Inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis

Gene 354 (2005) 140 – 146 www.elsevier.com/locate/gene

Inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis Keshav K. Singha,*, Mariola Kulawieca, Ivan Stilla, Mohamed M. Desoukia, Joseph Geradtsb, Sei-Ichi Matsuia a

Department of Cancer Genetics, Cell and Virus Building, Room 247, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA b Department of Pathology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA Received 2 February 2005; accepted 25 March 2005 Available online 24 June 2005 Received by K. Wolf

Abstract Mitochondrial dysfunction is a hallmark of cancer cells. Consistent with this phenotype mutations in mitochondrial genome have been reported in all cancers examined to date. However, it is not clear whether mitochondrial genomic status in human cells affects nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Using cell culture model and cybrid cell technology, we provide evidence that mitochondrial genetic status impacts nuclear genome stability in human cells. In particular our studies demonstrate 1) that depletion of mitochondrial genome (rho0) leads to chromosomal instability (CIN) reported to be present in variety of human tumors and 2) rho0 cells show transformed phenotype. Our study also demonstrates that mitochondrial genetic status plays a key role in regulation of a multifunctional protein APE1 (also known as Ref1 or HAP1) involved in transcription and DNA repair in the nucleus and the mitochondria. Interestingly we found that altered expression of APE1 in rho0 cells and tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in rho0 cells. Furthermore, we demonstrate that APE1 expression is altered in variety of primary tumors. Taken together, these studies suggest that inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis and that APE1 mediates this process. D 2005 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Inter-genomic; Nucleus; Chromosomal instability; Tumorigenesis; APE1

1. Introduction Mitochondria perform multiple essential cellular functions (Modica-Napolitano and Singh, 2002, 2004). Although the mitochondrial and nuclear genomes are physically distinct, there is a high degree of functional interdependence between the two genomes. Of the 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, the mitochondrial genome encodes 13 sub-

Abbreviations: mtDNA, Mitochondrial DNA; CIN, Chromosomal instability; SKY, Spectral karyotyping; IHC, Immunohistochemistry; APE1, Apurinic/apyrimidinic endonuclease 1; TARP, Tissue arrays research program. * Corresponding author. Tel.: +1 716 845 8017; fax: +1 716 845 1047. E-mail address: [email protected] (K.K. Singh). 0378-1119/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.03.027

units (see below) and the rest of the subunits are encoded by the nuclear genome. Correct mitochondrial functions depend on an orchestrated cross talk between the nuclear and mitochondrial genomes. The mitochondrial genome is a small 16.6 unit molecule that encodes 13 subunits of the respiratory chain complexes, 22 tRNAs and 2 ribosomal RNAs. Mammalian cells typically contain 103 –104 copies of mitochondrial DNA (mtDNA). Unlike nuclear DNA, mammalian mtDNA contains no introns, has no protective histones and is exposed to deleterious reactive oxygen species generated by oxidative phosphorylation. In addition, replication of mtDNA might be error prone. The accumulation of mutations in mtDNA is approximately tenfold greater than that in nuclear DNA (Grossman and Shoubridge, 1996, Johns, 1995, Penta et al., 2001). Mutations in mtDNA have been reported in mito-

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chondrial diseases and in a variety of cancers, including ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain, bladder, head and neck, and breast cancers, and leukemia (reviewed in Penta et al., 2001 and in ModicaNapolitano and Singh, 2002, 2004). Deletions, point mutations, insertions and duplications have been detected throughout the genome, and certain mutations in mtDNA are associated with specific cancers. For example, a 40 bp insertion localized in the COX I gene appears to be specific for renal cell oncocytomas (Welter et al., 1989), and a deletion mutation resulting in the loss of mtDNA within NADH dehydrogenase subunit III is a phenotype associated with renal cell carcinoma (Selvanayagam and Rajaraman, 1996). The D-loop region appears particularly susceptible to DNA mutations. Both hepatocellular carcinoma (Nomoto et al., 2002) and breast cancer (Parrella et al., 2001) are associated with certain deletion/insertion mutations in the Ctract, a hotspot and a potential replication start site within the D-loop of the mitochondrial genome. These studies suggest that mutations in mtDNA is a common feature of cancer cells. Unfortunately, to date it is not clear whether mutations in the mitochondrial genome affect nuclear genome stability and whether inter-genomic cross talk is involved in tumorigenesis. Our studies conducted in yeast Saccharomyces cerevisiae model system suggest that mutations in the mitochondrial genome cause nuclear genomic instability (Rasmussen et al., 2003). We also identified that nuclear genome stability was mediated by Rev1p dependent error prone repair pathway (Rasmussen et al., 2003). Using a human cell culture model our recent study provided evidence that mitochondrial genomic dysfunction leads to impaired oxidative DNA repair in the nucleus (Delsite et al., 2003). Our study also revealed that mitochondrial dysfunction leads to elevated expression of MnSOD which causes resistance to apoptosis (Park et al. 2004). In the present paper, we analyzed the importance of the mitochondrial genome in chromosomal instability (CIN) and its role in tumorigenesis. This study reveal that depletion of mitochondrial genome (rho0) leads to 1) chromosomal instability 2) altered expression of APE1, a DNA repair gene and 3) observed instability in nuclear genome plays a critical role in tumorigenesis. Furthermore, our study for the first time suggest that the tumorigenesis phenotype and the altered expression of APE1 can be reversed by transfer of exogenous wild type mitochondrial genome into rho0 cells. These studies suggest that intergenomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis.

