Potential conflict of interest: Nothing to report.
We thank professor Xiao fei Zheng (Beijing Institute of Radiation Medicine, China) for the pcDNA3.0-miR155 vector. We thank Dr Yan hong Tai (Chinese PLA General Hospital, China) for IHC analysis
Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. Conclusion: S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013)
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The tumor microenvironment plays an important role in modulating cancer and cancer stem cell progression.1, 2 Recently, mesenchymal stem cells (MSCs), as a pivotal part of the tumor stroma, have attracted great attention for their ability to participate in tumor proliferation3 and metastasis.4 Although several lines of evidence demonstrate that MSCs can be activated by cancer cells and contribute to tumor progression, the molecular mechanisms for MSCs driving cancer cell proliferation and invasion are not fully understood.
It has been previously demonstrated that cancer-associated MSCs could be educated by tumor cells and secreted many growth factors and chemokines, which play crucial roles in tumor progression.5, 6 This suggests that MSCs located in tumor tissues more appropriately reflect characteristics of the tumor microenvironment than MSCs from adjacent tumor-free tissues or adult bone marrow. Therefore, studying the characteristics of tumor-associated MSCs and exploring the interactions between MSCs and tumor cells are likely to offer critical insights into the crosstalk between niche and tumor cells.
To date, MSCs have been reported to be isolated from a few solid tumors, including ovarian cancer,6, 7 gastric cancer,8 osteosarcoma tissues,9 and breast cancer.10 McLean et al.6 isolated MSCs from ovarian carcinoma tissues and reported that they promoted cancer stem cell proliferation by way of altered bone morphogenetic protein (BMP) production. Our previous research demonstrated that breast cancer-associated MSCs promoted tumor and mammosphere formation partially by way of the EGF/EGFR/Akt pathway.10 Up to now, the characterization of liver cancer-associated MSCs has not been investigated, and their involvement in liver tumor pathophysiology remains unknown.
Here, for the first time, we report the identification of MSCs in primary hepatocellular carcinoma (HCC) tissues. We find that they significantly promote HCC cell proliferation and metastasis in vivo. We further demonstrate that liver cancer-associated MSCs (LC-MSCs) activated by HCC cells in vivo express more S100A4, which then triggers an intricate cascade that promotes HCC proliferation and metastasis. S100A4 up-regulates the expression of microRNA- 155 (miR-155) in HCC cells, which suppresses suppressor of cytokine signaling 1 (SOCS1), activates STAT3 signaling, and therefore enhances matrix metalloproteinases 9 (MMP9) expression, leading to increased tumor invasiveness. To summarize, our findings suggest that the LC-MSC microenvironment modulates HCC progression partially through the S100A4-miR-155-SOCS1-MMP9 axis, which may present a useful target for therapeutic interference of the HCC niche.
Materials and Methods
Isolation of MSCs from Primary HCC Tissues and Adjacent Tumor-Free Tissues.
Twenty-one pairs of matched primary hepatocarcinoma and adjacent tumor-free tissues were obtained from HCC patients at Beijing 302 Hospital. Immediately after liver surgical resection, tissues were minced with a scalpel and enzymatically dissociated in 10 mL of phosphate-buffered saline (PBS) supplemented with 0.1% collagenase I (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 hour with gentle agitation. The suspension was neutralized and cultured in alpha minimum essential medium (α-MEM) containing 10% selected fetal bovine serum (FBS) and 1 ng/mL basic fibroblast growth factor (bFGF, R&D Systems, Minneapolis, MN). Nonadherent cells and tissues were removed and washed twice with PBS after 24-48 hours, and the adherent fibroblast-like cells were further incubated for 4-5 days until 80%-90% confluence.
Intrahepatic Metastasis Model.
The intrahepatic metastasis procedure was used as described.11 Briefly, for each mouse, 2 × 106 MHCC97L cells with or without LC-MSCs were suspended in 30 μL PBS, including 50% Matrigel; the ratio of MHCC97L to LC-MSCs was 1:1. Each nude mouse (10 in each group, male nu/nu) was orthotopically inoculated in the left hepatic lobe under anesthesia, through a 1-cm transverse incision in the upper abdomen. Animals were sacrificed after 6-7 weeks, their livers and lungs were dissected, fixed in 10% buffered formalin, and prepared for histological analysis. Other methods are provided in detail in the Supporting Material.
cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; LC-MSCs, liver cancer-associated MSCs; LN-MSCs, liver normal MSCs; miRNA, microRNA; miR-155, microRNA-155; MMP9, matrix metalloproteinases 9; MSCs, mesenchymal stem cells; qRT-PCR, quantitative real time polymerase chain reaction; siRNA, small interfering RNA; SOCS1, suppressor of cytokine signaling 1; STAT, signal transducer and activator of transcription.
