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TUFM is a potential new prognostic indicator for colorectal carcinoma
Pathology (October 2012) 44(6), pp. 506–512
ANATOMICAL PATHOLOGY
TUFM is a potential new prognostic indicator for colorectal carcinoma HONGJUN SHI*, MARK HAYES*, CHANDRA KIRANA*, ROSEMARY MILLER{, JOHN KEATING{, DONIA MACARTNEY-COXSON§ AND RICHARD STUBBS* *Wakefield Biomedical Research Unit, and {Department of Pathology and Molecular Medicine, University of Otago (Wellington), zWellington Hospital, and §Institute of Environmental Science and Research (ESR), New Zealand
Summary Aims: Mitochondrial Tu translation elongation factor (TUFM) is a nuclear encoded protein that participates in mitochondrial polypeptide translation. TUFM has been reported to be overexpressed in many tumour types including colorectal carcinoma (CRC) by proteomics. The present study aims to examine the prognostic implication of TUFM in CRC. Methods: Immunohistochemical staining was performed in tissue microarrays composed of 123 cases of CRC using a polyclonal anti-TUFM antibody. Immunoreactivity was quantified using Image-Pro plus software, and analysed in association with patients’ clinicopathological parameters and survival time. Results: The immunoreactivity of TUFM was negative in 25%, weak in 50% and strong in 25% of CRC cases. TUFM immunoreactivity had no significant association with the clinicopathological parameters examined including TNM stage and grade. However, strong TUFM expression significantly correlated with a higher 5-year recurrence rate ( p ¼ 0.024). Kaplan–Meier analysis revealed that patients with strong TUFM expression had significantly shorter cancerspecific survival than patients with negative TUFM (log-rank test, p ¼ 0.038). In multivariate analysis, strong TUFM expression remained a stage-independent unfavourable prognostic indicator ( p ¼ 0.024). Conclusions: Increased expression of TUFM is a promising new prognostic indicator for CRC. Selective inhibition of TUFM in tumour cells may present a new avenue for the targeted therapy of this cancer. Key words: Colorectal carcinoma, mitochondria, prognosis, TUFM. Received 7 November 2011, revised 2 February, accepted 10 February 2012
INTRODUCTION Colorectal cancer is an important health problem both internationally and in New Zealand. Every year 2800 New Zealanders are diagnosed with CRC and approximately 1250 die from it, making CRC the second most common cancer registered and the second most common cause of death from cancer.1 As a result of advances in cancer screening and treatments, there has been a steady reduction in the death rate during the past few decades. However, at present, still more than 35% of CRC patients will not survive more than 5 years, mainly due to metastatic spread of the tumour to distant organs.2 Approximately 20% of CRC patients have detectable metastases at the time of their first consultation (synchronous metastasis)2 and a further one-third of the remaining patients Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e3283559cbe
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will go on to develop metastasis after surgical removal of their primary tumours.3 Thus, avenues for improving the prediction and prevention of metastatic spread may be the most promising approach to reducing the CRC mortality rate. Staging of CRC is useful in predicting the probability of cancer recurrence after surgical removal of the primary tumour and has become the gold standard in selecting patients who would benefit most from adjuvant chemotherapy.4 While patients with tumours limited to the bowel wall (TNM stage I) can be cured by surgery alone in more than 90% of cases, patients with tumours penetrating through the bowel wall (stage II) or involving the regional lymph nodes (stage III) have a substantial risk of relapse after surgical resection, with 5 year survival rates of approximately 80% and 60% for stage II and III, respectively.5 Although CRC prognosis is stage dependent, tumours of the same stage may have significantly different clinical outcomes.6 For stage III disease, clinical trials have consistently demonstrated a 10–13% absolute survival benefit from 5-fluorouracil based adjuvant chemotherapy which is now a standard of care around the world.7 However, approximately 30–44% of stage III patients do not develop metastases in the absence of chemotherapy and thus may have been overtreated.8 Stage II patients have a better prognosis. Current chemotherapy has shown a small but not consistently significant improvement in survival for stage II patients and is not routinely provided.9 Nonetheless, 20% of stage II patients develop secondary tumours that might have been prevented or delayed by adjuvant chemotherapy.9 Patients with high-risk stage II cancers, e.g., cancers with inadequately sampled nodes, T4 lesions, perforation or poorly differentiated histology, may be considered for adjuvant chemotherapy, although there has been no direct clinical trial evidence of benefit of adjuvant chemotherapy in such patients.10–12 Additional prognostic indicators are required to improve the accuracy of stratification of patients at increased risk of metastasis in whom more intensive therapy and monitoring are justified. CRC initiation, progression and metastasis are complex processes caused by multiple alterations within cells to ultimately influence change in cellular behaviour. Recent development in proteomic technology has allowed comparison of multiple proteins from different tissues to be accomplished with good reproducibility and sensitivity. This has led to identification of many putative protein markers with prognostic significance in CRC.13–16 In our previous proteomic study, we identified and validated the protein mitochondrial Tu translation elongation factor (TUFM) as being over-expressed in CRC compared to the patient matched normal colon mucosa. In addition, TUFM has also been reported to be increased in lung,
2012 Royal College of Pathologists of Australasia
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TUFM IN COLORECTAL CARCINOMA
gastric and oesophageal cancer by proteomics.17 – 19 Despite its frequent association with cancer in clinical onco-proteomic studies, little is known about its biological role in tumour development. TUFM has been most extensively studied as a fundamental molecule involved in GTP-dependent recruitment of codon-specified aminoacyl-tRNA to the ribosome during protein translation in mitochondria.20 TUFM along with a large number of other nuclear-encoded protein factors constitute the mitochondrial translation apparatus responsible for translation of all 13 mtDNA-encoded proteins that partially constitute the five respiratory chain complexes.21,22 Interestingly, in addition to its conventional role in peptide elongation in mitochondria, TUFM also displays chaperone-like properties in protein folding and renaturation under stress conditions, suggesting TUFM over-expression in CRC might also be a stress response. Since both the mitochondrial defects and the cellular stress such as hypoxia and oxidative stress have been shown to relate to carcinogenesis and tumour progression,23 – 26 the present study aims to evaluate the prognostic significance of TUFM in CRC.
