Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer-related deaths. Its incidence has more than doubled during the last two decades in the western world, where it is the fastest growing cause of cancer-related death (Llovet et al., 2003; Seeff and Hoofnagle, 2006; El Serag HB et al., 2007; Vivekanandan and Singh, 2008). Despite the well known etiology of HCC, the molecular mechanism underlying hepatocarcinogenesis is still not fully understood (Vivekanandan and Singh, 2008). LAPTM4B (lysosomal protein transmembrane 4 beta) is a newly identified cancer-associated gene (NM_018407, Gene ID=55353) and encodes a 35 kDa tetratransmembrance glycoprotein (Shao et al., 2003) designated as LAPTM4B-35. It has been reported that the LAPTM4B mRNA and the LAPTM4B-35 protein detected by Western blot (WB) or immunohistochemistry (IHC) are markedly upregulated in a wide variety of cancers, including HCC (more than 87%) (Shao et al., 2003; Peng et al., 2005; Yang et al., 2009), gallbladder carcinoma (76%) (Zhou et al., 2007), extrahepatic cholangiocarcinoma (72%) (Zhou et al., 2008), lung cancer (88%) (Kasper et al., 2005), breast cancer (50%) (Kasper et al., 2005), and ovarian cancer (69%) (Yang et al., 2008). It has been revealed that the levels of upregulated LAPTM4B mRNA and LAPTM4B-35 protein are correlated significantly with pathological grade/differentiation of cancers and prognosis of patients with HCC, gallbladder carcinoma, extrahepatic cholangiocarcinoma, and ovarian cancer, suggesting that it may be an independent prognostic marker in these cancers (Shao et al., 2003; Zhou et al., 2007; Yang et al., 2008; Zhou et al., 2008; Yang et al., 2009).
In this study, we investigated the effects of adenovirus-mediated upregulation of LAPTM4B-35 on cellular phenotypes in L02 human liver cell line, as well as on the tumorigenicity ex vivo of LAPTM4B-35 upregulating L02 cells. The results demonstrated that malignant cellular phenotypes, including deregulation of proliferation, evasion of cell death, and tumorigenicity, were promoted by upregulation of LAPTM4B-35. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes that included activation of the phosphoinositide 3-kinases (PI3K)/serine/threonine protein kinase B (PKB/AKT)/bcl-xL/bcl-2-associated death promoter homolog (Bad) signaling pathway, inhibition of caspase-3 activation, upregulation of prosurvival Bcl-2 protein, and deregulation of proapoptotic Bax protein. These data suggest that LAPTM4B-35 may be a potential target for cancer therapy.
MATERIALS AND METHODS
Construction of Recombinant Adenovirus Vectors
The pAdEasy-1 Vector System, an E1/E3-deleted replication-deficient adenovirus type 5 (Ad5) vector, was used to construct recombinant adenovirus vectors of Ad-AE. Ad-AE contains the full ORF (951 bp) of LAPTM4B cDNA that encodes LAPTM4B-35. This construction was performed by Vector Gene Technology.
Monoclonal antibodies anticaspase-3, antipoly(ADP-ribose)polymerase (anti-PARP), and anti-p-AKT (473) (Cell Signal Technology, CA), anti-Bcl-2 (Santa Cruz Biotechnology, CA), polyclonal anti-p-Bad (136) and anti-AKT (Cell Signal Technology, CA), and anti-Bax and antisurvivin (Santa Cruz Biotechnology, CA) were purchased commercially. Anti-LAPTM4B-N10 pAb was prepared and immunoaffinity purified in our laboratory.
Cell Culture and Virus Infection
The L02 cell line derived from human normal liver was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, and Chinese Academy of Sciences. Viral stocks were suitably diluted in serum-free medium to obtain the desired multiplicity of infection (MOI) and added to cell monolayers. The serum-free medium containing viruses was then replaced by culture medium with 10% newborn calf serum (NCS) after 2-hr incubation at 37°C. All infected cells in the present experiments were used 48 hr after initial infection and they were transient infected cells.
Protein Extraction and WB
Cells infected with Ad-AE or Ad-Null were harvested and lysed in RIPA buffer (Pierce, Rockford) containing protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), ultrasonicated in a iced water-bath, and incubated for 30 min on ice. After centrifugation at 12,000g for 15 min at 4°C, the supernatant was subjected to WB analysis. For detection of phosphorylation in signaling proteins, phosphatase inhibitor cocktail (Roche) was added to the lysis buffer. Anti-LAPTM4B-N10 pAb, which specifically recognizes LAPTM4B-35 or other appropriate antibodies, was used for blotting.