Characterization of MSCs Derived From Primary HCC Tissues.
To investigate whether MSCs exist in HCC stroma, we set out to isolate MSCs from HCC and adjacent tumor-free tissues. The adherent cells showed homogenous fibroblastic morphology (Fig. 1A). When cultured in appropriate induction medium, they differentiated into bone cells, which were confirmed by high alkaline phosphate (ALP) and Von kossa staining (Fig. 1A). They were also able to differentiate into adipose cells, which were verified by Oil Red O staining (Fig. 1A). Flow cytometry showed that LC-MSCs possessed uniform surface markers, and were positive for mesenchymal markers (CD29, CD73, CD166, CD90, and CD105), negative for hematopoietic and endothelial markers (CD45, CD14, CD144, and CD31), shown in Fig. 1B. Furthermore, immunofluorescence staining demonstrated that LC-MSCs were positive for myofibroblastic markers: vimentin, alpha smooth muscle actin (α-SMA), and N-cadherin, negative for epithelial markers: CK18 and E-cadherin (Fig. 1C). Liver normal MSCs (LN-MSCs) had the same phenotype and differential potential as LC-MSCs (Supporting Fig. 1).
LC-MSCs Promoted Hepatocarcinoma Growth and Increased Tumor Sphere Formation In Vitro.
To evaluate the function of LC-MSCs in tumor progress, we subcutaneously injected HCC cells mixed with LC-MSCs into nude mice. Compared with MHCC97H cells alone, LC-MSCs significantly accelerated the growth of tumors (Fig. 2A), and we got similar results with two other cell lines HepG2 and SMMC-7721 (Supporting Fig. 2). But as many as 1 × 107 LC-MSCs alone did not form visible tumors even after 7 weeks (data not shown). Furthermore, we established an MSC/tumor sphere coculture system to investigate whether LC-MSCs could modulate the proliferation of tumor spheres. The results revealed that LC-MSCs significantly accelerated the proliferation of tumor spheres (Fig. 2B).
LC-MSCs Enhanced HCC Cell Migration and Metastasis.
Transwell migration assays demonstrated that the conditioned medium from LC-MSCs significantly increased the migration potency of HCC cells (Fig. 3A). To further explore whether LC-MSCs promote tumor metastasis, we established an intrahepatic metastasis model in which MHCC97L cells mixed with LC-MSCs were used for orthotopic liver implantation.11 We found that when mixed with LC-MSCs, MHCC97L cells showed a dramatic increase in metastatic capacity. The number of metastatic nodules on the liver surface was increased when MHCC97L cells were cotransplanted with LC-MSCs (Fig. 3B). Furthermore, histological analysis demonstrated that MHCC97L with LC-MSCs group had more pulmonary metastasis sites (Fig. 3C). Taken together, these data suggest LC-MSCs play an important role in promoting HCC metastasis.
S100A4 Secreted from LC-MSCs Promoted HCC Cell Proliferation and Metastasis.
