α4 is highly expressed in carcinogen-transformed human cells and primary human cancers

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Abstract

A regulator of the protein phosphatase 2A (PP2A), α4, has been implicated in a variety of functions that regulate many cellular processes. To explore the role of α4 in human cell transformation and tumorigenesis, we show that α4 is highly expressed in human cells transformed by chemical carcinogens including benzo(a)pyrene, aflatoxin B1, N-methyl-N′-nitro-N-nitrosoguanidine, nickel sulfate and in several hepatic and lung cancer cell lines. In addition, overexpression of α4 was detected in 87.5% (74/80) of primary hepatocellular carcinomas, 84.0% (21/25) of primary lung cancers and 81.8% (9/11) of primary breast cancers, indicating that α4 is ubiquitously highly expressed in human cancer. Functional studies revealed that elevated α4 expression results in an increase in cell proliferation, promotion of cell survival and decreased PP2A-attributable activity. Importantly, ectopic expression of α4 permits non-transformed human embryonic kidney cells (HEKTER) and L02R cells to form tumors in immunodeficient mice. Furthermore, we show that the highly expressed α4 in transformed cells or human tumors is not regulated by DNA hypomethylation. A microRNA, miR-34b, that suppresses the expression of α4 through specific binding to the 3′-untranslated region of α4 is downregulated in transformed or human lung tumors. Taken together, these observations identify that α4 possesses an oncogenic function. Reduction of PP2A activity due to an enhanced α4–PP2A interaction contributes directly to chemical carcinogen-induced tumorigenesis.

Introduction

The protein phosphatase 2A (PP2A) holoenzyme contains the catalytic subunit C, the scaffold subunit A and a regulatory subunit B. The PP2A B subunit targets the AC core dimer to specific substrates and intracellular localizations, playing important roles in the regulation of many cellular processes, including transcription, translation, cell cycle progression, apoptosis, autophagy and cell transformation (Virshup, 2000; Janssens and Goris, 2001; Arroyo and Hahn, 2005; Yorimitsu et al., 2009). Many proteins have been found to form complexes with PP2A subunits and in turn regulate the enzyme activity and biological functions. As a regulatory protein of PP2A, α4 (Tap42 in yeast), encoded by human IGBP1 gene, directly binds to the PP2A catalytic subunit (PP2Ac) by its N-terminal domain (Smetana et al., 2006), altering catalytic activity and substrate specificity (Chen et al., 1998). Binding of α4 to PP2Ac displaces PP2Ac from A and B subunits owing to an overlap in the binding site on PP2Ac (Goldberg, 1999). Crystal structure analysis reveals a scaffolding function for Tap42, which interacts with PP2Ac at its N-terminus, promotes the dephosphorylation of substrates recruited to the C-terminal region of the molecule in Saccharomyces cerevisiae (Yang et al., 2007) and prevents the substrates from dephosphorylation by PP2A (Kong et al., 2009). Unlike the A subunit that binds only to PP2Ac, α4 also binds the related phosphatases PP4 and PP6 (Chen et al., 1998), suggesting that α4 and the PP2A A subunit have distinct functions in determining the substrate specificity or the activity of PP2A.

Although it is estimated that only a small amount of cellular PP2Ac is bound to α4 (Di Como and Arndt, 1996; Murata et al., 1997), α4 plays an essential role in regulating the assembly of PP2A complexes and PP2A activity (Kong et al., 2009). α4 interacts with ubiquitin and mediates PP2Ac polyubiquitination by the ubiquitin ligase Midline 1, which is pivotal in the pathogenesis of Opitz syndrome (Trockenbacher et al., 2001; McConnell et al., 2010), indicating that the α4–PP2Ac interaction leads to the inhibition of PP2A activity. The human IGBP1 gene has been mapped to q13.1–q13.3 on chromosome X (Onda et al., 1997). Genetic knockout of α4 is embryonic lethal in animals and deletion of α4 results in the expression of proapoptotic genes with concomitant apoptosis (Kong et al., 2004), providing strong evidence that α4 is an essential inhibitor of apoptosis. In contrast, overexpression of α4 results in an increase in the GTP-bound state of Ras-related C3 botulinum toxin substrate 1 (Rac1), promoting cell spreading and migration (Kong et al., 2007). Recent studies revealed that α4 is involved in DNA repair processes. α4 deletion results in sustained phosphorylation of a variety of DNA damage response proteins, including p53, histone H2AX and ataxia telangiectasia-mutated kinase (Kong et al., 2009). These observations suggest a role of α4 in cell proliferation. Previously, we showed that perturbation of PP2A activity or displacement of specific regulatory subunits by interaction with oncoproteins led to cell transformation (Chen et al., 2004; Arroyo and Hahn, 2005). However, the mechanism underlying malignant cell transformation remains obscure. Although it has been reported that PP4 is overexpressed in human breast and lung tumors (Ma et al., 2008), little is known with regard to the levels or functions of α4 in human cancers or malignant transformed cells. The understanding of the functions of α4 in malignant cells will help us to discover novel signaling pathways governed by α4–PP2A complexes.

