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Loss of BRCA1Gene Expression in Breast Carcinoma
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13 Loss of BRCA1 Gene Expression in Breast Carcinoma Wen-Ying Lee
Introduction BRCA1 (BReast CAncer 1) is a putative tumor suppressor gene located on chromosome 17q21, which encodes a nuclear protein of 220 kD consisting of 1863 amino acids (Chen et al., 1996; Miki et al., 1994). Many tumors with germline BRCA1 mutations display loss of heterozygosity at this locus, suggesting that BRCA1 is a tumor suppressor gene (Smith et al., 1992). Breast cancer occurs in familial (hereditary) and sporadic (nonhereditary) forms. Germline BRCA1 mutation accounts for 50% of breast cancer families (Ford et al., 1998), whereas none has currently been reported in sporadic breast cancers (Futreal et al., 1994). BRCA1 protein has been implicated in several important cellular functions, including regulation of transcription, repair of DNA damage, and cell cycle control. It has been reported that the BRCA1 gene has RING finger domain in its NH2 terminus (Miki et al., 1994) and BRCT motif in its COOH terminus (Koonin et al., 1996) that functions as a transactivator (Monteiro et al., 1996). BRCA1 interacts directly with p53 and transcriptionally activates p21waf1 (Zhang et al., 1998), suggesting a role in cell cycle control. BRCA1 also interacts with Rad51, a recA homologue of Escherichia coli that is involved in repair of double-strand breaks in Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 1: Molecular Genetics; Lung and Breast Carcinomas
DNA, suggesting a role in maintaining genomic stability (Koonin et al., 1996; Scully et al., 1997a, b). BRCA1 expression has shown to be associated with functional differentiation in multiple tissues (Marquis et al., 1995). It has been reported that BRCA1 transcripts are expressed in both alveolar and ductal epithelial cells of normal mammary gland, and breast cancer is frequently associated with loss of BRCA1 expression (Lane et al., 1995). This evidence suggests that the BRCA1 gene provides an important growth regulatory function in mammary epithelial cells. Although no somatic mutation of the BRCA1 gene has been detected (Futreal et al., 1994; Merajver et al., 1995), loss of heterozygosity at chromosome 17q21 (Jacobs et al., 1993) and decreased levels of the BRCA1 mRNA have been demonstrated in some sporadic cases of breast cancer (Thompson et al., 1995), indicating the involvement of BRCA1 even in the sporadic form. BRCA1 mRNA levels have been demonstrated to be markedly decreased during the transition from carcinoma in situ to invasive cancer in sporadic cases (Thompson et al., 1995). Furthermore, Chen et al. (1995) have demonstrated mislocalization of BRCA1 in the cytoplasm in breast cancer cells and cell lines from sporadic cases. Previous experiments have suggested that compromised BRCA1 function plays an
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362 important role in the pathogenesis and progression of sporadic breast cancer. However, controversy has existed during the last few years concerning the subcellular localization of BRCA1 (Chen et al., 1995; Scully et al., 1996; Jensen et al., 1996). Chen et al. (1995) reported that BRCA1 protein is localized in the nuclei of normal cells, but is aberrantly localized in the cytoplasm in breast cancer cell lines. They also demonstrated immunohistochemical staining of this protein in 50 fixed tissue sections of breast carcinomas and revealed a variable subcellular location, including 8 in the nuclei, 6 in the cytoplasm, 34 in both the nuclei and cytoplasm, and its absence in 2 cases. The observations reported by Chen et al. (1995) have been challenged by Scully et al. (1996), who stated that BRCA1 is exclusively nuclear regardless of cell type. Scully et al. (1996) reexamined the subcellular location of BRCA1 by using an affinity purified polyclonal antibody and a panel of monoclonal antibodies. Immunostaining with the various antibodies showed the BRCA1 nuclear signal in normal breast cells and breast cancer cell lines. Furthermore, all antibodies demonstrated a BRCA1 nuclear dot pattern in cells fixed with neutral paraformaldehyde, methanol, or 70% ethanol. Thus, the nuclear location of BRCA1 is not the result of a fixation artifact. BRCA1 concentrated in the nuclear but not in the cytoplasmic fraction was observed by biochemical extraction analysis in unfixed cells, indicating that BRCA1 nuclear subcellular location is independent of cell fixation conditions. Alcoholic, formalin-fixed, paraffin-embedded sections of breast cancer showed a variety of staining patterns, ranging from predominantly nuclear to mainly cytoplasmic. However, when these sections were treated with microwave heating, the staining was predominantly cytoplasmic. In cells fixed in neutral-buffered formalin and pretreated with microwave heating, the BRCA1 staining pattern was predominantly nuclear. Conversely, in neutral-buffered, formalin-fixed cells without microwave treatment, the signal was exclusively cytoplasmic. Thus, the subcellular location of BRCA1 was shown to be nuclear. The discrepancies of BRCA1 staining can be the result of variations in fixation, or staining conditions, or both (Scully et al., 1996). Studies by Chen et al. (1995) and Scully et al. (1996) indicate that BRCA1 is a 220-kD protein. However, Jensen et al. (1996) reported that BRCA1 as a 190-kD protein was located in both the cytoplasm and the cell membrane, and also suggested that BRCA1 with a granin motif functions as a secreted growth inhibitor but not a tumor suppressor gene product as previously described. Later studies reject the suggestion that BRCA1 is a granin (Koonin et al., 1996; Wilson et al., 1996).
IV Breast Carcinoma Currently, there has been no independent corroboration to suggest that BRCA1 is secreted. Jensen et al. (1996) used C-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) raised against residues 1843–1862, which has been proven to cross-react with epidermal growth factor receptor (EGFR) and HER-2 (BernardGallon et al., 1997; Wilson et al., 1996). Wilson et al. (1996) have confirmed the reactive band of C-20 antibody migrated at 190 kD, the size expected for EGFR. Interestingly, Wilson et al. (1997) and Thakur et al. (1997) have demonstrated BRCA1 splice variants that lack exon 11 including the nuclear localization signal (NLS) motifs. As a consequence of splice elimination of NLS, the proteins of splice variants cannot autonomously translocate to the nucleus. Thus, both studies have shown nuclear localization of full-length BRCA1, but cytoplasmic localization of splice variants (Thakur et al., 1997; Wilson et al., 1997). Wilson et al. (1999) have evaluated the specificity and use of 19 anti-BRCA1 antibodies directed against several different epitopes using immunoblotting, immunoprecipitation, immunocytochemical, and immunohistochemical techniques. Sixteen antibodies have identified BRCA1 220 kD in nuclear and total extracts, and several of these antibodies were highly specific (SD118, Ab-1, Ab-2, 17F8, and AP-16). Ab-1 (MS110) and Ab-2 (MS13) are commercially available (Oncogene Research Products, San Diego, California). SD118, AP16, and 17F8 are available from the academic laboratory (Livingston laboratory, DanaFarber Cancer Institute and Harvard Medical School, Boston, MA and Lee laboratory Cancer for Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, San Antonio, TX) (Chen et al., 1995; Scully et al., 1996). Other antibodies (C-20, D-20, and I-20; Santa Cruz Biotechnology) recognized additional proteins that are unlikely to be BRCA1 products of alternative splicing. Furthermore, only Ab-1 antibody (MS110), when combined with antigen retrieval, yielded consistent nuclear staining in formalin-fixed and paraffin-embedded tissues (Wilson et al., 1999). The descriptions of five highly specific antibodies are summarized in Table 24. Perez-Valles et al. (2001) have compared the immunohistochemistry of four commercially available anti-BRCA1 antibodies (D-20, I-20, K-18, and MS110) on paraffin-embedded tissue sections from breast cancers. All positive cases showed predominantly cytoplasmic staining with the polyclonal antibodies D-20, I-20, and K-18. After heating pretreatment, both nuclear and cytoplasmic stainings were found with the I-20 antibody. Only monoclonal antibody MS110 showed predominantly nuclear staining after microwave treatment. The results are consistent with the previous report (Wilson et al., 1999).