To explore the mechanisms by which LC-MSCs modulate cancer progression, we conducted complementary DNA (cDNA) microarray analysis, and found that there were about 2,121 genes that were significantly different between LC-MSCs and LN-MSCs (the gene expression profiles can be obtained at the web:http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42357). Many factors, including MMP1, S100A11, HGF, VEGF-C, FGF13, FGF5, CCL13, IL-24, S100A3, CXCL12, S100A4, MMP3, Gremlin2, Chordin, WNT2, and CCL8, were found to be significantly up-regulated in LC-MSCs than in LN-MSCs (Fig. 4A; Supporting Table S1). Among them, the aberrant expression of S100A4 has been linked to a more aggressive phenotype in prostate cancer,12 cholangiocarcinoma,13 and head and neck cancer.14 Whether LC-MSCs can modulate HCC progression by way of S100A4 remains unreported. We further confirmed the microarray results by enzyme-linked immunosorbent assay (ELISA) (Fig. 4B) and western blotting (Fig. 4C). Moreover, S100A4 expression was enhanced in LN-MSCs after activation with HCC-conditioned medium (Fig. 4C). The immunofluorescence staining of HCC tissues demonstrated S100A4 was highly expressed in tumor stroma, especially the stromal cells closely contacted by HCC cells (Fig. 4D). We then confirmed the expression of S100A4 in primary HCC and adjacent cancer-free tissues by immunohistochemistry (Supporting Fig. 3A). The results showed that there was a significant difference (P < 0.001, Fisher's exact test) between HCC and adjacent cancer-free tissues as follows: 84.2% (80 of 95) of HCC samples expressed S100A4, whereas only 26.8% (11 of 41) of adjacent cancer-free tissues were positive for S100A4 (Supporting Fig. 3B; Supporting Tables S2, S3). In the HCC samples, S100A4 was mainly expressed in stromal cells of HCC, including the stroma-only positive group (70.5%, 67 of 95), stroma and tumor double-positive group (8.4%, 8 of 95), 5.3% (5 of 95) of samples expressed S100A4 only in HCC cells, and 15.8% (15 of 95) samples were negative for S100A4 expression (Supporting Fig. 3C).
To further explore the function of S100A4 in HCC progression, we compared the expression of S100A4 in HCC cell lines (Fig. 5A), and ectopic expression and RNA interference (RNAi) strategies were used (Fig. 5B). We found that S100A4 knockdown in LC-MSCs significantly abolished their invasion-promoting capacity on HCC cells (Fig. 5C). Furthermore, overexpression of S100A4 in HCC cells could significantly promote their proliferation and invasion potency, while S100A4 knockdown in HCC cells significantly reduced these effects (Fig. 5D; Supporting Fig. 4). Intrahepatic metastasis assays showed that, compared with the control group, S100A4 overexpression increased the number of metastatic nodules on the liver surface and more pulmonary metastasis sites (Fig. 5E,F). Taken together, these results indicated that HCC cells activated S100A4 expression in LC-MSCs, which then play essential roles in enhancing cancer progression.
S100A4 From LC-MSCs Promoted miR-155 Expression in HCC Cells.
Hematoxylin and eosin (H&E) staining and α-SMA immunohistochemical staining showed that there were intensive interactions between hepatocarcinoma cells and stroma (Fig. 6A). Accumulating evidence demonstrates that aberrant expression of miRNAs plays important roles in cancer development.15, 16 To explore whether miRNAs are involved in LC-MSCs modulating tumor progression, we investigated the miRNA alterations in green fluorescent protein (GFP)-positive HCC cells cocultured with LC-MSCs (Fig. 6B). Immunofluorescence staining showed that GFP-positive HCC cells interacted with LC-MSCs (Fig. 6C). After coculture, GFP-positive tumor cells were sorted by fluorescent-activated cell sorter (FACS) analysis (Fig. 6D). Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed to investigate miRNA alterations in HCC cells activated by LC-MSCs (miRNAs primers in Supporting Table S4). The results showed that LC-MSCs could enhance the expression of several miRNAs (Supporting Fig. 5). Among them, miR-155 expression was dramatically increased (Fig. 6E). Importantly, we also demonstrated that S100A4 significantly enhanced the expression of miR-155 in HCC cells (Fig. 6F), suggesting that the S100A4-miR-155 axis may be involved in LC-MSCs mediated cell proliferation and invasion.
miR-155 Promoted HCC Cell Proliferation and Invasion.
To further evaluate the function of miR-155 in hepatocellular carcinoma, we established three stable miR-155-overexpressing HCC cell lines (Fig. 7A). We found that miR-155 overexpression enhanced HCC cell proliferation in vitro (Fig. 7B) and promoted tumor growth in vivo (Fig. 7C), while the miR-155 inhibitor reduced the proliferation of Huh7 cells (Fig. 7D). Transwell invasion assays showed that miR-155 significantly enhanced the invasive ability of HepG2 cells (Fig. 7E). Furthermore, miR-155 inhibitor could significantly abolish the invasion capacity of SMMC-7721 with ectopic S100A4 expression, or MHCC97L cells with high levels of endogenous S100A4 expression (Fig. 7F). Therefore, the results indicate that S100A4 affects tumor motility by modulating miR-155 expression in cancer cells.
miR-155 Induced by S100A4 Promoted HCC Invasion by Way of SOCS1-STAT3-MMP9 Axis.