The investigation of mechanisms by which chemical carcinogens transform human cells will lead to a greater understanding of the molecular events that program the malignant state. Previously we have created many human malignant transformed cells induced by various chemical carcinogens (Pang et al., 2008). To explore the role of the α4–PP2A interaction in the initiation of chemical carcinogenesis, in this study we examined the expression of α4 and found it highly expressed in all transformed cells. Furthermore, α4 is remarkably overexpressed in several types of primary human cancers. Functional studies reveal that overexpression of α4 directly contributes to the transformation of human cells.

Results

α4 is upregulated in malignant transformed cells and human tumors

We previously created chemical-transformed human cells that grow in an anchorage-independent manner and form tumors in immunodeficient mice (Pang et al., 2008). To investigate the role of α4–PP2A interaction on cell transformation, we first examined the expression of α4 in these transformed cells. Protein expression of α4 in aflatoxin B1 (AFB1)-transformed human hepatic cells (L02RT-AFB1) and hepatic tumor cell lines (SMMC, HepG2 and Bel7402) is 5- to 16-fold higher than that in non-transformed L02R hepatic cells. In contrast, the expression of PP2A Cα or Aα subunits were not significantly altered (data not shown). Similar results were shown in benzo(a)pyrene (BaP)-transformed human bronchial epithelial cells (HBERT-BaP) as well as lung tumor cells (SK, A549 and H226) when compared with non-transformed HBER cells (Figure 1a). Moreover, human embryonic kidney cells (HEKTER) transformed by BaP, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or nickel sulfate also exhibit high levels of α4 as shown in Figure 1a. These observations suggest that aberrant regulation of α4 is common in chemical-transformed human cells and indicates a role of α4 in human cell transformation.

Figure 1
figure1

α4 is highly expressed in carcinogen-transformed cells and human cancers. (a) Total cell lysates (50 μg) from indicated cell lines were immunoblotted with indicated antibodies. L02R, HEK, HEKTER and HBER cells are non-transformed cells, whereas L02RT-AFB1, HEKRT-BaP, HEKRT-MNNG, HEKRT-NiSO4 and HBERT-BaP are transformed human cells. Bel7402, HepG2 and SMMC are liver tumor cell lines, and SK, A549 and H226 are lung tumor cell lines. Cell lysates (80 μg) from patient specimens were immunoblotted with indicated antibodies. Representative images showed tumor tissues (T) versus adjacent normal tissue (N) in nine pairs of primary hepatocellular carcinoma (b), 10 pairs of primary lung cancers (c) and nine pairs of primary breast cancers (d). The degree of α4 overexpression (T/N) indicated was calculated as the fold of α4 in tumor tissues to the adjacent non-tumor tissues using densitometry analysis after normalized by internal control (β-actin).

To assess whether highly expressed α4 is a marker of malignancy of primary human cancers, we examined the expression of α4 in human cancer specimens and matched adjacent non-tumor tissues. Eighty paired primary hepatocellular carcinoma, 25 paired lung cancers and 10 paired breast cancers were examined by immunoblotting with an α4 antibody. Representative results are shown in Figures 1b–d. Overexpression of α4 was observed in 87.5% (74/80) of hepatocellular carcinoma, 84.0% (21/25) of primary lung cancer and 81.8% (9/11) of primary breast cancers. Clinical and pathological features of hepatocellular carcinoma samples are summarized in Supplementary Table 1. The levels of α4 overexpression is correlated to tumor node metastasis stage (P<0.05), whereas other parameters such as age, gender, tumor size and hepatitis B surface antigen+ did not correlate with the levels of α4 expression (Supplementary Table 1). Taken together, these results indicate that α4 is ubiquitously overexpressed in human cancers and may contribute to spontaneous cancer development.

