In this study, we investigated the effects of Annexin A7 on HCC and lymphatic metastasis in a mouse model of lymph node metastasis by using the two mice hepatocarcinoma ascites syngeneic cell lines Hca-F and Hca-P with high and low lymphatic metastasis potential, respectively. The data showed that the lymph node metastasis rate was decreased from 36% to 0% after upregulation of Annexin A7 in Hca-P cells, but it increased from 77% to 100% after downregulation of Annexin A7 expression in Hca-F cells. Thus, the
in vivo data implied that Annexin A7 may play an important role in HCC lymphatic metastasis and play a tumor suppressor function in HCC. A previous study has shown that Annexin A7 expression is lost in metastatic and local recurrent hormone-refractory prostate cancer compared to primary tumors [
10]. Srivastava
et al. reported the knockout of the Annexin A7 gene in mice to investigate the involvement of Annexin A7 in Ca
2+ signaling in secreting pancreatic β cells and its function in the control of cancer development [
11,
12]. Annexin A7 has been shown to be a tumor suppressor in hormone-relevant prostate and breast cancers [
10‐
15]. In prostate cancer, Annexin A7 as a tumor suppressor could be through inhibition of pathologic androgen signaling and dysfunctional retinoblastoma 1, PTEN, and p53 activity. Annexin A7 could also be associated with its mediation of exocytosis and secretion in prostate cells and possibly in other cancers [
14]. In addition, haplo-insufficiency of Annexin A7 expression appears to drive disease progression to cancer because the genomic instability could lead to a discrete signaling pathway to reduce expression of the other tumor suppressor genes, DNA-repair genes, or apoptosis-related genes [
12]. Some work regarding Annexin A7 from our laboratory clearly showed that the Annexin A7 gene is associated with lymph node metastasis and progression of HCC [
5‐
7,
16‐
19]. However, the tumor suppressor mechanisms of Annexin A7 in HCC have not yet been elucidated. Future studies will investigate Annexin A7 expression
ex vivo before Annexin A7 expression is used to control HCC progression in the clinic.
Immunohistochemistry experiments showed that the subcellular localization of Annexin A7 protein in both the primary and lymph node-metastasized tumors was mainly in the cytosol, with some in the nuclei and cell membrane; while the level of Annexin A7 expression in the tumors was associated with their metastasis potential. Our current study demonstrated that the subcellular localization of Annexin A7 protein may be involved with lymph node metastasis of HCC. Meanwhile, Asma
et al. found that Annexin A7 protein can be localized in the cytosol, on the cell membrane, or on the cytoskeleton [
17]. Furthermore, Rick
et al. detected both of the Annexin A7 isoforms (47 kDa and 51 kDa) in a diabetes-related animal model. Diabetic wild-type animals showed reduced levels of the 47 kDa protein isoform. During brain development, Annexin A7 expression changes from the cytoplasm to the nuclei, and the subcellular distribution of Annexin A7 protein depends on the cell type in the adult central nervous system [
20]. In this study, we found that Annexin A7 expression was different in metastasized lymph nodes and primeval tumor cells derived from Hca-P and Hca-F cells. This disparity illustrates that the Annexin A7 gene plays an important role in high and low lymph node metastasis. This result was supported by a study that disclosed that the loss of Annexin A7 is an important factor in distant metastasis of gastric cancer [
21]. In addition, altered expression of Annexin A7 could affect the tumor stage and survival in hormone-refractory human prostate and breast cancers [
22‐
24]. Molecularly, Annexin A7 can regulate cellular exocytosis [
25,
26], and the latter event was associated with tumorigenesis [
27]. Annexin A7 can also modulate neoangiogenesis and tumor invasiveness through its involvement in VEGFR1 signaling [
28]. Ras proteins control at least three crucial signaling networks, including anchorage independence, survival, and proliferation protein dysregulated pathways, such as Annexin A7 [
29]. Annexin A7 can translocate from the cytoplasm to the cellular membrane in cultured cells after damage, apoptosis, and treatment with Ca
2+-ionophore [
30]. The 47 kDa isoform of Annexin A7 is expressed in astrocyte-derived C6 rat glioblastoma cells, which is in contrast to human brain tissues [
31]. Both isoforms appear in red blood cells, heart muscle, and the brain [
31‐
35]; different isoforms with a tissue-specific distribution may indicate different functions of Annexin A7 [
34]. Our experiments showed that both the 47 kDa and 51 kDa isoforms of Annexin A7 occurred in hepatocarcinoma tissues. In Hca-F cells with a high metastasis potential, the 47 kDa isoform was abundant; whereas in Hca-P cells with a low metastasis potential, the 51 kDa isoform was dominant. In addition, the expression of the 47 kDa and 51 kDa isoforms varied over time; thus, these data suggest that both isoforms play different roles in HCC progression. Afterwards, we detected Annexin A7 expression in mouse xenografts from primary and secondary tumors and found that the expression levels of Annexin A7 in tumors were reversely associated with their metastasis potential, indicating that Annexin A7 does play a role in suppression of tumor metastasis
in vivo. These data demonstrate that Annexin A7 functions as a tumor suppressor gene in hepatocarcinoma and could be further evaluated as a novel therapeutic target for hepatocarcinoma.
In summary, our current data demonstrate that the dysregulation of Annexin A7 is an important factor associated with lymph node metastasis of HCC. Further mechanistic studies will provide more insight into Annexin A7 tumor suppressor function for potential diagnostic and therapeutic uses.