umbilical vein endothelial cells

IAP

integrin-associated protein

IHC

immunohistochemistry

IKKβ

inhibitor kappa B kinase beta

mRNA

messenger RNA

NF-κB

nuclear factor kappa B

NOD

nonobese diabetic

OS

overall survival

PAR2

protease-activated receptor 2

PBS

phosphate-buffered saline

qPCR

quantitative polymerase chain reaction

SC

subcutaneously

SCID

severe combined immunodeficiency

shRNA

short hairpin RNA

SIRP-α

signal-regulatory protein alpha

TICs

tumor-initiating cells

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer death worldwide[1] and is often associated with metastasis and recurrence, even after surgical resection, leading to a poor prognosis. Therefore, development of novel treatment regimens is urgently needed to improve the survival of these patients. Furthermore, HCC has heterogeneous pathologies, speculated to be the result of the existence of “cancer stem cells” (or tumor-initiating cells; TICs) within tumor bulk. These subsets of cancer cells possess stem cell features that are indispensable in fueling growth of tumor. They also provide an explanation for the failure of current chemotherapeutic treatments because they largely target at eradicating rapidly proliferating tumor bulk. Accumulating evidence suggests the involvement of TICs in the perpetuation of various cancers, including acute myeloid leukemia,[2] brain, [3] and colon.[4] Recently, liver TICs have been identified by several cell-surface antigens, such as CD133,[5, 6] CD90,[7] epithelial cell adhesion molecule (EpCAM),[8] CD13,[9] and CD24.[10] Furthermore, these TICs are capable of self-renewal and are resistant to chemotherapeutic drugs.[11] However, neutralizing antibodies (Abs) against these markers have not been effective in preclinical studies, and identification of therapeutic targets against liver TICs is needed.

As a continual pursuit in search of novel therapeutic targets against liver TICs, we cultured serial passages of hepatospheres combined with chemotherapeutic regimens as a strategy to enrich subpopulations of liver TICs, specifically by use of TIC properties, including self-renewal and chemoresistance. Using complementary DNA (cDNA) microarray, we compared expression profiles between the enriched TIC population and differentiated progeny, which differ in self-renewal and tumorigenicity. This analysis showed that CD47, an integrin-associated protein (IAP), is up-regulated in TIC-enriched hepatospheres. Recent reports have highlighted the superiority of CD47; because of its inhibitory role in macrophage-mediated phagocytosis, anti-CD47 monoclonal Ab therapy has shown positive results in various cancer types.[12-14] Based on the above data, we hypothesized that CD47 was preferentially expressed in liver TICs and that therapeutic targeting of CD47 suppressed the tumor growth in human HCC xenograft.

Freshly isolated CD47+ cells were found to possess a greater ability to form tumors in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, self-renew, and metastasize. CD47 messenger RNA (mRNA) expression levels were preferentially expressed in liver TICs marked by CD133+ and CD24+ HCC cells. In addition, increased CD47 mRNA expression levels in HCC clinical samples correlated with poor patient survival. Knockdown of CD47 by a lentiviral-based short hairpin RNA (shRNA) approach suppressed the stem/progenitor cell characteristics, suggesting a functional role of CD47 in regulating stem-like properties of HCC cells. Furthermore, we identified cathepsin S (CTSS) as an important downstream effector of CD47-mediated HCC tumorigenicity, metastasis, and self-renewal in nuclear factor kappa B (NF-κB)-dependent manner. CTSS was preferentially secreted by CD47+ HCC cells and directly regulated properties of liver TICs through activation of protease-activated receptor 2 (PAR2) by an autocrine loop. Clinically, serum levels of CTSS significantly correlated with advanced tumor behavior of human HCCs. Using the cell line and patient-derived xenograft models, suppression of CD47 by a morpholino approach inhibited growth of HCC and sensitized cells to the effect of chemotherapy through blockade of CTSS/PAR2 signaling. Our findings suggest a signaling function of CD47 and its role in cancer pathogenesis through the CTSS/PAR2 loop and provide a potential and attractive target for treatment of HCC patients.

Materials and Methods

Patient Samples

Human HCC and corresponding nontumor liver samples were collected at the time of surgical resection at Queen Mary Hospital, the University of Hong Kong, from 1991 to 2001. After collection from surgical resection, all samples were immediately snap-frozen in liquid nitrogen before storage at −80°C.