Effects of suppressing α4 expression

The remarkable upregulation of α4 in chemical carcinogen-transformed cells and primary human tumors prompted us to assess the impact of the α4-containing form of PP2A on the maintenance of malignant phenotypes of transformed cells. First, we generated a vector that drives the expression of a short hairpin RNA that targets α4 (SHα4) or a control vector encoding a short hairpin RNA specific for GFP (SHGFP). By infecting cells with increasing titers of SHα4 retroviruses in HEKTER cells, L02RT-AFB1 cells and the SMMC tumor cell line, we succeeded in generating stable cell lines expressing α4 protein at 50 and 20% of cells expressing a control vector. We named these cells HEKTERSHα4–50, HEKTERSHα4–20, L02RTSH-α4–50, L02RTSHα4–20, SMMCSHα4–50 and SMMC-SHα4–20, respectively. As seen in Figure 2a, suppression of α4 by 50 or 20% did not alter PP2A Cα subunit expression in either immortal or transformed cells. However, suppression of α4 resulted in increased PP2A Aα subunit protein levels in transformed cells (Figure 2a). Particularly, we observed that cells expressing low levels of α4 (HEKTERSHα4–20 cells) proliferated poorly. To determine whether these cells were apoptotic, we employed co-staining cells with Annexin V and propidium iodide. We found that 21.4% of the HEKTERSHα4–20 cells stained for Annexin V, whereas only 5.0% of HEKTERSHGFP and 4.5% of HEKTERSHα4–50 cells stained for Annexin V (data not shown). These findings indicate that suppression of α4 leads to apoptosis, consistent with previous observations from Drosophila (Bielinski and Mumby, 2007) and murine cells (Kong et al., 2004).

Figure 2
figure2

Effects of suppressing α4 expression. (a) Dose-dependent suppression of α4 expression was achieved by manipulating the titer of the SHα4 retrovirus. HEKTER, L02RT and SMMC cells were infected with different titers of SHα4 retrovirus, generating stable cell lines, SH-GFP, SHα4–50 and SHα4–20, respectively. Values reported represent the degree of α4 suppression in cells expressing SHα4. Expression of the indicated proteins was determined by immunoblotting with antibodies specific for PP2A Aα, Cα subunits and α4. (b) The effect of suppression of α4 on cell growth. A total of 1 × 104 cells were plated and counted at the time points indicated. Data are expressed as mean±s.e.m. for three independent experiments. (c) PP2A-attributable phosphatase activity in α4-suppressed cells. Cell lysates (500 μg) were subjected to immunoprecipitation with PP2Ac-specific antibody. PP2A activity from PP2Ac immune complexes was determined by measuring the release of phosphate from the substrate phosphopeptide (RRA(pT)VA) and defined as pmol free PO4 generated per μg protein per min. (d) Effect of α4 suppression on anchorage-independent cell growth. A total of 1 × 105 cells indicated were plated in soft agar and counted 28 days after seeding. Data were presented as mean±s.e.m. for three independent experiments.

In contrast to HEKTERSHα4–20 cells, cells in which α4 is suppressed to approximately 50% of wild-type levels proliferate 45% more slowly than control cells (Figure 2b). Meanwhile, we found that HEKTERSHα4–50 cells exhibited an increase in PP2A-attributable phosphatase activity by 30%. Similar results were found in L02RT-AFB1 and SMMC cells in which α4 levels were suppressed approximately by 50% of the controls (Figure 2c). In addition, such cells showed a decrease in cell proliferation (Figure 2b) and failed to grow in soft agar (Figure 2d).

Collectively, these results confirm that α4 is required for cell survival. Partial depletion of α4 increases PP2A phosphatase activity and suppresses cell proliferation.

