|Year : 2019 | Volume
| Issue : 5 | Page : 1162-1166
Aberrant signal transduction in Indian triple-negative breast cancer patients
Vasantha Kumar Bhaskara1, Chaitra Jayaram1, M Priyanga1, N H Thilak Nayaka1, A Shivakumara1, Nagarathna Amaresh1, Vijaya V Mysorekar2
1 Department of Biochemistry, RCASC, Bengaluru, Karnataka, India
2 Department of Pathology, RMCH, Bengaluru, Karnataka, India
|Date of Web Publication||4-Oct-2019|
Vasantha Kumar Bhaskara
Department of Biochemistry, RCASC, MSR Nagar, MSRIT Post, Mathikere, Bengaluru - 560 054, Karnataka
Source of Support: None, Conflict of Interest: None
Aim of Study: The aim of this study is to correlate the prominin-1 or CD133 association with functional pathway markers of cancer stemness in Indian triple-negative breast cancer (TNBC) patient samples.
Materials and Methods: TNBC samples were confirmed for the absence of hormone receptors (estrogen receptor–ER/progesterone receptor) and human epidermal growth factor receptor-2 or proto-oncogene neu or erbB2 or CD340 by immunohistochemical analysis. Formalin-fixed paraffin-embedded samples of patients were used to collect the total RNA. Then, one-step reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the cancer stemness-related transcript levels in the different samples. The RT-PCR products were analyzed semi-quantitatively on agarose gels. The band intensities of respective samples for different transcripts were analyzed by densitometry.
Results: TNBC-confirmed samples had shown increased levels of CD133 transcript than control tissues. Further, elevated CD133 transcripts are correlated with higher transcript levels of NOTCH1/FZD7/transforming growth factor-beta receptor Type III R/patched-1 pathway mediators.
Conclusions: This work has clearly indicated that there is a correlation between CD133 and functional pathways that control cancer stem cells in TNBC. These observations may indicate the possible association between cancer stemness and TNBC malignancy.
Keywords: Cancer stem cells, FZD7, NOTCH1, patched-1, signal transduction, transforming growth factor-beta receptor Type III R, triple-negative breast cancer
|How to cite this article:|
Bhaskara VK, Jayaram C, Priyanga M, Nayaka N H, Shivakumara A, Amaresh N, Mysorekar VV. Aberrant signal transduction in Indian triple-negative breast cancer patients. J Can Res Ther 2019;15:1162-6
|How to cite this URL:|
Bhaskara VK, Jayaram C, Priyanga M, Nayaka N H, Shivakumara A, Amaresh N, Mysorekar VV. Aberrant signal transduction in Indian triple-negative breast cancer patients. J Can Res Ther [serial online] 2019 [cited 2020 Apr 4];15:1162-6. Available from: http://www.cancerjournal.net/text.asp?2019/15/5/1162/243494
| > Introduction|| |
Breast cancers have been reported to be the second most common form of cancers among women with significant mortality in India and throughout the world. Breast cancers exhibit significant heterogeneity with almost 21 distinct histological subtypes. One of the classifications which is routinely used by clinicians that helps for planning effective treatment regimens is based on the presence or absence of hormone receptors (HR) (estrogen receptor-ER and progesterone receptor – PR) and human epidermal growth factor receptor-2 (HER2/neu). The important molecular subtypes of breast cancer include luminal A (HR +/HER2−), luminal B (HR +/HER2+), HER2-enriched (HR −/HER2+), and triple-negative (HR −/HER2−) breast cancers (TNBCs).
Among all different subtypes, TNBC occurs at an incidence rate of about 31.9% in India. TNBC incidence is reported to be more common in premenopausal women and those with BRCA1 gene mutation. Other risk factors of TNBC incidence include obesity (35%), and women who had never given birth have 40% lower risk for the disease than those who had full-term pregnancy. Women with more than three children were reported to be at a high risk of getting TNBC.
