of hereditary nonpolyposis colorectal cancer (HNPCC) and with approximately 10% to 15% of sporadic colorectal cancers. (CRCs). In HNPCC, MSI frequently ...
Anatomic Pathology / APOPTOSIS IN COLORECTAL CANCER WITH MSI
Role of bax Mutations in Apoptosis in Colorectal Cancers With Microsatellite Instability Catherine Miquel, MD,1,2 Francesco Borrini, MD,1 Sophie Grandjouan, MD,3,4 Anne Aupérin, MD,5 Jérôme Viguier, MD,2 Valérie Velasco,1 Pierre Duvillard, MD,1 Françoise Praz, PhD,2* and Jean-Christophe Sabourin, MD, PhD1* Key Words: bax; Colorectal cancer; Microsatellite instability; Apoptosis DOI: 10.1309/JQ2X3RV3L8F9TGYW
Abstract Half of colorectal tumors with microsatellite instability contain frameshift mutations in the (G)8 tract of bax, a major apoptosis effector, but their functional significance remains unclear. We studied the role of bax mutations on bax expression and apoptosis in 59 primary colorectal cancers of which 41 were microsatellite unstable. Tumors were screened for bax(G)8 mutations and evaluated immunohistochemically for bax, bcl-2, and p53 protein expression and apoptotic (M30 cytoDEATH) and proliferative (Ki-67) indexes. We identified bax(G)8 mutations in 20 (49%) of 41 unstable tumors; the mutations were associated significantly with proximal, poorly differentiated, or mucinous adenocarcinomas. Most bax-mutated cases displayed a bax-immunonegative zone in all or part of the tumor that was proved to correspond to biallelic bax(G)8 mutations by microdissection and to confer growth advantage to the tumor by decreasing apoptosis compared with adjacent bax-immunopositive tumor. Biallelic bax(G)8 mutations are subject to positive selection pressure and might disable apoptosis in colorectal cancer.
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Am J Clin Pathol 2005;123:562-570 DOI: 10.1309/JQ2X3RV3L8F9TGYW
There is substantial evidence that apoptosis has a role in the development of human cancer and that it might be a major mechanism in the action of current anticancer drugs.1 Members of the bcl-2 family of proteins that regulate apoptotic signaling through mitochondria are key players in the determination of cell survival and death.2,3 The proapoptotic protein bax, which belongs to the bcl-2 family, serves as an essential effector of the mitochondrial apoptotic pathways and participates in executing p53-mediated apoptosis.4,5 In response to the induction of wild-type p53, bax is translocated from the cytoplasm into the mitochondria and undergoes conformational changes indicative of activation. This results in the release of cytochrome c and Smac/DIABLO from mitochondria, which facilitates caspase activation and subsequent nuclear fragmentation.2 bax can heterodimerize with other proapoptotic and antiapoptotic bcl2 family members, and the balance between bax and its partners might act as a rheostat for apoptotic response to various apoptotic stimuli.6,7 Microsatellite instability in simple repeated sequences (referred to as MSI) has been associated with the vast majority of hereditary nonpolyposis colorectal cancer (HNPCC) and with approximately 10% to 15% of sporadic colorectal cancers (CRCs). In HNPCC, MSI frequently results from a germline mutation in one of the mismatch repair (MMR) genes, predominantly MLH1 and MSH2.8 In sporadic CRC, the silencing of MLH1 through aberrant methylation of its promoter is involved.8 CRC with MSI represents a distinct molecular pathway for colorectal carcinogenesis because, as a result of unrepaired replication errors, genes that contain microsatellite repeats in their coding sequences are the targets for MSI-driven frameshift mutations resulting in their inactivation.9 © American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
bax is one of the most frequently inactivated genes in CRC with MSI through frameshift mutations in the (G)8 microsatellite sequence inside its coding region, which leads to a synthesis of a truncated protein and reduced or undetectable bax expression.10 Furthermore, bax deficiency in transgenic mouse tumor models accelerates tumor growth and decreases apoptosis.11 These observations suggest that apoptosis might be disabled through bax inactivation during human tumor development. The observation that bax constitutes a critical checkpoint for the apoptosis response in epithelial cells is of much importance with regard to oncogenesis and cancer therapy.12 The role of bax mutations in human CRC is far from clear. Studies of bax mutations in CRC in relation to apoptosis are occasional and have contradictory results.13,14 This prompted us to examine the biologic significance of bax(G)8 mutations in CRC with MSI in apoptotic dysregulation. We hypothesized that the discrepancies of previous studies might be related to the underestimated importance of the intratumoral heterogeneity of bax gene and bax protein alterations. Thus, we postulated that the evaluation of apoptosis using M30 cytoDEATH (Roche, Mannheim, Germany), an apoptosis marker, and taking into consideration the heterogeneity of bax expression might provide additional information. We used polymerase chain reaction (PCR) to detect mutations in the (G)8 tract of bax and microdissection techniques to evaluate the biallelic or monoallelic nature of these mutations in some cases. Immunohistochemical analysis was performed to estimate bax, p53, and bcl-2 expression; apoptosis (M30 cytoDEATH); and cell proliferation (Ki-67). Our study included 59 cases of CRC, of which 41 were microsatellite unstable, and 3 human CRC cell lines, used as control samples. The group of CRCs with MSI first was compared with a control group consisting of microsatellite stable (MSS) CRCs, which allowed us to characterize both colorectal carcinogenesis pathways. To further assess the role of bax(G)8 mutations in CRC with MSI, we defined and compared 2 groups of CRCs with MSI according to the bax(G)8 mutational status: the MSI baxmut with a frameshift mutation and MSI baxwt devoid of mutation.
