• Open Access

Subtype-specific alterations of the Wnt signaling pathway in breast cancer: Clinical and prognostic significance

Authors


To whom correspondence should be addressed. E-mail: ckpanda.cnci@gmail.com

Abstract

The aim of the study is to understand the importance of the Wnt/β-catenin pathway in the development of breast cancer (BC) and its association with different clinicopathological parameters. Alterations (deletion/methylation/expression) of some Wnt/β-catenin pathway antagonists like APC, SFRP1/2, CDH1 and activator β-catenin (CTNNB1) were analyzed in primary BC in Indian patients. High frequencies (65–70%) of overall alterations (deletion/methylation) of the antagonists were seen in the BC samples. Also, 99% (156/158) of the samples showed alterations in any one of the genes, indicating the importance of this pathway in the development of this tumor. Co-alterations of these genes were observed in 30% of samples, with significantly high alterations in late-onset (37%) and estrogen receptor (ER)−/progesterone receptor (PR)− (37%) BC compared with early onset (21%) and ER/PR+ (18%) BC samples, respectively. Significantly high (P-value = 0.001–0.02) alterations of APC and CDH1 genes were seen in ER−/PR− BC compared with ER/PR+ BC. Immunohistochemical analysis showed reduced expression of the Wnt antagonists in BC concordant with their molecular alterations. Nuclear localization of β-catenin showed significant association with alterations in the antagonists and was also significantly high in the ER−/PR− BC samples. Alterations of SFRP2 coupled with a late clinical stage and low/nulliparity predicted the worst prognosis in BC patients. Therefore, the present study suggests that cumulative alterations in more than one Wnt antagonist along with increased nuclear accumulation of β-catenin play an important role in the development of BC and have significant clinical as well as prognostic importance. (Cancer Sci 2012; 103: 210–220)

Breast cancer (BC) is a frequently occurring cancer in women worldwide and accounts for 25% of cancer cases in the Eastern Indian population, especially in urban areas.(1) There are several known risk factors for BC.(2) In general, younger women with BC (age ≤ 40 years) exhibit more aggressive pathological features and lower survival rates compared with older women with BC.(3) The molecular subtypes of BC, which usually lack expression of the estrogen receptor (ER) and progesterone receptor (PR), also show the worst prognosis of BC.(4) It has been suggested that ER and PR play important roles in regulating the mammary stem cell population and its development.(5)

The stem cell population is regulated by a number of self-renewal pathways,(6) among which the Wnt/β-catenin pathway is quite important due to its cross-talk with the ER signaling pathway(7) and its alterations in several carcinomas including BC.(8) There are several antagonists of the Wnt/β-catenin pathway like SFRP1, SFRP2, APC and CDH1.(9,10) In different ways, these antagonists regulate the cytoplasmic and nuclear accumulation of β-catenin, the signal transducer of this pathway.(8,11)

Accumulation of data shows varying frequencies of deletion (23–73%) and frequent promoter methylation in the Wnt antagonists such as SFRP1/SFRP2,(9,12,13)APC(10,14–17) and CDH1(10,18–20) were present in BC. Moreover, loss of β-catenin membranous expression and nuclear accumulation of β-catenin have been reported to be associated with ER− and reduced CDH1 expression,(21,22) along with aggressive tumor phenotype and poor patient outcome in BC.(22,23) It has been reported that ERα could interact with β-catenin (CTNNB1) to activate different target genes in the nucleus.(24) In addition, there are several conflicting reports concerning the association of Wnt antagonists and β-catenin with conventional clinicopathological parameters of BC such as ER/PR status and clinical stage.(16,20,25,26) However, the molecular changes and mechanisms that regulate the development and progression of BC remain unclear.