Existence and association of cancer stem cells (CSCs) has been evidenced in many cancers including leukemia and many other solid tumors. The presence of CSCs is an ultimate reason for therapeutic resistance and tumor relapse. Thus, the CSCs will possess tremendous repopulation tendencies and can survive quiescently for extended periods. CSCs will act as the tumor-initiating cells that can Self-renew by pluripotency and with immortality. This subpopulation of cells has shown to be responsible for cancer initiation, progression, metastasis, recurrence, and drug resistance. Although CSC role in cancer malignancy is evidenced, the mechanisms of their formation, survival, and interactions with tumor-microenvironment are not yet clearly understood.
We proposed to correlate the transcript levels of cancer stemness marker (CD133) with that of the most possible signaling pathways associated with cancer stemness. Among TNBC patients, CD133 expression has been shown to be about 2%–20% and with highest expression in the tubulolobular carcinoma types. Further, disease prognosis and CD133 expression have been shown to be associated with NOTCH1, canonical β-catenin pathway,, transforming growth factor-beta (TGF-β), and sonic hedgehog (SHH), pathways in different cancer types. However, its occurrence and association with malignancy is not yet clearly understood. Although our study is limited by sample number, the observations are with significant conclusions.
| > Materials and Methods|| |
The present study is a retrospective pilot study conducted after obtaining the institutional ethical clearance. The formalin-fixed paraffin-embedded (FFPE) samples of nonmalignant breast tissue and TNBCs tissues were collected from the Department of Pathology, MS Ramaiah Medical College and Hospital (MSRMCH), Bangalore, India, for RNA isolation and for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Patient details of the samples used in the present study are presented in [Table 1]. The hematoxylin and eosin (H and E)-stained sections of normal and tumor breast tissues were studied in detail for the morphology.
Patient tissues used in the present study were analyzed by the immunohistochemical analysis to confirm the absence of HRs and HER2. Tissue sections were subjected to immunohistochemistry for analysis of ER/PR/HER2 expression. All the primary antibodies used were monoclonal and purchased from BioGenex, India. Sections were developed using horseradish peroxidase-conjugated detection system with 3,3'-Diaminobenzidine tetra hydrochloride as the chromogen. Positive and negative controls were simultaneously run along with the test samples.
Total RNA isolation
Using a sterilized RNase-free scalpel, excess paraffin was removed from the sample block. Samples were heated up to 60°C in water bath, and supernatant with liquid wax was removed. Then, the tissues were finely minced and transferred to an RNase-free 2 ml microcentrifuge tube. To this, 1 ml of xylene was added and vortexed vigorously and then centrifuged at 10,000 rpm for 2 min. The supernatant was discarded, and the pellet was resuspended in 1 ml ethanol (96%), mixed by vortexing, and then subjected to centrifugation at 10,000 rpm for 2 min. The supernatant was discarded, and pellet was air dried to remove the residual alcohol by keeping the lid open. The pellets were then incubated in RNA lysis solution with 10 μl proteinase K, and further steps of the protocol were followed as per the HiPurA total RNA miniprep purification kit data sheet (Cat#MB602, HIMEDIA, India). Final RNA samples were analyzed by ultraviolet (UV) spectrophotometer for their yield and quantifications.
Reverse transcription-polymerase chain reaction
The transcript levels of desired genes were analyzed using HiScript one-step RT-PCR kit. The following oligonucleotide primers (Xcelris Labs Ltd., India) were used for PCR amplification: CD-133, forward 5'-GCTTTGCAATCTCCCTGTTG-3', reverse 5'-TTGATCCGGGTTCTTACCTG-3'; NOTCH1, forward 5'-GTGACT GCTCCCTCAACTTCAAT-3', reverse 5'-CTGTCACAGTGGCCGTCACT-3'; Hes-1, forward 5'-AGGCGGACATTCCTGGAAATG-3', reverse 5'-CGGTACTTCCCCAGCACATT-3'; FZD7, forward 5'-TTCTCGGACGATGGCTACC-3', reverse 5'-GAACCAAGTGAGAGACAGATGACC-3'; β-catenin, forward 5'-AAAATGGCAGTGCGTTTAG-3', reverse 5'-TTTGAAGGCAGTCTGTCGTA-3'; TGF-βIII, forward 5'-TGGGGTCTCCAGACTGTTTTT-3', reverse 5'-CTGCTCCATACTCTTTTCGGG-3'; SMAD7, forward 5'-CCAACTGCAGACTGTCCAGA-3', reverse 5'-CAGGCTCCAGAAGAAGTTGG-3'; patched-1 (PTCH1), forward 5'-CTCCCAAGCAAATGTACGAGCA-3', reverse 5'-TGAGTGGAGTTCTGTGCGACAG-3'; glioma-associated homolog 1 (GLI-1), forward 5'-CTCCCGAAGGACAGGTATGTAAC-3', reverse 5'-CCCTACTCTTTAGGCACTAGAGTTG-3'; and β-actin, forward 5'-CTGGCACCACACCTTCTAC-3', reverse 5'-CATACTCCTGCTTGCTGATC-3'. The PCR reactions were conducted in total 50 μl reaction volumes as per the instructions of the HiScript one-step RT-PCR kit (Cat#MBT095; HiMedia, India).
