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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 1  |  Page : 13-18
Association of different patterns of expression of beta-catenin and cyclin D1 with pathogenesis of breast carcinoma


1 Department of Pathology and Surgery, M.L.N. Medical College, Allahabad, Uttar Pradesh, India
2 Department of Onco-Surgery, Kamla Nehru Memorial Hospital, Allahabad, Uttar Pradesh, India

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Date of Web Publication31-Jan-2020
 

   Abstract 


Background: Beta-catenin and cyclin D1 have attracted considerable attention in recent studies as potential proto-oncogenes in many human cancers especially colonic cancer. Beta-catenin plays multiple roles within the cell such as canonical Wnt signaling where cyclin D1 has been identified as one of its target genes. The role of beta-catenin and cyclin D1 in breast cancer has been evaluated in many studies but not established yet. Materials and Methods: The expression of beta-catenin and cyclin D1 was evaluated in 82 cases of breast carcinoma (BCa) and 32 cases of ductal carcinoma in situ(DCIS) by immunohistochemistry (IHC). Their relationship with clinicopathological features was also investigated. Statistical analysis was done to establish an association. Results: Abnormal expression of beta-catenin (ABE) was seen in 80.2% cases of invasive ductal carcinoma (IDC) and 47% cases of DCIS, while the cyclin D1 positive expression rate was 60.9% and 50%, respectively. In the cases showing ABE, cyclin D1 positivity was 88.1%. ABE showed significant association with high-grade BCa. The most common pattern of ABE was loss of membrane with nuclear positivity which is associated with worst prognosis. In addition, ABE in cases of BCa and DCIS showed concordant patterns. Conclusion: Therefore, an association exists between ABE and cyclin D1 in BCa and its precursor lesions implying that Wnt/beta-catenin oncogenic pathway may have a definite role in breast carcinogenesis and can be used for targeted therapy. Also, different patterns of beta-catenin expression may have prognostic and predictive value.

Keywords: Beta-catenin, breast carcinoma, cyclin D1, Wnt signaling pathway

How to cite this article:
Varma K, Chauhan A, Bhargava M, Misra V, Srivastava S. Association of different patterns of expression of beta-catenin and cyclin D1 with pathogenesis of breast carcinoma. Indian J Pathol Microbiol 2020;63:13-8

How to cite this URL:
Varma K, Chauhan A, Bhargava M, Misra V, Srivastava S. Association of different patterns of expression of beta-catenin and cyclin D1 with pathogenesis of breast carcinoma. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Jul 5];63:13-8. Available from: http://www.ijpmonline.org/text.asp?2020/63/1/13/277383





   Introduction Top


Breast cancer is one of the most common carcinomas in women. Its incidence in India is rising and it is associated with significant morbidity as it affects patients at a younger age and most of the cases are detected at an advanced stage.[1] Newer molecular markers have aided in the discovery of novel pathway-specific targeted therapeutics and “patient-tailored” treatment planning. Recent studies are targeting Wnt/beta-catenin signaling pathway as a therapeutic target in many tumors especially colorectal carcinomas.[2],[3]

Beta-catenin is a ubiquitous protein. In addition to its role in regulating cell–cell adhesion through interactions with E-cadherin, beta-catenin has a critical role in the highly conserved Wnt signaling pathway and carcinogenesis. The beta-catenin pool that functions in Wnt signaling is regulated in part by a multiprotein complex, consisting of the adenomatous polyposis coli (APC) tumor suppressor, Axin, and glycogen synthase kinase 3β (GSK3β) proteins. Mutations in Wnt pathway cause elevation of beta-catenin levels in the cytoplasm and nucleus which leads to altered expression of TCF-regulated target genes. The proteins encoded by these TCF-regulated genes presumably play important roles in affecting the neoplastic transformation. Certain studies have shown that c-myc and cyclin D1 are the target oncogenes in the Wnt/beta-catenin pathway.[4],[5],[6]

Of the published studies on the role of beta-catenin, Wnt pathway, and cyclin D1 in carcinogenesis, nearly all have focused on colorectal cancers; however, few studies have explored their role in breast carcinomas (BCas). Also, contradictory associations have been reported among beta-catenin expression, clinicopathological variables, and disease outcome.[4],[5],[6],[7],[8],[9] Therefore, this study was undertaken to establish the role of beta-catenin and cyclin D1 in BCa and its precursor lesions.


