Mortalin (mot-2/mthsp70/GRP75), a member of the hsp70 family of proteins, is an essential protein that performs various functions related to proliferation, functional maintenance and stress response of cells.1, 2, 3, 4, 5 It is located in mitochondria, endoplasmic reticulum (ER), plasma membrane, cytoplasmic vesicles and cytosol, and has a differential pattern of subcellular distribution in normal and transformed human cells.6, 7, 8 Mortalin has multiple binding partners, including p53, FGF-1, IL-1 receptor type 1, GRP94, VDAC, NADH dehydrogenase, MPD and Tim23, and is involved in a number of molecular pathways responsible for control of cell proliferation.9, 10, 11, 12, 13, 14 It interacts with p53 in the cytoplasm of transformed cells, resulting in cytoplasmic retention and transcriptional inactivation of p53, and thus provides a mechanism of inactivation of wild type p53 in tumors.9, 15, 16 Inactivation of p53 by mortalin explains, at least in part, its ability to (i) extend the proliferative lifespan of human fibroblasts17 and promote their immortalization,18 (ii) malignantly transform immortal murine cells,19 (iii) attenuate the differentiation of HL60 cells.20 The mortalin ortholog has been reported to extend the lifespan of nematodes.21 Other predicted functions of mortalin, such as intracellular trafficking, chaperone activity and mitochondrial import are more likely to contribute to its roles in promotion of cell proliferation and response to stress.22, 23, 24 Recently, we have reported that downregulation of mortalin expression suppresses the growth of human transformed cells.25, 26 In the present study, we have investigated the involvement of mortalin in tumorigenesis by examining its expression level in multiple clinical human tumor samples, tumor-derived cells, in vitro immortalized cells, and by determining the effects of upregulating its expression in human cell model systems. We have found that (i) mortalin expression is elevated in many human tumor tissues and cells, and (ii) its upregulation confers a range of malignant properties on human cells.
Mortalin, also known as mthsp70/GRP75/PBP74, interacts with the tumor suppressor protein p53 and inactivates its transcriptional activation and apoptotic functions. Here, we examined the level of mortalin expression in a large variety of tumor tissues, tumor-derived and in vitro immortalized human cells. It was elevated in many human tumors, and in all of the tumor-derived and in vitro immortalized cells. In human embryonic fibroblasts immortalized with an expression plasmid for hTERT, the telomerase catalytic subunit, with or without human papillomavirus E6 and E7 genes, we found that subclones with spontaneously increased mortalin expression levels became anchorage-independent and acquired the ability to form tumors in nude mice. Furthermore, overexpression of mortalin was sufficient to increase the malignancy of breast carcinoma cells. The study demonstrates that upregulation of mortalin contributes significantly to tumorigenesis, and thus is a good candidate target for cancer therapy. © 2006 Wiley-Liss, Inc.
Materials and methods
Human normal, in vitro immortalized and tumor-derived, cells were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM), supplemented with 10% fetal bovine serum, penicillin, streptomycin and fungizone (Life Technologies) at 37°C, in an atmosphere of 5% CO2 and 95% air in a humidified incubator. The origin of the cells and some of their characteristics are described in Table I. HCA2 cells (also called MJ cells) were obtained from James R. Smith (University of Texas Health Science Center, San Antonio, USA). Cells (500 cells/6-cm dish) were plated on 0.7% agar-DMEM plates, and incubated at 37°C in a CO2 incubator. Number of colonies formed was counted under a microscope after 15 days, and percent colony forming efficiency (CFE) was determined for each cell type by 3 independent experiments, in which triplicate dishes were used.