We studied the pathways associated with the S100A4-miR-155 axis mediated cell invasion. The expression of several proteolytic enzymes involved in degrading the basement membrane, including matrix metalloproteinases (MMPs) were analyzed by RT-PCR in HCC cells following LC-MSCs stimulation, S100A4, or miR-155 transfection (data not shown). MMP9 expression was found to be significantly enhanced following LC-MSCs treatment, S100A4, or miR-155 transfection, which was confirmed by western blotting and immunohistochemistry analysis (Fig. 8A). To investigate whether S100A4 promotes cell invasion by regulating MMP9 expression, RNAi was used to knockdown MMP9 expression in S100A4-expressing cancer cells (MHCC97H with high levels of endogenous S100A4 expression, and SMMC-7721 with ectopic S100A4 expression). We found that knockdown of MMP9 significantly suppressed the invasion of HCC cells (Supporting Fig. 6), suggesting that S100A4 facilitated the process by up-regulating MMP9.
Although the S100A4-miR-155 axis promoted cell invasion through MMP9 expression, MMP9 is not a direct target of miR-155. To further define the functional link between S100A4-miR-155 axis and MMP9-mediated cell invasion, we analyzed the expression of SOCS1, one of miR-155′s target genes. SOCS1 has been reported to regulate p-STAT3, one of the mediators involved in regulating MMP9 expression. Western blotting demonstrated that both ectopic S100A4 and miR-155 expression down-regulated SOCS1 expression (Fig. 8B). In contrast, miR-155 inhibitor resulted in up-regulation of SOCS1 with a corresponding down-regulation of MMP9 (Fig. 8C). Furthermore, we found that p-STAT3, one of the downstream effectors of SOCS1, was up-regulated in HCC cells following LC-MSCs stimulation or S100A4 overexpression (Fig. 8D). In addition, the STAT3 inhibitor, AG490, inhibited MMP9 expression (Fig. 8D) and reduced the invasive abilities of 7721-S100A4 cells and 7721-miR155 cells (Fig. 8E). All these results demonstrate that S100A4 from LC-MSCs up-regulates miR-155 expression in cancer cells, which then down-regulates SOCS1 and activates the STAT3 signaling pathway to facilitate MMP9 expression and stimulate HCC invasion (Supporting Fig. 7).
Cancer-associated MSCs from bone marrow of hematological malignancy patients have been fully characterized.17, 18 However, a thorough understanding of solid tumor-associated MSCs is lacking, due to the difficulty of primary culture and maintenance. In the present study we successfully identified LC-MSCs from HCC tissues. Under appropriate conditions, LC-MSCs could differentiate into adipose and bone lineages.19 Consistent with previous studies, we found LC-MSCs significantly promoted tumor formation and enhanced tumor sphere formation.6 Our study provides the first evidence that solid tumor-associated MSCs can promote cancer metastasis in vivo.
Next, we want to identify which factors secreted from LC-MSCs may participate in HCC progression. cDNA microarray analysis showed that LC-MSCs produce many trophic factors including S100A4, HGF, VEGF, SDF-1, MMP family, and BMP antagonists. Among these factors, we selected S100A4 for further investigation, largely because S100A4 has gained increasing attention for its metastasis- and proliferation-promoting properties. An elevated expression of S100A4 has been linked to a more aggressive phenotype in many cancers. Grum-Schwensen et al.20 reported a suppression of tumor metastasis in S100A4 (−/−) mice compared with wildtype controls. This suppression could be rescued by the coinjection of tumor cells with S100A4-expressing fibroblasts, pointing to an important role of S100A4 expressing stromal cells in tumor proliferation and metastasis. This suggested that S100A4 in the MSC microenvironment may modulate HCC progression. Up till now, the expression pattern and function of S100A4 in HCC have remained unclear, and the role of S100A4 in the interactions between stroma and HCC was unknown. We were interested in addressing whether S100A4 secreted from LC-MSCs could modulate HCC progression. Our experiments verified that S100A4 secreted from LC-MSCs could promote HCC progression. Except for S100A4, several other factors such as HGF, Gremlin, SDF-1, and so on, may be also involved in MSC promoting HCC progression. It has been reported that tumor stroma induced tumor cell drug resistance by way of HGF secretion.21 Gremlin was widely expressed by cancer-associated fibroblasts and promoted tumor cell proliferation. Cancer-associated fibroblasts and cancer cells-derived SDF-1 promoted cancer proliferation in a paracrine or autocrine manner, respectively.22 Whether S100A4 could synergize with other factors to regulate HCC progression requires further investigation.