   Materials and Methods Top


Tumor samples

A total of 82 cases of BCa histologically classified as invasive ductal carcinoma

(IDC) and 32 cases of ductal carcinoma in situ(DCIS) were studied in detail for histological and prognostic factors including tumor size, histological grade, axillary lymph node (ALN) status, estrogen receptor (ER), progesterone receptor (PR), and Her-2 neu status. Histological grading was done using Modified Bloom Richardson's Grading (MBR). Follow-up for survival, cancer recurrence, and distant metastasis was done wherever possible.

Immunohistochemical analyses of beta-catenin and cyclin D1

Immunohistochemistry (IHC) was done for beta-catenin and cyclin D1. Five-micrometer sections of paraffin-embedded tissue were deparaffinized in xylene and rehydrated into distilled water through graded series of ethanol solutions. The endogenous peroxidase activity was blocked by 10 min incubation in 3% hydrogen peroxide/methanol buffer, and the sections were then rinsed in phosphate-buffered saline (PBS). The slides were incubated with normal bovine serum for 30 min at room temperature to reduce the nonspecific background staining. To enhance the immunoreactivity, microwave antigen retrieval was performed for 15 min in a citrate buffer (pH 6.0). The sections were subsequently incubated with primary antibodies and were then incubated with a rabbit monoclonal anti-beta-catenin (EP 35; BioGenex, Fremont, USA) at a dilution of 1:500 and anti-cyclin D1 antibody (EPR 2241; BioGenex) at a dilution of 1:50 at room temperature. After washing the sections, they were incubated with biotinylated secondary antibody for 30 min at room temperature and were rinsed with PBS. Antigen–antibody complexes were visualized using a peroxidase-conjugated streptavidin with 3,3′-diaminobenzidine as a chromogen. The slides were counterstained with hematoxylin, rinsed in tap water, and mounted. Adequate control samples were put in parallel. Semi-quantitative grading of beta-catenin and cylin D1 was done as shown below. The adjacent normal breast tissue was used as an internal positive control for beta-catenin.

The beta-catenin expression on IHC was classified as normal and abnormal as follows:

Normal expression: The normal expression of beta-catenin was defined as exclusively complete membranous staining (>70%) of a similar intensity to that in the adjacent normal epithelia. The cases with strong membranous staining with faint granular cytoplasmic staining were considered normal [Figure 1]a.
Figure 1: Different patterns of beta-catenin and cyclin D1 expression. (a) Strong membranous staining with faint granular cytoplasmic staining (IHC, ×40). (b) Grade 1: Loss of membranous staining (IHC, ×40). (c) Grade 2: LOM with brown granular cytoplasmic staining and without nuclear staining (IHC, ×40). (d) Grade 3: LOM with brown nuclear staining (IHC, ×40). (e) Cyclin D1 overexpression: Intense brown nuclear positivity in 40% cells (IHC, ×40). (f) LOM with cytoplasmic positivity in foci of ductal carcinoma in situ(IHC, ×40)

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Abnormal beta-catenin expression (ABE) was graded as follows:[5]

  • Grade 1: Complete or partial loss of membranous staining (LOM) without cytoplasmic staining and nuclear staining [Figure 1]b
  • Grade 2: LOM with brown granular cytoplasmic staining and without nuclear staining [Figure 1]c
  • Grade 3: LOM with brown nuclear staining and with/without cytoplasmic staining [Figure 1]d.