Semi-quantitative analysis of reverse transcription-polymerase chain reaction amplicons
The final gene product amplicons were size-fractionated in a 1.8% (W/V) agarose gel, and DNA was visualized using ethidium bromide and UV light. The bands obtained were analyzed for their intensity by densitometry.
| > Results|| |
The patient samples used for RT-PCR were prior analyzed for ER, PR, and HER2 expressions to confirm triple-negative status of breast tumors using immunohistochemistry. The microscopic analysis of H- and E-stained sections of four different breast cancer patient tissues shows the infiltrating malignant epithelial cells arranged in tubular or syncytial pattern. Individual tumor cells are large, round to polygonal, or spindle-shaped with pleomorphic, hyperchromatic nuclei and amphophilic or eosinophilic cytoplasm. Some fields of the sections show cells with high nuclear-cytoplasmic (N/C) ratio, vesicular nucleus, prominent nucleoli, and with numerous atypical mitoses [Figure 1].
|Figure 1: H- and E-stained microphotographs. Fibrofatty mass of breast tissue section showing ductal epithelial component which retained normal bilayered epithelial lining (C). Syncytial sheets of tumor cells which are poorly differentiated and with high nuclear-cytoplasmic (N/C) ratio, vesicular nuclei, and with moderate eosinophilic cytoplasm (S1). Malignant infiltration of tumor cells which are well differentiated and arranged in irregular tubular pattern with large hyperchromatic nuclei and moderate eosinophilic cytoplasm (S2). Moderately differentiated tumor cells are arranged in sheets and cords and singly with pleomorphic hyperchromatic nuclei (S3). Poorly differentiated malignant tumor cells are arranged in sheets and cells invading adipose tissue are seen (S4)|
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Immunohistochemical staining of respective patient tissue sections was carried out for analyzing the status of HRs (ER/PR) and HER2. All the tissues used in the present study are prior confirmed that they are TNBCs [Figure 2].
|Figure 2: Immunohistochemical analysis of estrogen receptor/progesterone receptor/human epidermal growth factor receptor-2. Representative immunohistochemical micrographs of four tumor tissues showing the absence of ER−, PR−, and human epidermal receptor-2 (HER2−) proteins. Positive reactions of estrogen receptor/progesterone receptor antibodies used for this technique were observed as inbuilt positive nonmalignant cells, and a separate positive control section shows human epidermal growth factor receptor-2 reactivity|
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Semi-quantitative reverse transcription-polymerase chain reaction analysis
The product amplicons formed during RT-PCR of respective genes were analyzed by the agarose gel electrophoresis. The expression levels were normalized by comparing to the levels of β-actin as an internal control. All the transcripts of tumor tissues were compared to their corresponding levels in the nonmalignant breast tissue. The expression levels of transcripts in tumors were found to be elevated significantly over the control tissues [Figure 3]. Normalized densitometry has shown that the elevated CD133 transcripts are at the highest in S1 (sample 1) followed by S4 (Sample 4). The same trend was observed with transcripts of canonical Wnt, SHH, NOTCH1, and TGF-βIII R pathway mediators.