MATERIALS AND METHODS Patients Out of the 282 patients who had undergone partial colectomy or anterior resection of CRC performed by Dr John Keating from 1997 to 2005 at Wellington Hospital, 169 patients had archival tissue blocks available for investigation at the time of this study. A representative block from each patient was drawn and sectioned for H&E staining. On histological examination, 42 blocks were excluded from the cohort due to the absence or inadequacy of tumour cells in the sections from the blocks initially chosen from the tissue archives. Consequently, a total of 127 CRC cases were finally included in this study. Clinicopathological features of the resected CRC were obtained from a prospective surgical database according to the clinical and pathological reports held at Wellington Hospital. Pathological stages were classified according to the TNM staging system.27 Histological grading and typing of the tumour was determined according to the World Health Organization tumour classification system.28 Cancer specific survival was defined as the interval between the date of the first operation of the primary tumour to the date when the patient died from recurrent CRC. Cases were censored at the end of the follow-up or at the time of death due to other causes. Thirty-seven patients died of recurrent CRC during the follow-up period. Median follow-up time was 61 months (range 2–164 months). Construction of tissue microarray (TMA) and immunohistochemistry on the TMAs using archival human tissues was conducted with the approval of the New Zealand Central Regional Ethics Committee. Tissue microarray Five TMA blocks containing a total of 127 CRC cases were constructed. Each TMA consisted of up to 26 tissue cores with a single tissue core per patient’s tumour. Formalin fixed, paraffin embedded tissue blocks of CRC were obtained from the hospital tumour archive. Before constructing TMA, a 4 mm section was sliced from each tumour block for a routine H&E inspection by a pathologist. After histological confirmation of the tumours, areas of sampling (AOS) were defined and marked on the microscope slide by the pathologist. These microscope slides with the spotted AOS were used later as a reference to guide the location of tissue cores for punching. AOS was defined as the area of obvious invasive cancer close to the lumen, not including any potential adenomatous areas. TMA was constructed using the Beecher Automated Tissue Arrayer (ATA-27, Beecher Instruments, USA) through the Molecular and Clinical Pathology Research Laboratory, Clinical and Statewide Services, Princess Alexandra Hospital, Queensland, Australia. A tissue core with a diameter of 1 mm was punched from the donor tissue under the guidance of the reference slides, and transferred to a recipient paraffin block (array margin of 10 20 mm). Once the TMAs were made, they were heat
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cycled from 448C for 1 h and room temperature (RT) for 1 h for a total of 5 cycles to aid cutting. Immunohistochemistry A 4 mm thick tissue section was cut from each TMA block. All subsequent procedures were carried out at RT unless otherwise stated. Tissue sections were serially rehydrated. Antigen retrieval was performed by submerging the slides in sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) in a boiling water bath for 25 min. After 25 min cooling at RT, tissue sections were incubated in 3% H2O2 in PBS-T for 10 min to block endogenous peroxidase. A circle of hydrophobic barrier was drawn around the specimen by a Pap pen (Invitrogen, USA). Sections were then sequentially blocked in 2.5% normal horse serum in PBS-T for 30 min and avidin/biotin blocking reagents for 20 min according the manufacturer’s instruction (Invitrogen). Tissues were then incubated in a humidified chamber with the goat polyclonal anti-TUFM antibody (1:100, sc-12991; Santa Cruz Biotechnology, USA) diluted in 1.25% normal horse serum in PBS-T for 1 h. Specific bindings were detected using VECTASTAIN Universal Quick Kit (Vector Laboratories, USA) and DAB substrate (Invitrogen). A non-immune normal goat IgG (1:200, sc-2028; Santa Cruz Biotechnology) was used as a negative control. Image analysis and scoring Immunoreactivity of TUFM was quantified using the Image-Pro Plus software (version 6) (Media Cybernetics, USA). Briefly, a digital image (TIFF format) under 20 objective lens was taken near the centre of each tumour core (Eclipse 80i; Nikon, Japan). The imaging field under this objective covered approximately 40% of the entire core. Images of all tissue cores were acquired at the same time with a constant set of imaging parameters on the microscope and imaging software. The images were then subject to optical density analysis by the Image-Pro Plus software. Adjustments of background and colour intensity range were performed on a representative image showing high immunoreactivity. The black and incident level (representing the optical density of the maximal positive staining and white background, respectively) were determined from an immunostained region and a blank region in the image, respectively. Intensity range selection was based on histogram, with intensity (I) and saturation (S) set at maximum, and hue (H) set at a range where most of the brown DAB colour was selected while blue nuclei counterstain was excluded. These settings were saved and subsequently applied to all images analysed. On opening each image, an area of interest (AOI) was drawn to include most of the malignant epithelial cells in the image and exclude stroma, muscle, necrosis and other non-tumour components. After defining the AOI, the mean optical density of the selected area [integrated optical density (IOD)/unit area] was determined by the software. This represents the immunoreactivity of the candidate protein within tumour tissue. To facilitate statistical analysis, IOD/unit area was manually categorised into three classes of immunoreactivity (negative, weak and strong) as described in the Results section. Statistical analysis All statistical analyses were performed using SPSS (version 17; SPSS, USA). The association between TUFM immunoreactivity and patient clinicopathological parameters was assessed by x2 test. The impact of TUFM on patient survival was examined by Kaplan–Meier curve analysis and the statistical significance was determined using log-rank test. A multivariate analysis based on Cox proportional hazard regression model was applied to determine independent prognostic factors. Variables included in Cox regression analysis were histological grade, histological types, TNM stages, vascular invasion, perineural invasion, type of operation and TUFM immunoreactivity. A p value of less than 0.05 was considered significant.
RESULTS Expression pattern of TUFM in CRC tissues TUFM was generally homogeneously expressed in all neoplastic epithelial cells in the TUFM positive tumour cores. TUFM was nearly absent or only weakly expressed in the stroma. In positive neoplastic cells, TUFM exhibited a punctate staining pattern throughout the cytoplasm. A representative staining is shown in Fig. 1.
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Fig. 1 A representative photomicrograph of TUFM staining in a colorectal tumour. TUFM exhibited a punctate cytoplasmic staining pattern in neoplastic cells (T). Very few stroma cells (S) were positively stained.
Correlation of TUFM expression and tumour characteristics Of the 127 tissues core sections, four sections were lost during the sectioning procedure. A total of 123 tissue sections were included in the image and statistical analysis. The TUFM immunoreactivity varied considerably among the 123 patients (IOD/unit area ranged from 0 to 167, with the mean across samples of 27.56 36.65 SD). To facilitate statistical analysis, IOD/unit area was manually categorised into four groups of equal number of patients across the subgroups of immunoreactivity (negative, weakly positive, moderately positive and strongly positive, respectively). Because subsequent Kaplan– Meier curve analysis showed that subdivision of the weakly positive and moderately positive cases did not produce additional discriminatory power in predicting prognosis (data not shown), these two middle classes were combined as a single
class of ‘weakly positive’ cases in the final statistical analysis. This results in two cut-off points (1.5 and 41.0 IOD/unit area) which broke the entire cohort of patients into three categories with negative (score 0, 25%), weak (score 1, 50%) and strong immunoreactivity (score 2, 25%) respectively (Fig. 2). The relationship between TUFM immunoreactivity and tumour characteristics is shown in Table 1. No statistically significant correlation was seen between TUFM expression and age, gender, tumour location, histological type, histological grade, vascular invasion, perineural invasion, TNM stage, nodal status or depth of invasion. Increased TUFM expression tended to correlate with an increased probability of 5-year recurrence, although it did not reach statistical significance ( p ¼ 0.078). Notably, in this analysis one category has only three events, making the statistical analysis less accurate. To improve this, the number of tumours with negative and weak expression was combined, and the resulting two tiered grades were re-subjected to x2 tests in association with cancer recurrence. The analysis showed that the 5-year recurrence rate was significantly higher in patients with strong TUFM expression in tumours than those with weak or negative TUFM expression (39% versus 14%, p ¼ 0.024).
Survival analysis The correlation between TUFM expression and cancer specific survival was assessed by univariate analysis using Kaplan– Meier curve and log-rank test. As shown in Fig. 3, patients with strong TUFM expression showed a significantly shorter cancer specific survival than patients with negative TUFM expression (55% versus 80% 5 year cumulative probability of cancer specific survival, p ¼ 0.038). The difference in cancer specific survival between TUFM negative and weakly positive tumours, and between TUFM weakly positive and strongly positive tumours was not significant ( p ¼ 0.226 and 0.177, respectively).
A
B
0.03 Negative (score 0) 0–1.5 n = 31
25.25 Weak (score 1) 1.6–41.0 n = 62
148.14 IOD/unit area Strong (score 2) 41.1–166.8 IOD/unit area n = 30
Fig. 2 Representative CRC tissues showing negative, weak and strong TUFM expression. The brown immunostains in the original images (A) were detected by the Image-Pro Plus software (marked in red) based on a pre-defined colour intensity selection range (B). The staining intensity was only determined for the area of interest (AOI) (demarcated in green line), and the level of immunoreactivity was expressed as integrated optical density (IOD)/unit area.
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TUFM IN COLORECTAL CARCINOMA
Table 1
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Correlation between TUFM immunoreactivity and tumour characteristics
Clinicopathological parameters Age <65 65 Gender F M Tumour location Colon Rectum Histological type Non-mucinous Mucinous Histological grade High grade Low grade Vascular invasion Negative Positive Perineural invasion Negative Positive TNM stages I II III IV Lymph node metastasis Negative Positive Depth of invasion T1/T2 T3/T4 5 year recurrence§ Recurrence free Recurrent 5 year recurrence§ Recurrence free Recurrent
No. of cases (%*)
Negative (%{)
Weak (%{)
Strong (%{)
36 (29) 87 (71)
8 (22) 23 (26)
20 (56) 42 (48)
8 (22) 22 (25)
62 (50) 61 (50)
21 (34) 10 (16)
27 (44) 35 (57)
14 (23) 16 (26)
85 (69) 38 (31)
23 (27) 8 (21)
44 (52) 18 (47)
18 (21) 12 (32)
106 (87) 16 (13)
25 (24) 5 (31)
53 (50) 9 (56)
28 (26) 2 (13)
24 (20) 98 (80)
6 (25) 25 (26)
14 (58) 47 (48)
4 (17) 26 (27)
94 (76) 29 (24)
25 (27) 6 (21)
46 (49) 16 (55)
23 (24) 7 (24)
113 (93) 9 (7)
30 (27) 1 (11)
56 (50) 5 (56)
27 (24) 3 (33)
3 17 9 2
12 21 14 13
5 8 11 6
pz value 0.761
0.080
0.440
0.464
0.554
0.787
0.078
0.176 20 46 34 21
(17) (38) (28) (17)
(15) (37) (26) (10)
(60) (46) (41) (62)
(25) (17) (32) (29) 0.109
70 (60) 47 (40)
22 (31) 8 (17)
34 (49) 23 (49)
14 (20) 16 (34)
23 (19) 97 (81)
3 (13) 28 (29)
14 (61) 45 (46)
6 (26) 24 (25)
31 (53) 5 (33)
11 (19) 7 (47) Strong 11 (19) 7 (47)
0.271 0.078 59 (80) 15 (20) 59 (80) 15 (20)
17 (29) 3 (20) Negative þ weak 48 (81) 8 (53)
0.024jj
*
Percentage of the column. Percentage of the row. z p value based on Pearson’s x2 test. § Presence or absence of local or distant metachronous recurrence within 5 year follow-up. jj Test based on a two tiered system of TUFM expression: a combined category of negative and weak expression, and a category of strong expression. {
In multivariate Cox regression analysis, including known CRC prognostic factors such as histological grade, histological types, TNM stages, vascular invasion, perineural invasion, type of operation and TUFM immunoreactivity, the following variables were identified as independent prognostic indicators: perineural invasion, TNM stage and TUFM expression (Table 2).