Analysis of cyclin D1 expression

A brown intense nuclear positivity was considered positive. A semi-quantitative grading was done as follows:[5]

<10% regarded as negative,

10%–50% regarded as positive [Figure 1]e.

>50% regarded as overexpression.

Ethics

No ethical clearance was required for our study.

Statistical analysis

Statistical analysis for the relationship between the ABE and the clinicopathological parameters was performed using Chi-square test or Fisher's exact test. The results were considered to be statistically significant when the P values were less than 0.05. All statistical analyses were conducted using the SPSS 11.0 statistical software program (SPSS Inc., Chicago, IL, USA).


   Results Top


The mean age of patients was 51 years. In most of the cases, 91% tumors were high-grade (91%) IDC. ALN metastasis was present in 70% of our cases. In addition, IHC hormonal status showed 34% of cases to be ER-positive and 37% were triple-negative breast carcinoma (TNBC).

It was seen that normal breast epithelium showed strong normal membranous expression (>70%) of beta-catenin in the epithelium. Faint cytoplasmic staining was sometimes seen, but no nuclear staining was seen in normal ducts and lobules. ABE was seen in 80% of cases of BCa, and this value was statistically significant. This ABE showed different patterns which are shown in [Figure 1]a [Figure 1]b [Figure 1]c [Figure 1]d. The most common pattern observed was LOM with nuclear positivity (Grade 3) in 41% of cases followed by LOM with cytoplasmic positivity (Grade 2) in 35% of cases, whereas LOM (Grade 1) was seen in only 4% of cases as shown in [Table 1].
Table 1: Different patterns of beta-catenin expression on IHC

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The expression patterns of beta-catenin were compared between cases of DCIS and IDC as shown in [Figure 2]. Abnormal expression was seen in 15 of 32 (47%) cases of DCIS [Figure 1]f which was statistically significant when compared with controls. ABE was more commonly seen in high-grade DCIS and high MBR grade IDC, whereas normal beta-catenin expression was seen more commonly in low-grade DCIS and low-MBR grade IDC. DCIS and IDC showed concordance in 20 of 32 (62%) cases of ABE.
Figure 2: Normal and abnormal beta-catenin expression in different grades of DCIS and breast carcinoma

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Cyclin D1 positivity was seen in 50 of 82 (61%) cases of IDC [Figure 1]e. The association between beta-catenin and cyclin D1 was also evaluated in our study as shown in [Table 2]. Although not statistically significant, 88% of cases with cyclin D1 positivity had ABE. Cyclin D1 expression was graded and its different grades of expression were compared with different grades of beta-catenin expression as shown in [Table 3]. It was found that as cyclin D1 expression grades increased, there was a higher percentage of ABE. Hence, a positive association is seen with increasing degree of cyclin D1 positivity and loss of membrane with nuclear with/without cytoplasmic expression.
Table 2: Correlation between expression of ABE expression and cyclin D1 in breast carcinoma

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Table 3: Comparison of different patterns of beta-catenin expression with different grades of cyclin D1 positivity

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ABE and cyclin D1 expression was correlated with prognostic factors, that is, age, tumor size, histological grade, and ALN status as shown in [Table 4]. Significant association was found between tumor grade and ABE. All cases of MBR Grade 1 had normal expression of beta-catenin, whereas 56% and 60% of MBR Grades 2 and 3 had ABE and these findings were statistically significant. Hence, as the lesion progresses from benign, premalignant to malignant, ABE increases and normal membranous expression decreases [Table 2]. Only 25 (30%) cases were available for follow-up, of which 3 (12%) cases presented with a recurrence. Hence, comprehensive survival data could not be analyzed.
Table 4: Correlation between beta-catenin expression, cyclin D1, and clinicopathological factors of breast carcinoma

