Reduced mitochondrial DNA copy number in Chinese patients with osteosarcoma

Reduced mitochondrial DNA copy number in Chinese patients with osteosarcoma MAN YU, YANFANG WAN, and QINGHUA ZOU TORONTO, ON, CANADA; TIANJIN, CHINA; AND LANGFANG, HEBEI PROVINCE, CHINA

A plethora of somatic mutations and germline variations in mitochondrial DNA (mtDNA) have been increasingly reported in numerous cancer entities including osteosarcoma. However, it remains largely unclear whether mtDNA copy number changes occur during the multistep process of osteosarcoma carcinogenesis. For this purpose, we determined quantitative mtDNA levels in 31 primary osteosarcoma specimens and 5 normal bone tissue samples using a real-time polymerase chain reaction assay. Our data showed that the average mtDNA amount was significantly reduced in osteosarcoma tissues compared with normal bone controls. The copy number of mtDNA was statistically associated with tumor metastasis. There was an approximately 2-fold decrease of mtDNA quantity in tumors with metastasis than that in low-grade tumors without metastasis. Furthermore, change in mtDNA content was linked with somatic mutations in the D-loop regulatory region. Tumors carrying somatic D-loop mutations, at the polycytidine stretch between nucleotide positions 303 and 309 or close to the replication origin sites of the heavy strand, had significantly lowered mtDNA levels in comparison with those without mutations. Taken together, these results provide evidence for the first time that reduced mtDNA content may be critically implicated in the development and/or progression of osteosarcoma. Somatic D-loop mutation is likely one key factor among others leading to altered mtDNA amount in osteosarcoma. (Translational Research 2013;161:165–171) Abbreviations: CT ¼ computerized tomography; EWS ¼ Ewing’s sarcoma; HCC ¼ hepatocellular carcinoma; HVS ¼ hypervariable segment; mtDNA ¼ mitochondrial DNA; nDNA ¼ nuclear DNA

steosarcoma is the most common primary bone tumor in children and young adults, predominantly targeting the metaphysis of long bones in the appendicular skeleton.1 This highly aggressive cancer is histopathologically composed of spindle-shaped malignant cells producing aberrant osteoid and has a propensity for rapid local invasion and early pulmonary metastasis.2 Unlike many sarcomas ge-

O

netically featured by specific chromosomal translocations, osteosarcoma is hallmarked by the presence of complex genomic instability, including a high degree of aneuploidy, the accumulation of unbalanced chromosomal rearrangements, and multiple regions of amplifications and deletions.3 Currently, the overall 5-year survival rate of patients with localized disease is approximately 60%–75% but it falls to about 15%–30%

From the Ontario Cancer Institute/Princess Margaret Hospital, University Health Network and University of Toronto, Toronto, ON, Canada; Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Hospital and Institute, Tianjin, China; Department of Surgical Oncology, Central Hospital of China National Petroleum Corporation, Langfang, Hebei Province, China.

Submitted for publication October 13, 2012; revision submitted October 26, 2012; accepted for publication October 29, 2012.

This study was supported in part by an internal research grant (to Z.Q.) and a Chinese government award (to M.Y.).

Ó 2013 Mosby, Inc. All rights reserved.

Conflict of interest: All authors have carefully read the journal’s policy on disclosure of potential conflicts of interest and declare that there are no conflicts of interest in relation with this study.

Reprint requests: Man Yu, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network and University of Toronto, Room 10-610, 610 University Avenue, Toronto, ON, Canada M5G 2M9; e-mail: [email protected]. 1931-5244/$ - see front matter http://dx.doi.org/10.1016/j.trsl.2012.10.011

165

166

Translational Research March 2013

Yu et al

AT A GLANCE COMMENTARY Yu M, et al. Background

A wide range of somatic mutations and germline variations in mitochondrial DNA (mtDNA) have been increasingly reported in virtually all types of human malignancies including osteosarcoma. However, it still remains hitherto unknown whether mtDNA copy number changes occur during the multistep process of osteosarcoma carcinogenesis. Translational Significance