DISCUSSION In this study, we performed IHC staining of mitochondrial Tu translation elongation factor on five blocks of TMAs consisting of 123 CRC cases. The immunoreactivity was determined using computer assisted image analysis software. We found that increased expression of TUFM in CRC was associated with a significantly poorer cancer specific survival and the expression level of TUFM was not significantly correlated with stage. This suggests that the immunoexpression of TUFM may be an important independent prognostic factor for CRC. TMA is a highly efficient experimental tool to analyse the expression pattern of candidate markers in a large number of samples at once with proven reproducibility and correlation
with the data obtained from whole tissue sections.29 However, despite its reported advantages, TMA is often criticised for its small tissue size and its inability in revealing the complete features of the whole tumour section. This is particularly relevant to the present study as we only used a single core from each tumour. To assess the heterogeneity of TUFM expression within tumour, we have performed IHC staining on the whole tumour sections from nine of the blocks from which the cores for the TMAs had been taken (data not shown). The protein was uniformly negatively or positively stained with equal level of intensity across the entire tumour section in seven of nine tumours. Two of nine tumours displayed variability in the staining intensity within the section. Thus, staining heterogeneity is not a major feature for this protein. As all the tissue cores were randomly sampled in a blinded fashion to variability in immunoreactivity, we believe that the accuracy and representativeness of single tumour cores in validating the prognostic significance of TUFM can be assured by including a relatively large cohort of patients (biological replicates) in TMA as reported in this study. Nevertheless, we expect the positive findings in this pilot study to be further validated in a more extensive study involving a much larger number of
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just a result of increased mitochondrial biogenesis as this has been shown to be associated with less aggressive tumours. Therefore, we hypothesised that TUFM is actively up-regulated at the organelle level during the course of tumour progression. Up-regulation of TUFM protein in mitochondria might be a compensatory response to an impairment in mitochondrial activity. As originally suggested by Warburg37 and subsequently evidenced in most solid tumours,38 cancer cells often have an abnormally high rate of glucose uptake and glycolysis, while mitochondria respiration is suppressed even in the presence of adequate oxygen. This is termed the Warburg effect. Associated with this metabolic reprogramming are loss-offunction mutations and down-regulation of expression of many oxidative phosphorylation (OXPHOS) enzymes and electron transport chain complexes in the mitochondria of cancer cells.31,39–42 Although most experimental evidence supports the idea that a glycolytic shift will favour tumour growth by providing both an abundance of biosynthetic intermediate for sustained cell proliferation and sufficient energy under prolonged hypoxic environment,43 excessive mitochondrial damage is deleterious to cell survival.44–46 In particular, it has been reported that in prostate and renal cancer cells prolonged and continuous impairment of protein synthesis inside the mitochondria by tetracycline treatment leads to reduction of the OXPHOS components and proliferation arrest.47 Thus, over-expression of TUFM might be a cytoprotective mechanism that, through reinforcing the overall protein translation efficiency in the mitochondria, compensates for the defective OXPHOS system. In this regard, the level of TUFM over-expression may be considered an indicator of the degree of mitochondria dysfunction which is known be associated with colon tumour progression.23 Selective inhibition of TUFM in tumour cells may trigger mitochondrial collapse and tumour regression. However, this hypothesis remains to be tested. It is important to note that protein EF-Tu, the bacterial ortholog of human TUFM, has been shown to possess molecular chaperon and protein disulfide isomerise activity in protein folding and renaturation after stress, in addition to its role in protein translation.48–50 Consistent with this, EF-Tu expression was found to be inducible by heat shock in a heattolerant maize line,51 and by ischaemia in an ischaemic rat heart model.52 Microenvironmental stresses such as hypoxia, nutritional starvation, reactive oxygen species, acidosis, etc., are also present in solid tumours. Therefore, TUFM might be one of the molecular chaperones that are induced in response to stress. This is in line with the observations that many heat shock proteins also show prognostic implications in certain cancer types.53 In conclusion, our results indicate that increased expression of TUFM is correlated with CRC progression independent of tumour stage. This justifies a large scale study to further
Cum cancer specific survival
1.0 Score 0, n = 31 0.8 Score 1, n = 62 0.6
Score 2, n = 30
0.4
Score 0 vs 2, p = 0.038 Score 0 vs 1, p = 0.226 Score 1 vs 2, p = 0.177
0.2
0.0 0
50
100
150
200
Time (months) Fig. 3 Cancer specific survival of CRC patients in association with TUFM expression of the tumour. The difference in cancer specific survival between TUFM negative and strongly positive tumours was statistically significant ( p ¼ 0.038).
patients with replicate tumour cores taken from each patient. Further validation based on whole tumour sections and anatomical pathologists’ scores is also planned in the future. Despite intensive research on the biochemical properties of TUFM in mitochondrial protein translation, little is known about its specific roles in tumourigenesis, nor its regulatory networks under pathophysiological conditions. In CRC tissues, TUFM displayed a punctate cytoplasmic staining pattern, consistent with a mitochondrial localisation. The specific localisation and function of TUFM in mitochondria suggests that its altered expression may relate to mitochondria dysfunction which is known to be an important contributing factor to tumourigenesis in many types of tumours.30,31 Enhanced expression of TUFM may be a result of increased mitochondrial content in tumour tissues, since mtDNA copy number as a measurement of mitochondrial content has been reported to be increased in the majority of CRC tissues compared to the corresponding normal mucosa.32–34 In vitro experiments also showed that mitochondrial biogenesis may be activated by various endogenous and exogenous factors such as growth stimulations and oxidative stress.35,36 However, evidence has also shown that, although the majority of CRC harbour more mitochondria than normal tissues, mtDNA copy number in fact declines during tumour progression33 and decreased mtDNA is associated with an unfavourable 5 year disease free survival.34 These observations imply that increased TUFM staining in more aggressive tumours is unlikely to be Table 2
Cox regression analysis of tumour characteristics with respect to cancer specific survival
Variable Histological type (mucinous versus non-mucinous) Histological grade (high grade versus low grade) Vascular invasion (positive versus negative) Operation (emergency versus elective) Perineural invasion (positive versus negative) Stage (IV/III versus II/I) TUFM expression (strong versus negative/weak)
Hazard ratio
95% confidence interval
p value
1.25 1.73 1.61 1.62 2.86 7.29 2.36
0.32–4.89 0.69–4.32 0.71–3.62 0.74–3.58 1.08–7.56 2.68–19.82 1.12–4.97
0.751 0.242 0.251 0.230 0.035 <0.001 0.024
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TUFM IN COLORECTAL CARCINOMA
evaluate the usefulness of the immunoexpression of TUFM in stratifying patients for adjuvant chemotherapy. Functional investigations are warranted to explore the molecular mechanism underlying TUFM over-expression and ascertain whether TUFM has therapeutic potential in CRC. Acknowledgements: We are grateful to Dr John Groom at Wakefield Hospital who provided surgical samples for the initial proteomic analysis and optimisation of antibody stainings in this study, Ms Yvette Emmanuel at the Molecular and Clinical Pathology Research Laboratory, Clinical and Statewide Services, Princess Alexandra Hospital (Qld) for facilitating construction of the tissue microarrays used in this study, and Dr Bronwyn Kivell at Victoria University of Wellington for allowing us to use the ImagePro Plus software. Conflicts of interest and sources of funding: The authors wish to thank the Cancer Society of New Zealand (Wellington Division), Wellington Medical Research Foundation, Lotteries Health Research and University of Otago for providing funding in support of this research, and the Tertiary Education Commission (TEC) of New Zealand for providing a PhD scholarship which supported Mr Hongjun Shi. The authors have no conflicts of interest to declare. Address for correspondence: Mr H. Shi, The Wakefield Biomedical Research Unit, University of Otago (Wellington), Private Bag 7909, Wellington South 6242, New Zealand. E-mail: [email protected]
References 1. Ministry of Health New Zealand. Cancer: New Registrations and Deaths 2007. Wellington: Ministry of Health New Zealand, 2010. 2. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010; 60: 277–300. 3. Millikan KW, Staren ED, Doolas A. Invasive therapy of metastatic colorectal cancer to the liver. Surg Clin North Am 1997; 77: 27–48. 4. Aschele C, Bergamo F, Lonardi S. Chemotherapy for operable and advanced colorectal cancer. Cancer Treat Rev 2009; 35: 509–16. 5. O’Connell JB, Maggard MA, Ko CY. Colon cancer survival rates with the new American Joint Committee on Cancer sixth edition staging. J Natl Cancer Inst 2004; 96: 1420–5. 6. Chang GJ, Rodriguez-Bigas MA, Skibber JM, Moyer VA. Lymph node evaluation and survival after curative resection of colon cancer: systematic review. J Natl Cancer Inst 2007; 99: 433–41. 7. Sargent D, Sobrero A, Grothey A, O’Connell MJ, Buyse M, Andre T, et al. Evidence for cure by adjuvant therapy in colon cancer: observations based on individual patient data from 20,898 patients on 18 randomized trials. J Clin Oncol 2009; 27: 872–7. 8. Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Goodman PJ, et al. Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma. N Engl J Med 1990; 322: 352–8. 9. Gray R, Barnwell J, McConkey C, Hills RK, Williams NS, Kerr DJ. Adjuvant chemotherapy versus observation in patients with colorectal cancer: a randomised study. Lancet 2007; 370: 2020–9. 10. Jonker DJ, Spithoff K, Maroun J. Adjuvant systemic chemotherapy for Stage II and III colon cancer after complete resection: an updated practice guideline. Clin Oncol (R Coll Radiol) 2011; 23: 314–22. 11. Benson AB 3rd, Schrag D, Somerfield MR, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol 2004; 22: 3408–19. 12. O’Connor ES, Greenblatt DY, LoConte NK, et al. Adjuvant chemotherapy for stage II colon cancer with poor prognostic features. J Clin Oncol 2011; 29: 3381–8. 13. Lin YH, Friederichs J, Black MA, et al. Multiple gene expression classifiers from different array platforms predict poor prognosis of colorectal cancer. Clin Cancer Res 2007; 13: 498–507. 14. Dundas SR, Lawrie LC, Rooney PH, Murray GI. Mortalin is over-expressed by colorectal adenocarcinomas and correlates with poor survival. J Pathol 2005; 205: 74–81.