2. Method 2.1. Cell cultures Human osteosarcoma cell line 143B and 143B rho0 and derived cybrid cell lines were a kind gift of G. Manfredi

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(Columbia University, New York City). The cybrid cells were isolated by transfer of platelets from a normal volunteer into human mtDNA depleted (rho0) 143B cells (Gajewski et al., 2003). Cell lines were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 units/ ml penicillin and 100 Ag/ ml streptomycin (Invitrogen, Carlsbad, CA) and 50 Ag/ml uridine (Sigma, St. Louis, MO). All cell lines were maintained in a humidified 5% CO2 atmosphere at 37 -C (Singh et al., 1999). 2.2. Spectral karyotyping analysis (SKY) After mitotic arrest for 2 h with Colcemid, cells were harvested and treated with hypotonic solution according to the standard protocol. Chromosome slides were prepared using air-drying methods. After sequential digestion with RNAse and pepsin according to the procedure recommended by Applied Spectral Imaging, Inc. (ASI: Carlsbad, CA), the chromosomes were denatured in 70% formamide and hybridized with human SKY paint probes tagged with various nucleotide analogues (i.e., a mixture of individual chromosome DNAs prepared by flow-sorting and PCR amplification). The multiple fluorescence color images of chromosomes generated by Rhodamine, Texas-Red, Cy5, FITC and Cy5.5 were captured using a Nikon microscope equipped with a Spectral cube and Interferometer module and analyzed using SKY View software (version 1.62). Chromosome number and chromosomal rearrangements or alterations including simple balanced translocation or unbalanced (or non-reciprocal) translocation, deletion and duplication, were analyzed to determine the lineage of individual knock-out cell lines, compared to the original wild type counterpart (Matsui et al., 2002, 2003). 2.3. Assay for changes in tumorigenic phenotype In vitro assays for changes in tumorigenic phenotype was assayed as previously described in Lauffart et al. (2003). For soft agar assays, cells were seeded at 5000 cells per well in 0.35% agar in 12 well plates. Colonies 50 Am or greater were scored as positive after 2 weeks of growth. Invasion assay was performed using the Matrigel Invasion Chamber assay (Becton Dickinson, Bedford, MA, USA) according to manufacturer’s instructions. Cells (5  104) in culture medium lacking serum were introduced to the upper side of the invasion chamber. For the purpose of this experiment, culture medium containing 10% serum was used as the chemoattractant. After 24 h at 37 -C, the membrane was removed and invading cells on the lower surface fixed and stained. Cells were visualized by microscopy and counted. For all assays, each data point was performed in triplicate and differences between cell lines analyzed using one way ANOVA followed by Dunnett’s Multiple Comparison Post Test (Graphpad Prism Version 3.0, Graphpad Prism Software Inc.).

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lowed by incubation with secondary peroxidase labeled anti-mouse IgG (Vector Laboratories) in a conc. of 5 Ag/ml for 2 h at room temperature. Color was developed by incubating slides with DAB kit (Vector Laboratories) followed by counterstaining with Harris’ Hematoxylin. Slides were examined with light E600 Nikon microscope. Pictures were taken with Spot advanced software. Scoring of immunoreactivity was considered; negative, no positive cells, score + < 10% positive, score ++ 10– 50% positive and score ++ >50% positive cells.

2.4. Western blotting Total proteins (20 Ag) were resolved on one-dimensional 10% SDS-PAGE gels (10  8 cm) and transferred to Immun-Blot\ PVDF membrane (BioRad, Hercules, CA) using semi-dry transfer unit (BioRad, Hercules, CA). After 2 h incubation in blocking solution [5% nonfat milk 1  PBS 0.05 Tween-20], reaction with specific antibodies was performed. The APE1 antibody tested was from Novus Biologicals, Littleton, CO. Anti-tubulin (dilution 1 : 1000) (Molecular Probes, Eugene, OR) was used to assess equal protein loading. Secondary antibodies were horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (dilution 1 : 3000) (Vector Laboratories, Burlingame, CA). Reactions were revealed by the ECL method (Amersham Biosciences, Piscataway, NJ).