Human HCC Tissue Collection and Processing

Liver tumor and adjacent nontumor liver tissue specimens were collected from 13 patients (patient nos. 3, 18, 38, 73, 83, 98, 497, 498, 499, 500, 501, 502, and 503; ranging in age from 37 to 82 years) who underwent surgical hepatectomy for HCC between 2008 and 2012 in the Department of Surgery, Queen Mary Hospital, Hong Kong, with institutional review board approval. Tumor tissue from xenografts and fresh tumors was minced into 1-mm3 cubes and incubated with type IV collagenase (Sigma-Aldrich, St. Louis, MO) for 5-10 minutes at 37°C. A single-cell suspension was obtained by filtering the supernatant through a 100-μm cell strainer (BD Biosciences, San Jose, CA). Cell viability was assessed by trypan blue exclusion staining and counting using a hemocytometer. For fresh clinical tumors, removal of CD45+ cells from within the tumor was achieved with a CD45 depletion kit (Miltenyi Biotech, Bergisch Gladbach, Germany).

Isolation of CD47+ and CD47 Populations by Flow Cytometery

For isolation of CD47+ and CD47 cell populations, cells were stained with phycoerythrin-conjugated CD47 Ab (BD Biosciences, San Jose, CA) and with isotype-matched mouse immunoglobulin as a control. Samples were analyzed and sorted on a BD FACSAria (BD Biosciences). For the positive and negative populations, the top 10% most brightly stained cells and the bottom 10% most dimly stained cells were selected, respectively. Aliquots of CD47+ and CD47 sorted cells were evaluated for purity with a FACSCalibur machine and CellQuest software (BD Biosciences).

In Vivo Therapeutic Targeting Using CD47

The xenograft was established in 4- to 6-week-old male athymic nude mice (BALB/c-nu/nu) with HCC cell-line–labeled PLC/PRF/5 cells (PLC/PRF/5-luc). Treatment was started once the size of the xenograft reached approximately 4 × 4 mm (length × width) in size after subcutaneous (SC) injection of 3 × 106 of PLC/PRF/5-luc cells, during which mice were randomly assigned into five groups and each group consisted of 5 mice. For evaluation of the effect of CD47 suppression alone, we had three groups that received 100 μL of the following by direct injection surrounding tumor mass every 4 days: (1) 5 μM of anti-CD47 morpholino (group A; Gene Tools, LLC, Philomath, Oregon); (2) 5-mispair morpholino (5MM); and (3) phosphate-buffered saline (PBS) as a control (groups D and E). For evaluation of the combined effect of CD47 suppression with conventional chemotherapy, an additional two groups were included, consisting of intraperitoneal injections of either 2 mg/kg of doxorubicin (DOX) plus 5MM (group B) or 2 mg/kg of DOX (EBEWE Phama, Unterach am Attersee, Austria) plus anti-CD47 morpholino (group C). After 40 days of various treatments, effects on tumor growth were measured and recorded with Xenogen imaging. For patient-derived tumor xenograft PDTX #10, fresh patient tumor was inoculated into athymic nude mice (BALB/c-nu/nu). Treatment was started once the size of the xenografts reached approximately 6 × 6 mm (length × width). Mice were randomly assigned into four groups and each group consisted of 5 mice.

Results

CD47 Was Elevated in Self-Renewing Liver Cancer Cells Enriched Through In Vitro Serial Passage of Hepatospheres Combined With Chemotherapy

Based on the distinct properties of stem/progenitor cells, including self-renewal and chemoresistance, we enrich the liver TIC population from the PLC/PRF/5 HCC cell line upon 16 serial passages in serum-free medium combined with 2 mg/mL of DOX and 1 mg/mL of cisplatin (CIS; Fig. 1A). We observed an enrichment of a liver TIC population in chemoresistant hepatospheres, as reflected by an increased tumorigenic ability, self-renewal, expression of stemness-associated genes, and liver TIC markers (Fig. 1B,C and Supporting Fig. 1A). To further determine the markers elevated in self-renewing chemoresistant HCC cells, mRNA profiles from chemoresistant hepatospheres and their differentiated progenies were compared using a cDNA microarray (Gene Expression Omnibus [GEO] accession no.: GSE53005). Among all CD markers, CD47 was found to be up-regulated with highest fold change by 5.43-fold in chemoresistant hepatospheres, as compared with their differentiated progenies. Consistently, CD47 was found to be up-regulated ∼9-fold by quantitative polymerase chain reaction (qPCR; Supporting Fig. 1B). To exclude the possibility of a cell-type–specific effect, CD47 expression was further evaluated in the same in vitro model, but from the tumor sample of an HCC patient (patient 3). Consistently, CD47 expression was also significantly up-regulated in chemoresistant hepatospheres (P < 0.010, t test; Supporting Fig. 1C,D).

Figure 1.