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Table 24 Descriptions of Five Highly Specific BRCA1 Antibodies Name
Reference
N-terminus MS110 (Ab-1) Scully et al. (1996)
Antigen
Type
Source IP
IB
ICC
IHC/F
IHC/P
+++
+++
+++
+++
+++
+++
+++
+++
−
−
r 1-304
Monoclonal
Scully et al. (1996)
r 1-304
Monoclonal
Exon-11 17F8 SD118
Chen et al. (1995) Unpublished
r 762-1315 r 1005-1313
Monoclonal Monoclonal
Lee laboratoryb + + + Livingston +++ laboratoryc
+++ +++
++ +++
+ +++
− −
C-terminus AP-16
Scully et al. (1996)
r 1313-1863
Monoclonal
Livingston laboratoryc
+++
+++
+++
−
−
MS13 (Ab-2)
Oncogene Research Productsa Oncogene Research Productsa
Applications
IB, immunoblotting; ICC, immunocytochemistry on cultured cells; IHC/F, immunohistochemistry with frozen cryostat tissue sections; IHC/P, immunohistochemistry with formalin-fixed, paraffin-embedded tissue samples; IP, immunoprecipitation; −, no signal; +, ++, weak signals; +++, strong signals. aSan
Diego, California.
bCenter
for Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, San Antonio, TX.
cDana-Farber
Cancer Institute and Harvard Medical School, Boston, MA.
Thus, the confusing observations of BRCA1 subcellular localization may have resulted from the specificity of the antibodies used to detect BRCA1 protein. Certain technical treatment and splice variants of BRCA1 protein may further complicate the situation.
MATERIALS 1. Primary antibody: BRCA1 antibody (Ab-1, clone MS110; Oncogene Research Products). 2. Antibody diluent (Code No. S0809; Dako, Glostrup, Denmark), which contains 0.05 M Tris-HCl buffer (pH 7.2–7.6) and 1% bovine serum albumin. 3. Wash buffer solution: 0.02 M phosphate buffer saline (PBS) (Code No. S3024; Dako), which makes 1 liter of 0.02 mol/L sodium phosphate buffer, 0.15 mol/L NaCl (pH 7.0). 4. Dako LSAB2 system, peroxidase (Code No. K0675). 5. 3% hydrogen peroxide. 6. Methanol. 7. Chromogen: 3-amino-9-ethylcarbazole (AEC) or diaminobenzidine (DAB). 8. Antigen retrieval solution (Code No. S1699; Dako), a modified citrate buffer (pH 6.1). 9. Positive control tissue: normal breast tissue. 10. Counterstain: Mayer’s hematoxylin. 11. Ethanol, absolute and 95%. 12. Xylene. 13. Distilled water.
14. Mounting media, such as Dako Faramount (Code No. S3025) or Dako Glycergel (Code No. C0563). 15. Poly L–lysine–coated slides. 16. Coverslips. 17. Humid chamber. 18. Timer. 19. Staining jars. 20. Absorbent wipes. 21. Drying oven, capable of maintaining 60°C or less.
METHOD 1. Cut 4-μm-thick sections from neutral-buffered, formalin-fixed and paraffin-embedded tissue blocks on Poly-L-lysine–coated slides. 2. Place tissue slides in drying oven at 56°C for 20–30 min. 3. Store tissue slides at 2–25°C before immunohistochemical staining. 4. Place tissue slides in a xylene bath and incubate for 5 min. Change baths and repeat once. 5. Tap off excess liquid and place slides in absolute ethanol for 3–5 min. Change baths and repeat once. 6. Tap off excess liquid and place slides in 95% ethanol for 3–5 min. Change baths and repeat once. 7. Change xylene and alcohol solutions after 40 slides. 8. Tap off excess liquid and place slides in distilled water for 5–10 min. 9. Tap off excess liquid and place slides in PBS for 5 min.
364 10. Tap off excess liquid and place slides in 3% H2O2 in methanol for 10 min. 11. Rinse gently with distilled water or PBS. 12. Place in PBS bath for 5 min. 13. Tap off excess liquid. Using a lintless tissue, carefully wipe around the specimen to remove any remaining liquid. 14. Place slides in antigen retrieval solution and heat in microwave oven at 750 W for 10 min. 15. Place slides at room temperature for 20 min. 16. Place slides in PBS bath for 5 min. 17. Tap off excess liquid and wipe slides as done previously (step 13). 18. Dilute primary antibody (Ab-1 antibody) at 1:50 with Dako Antibody Diluent. 19. Apply enough primary antibody to cover specimen. 20. Incubate slides with primary antibody overnight at 4°C. 21. Rinse gently with distilled water or PBS. 22. Place in PBS bath for 5 min. 23. Tap off excess liquid and wipe slides as done previously (step 13). 24. Apply enough drops of biotinylated link (Bottle of Dako LSAB2 System) to cover specimen. 25. Incubate for 10 min. 26. Rinse gently with distilled water or PBS. 27. Place in PBS bath for 5 min. 28. Tap off excess liquid and wipe slides as done previously (step 13). 29. Apply enough drops of streptavidin–horseradish peroxidase (HRP) (Bottle2 of Dako LSAB2 System) to cover specimen. 30. Incubate for 10 min. 31. Rinse gently with distilled water or PBS. 32. Place in PBS bath for 5 min. 33. Tap off excess liquid and wipe slides as done previously (step 13). 34. Apply substrate-chromogen solution (AEC or DAB) to cover specimen. 35. Incubate for 5–10 min. 36. Rinse gently with distilled water or PBS. 37. Place in PBS bath for 5 min. 38. Immerse slides in a bath of hematoxylin. 39. Incubate for 1–3 min, depending on the strength of hematoxylin used. 40. Rinse gently in a distilled water bath. 41. Mount coverslips with an aqueous-based mounting medium. 42. Include positive control slide (normal breast tissue) on each run. 43. Include negative control slide (omitting the primary antibody in normal breast tissue) on each run.
IV Breast Carcinoma 44. Interpret tumors as BRCA1-positive staining if more than 10% of tumor cells have distinct nuclear staining.