Materials and Methods Cell Lines The human colon carcinoma cell lines HT29, HCT116, and LoVo were provided by Peter Karran, PhD (Imperial Cancer Research Fund, Hertz, England). The HT29 cells are MMR-proficient, whereas HCT116 and LoVo cell lines are deficient in MLH1 and MSH2, respectively. The cells were cultured at 37°C in a carbon dioxide–humidified atmosphere
and were maintained in Dulbecco modified essential medium supplemented with 10% heat-inactivated fetal calf serum, Lglutamine (2-mmol/L concentration), and antibiotics. For immunohistochemical analysis, centrifuged pellets were resuspended in formalin, incubated for 5 minutes, and embedded in paraffin. Patient Specimens The study was carried out on a selected series of samples from 59 patients with surgically resected primary CRC. Formalin-fixed, paraffin-embedded tissue samples were obtained following institutional ethics committee approval and French Medical Research Council guidelines. All tumors previously were characterized for MSI status using the 5 reference Bethesda panel markers and classified as MSI-high or MSS according to the National Cancer Institute recommendations.15 The clinicopathologic characteristics of the patients and the CRCs are shown in ❚Table 1❚. Of 59 patients, 41 (69%) had tumors with high MSI (ie, instability at ≥2 markers) and 18 patients (31%), selected for being younger than 50 years at the time of CRC diagnosis, had sporadic MSS CRC. Among the 41 patients with CRC with MSI, 32 (78%) were classified as meeting complete or incomplete criteria for HNPCC as defined by the International Collaborative Group on HNPCC.16 The histologic type and grading of differentiation were assessed, and the tumors were staged according to criteria defined by the World Health Organization and the International Union Against Cancer. Molecular Analysis Genomic DNA was extracted from 7-µm-thick, formalinfixed, paraffin-embedded tissue sections of primary CRC as described.17 The 94-base-pair region encompassing the (G)8 tract in the bax coding sequence was amplified by PCR on the CRCs with MSI, with 6-FAM- or HEX-labeled 5'ATCCAGGATCGAGCAGGGCGA-3' sense primer and 5'CACTCGCTCAGCTTCTTGGTGGAC-3' antisense primer. PCR was carried out in a 20-µL reaction volume containing approximately 100 ng of genomic DNA, a 200-µmol/L concentration of 2'-deoxynucleoside 5'-triphosphate (Amersham Biosciences, Uppsala, Sweden), and 0.5 U of HotStarTaq DNA Polymerase (QIAGEN, Hilden, Germany). Amplification consisted of a 15-minute denaturation step at 95°C, followed by 50 cycles of 30 seconds at 95°C, 30 seconds at 50°C, and 30 seconds at 72°C and a final extension step of 5 minutes at 72°C. Adequate dilutions of fluorescent PCR products were mixed with formamide and carboxy-X-rhodamine–labeled molecular weight standards (GS-HD400-ROX, Applied Biosystems, Foster City, CA), heat denaturated, and run on a 50-cm capillary array containing GS Performance Optimized Polymer 6 (Applied Biosystems), at a voltage of 15 kV on the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Am J Clin Pathol 2005;123:562-570
© American Society for Clinical Pathology 563
DOI: 10.1309/JQ2X3RV3L8F9TGYW
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Miquel et al / APOPTOSIS IN COLORECTAL CANCER WITH MSI
❚Table 1❚ Clinicopathologic Data for Patients and the Primary CRC* CRCs With MSI Variable
MSS CRC (n = 18)
baxmut (n = 20)
0.8 (8/10) 38.5 (20.5-50)
1.0 (10/10) 50.8 (28.1-72.1)
Sex ratio (M/F) Mean (range) age (y) Primary tumor site§ Proximal colon Distal colon or rectum Histologic grade|| G1 or G2 G3 or mucinous Wall invasion T1 or T2 T3 or T4 Lymph node metastases Negative (N0) Positive (N1 or N2) Distant metastases Negative (M0) Positive (M1) Stage¶ I II III IV
baxwt (n = 21) 2.0 (14/7) 41.7 (26.7-68.6)
2/18 (11) 16/18 (89)
18/20 (90) 2/20 (10)
12/21 (57) 9/21 (43)
15/18 (83) 3/18 (17)
9/19 (47) 10/19 (53)
18/21 (86) 3/21 (14)
2/18 (11) 16/18 (89)
6/20 (30) 14/20 (70)
4/21 (19) 17/21 (81)
6/17 (35) 11/17 (65)
11/19 (58) 8/19 (42)
13/21 (62) 8/21 (38)
10/17 (59) 7/17 (41)
16/18 (89) 2/18 (11)
15/19 (79) 4/19 (21)
2/17 (12) 3/17 (18) 5/17 (29) 7/17 (41)
6/18 (33) 3/18 (17) 7/18 (39) 2/18 (11)
2/19 (11) 8/19 (42) 5/19 (26) 4/19 (21)
P†
P‡
.32 .052