(A) Hepatospheres were developed from PLC/PRF/5 cells upon 16 serial passages in serum-free medium combined with chemotherapeutic drug. Morphological differences of hepatospheres and their differentiated progenies are shown. (B) Hepatospheres had higher tumorigenicity (left, chemoresistant hepatospheres; right, differentiated) and increased self-renewal ability and (C) stemness-associated genes. (D) MIHA expressed negligible levels of CD47, whereas HCC cell lines had varying expression levels, ranging from 40.6% ± 9.3% in PLC/PRF/5 to 81.5% ± 14.3% in Huh-7 cells. (E) CD47 expression was evaluated in HCC cells by FCM, relative to an isotype-matched control. Each dot represents a different HCC sample/xenograft. The blue dot represents xenograft of Huh-7; the orange dot represents xenograft of MHCC-97L; black dots represent clinical HCC samples. (F) Mean OS and DFS rates of HCC patients with high CD47 overexpression in their tumors were 37.0 and 12.3 months, as compared with 116.4 and 74.1 months in patients with low CD47 expression (P = 0.001 and P = 0.002, respectively; log-rank test). N indicates the number of patients at risk. For OS, patients with low CD47 expression: Month 0 = 21; month 50 = 16; month 100 = 9; month 150 = 5; month 200 = 1; patients with high CD47 expression: month 0 = 21; month 50 = 4; month 100 = 1. For DFS, patients with low CD47 expression: month 0 = 19; month 50 = 9; month 100 = 7; month 150 = 2; patients with high CD47 expression: month 0 = 14.

CD47 Was Sporadically Expressed in HCC Cell Lines and Human HCC Specimens and Preferentially Expressed in Liver TIC

To determine whether CD47-marked liver cells were more tumorigenic, we examined CD47 expression using flow cytometry (FCM) on a panel of liver cell lines, including the nontumorigenic immortalized cell line, MIHA, and the HCC cell lines, Huh-7, PLC/PRF/5, HLE, MHCC-97L, and MHCC-LM3. Expression of CD47 was variable in HCC cell lines with in vivo tumor-forming ability. In contrast, MIHA, which is incapable of tumor formation in vivo, had negligible CD47 expression (∼3%; Fig. 1D). Next, we evaluated CD47 expression of dissociated cells from patients' HCC samples using FCM. Percentage of CD47+ cells in xenografts/HCC specimens ranged from 3.93% to 98.5% (Fig. 1E). Next, we investigated the potential relationship between CD47 expression and clinical outcome of HCC patients. We analyzed CD47 expression in 42 HCC patients by qPCR. Patients whose tumors had CD47 overexpression had significantly shorter overall and disease-free survival (OS/DFS) rates than those with low CD47 expression, respectively (P = 0.001 and P = 0.002; Fig. 1F). To determine whether CD47 was preferentially expressed in liver TICs, we compared CD47 mRNA expression of CD133+/CD24+ HCC cells with CD133/CD24 cells. qPCR analysis revealed a significantly higher CD47 mRNA expression in CD133+/CD24+ liver TICs than in CD133/CD24 cells (Supporting Fig. 2A). In addition, FCM analysis showed that CD47 expression was found to highly overlap with that of CD133, CD24, and EpCAM in Huh-7 and Hep3B cells, suggesting that CD47 represents a high population of liver TICs (Supporting Fig. 2B).

CD47+ HCC Cells Possessed Characteristics of Stem/Progenitor Cells With Distinct Metastatic Features

Because CD47 is preferentially expressed in liver TICs, we first determined whether CD47+ HCC cells were more tumorigenic in vivo than their CD47 counterparts using a tumor-forming assay, with CD47+ and CD47 cells purified from PLC/PRF/5, patient 73, 83 and 98, and their CD47 expression was found to be 40%, 64%, 80%, and 50%, respectively. Purified cells were inoculated SC into NOD/SCID mice. A significant difference in tumor incidence was observed between CD47+ and CD47 cells (Fig. 2A,B). As few as 500 CD47+ cells were sufficient for consistent tumor development in NOD/SCID mice (Supporting Table 1A-D). After xenografts derived from cells sorted from patient 73 had formed, we excised the corresponding tumors from the primary recipients, dissociated them into single-cell suspensions, resorted them into CD47+ and CD47, and then reinjected them into secondary mouse recipients. As few as 500 CD47+ cells were sufficient for tumor formation (Fig. 2A,B; Supporting Table 1E). To obtain further evidence for the self-renewal ability of CD47+ cells, we performed the sphere formation assay. Compared with CD47 cells, significantly larger and more hepatospheres were observed in CD47+ cells isolated from both PLC/PRF/5 and patient 73 (Fig. 2C). Furthermore, we found that CD47+ fractions purified from both PLC/PRF/5 and patient 73 had a general overexpression of these genes (Supporting Table 2). Finally, CD47+ HCC cells derived from PLC/PRF/5 were more chemoresistant than CD47 cells in response to DOX and CIS treatments (Fig. 2D). Notably, lung metastasis was also observed in NOD/SCID mice after SC injection of CD47+ derived from PLC/PRF/5 and patient 73 (Supporting Fig. 3). Consistently, CD47+ cells from PLC/PRF/5 displayed approximately 2.4-fold higher cell migration efficiency, respectively (P < 0.005, t test; Fig. 2E). A similar observation was found with cells from patient 73 (data not shown).