511
15. Pei H, Zhu H, Zeng S, et al. Proteome analysis and tissue microarray for profiling protein markers associated with lymph node metastasis in colorectal cancer. J Proteome Res 2007; 6: 2495–501. 16. Ma YL, Peng JY, Zhang P, et al. Heterogeneous nuclear ribonucleoprotein A1 is identified as a potential biomarker for colorectal cancer based on differential proteomics technology. J Proteome Res 2009; 8: 4525–35. 17. Qi Y, Chiu JF, Wang L, Kwong DL, He QY. Comparative proteomic analysis of esophageal squamous cell carcinoma. Proteomics 2005; 5: 2960–71. 18. Li LS, Kim H, Rhee H, et al. Proteomic analysis distinguishes basaloid carcinoma as a distinct subtype of nonsmall cell lung carcinoma. Proteomics 2004; 4: 3394–400. 19. He QY, Cheung YH, Leung SY, Yuen ST, Chu KM, Chiu JF. Diverse proteomic alterations in gastric adenocarcinoma. Proteomics 2004; 4: 3276–87. 20. Spremulli LL, Coursey A, Navratil T, Hunter SE. Initiation and elongation factors in mammalian mitochondrial protein biosynthesis. Prog Nucleic Acid Res Mol Biol 2004; 77: 211–61. 21. Dakubo GD. Mitochondrial Genetics and Cancer. Berlin: Springer-Verlag, 2010. 22. Bellance N, Lestienne P, Rossignol R. Mitochondria: from bioenergetics to the metabolic regulation of carcinogenesis. Front Biosci 2009; 14: 4015– 34. 23. Sanchez-Arago M, Chamorro M, Cuezva JM. Selection of cancer cells with repressed mitochondria triggers colon cancer progression. Carcinogenesis 2010; 31: 567–76. 24. Inokuma T, Haraguchi M, Fujita F, Tajima Y, Kanematsu T. Oxidative stress and tumor progression in colorectal cancer. Hepatogastroenterology 2009; 56: 343–7. 25. Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996; 32A: 30–8. 26. Zhou J, Schmid T, Schnitzer S, Brune B. Tumor hypoxia and cancer progression. Cancer Lett 2006; 237: 10–21. 27. AJCC. AJCC Cancer Staging Handbook. New York: Springer, 2010. 28. Hamilton SR, Aaltonen LA. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Digestive System. Lyon: IARC Press, 2000. 29. Lee HS, Kim WH. Tissue array methods for high-throughput clinicopathologic research. Cancer Res Treat 2006; 38: 1–6. 30. Cuezva JM, Ortega AD, Willers I, Sanchez-Cenizo L, Aldea M, SanchezArago M. The tumor suppressor function of mitochondria: translation into the clinics. Biochim Biophys Acta 2009; 1792: 1145–58. 31. Cuezva JM, Krajewska M, de Heredia ML, et al. The bioenergetic signature of cancer: a marker of tumor progression. Cancer Res 2002; 62: 6674–81. 32. Lee HC, Yin PH, Lin JC, et al. Mitochondrial genome instability and mtDNA depletion in human cancers. Ann NY Acad Sci 2005; 1042: 109–22. 33. Lin PC, Lin JK, Yang SH, Wang HS, Li AF, Chang SC. Expression of betaF1-ATPase and mitochondrial transcription factor A and the change in mitochondrial DNA content in colorectal cancer: clinical data analysis and evidence from an in vitro study. Int J Colorectal Dis 2008; 23: 1223–32. 34. Chang SC, Lin PC, Yang SH, Wang HS, Liang WY, Lin JK. Mitochondrial D-loop mutation is a common event in colorectal cancers with p53 mutations. Int J Colorectal Dis 2009; 24: 623–8. 35. Dang CV, Li F, Lee LA. Could MYC induction of mitochondrial biogenesis be linked to ROS production and genomic instability? Cell Cycle 2005; 4: 1465–6. 36. Echave P, Machado-da-Silva G, Arkell RS, et al. Extracellular growth factors and mitogens cooperate to drive mitochondrial biogenesis. J Cell Sci 2009; 122: 4516–25. 37. Warburg O. On the origin of cancer cells. Science 1956; 123: 309–14. 38. Kim JW, Dang CV. Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 2006; 66: 8927–30. 39. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006; 3: 177–85. 40. Simonnet H, Alazard N, Pfeiffer K, et al. Low mitochondrial respiratory chain content correlates with tumor aggressiveness in renal cell carcinoma. Carcinogenesis 2002; 23: 759–68. 41. Chatterjee A, Mambo E, Sidransky D. Mitochondrial DNA mutations in human cancer. Oncogene 2006; 25: 4663–74. 42. Pollard PJ, Briere JJ, Alam NA, et al. Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol Genet 2005; 14: 2231–9. 43. Formentini L, Martinez-Reyes I, Cuezva JM. The mitochondrial bioenergetic capacity of carcinomas. IUBMB Life 2010; 62: 554–60. 44. Berridge MV, Tan AS. Effects of mitochondrial gene deletion on tumorigenicity of metastatic melanoma: reassessing the Warburg effect. Rejuvenation Res 2010; 13: 139–41. 45. Weinberg F, Hamanaka R, Wheaton WW, et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci USA 2010; 107: 8788–93.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
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46. Cavalli LR, Varella-Garcia M, Liang BC. Diminished tumorigenic phenotype after depletion of mitochondrial DNA. Cell Growth Differ 1997; 8: 1189–98. 47. van den Bogert C, Dontje BH, Holtrop M, et al. Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis. Cancer Res 1986; 46: 3283–9. 48. Kudlicki W, Coffman A, Kramer G, Hardesty B. Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing. J Biol Chem 1997; 272: 32206–10. 49. Caldas TD, El Yaagoubi A, Richarme G. Chaperone properties of bacterial elongation factor EF-Tu. J Biol Chem 1998; 273: 11478–82.
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50. Richarme G. Protein-disulfide isomerase activity of elongation factor EF-Tu. Biochem Biophys Res Commun 1998; 252: 156–61. 51. Bhadula SK, Elthon TE, Habben JE, Helentjaris TG, Jiao S, Ristic Z. Heatstress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line. Planta 2001; 212: 359–66. 52. Sakai J, Ishikawa H, Kojima S, Satoh H, Yamamoto S, Kanaoka M. Proteomic analysis of rat heart in ischemia and ischemia-reperfusion using fluorescence two-dimensional difference gel electrophoresis. Proteomics 2003; 3: 1318–24. 53. Ciocca DR, Calderwood SK. Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones 2005; 10: 86–103.
