Background
Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third leading cause of cancer-related deaths worldwide, with over a half million deaths per annum[
1]. The annual incidence of HCC in hepatitis B cirrhotic patients can run as high as 3–5%, and one-third will develop HCC in their lifetime [
2]. In China, an endemic area with almost one third of the HBsAg carriers found worldwide [
3]. Because of high infection rates with hepatitis B virus (HBV), 55% of world’s HCC cases occur in the country [
4]. Surgical resection provides an opportunity for cure, but frequent recurrence after surgery remains the major obstacle to long-term survival [
5]. It is estimated that approximately 70% of patients will relapse within 5 years after surgery and more than 80% of postoperative recurrence occurs in the remnant liver [
6], which can be either intrahepatic metastasis from the primary tumor or de novo multicentric tumors. Typically, recurrence in HCC follows a 2-peak distribution: the first peak, usually within 2 years after resection, is mostly related to true metastatic spread (i.e., early recurrence), whereas the second peak mainly results from de novo tumors as a consequence of the carcinogenic cirrhosis (i.e., late recurrence) [
7]. Vascular invasion (macroscopic and microscopic) is the strongest predictor of recurrence although other factors such as tumor size, number of nodules, α-fetoprotein (AFP) levels, degree of differentiation, and satellite lesions are also associated with recurrence [
6]. Unfortunately, microvascular invasion and satellites can be assessed only with the full pathologic specimen, which reduces the odds for an accurate preoperative prediction of HCC recurrence. In addition to cancer, another life-threatening condition (i.e., cirrhosis) is present in more than 80% of patients with HCC, which renders prognostic prediction a major challenge. Some clinical-based staging systems, especially the widely accepted Barcelona Clinic Liver Cancer (BCLC) algorithm [
8], establish a road map for routine clinical decision-making. However, these systems fail to provide molecular information, which can complement the portrait of prognosis in complex solid neoplasms. Therefore, elucidating the molecular mechanisms underlying recurrence is essential for identifying accurate predictive biomarkers and developing effective therapeutic modalities.
To date, some cancer cell-oriented predictive systems are neither superior to morphological classification nor display any overlapping predictor genes, and they include few disease-related genes [
9,
10]. It seems that high levels of HBV replication contribute to the recurrence and poor prognosis of HCC, which is linked to inflammatory cell infiltration. Thus, the liver inflammatory response and the whole-body immune status can largely influence the biological behavior of HCC. Peripheral blood mononuclear cells (PBMCs), the most common immune cell subsets, are transported throughout the entire body. Some PBMC genes may reflect behavior, especially that of HBV-related HCC, which is closely related to the inflammatory response. In addition, it has been reported that some related signals play crucial roles in cancer and inflammation by controlling the expression of certain cytokines [
11]. These cytokines, such as IL-6, are produced by lymphocytes in liver and peripheral blood. As a result, some characteristics of genes in PBMCs may be related to the pathogenesis and progression of HCC.
In this study, the whole-genome Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array was applied to define a comprehensive copy number profile in PBMCs that predicts HCC recurrence. The differentially expressed mRNAs were then selected, validated, and subjected to gene ontological (GO) and pathway analysis. The target genes predominating in the gene regulatory networks were further investigated in an attempt to provide better understanding of the biological features of HCC recurrence. Moreover, to ensure that the signature reflecting the profile of recurrence, we simultaneously tested the potential biomarkers from 2 different kinds of patient samples, including PBMCs and cancerous tissues.
Discussion
In the present study, we found cyclin B1, Sec62, and Birc3 were aberrantly expressed proteins in HCC patients. Highly expressed cyclin B1, Sec62, and Birc3 were associated with significantly reduced recurrence-free survival, and cyclin B1 and Sec62 were independent prognostic factors in this cohort. To our knowledge, this is the first detailed systematic investigation of the expression pattern in PBMCs and the roles of these 3 proteins, especially Sec62, in HCC recurrence.
