In the present study, we provide the first evidence that coexpression of HMGB1 and VEGF-C is associated with aggressive biological behavior and poor prognosis in a large series of human GCs and is a valuable prognostic marker.
HMGB1, the focus of many recent cancer studies, plays a critical role in cancer development, progression, and metastasis through its pro-angiogenic and pro-lymphangiogenic functions . Consistent with other studies, the HMGB1 protein was highly expressed in GC samples. We also observed that the positive staining signal was mainly localized to the nucleus in GC but was also found in the cytoplasm and stroma, in accordance with its biological function. Hypoxia results in necrotic or damage-induced cell death within the tumor when the growing tumor exceeds the capacity of the existing vasculature. HMGB1 not only activates vascular endothelial cell proliferation and neovascularization through the HMGB1-RAGE pathway, it can also stimulate inflammation . Damaged or necrotic cells can actively secrete or passively release HMGB1 into the extracellular milieu. The constant release of the pro-inflammatory cytokine HMGB1 from necrotic tumor cells creates a microenvironment similar to chronic inflammation and contributes to the development of epithelial malignancies . Furthermore, the rate of HMGB1 expression (48.80% of 166) is within the range of previously reported data [8–12]. Moreover, we observed that HMGB1 expression was closely correlated with TNM stage, nodal status, and survival rate but not with age, gender, or pathological type, as reported previously [8–12]. Controversially, we did not observe any significant differences in HMGB1 expression between well-differentiated tumors and poorly differentiated ones, similar to the results obtained by Hao et al., in which no correlation between HMGB1 expression and tumor differentiation was observed . Further studies of HMGB1 expression in GC and its function are needed.
As expected, the correlation of HMGB1 expression with GC prognosis is in accordance with most of the findings of previous studies. HMGB1 expression has been reported to be significantly associated with tumor invasion, lymph node metastasis, distant metastasis, and Duke’s stage and inversely associated with overall survival in human colorectal carcinoma . HMGB1 has been shown to be an independent prognostic factor for patients with squamouscell carcinoma of the head and neck , and its overexpression also plays a role in the progression of nasopharyngeal carcinoma (NPC) and is correlated with a poor clinical outcome . Serum HMGB1 is closely associated with the clinical and pathological features of GC and appears to be a useful serological biomarker for early diagnosis as well as the evaluation of tumorigenesis, stage, and prognosis in GC . Regarding the potential mechanism, Ohmori et al. demonstrated that HMGB1 enhances the proliferation, motility, invasion, and survival of cancer cells, induces apoptosis of macrophages, and suppresses the host anti-cancer immune system .
However, several studies differ from our conclusion. Bao et al. concluded that there was no significant association between HMGB1 expression and invasion depth, tumor stage, and lymph node metastases. Curiously, their results suggested that overexpression of HMGB1 was positively correlated with patient prognosis after curative resection and adjuvant chemotherapy . Akaike et al. reported that the prognosis of the low HMGB1 group was significantly poorer than that of the high HMGB1 group in GC . Similarly, Jiao et al. proposed that HMGB1 could function as a tumor suppressor and radiosensitizer in breast cancer  because oxidized HMGB1 increased the cytotoxicity of these agents and induced apoptosis via the mitochondrial pathway or the caspase-9/-3 intrinsic pathway . Furthermore, HMGB1 plays a critical role in tumor immunology. The interaction of the HMGB1 protein released from dying tumor cells with Toll-like receptor 4 on dendritic cells is required for the crosspresentation of tumor antigens and the promotion of tumor-specific cytotoxic Tcell responses [27, 28], which are selectively involved in the crosspriming of anti-tumor T lymphocytes in vivo[29, 30]. This discordance may be due to the various tumor types, intricate microenvironments, and different responses to follow-up treatment in these studies. Further study of the underlying mechanism of this discordance is a worthwhile and beneficial avenue to elucidate the precise role of HMGB1 in tumorigenesis.
VEGF-C is a classic specific growth factor of the lymphatic system that is also known to play several roles in tumor growth and metastasis to lymph nodes and distant organs. VEGF-C may induce angiogenesis and lymphangiogenesis in malignant tumors [31, 32]. Metastasis to regional lymph nodes and distant organs through the expression of VEGF-C has been identified in several cancers such as colon cancer, prostate cancer, and gastric cancer [16, 33–38]. Similarly, in our study, expression of VEGF-C was significantly associated with TNM stage, nodal status, and poorer overall survival [39–45].
Whether HMGB1 and VEGF-C act independently or cooperatively to increase the malignant potential of GC is not clear. We verified the high correlation between these two markers in GC tissues and found that HMGB1 could increase VEGF-C secretion in GC cells (Figure 2). We also demonstrated that HMGB1 could upregulate VEGF-C secretion in GC cell lines, suggesting that HMGB1 may enhance the invasion ability of tumor cells, at least in part, through VEGF-C-related pathways [17, 20]. As expected, Kaplan-Meier survival analysis indicated that patients with expression of both markers had the poorest prognoses when compared to all other groups. Correspondingly, individuals negative for both markers displayed the longest survival, while patients expressing a single marker had intermediate survival rates (Figure 4). Therefore, assessment of HMGB1 and VEGF-C in preoperative biopsies may assist in the stratification of GC patients according to different optimized treatment protocols, such as adjuvant radical or chemotherapy.
The application of molecular diagnosis can be beneficial for the earlier and more accurate identification of cancer prognosis or grade of malignancy, thus offering rapid and efficient therapy . Accumulating evidence suggests that HMGB1 and VEGF-C are useful adjunct markers for traditional prognostication indices [9–12, 39–45]. In this study, we further confirmed the predictive value of the molecular approach and verified the benefit by simultaneously evaluating the expression of HMGB1 and VEGF-C (hazard ratio 2.78, P = 0.0162). Nevertheless, the mechanism by which coexpression of HMGB1 and VEGF-C promotes GC progress and metastasis needs to be further investigated.