This is the first study reporting that reduced mtDNA content may be possibly involved in the occurrence and/or progression of osteosarcoma. Somatic D-loop mutation is possibly an important factor leading to altered mtDNA amount in osteosarcoma. Scrutinizing mtDNA quantitative changes might help offer valuable clues for more efficacious diagnostics and better management of osteosarcoma patients.

for those harboring distant metastases, with only minor therapeutic improvement over the past 2 decades mainly because of poor response to multi-agent chemotherapy.4 The prognosis for individuals encountering recurrence also remains quite dismal, with less than 20% longterm survival.4 Hence, the identification of novel molecular events governing osteosarcoma initiation and progression is still critical for exploiting alternative therapeutic targets of clinical intervention. Mitochondria are semi-autonomous organelles in eukaryotic cells and play prominent roles in energy production, free radical formation and apoptosis.5 Human mitochondrial DNA (mtDNA) is a 16,569 bp, compactly organized, double-stranded, circular DNA molecule that encodes 13 polypeptide subunits of respiratory enzyme complexes, 22 transfer RNAs, and 2 ribosomal RNAs.6 MtDNA contains a unique 1124 bp non-coding segment termed displacement (D)-loop with regulatory elements for control of mtDNA transcription and replication.7 Compared with nuclear DNA (nDNA), mtDNA is present at extremely high levels in each individual cell with up to 103–104 copies.8 It is established that mtDNA copy number is highly dynamic and varies drastically as a function of metabolic demand with tissue type and diverse internal or external microenvironments.8 Apart from a broad spectrum of sequence variations accumulated in the mitochondrial genome,7 especially in the

well-known mutational hotspot D-loop region, mtDNA quantitative alterations have been observed in a wide array of primary solid tumors, as either an increase in the majority of head and neck cancers, colorectal carcinoma, ovarian cancer, prostate cancer, endometrial carcinoma, and esophageal squamous cell carcinomas9-14 or a decline in advanced gastric cancer, breast cancer, Ewing’s sarcoma (EWS), hepatocellular carcinoma (HCC), non-small cell lung cancer, and renal cell carcinoma.5-21 More importantly, mtDNA content variations have been demonstrated to be linked with neoplastic transformation, tumor progression, and metastasis, as well as clinical outcome in patients with breast cancer, ovarian cancer, EWS, HCC, and non-small cell lung cancer.14,15,20-23 To our best knowledge, no previous research has investigated mtDNA copy number changes in osteosarcoma and its probable diagnostic or therapeutic values. To test this possibility, in the present study, we first performed a quantitative real-time polymerase chain reaction (PCR) experiment to measure and compare mtDNA levels in 31 cases of primary osteosarcoma and 5 normal bone tissues. Next, we explored whether any association exists between mtDNA content and various clinicopathologic factors of osteosarcoma in an attempt to elucidate the functional significance of quantitative mtDNA abnormalities in the pathogenesis of osteosarcoma. The potential relationship of altered mtDNA amount and somatic D-loop mutations was also evaluated. MATERIALS AND METHODS Study subjects and tumor samples. Thirty-one patients (18 males and 13 females) who were diagnosed with osteosarcoma and received surgical resection at the Department of Bone and Soft Tissue Tumor, Tianjin Medical University Cancer Hospital and at the Department of Surgical Oncology, Central Hospital of China National Petroleum Corporation from June 2005 to October 2010 were enrolled in this study. All these subjects were from Tianjin or Hebei Province, China, and no enrolled participants underwent preoperative treatment. The median age of patients were 15 years (range: 4–36). The histologic subtypes were 22 cases of osteoblastic osteosarcomas, 6 cases of chondroblastic osteosarcomas and 3 cases of fibroblastic osteosarcoma. Primary neoplasms were located in the distal femur (n 5 19), the proximal humerus (n 5 6), the proximal tibia (n 5 4), the proximal fibula (n 5 1), and the distal ulna (n 5 1). Eight people had metastasis in the lungs at initial diagnosis. All tumor specimens were histologically reviewed by 2 independent expert pathologists. The tumor size was measured as the maximum diameter

