Appraisal of the Relationships between Mitochondrial DNA Alterations and Invasiveness of Thoracic Esophageal Squamous Cell Carcinoma

• Main Authors: Chen-Sung Lin, 林振嵩
• Other Authors: Yau-Huei Wei
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/37640083410150389664

博士 === 國立陽明大學 === 臨床醫學研究所 === 98 === Thoracic esophageal squamous cell carcinoma (TESCC) has become one of the ten highly prevalent cancer-related deaths in Taiwan. Even with the rapid medical advances, the 5-year survival rate of TESCC patients after radical surgical resection has remained only around 30%. Thus, the issue to evaluate the prognostic variables related to the outcomes of TESCC patients is of great clinical relevance. In the first part of this thesis (Part I), I retrospectively collected 109 TESCC patients, who underwent surgical resection as the primary treatment modality, to evaluate the possible prognostic variables and their relative risks. After conducting the Cox’s regression model with stepwise analysis, four independent prognostic factors that were related to the poor outcomes of TESCC patients were demonstrated, including (1) clinical symptoms of swallowing difficulty (p=0.024), (2) cigarette smoking (p=0.003), (3) number of positive lymph nodes metastasis more than 4 (p<0.001) and (4) TESCC with gastric cardia invasion (p=0.012), and they had an elevated relative risk of 2.3, 2.5, 4.2 and 2.9, respectively. Cigarette smoking plays an important role not only in the pathogenesis but also in the progression and prognosis of TESCC as described in Part I study. According to the previous molecular analysis, cigarette smoking can also cause damage to mitochondrial DNA (mtDNA), including change of mtDNA copy number and mutation of mtDNA. As a result, an evaluation of the role of mtDNA alteration in TESCC is of great clinical importance. Guiding by the hematoxylin & eosin (H&E) stain under light microscopy, paired clinical samples, the normal esophageal muscle (as the reference), non-cancerous esophageal mucosa, cancerous TESCC nest and metastatic lymph node, were microdissected from the resected esophagus of TESCC patients to evaluate the alterations of mtDNA and their relations to the clinical relevance. Regarding the qualitative alterations, I focused on the somatic mutation over the polycytidine tract (PCT) and the changes of homoplasmy or heteroplasmy of mtDNA. Concerning the quantitative alterations, I mainly discussed the changes of mtDNA copy number. In the second part of this thesis (Part II), I pointed out four main conclusions, and they were (1) high percentage of homoplasmic PCT distribution in the normal esophageal muscles (43/72, 59.7% with homoplasmy) was changed to high percentage of heteroplasmic PCT distribution in the non-cancerous esophageal mucosa (46/72, 63.9% with heteroplasmy)(p=0.005), and this alteration can be explained by a result of accumulated damage over esophageal mucosa during the aging process; (2) subsequent somatic PCT mutations of mtDNA in cancerous TESCC nests were highly associated with a poor prognosis of TESCC patients (p=0.002); (3) progressive increase of the mtDNA copy number (when the mtDNA copy number of 143B cell line was defined as 1) from noncancerous esophageal mucosa of 0.159 to cancerous TESCC nest of 0.192 and then metastatic lymph node of 0.206 (p=0.024) and a phenomenon of heteroplasmic to homoplasmic PCT re-distribution from non-cancerous esophageal mucosa (46/72, 63.9% with heteroplasmy) to cancerous TESCC nest (41/72, 56.9% with homoplasmy) and then the metastatic lymph node (25/37, 67.6% with homoplasmy)(p<0.001) supported the theory of cancer clonal expansion during TESCC progression; and (4) both cigarette smoking (p=0.014) and alcohol drinking (p=0.005) were highly associated with the increase of mtDNA copy number. Although the phenomenon of cancer clonal expansion during TESCC evolution was validated from the viewpoint of mtDNA alterations, whether such a change also can be observed in nuclear DNA (nDNA), especially the well-known tumor suppressor gene TP53, has remained unclear. Thus, I further analyzed the TP53 alterations by means of loss of heterozygosity (LOH) or microsatellite instability (MSI) and correlated their associations with the alterations in mtDNA during TESCC