RESULTS AND DISCUSSION Using monoclonal antibody Ab-1 for BRCA1 (MS110) on formalin-fixed and paraffin-embedded tissue sections, BRCA1 was exclusively localized in the nuclei of normal ductal (Figure 55) and lobular epithelia, including the normal breast tissue adjacent to breast cancer. Using the same antibody, loss of BRCA1 nuclear expression has been reported in 20% (Lee et al., 1999), 27% (Yoshikawa et al., 1999), 34.3% (Yang et al., 2001), and 43.5% (Wilson et al., 1999) of sporadic breast carcinoma. Lee et al. (1999) reported that in 108 consecutive cases of sporadic breast cancer (invasive ductal carcinoma, not otherwise specified type), 72% had BRCA1 expression exclusively in the nucleus (Figure 56), 1.9% had BRCA1 expression exclusively in the cytoplasm, 2.8% had both nuclear and cytoplasmic expression, and 18.5% were without BRCA1 expression. Complete loss of nuclear BRCA1 expression was demonstrated in 20.4% of invasive ductal carcinomas. In 86 cases with BRCA1 nuclear expression, 96.5% showed more than 50% positive cells and 63.9% showed more than 75% positive cells. BRCA1 nuclear expression could be considered to represent the normal or physiologic phenotype. Altered BRCA1 protein expression may play an important role in sporadic breast carcinoma. A similar conclusion was suggested using other antibodies in other studies (Rio et al., 1999; Taylor et al., 1998; Yoshikawa et al., 1999). The lack of somatic mutations of BRCA1 in sporadic breast carcinoma (Futreal et al., 1994) suggests that BRCA1 expression might be down-regulated by epigenetic changes other than point mutations. One epigenetic alteration of BRCA1 inactivation that has been reported in sporadic breast carcinoma is hypermethylation of the BRCA1 promoter region (Rice et al., 1998; Catteau et al., 1999, 2002). It has been reported that aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells (Rice et al., 1998). Hypermethylation of CpG-rich areas located within the promoter of genes may be a common mechanism of silencing tumor suppressor genes. However, because methylation was detected only in 11% of breast cancer cases (Catteau et al., 1999), methylation cannot be the sole mechanism mediating the loss of BRCA1 expression in sporadic breast carcinoma. On the other hand, Baldassarre et al. (2003) reported that HMGA1b protein binds to and inhibits the activity of
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Figure 55 BRCA1 immunostaining. Strong nuclear immunostaining of normal ductal epithelia is visible (immunoperoxidase, original magnification X400).
the BRCA1 promoters both in vitro and in vivo. An inverse correlation between HMGA1 and BRCA1 mRNA and protein expression was also found in breast cancer cell lines and tissue. These results suggest that HMGA1 protein can negatively regulate BRCA1 gene expression in sporadic breast carcinoma. Thus, loss of BRCA1 expression in sporadic breast carcinoma might result from nonmutational mechanisms, such as
Figure 56 BRCA1 immunostaining. Strong nuclear immunostaining of an invasive ductal carcinoma, grade I (immunoperoxidase, original magnification X400).
changes in upstream regulatory pathways or altered expression of the regulatory gene. Although the mechanism of BRCA1-mediated tumor suppression and growth regulation is not fully elucidated, BRCA1 has been considered as a caretaker in maintaining genomic stability by cell cycle arrest and DNA repair (Scully et al., 1997b; Somasundaram et al., 1997). BRCA1-associated hereditary breast cancer
366 has been reported to have greater tumor cell proliferation rates (Marcus et al., 1996) and significant association with grade III tumor, indicating poor prognosis (Jacquemier et al., 1995). Loss of BRCA1 nuclear expression has also been demonstrated to be significantly associated with high Ki-67 index (Jarvis et al., 1998; Lee et al., 2002). Furthermore, complete loss of BRCA1 nuclear expression was significantly more frequent in high histologic grade tumors (3.2% in grade I, 26.2% in grade II, and 31.3% in grade III) in sporadic breast cancers (Lee et al., 1999). This observation is supported by other researchers (Taylor et al., 1998; Wilson et al., 1999; Yang et al., 2001). On the basis of these observations, it is suggested that loss of nuclear BRCA1 expression contributes to the pathogenesis and progression of sporadic breast carcinoma. Loss of BRCA1 nuclear expression in sporadic breast carcinomas causes highly proliferative tumor growth and genetic instability, which may trigger further genetic alterations and lead to a more aggressive behavior. Seery et al. (1999) have investigated 42 breast cancers and have reported that decreased BRCA1 mRNA expression is significantly associated with acquisition of metastatic capabilities, suggesting that BRCA1 expression might predict distant metastasis of sporadic breast cancers. Furthermore, BRCA1 expression has been correlated to disease-free survival (DFS) in a cohort of 175 sporadic breast carcinomas using immunohistochemical staining (Yang et al., 2001). Univariate analysis focusing on DFS has revealed axillary lymph node status, histologic grade, and BRCA1 expression as significant prognostic factors. Multivariate Cox regression analysis also demonstrated that axillary lymph node status, histologic grade, and BRCA1 expression are independent prognostic factors. The odds ratio for BRCA1 expression is 5.724, indicating that the risk for patients with BRCA1 negativity dying within a specific time is 5.7 times greater than that for patients with BRCA1 positivity (Yang et al., 2001). Thus, loss of BRCA1 nuclear expression appears to be a poor prognostic biomarker. It has been reported that patients with BRCA1 mutation have a much worse DFS rate compared with those with wild-type BRCA1 tumors (Chappuis et al., 2000a). Loss of BRCA1 expression by epigenetic mechanisms might confer a similar prognostic outcome to that observed as a consequence of BRCA1 mutation. Consistent with this hypothesis, van’t Veer et al. (2002) reported a sporadic breast cancer without BRCA1 mutation that had a BRCA1-mutant genetic profile and shared a similar prognosis. Conversely, the extent of apoptosis in tumors can be enhanced by the administration of tamoxifen and almost any kind of
IV Breast Carcinoma chemotherapeutic drug (Barry et al., 1990; Cameron et al., 2000), but loss of the genes inducing apoptosis or overactivation of the genes blocking apoptosis may make tumors resistant to anticancer agents. It has been reported that BRCA1 facilitates stressinduced apoptosis in breast and ovarian carcinoma cell lines (Thangaraju et al., 2000). Loss of BRCA1 expression might up-regulate the threshold for druginduced apoptosis, which results in worse prognosis. It is interesting to speculate that BRCA1 expression may be useful in the treatment of breast carcinoma as a decision-making biomarker for aggressive treatment after surgery. However, studies involving large population-based cohorts of patients with breast cancer are needed to definitively determine the prognostic impact of BRCA1 expression in sporadic breast carcinoma. Young women (< 35 or 40 years old) with breast cancer have a more advanced disease and worse prognosis than older women (Bonnier et al., 1995). It has been reported that breast cancer in women younger than 35 years has a significantly greater incidence of large tumor, high proliferative rate, high histologic grade, loss of BRCA1 