Thus, to understand the importance of the Wnt/β-catenin pathway in the development of BC and its association with different clinicopathological parameters, it is pertinent to analyze alterations of the important antagonists of this pathway and β-catenin in the same set of samples. In the present study, alterations (deletion/methylation) of SFRP1/2, APC and CDH1 were analyzed in 158 primary BC samples from Indian patients. Also, to understand the effect of alterations of these genes on β-catenin metabolism the expression of SFRP1, APC and CDH1 along with β-catenin were analyzed immunohistochemically in 115 primary BC samples. Our data suggest differential alterations of Wnt antagonists and nuclear β-catenin expression in ER/PR (+/−) and early/late onset BC, suggesting differences in the molecular pathogenesis of the different subtypes.

Materials and Methods

Sample collection and clinical data.  One hundred and fifty-eight invasive BC samples and their corresponding normal tissues or peripheral blood leukocytes were randomly collected from 155 unrelated and untreated BC patients undergoing surgery at the hospital section of Chittaranjan National Cancer Institute (CNCI), Kolkata, India, during the period 1999–2008. Informed consent from patients and approval from the Research Ethics Committee of the institute were obtained. All tumors were staged according to the International Union against Cancer (UICC) tumor–node–metastasis (TNM) classification.(27) Details regarding the work plan for the BC samples are presented in Data S1 and Figure S1. Detailed clinicopathological histories of the patients are presented in Table 1.

Table 1.   Clinicopathological features of breast cancer (BC) patients
Clinical featuresn (%)Mean age (years)ER and PR negative status (n = 114)P-value
−/−+%
  1. *Statistically significant (P < 0.05). ER, estrogen receptor; PR, progesterone receptor.

Age (years)
 ≤4067 (42)34 ± 63211720.171
 >4091 (58)53 ± 8442758
Sex
 Female153 (98)44 ± 107138650.95
 Male3 (2)50 ± 52167
Menopausal status
 Premenopausal79 (50)37 ± 63216670.74
 Postmenopausal79 (50)51 ± 7422464
Parity
 P ≤ 268 (49)42 ± 133415690.84
 P > 271 (51)46 ± 10251071
Familial BC
 Familial BC+15 (10)38 ± 653620.84
 Familial BC−140 (90)43 ± 9703666
Tumor grade
 Grade I18 (11)42 ± 1257420.003*
 Grade II91 (57)43 ± 11372134
 Grade III49 (31)49 ± 1226487
Stage
 Stage I and II60 (38)46 ± 131924440.0001*
 Stage III and IV98 (62)43 ± 10571480
Lymph node (LN)
 LN+104 (66)45 ± 115018730.07
 LN−54 (44)44 ± 12191556

Microdissection and DNA extraction.  The procedure is described in detail in Data S1. High-molecular-weight DNA was extracted according to the standard procedure.(28)

Deletion analysis.  Deletion of SFRP1/2, APC and CDH1 genes were analyzed using microsatellite and restriction fragment length polymorphism markers located in or around these genes (Table S1) according to the method previously described(1,29) (Data S1).

Promoter methylation analysis.  The promoter methylation status of APC, SFRP1, SFRP2 and CDH1 genes were determined by methylation-sensitive restriction analysis(30) (MSRA) using methyl-sensitive restriction enzyme HhaI with recognition site GCGC. Promoter regions of the genes harbored at least one HhaI site. Details of primer sequences are listed in Table S1. The 445-bp fragment of β-3A adaptin gene (K1) and 229-bp fragment of RARβ2 exon-1 (K2) was used as digestion and integrity controls, respectively (see Data S1 and Table S1 for details of PCR conditions).

Immunohistochemical analysis.  Of the 158 BC samples, 114 paraffin-embedded BC tissue sections were randomly selected for immunohistochemical assay to analyze the expression status of ER and PR in BC.(31) To verify the impact of gene alterations at the protein level, the expression status of APC, SFRP1, CDH1 and β-catenin were detected immunohistochemically in paraffin-embedded sections of five normal breast tissues and 115 BC samples(32) (randomly selected from 158 BC samples included in the present study). For color development, diaminobenzidine (sc-24982, SantaCruz Biotechnology, Santa Cruz, CA, USA) was used, with hematoxylin as a counterstain. The antibodies, dilutions and antigen retrieval methods used are summarized in Table S2 (see Data S1 for details).