Figure 2.

CD47+ HCC cells possessed stem/progenitor cell traits. (A) Representative images of tumors formed from primary-sorted PLC/PRF/5 cells, Patient 73, 83, and 98 cells and secondary-sorted patient 73 cells. Right flanks were injected with CD47+ cells and left flanks with CD47 cells. The red arrow indicates tumor formation. (B) Paraffin-embedded tissue of xenotransplanted tumors were processed for hematoxylin and eosin (H&E) staining. (C) In sphere forming assay, the in vitro self-renewal ability was significantly enhanced in CD47+ cells from PLC/PRF/5 (**P < 0.001) and patient 73 (**P < 0.001). (D) Methyl thiazol tetrazolium assay on sorted cells of PLC/PRF/5 demonstrated that CD47+ HCC cells were more chemoresistant to CIS and DOX, respectively (**P < 0.001 and **P < 0.001, respectively, t test). (E) Cell migration assay demonstrated that CD47+ HCC cells from PLC/PRF/5 were more migratory, when compared with their negative counterparts (*P < 0.005, t test).

CD47 Knockdown Reduced Stem/Progenitor Characteristics of HCC Cells

To examine whether CD47 functionally contributes to traits of stem/progenitor cells, we performed a CD47 knockdown experiment using a lentiviral-based approach (Huh-7 and MHCC-97L). After confirmation of successful CD47 knockdown (Fig. 3A), we examined the tumorigenicity of Huh-7 cells upon CD47 knockdown. We found that both the number and size of the tumors formed in NOD/SCID mice were lower in shCD47 clones, when compared with those in the nontarget control group (Fig. 3B; Supporting Table 3). A similar observation was found in MHCC-97L cells upon CD47 knockdown (data not shown). By sphere formation assay, we found that CD47 shCD47 cells led to generation of fewer and smaller hepatospheres (Fig. 3C). Next, we compared expression of stemness-associated genes between shCD47 and nontarget controls. Compared to nontarget controls, these genes, including ABCB1, NOTCH1, NANOG, ABCC1, ABCC2, and SMO, were down-regulated in shCD47 clones (Supporting Table 4). In addition, knockdown of CD47 in Huh-7 and MHCC-97L cells increased the sensitivity of cells to both CIS and DOX (Fig. 3D). Finally, we found that shCD47 cells displayed significantly lower migration and invasive efficiencies in both Huh-7 and MHCC-97L cells (Fig. 3E).

Figure 3.

CD47 knockdown reduced stem/progenitor characteristics of HCC cells. (A) Knockdown of CD47 in Huh-7 and MHCC-97L cells resulted in a 90% decrease (shCD47-773 and shCD47-998) in CD47 expression, compared to the nontarget control (NTC). (B) shCD47 cells exhibited reduced tumor-forming incidence, when compared to NTC cells. Right flanks were injected with NTC cells and left flanks with shCD47 cells. (C) Knockdown of CD47 also reduced the size and number of hepatospheres formed by Huh-7 and MHCC-97L cells (**P < 0.001, t test). (D) shCD47 cells were more chemosensitive to CIS (16.9% vs. 27.7% and 27.1%; 18.6% vs. 44.7% and 39.2%) and DOX (32.4% vs. 72.1% and 48%; 25% vs. 45.1% and 37.9%), when compared to control cells, after treatment for 24 hours at 10 and 4 µg/mL at a serum-free condition, respectively. (E) Transwell migration and invasion assays demonstrated the decreased migratory and invasive abilities of shCD47-transfected Huh-7 and MHCC-97L cells (*P < 0.05 and **P < 0.001, respectively, t test).