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ANATOMICAL PATHOLOGY
TUFM is a potential new prognostic indicator for colorectal carcinoma HONGJUN SHI*, MARK HAYES*, CHANDRA KIRANA*, ROSEMARY MILLER{, JOHN KEATING{, DONIA MACARTNEY-COXSON§ AND RICHARD STUBBS* *Wakefield Biomedical Research Unit, and {Department of Pathology and Molecular Medicine, University of Otago (Wellington), zWellington Hospital, and §Institute of Environmental Science and Research (ESR), New Zealand
Summary Aims: Mitochondrial Tu translation elongation factor (TUFM) is a nuclear encoded protein that participates in mitochondrial polypeptide translation. TUFM has been reported to be overexpressed in many tumour types including colorectal carcinoma (CRC) by proteomics. The present study aims to examine the prognostic implication of TUFM in CRC. Methods: Immunohistochemical staining was performed in tissue microarrays composed of 123 cases of CRC using a polyclonal anti-TUFM antibody. Immunoreactivity was quantified using Image-Pro plus software, and analysed in association with patients’ clinicopathological parameters and survival time. Results: The immunoreactivity of TUFM was negative in 25%, weak in 50% and strong in 25% of CRC cases. TUFM immunoreactivity had no significant association with the clinicopathological parameters examined including TNM stage and grade. However, strong TUFM expression significantly correlated with a higher 5-year recurrence rate ( p ¼ 0.024). Kaplan–Meier analysis revealed that patients with strong TUFM expression had significantly shorter cancerspecific survival than patients with negative TUFM (log-rank test, p ¼ 0.038). In multivariate analysis, strong TUFM expression remained a stage-independent unfavourable prognostic indicator ( p ¼ 0.024). Conclusions: Increased expression of TUFM is a promising new prognostic indicator for CRC. Selective inhibition of TUFM in tumour cells may present a new avenue for the targeted therapy of this cancer. Key words: Colorectal carcinoma, mitochondria, prognosis, TUFM. Received 7 November 2011, revised 2 February, accepted 10 February 2012
INTRODUCTION Colorectal cancer is an important health problem both internationally and in New Zealand. Every year 2800 New Zealanders are diagnosed with CRC and approximately 1250 die from it, making CRC the second most common cancer registered and the second most common cause of death from cancer.1 As a result of advances in cancer screening and treatments, there has been a steady reduction in the death rate during the past few decades. However, at present, still more than 35% of CRC patients will not survive more than 5 years, mainly due to metastatic spread of the tumour to distant organs.2 Approximately 20% of CRC patients have detectable metastases at the time of their first consultation (synchronous metastasis)2 and a further one-third of the remaining patients Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e3283559cbe
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will go on to develop metastasis after surgical removal of their primary tumours.3 Thus, avenues for improving the prediction and prevention of metastatic spread may be the most promising approach to reducing the CRC mortality rate. Staging of CRC is useful in predicting the probability of cancer recurrence after surgical removal of the primary tumour and has become the gold standard in selecting patients who would benefit most from adjuvant chemotherapy.4 While patients with tumours limited to the bowel wall (TNM stage I) can be cured by surgery alone in more than 90% of cases, patients with tumours penetrating through the bowel wall (stage II) or involving the regional lymph nodes (stage III) have a substantial risk of relapse after surgical resection, with 5 year survival rates of approximately 80% and 60% for stage II and III, respectively.5 Although CRC prognosis is stage dependent, tumours of the same stage may have significantly different clinical outcomes.6 For stage III disease, clinical trials have consistently demonstrated a 10–13% absolute survival benefit from 5-fluorouracil based adjuvant chemotherapy which is now a standard of care around the world.7 However, approximately 30–44% of stage III patients do not develop metastases in the absence of chemotherapy and thus may have been overtreated.8 Stage II patients have a better prognosis. Current chemotherapy has shown a small but not consistently significant improvement in survival for stage II patients and is not routinely provided.9 Nonetheless, 20% of stage II patients develop secondary tumours that might have been prevented or delayed by adjuvant chemotherapy.9 Patients with high-risk stage II cancers, e.g., cancers with inadequately sampled nodes, T4 lesions, perforation or poorly differentiated histology, may be considered for adjuvant chemotherapy, although there has been no direct clinical trial evidence of benefit of adjuvant chemotherapy in such patients.10–12 Additional prognostic indicators are required to improve the accuracy of stratification of patients at increased risk of metastasis in whom more intensive therapy and monitoring are justified. CRC initiation, progression and metastasis are complex processes caused by multiple alterations within cells to ultimately influence change in cellular behaviour. Recent development in proteomic technology has allowed comparison of multiple proteins from different tissues to be accomplished with good reproducibility and sensitivity. This has led to identification of many putative protein markers with prognostic significance in CRC.13–16 In our previous proteomic study, we identified and validated the protein mitochondrial Tu translation elongation factor (TUFM) as being over-expressed in CRC compared to the patient matched normal colon mucosa. In addition, TUFM has also been reported to be increased in lung,
2012 Royal College of Pathologists of Australasia
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TUFM IN COLORECTAL CARCINOMA
gastric and oesophageal cancer by proteomics.17 – 19 Despite its frequent association with cancer in clinical onco-proteomic studies, little is known about its biological role in tumour development. TUFM has been most extensively studied as a fundamental molecule involved in GTP-dependent recruitment of codon-specified aminoacyl-tRNA to the ribosome during protein translation in mitochondria.20 TUFM along with a large number of other nuclear-encoded protein factors constitute the mitochondrial translation apparatus responsible for translation of all 13 mtDNA-encoded proteins that partially constitute the five respiratory chain complexes.21,22 Interestingly, in addition to its conventional role in peptide elongation in mitochondria, TUFM also displays chaperone-like properties in protein folding and renaturation under stress conditions, suggesting TUFM over-expression in CRC might also be a stress response. Since both the mitochondrial defects and the cellular stress such as hypoxia and oxidative stress have been shown to relate to carcinogenesis and tumour progression,23 – 26 the present study aims to evaluate the prognostic significance of TUFM in CRC.
MATERIALS AND METHODS Patients Out of the 282 patients who had undergone partial colectomy or anterior resection of CRC performed by Dr John Keating from 1997 to 2005 at Wellington Hospital, 169 patients had archival tissue blocks available for investigation at the time of this study. A representative block from each patient was drawn and sectioned for H&E staining. On histological examination, 42 blocks were excluded from the cohort due to the absence or inadequacy of tumour cells in the sections from the blocks initially chosen from the tissue archives. Consequently, a total of 127 CRC cases were finally included in this study. Clinicopathological features of the resected CRC were obtained from a prospective surgical database according to the clinical and pathological reports held at Wellington Hospital. Pathological stages were classified according to the TNM staging system.27 Histological grading and typing of the tumour was determined according to the World Health Organization tumour classification system.28 Cancer specific survival was defined as the interval between the date of the first operation of the primary tumour to the date when the patient died from recurrent CRC. Cases were censored at the end of the follow-up or at the time of death due to other causes. Thirty-seven patients died of recurrent CRC during the follow-up period. Median follow-up time was 61 months (range 2–164 months). Construction of tissue microarray (TMA) and immunohistochemistry on the TMAs using archival human tissues was conducted with the approval of the New Zealand Central Regional Ethics Committee. Tissue microarray Five TMA blocks containing a total of 127 CRC cases were constructed. Each TMA consisted of up to 26 tissue cores with a single tissue core per patient’s tumour. Formalin fixed, paraffin embedded tissue blocks of CRC were obtained from the hospital tumour archive. Before constructing TMA, a 4 mm section was sliced from each tumour block for a routine H&E inspection by a pathologist. After histological confirmation of the tumours, areas of sampling (AOS) were defined and marked on the microscope slide by the pathologist. These microscope slides with the spotted AOS were used later as a reference to guide the location of tissue cores for punching. AOS was defined as the area of obvious invasive cancer close to the lumen, not including any potential adenomatous areas. TMA was constructed using the Beecher Automated Tissue Arrayer (ATA-27, Beecher Instruments, USA) through the Molecular and Clinical Pathology Research Laboratory, Clinical and Statewide Services, Princess Alexandra Hospital, Queensland, Australia. A tissue core with a diameter of 1 mm was punched from the donor tissue under the guidance of the reference slides, and transferred to a recipient paraffin block (array margin of 10 20 mm). Once the TMAs were made, they were heat