nuclear expression, and Bcl-2 negativity than older women (Lee, 2002). No difference is found in lymph node status and HER-2 and p53 gene expression between these age-groups. Bogdani et al. (2002) have also reported that nuclear BRCA1 expression is significantly less in young women with breast cancer. These findings suggest that young women with breast cancer have frequent loss of BRCA1 nuclear expression, which may be responsible for the specific tumor biology different from older women. However, the frequency of BRCA1 gene mutations in young women with breast cancer is low, from 6.2% to 10% in white women (Langston et al., 1996; Malone et al., 1998) and 8% in Chinese women (Tang et al., 2001), and is not significantly greater than in a general group of patients with breast cancer. Although BRCA1 mutation cannot be completely responsible for the more aggressive tumors in young women, loss of BRCA1 nuclear expression by epigenetic mechanisms might confer a similar prognostic outcome to the patients with breast cancer with BRCA1 mutation who have an aggressive cancer phenotype (Chappuis et al., 2000a). BRCA1-associated hereditary breast cancers are more frequently estrogen receptor (ER) negative (Chappuis et al., 2000b). Similarly, a significant correlation between expression of BRCA1 and ER mRNA has been observed (Seery et al., 1999) in sporadic breast carcinoma. Furthermore, loss of BRCA1 nuclear expression has been frequently found in tumors with negative ER in sporadic form (Lee et al., 1999),
13 Loss of BRAC1 Gene Expression in Breast Carcinoma suggesting a functional relationship between these two genes. The positive correlation between BRCA1 and ER expression is consistent with the results reported by Gudas et al. (1995), whose data suggest that BRCA1 expression is regulated by the steroid hormones in the human breast cancer cell. The increase in BRCA1 expression on stimulation with estrogen is not coordinated with the early induction of the estrogen-dependent p53 gene, but closely parallels the delayed increase in the S-phase–dependent marker cyclin A. Steroid hormones might induce BRCA1 transcription indirectly by altering the proliferative status of the cells rather than acting directly on DNA sequences in the BRCA1 gene itself (Gudas et al., 1995). Apoptosis is believed to act as a counterbalance to proliferation and is a critical factor in tissue homeostasis. Dysregulation of the apoptosis process may therefore play a crucial role in oncogenesis. It has been reported that BRCA1 is associated with the p53 tumor suppressor protein and functions as a transcriptional coactivator for p53 (Zhang et al., 1998). BRCA1 can induce apoptosis, which is enhanced by the coexpression of p53 (Zhang et al., 1998). Yang et al. (2002) have investigated the relationship between alterations of BRCA1 expression and the apoptosis-related proteins p53, Bcl-2, and bax in sporadic breast carcinomas. It has shown that BRCA1 expression was positively correlated with Bcl-2 expression, but not with BRCA1, p53, or bax expressions. These results are consistent with the report by Lee et al. (1999). These authors have demonstrated that loss of BRCA1 expression is significantly correlated with Bcl-2 negativity, but there was no correlation between BRCA1 and p53 expression. Furthermore, loss expression of Bcl-2 is also observed in BRCA1-associated breast cancers (Freneaux et al., 2000). Most BRCA1-associated breast carcinomas are characterized by high mitotic and apoptotic rates and a decrease of Bcl-2 expression. Therefore, the regulating relationship between Bcl-2 and BRCA1 genes may exit in both sporadic and hereditary breast carcinomas. The alterations of BRCA1 protein might be responsible for the down-regulation of Bcl-2, and thus for the high proliferative rate and increased apoptosis. However, the down-regulation of Bcl-2 in tumors with loss of BRCA1 expression might merely reflect the downregulation of negative ER. Because BRCA1-associated breast carcinomas are frequently ER negative (Chappuis et al., 2000b) and a positive association exists between the expression of ER and Bcl-2 (Bhargava et al., 1994), there remain many questions to be addressed in the regulation mechanism between BRCA1 and Bcl-2.
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CONCLUSION It is concluded that BRCA1 is located in nuclear foci of all cell types, including normal and malignant breast cells. BRCA1 is a tumor suppressor gene and acts as caretaker in maintaining genetic stability by cell cycle arrest and DNA repair. Although somatic BRCA1 mutation has not been reported in sporadic breast cancer, loss of nuclear BRCA1 expression may contribute to the pathogenesis and progression of sporadic breast carcinoma. Loss of BRCA1 protein expression in sporadic breast cancers causes highly proliferative tumor growth and genetic instability, which may trigger further genetic alterations and lead to a more aggressive behavior. Only monoclonal antibody Ab-1 for BRCA1 (MS110) (commercially available) is highly specific and can be applied on formalin-fixed, paraffin-embedded tissue sections that show a predominantly nuclear staining after microwave treatment.
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IV Breast Carcinoma Lee, W.Y., Jin, Y.T., Chang, T.W., Lin, P.W., and Su, I.J. 1999. Immunolocalization of BRCA1 protein in normal breast tissue and sporadic invasive ductal carcinomas: A correlation with other biological parameters. Histopathology 34:106–112. Malone, K.E., Daling, J.R., Thompson, J.D., O’Brien, C.A., Francisco, L.V., and Ostrander, E.A. 1998. BRCA1 mutations and breast cancer in the general population: Analyses in women before age 35 years and in women before age 45 years with first-degree family history. JAMA 279:922–929. Marcus, J.N., Watson, P., Page, D.L., Narod, S.A., Lenoir, G.M., Tonin, P., Linder-Stephenson, L., Salerno, G., Conway, T.A., and Lynch, H.T. 1996. Hereditary breast cancer: Pathobiology, prognosis, and BRCA1 and BRCA2 gene linkage. Cancer 77:697–709. Marquis, S.T., Rajan, J.V., Wynshaw-Boris. A., Xu, J., Yin, G.Y., Abel, K.J., Weber, B.L., and Chodosh, L.A. 1995. The developmental pattern of BRCA1 expression implies a role in differentiation of the breast and other tissues. Nat. Genet. 11:17–26. Merajver, S.D., Pham, T.M., Caduff, R.F., Chen, M., Poy, E.L., Cooney, K.A., Weber, B.L., Collins, F.S., Johnston, C., and Frank, T.S. 1995. Somatic mutations in the BRCA1 gene in sporadic ovarian tumours. Nat. Genet. 9:439–443. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P.A., Harshman, K., and Tavtigian, S. 1994. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66–71. Monteiro, A.N., August, A., and Hanafusa, H. 1996. Evidence for a transcriptional activation function of BRCA1 C-terminal region. Proc. Natl. Acad. Sci. USA 93:13595–13599. Perez-Valles, A., Martorell-Cebollada, M., Nogueira-Vazquez, E., Garcia-Garcia, J.A., and Fuster-Diana, E. 2001. The usefulness of anitbodies to the BRCA1 protein in detecting the mutated BRCA1 gene: An immunohistochemical study. J. Clin. Pathol. 54:476–480. Rice, J.C., Massey-Brown, K.S., and Futscher, B.W. 1998. Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells. Oncogene 17:1807–1812. Rio, P.G., Maurizis, J.C., Peffault de Latour, M., Bignon, Y.J., and Bernard-Gallon, D.J. 1999. Quantification of BRCA1 protein in sporadic breast carcinoma with or without loss of heterozygosity of the BRCA1 gene. Int. J. Cancer 80:823–826. Scully, R., Chen, J., Ochs, R.L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D.M. 1997a. Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90:425–435. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and Livingston, D.M. 1997b. Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 88:265–275. Scully, R., Ganesan, S., Brown, M., De Caprio, J.A., Cannistra, S.A., Feunteun, J., Schnitt, S., and Livingston, D.M. 1996. Location of BRCA1 in human breast and ovarian cancer cells. Science 272:123–126. Seery, L.T., Knowlden, J.M., Gee, J.M.W., Robertson, J.F.R., Kenny, F.S., Ellis, I.O., and Nicholson, R.I. 1999. BRCA1 expression levels predict distant metastasis of sporadic breast cancers. Int. J. Cancer 84:258–262. Smith, S.A., Easton, D.F., Evans, D.G., and Ponder, B.A. 1992. Allele losses in the region 17q12-21 in familial breast and ovarian cancer involve the wild-type chromosome. Nat. Genet. 2:128–131. Somasundaram, K., Zhang, H., Zeng, Y.X., Houvras, Y., Peng, Y., Zhang, H., Wu, G.S., Licht, J.D., Weber, B.L., and El-Deiry, W.S. 1997. Arrest of the cell cycle by the tumour-suppressor
13 Loss of BRAC1 Gene Expression in Breast Carcinoma BRCA1 requires the CDK-inhibitor p21WAF1/CiP1. Nature 389:187–190. Tang, N.L., Choy, K.W., Pang, C.P., Yeo, W., and Johnson, P.J. 2001. Prevalence of breast cancer predisposition gene mutations in Chinese women and guidelines for genetic testing. Clin. Chim. Acta. 313:179–185. Taylor, J., Lymboura, M., Pace, P.E., A’hern, R.P., Desai, A.J., Shousha, S., Coombes, R.C., and Ali, S. 1998. An important role for BRCA1 in breast cancer progression is indicated by its loss in a large proportion of non-familial breast cancers. Int. J. Cancer 79:334–342. Thakur, S., Zhang, H.B., Peng, Y., Le, H., Carroll, B., Ward, T., Yao, J., Farid, L.M., Couch, F.J., Wilson, R.B., and Weber, B.L. 1997. Localization of BRCA1 and a splice variant identifies the nuclear localization signal. Mol. Cell Biol. 17:444–452. Thangaraju, M., Kaufmann, S.H., and Couch, F.J. 2000. BRCA1 facilitates stress-induced apoptosis in breast and ovarian cancer cell lines. J. Biol. Chem. 275:33487–33496. Thompson, M.E., Jensen, R.A., Obermiller, P.S., Page, D.L., and Holt, J.T. 1995. Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nat. Genet. 9:444–450. van’t Veer, L.J., Dai, H., van de Vijver, M.J., He, Y.D., Hart, A.A., Mao, M., Peterse, H.L., van der Kooy, K., Marton, M.J., Witteveen, A.T., Schreiber, G.J., Kerkhoven, R.M., Roberts, C., Linsley, P.S., Bernards, R., and Friend, S.H. 2002. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415:530–536. Wilson, C.A., Payton, M.N., Elliott, G.S., Buaas, F.W., Cajulis, E.E., Grosshans, D., Ramos, L., Reese, D.M., Slamon, D.J., and Calzone, F.J. 1997. Differential subcellular localization,
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expression and biological toxicity of BRCA1 and the splice variant BRCA1-delta11b. Oncogene 14:1–16. Wilson, C.A., Payton, M.N., Pekar, S.K., Zhang, K., Pacifici, R.E., Gudas, J.L., Thukral, S., Calzone, F.J., Reese, D.M., and Slamon, D.I. 1996. BRCA1 protein products: Antibody specificity. Nat. Genet. 13:264–265. Wilson, C.A., Ramos, L., Villasenor, M.R., Anders, K.H., Press, M.F., Clarke, K., Karlan, B., Chen, J.J., Scully, R., Livingston, D., Zuch, R.H., Kanter, M.H., Cohen, S., Calzone, F.J., and Slamon, D.J. 1999. Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas. Nat. Genet. 21:236–240. Yang, Q., Sakurai, T., Mori, I., Yoshimura, G., Nakamura, M., Nakamura, Y., Suzuma, T., Tamaki, T., Umemura, T., and Kakudo, K. 2001. Prognostic significance of BRCA1 expression in Japanese sporadic breast carcinomas. Cancer 92:54–60. Yang, Q., Yoshimura, G., Nakamura, M., Nakamura, Y., Suzuma, T., Umemura, T., Mori, I., Sakurai, T., and Kakudo, K. 2002. Correlation between BRCA1 expression and apoptosis-related biological parameters in sporadic breast carcinomas. Anticancer Res. 22:3615–3619. Yoshikawa, K., Honda, K., Inamoto, T., Shinohara, H., Yamauchi, A., Suga, K., Okuyama, T., Shimada, T., Kodama, H., Noguchi, S., Gazdar, A.F., Yamaoka, Y., and Takahashi, R. 1999. Reduction of BRCA1 protein expression in Japanese sporadic breast carcinomas and its frequent loss in BRCA1-associated cases. Clin. Cancer Res. 5:1249–1261. Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Zhang, H., Bi, D., Weber, B.L., and El-Deiry, W.S. 1998. BRCA1 physically associates with p53 and stimulates its transcriptional activity. Oncogene 16:1713–1721.
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13 Loss of BRCA1 Gene Expression in Breast Carcinoma Wen-Ying Lee
Introduction BRCA1 (BReast CAncer 1) is a putative tumor suppressor gene located on chromosome 17q21, which encodes a nuclear protein of 220 kD consisting of 1863 amino acids (Chen et al., 1996; Miki et al., 1994). Many tumors with germline BRCA1 mutations display loss of heterozygosity at this locus, suggesting that BRCA1 is a tumor suppressor gene (Smith et al., 1992). Breast cancer occurs in familial (hereditary) and sporadic (nonhereditary) forms. Germline BRCA1 mutation accounts for 50% of breast cancer families (Ford et al., 1998), whereas none has currently been reported in sporadic breast cancers (Futreal et al., 1994). BRCA1 protein has been implicated in several important cellular functions, including regulation of transcription, repair of DNA damage, and cell cycle control. It has been reported that the BRCA1 gene has RING finger domain in its NH2 terminus (Miki et al., 1994) and BRCT motif in its COOH terminus (Koonin et al., 1996) that functions as a transactivator (Monteiro et al., 1996). BRCA1 interacts directly with p53 and transcriptionally activates p21waf1 (Zhang et al., 1998), suggesting a role in cell cycle control. BRCA1 also interacts with Rad51, a recA homologue of Escherichia coli that is involved in repair of double-strand breaks in Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 1: Molecular Genetics; Lung and Breast Carcinomas
DNA, suggesting a role in maintaining genomic stability (Koonin et al., 1996; Scully et al., 1997a, b). BRCA1 expression has shown to be associated with functional differentiation in multiple tissues (Marquis et al., 1995). It has been reported that BRCA1 transcripts are expressed in both alveolar and ductal epithelial cells of normal mammary gland, and breast cancer is frequently associated with loss of BRCA1 expression (Lane et al., 1995). This evidence suggests that the BRCA1 gene provides an important growth regulatory function in mammary epithelial cells. Although no somatic mutation of the BRCA1 gene has been detected (Futreal et al., 1994; Merajver et al., 1995), loss of heterozygosity at chromosome 17q21 (Jacobs et al., 1993) and decreased levels of the BRCA1 mRNA have been demonstrated in some sporadic cases of breast cancer (Thompson et al., 1995), indicating the involvement of BRCA1 even in the sporadic form. BRCA1 mRNA levels have been demonstrated to be markedly decreased during the transition from carcinoma in situ to invasive cancer in sporadic cases (Thompson et al., 1995). Furthermore, Chen et al. (1995) have demonstrated mislocalization of BRCA1 in the cytoplasm in breast cancer cells and cell lines from sporadic cases. Previous experiments have suggested that compromised BRCA1 function plays an
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Copyright © 2004 by Elsevier (USA) All rights reserved.