CD47 Drove Tumor Initiation, Self-Renewal, Chemoresistance, and Invasiveness Through Up-Regulation of CTSS

To determine the major downstream mediator of CD47, we employed cDNA microarray to compare gene expression profiles of shCD47 Huh-7 and nontarget control cells (GEO accession no.: GSE47563). We selected CTSS, which had a 4.57-fold decrease, for further analysis because it was one of the 10 most deregulated genes common to both sets of microarray profiling (chemoresistant hepatospheres vs. differentiated [3.27-fold] and CD47 knockdown vs. control; Supporting Table 5). Further analysis demonstrated down-regulation of CTSS in Huh-7 and MHCC-97L cells upon CD47 knockdown. A cell-sorting approach also revealed consistent up-regulation of the CTSS mRNA level in CD47+ HCC cells derived from PLC/PRF/5 (Supporting Fig. 4A). In six liver cell lines, CD47 expression positively correlated with that of CTSS (Supporting Fig. 4B). Consistently, CD47 significantly correlated with CTSS expression in 42 HCC patient samples (P < 0.0001 and R2 = 0.483, Pearson's correlation; Supporting Fig. 4C). To determine whether CD47 drove tumor initiation, stemness, and invasiveness through activation of CTSS gene expression, we overexpressed CTSS in shCD47-transfected cells of Huh-7 and MHCC-97L by lentiviral approach to investigate whether effects of CD47 knockdown could be eliminated upon transfection with CTSS (Fig. 4A). Tumorigenicity of shCD47 cells increased upon CTSS transfection and was comparable to that of nontarget controls in NOD/SCID mice (Fig. 4B; Supporting Table 6). In addition, shCD47 cells generated greater and larger hepatospheres upon CTSS overexpression (Fig. 4C). By Annexin V staining, shCD47-CTSS cells showed a decreased sensitivity to DOX and CIS treatments (Supporting Fig. 5). Using migration and invasion assays, we found that shCD47-CTSS cells had increased migratory and invasive abilities over those of shCD47 cells of both Huh-7 and MHCC-97L (Fig. 4D). This result has provided direct evidence that CD47 mediates tumor initiation, self-renewal, and invasiveness by up-regulating CTSS expression.

Figure 4.

CD47 regulated liver TIC function through NF-κB-mediated CTSS regulation. (A) CTSS ORF was successfully transfected into a CD47 knockdown clone of Huh-7 and MHCC-97L cells; the expression level was comparable to the nontarget control. (B) Tumorigenicity of CD47 knockdown cells increased upon CTSS transfection. (C) In addition, CTSS overexpression in shCD47 knockdown cells also increased the sizes and number of hepatospheres formed in Huh-7 and MHCC-97L cells, respectively (**P < 0.001 and **P < 0.001, respectively, t test). (D) In addition, CTSS overexpression increased the migratory (*P < 0.010 and *P < 0.050, respectively, t test) and invasive abilities (**P < 0.001 and *P < 0.010, respectively, t test) of shCD47 cells in Huh-7 and MHCC-97L, respectively. (E) Upon CD47 knockdown, nuclear, but not total, protein NF-κB expression decreased in Huh-7 and MHCC-97L.

CD47 Regulated CTSS Expression Through NF-κB Activation

To identify the mediator linking CD47 and CTSS, we performed an Ingenuity Pathway Analysis based on the cDNA microarray data (Supporting Table 5). Expression of several genes downstream of NF-κB was altered upon CD47 knockdown (Supporting Fig. 6A), suggesting the importance of NF-κB activation in CD47 signaling. To test this hypothesis, we examined expression of nuclear and total NF-κB in shCD47 cells from Huh-7 and MHCC-97L cells. We found lower levels of nuclear NF-κB upon CD47 knockdown, but not for total protein levels (Fig. 4E). To examine further whether regulation of CTSS expression in HCC cells was NF-κB dependent, we examined nuclear and total NF-κB levels and CTSS expression in MHCC-97L and Huh-7 cells upon knockdown of inhibitor kappa B kinase beta (IKKβ). Consistently, nuclear NF-κB and CTSS expression was found to be down-regulated upon knockdown of IKKβ (Supporting Fig. 6B). Next, we wished to examine whether NF-κB was crucial for CD47-mediated tumorigenicity and self-renewal. We found that both the number and size of tumors formed in NOD/SCID mice were lower in IKKβ clones, when compared to those in the nontarget control group (Supporting Figure 6C). In addition, we found that IKKβ knockdown cells led to generation of fewer and smaller hepatospheres (Supporting Fig. 6D).

