Background
Gallbladder cancer (GBC) represents the most common malignancy of the biliary tract with a poor prognosis. The median survival time is less than one year, and the 5-year overall survival rate is approximately 5 % [
1,
2]. Currently, radical resection at an early stage is considered to be potentially curative therapy for GBCs. However, due to the lack of typical symptoms and specific biomarkers, most patients are identified at advanced stages and miss the chance for curative resection. Moreover, palliative chemotherapy and radiation therapy only offer limited benefits to advanced GBCs [
3,
4]. Therefore, it is desirable to explore the molecular mechanisms involved in GBC progression and to develop effective therapeutic strategies for prognosis improvement.
Mutational activation of the Ras-raf-MEK-ERK signaling pathway is frequently observed in human cancers, including GBC, and plays a prominent role in the regulation of malignant cellular proliferation, migration, invasion and survival [
5‐
8]. These observations have promoted the development of new molecularly targeted therapies, such as Raf and MEK kinase inhibitors. Unfortunately, cancer cells quickly adapt to these new targeted agents, and tumors with acquired resistance can emerge within several months following the primary treatments [
9‐
11]. Alternatively, therapeutics targeting regions outside the kinase domain might provide a new paradigm in Ras-raf-MEK-ERK pathway-targeted therapy [
12,
13]. Recent studies have demonstrated that prohibitin (PHB), an evolutionarily conserved and ubiquitously expressed protein, is required for the membrane localization and activation of C-Raf by the oncogene Ras [
14]. Interestingly, our microarray analysis of differential gene expression has also revealed that PHB expression is substantially upregulated in GBCs compared with that in their adjacent normal gallbladder tissues (unpublished data). This finding prompted our interest in investigating PHB expression and its biological function in GBCs and led us to explore whether PHB could serve as a potential therapeutic target in GBC patients.
In the present study, we determined PHB protein expression in GBC tissue samples using immunohistochemistry (IHC) staining and subsequently analyzed its clinicopathologic significance. We also investigated the role of PHB in regulating cell proliferation and invasion through the extracellular signal-regulated kinase (ERK) pathway in K-ras mutant and wild-type GBC cells. Our findings suggest that PHB overexpression predicts poor prognosis in GBC patients and, more importantly, that PHB could potentially serve as a novel therapeutic target in the oncogenic Ras-driven GBCs.
Methods
Study population, GBC cell lines and chemicals
This study was reviewed and approved by the ethics committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. Written informed consent was obtained from all of the patients enrolled in this study. GBC tissue specimens were obtained from 74 patients who underwent radical cholecystectomy (without prior radiotherapy or chemotherapy) from 2004 to 2012 in the Department of General Surgery, Xinhua Hospital. Additionally, 60 patients with chronic cholecystitis who underwent simple cholecystectomy were included as controls. The tumor stage for the GBC participants was defined according to the 7th AJCC-TNM classification system. The present GBC study population included 21 males and 53 females with a mean age of 66 years (range 39–86 years). All patients were periodically followed up for survival data until July 2014. The human GBC cell lines NOZ (K-ras mutant) and SGC-996 (K-ras wild-type) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) [
5]. The NOZ cells were cultured in William’s medium E (Lonza, Belgium, WI), and the SGC-996 cells were maintained in RPMI 1640 medium (Gibco, Gaithersburg, MD) at 37°C in a humidified 5 % CO
2 incubator. Both media were supplemented with 10 % fetal bovine serum (FBS). PD0325901 was obtained from Selleckchem (Houston, TX, USA).
Quantitative immunohistochemistry assays
IHC staining was performed to investigate PHB, phosphorylated ERK (p-ERK), matrix metalloproteinase 9 (MMP-9) and proliferating cell nuclear antigen (PCNA) expression in the formalin-fixed and paraffin-embedded GBC tissues using a standard immunoperoxidase staining procedure. The primary antibodies used were PHB (Abcam, cat # ab1836), p-ERK (Cell Signaling, cat # 4370), MMP-9 (Santa Cruz Biotechnology, cat # sc-21733) and PCNA (Abcam, cat # ab19166). A semi-quantitative scoring system was employed to evaluate the protein expression based on the staining intensity and percentage of stained cells. Immunostaining intensity (i) was classified as lack of staining (0), mild staining (1), moderate staining (2) and strong staining (3). The percentage of stained cells (ii) was divided into five grades: ≤5 % (0), 6–25 % (1), 26–50 % (2), 51–75 % (3) and ≥75 % (4). The score for each section was calculated as (i) × (ii), and the result was then defined as negative (0), weakly positive (1–3), moderately positive (4–7) and strongly positive (8–12). A score of 0–3 was categorized as PHB/p-ERK-negative, and a score of 4–12 was classified as PHB/p-ERK-positive.