The current consensus is that surgery is one of the most important treatment options for patients with HCC. However, tumor recurrence remains one of the major challenges for those postoperative patients. Although increasing numbers of genes have been indentified, the molecular mechanism of HCC metastasis and recurrence are not fully understood. Based on the results of microarray analysis, cyclin B1, Sec62, Birc3 were chosen for subsequent research. Cyclin B1 is known to regulate the G2/M transition in the cell cycle. Recent studies have demonstrated aberrant expression of cyclin B1 in several malignant cancers, including breast cancer [
15], esophageal squamous cell carcinoma [
16], non-small cell carcinoma [
17], gastric cancer, and hepatocellular carcinoma [
18,
19]. But it remains unclear how cyclin B1 overexpression is involved in oncogenesis and tumor progression. Previous study demonstrated that cyclin B1 act as a promising prognostic and therapeutic target for HCC [
20]. However, Chae [
21] reported that the expression of cyclin B1 had no influence on the survival of patients with breast cancer. In the present study, elevated expressed cyclin B1 was found in the patients with recurrent HCC, contrary to that in non-recurrent patients and healthy volunteers. Moreover, there was no significant difference in cyclin B1 expression between the patients with non-recurrent HCC and healthy subjects. Through the univariate analysis, cyclin B1 expression was identified as an independent risk factor for recurrence in HCC patients after surgery. This discrepancy might be due to dissimilar expression of cyclin B1 in different tumor types.
Similar results were observed for Sec62, which is a member of the protein translocation apparatus in the endoplasmic reticulum membrane. Previous studies demonstrated the amplification and overexpression of Sec62 in prostate cancer cell lines, and described
SEC62 as a potential target gene in prostate cancer [
22]. Overproduction of Sec62 is also observed in other tumors, primarily in tumors of the lung and thyroid [
23]. In our study, it seems that Sec62 plays a significant role in HCC recurrence. Sec62 overexpression was found in the patients with recurrent HCC. Importantly, Sec62 was an independent risk factor for recurrence in HCC patients after surgery as evidenced by univariate analysis.
Although the expression of Birc3 was significantly higher in the recurrent HCC samples than that in the non-recurrent HCC and normal samples, a specific independent role in predicting HCC recurrence was not identified for Birc3. Consistently, DNA amplifications of Birc2 and Birc3 have been observed in mouse liver and human lung cancers [
24,
25], liver carcinoma [
24], oral squamous cell carcinoma [
26,
27], medulloblastoma [
28], glioblastoma [
29], and pancreatic cancer [
30]. The exact role of Birc3 in HCC must be verified through a larger prospective study.
In recent years, studies on malignant tumors has primarily focused on cell proliferation, migration, and apoptosis. Cyclin B1, Sec62, and Birc3, chosen in this study according to our microarray analysis, likely play important roles in cell proliferation and migration. They can exert a tumor-promoting effect on HCC by regulating cell cycle and protein translocation. In contrast to previous studies using only HCC tissues, we examined PBMCs and tumor tissues in the present study. Interestingly, the results obtained in PBMCs were consistent with those of the tumor tissues by immunohistochemical analysis for. As a result, elevated cyclin B1 and Sec62 expression in PBMCs had a significantly negative prognostic value in terms of recurrence-free survival, which hints the potential use of these molecular markers to predict the risk of tumor recurrence after surgery and to act as therapeutic targets to reduce tumor recurrence and improve clinical therapies.
The contribution of HBV to the current findings must be mentioned. China is one of the highest prevalent areas of HCC, mainly because chronic hepatitis B carriers account for more than 10% of the Chinese population [
31]. Over 85% of patients with HCC have HBV infection in China [
32]. At present, the studied population almost unavoidably consisted of patients with HBV-associated HCC because of the special situation in China. The induction of apoptosis and stimulation of cell cycle by the HBV X protein has been reported [
33,
34]. The analysis of cyclin B1, Sec62, and Birc3 expressions in HCC patients with other etiological backgrounds may be very useful to ascertain the real predictive value of cyclin B1 and Sec62 for HCC recurrence.
Despite the important roles of cyclin B1 and Sec62 in tumor recurrence and their predictive implications, this study should be viewed as a hypothesis-generating study. Prospective and animal studies are needed to confirm our findings and clarify the biological effects of these proteins in more detail.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Li W and Juan Du made the microarrays, performed the real-time, western-blot and immunohistochemical staining, collected the clinical data and contributed to the writing of the manuscript. Qinghui Z analyzed the statistical data and participated in writing the manuscript. Binbin C and Jun L participated in collecting the clinical data. Changquan L and Denghai Z participated in designing and coordinating the study, and in writing the manuscript. All of the authors read and approved the final manuscript.