Translational Research Volume 161, Number 3

on radiographic images including computerized tomography (CT) scans and magnetic resonance imaging. Tumor tissues obtained during surgical resection were immediately snap-frozen in liquid nitrogen and kept at –80 C prior to DNA extraction. As references, 5 normal human long and flat bone tissues obtained (all traces of soft tissue and bone marrow had been removed) at autopsy of 5 road traffic accident victims (3 males and 2 females; the median age: 27 years, range: 16-41) who were not affiliated with bone or soft tissue tumors as well as major metabolic diseases were collected at the tissue bank of Tianjin Orthopedic Hospital. To minimize the potential effects of demographic parameters, only subjects who were from either Tianjin or Hebei Province, China were selected. In addition, 5–10 mL of peripheral blood samples were drawn from our patient cohort for the D-loop mutation experiment. Written informed consent was obtained from all patients or their guardians, and this study was approved by the Review Board of the Hospital Ethics Committee. DNA extraction. Total DNA from osteosarcoma and normal bone tissues were prepared by digestion with 0.2 mg/mL proteinase K and 1% sodium dodecyl sulfate, followed by phenol/chloroform extraction and ethanol precipitation using standard protocols. Total DNA from 2 mL of blood samples was extracted by using a QIAamp DNA blood Midi Kit (Qiagen, Shanghai, China). Quantification of mtDNA copy number. To determine mtDNA copy number, real-time PCR assays were conducted on an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with a final volume of 50 mL reaction mixture containing 100 ng DNA template, 25 mL QuantiTect SYBR Green PCR Master Mix (Qiagen), 1.25 U Hot Start Taq DNA polymerase (Qiagen), and 20 pmol of each primer. The sequences of primer pairs designed to amplify the entire D-loop of mtDNA as well as a partial fragment of nDNA-encoded internal control gene b-actin were forward 5’-CTCCACCA TTAGCACCCAAAG-3’, reverse 5’-GTGATGTGAG CCCGTCTAAAC-3’ (D-loop, 1,232 bp); forward 5’ATCATGTTTGAGACCTTCAACA-3’, reverse 5’CATCTCTTGCTCGAAGTCCA-3’ (b-actin, 318 bp). The PCR cycling conditions were an initial ‘‘Hot Start’’ activation step at 95 C for 15 min, followed by 30 cycles of 30 s at 95 C, 30 s at 58 C and 90 s at 72 C. In each individual real-time PCR run, standard curves for mtDNA and b-actin were established using 5 serially diluted plasmids (pDrive TA cloning vector; Qiagen) containing the respective amplified fragment of either mtDNA D-loop or b-actin as templates. The copy number of mtDNA was then normalized against that of b-actin to calculate the relative value of mtDNA

Yu et al

167

content in each tested sample. All experiments were repeated at least twice in triplicate, and a non-template negative control was included in each reaction. Nucleotide sequencing analysis of mtDNA D-loop region. The complete D-loop segment of mtDNA was

specifically amplified by high-fidelity PCR employing the above-noted primer set. The resultant PCR products were purified and sequenced in both directions as previously described.24 The sequencing reads were compared against the revised Cambridge sequence in the Mitomap database (www.mitomap.org) using the MegAlin program of the DNAstar software package. The sequence alterations identified only in tumor tissues, but not in the matched peripheral blood samples, were recorded as somatic mutations. Statistical analysis. All statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS Inc, Chicago, Ill) 11.5 for Microsoft Windows. The differences in mtDNA copy number between tumor specimens and normal bone tissues, tumors with and without lung metastasis, and tumors with and without somatic D-loop mutations were compared by using the Student t test. The association between mtDNA copy number and clinicopathologic parameters of osteosarcoma was examined by using the Fisher exact test. All quantitative data were shown as means 6 SD. P values of ,0.05 were considered as statistically significant. RESULTS MtDNA copy number in osteosarcoma. After normalization against quantitative levels of the b-actin gene, we found that the average copy number ratios of mtDNA to nDNA were 26.3 6 9.5 and 49.4 6 10.8 in osteosarcoma and normal bone tissues, respectively. The difference between the 2 groups was statistically significant, around 46.8% decline in tumor tissues (P 5 0.046, Fig 1, A). Among the 31 tumor specimens, 20 cases (64.5%) displayed pronounced reduction of mtDNA content compared with the mean value of normal bone controls. Association between mtDNA content clinicopathologic variables of osteosarcoma. To