Immunofluorescence and western blotting analysis
Immunofluorescence staining was employed to investigate PHB protein expression in NOZ and SGC-996 cells. After the fixation and permeabilization, the cells were probed with the primary antibodies against PHB (Abcam, cat # ab1836) and were then incubated with Cy3 rabbit anti-mouse IgG. The cells were counterstained with DAPI and then imaged under a fluorescence microscope. For western blotting analysis, equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis. The proteins were then blotted onto a PVDF membrane and probed with the primary antibodies against PHB (Abcam, cat # ab1836), p-ERK (Cell Signaling, cat # 4370), ERK (Cell Signaling, cat # 4695), MMP-9 (Santa Cruz Biotechnology, cat # sc-21733) or β-actin (Sigma-Aldrich, cat # AC-15). Afterwards, the blots were incubated with HRP-conjugated secondary antibodies, followed by enhanced chemiluminescence (ECL) detection.
RNA interference, construction of plasmids and transfection
For pFH1UGW lentivirus-mediated silencing of PHB, the short hairpin RNA (shRNA) sequence that effectively targeted human PHB was 5′-CAGAAAUCACUGUGAAA UUTT-3′. Recombinant lentiviruses expressing PHB shRNA or negative control shRNA (sh-PHB or sh-NC) were produced by Genechem (Shanghai, China). The NOZ and SGC-996 cells were infected with concentrated virus according to the manufacturer’s instructions. The plasmids pCDNA3.1-Prohibitin was made by inserting human prohibitin cDNA into a pCDNA3.1 expression vector. Empty vector-transfected cells (MOCK) were used as control. Constructs were transfected into cells using Lipofectamine 2000. The PHB expression in the infected cells was validated by western blotting assay.
In vitro cell proliferation assay
Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions (Dojindo Laboratories, Kumamoto, Japan). The absorbance values of NOZ and SGC-996 cells at various time points after transfection were measured using a microplate reader. Moreover, DNA synthesis was determined by the percentage of cells showing 5-ethynyl-2’-deoxyuridine (Edu) incorporation into DNA. Briefly, the transfected cells were cultured with 10 μM Edu and then fixed in 4 % paraformaldehyde. After the permeabilization, the cells were reacted with 1× Apollo reaction cocktail (RiboBio, Guangzhou, China). The cells nuclei were counterstained with Hoechst 33342 and visualized under a fluorescence microscope.
Flow cytometric cell cycle and cell apoptosis analysis
The effect of PHB depletion on cell cycle progression was determined by flow cytometry. After fixation, the transfected cells were stained with propidium iodide (PI) solution (50 μg/ml PI and 100 μg/ml RNase A in PBS) and then subjected to cell cycle analysis. The extent of cell apoptosis was measured using Annexin V/PI double staining. Briefly, 100 μl of binding buffer containing 2.5 μl of Annexin V-FITC and 1μl of PI was added to the transfected cell suspension, which was then incubated for 30 min in the dark. The samples were analyzed with a FACScan flow cytometer.
In vitro cell migration and invasion assay
For the wound-healing migration assay, the transfected NOZ and SGC-996 cells were seeded into 6-well plates and grown to confluence. Wounds were created by scraping confluent cell monolayers with a 1ml pipette tip. Photomicrographs were taken at time points 0 and 24 h after wounding. The percentage (%) change in migration was determined via comparison of the differences in wound width. Moreover, the effects of PHB knockdown on cell migration and invasion were evaluated using 8-μm transwell filters (BD Biosciences, Franklin Lakes, NJ). Briefly, the transfected NOZ and SGC-996 cells were suspended in 0.5 ml serum-free media and added into the upper chamber with an uncoated or Matrigel-coated membrane, whereas medium containing 10 % FBS was added to the lower chamber. After incubation for 24 h, the cells that migrated or invaded through and adhered to the bottom of the membrane were fixed and stained. Five random fields (100× magnification) were captured for each membrane, and the migratory and invasive cells were counted and averaged.
Gelatin zymography
The conditioned media of the transfected NOZ and SGC-996 cells were collected and concentrated with centrifugal filters. A gelatin zymography assay was conducted to evaluate the influence of PHB depletion on the active MMP secretion as previously described [
15]. Briefly, equal amounts of protein were separated using 10 % SDS-PAGE co-polymerized with 0.1 % gelatin as a substrate (Invitrogen, CA). After electrophoresis, the gels were renatured for 1 h at room temperature in 1 × zymogram renaturing buffer (Invitrogen, CA) and incubated at 37°C overnight in 1 × zymogram developing buffer (Invitrogen, CA). The gels were stained with Coomassie brilliant blue and de-stained with 20 % methanol and 10 % acetic acid in distilled water until clear bands could be visualized. The activities of the MMP bands were quantified using densitometry.