362 important role in the pathogenesis and progression of sporadic breast cancer. However, controversy has existed during the last few years concerning the subcellular localization of BRCA1 (Chen et al., 1995; Scully et al., 1996; Jensen et al., 1996). Chen et al. (1995) reported that BRCA1 protein is localized in the nuclei of normal cells, but is aberrantly localized in the cytoplasm in breast cancer cell lines. They also demonstrated immunohistochemical staining of this protein in 50 fixed tissue sections of breast carcinomas and revealed a variable subcellular location, including 8 in the nuclei, 6 in the cytoplasm, 34 in both the nuclei and cytoplasm, and its absence in 2 cases. The observations reported by Chen et al. (1995) have been challenged by Scully et al. (1996), who stated that BRCA1 is exclusively nuclear regardless of cell type. Scully et al. (1996) reexamined the subcellular location of BRCA1 by using an affinity purified polyclonal antibody and a panel of monoclonal antibodies. Immunostaining with the various antibodies showed the BRCA1 nuclear signal in normal breast cells and breast cancer cell lines. Furthermore, all antibodies demonstrated a BRCA1 nuclear dot pattern in cells fixed with neutral paraformaldehyde, methanol, or 70% ethanol. Thus, the nuclear location of BRCA1 is not the result of a fixation artifact. BRCA1 concentrated in the nuclear but not in the cytoplasmic fraction was observed by biochemical extraction analysis in unfixed cells, indicating that BRCA1 nuclear subcellular location is independent of cell fixation conditions. Alcoholic, formalin-fixed, paraffin-embedded sections of breast cancer showed a variety of staining patterns, ranging from predominantly nuclear to mainly cytoplasmic. However, when these sections were treated with microwave heating, the staining was predominantly cytoplasmic. In cells fixed in neutral-buffered formalin and pretreated with microwave heating, the BRCA1 staining pattern was predominantly nuclear. Conversely, in neutral-buffered, formalin-fixed cells without microwave treatment, the signal was exclusively cytoplasmic. Thus, the subcellular location of BRCA1 was shown to be nuclear. The discrepancies of BRCA1 staining can be the result of variations in fixation, or staining conditions, or both (Scully et al., 1996). Studies by Chen et al. (1995) and Scully et al. (1996) indicate that BRCA1 is a 220-kD protein. However, Jensen et al. (1996) reported that BRCA1 as a 190-kD protein was located in both the cytoplasm and the cell membrane, and also suggested that BRCA1 with a granin motif functions as a secreted growth inhibitor but not a tumor suppressor gene product as previously described. Later studies reject the suggestion that BRCA1 is a granin (Koonin et al., 1996; Wilson et al., 1996).
IV Breast Carcinoma Currently, there has been no independent corroboration to suggest that BRCA1 is secreted. Jensen et al. (1996) used C-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) raised against residues 1843–1862, which has been proven to cross-react with epidermal growth factor receptor (EGFR) and HER-2 (BernardGallon et al., 1997; Wilson et al., 1996). Wilson et al. (1996) have confirmed the reactive band of C-20 antibody migrated at 190 kD, the size expected for EGFR. Interestingly, Wilson et al. (1997) and Thakur et al. (1997) have demonstrated BRCA1 splice variants that lack exon 11 including the nuclear localization signal (NLS) motifs. As a consequence of splice elimination of NLS, the proteins of splice variants cannot autonomously translocate to the nucleus. Thus, both studies have shown nuclear localization of full-length BRCA1, but cytoplasmic localization of splice variants (Thakur et al., 1997; Wilson et al., 1997). Wilson et al. (1999) have evaluated the specificity and use of 19 anti-BRCA1 antibodies directed against several different epitopes using immunoblotting, immunoprecipitation, immunocytochemical, and immunohistochemical techniques. Sixteen antibodies have identified BRCA1 220 kD in nuclear and total extracts, and several of these antibodies were highly specific (SD118, Ab-1, Ab-2, 17F8, and AP-16). Ab-1 (MS110) and Ab-2 (MS13) are commercially available (Oncogene Research Products, San Diego, California). SD118, AP16, and 17F8 are available from the academic laboratory (Livingston laboratory, DanaFarber Cancer Institute and Harvard Medical School, Boston, MA and Lee laboratory Cancer for Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, San Antonio, TX) (Chen et al., 1995; Scully et al., 1996). Other antibodies (C-20, D-20, and I-20; Santa Cruz Biotechnology) recognized additional proteins that are unlikely to be BRCA1 products of alternative splicing. Furthermore, only Ab-1 antibody (MS110), when combined with antigen retrieval, yielded consistent nuclear staining in formalin-fixed and paraffin-embedded tissues (Wilson et al., 1999). The descriptions of five highly specific antibodies are summarized in Table 24. Perez-Valles et al. (2001) have compared the immunohistochemistry of four commercially available anti-BRCA1 antibodies (D-20, I-20, K-18, and MS110) on paraffin-embedded tissue sections from breast cancers. All positive cases showed predominantly cytoplasmic staining with the polyclonal antibodies D-20, I-20, and K-18. After heating pretreatment, both nuclear and cytoplasmic stainings were found with the I-20 antibody. Only monoclonal antibody MS110 showed predominantly nuclear staining after microwave treatment. The results are consistent with the previous report (Wilson et al., 1999).
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Table 24 Descriptions of Five Highly Specific BRCA1 Antibodies Name
Reference
N-terminus MS110 (Ab-1) Scully et al. (1996)
Antigen
Type
Source IP
IB
ICC
IHC/F
IHC/P
+++
+++
+++
+++
+++
+++
+++
+++
−
−
r 1-304
Monoclonal
Scully et al. (1996)
r 1-304
Monoclonal
Exon-11 17F8 SD118
Chen et al. (1995) Unpublished
r 762-1315 r 1005-1313
Monoclonal Monoclonal
Lee laboratoryb + + + Livingston +++ laboratoryc
+++ +++
++ +++
+ +++
− −
C-terminus AP-16
Scully et al. (1996)
r 1313-1863
Monoclonal
Livingston laboratoryc
+++
+++
+++
−
−
MS13 (Ab-2)
Oncogene Research Productsa Oncogene Research Productsa
Applications
IB, immunoblotting; ICC, immunocytochemistry on cultured cells; IHC/F, immunohistochemistry with frozen cryostat tissue sections; IHC/P, immunohistochemistry with formalin-fixed, paraffin-embedded tissue samples; IP, immunoprecipitation; −, no signal; +, ++, weak signals; +++, strong signals. aSan
Diego, California.
bCenter
for Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, San Antonio, TX.
cDana-Farber
Cancer Institute and Harvard Medical School, Boston, MA.
Thus, the confusing observations of BRCA1 subcellular localization may have resulted from the specificity of the antibodies used to detect BRCA1 protein. Certain technical treatment and splice variants of BRCA1 protein may further complicate the situation.
MATERIALS 1. Primary antibody: BRCA1 antibody (Ab-1, clone MS110; Oncogene Research Products). 2. Antibody diluent (Code No. S0809; Dako, Glostrup, Denmark), which contains 0.05 M Tris-HCl buffer (pH 7.2–7.6) and 1% bovine serum albumin. 3. Wash buffer solution: 0.02 M phosphate buffer saline (PBS) (Code No. S3024; Dako), which makes 1 liter of 0.02 mol/L sodium phosphate buffer, 0.15 mol/L NaCl (pH 7.0). 4. Dako LSAB2 system, peroxidase (Code No. K0675). 5. 3% hydrogen peroxide. 6. Methanol. 7. Chromogen: 3-amino-9-ethylcarbazole (AEC) or diaminobenzidine (DAB). 8. Antigen retrieval solution (Code No. S1699; Dako), a modified citrate buffer (pH 6.1). 9. Positive control tissue: normal breast tissue. 10. Counterstain: Mayer’s hematoxylin. 11. Ethanol, absolute and 95%. 12. Xylene. 13. Distilled water.
14. Mounting media, such as Dako Faramount (Code No. S3025) or Dako Glycergel (Code No. C0563). 15. Poly L–lysine–coated slides. 16. Coverslips. 17. Humid chamber. 18. Timer. 19. Staining jars. 20. Absorbent wipes. 21. Drying oven, capable of maintaining 60°C or less.