CD47 Regulated Liver TICs Through Autocrine Secretion of CTSS

Notably, we found that NF-κB was reactivated in CD47 knockdown cells upon CTSS overexpression (Fig. 4E). This result suggests a possible positive regulatory loop between CD47 and CTSS. To test this hypothesis, we examined whether CTSS was preferentially secreted by CD47+ cells. Using enzyme-linked immunosorbent assay (ELISA) assay, we demonstrated that the level of secreted CTSS was decreased upon CD47 knockdown and increased upon CTSS overexpression (Fig. 5A). In addition, CTSS was also preferentially secreted by CD47+ cells derived from PLC/PRF/5 (data not shown). To confirm our hypothesis, we examined whether NF-κB was activated in shCD47 cells upon incubation with conditioned medium of shCD47-CTSS cells. An increase in nuclear NF-κB expression was observed in Huh-7 and MHCC-97L shCD47 cells upon incubation with conditioned medium from shCD47-CTSS cells, with a level similar to the effect from nontarget control cells (Fig. 5B). Next, we examined whether CD47 regulated liver TICs through secretion of CTSS in an autocrine manner. For this purpose, we examined the effect of conditioned medium on abilities of tube formation, invasiveness, and sphere formation from nontarget control shCD47 and shCD47-CTSS cells. We found that conditioned medium from shCD47-CTSS cells increased the abilities of Huh-7 shCD47 cells in tube formation, migration, invasion, and sphere formation (Fig. 5C). We also analyzed serum CTSS levels in 60 HCC patients and 31 healthy subjects. We found a step-wise increase in serum CTSS level in HCC progression from healthy subjects through early HCC to advanced HCC (Fig. 5D). Patients who had a higher serum CTSS level had advanced tumor stages (P = 0.002, chi-square test) and larger tumor size (P = 0.008, chi-square test; Table 1). Next, we analyzed CTSS expression in the same 42 HCC patients by qPCR. Patients whose tumors had CTSS overexpression had significantly shorter OS and DFS rates than those with low CTSS expression, respectively (P < 0.001 and P = 0.008; Supporting Fig. 7).

Figure 5.

CD47 regulated the function of liver TICs through the CTSS/PAR2 loop. (A) An ELISA assay demonstrated that secreted CTSS level was decreased in shCD47 cells in Huh-7 and MHCC-97L, whereas the level was increased upon CTSS overexpression. (B) Upon incubation of Huh-7 and MHCC-97L shCD47 cells with conditioned medium from shCD47-CTSS cells, an increase in nuclear NF-κB and CTSS expression was observed, which was comparable to that observed upon incubation with conditioned medium from nontarget control (NTC) cells. (C) In addition, conditioned medium from shCD47-CTSS cells increased migratory and invasive abilities (*P < 0.010 and **P < 0.001, respectively, t test) and sphere formation (**P < 0.001, t test) of Huh-7 shCD47 cells. In addition, conditioned medium from shCD47-CTSS cells increased the tube formation ability of HUVEC cells, when compared with that from shCD47 cells. White dotted circles represent tube formation. (D) There was a step-wise increase in serum CTSS correlating with HCC progression from healthy subjects from early HCC to advanced HCC (*P < 0.050 and **P < 0.001). (E) Ectopic CTSS transfection increased PAR2 expression in Huh-7 and MHCC-97L shCD47 cells. A similar result was observed when shCD47 cells were cultured with conditioned medium from shCD47-CTSS cells. (F) PAR2 was efficiently knocked down in Huh-7 cells with a lentiviral-based knockdown approach. In NTC cells of Huh-7, up-regulation of both nuclear NF-κB and CTSS was observed in response to conditioned medium of shCD47-CTSS. In contrast, no change in nuclear NF-κB and CTSS expression was observed in PAR2 knockdown cells.

Table 1. Clinicopathologic Correlation of CTSS Expression in HCC Patients
 CTSS Serum Level 
Clinicopathological Variables<7,500 pg/mL≥7,500 pg/mLP Value
  1. Abbreviations: TNM, tumor node metastasis; UICC, Union for International Cancer Control; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; AFP, alpha-fetoprotein.

  2. a

    Significant difference.

Gender
Male10390.275
Female47
Age
<608260.764
≥60719
TNM stage
Early stage (I-II)12180.002*
Late stage (III-IV)228
UICC stage
Early stage (I-II)10110.003*
Late stage (III-IV)534
Tumor size (cm)
Small (≤5)11170.008*
Large (>5)223
Venous invasion
Absent13330.185
Present110
HBV association
Negative of HBsAg340.157
Positive of HBsAg1042
Serum AFP level (ng/mL)
<4001440.319
≥4001442

