1.Introduction
c-MYC proto-oncogene is involved in the control of cell cycle progression, proliferation, metabolism, and apoptosis [1,2]. The expression of c-MYC protein is tightly controlled during cell proliferation and differentiation [1,2]. c-MYC is one of the most frequently altered genes in human cancerand deregulation of c-MYC is constantly implicated in tumorigenesis [1,3,4]. Many c-MYC target genes were identified which are linked to proliferation, transformation, and metabolism [2]. Although many c-MYC target genes were identified, genes responsible for inducing transformation still remain to be explored.
Heat shock protein 60 (HSP60) is the major component of the chaperonins complex which weighs nearly a million Daltons and is composed of back-to-back rings of identical or closely related rotationally symmetric subunits [5,6]. HSP60 plays an essential role in assisting a large variety of newly synthesized proteins to reach their native forms by binding and facilitating their folding into native status inside a large central channel within each ring, as demonstrated in the bacterial GroEL-GroES-ATP complex [5-7]. However, folding of native proteins by a single domain was demonstrated in the mammalian HSP60 protein [8], which is different from the bacterial model [5-7]. HSP60 was mostly shown to be involved in innate immunity and cardiac diseases [9,10]. Increased HSP60 expression is observed in different types of human cancers (ovarian cancer, pancreatic cancer, large bowel carcinoma, etc) [11-13]. However, the role of HSP60 in tumorigenesis remains relatively unknown.
In this report, we demonstrated the tight correlation between c-MYC and HSP60 expression and direct regulation of HSP60 expression by c-MYC through an E-box site located in the proximal promoter of the HSP60 gene. Overexpression of HSP60 induces transformation in a Rat1a cell line. siRNA (short-interference RNA) mediated repression of endogenous HSP60 expression in RatMyc cells causes a significant decrease in transformation. These results indicate that activation of HSP60 expression by c-MYC is critical for c-MYC mediated transformation.
2.Materials and Methods
2.1.Cell lines, plasmids, and reagents
The lymphoblastoid cell lines (CB33 control, CBMyc.Max, and CBMax), U937, EREB.TCMyc, Rat.MycERTM, RatMyc, 293TMyc-si and HeLaMyc-si cell lines were previously described [14,15]. The construction of different plasmids including the vector backbones, restriction sites, inserted fragments, and the oligonucleotides used in the construction of plasmids were described (Supplementary Tables 1 & 2). The generation of stable clones by calcium phosphate transfection was described [14]. All the stable clones were established by transfection of plasmids as designated by the name of the clones. The construction of pSUPER-siRNA plasmids were performed using the oligonucleotides as described (Supplementary Table 3). All the siRNA stables clones were designated by the suffix “-si”. 4-Hydroxytamoxifen and cycloheximide were previously described [14].
2.2.RNA purification, Northern blot analysis, protein extraction, and Western blot analysis
RNA purification and Northern blot analysis were previously described [14-16]. The HSP60 probe was generated from the full length cDNA of HSP60 (Supplementary Table 1). The -actin, GAPDH, vimentin, c-MYC exon 1 and 3 probes were described [14-16]. The extraction of proteins from cells and Western blot analysis were performed as described [14,15]. The characteristics of the antibodies used were listed (Supplementary Table 4). Data shown here are representative of two or more experimentsfrom independent cell cultures.
2.3.Cloning of the human HSP60 promoter region, generation of HSP60 promoter-driven reporter constructs, transient transfection, and luciferase assays
The HSP60 promoter region (-541 to +16 bp surrounding the transcription start site) was cloned by PCR amplification of 293T genomic DNA and it was inserted into the HindIII/BglII sites of the pXP2 vector to generate the pHSP60PWLuc parental construct (Fig. 2B) [14,16]. The pHSP60PMLuc mutant construct was made by site-directed mutagenesis of the pHSP60PWLuc vector (Fig. 2B). The reporter constructs were co-transfected into 293T cells with pMT2T, pMT2TMyc, or its deletion mutants [14-16]. A pCMV--gal plasmid was used as an internal control. The luciferase activities were assayed as described [14].
2.4.Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed as described [15]. Briefly, cells were cross-linked and harvested. The lysates were incubated with no antibody or antibodies specific for Rel or c-MYC. The DNA fragments were extracted and resuspended for PCR analysis. The PCR reaction generated a 220 bp product from HSP60 proximal promoter (-550 to -331 bp) containing E-box (-523 to -518 bp), two 220 bp products from the HSP60 intron 1 region containing E-boxes(+232 to +237 bp, +460 to +465 bp), or a 265 bp product from the distal promoter region (-1844 to -1580 bp) without E-box. The regions in the HSP90A gene promoter which were used in the control ChIP experiments were described [17]. The primers and antibodies used in the ChIP assays were listed (Supplementary Tables 3 & 4).