Elevated α4 expression contributes directly to cell transformation

To assess the effects of high expression of α4 on cell transformation, we introduced an expression vector carrying a wild-type version of α4 or a control vector into the non-transformed human cell lines L02R and HEKTER, generating stable cell lines L02R-α4 and HEKTER-α4. As shown in Figure 3a, the levels of α4 in L02R-α4 and HEKTER-α4 cells are three- to fourfold higher than cells expressing control vectors. Ectopic expression of α4 promotes increased cell growth by 40% in L02R-α4 cells and 28% in HEKTER-α4 cells (Figure 3b). In addition, high level of α4 resulted in a 30–35% decrease in PP2A-attributable phosphatase activity in L02R-α4 and HEKTER-α4 cells (Figure 3c), confirming that the interaction of α4 with PP2Ac leads to the inhibition of enzyme activity. Also, we observed a greater amount of α4 bound to PP2Ac in L02RT-AFB1 cells compared with L02R cells by immunoprecipitation with antibody against PP2Ac (Figure 3d). Moreover, ectopic expression of α4 conferred resistance to chemical-induced cell death as L02R-α4 cells exhibited increased cell viability relative to the control upon exposure to the DNA-damaging reagents camptothecin or MNNG (Supplementary Figure 1a). Similar results were found when L02R-α4 cells were treated with the phosphatidylinositol 3-kinase inhibitor wortmannin or the mammalian target of rapamycin (mTOR) inhibitor rapamycin, particularly under low serum conditions (Supplementary Figure 1b and c). More interestingly, ectopic expression of α4 in L02R cells or HEKTER cells led to cell transformation as shown by growth in an anchorage-independent manner and formation of tumors in immunodecient mice (Figures 3e and f). These findings show that high expression of α4 induces malignant transformation of human cells.

Figure 3
figure3

Elevated α4 expression contributes directly to cell transformation. (a) Immunoblotting analysis of α4, Aα and Cα at L02R and HEKTER cells expressing wild type of α4. (b) The effect of upregulation of α4 on cell proliferation. (c) PP2A-attributable phosphatase activities in L02R-α4 and HEKTER-α4 cells. The results are expressed as the mean±s.e.m. for three experiments. (d) Immunoprecipitation was performed on 3 mg of whole-cell lysates with a PP2A Cα-specific antibody, followed by immunoblotting using specific antibodies against PP2A Aα, α4 and Cα. Representative results of three independent experiments are shown. (e) Effect of α4 overexpression on anchorage-independent cell growth and tumor formation in immunodeficient mice. Transformed cells L02RT-AFB1 and HEKTERT-BaP were served as positive controls. Data were presented as mean±s.e.m. For tumor formation, 2 × 106 cells were subcutaneously injected into immunodeficient mice. Data are reported as the number of tumors formed per number of injection sites.

α4 expression was not controlled by DNA methylation

To explore why α4 was upregulated in transformed cells or human cancers, we first examined carcinogen-transformed cells and 10 paired human lung tumors in which α4 were highly expressed for mutations in the α4 gene. However, no mutations in the coding region of α4 in these cells or primary lung tumors were identified by direct sequencing (data not shown).

Next, we speculated that hypomethylation of CpG islands of the α4 gene might be responsible for α4 overexpression. To test this hypothesis, we analyzed the status of α4 DNA methylation in carcinogen-transformed cells and 10 paired human lung cancer specimens using bisulte sequencing. Using the UCSC Genome Browser, we located CpG islands at positions −694 to 106 relative to the transcriptional start of the α4 gene. However, we did not find any difference in the status of DNA methylation of this α4 promoter between the transformed cells and control cells or between lung cancer tissues and matching non-tumor tissues (data not shown). To further eliminate the possibility that α4 expression was controlled by DNA methylation, we generated stable cells, L02SHGFP and L02SHDNMT1, by infecting L02R cells with vectors carrying SHGFP or a short hairpin RNA against the DNA 5′-cytosine-methyltransferase 1 (SHDNMT1). As seen in Figure 4a, we did not observe alteration of α4 expression at the RNA or protein level in L02SHDNMT1 cells, despite a 50% reduction in DNMT1 expression. These results were further confirmed when we treated the transformed L02RT-AFB1 with the demethylating reagent 5-aza-deoxycytidine-CdR for 72 h (Figure 4b) and found no difference of α4 mRNA levels in transformed or tumor cells (Figure 4c) or 10 paired primary lung tumors (Figure 4d) in comparison to their counterparts. Collectively, these observations show that overexpression of α4 is not the consequence of DNA hypomethylation.

Figure 4
figure4

α4 expression was not controlled by DNA methylation. (a) Immunoblotting analysis of α4 in L02 cells transfected with vectors carrying SHGFP or SHDNMT1. (b) RT–PCR and immunoblotting were performed to analyze the levels of mRNA and protein of α4 in L02 and L02RT-AFB1 cells with or without treatment of 5 μM 5-aza-deoxycytidine. The levels of mRNA and protein of α4 in cell lines L02, L02RT-AFB1 and SMMC (c) or in 10 pairs of primary lung cancer (T) and adjacent normal tissues (N), respectively (d).