3. Results 3.1. Chromosomal changes associated with depletion of mitochondrial genome Our previous analysis conducted in yeast model system revealed that inhibition of mitochondrial function leads to mutator phenotype in the nuclear genome (Rasmussen et al., 2003). We also found frequency of mutations in nuclear genome was significantly higher in rho0 and rho cells. Analysis of mutational spectrum of rho0 and rho cells revealed deletions and insertions in the nuclear DNA (Rasmussen et al., 2003). We therefore envisioned that depletion of mitochondrial genome in mammalian cells should lead to chromosomal instability (CIN) in the nucleus. To address this possibility, we carried out SKY analysis of parental 143 B, 143 B rho0 and 143 B rho0 cells containing wild type mitochondria (cybrid cells). Sky analysis revealed specific chromosomal changes in rho0 cells (Fig. 1). These were translocations at

2.5. Immunohistochemistry (IHC) Tissue array slides from the Cooperative Human Tissue Network (CHTN) and Tissue Array Research Program (TARP2) of the National Cancer Institute, National Institutes of Health, Bethesda, MD were used in the present study. Also, two-breast carcinoma tissue sections with matched benign ones obtained from RPCI were included in the present work. Standard protocol for IHC was applied. Briefly, the slides were deparaffinized by heating and incubation with xylene, antigenically retrieved by heating in 10 mM sodium citrate buffer, blocked with 10% goat serum, incubated overnight at 4 -C with 1 : 250 dilution of human AP Endonuclease-1 (APE-1) (NovoCastra Laboratories, Newcastle, UK), folCell Line 143B Parental

t(1;21) t(1;21;3;7;15) t(2;16;5;2) x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

Rho Zero

Cybrid

0

t(9;21;1)

x x x x x x x x x x x x x

t(12;14) t(12;22;14) x x x x x x x x x x x x x x x x x x x x x x x x x x x x

x x x

x x 0

t(14;8;14) t(14;8;14;4) x

t(21;6)

x x x x

x x x x x x x x x x x x x

x x x x x x x x x x x x

x x x x x x x x x x x x x

Fig. 1. Chromosomal instability in rho cells. Chromosomal instability in parental, rho and cybrid cells was analyzed as described in Methods. Translocations present in all three cell lines are presented in gray column whereas specific translocation found in rho0 cells are presented in red columns. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Chromosomal translocation t(1;21;3) t(9;21;1) t(9;21)

t(12;22)

t(14;8;14;4) t(14;8;14) t(21;6)

Organ site

Bone marrow Brain Lymph node Skeleton Tongue Adrenal Bone marrow Brain Intra-abdominal Kidney Lymph node Salivary gland Skeleton Skin Small intestine Soft tissue Stomach Uterus-corpus Bone marrow Salivary gland Bone marrow Larynx Lymph nodes Nasal cavity/paranasal sinuses Salivary gland Skin Soft tissue Uterus-corpus

Type of cancer Unknown Unknown Chronic myeloproliferative disorder, AML, ALL, refractory anemia, adult T-cell lymphoma Benign epithelial tumor Follicular lymphoma, splenic marginal zone B-cell lymphoma Osteosarcoma Squamous cell carcinoma Peripheral neuroepithelioma, B-lineage or biphenotypic leukemia, AML, CML, ALL, adult T-cell Lymphoma – leukemia, multiple myeloma, peripheral B-cell neoplasm, acute megakaryoblastic leukemia Rhabdoid tumor Clear cell sarcoma Clear cell sarcoma Diffuse large B-cell lymphoma, peripheral B-cell neoplasm Adenocarcinoma Ewing tumor/peripheral primitive neuroectodermal tumor Malignant epithelial tumor Clear sarcoma Clear cell sarcoma, liposarcoma Clear cell sarcoma Leiomyoma Unkonwn ALL Adenoma AML, CML, ALL, refractory anemia Squamous cell carcinoma Follicular lymphoma Squamous cell carcinoma Acinic cell carcinoma Basal cell carcinoma Atypical lipomatous tumor – atypical lipoma – liposarcoma Leiomyoma