The stable clones were plated at three different cell density (5x103, 104, 2x104) using standard assay conditions as mentioned except that 15% fetal calf serum was used [15,17]. Data shown here are representative of two or more experiments from independent cell cultures.
3.Results
3.1.Correlation between c-MYC and HSP60 expression
To identify c-MYC target genes involved in transformation, we screened a cDNA microarray filter (Atlas Human array, Clontech) using cDNA probes generated from a previously characterized B cell line (EREB.TCMyc) [14-17]. Among numerous genes overexpressed in c-MYC expressing cells, we focused on the gene HSP60 due to the increased HSP60 expression in different tumor types [11-13]. Northern blot analysis showed that withdrawal of estrogen and tetracycline (causing induction of exogenous c-MYC) caused a 2-fold increase in the expression of HSP60 (Fig. 1A). HSP60 expression significantly increased (~2.5 fold) in CBMyc.Max cells when compared to the control lymphoblastoid cells (CB33 control) or CBMax cells (Fig. 1B). Western blot analysis showed that the increase in HSP60 protein expression also coincided with the increase in its mRNA levels (Fig. 1C). To confirm this correlation in different cell types, mRNA levels of HSP60 were shown to decrease in U937 cells induced into differentiation by TPA, which correlated with the decline in c-MYC expression (Fig. 1D). Rat HSP60 protein levels also increased (~2 fold) in RatMyc cells overexpressing c-MYC compared with the control RatCMV cells (Fig. 1E). To test whether HSP60 expression is regulated by endogenous c-MYC, we performed siRNA experiments on 293T and HeLa cells to knockdown endogenous c-MYC expression [15,17]. The results
showed that endogenous c-MYC expression was repressed in the 293TMyc-si and HeLaMyc-si clones followed by a corresponding decrease in the HSP60 protein levels compared to the control 293T and HeLa clones (Fig. 1F, G), demonstrating the regulation of HSP60 by endogenous c-MYC. All the results demonstrated the tight correlation between c-MYC and HSP60 expression.
3.2.Direct regulation of HSP60 expression by c-MYC
To investigate whether c-MYC-mediated upregulation of HSP60 expression was direct, we studied HSP60 expression in a Rat.MycERTM cell line previously described [14,15,17].
By treating the cells with 4-hydroxytamoxifen (TM), which specifically activates MycERTM,
and by simultaneous treatment with cyclohexamide (CX) to inhibit new protein synthesis, Fig. 2A showed that TM treatment led to a significant (~4 fold after 4 hours) and rapid (detectable after 1/2 hour) increase in HSP60 mRNA levels (left panel). This induction cannot be abolished by co-treatment with CX (CX+TM) (Fig. 2A, middle panel), indicating that induction of HSP60 expression involves a mechanism that is independent of de novo protein synthesis in the presence of MycER proteins and is consistent with a direct effect of c-MYC. To ascertain whether c-MYC binds to the HSP60 promoter, we investigated whether the HSP60 promoter contains c-MYC binding sites. An E-box site (CACGTG) was identified ~1.5 kb upstream from the HSP60 translation start site (ATG) (Fig. 2B). A reporter vector
containing the HSP60 promoter with the E-box site (pHSP60PWLuc; Fig. 2B) was then co-transfected with either the control pMT2T or the pMT2TMyc expression vector into 293T cells. Fig. 2C showed that c-MYC was able to activate the expression of pHSP60PWLuc about 2.5-fold. However, c-MYC was not able to activate the pHSP60PMLuc construct whose E-box was mutated to CACCTG (Fig. 2B,C), indicating that the E-box in the HSP60 promoterresponded to transcriptional activation by c-MYC. In addition, the c-MYC mutants lacking the transactivation domain [pMT2TMyc(7-91)] or heterodimerization domain [pMT2TMyc(371-412)] were defective in activating the expression of pHSP60PWLuc (Fig. 2D). However, the Myc BoxII deletion mutant [pMT2TMyc(122-140)] was able to activate the expression of pHSP60PWLuc (Fig. 2D). These results indicated that c-MYC-mediated activation of HSP60 transcription required both the transactivation and heterodimerization domains, but not the Myc BoxII domain [18]. To determine whether c-MYC binds to the E-box in vivo, a chromatin immunoprecipitation (ChIP) assay using CBMyc.Max cells was performed (Fig. 2E under the CBMycMax column). The results showed that c-MYC, but not c-Rel, bound to the E-box as demonstrated by the c-MYC immunoprecipitated chromatin amplifiable to generate a 220 bp fragment (depicted in Fig. 2B and labeled as E-box (promoter) in Fig. 2E). Control experiment to amplify another HSP60 promoter fragment (265 bp as depicted in Fig. 2B) which did not contain an E-box failed to generate an amplifiable fragment (labeled as control in Fig. 2E). The 220 bp fragment was also amplifiable from the
c-MYC immunoprecipitated chromatin prepared from EREB.TCMyc(+TC/+E2) cells, demonstrating the binding of endogenous c-MYC to the E-box site (Fig. 2E under the EREB.TCMyc(+TC/+E2) column). In addition, the 220 bp fragment was not amplifiable in c-MYC immunoprecipitated chromatin prepared from CB33 control cells or quiescent EREB.TCMyc(+TC/-E2) cells (Fig. 2E under the CB33 or the EREB.TCMyc(+TC/-E2) column). A positive control experiment using the E-box located in the HSP90A gene promoter [17] showed a similar intensity of amplified PCR fragment by ChIP experiment (labeled as E-box in Fig. 2E under the HSP90A section). Taken together, these results indicated that c-MYC directly activates HSP60 transcription by binding to the E-box site located in the HSP60 proximal promoter.