Statistical analysis.  Probability value (P-value) ≤ 0.05 was considered statistically significant. All statistical analysis was performed using statistical programs EpiInfo (version 6.04b, 1997; Centers for Disease Control and Prevention, Atlanta, GA, USA) and SPSS 10.0 (SPSS Inc. Chicago, IL, USA) (see Data S1 for details).

Results

ER/PR status of the BC samples.  Positive staining with ER/PR was localized to the nuclei of BC cells. Also, heterogeneity of staining was frequently observed. The majority (66%; 76/114) of the BC samples showed no expression of ER and PR (Fig. S2). The ER and PR negative status of the samples showed a significant association with late tumor stage (P = 0.003) and increased grade (P = 0.004) in BC patients, along with borderline significance with nodes at pathology (P = 0.07) (Table 1). However, no such correlation was seen with age of onset, menopausal status and parity.

Deletion analysis of the candidate Wnt/β-catenin antagonists.  After excluding non-informative tumor samples (35 of 158 at SFRP1, 14 of 158 at APC and 79 of 158 at the CDH1 locus), deletion analysis of the Wnt/β-catenin antagonists revealed the highest deletion frequency in SFRP1 (29%, 36/123) followed by APC (26%, 38/144), CDH1 (23%, 18/79) and SFRP2 (21%, 33/158) (Fig. 1). No significant differences were observed in the deletion frequencies of these genes between early/late-onset BC. However, significantly higher deletion frequencies of APC and CDH1 were observed in ER−/PR− BC than ER/PR+ BC samples (P = 0.029, 0.048, 0.023, respectively). Two samples showed homozygous deletions in the SFRP2 locus (#4187, #5051). A low frequency of microsatellite alterations (MA) (1–6%) and biallelic alterations (LMA) (3–5%) were observed in the samples (Table S3).

Figure 1.

 Deletion status of the Wnt antagonists in breast cancer (BC). (A) Representative autoradiograph showing: (a) loss of heterozygosity (LOH); (b) MA-1, microsatellite size alteration of one allele; (c) LOH + MA, loss of one allele and size alteration of the other; (d,e) homozygous/hemizygous deletion (HD/HED); and (f) LOH analysis of exonic markers. Arrow indicates loss of corresponding alleles. N, normal DNA; T, tumor DNA. (B) Pattern of deletion of Wnt/β-catenin antagonists in early/late-onset BC and estrogen receptor/progesterone receptor (ER/PR) (+/−) BC. *Statistically significant difference in the deletion frequency of Wnt antagonists between ER/PR (+/−) BC.

Overall 44% (69/158) of the samples harbored deletions in at least one of the antagonists and 6 (4%) samples harbored deletions in all genes. Deletion of these genes showed a significant association with each other in the overall BC samples. A similar trend was observed in early/late-onset BC and ER−/PR− BC (Table S4).

Promoter methylation analysis of candidate Wnt/β-catenin antagonists.  The differential methylation pattern in promoter regions of the antagonists was seen in the following order: APC (65%, 103/158) > CDH1 (63%, 100/158) > SFRP1 (56%, 85/158) > SFRP2 (54%, 82/158). No significant difference was observed in the promoter methylation frequencies of the candidate genes in the early/late-onset BC samples. However, a significant increase in promoter methylation was seen in APC and CDH1 in the ER−/PR− BC samples compared with the ER/PR+ BC samples (P = 0.0008, 0.042) (Fig. 2).

Figure 2.

 Promoter methylation status of Wnt antagonists analyzed by methylation-sensitive restriction analysis. (A) Schematic representation of promoter regions of candidate genes revealing distribution of HhaI (GCGC: arrowhead) restriction sites. (→), Location of methylation primers; +1, transcription start site. (B) Methylation status of antagonists in breast cancer (BC). H, HhaI digested DNA; U, undigested DNA; K1 and K2, controls for DNA digestion and integrity check, respectively. (C) Pattern of promoter methylation of different Wnt antagonists in early/late-onset BC and ER/PR (+/−) BC. *Significant difference in methylation frequency of the genes between ER/PR (+/−) BC.