α4 expression regulated by miR-34b

Given the results that overexpression of α4 is not the outcome of gene mutation or DNA hypomethylation, we sought to determine whether downregulation of specific microRNA (miRNAs) is involved in the regulation of α4 expression. As predicted by TargetScan algorithms, miR-34b was identified as a potent regulator of α4 expression. Previously, downregulation of miR-34b had been reported in non-small-cell lung cancer and leukemia, suggesting a role of miR-34b in tumor suppression (Bommer et al., 2007; Corney et al., 2007; Pigazzi et al., 2009). We determined the levels of miR-34b in 10 paired primary lung tumors by quantitative reverse transcription–PCR and found that miR-34b expression was downregulated in nine out of 10 paired primary lung cancers. In addition, the levels of α4 protein were found to be inversely correlated to miR-34b expression in nine out of 10 pairs of lung tumors (Figure 5a). Moreover, the transfection of an miR-34b mimic into L02RT-AFB1 cells and tumor cell line SMMC led to a decrease of α4 expression by 50% compared with cells transfected with mimic of normal control (Figure 5b), suggesting that miR-34b targets α4 mRNA for degradation.

Figure 5
figure5

α4 expression is regulated by miR-34b. (a) Expression of miR-34b in 10 paired primary lung cancer tissues (T) and adjacent non-tumor tissues (N) was analyzed by real-time RT–PCR, and α4 protein expression was assessed by western blot analysis. (b) Control RNA duplex (NC) and miR-34b mimic were transfected into L02, L02RT-AFB1 and SMMC cells, respectively, followed by immunoblotting with specific antibodies against α4. (c) The seed region (147–153) of miR-34b and putative binding site on the 3′-UTR of α4. The mutant generated at the α4 3′-UTR complementary to the seed region of miR-34b is underlined and serves as a control. (d) An α4 3′-UTR fragment containing wild type or mutant of the α4 binding sequence was cloned downstream of the luciferase reporter gene. A549 cells were co-transfected firefly luciferase reporter containing either wild-type or mutant α4 3′-UTR with miR-34b or control duplex and pRL-TK. The normalized luciferase activity for the cells indicated was shown as relative luciferase activity.

We further assessed whether miR-34b suppressed α4 expression through specific binding to the 3′-untranslated region (UTR) of α4 mRNA, using a dual-luciferase reporter system. A firefly luciferase reporter containing a wild-type α4 3′-UTR or an α4 3′-UTR mutated at the miR-34b seed region (Figure 5c) was co-transfected with a Renilla luciferase reporter and with either an miR-34b or normal control duplex. Co-transfection of miR-34b with the wild-type α4 3′-UTR significantly inhibited firefly luciferase activity by 28%, indicating that specific binding existed. In contrast, miR-34b failed to bind to the mutated 3′-UTR and exhibited no inhibitory effect on luciferase activity (Figure 5d). These observations show that miR-34b negatively regulates α4 expression.

Discussion

Chronic exposure to exogenous chemical carcinogens is fundamental for the initiation of most human cancers. Chemical carcinogenesis is a multistage and multifactor process that progressively converts normal cells into malignant cancer cells (Irigaray and Belpomme, 2010). Previously, we developed a series of human cell transformation models induced by a variety of chemical carcinogens (Pang et al., 2008). Using these cell models, we evaluated the potential of both genotoxic and non-genotoxic carcinogens within a relatively short time period and revealed an aberrant epigenetic pattern in carcinogen-transformed cells (Ji et al., 2008). In this study, we presented that α4 was highly expressed in all carcinogen-transformed cells examined and in more than 80% of human cancer tissues of different origins, suggesting that α4 might be involved in carcinogenesis. The hypothesis that α4 exerted an oncogenic function was supported by observations that elevated α4 promoted cell survival, stimulated cell proliferation and increased colony formation in vitro. Importantly, the tumor-promoting potential of α4 was further shown in vivo, in which ectopic expression of α4 allowed cells to grow in an anchorage-independent manner and form tumors in immunodeficient mice. These observations show that α4 promotes spontaneous or chemical-driven human cancer development through functioning as a negative regulator of PP2A activity owing to an enhanced α4–PP2A interaction.