3.3.Induction of transformation by HSP60 in a Rat1a cell line
To test whether HSP60 contributes to transformation activity caused by c-MYC, Rat1a stable clones overexpressing HSP60 were generated (Fig. 3A). Soft agar colony formation assay was performed. The results showed that Rat1a clones overexpressing HSP60 (RatHSP60) had increased soft colony formation activity compared to the control Rat1a clones (RatCMV) (Fig. 3B), supporting the role of HSP60 in the induction of transformation mediated by c-MYC.
To determine whether the role of HSP60 is critical in the induction of transformation caused by c-MYC, RatMyc cells were transiently transfected with pSUPER-rhsp60i plasmid to repress endogenous HSP60 (Fig. 4A). Soft agar colony formation assays were performed comparing the RatMyc cells receiving siRNA against HSP60 vs. the control RatMyc clone. The results showed that there was a significant decrease in transformation activity in RatMyc cells receiving siRNA against HSP60 (Fig. 4B), demonstrating the critical role of HSP60 in the induction of transformation by c-MYC. Control experiments using pSUPER plasmids expressing mismatched siRNA rhsp60iM) or scrambled siRNA (pSUPER-scrambled) did not cause a decrease in HSP60 protein levels (Fig. 4C) or transformation activity of RatMyc cells (Fig. 4D), supporting the specificity of pSUPER-rhsp60i plasmid used to repress endogenous HSP60 in RatMyc cells. These results demonstrated the critical role of HSP60 in c-MYC mediated transformation.
4.Discussion
Collectively, our results demonstrated that HSP60 expression is regulated by c-MYC and overexpression of HSP60 induces transformation. HSP60 was shown to be a putative c-MYC target gene [19-23]. Our detailed characterization using different cell lines and identification of an E-box site in the proximal promoter of the HSP60 genes further demonstrated that HSP60 is a direct c-MYC target gene. Although the Myc BoxII deletion was shown to impair most of the transactivation activity mediated by wild type Myc, it was shown that a small proportion of c-Myc target genes could still be activated by Myc BoxII deletion mutant [18]. From our results, it was possible that HSP60 may belong to this kind of target genes not requiring the transactivation function of Myc BoxII domain (Fig. 2D). Soft agar colony formation assays supported its possible role in c-MYC mediated transformation. siRNA mediated repression of HSP60 in RatMyc cells significantly decreased the transformation activity. HSP60 activation may be a crucial component of the program by which constitutive c-MYC expression induces transformation.
Increased HSP60 expression is observed in different types of human cancers (ovarian cancer, pancreatic cancer, large bowel carcinoma, etc) [11-13]. However, the role of increased HSP60 expression in tumorigenesis is relatively unknown. Our demonstration that overexpression of HSP60 induces soft agar colony formation in a Rat1a cell line is consistent with its role to contribute to transformation and tumorigenesis in different types of human
cancers. Recently, HSP60 was shown to stabilize mitochondrial survivin and inhibit apoptosis in tumor cells, which also supports the tumorigenic role of HSP60 [24]. HSP60 was mostly shown to be involved in innate immunity and cardiac diseases [9,10] in contrast to our demonstration of its role in transformation. Taken together, our results demonstrated the direct regulation of HSP60 expression by c-MYC and the critical role of HSP60 in c-MYC mediated transformation.
Acknowledgements
Part of the initial characterizations of HSP60 was performed in R. Dalla-Favera’s lab. This work was supported in part by National Science Council (2311-B-010-007, NSC97-2320-B-010-029), National Research Program for Genomic Medicine (NRPGM-DOH97-TD-G-111-038), a grant from Ministry of Education, Aim for the Top University Plan (97A-C-D104, 97A-C-T509) and National Health Research Institutes (NHRI-EX97-9611BI) (all to K.J.W.).