Simultaneous promoter methylation in the four genes was seen in 18% (28/158) of the total BC samples (Table S3). Significant association was observed among promoter methylation of APC and SFRP2 with CDH1, and SFRP1 with SFRP2 in the total BC. A differential pattern of correlation was observed in these genes in early/late-onset BC and in the ER/PR(+/−) BC samples (P = 0.001–0.03) (Table S5).

Overall alterations of the Wnt/β-catenin antagonists.  Overall alterations (deletion/methylation) of the Wnt/β-catenin antagonists were seen in the following order: APC (70%, 111/158) > SFRP1 (67%, 106/158) > CDH1 (66%, 104/158) > SFRP2 (65%, 103/158) (Fig. 3). Alterations in any one of these genes were seen in approximately 99% (157/158) of the BC samples indicating the importance of these genes in the development of this tumor.

Figure 3.

 Pattern of overall alterations (deletion/methylation) of different Wnt/β-catenin antagonists in early/late-onset breast cancer (BC) and ER/PR (+/−) BC. # and *Significant difference (P < 0.05) in the two subsets of BC; A, alteration.

Biallelic alterations of these genes by deletion and methylation were observed in the following order: APC (27%, 30/111) > SFRP2 (18%, 17/103) > SFRP1 (20%, 17/106) > CDH1 (13%, 13/104). Co-alterations of these genes were observed in 30% of the BC samples with significantly high alterations in the late-onset (37%) and ER−/PR− (37%) BC samples compared with early onset (21%) and ER/PR+ (18%) BC. Significantly higher alterations of APC and CDH1 were observed in ER−/PR− BC compared with ER/PR+ BC samples, but no such difference was seen with age of onset of the disease (Table 2A).

Table 2.    (A) Summary of molecular alterations of Wnt/β-catenin antagonists in breast cancer (BC). Association of alterations in the candidate genes in (A) total BC, (B) early and late-onset BC, (C) ER/PR+ and (D) ER−/PR− BC samplesThumbnail image of

Interestingly, in the absence of APC alterations, significantly high alterations of the other three genes were observed in the ER/PR+ BC samples compared with the ER−/PR− BC samples. A similar phenomenon was also evident in the samples without CDH1 alterations, but no such association was seen in the samples without SFRP1/2 alterations. Also, 13% of the samples (23/158) showed alterations in SFRP1/2 only and were prevalent in the ER/PR+ BC samples (Fig. 3).

Chi-squared analysis of the alterations of the genes revealed alterations of APC were significantly associated with CDH1 (P = 0.003) in the BC samples (Table 2B). However, differential association among alterations of these genes was observed in early/late-onset BC and ER/PR (+/−) BC (Table 2C,D).

Immunohistochemical analysis.  In normal breast tissue, predominant membrane expression of CDH1, β-catenin and SFRP1 was observed in luminal epithelial and myoepithelial cells (Fig. 4). In contrast, cytoplasmic expression of APC and SFRP1 was evident in both luminal and myoepithelial cells, while cytoplasmic expression of β-catenin was present in luminal epithelial cells only.

Figure 4.

 Representative immunohistochemical staining patterns of CDH1, SFRP1, APC and β-catenin proteins in breast tissues. (A,D,G,J) Normal breast tissue showing the expression pattern of CDH1, SFRP1, APC and β-catenin. (B,C) Reduced/negative expression of CDH1 in breast cancer (BC). (E,F) Reduced/negative, predominantly cytoplasmic expression of SFRP1 in BC. (H,I) Reduced/negative cytoplasmic expression along with nuclear expression of APC in BC. (K,L) Intense nuclear and/or cytoplasmic staining of β-catenin in BC. Arrows indicate membrane/cytoplasmic expression. Arrowheads indicate nuclear expression pattern of these genes in primary BC. Bar, 25 μm.