and

further uncover the possible correlation between mtDNA copy number and clinicopathologic characteristics of osteosarcoma, we calculated the ratio of relative mtDNA levels in tumor samples to that in normal tissues, designated as the T/N ratio (mtDNA copy number/b-actin in tumor tissues divided by the average of mtDNA copy number/b-actin in normal controls 3100%). According to the T/N values, the patient cohort was divided into 2 subgroups: 16 individuals with low mtDNA copy number and 15 with high copy number, using a cutoff level of 70.9%, the median value of all T/N ratios. As

168

Translational Research March 2013

Yu et al

Fig 1. Comparison of quantitative mtDNA levels between different subject groups. A, MtDNA copy number ratio in osteosarcoma tissues was significantly lower than that in normal bone controls (P 5 0.046, Student t test). B, A significant decrease of relative mtDNA content was observed in tumors harboring lung metastasis at the time of diagnosis when compared with those without metastasis (P 5 0.037, Student t test). Data are presented as means 6 SD.

shown in Table I, mtDNA quantitative level was significantly associated with the status of tumor metastasis (P 5 0.037). Seven of the 8 patients suffering from metastatic disease were categorized into the low mtDNA content group. Indeed, our quantitative analysis revealed that the relative mtDNA copy number ratio in patients with tumor metastasis was markedly lower (16.8 6 5.9) than that in the nonmetastatic ones (37.6 6 9.2, P 5 0.037, Fig 1, B). However, there was no significant relationship between mtDNA amount and other clinicopathologic parameters, including age, sex, primary tumor site, tumor size, and histologic subtypes (Table I). Association between mtDNA copy number and somatic D-loop mutations. Given that the D-loop contains essen-

tial regulatory sequences responsible for maintaining mtDNA expression and is considerably more prone to oxidative damage and electrophilic attack than other domains of mtDNA because of inefficient repair mechanisms, high frequency of sequence variations in this region has been long thought to contribute to mtDNA content changes, at least, in some tumor types.25,26 Therefore, we next screened somatic D-loop mutations in our tumor series by using a direct DNA sequence approach to probe if diminished mtDNA quantity happens along with the presence of these mutations. Overall, a total of 29 somatic D-loop mutations were identified in 20 out of the 31 osteosarcoma samples (64.5%). The 29 somatic mutations included 18 single nucleotide substitutions, 7 deletions, and 4 insertions (Table II). The vast majority of the mutations were located within 2 hypervariable segments HVS1 (np 16,024–16,383, 17/ 29, 58.6%) and HVS2 (np 57–372, 10/29, 34.5%). Except for 1 deletion mutation found at the nucleotide

Table I. Association between mtDNA D-loop copy number and diverse clinicopathologic factors in 31 patients with primary osteosarcoma mtDNA copy number Parameters

Group

P Low (n, %) High (n, %) value†

#15 9 (56.3%) 7 (43.7%) 0.724 .15 7 (46.7%) 8 (53.3%) Sex Male 8 (44.4%) 10 (55.6%) 0.473 Female 8 (61.5%) 5 (38.5%) Primary location Femur 10 (52.6%) 9 (47.4%) 1.000 Others 6 (50.0%) 6 (50.0%) Tumor size (cm) #8 9 (45.0%) 11 (55.0%) 0.458 .8 7 (63.6%) 4 (36.4%) Histologic subtype Osteoblastic 12 (54.5%) 10 (45.5%) 0.704 Others 4 (44.4%) 5 (55.6%) Tumor metastasis Yes 7 (87.5%) 1 (12.5%) 0.037 No 9 (39.1%) 14 (60.9%) Age* (y)

*Divided into 2 groups by the median age. † The Fisher exact test was used to compare the difference between groups.