METHOD 1. Cut 4-μm-thick sections from neutral-buffered, formalin-fixed and paraffin-embedded tissue blocks on Poly-L-lysine–coated slides. 2. Place tissue slides in drying oven at 56°C for 20–30 min. 3. Store tissue slides at 2–25°C before immunohistochemical staining. 4. Place tissue slides in a xylene bath and incubate for 5 min. Change baths and repeat once. 5. Tap off excess liquid and place slides in absolute ethanol for 3–5 min. Change baths and repeat once. 6. Tap off excess liquid and place slides in 95% ethanol for 3–5 min. Change baths and repeat once. 7. Change xylene and alcohol solutions after 40 slides. 8. Tap off excess liquid and place slides in distilled water for 5–10 min. 9. Tap off excess liquid and place slides in PBS for 5 min.
364 10. Tap off excess liquid and place slides in 3% H2O2 in methanol for 10 min. 11. Rinse gently with distilled water or PBS. 12. Place in PBS bath for 5 min. 13. Tap off excess liquid. Using a lintless tissue, carefully wipe around the specimen to remove any remaining liquid. 14. Place slides in antigen retrieval solution and heat in microwave oven at 750 W for 10 min. 15. Place slides at room temperature for 20 min. 16. Place slides in PBS bath for 5 min. 17. Tap off excess liquid and wipe slides as done previously (step 13). 18. Dilute primary antibody (Ab-1 antibody) at 1:50 with Dako Antibody Diluent. 19. Apply enough primary antibody to cover specimen. 20. Incubate slides with primary antibody overnight at 4°C. 21. Rinse gently with distilled water or PBS. 22. Place in PBS bath for 5 min. 23. Tap off excess liquid and wipe slides as done previously (step 13). 24. Apply enough drops of biotinylated link (Bottle of Dako LSAB2 System) to cover specimen. 25. Incubate for 10 min. 26. Rinse gently with distilled water or PBS. 27. Place in PBS bath for 5 min. 28. Tap off excess liquid and wipe slides as done previously (step 13). 29. Apply enough drops of streptavidin–horseradish peroxidase (HRP) (Bottle2 of Dako LSAB2 System) to cover specimen. 30. Incubate for 10 min. 31. Rinse gently with distilled water or PBS. 32. Place in PBS bath for 5 min. 33. Tap off excess liquid and wipe slides as done previously (step 13). 34. Apply substrate-chromogen solution (AEC or DAB) to cover specimen. 35. Incubate for 5–10 min. 36. Rinse gently with distilled water or PBS. 37. Place in PBS bath for 5 min. 38. Immerse slides in a bath of hematoxylin. 39. Incubate for 1–3 min, depending on the strength of hematoxylin used. 40. Rinse gently in a distilled water bath. 41. Mount coverslips with an aqueous-based mounting medium. 42. Include positive control slide (normal breast tissue) on each run. 43. Include negative control slide (omitting the primary antibody in normal breast tissue) on each run.
IV Breast Carcinoma 44. Interpret tumors as BRCA1-positive staining if more than 10% of tumor cells have distinct nuclear staining.
RESULTS AND DISCUSSION Using monoclonal antibody Ab-1 for BRCA1 (MS110) on formalin-fixed and paraffin-embedded tissue sections, BRCA1 was exclusively localized in the nuclei of normal ductal (Figure 55) and lobular epithelia, including the normal breast tissue adjacent to breast cancer. Using the same antibody, loss of BRCA1 nuclear expression has been reported in 20% (Lee et al., 1999), 27% (Yoshikawa et al., 1999), 34.3% (Yang et al., 2001), and 43.5% (Wilson et al., 1999) of sporadic breast carcinoma. Lee et al. (1999) reported that in 108 consecutive cases of sporadic breast cancer (invasive ductal carcinoma, not otherwise specified type), 72% had BRCA1 expression exclusively in the nucleus (Figure 56), 1.9% had BRCA1 expression exclusively in the cytoplasm, 2.8% had both nuclear and cytoplasmic expression, and 18.5% were without BRCA1 expression. Complete loss of nuclear BRCA1 expression was demonstrated in 20.4% of invasive ductal carcinomas. In 86 cases with BRCA1 nuclear expression, 96.5% showed more than 50% positive cells and 63.9% showed more than 75% positive cells. BRCA1 nuclear expression could be considered to represent the normal or physiologic phenotype. Altered BRCA1 protein expression may play an important role in sporadic breast carcinoma. A similar conclusion was suggested using other antibodies in other studies (Rio et al., 1999; Taylor et al., 1998; Yoshikawa et al., 1999). The lack of somatic mutations of BRCA1 in sporadic breast carcinoma (Futreal et al., 1994) suggests that BRCA1 expression might be down-regulated by epigenetic changes other than point mutations. One epigenetic alteration of BRCA1 inactivation that has been reported in sporadic breast carcinoma is hypermethylation of the BRCA1 promoter region (Rice et al., 1998; Catteau et al., 1999, 2002). It has been reported that aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells (Rice et al., 1998). Hypermethylation of CpG-rich areas located within the promoter of genes may be a common mechanism of silencing tumor suppressor genes. However, because methylation was detected only in 11% of breast cancer cases (Catteau et al., 1999), methylation cannot be the sole mechanism mediating the loss of BRCA1 expression in sporadic breast carcinoma. On the other hand, Baldassarre et al. (2003) reported that HMGA1b protein binds to and inhibits the activity of
13 Loss of BRAC1 Gene Expression in Breast Carcinoma
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Figure 55 BRCA1 immunostaining. Strong nuclear immunostaining of normal ductal epithelia is visible (immunoperoxidase, original magnification X400).
the BRCA1 promoters both in vitro and in vivo. An inverse correlation between HMGA1 and BRCA1 mRNA and protein expression was also found in breast cancer cell lines and tissue. These results suggest that HMGA1 protein can negatively regulate BRCA1 gene expression in sporadic breast carcinoma. Thus, loss of BRCA1 expression in sporadic breast carcinoma might result from nonmutational mechanisms, such as
Figure 56 BRCA1 immunostaining. Strong nuclear immunostaining of an invasive ductal carcinoma, grade I (immunoperoxidase, original magnification X400).