In the BC samples, reduced or absence of expression of CDH1, SFRP1 and APC proteins was seen in 58% (67/115), 43% (49/115) and 64% (74/115) of the samples, respectively, while overexpression of β-catenin in the nucleus and cytoplasm was seen in 47% (54/115) and 54% (62/115) of the samples, respectively (Table S3). In addition to reduced cytoplasmic expression of APC seen in the breast tissue samples, nuclear expression of APC was evident in 33% (38/115) of the BC samples (Table 3). Expression of APC, SFRP1 and CDH1 proteins showed concordance with their molecular alterations in the samples. A statistically significant correlation was observed between β-catenin nuclear staining and alterations in APC, SFRP2 and CDH1 genes (P = 0.013–0.048) in the BC samples (Table 4). Interestingly, in the ER−/PR− BC samples, significantly low expression of CDH1, SFRP1 and APC along with a significant increase in nuclear expression of β-catenin were observed (P = 0.000001–0.032), pointing towards differences in the molecular pathogenesis of the ER/PR+ and ER−/PR− BC samples (Table 3).

Table 3.   Expression status of the Wnt/β-catenin pathway-associated genes in breast cancer (BC) samples
Expression statusTotal BC, % (n = 115)ER/PR+ BC, % (n = 38)ER−/PR− BC, % (n = 76)P-valueEarly onset, % (n = 49)Late-onset, % (n = 66)P-value
  1. *Statistically significant (P < 0.05). †Reduced expression (−/+). ‡Positive expression of APC in nucleus. §High (+++) nuclear expression of β-catenin. cyto, cytoplasmic expression; ER, estrogen receptor; mem, membrane expression; nuc, nuclear expression.

CDH1 (mem/cyto)†58 (67/115)26 (10/38)74 (56/76)0.000001*63 (31/49)61 (40/66)0.771
SFRP1 (cyto)†43 (49/115)29 (11/38)50 (38/76)0.032*51 (25/49)41 (27/66)0.281
APC (cyto)†64 (74/115)34 (13/38)80 (61/76)0.000001*61 (30/49)67 (44/66)0.546
APC (nuc)‡33 (38/115)18 (7/38)29 (22/76)0.22326 (13/49)21 (14/66)0.169
β-catenin (cyto)†54 (62/115)66 (25/38)49 (37/76)0.3353 (26/49)54 (36/66)0.874
β-catenin (nuc)§47 (54/115)16 (6/38)63 (48/76)0.000001*53 (26/49)42 (28/66)0.252
Table 4.   Correlation between the genetic status and protein expression of APC, SFRP1 and CDH1 genes in breast cancer
 CDH1 (mem/cyto)†SFRP1 (cyto)†APC (cyto)†APC (nuc)†β-catenin (cyto)†β-catenin (nuc)†
−/+++/ňP-value−/+++/ňP-value−/+++/ňP-value−/+++/+++P-value−/+++/+++P-value−/+++++P-value
  1. *Statistically significant (P < 0.05). †In the case of CDH1, SFRP1, APC and β-catenin (cytoplasm), negative/low expression of proteins was considered as an event while high expression of nuclear β-catenin was considered as an event. −/+, negative/low expression; ++/+++/ň, moderate/high/normal expression; ň, normal expression compared with normal breast tissue. cyto, cytoplasmic; mem, membrane expression; nuc, nuclear expression.

CDH1 alterations
 Positive53260.004*37420.17457220.01*55240.14136430.007*37420.048*
 Negative142212241719201626102412
SFRP1 alterations
 Positive42290.80438330.002*50210.15148210.2339320.78138330.896
 Negative251911332418271923212321
SFRP2 alterations
 Positive43250.19332360.24547210.249190.11534340.08630380.021*
 Negative242317302720261928193116
APC alterations
 Positive52240.002*40360.002*59180.0001*59170.69534420.005*34420.013*
 Negative15249301523291028112712

Clinicopathological association and patient survival.  Alterations (deletion/methylation) of these genes were comparatively high (63–68%) in stage I and II tumor samples and became comparable in the later stages (stages III and IV) (60–71%). A similar trend was also evident in tumor grades, except for significantly high alterations of CDH1 in higher grades of BC (P = 0.029). Alterations of these genes showed no association with the lymph node status of the patients (Table S6; Fig. S3).