Cell Proliferation Analysis by Acid Phosphatase Assay for Growth Curve and 5-Bromo-2′-Deoxy-Uridine Assay for DNA Synthesis
Infected cells (2 × 103) were seeded in 96-well plates for various lengths of time, and the viable cells were identified by acid phosphatase assay as previously described (Yang et al., 1996). The absorbance was determined at 405 nm with a microplate absorbance reader (Tecan Austria GmbH). DNA synthesis was evaluated by BrdU Labeling and Detection Kit III (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions (Gratzner, 1982).
Soft-Agar Colony Formation Assay
Dulbecco's Modified Eagle Medium (DMEM) (1.5 mL) containing 0.8% agarose (Sigma, St. Louis) and 10% NCS was plated onto each well of a six-well plate as the basal layer. DMEM (1.5 mL) containing 0.35% agarose, 10% NCS, and 1000 cells was overlaid on this basal agarose layer. Three weeks later, photographs were taken under microscopy (Olympus, Japan).
Cell Migration Assay
Cell migration was measured via the Boyden chamber assay (Albini et al., 1987). Infected cells (3 × 104) were seeded on the surface of upper chamber, and the chemoattractant (composed of conditioned serum-free medium of NIH3T3 cells and supplemented with 5 μg/mL of fibronectin) was added to the lower chamber. A PVP-free polycarbonate filter (13-mm diameter, 8-μm pore size, OSMONICS) was assembled between the upper and lower chamber. Cells in Boyden chamber were incubated at 37°C for 12 hr. The membranes were rinsed three times on the both sides with phosphate buffered saline (PBS). The upper surface of the membrane was scrubbed with a cotton swab to totally remove the remaining cells. Cells on the lower surface of the membranes were fixed with 100% methanol and stained with hematoxylin and eosin, and the cells in four random fields were counted under the light microscope.
LIVE/DEAD® Viability/Cytotoxicity Kit Assay
Infected cells (1 × 104) were seeded in 96-well plates, and then, apoptosis was induced by adriamycin (ADM) (WanLe Pharmacy, ShenZhen, China) addition into the medium (1 μg/mL) in L02-Ad-AE and L02-Ad-Null cells for 18 hr. The tested cells were stained with Calcein AM/EthD-1 reagent in LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen) and observed under a fluorescence microscope after 30-min incubation at 37°C (Jacobsen et al., 1996).
Detection of Apoptosis by FACS
Infected cells (2 × 105) were seeded in six-well plates for various lengths of time as indicated. Apoptosis was induced by adding adriamycin into the medium (1 μg/mL) (WanLe Pharmacy, ShenZhen, China) in L02-Ad-AE and L02-Ad-Null cells for 18 hr. Both adherent and floating cells were collected. Apoptosis was analyzed using an Annexin V Apoptosis Detection kit (Biosea, Beijing, China) with a FACSCalibur flow cytometer (Becton Dickinson).
Evaluation of Caspase-3 Activity
Infected cell lysates containing 20–100 μg protein were incubated at 37°C in a buffer (pH 7.5, 25 mmol/L 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mmol/L Ethylene Diamine Tetraacetic Acid (EDTA), 100 mmol/L NaCl, 0.1% 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), and 10 mmol/L dithiothreitol) containing the fluorogenic substrate Ac-DEVD-AMC (ALEXIS Biochemicals, Switzerland). The FLUOStar fluorometer (BMG Labtechnologies, Germany) with an excitation filter of 380 nm and emission filter of 460 nm was used to measure the signal (Rehm et al., 2002).
Transmission Electron Microscopy
The morphological changes of apoptosis were observed under transmission electron microscopy (Philips CM-120). Apoptosis was induced by adding doxorubicin into medium for 16 hr. Cells were then treated routinely.
Tumorigenicity of L02-Ad-AE Cells In Vivo
Six-week-old female BALB/c nude mice were obtained from the Department of Laboratory Animal Science, Health Science Center, Peking University. L02 cells (1 × 106) infected with Ad-Null or Ad-AE were inoculated subcutaneously into the right or left axilla of the same nude mice, respectively. Resultant tumors were measured, and tumor volume was calculated according to the formula: π/6 × length × width2. The mice were sacrificed 6 weeks after inoculation. The tumor sections were stained with conventional H&E staining and immunostained with purified anti-LAPTM4B-N10 pAb.