In vivo subcutaneous xenograft, peritoneal invasion and tail vein-lung metastasis tumor models
The use of animals and the experimental protocol were approved by the Institutional Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. All experiments were performed in accordance with relevant guidelines and regulations for the welfare and use of animals in cancer research.
BAL B/C nude mice were randomly divided into the Lv-sh NC group and the Lv-sh PHB group. Subcutaneous xenograft, peritoneal invasion and tail vein-lung metastasis tumor models were established as previously described [
16‐
18]. For the subcutaneous xenograft assay, tumor growth was monitored every 4 days. On day 28, the tumor tissues were harvested for further IHC staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis analysis. For the peritoneal invasion assay, on day 28 after the tumor inoculation, the peritoneal invasion rate was calculated based on the invasive tumors that appeared on the peritoneal cavity. In the tail vein-lung metastasis assay, the lung metastasis rate was quantified based on the metastatic foci that appeared on the lungs.
TUNEL apoptosis assay
The extent of cell apoptosis in the tumor specimens from the subcutaneous xenograft models was evaluated using a TUNEL apoptosis assay according to the manufacturer’s instructions (Roche, Basel, Switzerland). For quantitative analysis, the TUNEL-positive cells that fulfilled the morphological criteria of apoptosis were counted in 5 randomly chosen fields (400 × magnification). The results are expressed as the mean percentage of apoptosis cells.
Statistical analysis
All statistical analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL). The data are expressed as the mean ± standard deviation (SD). Statistical significance for the measurement data was determined using an independent Student’s t-test. The categorical variables were analyzed employing a Pearson’s chi-square test. The Kaplan-Meier test was performed for univariate survival analysis. Multivariate analysis was determined using the Cox proportional hazards model. Treatment differences with a two-sided p value < 0.05 were considered significantly different.
Discussion
Recent work has reported that PHB is overexpressed in several tumor types and plays crucial roles in cancer development and progression [
19‐
21]. Interestingly, our microarray analysis of differential gene expression has also demonstrated that PHB expression is considerably upregulated in GBCs compared with that in their adjacent normal gallbladder tissues. In the present study, we investigated PHB protein expression and its biological functions in GBCs. We found that PHB protein was overexpressed in the plasma membrane and cytoplasm of GBC cells and was significantly associated with histological grade, tumor stage and perineural invasion. Furthermore, PHB overexpression was associated with worse survival in GBC patients. In vitro experiments indicated that downregulation of PHB expression dramatically reduced cell proliferation and invasion in human GBC cell lines (NOZ and SGC-996). Subsequently, we explored the potential mechanism underlying the PHB-mediated aggressive behavior in GBCs. We observed that PHB expression in NOZ cells carrying the K-ras mutation was much higher than that in SGC-996 cells (K-ras wild-type). Moreover, PHB expression was concordantly associated with p-ERK basal levels in NOZ and SGC-996 cells. Furthermore, silencing of PHB expression contributed to a dramatic reduction in the activation of ERK protein kinase, as reflected by the decreased p-ERK expression. Additionally, PD0325901, a specific inhibitor of MEK, markedly impaired PHB- mediated phosphorylation of ERK protein. In human GBC tissue samples, we identified a positive association between PHB expression and the phosphorylation levels of ERK protein. In vivo, PHB depletion not only inhibited the growth and metastasis of GBC cells but also reduced the p-ERK expression level. Collectively, these findings suggest that PHB overexpression might promote cell proliferation and invasion through activating the ERK pathway, which plays a crucial role in GBC progression.