|Normal cell lines|
|Bre-40||Breast epithelial cells-normal||0.59|
|HFF5||Normal foreskin fibroblasts||0.63|
|IMR90||Normal fetal lung fibroblasts||0.47|
|MRC-5||Normal fetal lung fibroblasts||0.61|
|TIG-1||Normal skin fibroblasts||0.63|
|WI-38||Normal fetal lung fibroblasts||0.41|
|Immortalized cell lines|
|HB56B/5T||SV40-immortalized bronchial epithelial cells||1.32|
|MRC5-SV2||SV40-immortalized MRC-5 cells||1.36|
|WI-38/VA132RA||SV40-transformed WI-38 cells||0.94|
|PE01||Ovarian carcinoma cells||0.79|
|SW 480||Colon, adenocarcinoma||1.30|
|143B TK-||TE-85 osteosarcoma transformed by Kirsten mouse sarcoma virus||1.32|
|A-498||Renal cell carcinoma||1.42|
|BEJ-T35||Tumorigenic BEAS-2B (mutant H-ras gene)—nude mouse tumor line||1.56|
|BES-1A1||NHBE cells immortalized with SV40||1.34|
|BES-1A1 neo||NHBE cells immortalized with SV40 (neo control for BES-1A1 Ras)||1.33|
|BES-1A1 Ras||BES-1A1 cells transfected with mutant H-ras gene||1.42|
|BhaKi-T12||RAS transformed tumorigenic BEAS-2B— nude mouse tumor line||1.35|
|BVK-T11||BEAS-2B cells infected with Kirsten mouse sarcoma virus—nude mouse tumor line||1.83|
|Caski||Epidermoid carcinoma of uterine cervix||1.17|
|COLO 16||Squamous cell carcinoma from lung||1.33|
|DU-145||Prostate carcinoma, brain metastasis||1.31|
|HeLa||Epithelioid cervical carcinoma||1.10|
|KM12SM||Colon carcinoma, spontaneous metastasis (SM)||1.41|
Retroviral expression of mortalin
For construction of retrovirus driven expression of mortalin, pCX4neo (provided by Dr. Tsuyoshi Akagi, Osaka, Japan) was used. cDNA encoding myc-tagged full-length mortalin protein was cloned into the BamHI site of the vector. For production of retroviruses, Plat-E, an ecotropic murine leukemia virus-packaging cell line was transfected with the pVPack-GP (expression gal and pol) and pVPack VSVG (expressing env) vectors (Stratagene, CA), and with either pCXneo retroviral vector or pCX4neo/mot-2-myc using FuGENE6 (Boehringer Mannheim). After 48 h, culture supernatants were collected, filtered through 0.45-μm filters and used as viral stocks for infection. MCF7 cells (2 × 105/well in 6-well dishes) were treated with 8 μg/ml polybrene for 1 h at 37°C, following which cells were infected with 200 μl of filtered viral stock for 1 h. The plates were tilted after every 15 min to spread viral particles over the cells. After 1 h, 2 ml of DMEM was added to the culture to dilute the viral stock, and the plates were incubated at 37°C for a further 48 h. Cells were selected in a medium containing G418 (1 mg/ml), until stable expressing cell lines were obtained. The lines were tested for expression of mortalin-myc by Western blotting, as described later, and were maintained in 100 μg/ml G418-supplemented medium. Retroviral expression of Hsp60 was used as control.
Total RNA was prepared from 70% confluent cells in 10-cm dishes using Trizol (Life Technologies). The RNA was denatured and size-fractionated on 1% agarose gels, containing 2.2 M formaldehyde, and transferred to Hybond N+ membrane (Amersham). A 1.6 kb fragment of the human mortalin cDNA was used as a probe. Hybridization was performed at 65°C in Express™ hybridization buffer (Clontech). The membrane was washed for 10 min, each in 2× SSC and 2× SSC/0.1% SDS, and then washed twice in 1× SSC/0.1% SDS and autoradiographed. RNA loading on the blots was determined by hybridization with an actin or 18S probe. Human tumor tissue total RNA dot blots and human single tumor multisample total RNA Northern blots were purchased from Biochain, USA and BD Biosciences Clontech, USA.
Single locus DNA fingerprinting
Genomic DNA was prepared using the Stratagene DNA extraction kit. Ten micrograms of genomic DNA was incubated with 40U HinfI restriction endonuclease in the supplied buffer at 37°C overnight. DNA fragments were separated by electrophoresis in a 0.8% agarose gel in 1× TAE buffer for 16 h, and transferred to a Hybond N+ nylon membrane by Southern blotting. DNA was fingerprinted with 3 multi-allelic single-locus probes, all of which were derived from the multilocus 33.15 probe: MS1 (locus D1S7 on chromosome 1p), g3 (locus D7S22 on chromosome 7q) and NS43 (locus D12S11 on chromosome 12) using the NICE™ Probe Kit, Cell Mark Diagnostics, UK). Hybridization was performed at 50°C for 20 min, and the probe was detected by conjugated alkaline phosphatase activity.