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cycled from 448C for 1 h and room temperature (RT) for 1 h for a total of 5 cycles to aid cutting. Immunohistochemistry A 4 mm thick tissue section was cut from each TMA block. All subsequent procedures were carried out at RT unless otherwise stated. Tissue sections were serially rehydrated. Antigen retrieval was performed by submerging the slides in sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) in a boiling water bath for 25 min. After 25 min cooling at RT, tissue sections were incubated in 3% H2O2 in PBS-T for 10 min to block endogenous peroxidase. A circle of hydrophobic barrier was drawn around the specimen by a Pap pen (Invitrogen, USA). Sections were then sequentially blocked in 2.5% normal horse serum in PBS-T for 30 min and avidin/biotin blocking reagents for 20 min according the manufacturer’s instruction (Invitrogen). Tissues were then incubated in a humidified chamber with the goat polyclonal anti-TUFM antibody (1:100, sc-12991; Santa Cruz Biotechnology, USA) diluted in 1.25% normal horse serum in PBS-T for 1 h. Specific bindings were detected using VECTASTAIN Universal Quick Kit (Vector Laboratories, USA) and DAB substrate (Invitrogen). A non-immune normal goat IgG (1:200, sc-2028; Santa Cruz Biotechnology) was used as a negative control. Image analysis and scoring Immunoreactivity of TUFM was quantified using the Image-Pro Plus software (version 6) (Media Cybernetics, USA). Briefly, a digital image (TIFF format) under 20 objective lens was taken near the centre of each tumour core (Eclipse 80i; Nikon, Japan). The imaging field under this objective covered approximately 40% of the entire core. Images of all tissue cores were acquired at the same time with a constant set of imaging parameters on the microscope and imaging software. The images were then subject to optical density analysis by the Image-Pro Plus software. Adjustments of background and colour intensity range were performed on a representative image showing high immunoreactivity. The black and incident level (representing the optical density of the maximal positive staining and white background, respectively) were determined from an immunostained region and a blank region in the image, respectively. Intensity range selection was based on histogram, with intensity (I) and saturation (S) set at maximum, and hue (H) set at a range where most of the brown DAB colour was selected while blue nuclei counterstain was excluded. These settings were saved and subsequently applied to all images analysed. On opening each image, an area of interest (AOI) was drawn to include most of the malignant epithelial cells in the image and exclude stroma, muscle, necrosis and other non-tumour components. After defining the AOI, the mean optical density of the selected area [integrated optical density (IOD)/unit area] was determined by the software. This represents the immunoreactivity of the candidate protein within tumour tissue. To facilitate statistical analysis, IOD/unit area was manually categorised into three classes of immunoreactivity (negative, weak and strong) as described in the Results section. Statistical analysis All statistical analyses were performed using SPSS (version 17; SPSS, USA). The association between TUFM immunoreactivity and patient clinicopathological parameters was assessed by x2 test. The impact of TUFM on patient survival was examined by Kaplan–Meier curve analysis and the statistical significance was determined using log-rank test. A multivariate analysis based on Cox proportional hazard regression model was applied to determine independent prognostic factors. Variables included in Cox regression analysis were histological grade, histological types, TNM stages, vascular invasion, perineural invasion, type of operation and TUFM immunoreactivity. A p value of less than 0.05 was considered significant.
RESULTS Expression pattern of TUFM in CRC tissues TUFM was generally homogeneously expressed in all neoplastic epithelial cells in the TUFM positive tumour cores. TUFM was nearly absent or only weakly expressed in the stroma. In positive neoplastic cells, TUFM exhibited a punctate staining pattern throughout the cytoplasm. A representative staining is shown in Fig. 1.
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Fig. 1 A representative photomicrograph of TUFM staining in a colorectal tumour. TUFM exhibited a punctate cytoplasmic staining pattern in neoplastic cells (T). Very few stroma cells (S) were positively stained.
Correlation of TUFM expression and tumour characteristics Of the 127 tissues core sections, four sections were lost during the sectioning procedure. A total of 123 tissue sections were included in the image and statistical analysis. The TUFM immunoreactivity varied considerably among the 123 patients (IOD/unit area ranged from 0 to 167, with the mean across samples of 27.56 36.65 SD). To facilitate statistical analysis, IOD/unit area was manually categorised into four groups of equal number of patients across the subgroups of immunoreactivity (negative, weakly positive, moderately positive and strongly positive, respectively). Because subsequent Kaplan– Meier curve analysis showed that subdivision of the weakly positive and moderately positive cases did not produce additional discriminatory power in predicting prognosis (data not shown), these two middle classes were combined as a single
class of ‘weakly positive’ cases in the final statistical analysis. This results in two cut-off points (1.5 and 41.0 IOD/unit area) which broke the entire cohort of patients into three categories with negative (score 0, 25%), weak (score 1, 50%) and strong immunoreactivity (score 2, 25%) respectively (Fig. 2). The relationship between TUFM immunoreactivity and tumour characteristics is shown in Table 1. No statistically significant correlation was seen between TUFM expression and age, gender, tumour location, histological type, histological grade, vascular invasion, perineural invasion, TNM stage, nodal status or depth of invasion. Increased TUFM expression tended to correlate with an increased probability of 5-year recurrence, although it did not reach statistical significance ( p ¼ 0.078). Notably, in this analysis one category has only three events, making the statistical analysis less accurate. To improve this, the number of tumours with negative and weak expression was combined, and the resulting two tiered grades were re-subjected to x2 tests in association with cancer recurrence. The analysis showed that the 5-year recurrence rate was significantly higher in patients with strong TUFM expression in tumours than those with weak or negative TUFM expression (39% versus 14%, p ¼ 0.024).
Survival analysis The correlation between TUFM expression and cancer specific survival was assessed by univariate analysis using Kaplan– Meier curve and log-rank test. As shown in Fig. 3, patients with strong TUFM expression showed a significantly shorter cancer specific survival than patients with negative TUFM expression (55% versus 80% 5 year cumulative probability of cancer specific survival, p ¼ 0.038). The difference in cancer specific survival between TUFM negative and weakly positive tumours, and between TUFM weakly positive and strongly positive tumours was not significant ( p ¼ 0.226 and 0.177, respectively).
A
B
0.03 Negative (score 0) 0–1.5 n = 31
25.25 Weak (score 1) 1.6–41.0 n = 62
148.14 IOD/unit area Strong (score 2) 41.1–166.8 IOD/unit area n = 30
Fig. 2 Representative CRC tissues showing negative, weak and strong TUFM expression. The brown immunostains in the original images (A) were detected by the Image-Pro Plus software (marked in red) based on a pre-defined colour intensity selection range (B). The staining intensity was only determined for the area of interest (AOI) (demarcated in green line), and the level of immunoreactivity was expressed as integrated optical density (IOD)/unit area.
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TUFM IN COLORECTAL CARCINOMA
Table 1
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Correlation between TUFM immunoreactivity and tumour characteristics
Clinicopathological parameters Age <65 65 Gender F M Tumour location Colon Rectum Histological type Non-mucinous Mucinous Histological grade High grade Low grade Vascular invasion Negative Positive Perineural invasion Negative Positive TNM stages I II III IV Lymph node metastasis Negative Positive Depth of invasion T1/T2 T3/T4 5 year recurrence§ Recurrence free Recurrent 5 year recurrence§ Recurrence free Recurrent
No. of cases (%*)
Negative (%{)
Weak (%{)
Strong (%{)
36 (29) 87 (71)
8 (22) 23 (26)
20 (56) 42 (48)
8 (22) 22 (25)
62 (50) 61 (50)
21 (34) 10 (16)
27 (44) 35 (57)
14 (23) 16 (26)
85 (69) 38 (31)
23 (27) 8 (21)
44 (52) 18 (47)
18 (21) 12 (32)
106 (87) 16 (13)
25 (24) 5 (31)
53 (50) 9 (56)
28 (26) 2 (13)
24 (20) 98 (80)
6 (25) 25 (26)
14 (58) 47 (48)
4 (17) 26 (27)
94 (76) 29 (24)
25 (27) 6 (21)
46 (49) 16 (55)
23 (24) 7 (24)
113 (93) 9 (7)
30 (27) 1 (11)
56 (50) 5 (56)
27 (24) 3 (33)
3 17 9 2
12 21 14 13
5 8 11 6
pz value 0.761
0.080
0.440
0.464
0.554
0.787
0.078
0.176 20 46 34 21
(17) (38) (28) (17)
(15) (37) (26) (10)
(60) (46) (41) (62)
(25) (17) (32) (29) 0.109
70 (60) 47 (40)
22 (31) 8 (17)
34 (49) 23 (49)
14 (20) 16 (34)
23 (19) 97 (81)
3 (13) 28 (29)
14 (61) 45 (46)
6 (26) 24 (25)
31 (53) 5 (33)
11 (19) 7 (47) Strong 11 (19) 7 (47)
0.271 0.078 59 (80) 15 (20) 59 (80) 15 (20)
17 (29) 3 (20) Negative þ weak 48 (81) 8 (53)
0.024jj
*
Percentage of the column. Percentage of the row. z p value based on Pearson’s x2 test. § Presence or absence of local or distant metachronous recurrence within 5 year follow-up. jj Test based on a two tiered system of TUFM expression: a combined category of negative and weak expression, and a category of strong expression. {
In multivariate Cox regression analysis, including known CRC prognostic factors such as histological grade, histological types, TNM stages, vascular invasion, perineural invasion, type of operation and TUFM immunoreactivity, the following variables were identified as independent prognostic indicators: perineural invasion, TNM stage and TUFM expression (Table 2).