changes in upstream regulatory pathways or altered expression of the regulatory gene. Although the mechanism of BRCA1-mediated tumor suppression and growth regulation is not fully elucidated, BRCA1 has been considered as a caretaker in maintaining genomic stability by cell cycle arrest and DNA repair (Scully et al., 1997b; Somasundaram et al., 1997). BRCA1-associated hereditary breast cancer
366 has been reported to have greater tumor cell proliferation rates (Marcus et al., 1996) and significant association with grade III tumor, indicating poor prognosis (Jacquemier et al., 1995). Loss of BRCA1 nuclear expression has also been demonstrated to be significantly associated with high Ki-67 index (Jarvis et al., 1998; Lee et al., 2002). Furthermore, complete loss of BRCA1 nuclear expression was significantly more frequent in high histologic grade tumors (3.2% in grade I, 26.2% in grade II, and 31.3% in grade III) in sporadic breast cancers (Lee et al., 1999). This observation is supported by other researchers (Taylor et al., 1998; Wilson et al., 1999; Yang et al., 2001). On the basis of these observations, it is suggested that loss of nuclear BRCA1 expression contributes to the pathogenesis and progression of sporadic breast carcinoma. Loss of BRCA1 nuclear expression in sporadic breast carcinomas causes highly proliferative tumor growth and genetic instability, which may trigger further genetic alterations and lead to a more aggressive behavior. Seery et al. (1999) have investigated 42 breast cancers and have reported that decreased BRCA1 mRNA expression is significantly associated with acquisition of metastatic capabilities, suggesting that BRCA1 expression might predict distant metastasis of sporadic breast cancers. Furthermore, BRCA1 expression has been correlated to disease-free survival (DFS) in a cohort of 175 sporadic breast carcinomas using immunohistochemical staining (Yang et al., 2001). Univariate analysis focusing on DFS has revealed axillary lymph node status, histologic grade, and BRCA1 expression as significant prognostic factors. Multivariate Cox regression analysis also demonstrated that axillary lymph node status, histologic grade, and BRCA1 expression are independent prognostic factors. The odds ratio for BRCA1 expression is 5.724, indicating that the risk for patients with BRCA1 negativity dying within a specific time is 5.7 times greater than that for patients with BRCA1 positivity (Yang et al., 2001). Thus, loss of BRCA1 nuclear expression appears to be a poor prognostic biomarker. It has been reported that patients with BRCA1 mutation have a much worse DFS rate compared with those with wild-type BRCA1 tumors (Chappuis et al., 2000a). Loss of BRCA1 expression by epigenetic mechanisms might confer a similar prognostic outcome to that observed as a consequence of BRCA1 mutation. Consistent with this hypothesis, van’t Veer et al. (2002) reported a sporadic breast cancer without BRCA1 mutation that had a BRCA1-mutant genetic profile and shared a similar prognosis. Conversely, the extent of apoptosis in tumors can be enhanced by the administration of tamoxifen and almost any kind of
IV Breast Carcinoma chemotherapeutic drug (Barry et al., 1990; Cameron et al., 2000), but loss of the genes inducing apoptosis or overactivation of the genes blocking apoptosis may make tumors resistant to anticancer agents. It has been reported that BRCA1 facilitates stressinduced apoptosis in breast and ovarian carcinoma cell lines (Thangaraju et al., 2000). Loss of BRCA1 expression might up-regulate the threshold for druginduced apoptosis, which results in worse prognosis. It is interesting to speculate that BRCA1 expression may be useful in the treatment of breast carcinoma as a decision-making biomarker for aggressive treatment after surgery. However, studies involving large population-based cohorts of patients with breast cancer are needed to definitively determine the prognostic impact of BRCA1 expression in sporadic breast carcinoma. Young women (< 35 or 40 years old) with breast cancer have a more advanced disease and worse prognosis than older women (Bonnier et al., 1995). It has been reported that breast cancer in women younger than 35 years has a significantly greater incidence of large tumor, high proliferative rate, high histologic grade, loss of BRCA1 nuclear expression, and Bcl-2 negativity than older women (Lee, 2002). No difference is found in lymph node status and HER-2 and p53 gene expression between these age-groups. Bogdani et al. (2002) have also reported that nuclear BRCA1 expression is significantly less in young women with breast cancer. These findings suggest that young women with breast cancer have frequent loss of BRCA1 nuclear expression, which may be responsible for the specific tumor biology different from older women. However, the frequency of BRCA1 gene mutations in young women with breast cancer is low, from 6.2% to 10% in white women (Langston et al., 1996; Malone et al., 1998) and 8% in Chinese women (Tang et al., 2001), and is not significantly greater than in a general group of patients with breast cancer. Although BRCA1 mutation cannot be completely responsible for the more aggressive tumors in young women, loss of BRCA1 nuclear expression by epigenetic mechanisms might confer a similar prognostic outcome to the patients with breast cancer with BRCA1 mutation who have an aggressive cancer phenotype (Chappuis et al., 2000a). BRCA1-associated hereditary breast cancers are more frequently estrogen receptor (ER) negative (Chappuis et al., 2000b). Similarly, a significant correlation between expression of BRCA1 and ER mRNA has been observed (Seery et al., 1999) in sporadic breast carcinoma. Furthermore, loss of BRCA1 nuclear expression has been frequently found in tumors with negative ER in sporadic form (Lee et al., 1999),
13 Loss of BRAC1 Gene Expression in Breast Carcinoma suggesting a functional relationship between these two genes. The positive correlation between BRCA1 and ER expression is consistent with the results reported by Gudas et al. (1995), whose data suggest that BRCA1 expression is regulated by the steroid hormones in the human breast cancer cell. The increase in BRCA1 expression on stimulation with estrogen is not coordinated with the early induction of the estrogen-dependent p53 gene, but closely parallels the delayed increase in the S-phase–dependent marker cyclin A. Steroid hormones might induce BRCA1 transcription indirectly by altering the proliferative status of the cells rather than acting directly on DNA sequences in the BRCA1 gene itself (Gudas et al., 1995). Apoptosis is believed to act as a counterbalance to proliferation and is a critical factor in tissue homeostasis. Dysregulation of the apoptosis process may therefore play a crucial role in oncogenesis. It has been reported that BRCA1 is associated with the p53 tumor suppressor protein and functions as a transcriptional coactivator for p53 (Zhang et al., 1998). BRCA1 can induce apoptosis, which is enhanced by the coexpression of p53 (Zhang et al., 1998). Yang et al. (2002) have investigated the relationship between alterations of BRCA1 expression and the apoptosis-related proteins p53, Bcl-2, and bax in sporadic breast carcinomas. It has shown that BRCA1 expression was positively correlated with Bcl-2 expression, but not with BRCA1, p53, or bax expressions. These results are consistent with the report by Lee et al. (1999). These authors have demonstrated that loss of BRCA1 expression is significantly correlated with Bcl-2 negativity, but there was no correlation between BRCA1 and p53 expression. Furthermore, loss expression of Bcl-2 is also observed in BRCA1-associated breast cancers (Freneaux et al., 2000). Most BRCA1-associated breast carcinomas are characterized by high mitotic and apoptotic rates and a decrease of Bcl-2 expression. Therefore, the regulating relationship between Bcl-2 and BRCA1 genes may exit in both sporadic and hereditary breast carcinomas. The alterations of BRCA1 protein might be responsible for the down-regulation of Bcl-2, and thus for the high proliferative rate and increased apoptosis. However, the down-regulation of Bcl-2 in tumors with loss of BRCA1 expression might merely reflect the downregulation of negative ER. Because BRCA1-associated breast carcinomas are frequently ER negative (Chappuis et al., 2000b) and a positive association exists between the expression of ER and Bcl-2 (Bhargava et al., 1994), there remain many questions to be addressed in the regulation mechanism between BRCA1 and Bcl-2.
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CONCLUSION It is concluded that BRCA1 is located in nuclear foci of all cell types, including normal and malignant breast cells. BRCA1 is a tumor suppressor gene and acts as caretaker in maintaining genetic stability by cell cycle arrest and DNA repair. Although somatic BRCA1 mutation has not been reported in sporadic breast cancer, loss of nuclear BRCA1 expression may contribute to the pathogenesis and progression of sporadic breast carcinoma. Loss of BRCA1 protein expression in sporadic breast cancers causes highly proliferative tumor growth and genetic instability, which may trigger further genetic alterations and lead to a more aggressive behavior. Only monoclonal antibody Ab-1 for BRCA1 (MS110) (commercially available) is highly specific and can be applied on formalin-fixed, paraffin-embedded tissue sections that show a predominantly nuclear staining after microwave treatment.
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