CD47 Regulated Liver TICs Through Activation of the CTSS/PAR2 Loop

CTSS has been demonstrated to be a ligand of PAR2 in the itching process.[15] In addition, PAR2 was found to activate NF-κB signaling during inflammation.[16] These results, together with the cDNA microarray data showing consistent alterations of PAR2 in chemoresistant hepatospheres (up 2.62-fold) and cells with CD47 knockdown (down 2.15-fold; Supporting Table 5), suggest that CD47 regulates liver TIC function through the CTSS/PAR2 loop. Furthermore, PAR2 expression was consistently down-regulated upon CD47 knockdown, whereas its expression was increased upon CTSS overexpression (Fig. 5E). A similar observation was found when shCD47 cells were incubated with conditioned medium of shCD47-CTSS cells (Fig. 5E). Next, we examined whether CD47 regulated liver TIC through the CTSS/PAR2 loop in an autocrine manner. First, using FCM analysis, we found that CD47 expression highly overlapped with that of PAR2 in Huh-7 and MHCC-97L cells (Supporting Fig. 8A). Second, CD47+ HCC cells from PLC/PRF/5 and patient 73 preferentially expressed PAR2, when compared with their negative counterparts (Supporting Fig. 8B). The role of PAR2 in autocrine regulation in CD47 signaling was further confirmed by knocking down PAR2 expression in Huh-7 cells (Fig. 5F). Compared to nontarget control cells, PAR2 knockdown cells showed no up-regulation of NF-κB or CTSS in response to conditioned medium of shCD47-CTSS (Fig. 5F). This result showed that CD47 regulated liver TICs through activation of the CTSS/PAR2 loop.

CD47 Targeting Is a Novel Strategy Against HCC

We examined the therapeutic role of targeting CD47 and its combined effect with DOX in vivo using PLC/PRF/5 cells and patient-derived xenograft through use of a CD47 antisense morpholino oligonucleotide.[17] Corresponding tumors and their volumes of these animals are shown in Fig. 6A. CD47 antisense morpholino significantly reduced tumor volumes in a manner approximately 2-fold more potent than DOX. In addition, CD47 antisense morpholino exerted a synergistic effect with DOX, resulting in complete eradication of tumors. Therapeutic efficacy of CD47 antisense morpholino oligonucleotide was also examined in nude mice bearing patient-derived xenograft (PDTX 10). Treatment was started once the size of the xenografts reached approximately 6 × 6 mm. Similarly, we found that CD47 antisense morpholino significantly reduced tumor volumes in a manner approximately 2-fold more potent than DOX (Fig. 6B). In addition, CD47 antisense morpholino exerted a synergistic effect with DOX. To examine whether antitumor effect of CD47 antisense morpholino was the result of inhibition of CTSS/PAR2 signaling, we further evaluated CTSS expression within the tumor and its serum level between control and treatment groups. We found that CD47 morpholino decreased both CTSS expression and its serum level in nude mice bearing PDTX 10 (Fig. 6C). Next, we sought to examine whether antitumor activity of CD47 Ab was also the result of inhibition of CTSS/PAR2 signaling. To this goal, we examined CTSS expression of Huh-7 and MHCC-97L cells upon administration of anti-CD47 Ab. With western blotting analysis, we found that anti-CD47 Ab decreased CTSS expression dramatically in these two cell lines (Fig. 6D).

Figure 6.

CD47 targeting for HCC cancer therapy. In vivo therapeutic effect of CD47 suppression was examined in a nude mouse model with PLC/PRF/5 cells. Nude mice were randomized into five groups, each consisting of 5 animals. Each group was treated every 4 days for 40 days with either 100 µL of 5 µM of anti-CD47 morpholino (CD47morpho; group A), 2 mg/kg of DOX and 5MM; group B), 2 mg/kg of DOX and 5 µM of anti-CD47 morpholino (CD47morpho; group C), or PBS and 5MM control (groups D and E) as the control group. (A) Tumor volume in each group was evaluated using a Xenogen imaging system. Three representative mice in each group are shown. CD47morpho administration significantly reduced tumor size by more than 3-fold, as compared to PBS control (*P < 0.010, t test) and its effect was more potent than DOX alone. The combination of DOX and anti-CD47 morpholino exhibited a maximal effect on tumor suppression because tumors were undetectable after the treatment. (B) In vivo therapeutic effect of CD47 suppression was also examined in a nude mouse model with patient-derived tumor xenograft (PDTX 10). CD47morpho administration significantly reduced tumor size by 3-fold, as compared to 5MM control (*P < 0.010, t test), and its effect was more potent than DOX alone. Similarly, the combination of DOX and CD47morpho exhibited a synergistic effect on tumor suppression. Four representative mice in each group are shown. (C) By western blotting analysis, CD47morpho decreased CTSS expression within the tumor, and its expression paralleled the tumor volume. By ELISA assay, we found that mouse serum CTSS level was significantly decreased upon administration of CD47morpho (*P < 0.001, respectively, t test). (D) Upon administration of anti-CD47 Ab (B6H12; BioXCell, Lebanon, NH) at 4 µg/mL for 48 hours, CTSS expression was dramatically decreased in Huh-7 and MHCC-97L cells.