t(1;21;3;7;15), t(9;21;1), t(12;22;14), t(14;8;14;4), and t(21;6). Some abnormal chromosomes with other structural changes were also observed but were sporadic and not of clonal origin. The significance of genomic changes that enabled the viability of rho0 cells seems to rest on 5 translocations found in the chromosomes during progression to rho0 state. Among these five translocations in question, t(9;21;1), t(14;8;14;4), and t(21;6) are known to appear frequently in several tumors (see Table 1). For example, t(21;6) is associated with hemopoietic disorders such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymph node follicular lymphomas, nasal cavity squamous cell carcinoma, skin basal cell carcinoma and soft tissue liposarcoma, and uterus leiomyoma. t(9;21) and t(21;1) are associated with AML, T-cell lymphomas and ALL. Our analysis also revealed complex translocation involving multiple chromosomes. These include t(1;21;3;7;15), t(12;22;14) and t(14;8;14;4) translocation which have not been reported in the literature (Mittleman Data Base). Nevertheless, these translocations consist of partial chromosomal rearrangements that are thought to play important roles in carcinogenesis. Most interesting among several translocation is t(12;22) which is specifically associated with the emergence of

malignant forms such as clear cell sarcoma found in soft tissues and small intestines as well as AML, ALL, CML and CLL (chronic lymphocytic leukemia). Whereas we do not yet know the precise identity of genes involving structural rearrangements it is clear that nuclear gene alteration is responsible for the survival of cells lacking mtDNA. We conclude that depletion of mitochondrial genome leads to specific chromosomal translocations that are involved in tumorigenesis.

Parental

Rho 0

Cybrid

Ape1

Tubulin

Fig. 2. Mitochondria regulate APE1 expression: protein lysates (20 Ag) was separated in a 10% SDS-PAGE gel and transferred. Western blot analysis was performed using antibody against APE1 protein. Equal loading of protein was confirmed by anti-tubulin antibody. Figure shows decreased APE1 expression level in rho0 cells but equal expression in both parental and cybrid cells.

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Fig. 3. APE1 is involved in tumorigenesis. APE1 expression was analyzed in normal and carcinomas tissues. IHC analysis was done on tissue array (TARP2) containing variety of carcinomas tissues. Bar graph shows the percent of positive and negative carcinoma cases as a whole. Each panel shows a representative positive and negative carcinoma case as well expression in normal tissue. APE1 protein was visualized using DAB with hematoxylin counterstain. Bar = 50 Am.

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3.2. APE1 mediates inter-genomic cross talk between mitochondria and the nucleus The above studies suggest that mitochondrial genomic defect leads to chromosomal instability in the nucleus. To understand the underlying reason for chromosomal instability in the nucleus we performed cellular proteomic analysis of extracts obtained from parental 143 B, 143 B rho0 and cybrid cells containing wild type mitochondria (data not shown). The proteomic studies identified down regulation of apurinic/apyrimidinic endonuclease (APE1), also known as (redox effector factor (Ref1) protein (Fig. 2). APE1 is a multifunctional nuclear and mitochondrial protein. It acts as a DNA repair protein, functions as a transcriptional co-factor to stimulates the DNA binding activity of AP-1 (Fos, Jun) proteins, as well as nuclear factor-kB (NF-kB), polyoma virus enhancer-binding protein, early growth response-1, Myb members of the ATF/ CREB family, HIF-1a, HIF-like factor, Oax5 and Pax-8 (Evans et al., 2000). The DNA binding activity of these proteins is sensitive to reduction – oxidation (redox). Recently APE1 was also shown to control the p53 activity through redox dependent and independent mechanism (Evans et al., 2000). APE1 is also closely linked to apoptosis. Western blot analysis presented in Fig. 2 confirms proteomic data and shows that APE1 expression was down regulated in rho0 when compared to the parental cell line and expression was reversed to parental level in cybrid cells. This study suggests that APE1 is involved in inter-genomic cross talk between mitochondria and nucleus.

145

150

100

50

0 143B

rho0

Cybrid

Fig. 4. Rho0 cells show increased anchorage independent growth. Anchorage independent assay was carried out as described in Methods.

Napolitano and Singh, 2002, 2004). However, it is not clear whether mitochondrial genomic dysfunction caused by mutations affect tumorigenic properties of cells. To investigate the consequences of mitochondrial genomic dysfunction on tumorigenic properties, we assessed whether mitochondrial DNA deficient 143 B cells exhibited anchorage independent growth compared to the parental cell line (Fig. 4). Rho0 cells exhibited an increased ability to form colonies in soft agar ( P < 0.05), compared to wild type 143 B. In order to address whether repletion of mitochondrial genome in rho0 cells affects tumorigenic phenotype we conducted soft agar assays using the transmitochondrial cybrid. Significantly, the parental phenotype was restored in transmitochondrial cybrid cells, which showed a reduction in anchorage independent growth. We conclude that mitochondrial genome plays an important role in tumorigenic phenotype.