position 16,132, all the remaining deletions and insertions (10/29, 34.5%) were accumulated in the homopolymeric C stretch between nucleotide positions 303 and 309 of the D-loop. Interestingly, compared with the mean mtDNA level of ones without D-loop mutations (102.9% 6 10.1%), the average mtDNA quantity in tumors bearing mutations at the homopolymeric C repeat between nucleotide position 303 and 309 (62.5 6 7.7%, n 5 10) or at the heavy strand replication origin sites (nucleotide positions 146, 150, 152, and 189, 59.4% 6 8.3%, n 5 4) were significantly decreased (P 5 0.006 and P 5 0.004, respectively, Fig 2). Such apparent changes in mtDNA content were not identified

Translational Research Volume 161, Number 3

Yu et al

169

Table II. Somatic D-loop mutations of mtDNA in 20 of 31 osteosarcoma patients Patient code

1 3 4 5 7 8

9 12 13 14 16 18 19 21 23 24 26 27 30

31

Position

Nucleotide change*

Cambridge sequence†

16,293 150 544 16,257 303-309 16,319 16,223 16,132 303–309 548 189 303–309 303–309 207 73 146 16,362 303–309 303–309 16,217 16,293 263 303–309 303–309 152 16,145 16,183 303–309 303–309

A/G C/T C/T C/T 8C/9C G/A C/T A deletion 7C/9C C/T A/G 8C/7C 9C/8C G/A A/G T/C T/C 7C/8C 9C/8C T/C A/G A/G 9C/8C 8C/7C T/C G/A A/C 9C/8C 7C/8C

A C C C 7C G C A 7C C A 7C 7C G A T T 7C 7C T A A 7C 7C T G A 7C 7C

*Normal blood controls / tumor tissues. † Original nucleotide(s) in the revised Cambridge Reference Sequence of human mtDNA.

between tumor tissues with D-loop mutations located at other positions and the controls (data not shown). DISCUSSION

Abnormal genetic and epigenetic alterations of several nDNA-encoded oncogenes and tumor suppressors, such as C-MYC, C-MET, MDM2, P53, and RB, have been proposed to be implicated in driving the oncogenesis of osteosarcoma.3,27-29 Yet, little attention has been paid hitherto to the involvement of mtDNA content changes in the pathobiology of osteosarcoma. In this study, we showed that 64.5% (20/31) of our osteosarcoma series carried a significantly lower mtDNA copy number compared with noncancerous bone tissues, which is in line with previous work in multiple solid tumors and our recent finding in EWS.16-21 These data indicate that reduced content in mtDNA molecule may exert an active role in the commencement and/or development of many malignancies including bone

Fig 2. Reduced mtDNA copy number in osteosarcoma tissues harboring somatic D-loop mutations. Compared with the average mtDNA content in tumors lacking D-loop mutations (102.9% 6 10.1%), relative levels of mtDNA amount in samples with mutations at the poly-C tract (310Cn) (62.5 6 7.7%) or at the heavy-strand replication origin site (nucleotide positions 146, 150, 152, and 189; 59.4% 6 8.3%) were significantly lowered (P 5 0.006 and P 5 0.004, respectively, Student t test). Data are shown as means 6 SD and were examined by Student t test. OH 5 replication origin of the heavy strand.

tumor. On the contrary, Wang et al13 observed a 2fold increase of mtDNA quantity in pure endometrial adenocarcinoma cells compared with normal endometrial glandular epithelium. Likewise, an increment in mtDNA content was identified in cancerous esophageal squamous cell carcinomas nests relative to that in surrounding normal esophageal mucosa.11 These evidences favor an idea that mtDNA quantitative levels in cancer cells may be mastered by complex regulatory machinery, rather than simply interpreted as an outcome of abnormal cell growth. It has been gradually established that reduced mtDNA copies in certain tumor types may be related to somatic mutations in the D-loop segment of mtDNA or defective P53-mediated signaling (see further discussion below), whereas increment of mitochondrial mass and mtDNA content in some forms of cancer is most likely derived from elevated oxidative stress and mitochondrial dysfunction.7 In addition, the causative roles of mtDNA content among different cancers appear to have some degree of specificity for particular tumor types or sites. The detailed reasons why distinct tumor types exhibit differential response to mtDNA content changes deserves further investigation. A growing body of literature has illustrated that reduction in mtDNA quantity is sufficient enough to influence many aspects of cancer cell behavior, including cell proliferation and apoptosis, hormone dependence