As an evolutionarily conserved and ubiquitously expressed protein, PHB contains an N-terminal transmembrane domain, an evolutionarily conserved PHB domain that is similar to that of lipid raft associated proteins, and a C-terminal coiled-coil domain that is involved in the regulation of protein-protein interactions [
22‐
24]. Recent studies in human cervical and pancreatic cancers have demonstrated that the direct interaction of PHB with C-Raf is required for the localization and phosphorylation of C-Raf at serine 338 at the plasma membrane and results in the RAS-mediated activation of Raf and the downstream activation of the ERK pathway [
13,
14,
25]. Here, we observed that PHB protein was primarily localized to the plasma membrane and cytoplasm in human GBC tissues and cultured GBC cell lines (NOZ and SGC-996). Intriguingly, the PHB expression level in NOZ cells carrying the K-ras mutation was much higher than that in SGC-996 cells (K-ras wild-type). Furthermore, PHB expression was concordantly associated with the proportion of p-ERK among total ERK protein. Upon depletion of PHB in GBC cells, we observed a dramatic reduction in the ERK pathway activation, as reflected by the decreased p-ERK levels. The statistical analysis of the IHC staining results validated that PHB overexpression was frequently accompanied by the upregulation of p-ERK expression in GBC tissue specimens. Based on these observations, we postulated that the localization of PHB within the plasma membrane might be critically involved in ERK pathway modulation in GBCs. More recently, rocaglamide, a natural anticancer compound derived from the traditional Chinese medicinal plant
Aglaia, has been shown to selectively bind to PHB protein with nanomolar affinity in human cervical cancer cell line HeLa and human T cell leukemic cell line Jurkat. In turn, this binding disrupts the C-Raf-PHB interaction at the plasma membrane, thus leading to the inactivation of the oncogenic Raf-MEK-ERK signaling pathway [
26]. Whether rocaglamide exhibits similar anticancer effects in GBCs, especially the ones harboring RAS mutations, needs to be further explored. It is also worth noting that the inhibitory effects of PHB depletion on cell proliferation and invasion were more pronounced in NOZ cells that harbored the K-ras mutation than in SGC-996 cells (K-ras wild-type). This observation implies that prospective selection of patients with tumors carrying genetic alterations in the ERK pathway is likely to identify a subgroup of individuals who may benefit from the C-Raf -PHB interaction-targeted therapy.
Although PHB expression has been demonstrated to be considerably upregulated in several types of human cancers, the role of PHB in tumorigenesis remains controversial. PHB protein was initially found in the mitochondrial inner membrane and plays a central role in maintaining mitochondrial morphology and normal functions, thus preventing apoptosis in malignant cells against metabolic stress [
27‐
29]. Recently, PHB has been revealed to be indispensable for Raf-MEK-ERK pathway activation by the oncogene Ras, supporting the pro-tumorigenic role of PHB in cancer progression [
14,
30,
31]. Nevertheless, accumulating evidence has also highlighted the anti-tumorigenic properties of PHB localized within the nucleus. Through the interaction with the retinoblastoma in the nucleus, PHB could suppress E2F-mediated transcription for cell cycle progression, thereby resulting in the inhibition of malignant cellular growth [
32,
33]. In this study, we found that PHB protein was primarily localized to the plasma membrane and cytoplasm in GBC cells. Furthermore, PHB overexpression was associated with poor outcome in GBC patients. More importantly, silencing of PHB expression potently suppressed GBC cell proliferation and invasion in vivo and in vitro. These observations indicated that PHB may be pro-tumorigenic rather than a tumor suppressor in GBCs. Based on these findings, we speculated that the paradoxical anti-tumorigenic or pro-tumorigenic effect of PHB on different cell types might be determined by protein-protein interactions in different subcellular localizations. Moreover, we also observed that PHB depletion induced a significant increase in apoptosis in the NOZ cells’ subcutaneous xenografts. Given the previously reported function of mitochondrial PHB in enhancing cellular survival against metabolic stress, we postulated that PHB protein expressed in the cytoplasm of GBC cells might be at least partially restricted to the mitochondria and that silencing of the mitochondrial PHB weakened the capacity of GBC cells to survive in an intratumoral malnutrition microenvironment induced by the rapid proliferation of malignant cells.
Conclusions
In summary, we demonstrated that PHB overexpression was associated with an unfavorable prognosis in GBC patients. Furthermore, downregulation of PHB expression reduced proliferation and invasion in GBC cells via the ERK pathway. Therefore, PHB may be a potential prognostic and therapeutic biomarker in GBC patients.
Ethics approval and consent to participate
This study was approved by the ethics committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. Written informed consent was obtained from all of the patients enrolled in this study.
The use of animals and the experimental protocol were approved by the Institutional Animal Care and Use Committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. All experiments were performed in accordance with relevant guidelines and regulations for the welfare and use of animals in cancer research.
Consent for publication
Not applicable.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 81172029, 91440203 and 81402403), the National High Technology Research and Development Program (863 Program) (No. 2012AA022606), the Program for Changjiang Scholars, the Natural Science Research Foundation of Shanghai Jiao Tong University School of Medicine (No. 13XJ10037), and the Interdisciplinary Program of Shanghai Jiao Tong University (No.14JCRY05).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YBL, JZ and YC conceived and designed the experiments. YC, HBL and FZ performed the experiments and wrote the paper. XAW and SBL prepared the patient samples. SZ and YC collected and analyzed the data. RFB, YJS, QM and ZL provided technical expertise and performed the experiments. YBL and ZL provide assistance with revising this manuscript. All authors read and approved the manuscript.