Life-Span of Nude Mice Inoculated With L02-Ad-AE Cells or L02-Ad-Null Cells
Six-week-old female BALB/c nude mice (obtained from the same department as above) were randomly divided into three groups of six mice each. L02 (2.5 × 106) cells infected without or with Ad-Null or Ad-AE were inoculated subcutaneously into the abdomen of the nude mice. The survival time of the animals was observed for 6 months, and animal survival was determined by Kaplan–Meier method.
All animal experiments were performed with the approval of the Ethics Committee of Peking University Health Science Center.
The data are presented as mean ± SD of three independent experiments. The Student's t-test was used to analyze the statistical differences in the samples. Results were considered statistically significant, if the P value was less than 0.05.
Upregulated Expression of LAPTM4B-35 in Ad-AE-Infected Cells
The replication-deficient recombinant adenoviral vector Ad-AE was constructed using a pAdEasy-1 vector, an E1/E3 deleted Ad5 vector. Ad-AE contains the entire open reading frame (ORF) (951 bp) of LAPTM4B cDNA encoding LAPTM4B-35; Ad-Null, an empty adenoviral vector, was employed as the control. The L02 cell lines, in which expression of LAPTM4B-35 is relatively low, were infected with Ad-AE or Ad-Null. The efficiency of adenoviral infection was evaluated by Ad-GFP at 100 MOI. The GFP-expressing cells reached a maximum of about 80% at 48–72 hr after infection (data not shown). The expression of LAPTM4B-35 (Fig. 1) increased in a dose-dependent manner and reached a maximum analyzed by WB in the Ad-AE infected cells, but not in Ad-Null infected cells. The infected cells were designated as L02-Ad-AE and L02-Ad-Null, respectively. Therefore, L02-Ad-AE cells could be used to investigate the functions of LAPTM4B-35.
To examine the effect of LAPTM4B-35 on cell proliferation, three approaches were applied. Cell proliferation was first measured by growth curves. As shown in Fig. 2A, the number of L02-Ad-AE cells was being increased faster than that of L02-Ad-Null cells, indicating that upregulation of LAPTM4B-35 significantly accelerated the raising of cell number. DNA synthesis was then detected by 5-bromo-2′-deoxy-uridine (BrdU) incorporation assay. As shown in Fig. 2B, incorporation rate of BrdU increased to ∼1.5-fold in L02-Ad-AE cells compared with that in L02-Ad-Null cells, indicating that the DNA synthesis in LAPTM4B-35-upregulating cells increased. Moreover, to assess the effect of LAPTM4B-35 on malignant transformation in L02 cells, anchorage-independent proliferation was applied by colony formation in soft agar. Anchorage-independent proliferation in vitro is a characteristic phenotype of malignant transformed cells, which is closely associated with tumorigenesis in vivo. As shown in Fig. 2C, upregulation of LAPTM4B-35 significantly promoted colony formation of L02-Ad-AE cells in soft agar. More and larger colonies were formed from L02-Ad-AE cells compared with the L02-Ad-Null control, indicating that the proliferation promoted by upregulation of LAPTM4B-35 possessed characteristics of malignancy.
LAPTM4B-35 Upregulation Promotes Cell Migration
Enhanced potential of migration is one of the hallmarks of metastatic malignant cells. The effect of LAPTM4B-35 on cell migration was evaluated by Boyden chamber assay. As shown in Fig. 2D, the number of migratory cells increased potently in Ad-AE-infected L02 cells, demonstrating that overexpression of LAPTM4B-35 promoted cell migration.
LAPTM4B-35 Upregulation Protects Cells From Adriamycin-Induced Apoptosis
To investigate the effect of LAPTM4B-35 on cell survival and apoptosis in L02 cells, several approaches were used. In terms of cell number, the alterations in growth curves plotted with the acid phosphatase assay data were derived from either proliferation or death of cells. We first determined the rate of cell survival and death caused by upregulation of LAPTM4B-35 using the LIVE/DEAD Viability/Cytotoxicity Kit. The living cells showed intense uniform green fluorescence in cytoplasm and the dead cells, including late apoptotic cells and nonapoptotic dead cells showed bright red fluorescence in the nucleus (Jacobsen et al., 1996). Our results (Fig. 3A) showed that the percentage of dead cells induced by adriamycin (ADM) (1 μg/mL) in L02-Ad-Null cells were ∼2.5-fold of that in L02-Ad-AE cells, indicating that LAPTM4B-35 plays an important role in maintaining cell survival in the presence of ADM.