Alterations in PP2A function have been implicated in human cell transformation and cancer development (Arroyo and Hahn, 2005; Mumby, 2007; Westermarck and Hahn, 2008). Many cellular PP2A-interacting proteins, including PP2A regulators and substrates, contribute to the specificity of PP2A signaling and the complexity of its regulation (Janssens and Goris, 2001). Accumulating evidence indicates that PP2A acts as a tumor suppressor (Mumby, 2007; Westermarck and Hahn, 2008; Eichhorn et al., 2009). Previously, we showed that SV40 ST induces cell transformation by disrupting PP2A complexes containing B56γ (Chen et al., 2004) and identified both α and β isoforms of PP2A A subunits as tumor suppressors (Chen et al., 2005; Sablina et al., 2007). A recent study shows that a mutation at PP2A B56γ subunit in lung cancer disrupts the p53-dependent function of PP2A (Shouse et al., 2010). In addition to mutations, alterations in the expression levels of PP2A regulatory subunits are also found in human cancers. Reduction of PP2A Aα expression was found in 43% of primary human gliomas (Colella et al., 2001) and downregulation of B56γ was observed in human primary melanoma specimens (Deichmann et al., 2001). In addition, three subunits of the type 2A group of phosphatases, PP4 (PP4C, PP4R2 and PP4R3), are highly expressed in human primary breast and lung tumors (Wang et al., 2008). In this study, we found that overexpression of α4 is present not only in chemical carcinogen-transformed cells, but also in most human primary tumors, indicating that overexpression of α4 is ubiquitous and essential in tumorigenesis. The findings that the levels of α4 protein were correlated with the grade of malignancy in hepatocellular carcinoma indicate that α4 might be a marker of prognosis and could be used as a therapeutic target. Collectively, these data provide compelling evidence that deregulation of PP2A in cancer is mostly achieved by the loss of PP2A function resulting from PP2A subunit mutations or the aberrant expression of PP2A regulators.

A previous study revealed that α4 regulates the ubiquitination of PP2Ac and alters catalytic activity (Trockenbacher et al., 2001). In this study, we found that phosphatase activity of PP2A in transformed cells is decreased and suppression of α4 leads to partial restoration of this phosphatase activity. The observation that a greater amount of α4 binds to PP2Ac in transformed cells further show that highly expressed α4 negatively regulates the activities of PP2A, consistent with a recent study showing that α4 associates with C subunits rendering them enzymatically inactive (Kong et al., 2009). In addition to the suppression of PP2A-attributable enzyme activity, ectopic expression of α4 in HEKTER cells resulted in an acceleration of cell proliferation and resistance to chemical-induced cytotoxicity. Notably, ectopic expression of α4 in HEKTER cells led to cell transformation, providing strong evidence that α4 possesses oncogenic activity. This notion is supported by a previous report that the growth of transformed cells in soft agar was enhanced by elevated α4 and attenuated by α4 suppression (Prickett and Brautigan, 2007). Collectively, these observations show that high expression of α4 plays a causal role in human tumorigenesis.

Individual roles of the PP2A holoenzymes and their specific roles in cancer-associated signaling pathways, including mitogen-activated protein kinase and WNT signaling, have been dissected (Eichhorn et al., 2009). Recent study reveals that α4 is required to maintain a basal dephosphorylated state of a wide variety of proposed PP2A targets (Kong et al., 2009). The formation of the α4–PP2Ac complexes can be stimulated by mTOR, a highly conserved protein that controls cell growth in response to mitogens and changes in cellular metabolism (Wullschleger et al., 2006). The effects of mTOR on cell growth involve the phosphorylation of S6K and 4E-BP1 (Inui et al., 1998). We found that high expression of α4 not only led to the activation of mTOR signaling as presented by sustaining phosphorylation of S6K1 and 4E-BP1 in transformed and human tumor cells, but also rendered transformed cells resistant to rapamycin suppression of the mTOR pathway (data not shown). As the mTOR pathway is frequently hyperactivated in a number of human malignancies (Dowling et al., 2010) and rapamycin homologs are tested as anticancer drugs in a large variety of tumors, yielding promising results in clinical trials (Bjornsti and Houghton, 2004; Gibbons et al., 2009; Meric-Bernstam and Gonzalez-Angulo, 2009), we propose that α4 may be an attractive target for cancer therapy.