170

Translational Research March 2013

Yu et al

and their invasive and metastatic potency, eventually prompting the manifestation of tumor formation. Amuthan and colleagues30 showed that partial elimination of mtDNA molecules resulted in morphologic changes, markedly increased aggressiveness and upregulation of invasion-associated markers (CATHEPSIN L and TGF-b) in low-invasive lung cancer A549 cells. Similarly, mtDNA-depleted prostate cancer LNCaP and breast cancer MCF-7 cells underwent the epithelialmesenchymal transition and gained mesenchymal phenotypes, such as higher invasiveness and loss of hormone dependent growth, through activating the Raf/MAPK and TGF-b signaling pathways.31 Singh’s32 group recently reported that mtDNA loss and decreased mitochondrial activity invoked by mutations in mtDNA polymerase g could significantly increase Matrigel invasion of breast cancer cells. Moreover, mtDNA reduction was also revealed to be coupled with an increased migration onto the basement membrane protein laminin-1 in prostate cancer PC3 cells, reflecting a possible link between mtDNA content and the metastatic potential.33 Intriguingly, we found that reduced mtDNA copy number was intimately associated with the metastatic status of osteosarcoma, consistent with our earlier observation in EWS.21 One plausible hypothesis for these phenomena would be that the stepwise decrease of mtDNA amount from normal bone tissues to nonmetastatic and then to metastatic lesions may, at least to some extent, mirror a progressive accrual of malignant potential during osteosarcoma onset and advancement. As tumor spread is a principal prognostic factor in osteosarcoma, continuous studies are required to deeply dissect how reduced cellular mtDNA quantity confers the tumorigenic phenotype in osteosarcoma. The noncoding D-loop region contains the leadingstrand for the origin of mtDNA replication and a set of transcriptional promoters for both heavy and light strands.7 Although the hypervariability of this transcriptionally-active region does not necessarily cause major mitochondrial dysfunction, some specific D-loop mutations might intervene with the proper activity of major promoters and thus alter the rate of mtDNA replication and transcription.34 Consequently, high prevalence of sequence variations in this area might be deleterious to the copy number of the mitochondrial genome or leads to a repression of mtDNA gene expression. Coincident with findings in HCC, breast cancer, and EWS,16,20,21 mtDNA content was remarkably downregulated in osteosarcoma with somatic D-loop mutations compared with those without mutations. Further, a significant lower level of mtDNA was seen in tumors with somatic mutations at the polycytidine mononucleotide repeat or near the replication origin sites of the heavy-strand. Since these sequences participate in the formation of a RNA/

DNA hybrid imperative for priming mtDNA synthesis, enriched mutational changes at these specific locations are assumed to disturb the binding capacities of certain nDNA-encoded inducers and/or modulators (eg, mitochondrial transcription factor A) of mtDNA transcription and replication, ultimately giving rise to reduction in mtDNA content.34,35 Descending mtDNA copy number was also documented to emerge from deficient P53 pathway signaling. P53 not only sustains the stability of mtDNA via its translocation into mitochondria and interaction with mtDNA polymerase g but also physically mediates mitochondrial biogenesis and homeostasis as a mito-checkpoint protein.36,37 In this regard, disruption of p53 function may induce a noticeable increase of mtDNA’s sensitivity to oxidative stress, compromise the process of mitochondrial biogenesis process, and then elicit the reduction of mtDNA quantity. Since mutations of the P53 gene are present in approximately 30% of osteosarcoma,38,39 we are currently examining the relevance between P53 abnormalities and mtDNA quantitative changes in this cohort of osteosarcoma patients. In summary, despite the small sample size, we were able to show for the first time that decline in mtDNA content, which may potentially arise from somatic mutations in the D-loop region, are frequent events in osteosarcoma, and decreased mtDNA copy number has a significant association with tumor metastasis. These findings may shed more light upon the involvement of mtDNA alterations in osteosarcoma tumorgenesis. Considering that the mitochondrial genome has many unique advantages (eg, short length, simple structure, and high abundance) over nDNA,7 future studies involving a larger population of osteosarcoma patients, ideally from a different ethnic background, are warranted to adequately define the clinical applicability of monitoring aberrant turnovers of mtDNA quantity as a novel molecular biomarker for early diagnosis and tracking malignant transformation and progression of this fatal disease. The authors thank Yu Jiang for her kind technical assistance. The authors are grateful to Dr Ian Tannock and Carol M. Lee for critical reading of the manuscript and helpful discussions.