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   Discussion Top


It has been found that beta-catenin causes tumorigenesis by Wnt pathway. Normally, in the absence of Wnt signaling, cytoplasmic beta-catenin is maintained at a low level through ubiquitin-proteasome-mediated degradation. When Wnt pathway is activated through its receptors and dishevelled (Dvl), there is formation of a multiprotein destruction complex comprising Axin, APC, GSK, and dvl which leads to inactivation of GSK3B, and hence, beta-catenin degradation does not occur and its cytoplasmic level increases.[10] This beta catenin translocates to the nucleus where it forms a complex with a T-cell factor that upregulates cellular proliferation by increasing the transcription of several genes like cyclin D1 involved in the cell cycle.[3],[11],[12],[13]

Although many studies have found ABE in BCa, its role in this disease is not established yet and needs extensive research. In the studies by Lin et al., Lim and Lee, and López-Knowles et al.,[9] Lin et al. detected the beta-catenin expression levels of 123 breast cancer samples using an immunohistochemical method and found that ABE represented the activated state of beta-catenin and that beta-catenin was an independent prognostic factor for breast cancer survival. Lin et al. did a study of 123 BCa cases and evaluated both cyclin D1 and beta-catenin activity. In their study, multivariate analysis was done to look for the survival rate which showed that beta-catenin was an independent prognostic and a predictive marker for BCa.[12] However, Chung et al. indicated that abnormal expression of beta-catenin was not associated with prognosis. ABE was noted in more than 80% of our cases which is similar to other studies where its incidence varies from 66% to 79%.[7],[8],[9],[10] This fact implies that the Wnt/beta-catenin pathway is activated in breast cancers.

ABE shows different patterns of expression in the cells which are determined by the subcellular localization.[4] Normally, it is present in the membrane and this is seen as exclusive membranous staining on IHC which was the pattern we observed in normal ductal epithelium, low-grade DCIS and 20% of cases of IDC. As its level increases, the accumulated beta-catenin localizes in the cytoplasm (Grade 2) and then in nucleus (Grade 3). A higher percentage of nuclear positivity was noted as most of our cases were high-grade BCa and TNBC. Prasad et al. in their study have demonstrated an association between nuclear localization of Dvl protein and beta-catenin which supports the hypothesis that Dvl is responsible for nuclear translocation of beta-catenin and consequent activation of the Wnt/beta-catenin pathway. Dvl induces stabilization of beta-catenin and subsequent nuclear translocation.[10]

Brabletz et al. postulated that abnormal accumulation of beta-catenin in the nucleus resulted in the loss of E-cadherin and consequent loss of cell polarity and cell adhesion, and hence shows tumor progression.[14] Nakopoulou et al. examined 141 breast cancer specimens and reported that nuclear beta-catenin expression correlated with reduced overall survival (OS) and disease-free survival (DFS) times, while cytoplasmic beta-catenin expression was associated with longer OS and DFS times.[15] Extensive literature review shows that ABE increases with increasing MBR grade. Increased beta-catenin expression is seen in TNBC and basal-like phenotype.[6] These findings suggest that beta-catenin may be a marker of advanced breast cancer. This may represent a new diagnostic or therapeutic target for detection of advanced cancers especially TNBC. Niclosamide is being used as an effective inhibitor of Wnt/β-catenin signaling in several cancers, including colon, breast, and myeloma. Newer agents like benzimidazoles with improved specificity are now under trials.[16] Wnt/beta-catenin pathway can also contribute to immune invasion in BCa and studies have implicated that stromal infiltration by lymphocytes (TILs) may be associated with beta-catenin overexpression in BCa. TILs may also limit response to treatment in such cases.[17],[18]

ABE was also significantly increased in cases of DCIS. In a study of 121 cases by Karayiannakis et al., ABE was observed in 54% of DCIS with highly concordant beta-catenin expression patterns in the nearby in situ and invasive components.[19] In our study, the expression pattern of beta-catenin in DCIS when compared with IDCs showed concordance in 62% of cases. Similar expression patterns of β-catenin in BCa and DCIS suggest that changes in beta-catenin expression occur early in carcinogenesis. To date, several genetic analysis have been done on potential precursor lesion of IDC specially DCIS, and hence there may be similar genetic alterations in these two lesions.