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Figure legends
Figure 1. Activation of HSP60 expression by c-MYC in a variety of cell lines. (A) Northern blot analysis of RNA from EREB.TCMyc cells upon estrogen (E2) withdrawal (lanes 2-4) and c-MYC activation by tetracycline (TC) withdrawal (lane 4) showed the activation of HSP60 expression by c-MYC. The same blot was sequentially hybridized with the indicated probes (see Materials and methods section). (B) Northern blot analysis of RNA from different CB33 lymphoblastoid cell lines (CB33 control, CBMyc.Max, and CBMax) showed the correlation between c-MYC and HSP60 gene expression. The same blot was sequentially hybridized with the indicated probes. (C) Western blot analysis of the cell lysates from the CB33 control, CBMyc.Max, and CBMax cells using an anti-HSP60 antibody and an anti--actin antibody as a control for protein loading. (D) Correlation of HSP60 vs. c-MYC expression in U937 cells with or without TPA induction by Northern blot analysis. (E) Increased rat HSP60 protein levels in Rat1a cells overexpressing c-MYC (RatMyc) vs. the control RatCMV cells by Western blot analysis. (F) & (G) Western blot analysis showed the decrease in HSP60 levels in the 293T or HeLa clone expressing siRNA against c-MYC (293TMyc-si or HeLaMyc-si) vs. the control 293T or HeLa clone.
Figure 2. Direct regulation of HSP60 expression by c-MYC. (A) Northern blot analysis of Rat.MycERTM cells upon activation of MycERTM by 4-hydroxytamoxifen
(TM; 500 nM) alone, by TM in the presence of cyclohexamide (CX; 10 g/ml), or with CX alone. The same blots were sequentially hybridized to a HSP60 probe and a vimentin probe as a control for RNA loading. Quantification of the ratio of HSP60 vs. vimentin was shown underneath each panel. (B) Schematic representation of the genomic region encompassing the 5’ flanking region of the human HSP60 promoter and the reporter constructs used for analysis of transcriptional activation by c-MYC. E-boxes located in the proximal promoter and intron 1 (represented by open and closed triangles) were depicted. The reporter constructs (pHSP60PWLuc and pHSP60PMLuc) were made as described (Materials and Methods). (C) Transcriptional activation of pHSP60PWLuc, but not pHSP60PMLuc, by c-MYC using transient transfection assays. (D) Co-transfection of pHSP60PWLuc and expression vectors of c-MYC wild type or its different deletion mutants showed that the transactivation or heterodimerization deletion mutant was defective in activating pHSP60PWLuc using transient transfection assays. (E) PCR amplification of immunoprecipitated chromatin prepared from different cell lines. Chromatin was incubated without antibody (-), with an anti-c-Rel Rel) or with an anti-c-MYC (c-MYC) antibody. Different columns represented experiments using different cell lines
such as CB33 control cells, CBMyc.Max cells, EREB.TCMyc(+TC/-E2) cells, and EREB.TCMyc(+TC/+E2) cells. The lengths of different PCR fragments were shown in Supplementary Table 3. The labeling of control represented the promoter fragments in the HSP60 or HSP90A gene without box. Different boxes represented the E-box located in the promoter region of HSP60 or HSP90A gene or in the intron 1 region of HSP60 gene. Input is 2% of total input lysate.
Figure 3. Overexpression of HSP60 induces transformation in a Rat1a cell line in
vitro. (A) Western blot analysis showed the increased HSP60 protein levels in Rat1a
clones overexpressing HSP60 (RatHSP60 clones). (B) RatHSP60 clones had increased soft agar colony formation activity compared to Rat1a control clones (RatCMV). The numbers (5000, 10000, 20000) in the inset indicated the number of cells plated in each plate. Error bars indicated standard deviations (S.D.)
Figure 4. The critical role of HSP60 in the transformation activity of RatMyc cells. (A) Western blot analysis showed the decreased HSP60 levels in RatMyc cells receiving siRNA against endogenous HSP60. Different amounts of pSUPER-rhsp60i plasmids were transfected into RatMyc cells. (B) Significantly decreased soft agar colony formation activity in RatMyc cells receiving siRNA against endogenous HSP60. The numbers (5000, 10000, 20000) in the inset indicated the number of cells
plated in each plate. Error bars indicated standard deviations (S.D.) (C) Western blot analysis showed no significant decrease in HSP60 protein levels in RatMyc cells receiving mismatched or scrambled siRNA as controls. (D) No significant decrease in soft agar colony formation activity in RatMyc cells receiving mismatched or scrambled siRNA as controls. The numbers (5000, 10000, 20000) in the inset indicated the number of cells plated in each plate. Error bars indicated standard deviations (S.D.)