DISCUSSION In this study, we performed IHC staining of mitochondrial Tu translation elongation factor on five blocks of TMAs consisting of 123 CRC cases. The immunoreactivity was determined using computer assisted image analysis software. We found that increased expression of TUFM in CRC was associated with a significantly poorer cancer specific survival and the expression level of TUFM was not significantly correlated with stage. This suggests that the immunoexpression of TUFM may be an important independent prognostic factor for CRC. TMA is a highly efficient experimental tool to analyse the expression pattern of candidate markers in a large number of samples at once with proven reproducibility and correlation
with the data obtained from whole tissue sections.29 However, despite its reported advantages, TMA is often criticised for its small tissue size and its inability in revealing the complete features of the whole tumour section. This is particularly relevant to the present study as we only used a single core from each tumour. To assess the heterogeneity of TUFM expression within tumour, we have performed IHC staining on the whole tumour sections from nine of the blocks from which the cores for the TMAs had been taken (data not shown). The protein was uniformly negatively or positively stained with equal level of intensity across the entire tumour section in seven of nine tumours. Two of nine tumours displayed variability in the staining intensity within the section. Thus, staining heterogeneity is not a major feature for this protein. As all the tissue cores were randomly sampled in a blinded fashion to variability in immunoreactivity, we believe that the accuracy and representativeness of single tumour cores in validating the prognostic significance of TUFM can be assured by including a relatively large cohort of patients (biological replicates) in TMA as reported in this study. Nevertheless, we expect the positive findings in this pilot study to be further validated in a more extensive study involving a much larger number of
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just a result of increased mitochondrial biogenesis as this has been shown to be associated with less aggressive tumours. Therefore, we hypothesised that TUFM is actively up-regulated at the organelle level during the course of tumour progression. Up-regulation of TUFM protein in mitochondria might be a compensatory response to an impairment in mitochondrial activity. As originally suggested by Warburg37 and subsequently evidenced in most solid tumours,38 cancer cells often have an abnormally high rate of glucose uptake and glycolysis, while mitochondria respiration is suppressed even in the presence of adequate oxygen. This is termed the Warburg effect. Associated with this metabolic reprogramming are loss-offunction mutations and down-regulation of expression of many oxidative phosphorylation (OXPHOS) enzymes and electron transport chain complexes in the mitochondria of cancer cells.31,39–42 Although most experimental evidence supports the idea that a glycolytic shift will favour tumour growth by providing both an abundance of biosynthetic intermediate for sustained cell proliferation and sufficient energy under prolonged hypoxic environment,43 excessive mitochondrial damage is deleterious to cell survival.44–46 In particular, it has been reported that in prostate and renal cancer cells prolonged and continuous impairment of protein synthesis inside the mitochondria by tetracycline treatment leads to reduction of the OXPHOS components and proliferation arrest.47 Thus, over-expression of TUFM might be a cytoprotective mechanism that, through reinforcing the overall protein translation efficiency in the mitochondria, compensates for the defective OXPHOS system. In this regard, the level of TUFM over-expression may be considered an indicator of the degree of mitochondria dysfunction which is known be associated with colon tumour progression.23 Selective inhibition of TUFM in tumour cells may trigger mitochondrial collapse and tumour regression. However, this hypothesis remains to be tested. It is important to note that protein EF-Tu, the bacterial ortholog of human TUFM, has been shown to possess molecular chaperon and protein disulfide isomerise activity in protein folding and renaturation after stress, in addition to its role in protein translation.48–50 Consistent with this, EF-Tu expression was found to be inducible by heat shock in a heattolerant maize line,51 and by ischaemia in an ischaemic rat heart model.52 Microenvironmental stresses such as hypoxia, nutritional starvation, reactive oxygen species, acidosis, etc., are also present in solid tumours. Therefore, TUFM might be one of the molecular chaperones that are induced in response to stress. This is in line with the observations that many heat shock proteins also show prognostic implications in certain cancer types.53 In conclusion, our results indicate that increased expression of TUFM is correlated with CRC progression independent of tumour stage. This justifies a large scale study to further
Cum cancer specific survival
1.0 Score 0, n = 31 0.8 Score 1, n = 62 0.6
Score 2, n = 30
0.4
Score 0 vs 2, p = 0.038 Score 0 vs 1, p = 0.226 Score 1 vs 2, p = 0.177
0.2
0.0 0
50
100
150
200
Time (months) Fig. 3 Cancer specific survival of CRC patients in association with TUFM expression of the tumour. The difference in cancer specific survival between TUFM negative and strongly positive tumours was statistically significant ( p ¼ 0.038).
patients with replicate tumour cores taken from each patient. Further validation based on whole tumour sections and anatomical pathologists’ scores is also planned in the future. Despite intensive research on the biochemical properties of TUFM in mitochondrial protein translation, little is known about its specific roles in tumourigenesis, nor its regulatory networks under pathophysiological conditions. In CRC tissues, TUFM displayed a punctate cytoplasmic staining pattern, consistent with a mitochondrial localisation. The specific localisation and function of TUFM in mitochondria suggests that its altered expression may relate to mitochondria dysfunction which is known to be an important contributing factor to tumourigenesis in many types of tumours.30,31 Enhanced expression of TUFM may be a result of increased mitochondrial content in tumour tissues, since mtDNA copy number as a measurement of mitochondrial content has been reported to be increased in the majority of CRC tissues compared to the corresponding normal mucosa.32–34 In vitro experiments also showed that mitochondrial biogenesis may be activated by various endogenous and exogenous factors such as growth stimulations and oxidative stress.35,36 However, evidence has also shown that, although the majority of CRC harbour more mitochondria than normal tissues, mtDNA copy number in fact declines during tumour progression33 and decreased mtDNA is associated with an unfavourable 5 year disease free survival.34 These observations imply that increased TUFM staining in more aggressive tumours is unlikely to be Table 2
Cox regression analysis of tumour characteristics with respect to cancer specific survival
Variable Histological type (mucinous versus non-mucinous) Histological grade (high grade versus low grade) Vascular invasion (positive versus negative) Operation (emergency versus elective) Perineural invasion (positive versus negative) Stage (IV/III versus II/I) TUFM expression (strong versus negative/weak)
Hazard ratio
95% confidence interval
p value
1.25 1.73 1.61 1.62 2.86 7.29 2.36
0.32–4.89 0.69–4.32 0.71–3.62 0.74–3.58 1.08–7.56 2.68–19.82 1.12–4.97
0.751 0.242 0.251 0.230 0.035 <0.001 0.024
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TUFM IN COLORECTAL CARCINOMA
evaluate the usefulness of the immunoexpression of TUFM in stratifying patients for adjuvant chemotherapy. Functional investigations are warranted to explore the molecular mechanism underlying TUFM over-expression and ascertain whether TUFM has therapeutic potential in CRC. Acknowledgements: We are grateful to Dr John Groom at Wakefield Hospital who provided surgical samples for the initial proteomic analysis and optimisation of antibody stainings in this study, Ms Yvette Emmanuel at the Molecular and Clinical Pathology Research Laboratory, Clinical and Statewide Services, Princess Alexandra Hospital (Qld) for facilitating construction of the tissue microarrays used in this study, and Dr Bronwyn Kivell at Victoria University of Wellington for allowing us to use the ImagePro Plus software. Conflicts of interest and sources of funding: The authors wish to thank the Cancer Society of New Zealand (Wellington Division), Wellington Medical Research Foundation, Lotteries Health Research and University of Otago for providing funding in support of this research, and the Tertiary Education Commission (TEC) of New Zealand for providing a PhD scholarship which supported Mr Hongjun Shi. The authors have no conflicts of interest to declare. Address for correspondence: Mr H. Shi, The Wakefield Biomedical Research Unit, University of Otago (Wellington), Private Bag 7909, Wellington South 6242, New Zealand. E-mail: [email protected]
References 1. Ministry of Health New Zealand. Cancer: New Registrations and Deaths 2007. Wellington: Ministry of Health New Zealand, 2010. 2. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010; 60: 277–300. 3. Millikan KW, Staren ED, Doolas A. Invasive therapy of metastatic colorectal cancer to the liver. Surg Clin North Am 1997; 77: 27–48. 4. Aschele C, Bergamo F, Lonardi S. Chemotherapy for operable and advanced colorectal cancer. Cancer Treat Rev 2009; 35: 509–16. 5. O’Connell JB, Maggard MA, Ko CY. Colon cancer survival rates with the new American Joint Committee on Cancer sixth edition staging. J Natl Cancer Inst 2004; 96: 1420–5. 6. Chang GJ, Rodriguez-Bigas MA, Skibber JM, Moyer VA. Lymph node evaluation and survival after curative resection of colon cancer: systematic review. J Natl Cancer Inst 2007; 99: 433–41. 7. Sargent D, Sobrero A, Grothey A, O’Connell MJ, Buyse M, Andre T, et al. Evidence for cure by adjuvant therapy in colon cancer: observations based on individual patient data from 20,898 patients on 18 randomized trials. J Clin Oncol 2009; 27: 872–7. 8. Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Goodman PJ, et al. Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma. N Engl J Med 1990; 322: 352–8. 9. Gray R, Barnwell J, McConkey C, Hills RK, Williams NS, Kerr DJ. Adjuvant chemotherapy versus observation in patients with colorectal cancer: a randomised study. Lancet 2007; 370: 2020–9. 10. Jonker DJ, Spithoff K, Maroun J. Adjuvant systemic chemotherapy for Stage II and III colon cancer after complete resection: an updated practice guideline. Clin Oncol (R Coll Radiol) 2011; 23: 314–22. 11. Benson AB 3rd, Schrag D, Somerfield MR, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol 2004; 22: 3408–19. 12. O’Connor ES, Greenblatt DY, LoConte NK, et al. Adjuvant chemotherapy for stage II colon cancer with poor prognostic features. J Clin Oncol 2011; 29: 3381–8. 13. Lin YH, Friederichs J, Black MA, et al. Multiple gene expression classifiers from different array platforms predict poor prognosis of colorectal cancer. Clin Cancer Res 2007; 13: 498–507. 14. Dundas SR, Lawrie LC, Rooney PH, Murray GI. Mortalin is over-expressed by colorectal adenocarcinomas and correlates with poor survival. J Pathol 2005; 205: 74–81.