REFERENCES

1. Hayden JB, Hoang BH. Osteosarcoma: basic science and clinical implications. Orthop Clin North Am 2006;37:1–7. 2. Unni KK. Osteosarcoma of bone. J Orthop Sci 1998;3:287–94. 3. Martin JW, Squire JA, Zielenska M. The genetics of osteosarcoma. Sarcoma 2012;627254. 4. Geller DS, Gorlick R. Osteosarcoma: a review of diagnosis, management, and treatment strategies. Clin Adv Hematol Oncol 2010; 8:705–18. 5. Wallace DC, Fan W, Procaccio V. Mitochondrial energetics and therapeutics. Annu Rev Pathol 2010;5:297–348.

Translational Research Volume 161, Number 3

6. Chen XJ, Butow RA. The organization and inheritance of the mitochondrial genome. Nat Rev Genet 2005;6. 81–25. 7. Yu M. Somatic mitochondrial DNA mutations in human cancers. Adv Clin Chem 2012;57:99–138. 8. Clay Montier LL, Deng JJ, Bai Y. Number matters: control of mammalian mitochondrial DNA copy number. J Genet Genomics 2009;36:125–31. 9. Chen T, He J, Shen L, et al. The mitochondrial DNA 4,977-bp deletion and its implication in copy number alteration in colorectal cancer. BMC Med Genet 2011;12:8. 10. Kim MM, Clinger JD, Masayesva BG, et al. Mitochondrial DNA quantity increases with histopathologic grade in premalignant and malignant head and neck lesions. Clin Cancer Res 2004;10: 8512–5. 11. Lin CS, Chang SC, Wang LS, et al. The role of mitochondrial DNA alterations in esophageal squamous cell carcinomas. J Thorac Cardiovasc Surg 2010;139:189–97. 12. Mizumachi T, Muskhelishvili L, Naito A, et al. Increased distributional variance of mitochondrial DNA content associated with prostate cancer cells as compared with normal prostate cells. Prostate 2008;68:408–17. 13. Wang Y, Liu VW, Xue WC, Tsang PC, Cheung AN, Ngan HY. The increase of mitochondrial DNA content in endometrial adenocarcinoma cells: a quantitative study using laser-captured microdissected tissues. Gynecol Oncol 2005a;98:104–10. 14. Wang Y, Liu VW, Xue WC, Cheung AN, Ngan HY. Association of decreased mitochondrial DNA content with ovarian cancer progression. Br J Cancer 2006;95:1087–91. 15. Bai RK, Chang J, Yeh KT, et al. Mitochondrial DNA content varies with pathological characteristics of breast cancer. J Oncol 2011;496189. 16. Lee HC, Li SH, Lin JC, Wu CC, Yeh DC, Wei YH. Somatic mutations in the D–loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma. Mutat Res 2004;547:71–8. 17. Meierhofer D, Mayr JA, Foetschl U, et al. Decrease of mitochondrial DNA content and energy metabolism in renal cell carcinoma. Carcinogenesis 2004;25:1005–10. 18. Wu CW, Yin PH, Hung WY, et al. Mitochondrial DNA mutations and mitochondrial DNA depletion in gastric cancer. Genes Chromosomes Cancer 2005;44:19–28. 19. Wang H, Dai J. Changes on mitochondrial DNA content in nonsmall cell lung cancer. Zhongguo Fei Ai Za Zhi 2011;14:141–5. 20. Yu M, Zhou Y, Shi Y, et al. Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients. IUBMB Life 2007;59:450–7. 21. Yu M, Wan Y, Zou Q. Decreased copy number of mitochondrial DNA in Ewing’s sarcoma. Clin Chim Acta 2010;411:679–83. 22. Lin CS, Wang LS, Tsai CM, Wei YH. Low copy number and low oxidative damage of mitochondrial DNA are associated with tumor progression in lung cancer tissues after neoadjuvant chemotherapy. Interact Cardiovasc Thorac Surg 2008;7:954–8. 23. Yamada S, Nomoto S, Fujii T, et al. Correlation between copy number of mitochondrial DNA and clinicopathologic parame-