The roles of LAPTM4B-35 in cell survival and apoptosis were further confirmed by flow cytometry. As shown in Fig. 3B, the number of Annexin V single-positive cells and Annexin V/PI double-positive cells were much lower (less than 50%) in L02-Ad-AE cells than in the control cells.
Moreover, observation of cellular morphology by transmission electron microscopy showed that typical apoptotic-like morphology such as chromatin condensation, and apoptotic bodies were present in L02-Ad-Null cells (Fig. 3C), whereas L02-Ad-AE cells displayed no sign of apoptosis in Fig. 3C.
Taken together, these results demonstrated that LAPTM4B-35 plays a significant role in preventing cells from apoptosis.
Involvement of Caspase-Dependent Pathway in LAPTM4B-35-Regulated Survival and Antiapoptosis
Caspase-3 is the executor caspase and is responsible for the proteolytic cleavage of a range of structural and regulatory proteins in apoptosis (Salvesen and Dixit, 1997; He et al., 2003). Caspase-3 activation was measured by the DEVD cleavage assay, a quantitative method used to detect caspase-3 activity (Rehm et al., 2002), and the levels of procaspase-3 and cleaved caspase-3 were also evaluated by WB analysis. In L02-Ad-Null cells, ADM treatment induced the activation of caspase-3 in a time-dependent manner, but this effect of ADM was completely blocked in LAPTM4B-35-upregulating L02-Ad-AE cells (Fig. 4A). In addition, a substantial amount of cleaved/activated caspase-3 appeared in L02-Ad-Null cells but none in L02-Ad-AE cells (Fig. 4B). Moreover, cleavage of poly(ADP-ribose)polymerase, a substrate of caspase-3, was also decreased in L02-Ad-AE cells compared with L02-Ad-Null cells (Fig. 4B). These results demonstrated that the caspase cascade is inhibited by LAPTM4B-35-upregulation.
Involvement of the Bcl-2 Family in LAPTM4B-35-Regulated Survival and Antiapoptosis
As proteins of the Bcl-2 family are critical apoptotic regulators that control the mitochondria-dependent apoptosis pathway, levels of Bcl-2 and Bax proteins were tested by WB. The results (Fig. 4C) showed that in L02-Ad-AE cells, the antiapoptotic Bcl-2 was increased, and the proapoptotic Bax was decreased. These results demonstrated that the apoptosis-related cellular levels of Bcl-2 family proteins are controlled by LAPTM4B-35.
Involvement of PI3K/AKT/Bad Signaling Cascade in LAPTM4B-35-Regulated Survival and Antiapoptosis
The PI3K/AKT signaling pathway is of great importance for maintaining cell survival in a variety of cells and over activated in a large number of cancers. The phosphorylation of AKT is an indication of PI3K/AKT signaling activation. Bad is the downstream targeting effector of AKT (Datta et al., 1997; Nicholson and Anderson, 2002; Samuels and Ericson, 2006). Our results (Fig. 4D) from Western blotting showed that phosphorylations of both AKT and Bad were increased in L02-Ad-AE cells compared with L02-Ad-Null cells, indicating involvement of PI3K/AKT/Bad signaling cascade in the regulation of survival and antiapoptosis controlled by LAPTM4B-35.