|Figure 3: Semi-quantitative reverse transcription-polymerase chain reaction analysis of stemness genes. Semi-quantitative analysis of amplicon products of respective genes during reverse transcription-polymerase chain reaction is analyzed on agarose gels. Densitometry of band intensities represents normalized values of β-actin in respective samples against the control tissue for each gene|
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| > Discussion|| |
Breast cancer classification based on ER/PR/HER2 expression status is the fundamental strategy followed for an effective treatment regimen. Among the different types of breast cancers, about 68.9% are ER/PR+; Her2-types, 10.2% are ER/PR+; Her2+ types, and 7.5% are ER/PR- types; Her2+ types. Although TNBC has been reported to occur at a lesser incidence rate of about 13.4%, it is a highly aggressive and least responsive form of breast cancer. Most commonly TNBC incidence is associated with patients younger than 50 years and with BRCA1 gene mutation. TNBC subset represents highly aggressive forms, and once metastasis has occurred, a median survival of patients will be approximately 1 year. Currently, chemotherapy is similar to HER2-negative forms but with least response. Hence, there is a need for understanding the mechanism of TNBC formation to target more effectively.
Recent advances in cancer biology have evidenced that CSCs are the subset of cancer cells, which are the major cause of tumor repopulation tendencies and therapeutic resistance. Although there is an ample evidence showing the involvement of CSCs in the pathology of many cancer types, the exact mechanisms that control their survival and therapeutic resistance mechanisms have not been clearly understood. Hence, understanding the mechanism and finding the key participants in CSC maintenance is an important objective for an effective drug targeting.
Although the present study is limited by number of samples used, the changes observed in the TNBC samples are significant. The early detection method for ER and PR was on homogenized frozen breast tumor tissues using ligand binding assay and then was started the classification of breast tumors based on the presence or absence of HRs or HER2. Studies have shown that receptor status is related to disease prognosis and it is helpful in treatment regimen. Among all types, breast cancers without having expression for any of the receptors, named as triple-negative breast cancers, are most aggressive form. Immunohistochemistry is an ultimate method to categorize breast tumors as triple negative forms. All the breast tumor tissues used in the present study were diagnosed as triple-negative forms by following standard method of classification.
We have isolated total RNA from FFPE TNBC samples using the standard procedure. The levels of transcripts for different pathways were evaluated by analyzing the membrane receptor and its downstream transcription factor of respective pathways. Our analyses have revealed that in all TNBC tissues, the transcript levels of CD133 is overexpressed and higher levels were found in S1 followed by S4. Importantly, our results indicated that samples #S1 and S4 with higher CD133 transcript levels are with higher transcript expression levels of NOTCH1/Hes-1; FZD7/β-catenin; PTCH1/Gli-1; and TGF-β/SMAD7. These observations may indicate the correlation between CD133 and functional pathway activation in TNBC samples. In glioblastoma multiforme cell lines, it was reported that cancer stem-like side population cells have positive correlation with their CD133 and Oct4 expression and with self-renewal-related stemness genes such as smoothened (SMO), NOTCH3, and Indian hedgehog. Authors have reported that in the same cells, the treatment with honokiol inhibitor has shown induction of apoptosis and downregulation of NOTCH3 and Hes-1 genes. In gastric cancers, FZD7-mediated canonical Wnt/β-catenin along with CD133 was shown to be associated with patient prognosis. Overexpression of SHH pathway mediators including SHH, PTCH1, SMO, and glioma-associated homolog 1 (GLI-1) has been reported to be associated with triple-negative breast cancer patients. The transcription factor GLI-1 has shown to be a critical regulator in CSC survival. Thus, targeting SHH is an important strategy to target CSC pathways. Transforming growth factor-beta receptor Type III (TGF-βIII) is one type of TGF-β receptors which is recently found to be having a role in cancer rather than its tumor suppressor roles. It has been reported that in triple-negative breast cancer cell line of mesenchymal stem-like cells, TGF-βIII receptor exhibits tumor promoter activities.,
| > Conclusion|| |
Our work has clearly evidenced the critical role of functional pathways in the pathology of TNBC mediated by CD133-associated CSCs. This work indicates the necessity of further focus on understanding the role of functional pathways in TNBC stemness.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]