The remarkable upregulation of α4 in carcinogen-transformed cells and most human tumors might result from dysregulation at the transcriptional and/or post-translational levels. In our attempt to understand the dysregulation of α4 expression, we first explored whether hypomethylation of CpG islands existed and was responsible for highly expressed α4 in transformed cells. As a result, we failed to show that the status of DNA methylation correlated to α4 expression. In addition to methylation analysis, we found no mutations in the α4 coding sequence in transformed and lung cancer cells. However, we identify that α4 is a direct target of miR-34b. Reduction of miR-34b expression was observed in transformed and primary human lung cancers. Our observations are consistent with a previous study showing that miR-34b was downregulated in non-small-cell lung cancers and lymphocytic leukemia (Corney et al., 2007). Although we elucidated a role of miR-34b on the regulation of α4 expression in this study, we speculate that other post-translational mechanisms might participate in the control of α4 expression. α4 has been shown to serve as an adaptor protein to bind directly to Midline 1, which is responsible for ubiquitination of PP2Ac (Liu et al., 2001). Moreover, α4 interacts with isolated by differential display (EDD) E3 ubiquitin ligase independent of the α4–PP2Ac interaction (McDonald et al., 2010). Further studies are required to address whether the stability of α4 protein is regulated by other post-translational modifications, including ubiquitination, phosphorylation and glycosylation.

In this study, we show a causal role of α4 in chemical carcinogenesis. The decrease in PP2A-attributable phosphatase activity mediated by increasing the α4–PP2Ac interaction promotes malignant cell transformation. Adding to previous observations that α4 is an essential inhibitor of apoptosis, it is clear that α4 is oncogenic in human cells. Protein phosphatases are being recognized as promising therapeutic targets, and our results suggest that α4 may be one such promising target.

Materials and methods

Tissue samples

Primary lung/liver/breast tumors and paired adjacent non-tumor tissues were obtained from the Department of Thoracic Surgery, Sun Yat-Sen University Cancer Center and the Department of Thoracic Surgery, the Affiliated Cancer Hospital of Guangzhou Medical University, Guangzhou, China. Patients were undergoing tumor operation without any anticancer treatment. Tissue samples were collected in freezing tubes and preserved in liquid nitrogen until protein/DNA/RNA extraction. Informed consent from each patient and the study were approved by the institute research ethics committee of the hospital.

Cell lines and establishments of stable cell lines

The human embryonic kidney cells expressing Simian virus 40 LT antigen (LT) and the telomerase catalytic subunit (hTERT) (HEK cells), or expressing LT, hTERT and G12V H-Ras (HEKTER cells) were gifts from Dr WC Hahn (Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA). The HBE was a gift from Dr DC Gruenert (University of California, San Francisco, CA, USA) and human hepatocytes (L02) was purchased from Cell Bank, Chinese Academy of Sciences, Shanghai, China. Stable HBER cells and L02R were generated by infection with amphotropic retroviruses carrying H-Ras as described previously (Pang et al., 2008). The human hepatic tumor cell lines, SMMC, HepG2 and Bel7402 and lung tumor cell lines, SK, A549 and H226, were obtained from the American Type Culture Collection (Manassas, VA, USA). AFB1-induced transformed cells (L02RT-AFB1), BaP-transformed human bronchial epithelial cells HBERT-BaP, HEKTER cells transformed by BaP (HEKRT-BaP), MNNG (HEKRT-MNNG) and nickel sulfate (HEKRT-NiSO4) were generated as described previously (Pang et al., 2008). The pWZL-α4 plasmid was constructed by reverse transcription–PCR (the sequences of primers are provided in Supplementary Table 2). pLKO-SHα4 and control pLKO-GFP vectors were provided by Dr WC Hahn.

Cell proliferation

A total of 2 × 104 cells were plated in triplicate and harvested at the indicated time. The number of cells was determined by a Z2 Particle Count and Size Analyzer (Beckman-Coulter, Miami, FL, USA).