511
15. Pei H, Zhu H, Zeng S, et al. Proteome analysis and tissue microarray for profiling protein markers associated with lymph node metastasis in colorectal cancer. J Proteome Res 2007; 6: 2495–501. 16. Ma YL, Peng JY, Zhang P, et al. Heterogeneous nuclear ribonucleoprotein A1 is identified as a potential biomarker for colorectal cancer based on differential proteomics technology. J Proteome Res 2009; 8: 4525–35. 17. Qi Y, Chiu JF, Wang L, Kwong DL, He QY. Comparative proteomic analysis of esophageal squamous cell carcinoma. Proteomics 2005; 5: 2960–71. 18. Li LS, Kim H, Rhee H, et al. Proteomic analysis distinguishes basaloid carcinoma as a distinct subtype of nonsmall cell lung carcinoma. Proteomics 2004; 4: 3394–400. 19. He QY, Cheung YH, Leung SY, Yuen ST, Chu KM, Chiu JF. Diverse proteomic alterations in gastric adenocarcinoma. Proteomics 2004; 4: 3276–87. 20. Spremulli LL, Coursey A, Navratil T, Hunter SE. Initiation and elongation factors in mammalian mitochondrial protein biosynthesis. Prog Nucleic Acid Res Mol Biol 2004; 77: 211–61. 21. Dakubo GD. Mitochondrial Genetics and Cancer. Berlin: Springer-Verlag, 2010. 22. Bellance N, Lestienne P, Rossignol R. Mitochondria: from bioenergetics to the metabolic regulation of carcinogenesis. Front Biosci 2009; 14: 4015– 34. 23. Sanchez-Arago M, Chamorro M, Cuezva JM. Selection of cancer cells with repressed mitochondria triggers colon cancer progression. Carcinogenesis 2010; 31: 567–76. 24. Inokuma T, Haraguchi M, Fujita F, Tajima Y, Kanematsu T. Oxidative stress and tumor progression in colorectal cancer. Hepatogastroenterology 2009; 56: 343–7. 25. Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996; 32A: 30–8. 26. Zhou J, Schmid T, Schnitzer S, Brune B. Tumor hypoxia and cancer progression. Cancer Lett 2006; 237: 10–21. 27. AJCC. AJCC Cancer Staging Handbook. New York: Springer, 2010. 28. Hamilton SR, Aaltonen LA. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Digestive System. Lyon: IARC Press, 2000. 29. Lee HS, Kim WH. Tissue array methods for high-throughput clinicopathologic research. Cancer Res Treat 2006; 38: 1–6. 30. Cuezva JM, Ortega AD, Willers I, Sanchez-Cenizo L, Aldea M, SanchezArago M. The tumor suppressor function of mitochondria: translation into the clinics. Biochim Biophys Acta 2009; 1792: 1145–58. 31. Cuezva JM, Krajewska M, de Heredia ML, et al. The bioenergetic signature of cancer: a marker of tumor progression. Cancer Res 2002; 62: 6674–81. 32. Lee HC, Yin PH, Lin JC, et al. Mitochondrial genome instability and mtDNA depletion in human cancers. Ann NY Acad Sci 2005; 1042: 109–22. 33. Lin PC, Lin JK, Yang SH, Wang HS, Li AF, Chang SC. Expression of betaF1-ATPase and mitochondrial transcription factor A and the change in mitochondrial DNA content in colorectal cancer: clinical data analysis and evidence from an in vitro study. Int J Colorectal Dis 2008; 23: 1223–32. 34. Chang SC, Lin PC, Yang SH, Wang HS, Liang WY, Lin JK. Mitochondrial D-loop mutation is a common event in colorectal cancers with p53 mutations. Int J Colorectal Dis 2009; 24: 623–8. 35. Dang CV, Li F, Lee LA. Could MYC induction of mitochondrial biogenesis be linked to ROS production and genomic instability? Cell Cycle 2005; 4: 1465–6. 36. Echave P, Machado-da-Silva G, Arkell RS, et al. Extracellular growth factors and mitogens cooperate to drive mitochondrial biogenesis. J Cell Sci 2009; 122: 4516–25. 37. Warburg O. On the origin of cancer cells. Science 1956; 123: 309–14. 38. Kim JW, Dang CV. Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 2006; 66: 8927–30. 39. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006; 3: 177–85. 40. Simonnet H, Alazard N, Pfeiffer K, et al. Low mitochondrial respiratory chain content correlates with tumor aggressiveness in renal cell carcinoma. Carcinogenesis 2002; 23: 759–68. 41. Chatterjee A, Mambo E, Sidransky D. Mitochondrial DNA mutations in human cancer. Oncogene 2006; 25: 4663–74. 42. Pollard PJ, Briere JJ, Alam NA, et al. Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol Genet 2005; 14: 2231–9. 43. Formentini L, Martinez-Reyes I, Cuezva JM. The mitochondrial bioenergetic capacity of carcinomas. IUBMB Life 2010; 62: 554–60. 44. Berridge MV, Tan AS. Effects of mitochondrial gene deletion on tumorigenicity of metastatic melanoma: reassessing the Warburg effect. Rejuvenation Res 2010; 13: 139–41. 45. Weinberg F, Hamanaka R, Wheaton WW, et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci USA 2010; 107: 8788–93.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
512
SHI et al.
46. Cavalli LR, Varella-Garcia M, Liang BC. Diminished tumorigenic phenotype after depletion of mitochondrial DNA. Cell Growth Differ 1997; 8: 1189–98. 47. van den Bogert C, Dontje BH, Holtrop M, et al. Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis. Cancer Res 1986; 46: 3283–9. 48. Kudlicki W, Coffman A, Kramer G, Hardesty B. Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing. J Biol Chem 1997; 272: 32206–10. 49. Caldas TD, El Yaagoubi A, Richarme G. Chaperone properties of bacterial elongation factor EF-Tu. J Biol Chem 1998; 273: 11478–82.
Pathology (2012), 44(6), October
50. Richarme G. Protein-disulfide isomerase activity of elongation factor EF-Tu. Biochem Biophys Res Commun 1998; 252: 156–61. 51. Bhadula SK, Elthon TE, Habben JE, Helentjaris TG, Jiao S, Ristic Z. Heatstress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line. Planta 2001; 212: 359–66. 52. Sakai J, Ishikawa H, Kojima S, Satoh H, Yamamoto S, Kanaoka M. Proteomic analysis of rat heart in ischemia and ischemia-reperfusion using fluorescence two-dimensional difference gel electrophoresis. Proteomics 2003; 3: 1318–24. 53. Ciocca DR, Calderwood SK. Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones 2005; 10: 86–103.
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