Significant association was observed in promoter methylation of SFRP2 and CDH1 with a poor patient outcome for BC patients. The BC patients with alterations in CDH1, SFRP2 or APC showed significantly poor overall survival (P = 0.002–0.052) (Fig. 5). Importantly, significantly poor survival was seen in the ER−/PR− BC patients harboring alterations in any of the Wnt pathway antagonists (Fig. 5H). Interestingly, BC patients with high nuclear β-catenin expression in the samples also showed significantly poor overall survival (Fig. 5G).

Figure 5.

 Kaplan–Meier analysis of survival (up to 5 years) of breast cancer (BC) patients. Molecular alterations (deletion and methylation) of APC, SFRP1/2 and CDH1 genes and nuclear accumulation of β-catenin (CTNNB1) were significantly associated with poor overall survival (OS) (A–G). Survival of BC patients with/without alteration of Wnt antagonists stratified for estrogen receptor/progesterone receptor (ER/PR) status (H). A, alteration; n, sample size.

The univariate Cox model (Table 5) showed a significant association (P = 0.00026–0.0065) with alterations in SFRP2 and CDH1 genes, negative ER/PR status, positive lymph node status, late stage (III and IV) and low parity (≤2) with the overall survival of BC patients (Table 5). However, Cox multivariate analysis revealed that SFRP2 alterations, negative ER/PR status, positive lymph node status, low/nulliparity (parity ≤2) and late tumor stage were significantly associated with overall survival of BC patients (P = 0.0133–0.038) (Table 5).

Table 5.   Univariate (A) and multivariate (B) analyses of genetic, clinical and etiological parameters in predicting overall survival (OS) of breast cancer (BC) patients
VariableP-valueHR95% CI
(A)
  1. *P < 0.05, statistically significant. CI, confidence interval; ER, estrogen receptor; HR, hazard ratio; LN, lymph node; PR, progesterone receptor.

APC alteration+0.12591.68240.86423.2753
CDH1 alteration+0.0065*2.72351.32315.6062
SFRP1 alteration+0.83341.06550.59011.9239
SFRP2 alteration+0.0078*2.17941.22723.8706
ER/PR−0.0002*5.19602.199412.2758
LN+0.0029*2.86501.43305.7280
Grade III0.18601.46510.83182.5805
Stage III and IV0.0025*2.54041.38674.6541
Parity ≤20.0026*3.23051.50726.9240
Age ≤40 years0.10191.57690.91362.7217
Premenopausal status0.38070.76450.41931.3937
(B)
APC alteration+0.0133*0.33250.13910.795
CDH1 alteration+0.44890.71860.30551.6902
SFRP1 alteration+0.53321.33340.53953.2958
SFRP2 alteration+0.0353*2.19281.05564.5554
ER/PR−0.0387*3.20601.06259.6742
LN+0.0263*4.54711.195417.2966
Grade III0.96340.98330.47822.0216
Stage III and IV0.0325*2.68251.08556.6290
Parity ≤20.0388*2.59551.05016.4152
Age ≤40 years0.43480.69300.27611.7393
Premenopausal status0.15310.51090.20331.2837

Discussion

The aim of the present study was to understand the importance of the Wnt/β-catenin pathway in the development of BC. To begin, alterations of some antagonists of the Wnt/β-catenin pathway, namely APC, SFRP1, SFRP2 and CDH1, were analyzed in primary BC of Indian patients. Alterations of these genes were then correlated with different clinicopathological parameters such as age of onset, ER/PR expression status, nodes at pathology and parity. The majority of samples (67%) was ER−/PR− and showed a positive correlation with high tumor grade and stage of BC. A similar expression pattern of ER and PR has been reported in Indian patients(33,34) unlike European and North American populations.(33) This could be due to differences in etiological factors and probably ethnicity as suggested by other investigators.(5,34) Association of ER−/PR− status of BC with a higher tumor grade in ductal carcinoma of the breast has also been reported.(35)