Western blot analysis
The protein sample (10–20 μg) was separated on an SDS-polyacrylamide gel, and electroblotted onto a nylon membrane (Millipore) using a semidry transfer blotter. Immunoassays were performed with anti-mortalin,1 anti-myc (Santa Cruz, sc-A14), anti-p53 (Santa Cruz, sc-126), anti-p21 (Santa Cruz, sc-397), anti-pRB, anti-E7 (Santa Cruz, sc-1583), anti-mdm2 (Santa Cruz, sc-965) or anti-actin (Chemicon International-MAB1501R) antibodies. The immunocomplexes formed were visualized with horseradish peroxidase-conjugated secondary antibodies (sc-2004 and sc-2005), using an ECL kit (Amersham Pharmacia Biotech).
Chemotaxis assays were performed on control and mortalin-transduced MCF7 cells. Cells at 60–70% confluency were washed with cold PBS, trypsinized and resuspended in DMEM supplemented with 0.5% bovine serum albumin (Sigma) at 2 × 105 cells/ml. Cells (2 × 104) were plated in transwell inserts (12 mm-pore, Costar), and the invasion assay was performed following the manufacturer's instructions. Fibronectin from human plasma (Sigma) was used as a chemoattractant. Cells that moved through the transwell were fixed with 4% formaldehyde in PBS and stained with crystal violet (0.2% in PBS).
Nude mice assay
Balb/c nude mice (4 weeks old, female) were bought from Nihon Clea (Japan). WET and HCT, and MCF7 (wild type p53) cells and their mortalin-overexpressing derivatives (1 × 106 suspended in 0.3 ml of growth medium) were injected subcutaneously into the flank of nude mice (1 site per mouse). Tumor formation was monitored for 1 month. The assay was regarded as positive, if tumors appeared and grew progressively. The experiment was repeated twice, using 3 mice for each injection.
Mortalin is upregulated in tumor tissues
Tumors and their matched normal tissues were analyzed for expression level of mortalin by using total RNA dot blots. Sixteen of the 24 tumor tissues showed a higher level of mortalin expression than the normal control from the same individual (Fig. 1a). Eight tumor tissues (lung, oesophagus, stomach, rectum, ovary, adrenal gland, testis and lymphoma) did not show any change in the level of mortalin expression level (Fig. 1a). Equal loading of RNA was ensured by 18S probing of the blots by the suppliers (data not shown). We next extended the expression analysis to breast, kidney, brain, ovary, lung and colon tumors and their matched normal tissues. 6/8 breast, 1/8 kidney, 5/8 brain, 3/8 ovary, 4/8 lung and 6/8 colon tumors showed significantly elevated mortalin expression when compared with their matched normal controls (Fig. 1b). Equal loading of RNA, in all the lanes, was ensured by 18S probing of the blots (data not shown). Notably, mortalin expression appeared to be frequently upregulated in breast, brain and colon tumor tissues.
Mortalin is upregulated in tumor-derived cells
Tumor-derived cells provide a reliable material for analysis of tumor properties. We next analyzed mortalin expression in a large number of cell lines, established from tumors. The level of mortalin expression was quantitated against an internal control (18S RNA) and was expressed in relative units of mortalin expression (Table I). All of the tumor-derived or in vitro immortalized cells showed a higher level of expression of mortalin than the normal primary cells (MRC-5, TIG-1, WI-38, IMR90 and HFF5) (Table I). Upregulation of mortalin, in tumor derived cells and tissues, was also detected by Western blotting (data not shown).