Recently, important associations between miRNA expression and tumorigenesis have been implicated in many cancers.23 Furthermore, miRNAs have been identified as classical oncogenes or tumor suppressor genes.24 Thus, we were interested in whether S100A4 secreted from LC-MSCs could promote tumor progression by way of modulation of miRNAs. A coculture system was established to explore miRNA alterations in HCC cells interacting with LC-MSCs. For presumptive miRNA analysis, we mainly focused on those oncogenous miRNAs that were significantly up-regulated in HCC compared with adjacent nontumor tissues, by way of miRNA microarray profiling or quantitative RT-PCR assays.24-27 We found that miR-155 was dramatically up-regulated in HCC cells by coculture with LC-MSCs or S100A4 ectopic expression. MiR-155 is frequently up-regulated in many hematological malignancies and solid tumors, such as B-cell malignancies,28 breast cancer,29 colon cancer,30 and hepatocellular carcinoma.31, 32 Moreover, a possible link between miR-155 and inflammation in cancer has been reported.33 However, the role of miR-155 in a tumor microenvironment regulating HCC progression remains unreported. Our results indicate that miR-155 significantly promotes HCC cell proliferation and invasion. This is consistent with a previous report that miR-155 was up-regulated at early stages of experimental hepatocarcinogenesis in mice.31 Furthermore, Xie et al.32 found that aberrant miR-155 expression could accelerate HCC proliferation. Taken together, our results indicate that miR-155 may play an important role in the interactions of LC-MSCs and HCC cells, and S100A4 secreted from LC-MSCs may present a close link between LC-MSCs and the miR-155 expression in HCC cells.
To further define the downstream mechanism underlying S100A4-miR-155 axis-mediated cancer cell progression, we analyzed the expression of the MMP family. Western blotting and immunohistochemistry showed that both S100A4 and miR-155 could promote the expression of MMP9, which was consistent with previous results showing that the overexpression of S100A4 could enhance the expression of MMP9 in prostate cancer.12 However, MMP9 is not a direct target gene of miR-155. We analyzed the expression of SOCS1, one of the target genes of miR-155, a tumor suppressor that normally functions as a negative modulator of JAK/STAT3 signaling, and is involved in regulation of MMP9 expression. Our results further verified that S100A4 and miR-155 overexpression significantly suppressed the expression of SOCS1, leading to the activation of p-STAT3, the increased expression of MMP9, and the promotion of tumor cell invasion. In the presence of a JAK/STAT3 signaling inhibitor (AG490), both the invasion capacity and MMP9 expression were significantly repressed. These data show that the S100A4-miR-155-SOCS1-STAT3-MMP9 pathway may play an important role in LC-MSCs modulating HCC progression.
Other factors derived from LC-MSCs may also be implicated in modulating STAT3 activity. It has been reported that IL-6/gp130/JAK/STAT3 was a principal pathway in promoting tumorigenesis, but interleukin (IL)-6 expression in LC-MSCs is decreased than LN-MSCs in our system. We also demonstrate that SDF-1a significantly activates p-STAT3 (data not shown). Whether S100A4 can synergize with SDF-1a or other factors to regulate STAT3 activity needs further investigation.
In summary, our work sheds light on a poorly understood cascade of events involved in the LC-MSC modulation of HCC progression. We identified cancer-associated MSCs in hepatocarcinoma tissues, and demonstrated that S100A4 from LC-MSCs could significantly promote tumor proliferation and metastasis. Importantly, there was an intricate multistep cascade involved during this process: S100A4 up-regulated the expression of miR-155 in HCC cells, which suppressed SOCS1 leading to activation of STAT3 signaling. This in turn led to the up-regulation of MMP9 expression and increased tumor invasiveness. These results provide a foundation for further investigation into the precise mechanisms behind S100A4 regulation of tumorigenesis. We propose that interruption of the S100A4-miR-155-SOCS1-STAT3-MMP9 pathway may present a useful therapeutic approach for controlling HCC proliferation and metastasis.