The majority of samples (99%; 157/158) showed genetic and epigenetic alterations (deletion/methylation) in at least one of the antagonists of the Wnt/β-catenin pathway. This suggests that alterations in different antagonists might act in a complementary fashion to make comparable contributions to the process of breast carcinogenesis. A comparatively low frequency of deletion (21–26%) compared with methylation (65–67%) was observed in the Wnt antagonists in the samples, suggesting epigenetic alteration as the primary mechanism of inactivation of these genes in BC. Deletions of these genes showed a significant association with each other in the total BC samples, but showed differential association either in early/late-onset BC or in ER/PR(−/+) BC samples, indicating differences in the subtype-specific deletion pattern. A variable frequency of deletion (23–73%) in APC(10,15–17) and CDH1(10,18,19) genes has been reported by other investigators in BC, as well as in a variety of other human cancers.(36–38) This difference might be due to differences in sample size, methodology used in the analysis and ethnicity of BC patients. In addition, deletions in APC and SFRP2 associated with a poor overall survival of BC patients suggest their prognostic importance.

Like deletion, methylation of these genes showed a significant association with each other, suggesting the necessity of cumulative epigenetic hits for this tumorigenesis. Moreover, differential association in methylation of these genes was observed in either early/late onset of the disease or ER/PR (+/−) status of the samples. Varying frequencies of methylation in APC (36–71%), SFRP1 (40–61%), SFRP2 (77–83%) and CDH1 (41–72%) have been reported in BC by other investigators.(11,13,16–19) Like our data, association of CDH1 methylation with APC methylation and reduced ER expression has been reported in BC.(10,20,39) However, some investigators(16,25) did not find any association between methylation of APC and SFRP2 with the ER/PR status of the BC samples. The prognostic significance of CDH1, SFRP1 and APC methylation was also reported in BC and other cancers,(12,40,41) while promoter methylation of SFRP2 was reported to show no correlation with clinical patient outcome in BC.(25)

Besides, a significant difference in co-alterations (deletion/methylation) of these genes was observed in ER−/PR− BC compared with early onset and ER/PR+ BC samples, suggesting differences in the molecular pathogenesis of the subtypes. This was also evident from the significant association of APC/CDH1 alterations with ER/PR− BC samples and the prevalence of SFRP1/2 alterations alone in ER/PR+ BC samples. Similar differences in the molecular pathogenesis of the different subtypes of BC were observed in the Chi-squared analysis of alterations of these genes.

In the present study we did not find any homozygous deletion of these genes, but their bi-allele alterations were evident in 13–27% of samples, suggesting these as candidate tumor suppressor genes (TSGs) according to the Knudson’s two-hit hypothesis,(42) although other types of gene inactivation mechanisms such as mutation and splice variations of these genes cannot be ruled out in this tumor.(43,44)

Clinicopathological analysis suggests that alterations of the Wnt antagonists are needed for early invasiveness of the disease and have prognostic importance in either BC or ER−/PR− BC patients. Moreover, association of CDH1 alterations and higher histological grades of BC suggest association with tumor progression. In addition, univariate analysis suggests that ER−/PR− patients with parity ≤2, high clinical stage and nodes at pathology have a worst prognosis having alterations of any of the APC/CDH1/SFRP2 genes. In contrast, in multivariate analysis, SFRP2 alterations are suggested to be an important risk factor in patients with parity ≤2 and higher histological grades. The reason for the association of SFRP2 alterations with the above etiological factors in BC is not clear. Similar to our findings, Hill et al.(45) have reported a significant association between methylation of SFRP2 and relapse-free survival of BC patients.