Mortalin is upregulated in cells that underwent spontaneous malignant transformation
We next immortalized human embryonic fibroblasts by introduction of expression plasmids encoding the human papillomavirus-16 E6 and E7 genes and the catalytic subunit human telomerase reverse transcriptase (hTERT) of human telomerase. Emerging immortal clones were analyzed for expression of E7 by Western blotting (Fig. 2) and for telomerase activity by TRAP assays (data not shown). One such cell clone (WET: WI-38/E6E7/hTERT) was passaged for 150 population doublings. During this serial passaging, we observed the emergence of morphologically distinct cells that grew as shiny dense colonies in contrast to the flat and density-inhibited parental cultures. To analyze these colonies, we isolated visually distinct subclones by ring isolation. These were analyzed for expression of E7, to confirm their identity, and for expression levels of p53, p21, mdm-2, pRb and mortalin. In the parental cell line, WET, as expected, p53, mdm-2 and p21 levels were decreased in response to the introduction of E6E7 when compared with the normal WI-38 cells. Four subclones (WET-1, -6, -7 and -11) showed different expression levels of p53 and its downstream genes, mdm-2 and p21, and mortalin (Fig. 3a). To study the functional significance of these changes, we examined the malignant properties of the subclones by soft agar colony forming assay and nude mice assay. The subclone with the highest mortalin expression, WET-6, had a greatly enhanced ability to grow in soft agar and formed tumors in nude mice (Fig. 2a). WET-6 cells also exhibited decreased density-dependent inhibition of monolayer growth (Fig. 2b). Similar results were found in another strain (HCA2) of fibroblasts immortalized by hTERT alone: the 2 subclones with the highest mortalin expression level, HCT-62 and -64, exhibited anchorage-independent growth and tumorigenicity in nude mice. Identity of the cells was confirmed by single locus DNA fingerprinting (Figs. 2c and 2e). The data showing that subclones with spontaneously elevated levels of mortalin acquired malignant properties suggest that expression of this protein is involved in tumorigenesis.
Mortalin promotes malignant properties of cancer cells
Since mortalin expression was upregulated in all transformed and tumor-derived cells (Table I and Fig. 3a), we next investigated whether it contributes to the malignant properties of tumor cells. MCF7 breast carcinoma cells that do not form tumors in nude mice were used. Derivatives of MCF7 cells expressing retroviral-driven mortalin were generated. Stable expression of exogenous mortalin was detected by Western blotting with anti-myc antibody (Fig. 3b). Control (vector-infected and Hsp60-infected MCF7 cells) and mortalin-overexpressing derivatives were subjected to nude mice assays. Of note, 6/6 nude mice injected with mortalin, but not Hsp60, overexpressing cells developed tumors of variable sizes (Fig. 3c and data not shown). Mortalin-overexpressing cells also showed migration in the chemotaxis assay (Fig. 3d), confirming that an overexpression of mortalin increases the malignancy of cancer cells. Furthermore, chemotaxis of mortalin-overexpressing cells was abrogated when expression of mortalin was repressed with hybrid ribozymes (data not shown).
The prevailing paradigm of carcinogenesis asserts that normal cells progressively accumulate multiple genetic and epigenetic lesions, leading to their uncontrolled proliferation (hyperplasia), disorganization of tissue morphology (dysplasia) and, ultimately, acquisition of invasive and metastatic phenotypes. Expression of the catalytic component of human telomerase, hTERT, extended the lifespan of a variety of human cell types beyond senescence, without causing neoplastic transformation.27, 28, 29, 30 Additional genetic alterations, such as overexpression of the c-myc and Bmi-1 oncogenes, loss of p16INK4A, p14ARF and wild type p53 tumor suppressor proteins, were shown to be responsible for acquisition of malignant properties in hTERT-immortalized human fibroblasts.31, 32 In the present study, we detected upregulation of mortalin expression in a large variety of human transformed cells and tissues. In contrast to most of the other tumors, kidney tumors showed a low frequency of upregulation of mortalin expression. The reason for such tumor-specific difference remains to be known. To rule out the possibility that the differences were not due to differences in growth rate of cells or cell cycle progression, we examined the expression of mortalin in young (rapidly dividing) and old (slow dividing) human fibroblasts and during cell-cycle progression of human-transformed cells subsequent to double thymidine block. In both cases, the level of mortalin expression did not change significantly, suggesting that an upregulation of mortalin expression in transformed cells is not related to their growth rate (data not shown). This was in contrast to the regulation of Hsp60 that increases during the initial stage of senescence in fibroblasts, relating to cell-cycle progression, and has been implicated in carcinogenesis.33, 34, 35, 36, 37 The fact that all of the tumor-derived cells showed upregulation of mortalin, while some of the tumor tissues did not, might relate to (i) the clinical history of the tumors, which is not known in the present case, as the source of tissues was commercial and (ii) tumor cells with high level of mortalin expression may have selective advantage to proliferate and override the population in vitro.