Extensive literature search shows that the association between beta-catenin and cyclin D1 expression in breast cancer tissue remains controversial. The studies by Ozaki et al., Yang et al., and Lin et al. showed that ABE was associated with overexpression of cyclin D1. Lin et al. demonstrated that beta-catenin can function as an oncogene and correlated significantly with beta-catenin activation in cell cultures in vitro as well as in samples from breast cancer. They also found that high beta-catenin activity significantly correlated with poor prognosis of those cases. Similarly, Ozaki et al. did mutational analysis of cyclin D1, c-myc, Axin 1 genes, and beta-catenin which are components of the Wnt signaling pathway and APC gene. They concluded that the reduced expression of APC and overexpression of cyclin D1 and c-myc were statistically correlated with overexpression of beta catenin, and hence alterations in Wnt signaling pathway may be associated with carcinogenesis of breast cancer. However, Lim and Lee indicated that ABE was not associated with cyclin D1 overexpression.[8],[9],[10],[20],[21] Studies have shown that ER-β expression is associated with higher cyclin D1 expression and this could be connected with the Wnt signaling pathway through beta-catenin.[9] In our study, ABE was seen in 66 (80%) cases, among which 44 (66%) cases were cyclin-D1-positive. In a similar study by Prasad et al., cyclin D1 overexpression was found in 60% of IDC. These results indicated that Wnt signaling is overactivated in breast cancer tissues, resulting in ABE which may induce or activate cyclin D1 overexpression, leading to the occurrence and development of breast cancer. We also did a comparative study of grades of cyclin D1 expression with patterns of expression of ABE and found a positive association between increasing degree of cyclin D1 positivity and loss of membrane with nuclear pattern. This was a novel finding in our study and it suggests that cyclin D1 expression and higher grades of beta catenin expression could be poor prognostic indicators.


   Conclusion Top


In conclusion, an association exists between ABE and, cyclin D1 in BCa and its precursor lesions. Hence, Wnt/beta-catenin oncogenic pathway may have a definite role in breast carcinogenesis and may have a role in targeted therapy. ABE pattern, that is, LOM with nuclear expression of beta-catenin and overexpression of Cyclin D1 are associated with higher grades and TNBC and may be used as prognostic and predictive markers. We believe that our findings provide important information about potential prognostic breast cancer marker, that is, beta-catenin and cyclin D1 in BCa. However, owing to the small sample size and advanced stage of disease in most of our cases, more studies are needed to elucidate the underlying mechanisms of breast cancer and the impact of above markers in BCa.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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López-Knowles E, Zardawi SJ, McNeil CM, Millar EK, Crea P, Musgrove EA, et al. Cytoplasmic localization of beta-catenin is a marker of poor outcome in breast cancer patients. Cancer Epidemiol Biomarkers Prev 2010;19:301-9.  Back to cited text no. 9
    
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Prasad CP, Gupta SD, Rath G, Ralhan R. Wnt signaling pathway in invasive ductal carcinoma of the breast: Relationship between beta-catenin, dishevelled and cyclin D1 expression. Oncology 2007;73:112-7.  Back to cited text no. 13
    
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Brabletz T, Jung A, Reu S, Porzner M, Hlubek F, Kunz-Schughart LA, et al. Variable beta-catenin expression in colorectal cancers indicates tumour progression driven by tumour microenvironment. Proc Natl Acad Sci U S A 2001;98:10356-61.  Back to cited text no. 14
    
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Ozaki S, Ikeda S, Ishizaki Y, Kurihara T, Tokumoto N, Iseki M, et al. Alterations and correlations of the components in the Wnt signaling pathway and its target genes in breast cancer. Oncol Rep 2005;14:1437-43.  Back to cited text no. 21
    

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Correspondence Address:
Kachnar Varma
Department of Pathology, M.L.N. Medical College, Allahabad, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_419_19

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