Yu et al

24.

25.

26.

27. 28.

29.

30.

31.

32.

33.

34.

35.

36. 37.

38.

39.

171

ters of hepatocellular carcinoma. Eur J Surg Oncol 2006;32: 303–7. Yu M, Wan Y, Zou Q, Xi Y. High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma. Biochem Biophys Res Commun 2009;390:447–50. Mambo E, Gao X, Cohen Y, Guo Z, Talalay P, Sidransky D. Electrophile and oxidant damage of mitochondrial DNA leading to rapid evolution of homoplasmic mutations. Proc Natl Acad Sci USA 2003;100:1838–43. Yu M. Generation function and diagnostic value of mitochondrial DNA copy number alterations in human cancers. Life Sci 2011; 89:65–71. Cui J, Wang W, Li Z, Zhang Z, Wu B, Zeng L. Epigenetic changes in osteosarcoma. Bull Cancer 2011;98:E62–8. Letson GD, Muro-Cacho CA. Genetic and molecular abnormalities in tumors of the bone and soft tissues. Cancer Control 2001;8: 239–51. Oda Y, Naka T, Takesita M, Iwamoto Y, Tsuneyoshi M. Comparison of histological changes and changes in nm23 and c-met expression between primary and metastatic sites in osteosarcoma: a clinicopathologic and immunohistochemical study. Hum Pathol 2000;31:709–16. Amuthan G, Biswas G, Ananadatheerthavarada HK, et al. Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene 2002;21:7839–49. Naito A, Cook CC, Mizumachi T, et al. Progressive tumor features accompany epithelial-mesenchymal transition induced in mitochondrial DNA-depleted cells. Cancer Sci 2008;99:1584–8. Singh KK, Ayyasamy V, Owens KM, Koul MS, Vujcic M. Mutations in mitochondrial DNA polymerase-gamma promote breast tumorgenesis. J Hum Genet 2009;54:516–24. Moro L, Arbini AA, Marra E, Greco M. Mitochondrial DNA depletion reduces PARP-1 levels and promotes progression of the neoplastic phenotype in prostate carcinoma. Cell Oncol 2008; 30:307–22. Xu B, Clayton DA. A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence. Mol Cell Biol 1995;15:580–9. Wang Y, Liu VW, Ngan HY, Nagley P. Frequent occurrence of mitochondrial microsatellite instability in the D-loop region of human cancers. Ann N Y Acad Sci 2005b;1042:123–9. Kulawiec M, Ayyasamy V, Singh KK. p53 regulates mtDNA copy number and mitocheckpoint pathway. J Carcinog 2009;8:8. Lebedeva MA, Eaton JS, Shadel GS. Loss of p53 causes mitochondrial DNA depletion and altered mitochondrial reactive oxygen species homeostasis. Biochim Biophys Acta 2009;1787: 328–34. Toguchida J, Yamaguchi T, Ritchie B, et al. Mutation spectrum of the p53 gene in bone and soft tissue sarcomas. Cancer Res 1992; 52:6194–9. Wadayama B, Toguchida J, Yamaguchi T, Sasaki MS, Yamamuro T. p53 expression and its relationship to DNA alterations in bone and soft tissue sarcomas. Br J Cancer 1993;68:1134–9.