Furthermore, we immortalized 2 strains of normal human fibroblasts with the catalytic subunit (hTERT) of human telomerase or its combination with human papillomavirus-16 E6 and E7 genes. Apparently, the immortal cells accumulated additional genetic changes, resulting in altered phenotype. Examination of protein profiles in morphologically distinct clones revealed that the clones that acquired malignant properties also had upregulated mortalin in both cell strains. It is evident that the genetic alterations in carcinogenesis are accompanied by overexpression of molecular chaperones, which have a prosurvival function amidst the accrual of genetic mutations.38 Constitutive expression of stress-responsive Hsps, Hsp90, Hsp70 (heat-inducible) and Hsp27, has been detected in breast, uterine, renal, osteosarcoma and endometrial cancers. The overabundance of these Hsps in biopsy samples generally predicts poor patient outcomes.39
The heat noninducible mitochondrial hsp70, mortalin, has recently emerged to be another important player in human carcinogenesis. Functioning in the inner mitochondrial matrix, mortalin is a major component of the preprotein mitochondrial import complex essential for translocating mitochondrial-targeted proteins.40, 41, 42 Other activities associated with mortalin, such as inactivation of the wild-type tumor suppressor protein p53 in cancer cells,9, 15, 16, 17 modulation of the Ras-Raf-MAPK pathway,14 and its intracellular trafficking function,10, 43 are more likely to contribute to its proproliferative and tumorigenic properties. In a recent study using comparative proteomic analysis of cancer tissue arrays, it was found that an upregulation of mortalin is involved in colorectal neoplasia.44 Consistent with the premise that increased mortalin expression is associated with malignancy, it was observed that reduction of mortalin levels in some tumor cell lines, using ribozymes and antisense plasmids, resulted in loss of proliferative capacity, senescence and cell death.25, 26
Here, we report upregulation of mortalin mRNA and protein levels in in vitro immortalized and tumor-derived cells and, to a greater degree, in highly proliferative and aggressive tumor cells. By virtue of its role in mitochondrial biogenesis, it might be predicted that an upregulation of mortalin would parallel the increasing metabolic demand that accompanies increased proliferation. Some of the enhancing effect on tumorigenesis by overexpression of mortalin may also be attributed to its extramitochondrial chaperone activity. More than 30% of cellular mortalin is distributed in cellular sites other than the mitochondrion, namely, early endocytic vesicles, plasma membrane, ER and membranes proximal to the Golgi.4, 7, 45 An overabundance of mortalin expression on the cell surface was found by comparative proteome profiling of the cell surface and plasma membrane proteomes of various cancer and normal cells.46 It is possible that the presence of mortalin on the cell membrane may, in part, contribute to enhance metastatic and colonization potential in tumor cells as a result of altered membrane biophysical properties, such as membrane fluidity and deformability. We have found (Deocaris et al., unpublished data) that the cell membranes of mortalin overexpressing cells have altered biophysical properties. Furthermore, an enhanced chaperone loading may enhance cell survival during the stresses of the metastatic process.
In summary, in addition to its differential distribution in normal and tumor cells,8 we have shown here that it is highly expressed in neoplastic cells and tissues. As both parameters (subcellular localization and expression levels) could correlate with cellular proliferation and tumor invasiveness, mortalin may potentially be a suitable clinical marker for gauging aggressiveness of a tumor. Hence, determining relative levels of mortalin by ELISA or IHC from biopsy samples may aid clinical decision-making. In addition to serving as a facile marker capable of discriminating normal cells from tumor cells, we report here that an increased mortalin expression contributes to tumorigenesis, and is an attractive target for cancer diagnostics and therapeutics.
We thank Katherine Jeffrey for help in Northern analysis.