In immunohistochemical analysis, reduced expression of the APC, CDH1 and SFRP1 genes showed concordance with their alterations, suggesting inactivation of these genes was needed for the development of tumor. Also, alterations of some of these genes showed a significant association with nuclear β-catenin expression, indicating stabilization of cytoplasmic β-catenin leading to its migration in the nucleus for activation of the target genes. However, in normal breast tissue β-catenin expression was evident in the cytoplasm of luminal epithelia and the membrane of luminal and myoepithelial cells and was suggested to be associated with the development of breast by controlling several signal cascades such as the CDH1-β-catenin-α-catenin-p120 and SFRP1-Wnt-APC-β-catenin pathways.(46) Thus, aberrant activation of the Wnt/β-catenin pathway in BC might be due to cumulative alterations of CDH1, SFRP1 and APC genes. To the best of our knowledge, this is the first report of this kind on BC. However, reduced CDH1 expression and increased nuclear expression of β-catenin was reported in BC and was suggested to be associated with increased invasiveness of this tumor and poor overall survival in BC patients.(20,21) Reduced expression of SFRP1 has also been reported in a multitude of cancers including BC.(9,12) In addition, nuclear accumulation of β-catenin was evident in the absence of SFRP1 in mammary epithelial cells.(12) Inactivation of APC by genetic or epigenetic events was reported to result in elevated β-catenin levels and subsequent signaling in some tumors;(10) however, no association was found between methylation of APC and nuclear accumulation of β-catenin in breast lobular carcinomas.(16) In contrast, in the BC samples, nuclear expression of APC along with reduced cytoplasmic expression was also observed in 33% of samples. The reason for nuclear expression of APC in BC is not known. It seems that nuclear localization of APC could be due to the presence of its two nuclear export signals and might regulate the function of β-catenin,(47) although another function of APC in the nucleus is not ruled out. Significant reduced expression of APC and CDH1 in the ER−/PR− BC samples along with increased nuclear expression of β-catenin suggests a subtype-specific alteration of Wnt-associated genes in BC. A similar subtype-specific expression of CDH1 was also reported in BC, whereas other studies have failed to confirm these findings.(20,21) A prognostic implication of nuclear β-catenin was also evident in the present study. However, there are conflicting reports regarding the frequency of nuclear β-catenin expression in BC.(48,49) Khramtsov et al.(48) have reported cytosolic and nuclear β-catenin positively correlated with high tumor grade and was negatively associated with ER/PR status of BC patients. Several investigators have also shown association of high β-catenin activity with poor survival in BC patients.(25,50) Recently, phosphorylation/dephosphorylation of β-catenin at specific serine/threonine residues was reported to regulate the nuclear localization and function of β-catenin.(51) Also, ER−/PR− BC patients showed poor survival with alterations in Wnt antagonists indicating subtype-specific alterations of these genes in BC.

Thus, it might be concluded that a differential pattern of alterations as well as association between the alterations of Wnt antagonists exists in ER/PR (+/−) and early/late-onset BC samples, suggesting differences in the mechanism of molecular pathogenesis in various subtypes of BC (Fig. 6). Alterations of Wnt antagonists in 99% of the BC samples suggest that aberrant activation of this pathway plays an important role in the development of this tumor. Also, cumulative alterations in more than one Wnt antagonist are needed for tumorigenesis in BC. Therefore, to understand the precise mechanisms of BC progression, further studies are needed to delineate the effects of the Wnt/β-catenin pathway in ER- and PR-dependent mechanisms.

Figure 6.

 Summary of overall alterations in early/late-onset breast cancer (BC) and estrogen receptor/progesterone receptor (ER/PR) (+/−) BC patients. *P < 0.05 (statistically significant). A, alterations.

Acknowledgments

The authors are thankful to the Director, CNCI, Kolkata, India for active encouragement during this work. Financial support was provided by CSIR grant no. 60(0077)/06/EMR-II to C. K. P. and institutional funding provided by the Director, CNCI, Kolkata, India.

Disclosure Statement

The authors declare that they have no conflict of interests regarding this work.

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