3.3. APE1 expression is altered in variety of primary tumors

4. Discussion

APE1 is a multifunctional protein that plays important roles in maintaining the stability of nuclear and mitochondrial genomes, gene expression and apoptosis (Evans et al., 2000). Our studies described above suggest that mitochondrial genetic defect leads to down regulation of APE1 expression. Since mtDNA genetic defects has been described in almost all cancers examined to date, we therefore examined whether APE1 expression profile was altered in primary tumors. We examined the APE1 expression by IHC in tissue array slides containing a variety of tumors (Fig. 3). Figure shows the APE1 expression profile. It is evident that 68% of CNS tumors do not express APE1. In contrast 22% of breast, 13% of prostate, 27% lung, 20% melanomas, 19% ovary, 33% lymphomas did not express. These results suggest that inactivation of APE1 is a frequent event during tumorigenesis.

Mitochondrial dysfunction is a hallmark of cancer cells. Consistently, mutations in mtDNA has been described in all cancer examined to date. However, the significance of mtDNA mutation in tumorigenesis is unclear. Our recent study conducted in yeast model system has identified a novel function of mitochondria in maintaining nuclear genome integrity (Rasmussen et al., 2003). Recently we extended our study to a human cell model and found that like yeast, mitochondria in human cells also plays a critical role in the stability of the nuclear genome as cells without mtDNA showed extensive damage to nuclear genome due to oxidative stress (Delsite et al., 2003). Indeed, studies in both model systems revealed an important role of mitochondria derived oxidative stress in nuclear genome instability. Together, these studies emphasize the importance of intergenomic cross talk between the mitochondria and the nucleus and the role this cross talk may play in cancer. To further understand the importance of cross talk between the mitochondria and the nucleus, we utilized 143 B parental, its rho0 derivative and the cybrid containing wild type mitochondria. The study presented in this paper show for the first time that rho0 cells contain signature

3.4. Transfer of wild type mitochondrial genome reverses the tumorigenic phenotype Mutations in mitochondrial genome of cancer cells have been found in all most all cancer examined to date (Modica-

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chromosomal instability that are distinctly different when compared to the parental cell line. Analysis of chromosomal translocations in rho0 cells suggests that these identified regions are known to be involved in tumorigenesis (Table 1). Although it not clear how these multiple chromosomal translocation take place in rho0 cells, it is likely that double strand breaks are introduced during the progression of parental cells to rho0 state. Consistent with this suggestion, we find that APE1 was down regulated in rho0 cells. Interestingly, APE1 expression can be reversed to parental level by transfer of wild type mitochondria to rho0 cells (cybrid). APE1 is involved in redox regulation and apoptosis. APE1 also repairs DNA and interacts with p53, a protein which is intimately involved in DNA double strand break and other types of DNA repair. It is therefore likely that APE1 through its interaction with p53 plays a significant role in CIN in rho0 cells. Rho0 cells show increased anchorage independent growth, a characteristic feature of tumorigenic phenotype. Consistent with our reports Amuthan et al. (2001, 2002) have reported that partial depletion of human lung carcinoma A549 cells and murine C2C12 myoblast cells also leads to increased tumorigenic phenotype. Although not clear at this time, it is likely that depletion of mitochondrial genome in cells used by Amuthan et al. (2001, 2002) leads to CIN that may cause increased tumorigenic changes. Interestingly, our study demonstrates that tumorigenic phenotype is a reversible phenomenon because transfer of wild type mitochondria to rho0 cells reverts the anchorage independent growth and invasion (data not shown) to parental level. Together, these results suggest that expression of select nuclear genes play a key role in mitochondria mediated tumorigensis. We have identified APE1 as one such gene that is involved in tumorigenesis at various organ sites including breast, ovary, prostate, melanoma, CNS, lymphoma, lung, and colon. This observation is consistent with previously described role of APE1 in other tumors (Evans et al., 2000). It is tempting to focus the future efforts of gene search on the breakpoint of chromosomes involved in the specific genomic rearrangements. The genes in these particular break/fusion sites and/or chromosomal regions flanking the break/fusion points appear to be most likely affected during tumorigenesis and likely to be the key in cross talk between the mitochondria and the nucleus.

Acknowledgement This study was supported by grants from National Institutes of Health (RO1-097714), Elsa Pardee Foundation and a grant from Breast Cancer Coalition of Rochester.

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