evaluation. Microsatellite D17S960 that flanks near the TP53 gene was chosen as a marker to address the LOH or MSI of TP53 gene. In the third part of this thesis (Part III), I demonstrated that during TESCC evolution, the incidence of LOH was increased from the non-cancerous esophageal mucosa of 19.7% (13/66) to cancerous TESCC nest of 34.8% (23/66) and then the metastatic lymph node of 37.1% (13/35) progressively (p=0.023). Concerning the T status, the incidence of LOH was increased from T1 of 12.5% (1/8), T2 of 16.7% (2/12), to T3 of 34.8% (8/23) and then T4 of 52.2% (12/23) gradually (p=0.037). The progressive increase of TP53 LOH frequencies during TESCC evolution and invasion is consistent with the theory of cancer clonal expansion. Like the effect of cigarette smoking on mtDNA alterations, cigarette smokers (15/51, 29.4%) had a higher incidence of TP53 LOH or MSI over the non-cancerous esophageal mucosa than non-smokers (0/15, 0%). And patients who had a TP53 LOH or MSI over the non-cancerous esophageal mucosa also had a higher tendency to subsequent TP53 LOH or MSI over the corresponding cancerous TESCC nests (p<0.001). Applying immunohistochemistry (IHC) staining, the expression of p53 protein over the TESCC cancerous nest was detected, and I found that the positive rate of the p53 protein was decreased from T1 of 50% (4/8), T2 of 33.3% (4/12), to T3 of 26.1% (6/23) and then T4 of 21.7% (5/23) stepwise (p=0.058), which was inversely related to the incidences of TP53 LOH. It is reasonable to explain that the LOH on TP53 may result in impaired p53 protein expression. Interestingly, a condition of concurrent TP53 LOH and increasing mtDNA copy ratio (p=0.073) or concurrent TP53 MSI and mtDNA with PCT mutation (p=0.074) was detected among the 66 cancerous TESCC nests. Taken together, these alterations on mtDNA and TP53 during TESCC evolution are highly related and both of them validate the theory of cancer clonal expansion. Because progressive increase of mtDNA copy number was observed during TESCC evaluation, and such an increase was supposed to compensate for the damaged mtDNA and to keep mitochondrial function in proper. Thus, whether the mtDNA copy number alteration affects mitochondrial function in TESCC deserved further study. As we know, mtDNA copy number is mainly regulated by the mitochondrial transcription factor A (mtTFA). I adopted a small hairpin RNA (shRNA) knock-down system to down-regulate the expression of mtTFA in TESCC cell line, the CE-48T/VGH, to decrease the mtDNA copy number that mimics the de-compensation process, and then to evaluate its consequence. After constructing the double stranded oligonucleotides, sh-mtTFA(96), sh-mtTFA(97) and sh-Luc (Luc, gene coding for luciferase, control group) that contained sequences compensatory to mtTFA and Luc, into the pLKO.1 DNA backbone, they were packaged separately as virus particles for further viral infection. The CE-48T/VGH cells was infected with 2 MOI (multiplicity of infection) and 4 MOI of virus particles to establish new clones of 48T-sh-Luc-2, 48T-sh-Luc-4, 48T-sh-mtTFA(96)-2, 48T-sh-mtTFA(96)-4, 48T-sh-mtTFA(97)-2, and 48T-sh-mtTFA(97)-4 that could transcribe shRNA against the mRNA of mtTFA and Luc, respectively. Significant decreases in the expression of mtTFA protein and mRNA (p=0.017) were noted in clones of 48T-sh-mtTFA(96)-2 and 48T-sh-mtTFA(96)-4, but not in clones of 48T-sh-mtTFA(97)-2 and 48T-sh-mtTFA(97)-4. Further compares were evaluated among CE-48T/VGH, 48T-sh-Luc-2, 48T-sh-Luc-4, 48T-sh-mtTFA(96)-2 and 48T-sh-mtTFA(96)-4 cell lines. In the fourth part of this thesis (Part IV), I demonstrated that a decreased of mtTFA mRNA level was highly related to a decrease of mtDNA copy number (p=0.001), a decrease in the mRNA of mtDNA-encoded polypeptides of ND1 (p<0.001), ND6 (p=0.007), and COX III (p<0.001), respectively. Moreover, mtTFA knockdown was also associated with a decrease of coupling efficiency between respiration and oxidative-phosphorylation (p=0.007) and a decrease of horizontal migration activity (p=0.050) of TESCC cells. Taken together, these results suggest that the decrease of mtDNA copy number with subsequent mitochondrial dysfunction may affect cell migration and play an important role in the pathophysiology of the TESCC cell lines.