Immunoblotting

The frozen tissue samples were pulverized by mortar and pestle in liquid nitrogen, and reconstituted in ice-cold radioimmunoprecipitation assay lysis buffer (150 mmol/l NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecylsulfate and 50 mmol/l Tris (pH 7.4)) containing protease inhibitors. The lysates were centrifuged at 12 000 g for 20 min at 4 °C. For the analysis of α4 levels, cells were lysed directly on the plate using 2 × sodium dodecylsulfate sample buffer (125 mM Tris-base, 138 mM sodium dodecylsulfate, 10% β-mercaptoethanol, 20% glycerol, bromophenol blue (pH 6.8)). Soluble proteins (50 μg) were subjected to 8–16% gradient acrylamide gel for sodium dodecylsulfate–polyacrylamide gel electrophoresis before immunoblotting. Antibodies used include α4 (rabbit monoclonal; Novus, Littleton, CO, USA), PP2A Cα (clone 1D6; Upstate Biotech, Lake Placid, NY, USA), Cα (BD Biosciences, San Diego, CA, USA) and Aα (clone 6F9) (Covance, Richmond, CA, USA).

Protein phosphatase activity

The protein phosphatase activity in PP2A C immune complexes was determined as described previously (Chen et al., 2004).

Vector construction and luciferase reporter assay

To create pGL3cm-α4 3′-UTR-WT, cDNA (100 ng) from HEK cell line served as template to amplify α4 3′-UTR (1–340 nt, NM_001551) and cloned into the EcoRV and XbaI sites downstream of the luciferase reporter gene in pGL3cm plasmid (the sequences of primers are provided in Supplementary Table 2). pGL3cm-α4 3′-UTR-MUT, which carried the mutated sequence at the seed region of miR-34b indicated in Figure 5c.

For luciferase reporter assay, L02RT-AFB1 cells grown in a 96-well plate were co-transfected with 10 nM of either normal control (control RNA duplex) or miR-34b mimic (RIBOBIO, Guangzhou, China), 10 ng of firefly luciferase reporter comprising wild-type or mutant 3′-UTR of α4, and 2 ng of pRL-TK (Promega, Madison, WI, USA). Cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was detected by M200 microplate fluorescence reader (Tecan). The pRL-TK vector that provided the constitutive expression of Renilla luciferase was co-transfected as an internal control to correct the differences in both transfection and harvest efficiencies. Transfections were carried out in duplicates and repeated at least three times in independent experiments.

Bisulte sequencing

Genomic DNA from cell lines or tissue samples was isolated with the TaKaRa Universal Genomic DNA Extraction Kit. DNA (2 μg) was bisulte converted using previously published methods with minor modications (Zhang et al., 2009). In brief, α4 promoter sequences were amplied from bisulte-converted DNA by PCR (the sequences of primers are provided in Supplementary Table 2). The predicted fragments were purified and subcloned into pMD19-T vector (TaKaRa). Positive clones were selected by PCR using TA primer and electrophoresis to verify incorporation of insert of the correct sequences. Individual clones were sequenced by ABI PRISM 3130XL2 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). For each sample, at least 10 clones were sequenced to identify methylated cytosine residues.

Soft agar assay

A total of 1 × 105 cells were plated in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum in 0.4% agar above a layer of 0.6% agar in 6 cm2 dish. After 4 weeks, colonies were visualized and counted. All experiments were carried out in triplicate and repeated three times.

Tumorigenicity assay

A total of 2 × 106 cells were injected subcutaneously into BALBnu/nu mice at 4–6 weeks old. The number of tumors formed was determined at 40 days after injection. The mice were purchased from Experimental Animal Center of Guangdong (Guangzhou, China). All the procedures herein were approved by the Animal Care and Use Committee of Sun Yat-Sen University.

Statistical analysis

Data were presented as the mean±standard error (s.e.) from at least three separate experiments. The differences between groups were analyzed using SPSS 13.0. Unless otherwise indicated, the differences between two groups were analyzed using the Student's t-test, and the differences among more than two groups were assessed using a one-way analysis of variance, followed by a Mann–Whitney test. Differences were considered statistically significant at P<0.05.

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Acknowledgements

We thank Dr Richard Possemato for critical reading of the manuscript. This work was supported by a Distinguished Young Scholar of NSFC (30925029), Key Program of NSFC (30630055) and NSFC (30800930, 30771832, 30901211), National Key Basic Research and Development Program (2010CB912803), National High Technology Research and Development Key Program of China (2008AA062504), Ministry of Health of China (200902006), the Fundamental Research Funds for the Central Universities (10ykjc05 and 10lgzd10) and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme GDUPS (2010).

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Correspondence to W Chen.

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Supplementary Information accompanies the paper on the Oncogene website

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Keywords

  • α4
  • protein phosphatase 2A
  • cell transformation
  • human cancers

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