Discussion

In this study, we adopted serial passages of hepatospheres combined with anticancer chemotherapeutic drugs as a strategy to enrich a TIC population. Serial passages of the spheres could enrich TIC populations in vitro in various cancer types, including breast cancer.[18] In addition, administration of chemotherapeutic drugs could also enrich the TIC populations.[19, 20] Using this strategy, we successfully enriched liver TICs that had enhanced self-renewal capacity and tumorigenicity, when compared with differentiated progeny. Using cDNA microarray confirmed by qPCR, we found that CD47 was up-regulated in the enriched TIC population.

CD47, an integrin-associated protein (IAP) is a membrane receptor associated with various integrins through their beta3 subunit. CD47 exerts its antiphagocytic role through binding to phagocytic cells that express signal-regulatory protein alpha (SIRP-α).[21] Upon binding, CD47 initiates a signal transduction cascade resulting in inhibition of phagocytosis. The role of CD47 in cancer remains controversial. CD47 activation stimulates the spread of melanoma,[22] but induces apoptosis of B-cell chronic lymphocytic leukemia cells through a caspase-independent mechanism.[23] Recently, CD47 was reported to be a marker of TICs in leukemia and bladder cancer.[14, 24] In this study, we found that CD47+ HCC cells have higher capacity in tumorigenicity, self-renewal, and metastasis, when compared with CD47 counterparts. Similar to the studies in glioma and ovarian cancer,[16] we found that a higher CD47 mRNA level in HCC clinical samples correlated with a poorer clinical outcome. In addition, a significant higher CD47 mRNA level was found in CD133+ and CD24+ liver TICs. Using FCM analysis, CD47 was detected in all primary and xenograft samples from patients and HCC cell lines, with expression ranging from 3.93% to 98.5%. These results have demonstrated that a CD47 population in HCC is not rare. Notably, CD47 is expressed in a relatively high percentage of cells analyzed, but at an even higher level in CD133+ and CD24+ T-ICs with stem cell properties. These observations suggest that CD47 is an attractive target for potential therapeutic intervention against both liver TICs and differentiated cells.

Using a lentiviral-based shRNA approach, knockdown of CD47 suppressed traits of liver TICs. cDNA microarray analysis showed that CTSS is a crucial effector of CD47-mediated tumor formation, metastasis, and self-renewal. CTSS encodes for a cysteine protease, which has recently been shown to be overexpressed in cancers and plays a role in tumor growth,[25] metastasis,[26] and chemoresistance.[27] In our HCC patient cohort, we found higher serum CTSS levels in HCC patients, when compared to normal healthy subjects and this elevated level was associated with advanced tumor stage in HCC patients. These data support the finding of a previous study showing an association of high serum CTSS levels with an increased cancer mortality risk.[28] Subsequent studies showed that CD47+ HCC cells enhanced CTSS secretion, which, in turn, activate a CTSS-dependent autocrine feedback loop that signals through the NF-κB pathway. Further analysis showed the involvement of PAR2 in the CTSS-positive feedback loop in CD47 signaling (Supporting Fig. 9). However, secretory CTSS may affect CD47 HCC in a paracrine manner to a much lesser extent. In addition, PAR2 was also found to be expressed in human umbilical vein endothelial cells (HUVECs; data not shown). Secretory CTSS may also affect tumor angiogenesis through interaction of PAR2+ HUVEC cells.

In an in vivo animal model, we found that suppression of CD47 by use of morpholino suppressed tumor growth, with effects more potent than that of DOX. The decrease in tumor growth was parallel with CTSS expression within tumor and its secreted level. Therefore, the CD47-CTSS/PAR2 paracrine loop may account for the growth-suppressive effect of CD47 morpholino. Most important, CD47 morpholino exerted a synergistic effect with chemotherapy. Recently, CD47-SIRP-α interaction was found to be a novel therapeutic target for human solid tumors.[16] Sota-Pantoja et al. have reported that inhibitory signaling through SIRP-α is not sufficient to explain antitumor activities of CD47 Abs.[29] In this study, we found dramatic decrease in CTSS expression upon administration of anti-CD47 Ab. This result may suggest that antitumor activities of CD47 Ab may be the result of inhibitory signaling through CTSS/PAR2 signaling. In conclusion, identification of CD47-signaling pathways provides an attractive therapeutic strategy against this deadly disease.

Acknowledgment

The authors thank the LKS Faculty of Medicine at The University of Hong Kong for use of the Faculty Core Facility.

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