LAPTM4B-35 Promotes Tumor Growth in Immunodeficiency Nude Mice
Based on the stimulatory effects of LAPTM4B-35 on malignant phenotypes in vitro, we further evaluated the tumorigenicity of L02-Ad-AE cells in immunodeficiency nude mice. L02-Ad-Null or L02-Ad-AE cells were inoculated subcutaneously into the right or left axilla of the same mice, respectively. All six mice inoculated with L02-Ad-AE cells developed malignant tumors, and these tumors grew very rapidly (Fig. 5A,B). In all six animals, palpable tumors appeared on day 10 after inoculation with L02-Ad-AE cells, but only very small tumors appeared in two of the animals (Fig. 5A, indicated with white arrow) with a delayed latency of 36 days after inoculation with L02-Ad-Null cells. On the 44th day, after inoculation, L02-Ad-AE tumors reached an average weight of 1.076 ± 0.248 g, whereas the average weight of L02-Ad-Null tumors was only 0.045 ± 0.01 g. Histopathologic examination showed that tumors formed after inoculation of L02-Ad-AE cells in nude mice were carcinoma (Fig. 5C). Notably, the tumors formed by L02-Ad-AE cells contained plenty of blood vessels, and a number of cancer cells were inside these vessels; whereas the tumors formed by L02-Ad-Null cells were poor of blood vessels (Fig. 5C), indicating the high malignancy of tumor from L02-Ad-AE cells. In the meantime, high expression of LAPTM4B-35 protein in the tumor tissues formed from L02-Ad-AE cells was confirmed by IHC (Fig. 5C). Next, the effect of LAPTM4B-35 on the life-span of mice inoculated with L02 cells, L02-Ad-Null cells, or L02-Ad-AE cells was evaluated. The data showed that upregulation of LAPTM4B-35 accelerated the death of mice with tumor (Fig. 5D). The results obtained from these experiments indicated that upregulation of LAPTM4B-35 can promote malignant transformation of L02 cell line and induce tumorigenesis with invasive potential in nude mice.
In this study, we showed that upregulation of LAPTM4B-35 promotes malignant transformation in L02 human liver cell line, as evidenced by resistance to apoptosis, promotion of proliferation, enhancement of colony formation in soft agar, and tumorigenesis in nude mice. In addition, these effects have been identified in our laboratory via knockdown of endogenous LAPTM4B-35 of HepG2 and BEL7402 cell lines from hepatocellular carcinoma by RNAi (Yang et al., 2010). Our investigations clearly demonstrated the LAPTM4B-35 ability to promote xenograft tumor growth in nude mice. Therefore, it is reasonable to propose that overexpressed LAPTM4B-35 may act as an oncogenic protein.
Resistance to apoptosis is one of the most fundamental characteristics of malignancy and sensitization of tumor cells to apoptosis is an important strategy for cancer therapy (Pelengaris et al., 2002). Our data obtained from this study demonstrate that LAPTM4B-35 plays a significant role in preventing L02 cells from apoptosis induced by adriamycin. It is reported that when apoptosis is suppressed by upregulation of antiapoptotic proteins such as Bcl-2 or Bcl-XL, acceleration of tumorigenesis in transgenic mice is observed (Pelengaris et al., 2002). Based on our results, we propose that LAPTM4B-35 contributes to the control of tumorigenesis by regulating expression of antiapoptotic and proapoptotic proteins in the Bcl-2 family and modifying the caspase-dependent apoptosis pathways.
The PI3K/AKT signaling pathway plays an important role in maintaining cell survival and promoting proliferation, and is constitutively activated in a large number of cancers, thereby contributing to tumorigenesis and tumor progression (Nicholson and Anderson, 2002; Stokoe, 2005; Samuels and Ericson, 2006). We have demonstrated that upregulation of LAPTM4B-35 in HeLa cells can markedly activate the PI3K/AKT signaling pathway, which is one of the mechanisms of multidrug resistance in cancer (Li et al., 2010). Consistent with this concept, we demonstrated in this study that upregulation of LAPTM4B-35 promotes phosphorylation/activation of AKT and Bad, a downstream target of activated AKT. Nonphosphorylated Bad is thought to induce cell death, whereas its phosphorylation catalyzed by activated AKT would promote cell survival (Datta et al., 1997). Therefore, our data in this study confirmed that the PI3K/AKT/Bad cascade signaling pathway contributes to survival and apoptosis controlled by LAPTM4B-35.
Survivin is one of the members of the inhibitor of apoptosis family and is expressed in many human cancers, but not in normal tissues (Salvesen and Duckett, 2002). In our study, the expression of survivin in Ad-AE-infected cells was not altered as shown by WB analysis, indicating that the survivin pathway may not be involved in LAPTM4B-35-regulated apoptosis/survival.
Taken together, the findings in this study show that upregulation of LAPTM4B-35 promotes tumorigenesis in L02 cell line. Understanding the roles of LAPTM4B-35 in promoting tumorigenesis may provide novel strategies for cancer therapy.